REVIEW | doi:10.20944/preprints202001.0243.v1
Subject: Biology, Plant Sciences Keywords: DOF proteins; DELLA proteins; seed germination; seedling development; seed maturation
Online: 21 January 2020 (11:16:52 CET)
The DOF (DNA binding with one finger) family of plant-specific transcription factors (TF) was first identified in maize in 1995. Since then, DOF proteins have been shown to be present in the whole plant kingdom including the unicellular alga Chlamydomonas reinhardtii. The DOF TF family is characterised by a highly conserved DNA binding domain (DOF domain), consisting of a CX2C-X21-CX2C motif which is able to form a zinc finger structure. Early in the study of DOF proteins it became clear their relevance for seed biology. Indeed, the Prolamine Binding Factor (PBF), one of the first DOF proteins characterised, controls the endosperm-specific expression of the zein genes in maize. Subsequently, several DOF proteins from both monocots and dicots have been shown to be primarily involved in seed development, dormancy and germination, as well as in seedling development and other light-mediated processes. In the last two decades the molecular network underlying these processes have been outlined, and the main molecular players and their interactions have been identified. In this review, we will focus on the DOF TFs involved in these molecular networs, and on their interaction with other proteins.
ARTICLE | doi:10.20944/preprints201905.0203.v1
Subject: Biology, Agricultural Sciences & Agronomy Keywords: Bt toxins; insecticidal proteins; trypsin cleavage; tetrameric proteins; domain map
Online: 16 May 2019 (10:25:26 CEST)
Vip3 proteins are increasingly used in insect control in transgenic crops. To shed light on the structure of these proteins, we used the approach of trypsin fragmentation of mutants altering the conformation of the Vip3Af protein. From an alanine scanning on Vip3Af, we selected mutants with an altered proteolytic pattern. Based on the protease digestion patterns, their effect on oligomer formation, and theoretical cleavage sites, we generated a map of the Vip3Af protein with five domains, which match some of the domains proposed independently by two in silico models. Domain I ranges from aa12-198, domain II from aa199-313, domain III from aa314-526, domain IV from aa527-668 and domain V from aa669-788. The effect of some of the mutations on the ability to form a tetrameric molecule revealed that domains I-III are required for tetramerization, while domain V is not. The involvement of domain IV in the tetramer formation is not clear. Some mutations distributed from near the end of domain I up to the end of domain II affect the stability of the first three domains of the protein and negatively impact oligomerization upon trypsin treatment. Because of the high sequence similarity among Vip3 proteins, we propose that our domain map can be extended to many other members of the Vip3 family of proteins.
ARTICLE | doi:10.20944/preprints201801.0137.v1
Subject: Life Sciences, Biotechnology Keywords: ice-binding proteins; antifreeze proteins, cold finger, ice affinity purification.
Online: 16 January 2018 (07:56:40 CET)
Ice-binding proteins (IBPs) have several functions that permit their hosts to thrive in the presence of ice. The ability of IBPs to control ice growth makes them potential additives in various industries ranging from food storage and cryopreservation to anti-icing systems. For IBPs to be used in commercial applications, however, methods are needed to produce sufficient quantities of high-quality proteins. Here, we describe a new method for IBP purification, termed falling water ice purification (FWIP). The method is based on the affinity of IBPs for ice. A crude IBP solution is allowed to flow continuously over the large chilled vertical surface of a commercial ice machine. The temperature of the surface is lowered gradually until ice crystals are produced, to which the IBPs bind but other solutes do not. As in other ice affinity methods, FWIP does not require molecular tags and is suitable for purifying recombinant IBPs as well as IBPs from natural sources. The advantage of FWIP over other ice affinity methods is that it exploits an ice machine designed to produce large volumes of clear ice daily. This system can be easily scaled up and suits the purification of industrial quantities of IBPs. The FWIP method significantly advances the use of IBPs in research and industry.
REVIEW | doi:10.20944/preprints202008.0225.v1
Subject: Biology, Other Keywords: Heat-stable proteins; Heat treatment; Hyperthermophilic proteins; Heat stability; Protein purification
Online: 9 August 2020 (22:00:46 CEST)
Proteins possess complex three-dimensional structures, and these structures are stable only within specific ranges of temperature which mostly correspond to the temperature ranges of the host organisms. However, few exceptional proteins, called heat-stable proteins, are stable at temperatures that are substantially higher than those tolerated by the host organisms themselves. Most of the heat-stable proteins possess heat stability to perform their functions at high temperatures, but some of them are intrinsically heat-stable due to their structure. Heat-stable proteins are usually divided into three or four groups depending upon the intricacies of their structures and thermal behaviors. Their peculiar property, i.e. heat-stability, makes them very valuable in applications such as polymerase chain reaction, industrial processes requiring high temperature, and protein engineering. Heat-stability also makes it feasible to purify such proteins, from the rest of the heat-labile proteins, using a simple heat-treatment method. Moreover, heat treatment can be used as a combined cell-lysis and protein purification step which, as compared to conventional methods, can result in a higher yield of heat-stable proteins. Furthermore, some special heat-stable proteins, i.e. intrinsically disordered proteins (which include the proteins involved in important neurodegenerative diseases), need heat-treatment step, in some cases, as the only way for their successful purification and study. Hence, this paper provides a first-ever comprehensive review of all major aspects of heat-stable proteins, i.e., their structure, evolution, classification, significance, and heat-treatment mediated purification.
Subject: Life Sciences, Biochemistry Keywords: Insecta; Chemosensory proteins; Odorant binding proteins; Peptide mutation; Cell evolution; Abiogenesis
Online: 30 January 2020 (03:02:21 CET)
We remind about the dogma initially established with the nucleic acid double helix, i.e. the DNA structure as the primary source of life. However, we bring into the discussion those additional processes that were crucial to enable life and cell evolution. Studying chemosensory proteins (CSPs) and odor binding proteins (OBPs) of insects, we have found a high level of pinpoint mutations on the RNA and peptide sequences. Many of these mutations are found to be tissue-specific and induce subtle changes in the protein structure, leading to a new theory of cell multifunction and life evolution. Here, attention is given to RNA and peptide mutations in small soluble protein families known for carrying lipids and fatty acids as fuel for moth cells. A new phylogenetic analysis of mutations is presented and provides even more support to the pioneer work, i.e. the finding that mutations in binding proteins have spread through moths and various groups of insects. Then, focus is given to specific mechanisms of mutations that are not random, change α-helical profilings and bring new functions at the protein level. In conclusion, RNA and peptide mutations are not seen as representative of a multitude of diseases, but rather as an alternative way by which protocells developed to acquire multifunction and totipotency. This provides a basis for the theory of RNA/peptide mutations for birth and evolution of life on earth’s crust proposed here.
REVIEW | doi:10.20944/preprints202107.0218.v1
Subject: Keywords: arabinogalactan proteins; proton pump; auxin; calcium signaling; Hechtian oscillator; PIN proteins; morphogenesis.
Online: 9 July 2021 (12:52:11 CEST)
Novel molecular pinball machines of the plasma membrane control cytosolic Ca2+ levels that regulate plant metabolism. [https://youtu.be/zABg7LiBk88] Essential components involve: 1. an auxin-activated proton pump; 2. arabinogalactan glycoproteins (AGPs); 3. Ca2+ channels; 4. auxin-efflux “PIN” proteins. Typical pinball machines release pinballs that trigger various sound and visual effects. However, in plants “proton pinballs” eject Ca2+ bound by paired glucuronic acid residues of numerous glycomodules in periplasmic AGP-Ca2+. Freed Ca2+ ions flow down the electrostatic gradient through open Ca2+ channels into the cytosol thus activating numerous Ca2+-dependent activities.Clearly cytosolic Ca2+ levels depend on activity of the proton pump, the state of Ca2+ channels and size of the periplasmic AGP-Ca2+ capacitor: Proton pump activation is a major regulatory focal point tightly controlled by the supply of auxin: auxin efflux carriers conveniently known as “PIN” proteins [null mutants are pin-shaped!] pump auxin from cell to cell. Mechanosensitive Ca2+ channels and their activation by reactive oxygen species (ROS) are yet another factor regulating cytosolic Ca2+.Cell expansion also triggers proton pump/pinball activity by mechanotransduction of wall stress via Hechtian adhesion thus forming a Hechtian oscillator that underlies cycles of wall plasticity and oscillatory growth.Finally, Ca2+ homeostasis of plants depends on cell surface external storage as source of dynamic Ca2+, unlike the internal ER storage source of animals where the added regulatory complexities ranging from vitamin D to parathormone contrast with the elegant simplicity of plant life. This paper summarises a sixty year Odyssey.
REVIEW | doi:10.20944/preprints202002.0290.v1
Subject: Life Sciences, Biophysics Keywords: protein folding; molten globule state; two-state proteins; non-two-state proteins
Online: 20 February 2020 (07:12:46 CET)
From experimental studies of protein folding, it is now clear that there are two types of folding behavior, i.e., two-state folding and non-two-state folding, and understanding the relationships between these apparently different folding behaviors is essential for fully elucidating the molecular mechanisms of protein folding. This article describes how the presence of the two types of folding behavior has been confirmed experimentally, and discusses the relationships between the two-state and the non-two-state folding reactions, on the basis of available data on the correlations of the folding rate constant with various structure-based properties, which are determined primarily by the backbone topology of proteins. Finally, a two-stage hierarchical model is proposed as a general mechanism of protein folding. In this model, protein folding occurs in a hierarchical manner, reflecting the hierarchy of the native three-dimensional structure, as embodied in the case of non-two-state folding with an accumulation of the molten globule state as a folding intermediate. The two-state folding is thus merely a simplified version of the hierarchical folding caused either by an alteration in the rate-limiting step of folding or by destabilization of the intermediate.
Subject: Biology, Other Keywords: nucleoid-associated proteins (NAPs); moonlighting proteins; drug target; biofilm; specificity determination; phylogenetic analysis
Online: 7 June 2020 (09:07:56 CEST)
Nucleoid-associated proteins (NAPs) play an architectural role by bending, bridging, and wrapping the DNA along with a regulatory role of controlling various transcriptional units in the cell. Previews reviews have highlighted the role of HU and its paralog IHF plays in intracellular function as a transcriptional regulator, nucleoid bending protein and sometimes also moonlights in other functions. This review highlights along with the canonical functions of HU and IHF which affects genes responsible for translational machineries, cell wall biosynthesis, aerobic respiration and virulence ; other non-canonical roles which HU plays outside the cellular milieu, notably in acting as an adhesin and playing role in host-cell adhesion, its role in biofilm architecture and its association with cationic low complexity region, resembling histone like H1 proteins. HU and IHF thus has evolved as a hub protein performing a vast type of functions which makes it a important drug target for antibacterial therapy.
ARTICLE | doi:10.20944/preprints202301.0221.v1
Subject: Mathematics & Computer Science, Artificial Intelligence & Robotics Keywords: Ion channels; Membrane proteins; Transmembrane proteins; Drug discovery; Protein language models; Convolutional Neural Network
Online: 12 January 2023 (09:21:08 CET)
Ion channels are integral membrane proteins that facilitate the movement of ions across cell membranes, playing a key role in a range of biological processes. The high cost and time required for wet lab experiments to characterize ion channels has spurred the development of computational methods for this purpose. In our previous work, we demonstrated the effectiveness of protein language models for ion channel prediction, using a logistic regression classifier to distinguish ion channels from non-ion channels (TooT-BERT-C) and transporters from non-transporters (TooT-BERT-T). In this study, we build upon this approach by using a combination of classical machine learning classifiers and a Convolutional Neural Network (CNN) with fine-tuned representations from ProtBERT, ProtBERT-BFD, and MembraneBERT to discriminate ion channels from non-ion channels. The results of our experiments demonstrate that TooT-BERT-CNN-C, a combination of the representations from ProtBERT-BFD and a CNN, outperforms existing state-of-the-art methods for predicting ion channels, with a Matthews Correlation Coefficient (MCC) of 0.86 and an accuracy of 98.35% on an independent test set.
ARTICLE | doi:10.20944/preprints201705.0015.v1
Subject: Life Sciences, Molecular Biology Keywords: fusion proteins; protein therapeutics; ricin; pokeweed antiviral protein; protein engineering; immunotoxins; ribosome-inactivating proteins.
Online: 1 May 2017 (10:51:21 CEST)
Fusion protein therapeutics engineering is advancing to meet the need for novel medicine. Herein, we further characterize the development of novel RTA & PAP-S1 antiviral fusion proteins. In brief, RTA/PAP-S1 and PAP-S1/RTA fusion proteins were produced in both cell free and E. coli in vivo expression systems, purified by His-tag affinity chromatography, and protein synthesis inhibitory activity assayed by comparison to the production of a control protein, CalmL3. Results showed that the RTA/PAP-S1 fusion protein is amenable to standardized production and purification and has both increased potency and less toxicity compared to either RTA or PAP-S1 alone. Thus, this research highlights the developmental potential of novel fusion proteins with reduced cytotoxic risk and increased potency.
REVIEW | doi:10.20944/preprints202101.0582.v1
Subject: Medicine & Pharmacology, Allergology Keywords: Breast cancer; CCN proteins; Metastasis; Tumorigenesis
Online: 28 January 2021 (12:38:09 CET)
CCNs are specific type of matricellular proteins, which are essential signaling molecules, and play multiple roles in multicellular eukaryotes. This family of proteins consists of six separate members in mammals. The architecture of CCN proteins is multimodular and comprises four distinct motifs. CCN proteins achieve their specific physiological functions by binding to integrin receptors. The CCN family has been implicated in both cure and disease with impacts on biological interactions, such as cell adhesion, chemotaxis and migration, mitogenesis, cell survival, angiogenesis, differentiation, tumorigenesis, immune functions, chondrogenesis, and wound healing. Breast cancer is the most commonly diagnosed cancer worldwide and the leading cause of cancer mortality among women triggered by atypical expression of CCNs. A favorable or unfavorable association between various CCNs has been reported in patients with breast carcinomas. Aberrant expression of CCN1 intensifies the proliferation of epithelial cells that line the lobes and ducts of the breast. Evidence also shows that the expression of CCN2 can ameliorate tumor growth and metastasis. However, CCN3 (NOV), CCN5 (WISP-2), and CCN6 (WISP-3) are consistent with neoplastic development and metastasis repression. Particular CCN members can develop tumors and cancer progression, whereas others can competitively counter the processes. Several studies have been conducted on CCN proteins and cancer in recent years. In our study, we intend to provide an overview of those research works while keeping breast carcinoma on focus. We believe that the importance of the CCN protein family in breast cancer should be reconsidered.
REVIEW | doi:10.20944/preprints201901.0115.v1
Subject: Life Sciences, Biotechnology Keywords: Motion; Inert; Mechanical stimulation; Sensory proteins
Online: 11 January 2019 (15:51:51 CET)
Thought runs through the mind like blood runs through our body to keep us alive. Like the mind, the body does not stay inert and is in constant motion. Not a single cell in our body is left inert unless cell is under stress or dying. These scenarios are reflected upon when a person is sick, the person lies in bed with less movement; however, is active when the person is healthy. The topic of mechanical stimulation has emerged due to the increasing understanding of the physical stimulations we face each day. Further understanding of the mechanically-regulated mechanism can help us explore the pathological events in a disease. Here, we reviewed the role of sensory proteins in pathological events that are observed in cardiomyopathy, cancer, respiratory, renal, obesity, genetics, physical injury and bacterial infection. Taken together, sensory proteins are mechanically-activated which assist reception of external physical stimulation and convert into biochemical to trigger intracellular signaling cascade.
ARTICLE | doi:10.20944/preprints202212.0323.v2
Subject: Life Sciences, Virology Keywords: Apoptosis; Dengue Virus; microRNAs; Viral Nonstructural Proteins
Online: 26 January 2023 (10:47:59 CET)
The World Health Organization has estimated an annual occurrence of approximately 392 million Dengue virus (DENV) infections in more than 100 countries where the virus is endemic, and this represents a serious threat to humanity. DENV is a serologic group with four distinct serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) belonging to the genus Flavivirus, in the family Flaviviridae. Dengue is the most widespread mosquito-borne disease in the world. The ~10.7 kb DENV genome encodes three structural proteins (capsid [C], pre-membrane [prM], and envelope [E]) and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The NS1 protein is a membrane-associated dimer and a secreted, lipid-associated hexamer. Dimeric NS1 is found on membranes both in cellular compartments and cell surfaces. Secreted NS1 (sNS1) is often present in patient serum at very high levels, which correlates with severe dengue symptoms. This study was conducted to discover how NS1 protein, microRNAs-15/16 (miRNAs-15/16), and apoptosis are related during DENV-4 infection in human liver cell lines. Huh 7.5 and HepG2 cells were infected with DENV-4, and miRNAs-15/16, viral load, NS1 protein, and caspases-3/7 were quantified after different times of infection. This study demonstrated that miRNAs-15/16 are overexpressed during infection of HepG2 and Huh 7.5 cells by DENV-4 and have a relationship with NS1 protein expression, viral load, and activity of caspases-3/7, thus making these miRNAs potential injury markers during DENV infection in human hepatocytes.
ARTICLE | doi:10.20944/preprints201810.0355.v1
Subject: Chemistry, Medicinal Chemistry Keywords: assay; diarrhea; isolate; hydrolysis; proteins; inhibition zone
Online: 16 October 2018 (11:26:59 CEST)
The study compared antibacterial potential of hydrolysates of casein and alpha-lactalbumin from cow and goat milk on diarrhea-causing Escherichia coli and Staphylococcus aureus. Milk samples were aseptically obtained from lactating cows and goats. The samples were skimmed; casein was isolated using acetic acid and alpha-lactalbumin by filtrate thermoprecipitation at 75 °C. 50% of each isolate was reconstituted in a buffer and hydrolyzed with papain at 55 °C for 2 hours. The hydrolysates were heated to 75 °C to inactivate papain, cooled and their antibacterial activity determined by disc diffusion method. Results showed alpha-lactalbumins had higher degrees of hydrolysis and antibacterial activity than caseins; goat alpha-lactalbumin had the highest antibacterial activity with mean inhibition zones of 19.60 mm and 19.50 mm on E. coli and S. aureus. Cow alpha-lactalbumin inhibited E. coli more than S. aureus with inhibition zones of 16.80 mm and 12.50 mm. Cow and goat milk casein hydrolysates inhibited E. coli with mean inhibition zones of 8.00 mm and 10.90 mm and inhibited S. aureus with inhibition zones of 4.13 mm and 1.90 mm respectively. The research showed that the milk hydrolysates can be a source of antibiotics for diarrhea treatment. Research should be done to establish the peptide fractions associated with the observed bioactivity.
REVIEW | doi:10.20944/preprints202301.0487.v1
Subject: Life Sciences, Biochemistry Keywords: Plant proteins; legumes; protein digestibility; germination; peptidomics; proteomics
Online: 27 January 2023 (06:14:36 CET)
Legume seed protein is an important source of nutrition, but it is less digestible than animal protein. Poor protein digestibility in legume seeds and seedlings may partly reflect defences against herbivores. Protein changes during germination typically increase proteolysis and digestibility, by lowering the levels of anti-nutrient protease inhibitors, activating proteases, and breaking down storage proteins (including allergens). Germinating legume sprouts also show striking increases in free amino acids (especially asparagine), but their roles in host defence or other processes are not known. While the net effect of germination is generally to increase the digestibility of legume seed proteins, the extent of improvement in digestibility is species and strain dependent. Further research is needed to highlight which changes contribute the most to improved digestibility of sprouted seeds. Such knowledge could guide the selection of varieties that are more digestible, and also guide the development of food preparations that are more digestible, potentially combining germination with other factors altering digestibility, such as heating and fermentation. Techniques to characterize the shifts in protein make-up, activity and degradation during germination need to draw on traditional analytical approaches, complemented by proteomic and peptidomic analysis of mass spectrometry identified peptide breakdown products.
REVIEW | doi:10.20944/preprints202209.0099.v1
Subject: Life Sciences, Virology Keywords: enterovirus; viral proteins; signalling pathways; host-pathogen interaction
Online: 7 September 2022 (04:41:56 CEST)
Enteroviruses are members of Pichornaviridae family consisting of human enterovirus group A, B, C, and D as well as nonhuman enteroviruses. Human enterovirus type 71 (EV71) has emerged as a major cause of viral encephalitis Hand, foot, and mouth disease (HFMD) in children worldwide especially in the Asia‐Pacific region. EV71 and coxsackievirus A16 are two viruses responsible for HFMD which are members of group A enterovirus. The identified EV71 receptors provide useful information for understanding viral replication and tissue tropism. Host factors interact with the internal ribosome entry site (IRES) of EV71 to regulate viral translation. However, the specific molecular features of the EV71 genome that determine virulence remain unclear. Although an EV71 vaccine has been currently approved, there is no effective therapy for treating EV71 infected patients. Therefore, understanding the host-pathogen interaction could provide the knowledge in viral pathogenesis and further benefit in the anti-viral therapy development. The aim of this study was to investigate the latest findings about the interaction of viral ligands to the host receptor as well as the activation of immune related signalling pathways for the activation of innate immunity and involvement of different cytokines and chemokines in the host pathogen interaction of EV71 along with interaction of viral proteins, mainly 2A and 3C protease, and Interferons production/signaling pathway and their inhibitory effects.
REVIEW | doi:10.20944/preprints202110.0342.v2
Subject: Life Sciences, Biochemistry Keywords: kininogen; structure; AlphaFold; cryo-EM; proteins; kinin; bradykinin
Online: 1 November 2021 (13:50:19 CET)
Kininogens are multidomain glycoproteins found in the blood of most vertebrates. They are important in the blood coagulation cascade pathways - in intrinsic pathway activation leading to thrombin generation partially independently from tissue factor dependent extrinsic pathway, connecting blood coagulation with the kallikrein-kinin system. Nothing is known about the shape on atomic level therefore the endeavor to obtain the good-quality spatial structure of kininogens is important for a better understanding of their role in disease and treatment. Application of cryo-EM is important for solving the spatial structure of kininogens, drawing new frontiers in understanding the function and opening new pathways for drug development.
ARTICLE | doi:10.20944/preprints202110.0306.v1
Subject: Biology, Plant Sciences Keywords: Sulfur; Sulfotransferase; Potato; Bioinformatics; Proteins structure; Stimuli coping
Online: 21 October 2021 (12:41:52 CEST)
Various kinds of primary metabolisms in plants are modulated through sulfate assimilation that the uptake of this inorganic compound can be regulated via the sulfate transporters, such as sulfotransfer-ases (SOTs), engaged in the sulfur metabolism. In the current study a genome-wide approach has been utilized for recognition and characterization of SOT family genes in the significant nutritional crop po-tato (Solanum tuberosum L.). As a result, 29 StSOT genes were identified in the potato genome, which were mapped onto the nine S. tuberosum chromosomes. The protein motifs structure demonstrated two highly conserved 5' PSB region and 3' PB motif that are essential for sulfotransferase and catalytic ac-tivities. The protein-protein interaction networks also significantly demonstrated an interesting collabo-ration between SOTs and the other genes, such as PRTase, APS-kinase, protein phosphatase and APRs, in sulfur compounds biosynthesis and regulation of the flavonoid and brassinosteroid metabolic pro-cesses, which clearly detected the importance of sulfotransferases for potato proper growth/development and stress dealing. Notably, the homology modeling of StSOT proteins and dock-ing analysis of their ligand-binding sites revealed the presence of some stress-responsive residues, such as proline, glycine, serine and lysine, in their active sites. The expression assay of StSOT genes via the potato RNA-seq data clearly suggested the engagements of these gene family members in plants growth and extension as well as responses to various hormones and biotic/abiotic stimulus circum-stances. Our predictions can be informative for the functional characterization of the SOT genes in po-tato and may the other nutritional crops.
BRIEF REPORT | doi:10.20944/preprints202103.0755.v1
Subject: Life Sciences, Biochemistry Keywords: evolutionary divergence; phylogenetic analysis; transporter proteins; Leishmania major
Online: 31 March 2021 (09:55:35 CEST)
Transporter proteins, P-glycoprotein (P-gp) and P4ATPase-CDC50, are responsible for the transport of Miltefosine drug across cell membrane of a protozoan parasite Leishmania major. Mutations or change in activity of these proteins may lead to emergence of resistance in the parasite. Owing to the structural and functional importance of these transporter proteins, in this ppaper, we have tried to decipher the evolutionary divergence of these Miltefosine transporter proteins across different forms of life including Protists, Fungi, Plants and Animals. We retrieved 96, 207, and 189 sequences of P-gp, P4ATPase and CDC50 proteins respectively, across diverse variety of organisms for the conserved analysis. Phylogenetic trees were constructed for these three transporter proteins based on Bayesian posterior probability inference. The evolutionary analysis concluded that these proteins remain highly conserved throughout the species diversity but still substantial differences in the proteins for host (Homo sapiens) and parasite (L. major) were observed which have led in targeting these Miltefosine transporter proteins in a parasite specific manner.
ARTICLE | doi:10.20944/preprints202102.0204.v1
Subject: Life Sciences, Biochemistry Keywords: Lipidome; High Five insect cells; Membrane proteins; Biomembranes
Online: 8 February 2021 (12:23:09 CET)
The lipid composition of biomembranes influence the properties of the lipid bilayer as well as that of the proteins. In this study, the lipidome and the lipid/protein ratio of membranes from High FiveTM insect cells overexpressing mouse P-glycoprotein was characterized. This provides a better understanding of the lipid environment in which P-glycoprotein is embedded, and thus of its functional and structural properties. The relative abundance of the distinct phospholipid classes and their acyl chain composition was characterized. A mass ratio of 0.57 +/- 0.11 phospholipids to protein was obtained. Phosphatidylethanolamines are the most abundant phospholipids, followed by phosphatidylcholines. Membranes are also enriched in negatively charged lipids (phosphatidylserines, phosphatidylinositols and phosphatidylglycerols), and contain small amounts of sphingomyelins, ceramides and monoglycosilatedceramides. The most abundant acyl chains are monounsaturated, with significant amounts of saturated chains. The characterization of the phospholipids by HPLC-MS allowed identification of the combination of acyl chains, with palmitoyl-oleoyl being the most representative for all major phospholipid classes except for phosphatidylserines, which are mostly saturated. A mixture of POPE:POPC:POPS in the ratio 45:35:20 is proposed for the preparation of simple representative model membranes. The adequacy of the model membranes was further evaluated by characterizing their surface potential and fluidity.
ARTICLE | doi:10.20944/preprints202012.0325.v1
Subject: Social Sciences, Accounting Keywords: meat substitute; meathybrid; consumer preference, plant-based proteins
Online: 14 December 2020 (11:44:14 CET)
High levels of meat consumption are increasingly being criticised for ethical, environmental, and social reasons. Plant-based meat substitutes have been identified as healthy sources of protein in comparison to meat. This alternative offers several social, environmental and health benefits and may play a role in reducing meat consumption. However, there has been a lack of research on how specific meat substitute attributes can influence consumers to replace or partially replace meat in their diets. Research demonstrates that in many countries consumers are highly attached to meat. They consider it as an essential and integral element of their daily diet. For these consumers which are not interested in vegan or vegetarian alternatives to meat, so-called meathybrids could be a low-threshold option for a more sustainable food consumption behaviour. In meathybrids only a fraction of the meat product (e.g. 20% to 50%) is replaced with plant-based proteins. In this paper, the results of an online survey with 501 Belgium consumers are presented with focus on preferences and attitudes relating to meathyrids. The results show that more than fifty percent of consumers substitute meat at least occasionally. Thus, about half of the respondents reveal an eligible consumption behaviour in respect to sustainability and healthiness to a certain degree. Concerning the determinants of choosing either meathybrid or meat it becomes evident that a strong effect is exerted by the health perception. The healthier meathybrids are perceived, the higher is the choice probability. Thus, this egoistic motive seems to outperform altruistic motives like animal welfare or environmental concerns when it comes to choice for this new product category.
ARTICLE | doi:10.20944/preprints202012.0241.v1
Subject: Behavioral Sciences, Applied Psychology Keywords: meat substitute; meathybrid; consumer preference, plant-based proteins
Online: 10 December 2020 (09:22:00 CET)
High levels of meat consumption are increasingly being criticised for ethical, environmental, and social reasons. Plant-based meat substitutes have been identified as healthy sources of protein that, in comparison to meat, offer a number of social, environmental and health benefits and may play a role in reducing meat consumption. However, there has been a lack of research on the role they can play in the policy agenda and how specific meat substitute attributes can influence consumers to replace partially replace meat in their diets.
ARTICLE | doi:10.20944/preprints202011.0677.v1
Online: 26 November 2020 (23:08:59 CET)
High levels ofmeat consumption are increasingly being criticised for ethical, environmental, 2 and social reasons. Plant-based meat substitutes have been identified as healthy sources of protein in 3 comparison to meat. This alternative offers several social, environmental and health benefits and may 4 play a role in reducing meat consumption. However, there has been a lack of research on how specific 5 meat substitute attributes can influence consumers to replace or partially replace meat in their diets. 6 Research demonstrates that in many countries consumers are highly attached to meat.They consider 7 it as an essential and integral element of their daily diet. For these consumers which are not interested 8 in vegan or vegetarian alternatives to meat, so-called meathybrids could be a low-threshold option 9 for a more sustainable food consumption behaviour. In meathybrids only a fraction of the meat 10 product (e.g. 20% to 50%) is replaced with plant-based proteins. In this paper, the results of an online 11 survey with 500 German consumers are presented with focus on preferences and attitudes relating 12 to meathyrids. The results show that more than fifty percent of consumers substitute meat at least 13 occasionally. Thus, about half of the respondents reveal an eligible consumption behaviour in respect 14 to sustainability and healthiness to a certain degree. Concerning the determinants of choosing either 15 meathybrid or meat it becomes evident that the highest effect is exerted by the health perception. The 16 healthier meathybrids are perceived, the higher is the choice probability. Thus, this egoistic motive 17 seems to outperform altruistic motives like animal welfare or environmental concerns when it comes 18 to choice for this new product category.
ARTICLE | doi:10.20944/preprints202006.0352.v1
Online: 29 June 2020 (10:40:07 CEST)
Mitochondria are classically termed as powerhouse of a mammalian cell. Most of the cellular chemical energy in the form of adenosine tri phosphate (ATP) is generated by mitochondria and dysregulation of mitochondrial functions thus can be potentially fatal of cellular homeostasis and health. Acute respiratory distress has been earlier linked to mitochondrial dysfunction. SARS-CoV-2 infection severity leads to acute respiratory distress syndrome (ARDS) and can be fatal. We tried to investigate possible connection between SARS-CoV-2, ARDS and mitochondria. Here, we report identification of SARS-CoV-2 non-structural proteins (particularly Nsp12 and 13) that have recognition sequence with respect to mitochondrial entry. We also report that these proteins can potentially shuttle between cytoplasm and mitochondria based on the localization signals and help in downstream maintenance of the virus. Their properties to use ATP for enzymatic activities may cause ATP scavenging allowing viral RNA functions in lieu of host cell health.
ARTICLE | doi:10.20944/preprints202004.0463.v1
Subject: Medicine & Pharmacology, General Medical Research Keywords: diabetes mellitus; insulin resistance; cytokines; adaptor proteins; CLNK
Online: 25 April 2020 (11:32:48 CEST)
Type 2 diabetes mellitus (T2DM) is an endocrine illness associate with various changes in the immune system and adaptor protein levels. Cytokine dependent hematopoietic cell linker (CLNK) is an adapter protein that regulates immune receptor signaling and acts as a regulator of the receptor signaling of T-cells and natural killer T-cell. The role of CLNK in T2DM is not studied previously. In the present study, serum CLNK level was measured and correlated with some sociodemographic and insulin resistance (IR) parameters. This is achieved by performing measurement of CLNK and insulin parameters (glucose, insulin, and HbA1c in addition to the calculation of the functions of IR (HOMA2IR), insulin sensitivity (HOMA%S), and beta-cell function (HOMA%B)) in 60 T2DM patients and 30 controls. The results indicated a significant increase (p=0.025) in serum CLNK in patients group in comparison with the controls. Multivariate generalized linear model (GLM) analysis revealed no significant effect of age, BMI, and sex on the CLNK level. The results of tests for between-subjects showed that the CLNK affects diagnosis significantly (F=7.445, p=0.008, partial η2 =0.081) and its effect is approximately the same as the effect of insulin (F=8.107, p=0.006, partial η2 =0.087). The correlation study showed a highly significant positive correlation between CLNK and the duration of disease (rho=0.420, p<0.001). It can be concluded that the increase CLNK in T2DM revealing the role of the adaptor proteins level in the nature of disease. Elevation of CLNK level may be used as a predictor for diabetes complications, which needs more investigations.
CONCEPT PAPER | doi:10.20944/preprints201911.0372.v1
Subject: Biology, Plant Sciences Keywords: arabinogalactan proteins; phyllotaxis; Hechtian Oscillator; calcium homeostasis; auxin
Online: 29 November 2019 (08:22:04 CET)
Sixty years ago in the lab adjacent to Fred Sanger (1958 Nobel Prize for protein chemistry), I discovered the cell surface hydroxyproline-rich glycoproteins. Nature keeps some of her secrets longer than others. It has taken many years to dissect the molecular function and biological role of extensins and arabinogalactan proteins (AGPs). Extensins template the formation of new cell walls. AGPs remained baffling and enigmatic until a Eureka moment when computer prediction of AGP calcium binding depicted paired glucuronic acid residues and thus the likely role of a cell surface AGP-Ca2+capacitor: In conjunction with the auxin-activated proton pump that releases bound Ca2+ it led us to formulate the Hechtian Growth Oscillator as A Global Paradigm with a pivotal role in Ca2+ homeostasis. The ramifications are profound. They cannot be shrugged off with sceptical disdain but demand critical reappraisal of current dogma. Phyllotaxis is an ancient problem; it involves an essential role for auxin and the auxin efflux “PIN” proteins together with mechanotransduction of stress-strain as phyllotactic determinants. However, a general explanation remains elusive despite much effort, particularly by mathematicians. Here we propose a novel biochemical algorithm: Hechtian oscillator transduction of cell wall stress generates phyllotactic patterns quite independent of a mathematical approach. Plants simply use different rules and follow a different route.
ARTICLE | doi:10.20944/preprints201809.0024.v1
Subject: Earth Sciences, Other Keywords: oral mucositis; glycine; intercellular signaling peptides and proteins
Online: 3 September 2018 (11:09:30 CEST)
Oral mucositis is most frequently a toxic effect of chemotherapeutic and/or radiotherapeutic treatment, resulting from complex multifaceted biological events involving DNA damage. The clinical manifestations have a negative impact on the life quality of cancer patients. Preventive measures and curative treatment of mucositis are still not well established. The glycine has anti-inflammatory, immunomodulatory and cytoprotective actions, being a potential therapeutic in mucositis. The objective was to evaluate the effects of glycine on the expression of collagen and growth factors, platelet and epidermal in oral mucositis. The mucositis of which was induced by the protocol of Sonis. 40 hamsters were used, divided into two groups: Group I- control; Group II- supplemented with 5% intraperitoneal glycine, 2,0 mg/g diluted in hepes. Histopathological sections were used to perform the immune-histochemical method, the evaluation collagen expression and the growth factors: EGF and PDGF. It was observed that the group supplemented with glycine higher amounts of collagen expression and predominance type of collagen I. The glycine group presented lower immunoexpression of the growth factors, EGF and PDGF. The group supplemented with glycine showed a marked healing process of the oral mucosite, demonstrated by the predominance of collagen type I and reduction of growth factors, EGF and PDGF.
ARTICLE | doi:10.20944/preprints201608.0203.v1
Subject: Biology, Entomology Keywords: Sitophilus zeamais; COXⅡ; Soluble proteins; Enzyme activity; AITC
Online: 25 August 2016 (10:11:13 CEST)
COX II containing a dual core CuA active site is one of the three core subunits of mitochondrial Cco, which plays a significant role in the physiological process. In this report, the full-length cDNA of COXⅡ gene was cloned from Sitophilus zeamais, which had an ORF of 684 bp encoding 227 amino acids residues. The predicted COXⅡ protein had a molecular mass of 26.2 kDa with pI value of 6.37, and multiple sequence alignment and phylogenetic analysis indicated that Sitophilus zeamais COXⅡ had high sequence identity 78.51% with the COXⅡ of other insect species, especially similarity to sitophilus oryzae. This gene was subcloned into the prokaryotic expression vector pET-32a, and induced by IPTG in E.coli Transetta (DE3) expression system. Finally the COXⅡ with 6-His tag was purified using affinity chromatography with Ni2+-NTA agarose. WB showed the recombinant COXⅡ was about 44 kD, and the concentration of fusion protein was 50μg/mL. UV-spectrophotometer and infrared spectrometer analysis showed that recombinant COXⅡ could catalyze the oxidation of substrate Cytc, and influenced by AITC. It was found that AITC could form a hydrophobic region with COXⅡ protein via molecular docking, besides, a sulfur atom of AITC structure could form a length of 2.9 Å hydrogen bond with Leu-31. These results will provide valuable information for elucidating the role of COXⅡ in Sitophilus zeamais responses to AITC, meanwhile, it will helpful to carry out a point mutation in AITC binding sites for the future research.
ARTICLE | doi:10.20944/preprints201806.0042.v1
Subject: Biology, Plant Sciences Keywords: epigenetics; H3K9 methylation; H3K4 methylation; DNA methylation; seasonal climate change; summer dormancy; heat shock proteins; ribosomal proteins; Zygophyllum dumosum Boiss
Online: 4 June 2018 (12:49:59 CEST)
Plants thriving in desert environments are suitable for studying mechanisms for plant survival under extreme seasonal climate variation. Zygophyllum dumosum Boiss, like many other Zygophyllaceae species, displays a unique epigenetic mechanism whereby the repressive markers di- and tri-methyl of H3K9 do not exist. We studied epigenetic mechanisms underlying seasonal growth cycles in Z. dumosum and their association with factors regulating basic cell functions. We showed strong association between rainfall and seasonal growth and the epigenetic marker of dimethyl H3K4, which disappears on entry into the dry season and the acquisition of dormant state. DNA methylation is not affected by lack of H3K9 di and tri methyl and changes in methylation pattern are apparent on entry into the dry season. Proteome analysis of acid soluble fractions revealed extensive reduction in ribosomal proteins and in proteins involved in chloroplasts and mitochondria activities during the dry seasons concomitantly with up-regulation of molecular chaperone HSPs. Our results highlight mechanisms underlying Z. dumosum adaptation to seasonal climate variation. Particularly, summer dormancy is associated with loss of the permissive epigenetic marker dimethyl H3K4, which might facilitate genome compaction, concomitantly with significant reduction in proteins involved in basic cell functions (i.e., protein synthesis, photosynthesis and respiration).
REVIEW | doi:10.20944/preprints202302.0054.v1
Subject: Life Sciences, Microbiology Keywords: Chagas Disease; surface proteins; disperse protein family; Trypanosoma cruzi
Online: 3 February 2023 (03:11:08 CET)
Abstract Chaga´s disease caused by Trypanosoma cruzi infections is included in the group of neglected diseases, and efforts to develop new therapeutic or immunoprevention approaches have not been successful. After the publication of the T. cruzi genome, the number of molecular and biochemical studies on this parasite have increased considerably, many of which are focused on families of variant-surface-proteins, especially the trans-sialidases, mucins and mucin-associated proteins. The disperse gene protein 1 family (DGF-1) is one of the most abundant families in the T. cruzi genome, however, the large gene size, high copy numbers, and low antibody titers detected in infected humans make it an unattractive study target. Here, we argue that the DGF-1 gene family although not being the most obvious participant of the host-parasite immunological gamble, where T. cruzi appears to have the upper hand, it may play an important role in more basic host-parasite interactions that deserve further examination.
ARTICLE | doi:10.20944/preprints202209.0237.v1
Subject: Biology, Agricultural Sciences & Agronomy Keywords: malondialdehyde; duck meat; myofibrillar proteins; physicochemical changes; gel properties
Online: 16 September 2022 (03:50:02 CEST)
This paper focuses on the effect of malondialdehyde-induced oxidative modification (MiOM) on the gel properties of duck myofibrillar proteins (DMPs). DMPs were first prepared and treated with oxidative modification at different concentrations of malondialdehyde (0, 0.5, 2.5, 5.0 and 10.0 mmol/L). The physicochemical changes (carbonyl and free thiol group content) and gel properties (gel whiteness, gel strength, water holding capacity, rheological properties and mi-cro-structural properties) were then investigated. The results showed that the content of protein carbonyl groups increased with increasing MDA oxidation (P<0.05), while the content of free thiol groups decreased significantly (P<0.05). Meanwhile, there was a significant trend of decrease in gel whiteness; the hardness and water-holding capacity of protein gels increased significantly under the oxidation of low concentration of MDA (0-5 mmol/L), while the hardness of gels decreased under the oxidation of high concentration (10 mM). The storage modulus and loss modulus of oxidized DMPs also increased with increasing concentration; moreover, microstructural analysis confirmed that the gels oxidized at low concentrations were more compact and homogeneous in terms of pore size compared to the high concentration or blank group. In conclusion, moderate oxidation of malondialdehyde was beneficial to improve the gel properties of duck; however, excessive oxidation was detrimental to the formation of dense structured gels.
REVIEW | doi:10.20944/preprints202203.0324.v1
Subject: Biology, Animal Sciences & Zoology Keywords: metazoans; adhesive organs; suction organs; functional morphology; adhesive proteins
Online: 24 March 2022 (07:20:55 CET)
To resist hydrodynamic forces, two main underwater attachment strategies have evolved multiple times in aquatic animals: glue-like “bioadhesive secretions” and pressure-driven “suction attachment”. In this review, we use a multi-level approach to highlight convergence in underwater attachment mechanisms across four different length-scales (organism, organ, microscopic and molecular). At the organism level, the ability to attach may serve a variety of functions, the most important being: (i) positional maintenance, (ii) locomotion, (iii) feeding, (iv) building, and (v) defense. Aquatic species that use bioadhesive secretions have been identified in 28 metazoan phyla out of the 34 currently described, while suction organs have a more restricted distribution and have been identified in five phyla. Although biological adhesives are highly diverse, it is possible to categorize them into four main types according to the time scale of operation: permanent, temporary, transitory, and instantaneous adhesion. At the organ level some common principles have evolved independently in different biological lineages: for example, animals with single-unit attachment organs can be distinguished from those with multi-unit organs. Fundamental design elements can also be recognized for both types of attachment mechanisms. Suction attachment systems comprise a circular or elliptical attachment disc, a sealing rim to prevent leakage and a mechanism to lower the internal pressure. Bioadhesive-producing organs, on the other hand, usually contain a glandular tissue associated with connective tissues or other types of load-bearing support structures and muscles that facilitate locomotion or mechanical detachment. At the microscopic level, similar designs and organizations appear once again to have emerged independently in different phylogenetic lineages. Independent of the taxon and type of adhesion, there are species in which the biosynthesis, packaging and release of adhesive secretions takes place at the level of a single type of secretory cell, whereas in others these secretions are produced by two or more secretory cell types. Duo-gland adhesive systems involved in temporary adhesion present an additional level of complexity as they also exhibit de-adhesive secretory cells. Yet, strikingly similar cellular organizations have been reported in highly disparate species. In the case of biological suction organs, regions of the organ that contact the substratum are highly textured with stiff microstructures. Although clearly non-homologous in different animals, these microstructures are thought to enhance friction on rough surfaces. At the molecular level, proteins are the main organic constituent of adhesive secretions in aquatic animals. We compared the global amino acid compositions of bioadhesives using principal component analysis to show that homologous adhesives from phylogenetically related species cluster together, and there is little overlap between taxonomic groups. However, several non-permanent adhesives are grouped together even though they belong to disparate phyla, indicating convergence in amino acid composition. We also investigated relatedness among individual adhesive proteins using a sequence similarity-based clustering analysis. While many proteins appear taxon-specific, some have clear sequence homologies based on shared protein domains between phylogenetically distant organisms. However, it is highly probable that these domains, which are also present in many non-adhesive proteins, were convergently acquired from ancestral proteins with unrelated general functions. We herein present morphological, structural, and molecular convergences between different attachment mechanisms in aquatic animals that likely arose in response to shared functional and selective pressures.
REVIEW | doi:10.20944/preprints202012.0184.v1
Subject: Life Sciences, Biochemistry Keywords: C2H2 proteins; CTCF; LDB1; chromatin insulator; long-distance interactions
Online: 8 December 2020 (08:29:36 CET)
In higher eukaryotes, enhancers determine the activation of developmental gene transcription in specific cell types and stages of embryogenesis. Enhancers transform the signals produced by various transcription factors within a given cell, activating the transcription of the targeted genes. Often, developmental genes can be associated with dozens of enhancers, some of which are located at large distances from the promoters that they regulate. Currently, the mechanisms that underly the specific distance interactions between enhancers and promoters remain unknown. This review describes the properties and activities of enhancers and discusses the mechanisms of distance interactions and potential proteins involved in this process.
ARTICLE | doi:10.20944/preprints201909.0078.v1
Subject: Life Sciences, Molecular Biology Keywords: ZIKV; protein-protein interaction; non-structural viral proteins; network
Online: 7 September 2019 (00:18:39 CEST)
The Zika virus (ZIKV) is a mosquito-borne Flavivirus and can be transmitted through an infected mosquito bite or through human-to-human interaction by sexual activity, blood transfusion, breastfeeding or perinatal exposure. After the 2015-2016 outbreak in Brazil, a strong link between ZIKV infection and microcephaly emerged. ZIKV specifically targets human neural progenitor cells, suggesting that proteins encoded by ZIKV bind and inactivate host cell proteins leading to microcephaly. Here, we present a systematic annotation of interactions between human proteins and the seven non-structural ZIKV proteins corresponding to a Brazilian isolate. The interaction network was generated by combining tandem-affinity purification followed by mass spectrometry with yeast two-hybrid screens. We identified 150 human proteins, involved in distinct biological processes, as interactors to ZIKV non-structural proteins. Our interacting network is composed of proteins that have been previously associated with microcephaly in human genetic disorders and/or animal models. This study builds on previously published interacting networks of ZIKV and genes related to autosomal recessive primary microcephaly to generate a catalog of human cellular targets of ZIKV proteins implicated in processes related to microcephaly in humans. Collectively, this data can be used as a resource for future characterization of ZIKV infection biology and help create a basis for the discovery of drugs which may disrupt the interaction and reduce the health damage to the fetus.
REVIEW | doi:10.20944/preprints201710.0178.v1
Subject: Materials Science, Nanotechnology Keywords: hydrogels; peptides; proteins; crosslinked networks; controlled release; biodegradable polymers
Online: 30 October 2017 (03:39:55 CET)
Hydrogels evolved as an outstanding carrier material for local and controlled drug delivery that tend to the shortcomings of old conventional dosage forms for small drugs (NSAIDS) and large peptides and proteins. Aqueous swellable and crosslinked polymeric network structure of hydrogels is composed of various natural, synthetic and semisynthetic biodegradable polymers. Hydrogels have remarkable properties of functionality, reversibility, sterilizability, and biocompatibility. All these dynamic properties of hydrogels have increased the interest in their use as a carrier for peptides and proteins to be released slowly in a sustained manner. The therapeutical peptide and proteins are remarkable therapeutic agents in today’s world that allows the treatment of severe, chronic and life‐threatening diseases, such as diabetes, rheumatoid arthritis, hepatitis in an easy manner. Despite few limitations, hydrogels provide fine tuning of proteins and peptides delivery with enormous impact in clinical medicine. The primary objective of this article is to review current issues concerned with the therapeutics peptides and proteins and impact of remarkable properties of hydrogels on these therapeutic agents. Different routes for pharmaceutical peptides and proteins and superiority over other drugs candidates are presented. The article will also review literature concerning classification of hydrogels on different basis, polymers used, release mechanisms their physical and chemical characteristics and diverse applications.
ARTICLE | doi:10.20944/preprints202212.0431.v1
Subject: Life Sciences, Immunology Keywords: transcriptomics; COVID-19; tuberculosis; progression; non-coding RNA; hub proteins
Online: 23 December 2022 (01:24:48 CET)
The pandemic of COVID-19 ravaged most countries and made the healthcare system go for a toss. The impact of the disease is different in each patient and it progresses differently. Based on the severity, the COVID-19 infection is stratified into three main categories- mild, moderate, and severe. In this study, we performed a transcriptomic study of different stages and studied the progression of the disease. The study was based on an Indian population of 28 COVID-19 patients, which were classified into different groups. Our analysis has shown that as the disease progresses, the genes involved in the degranulation of the neutrophils and galactose metabolism increase. Furthermore, we identified the hub proteins in each stage. TB is one of the comorbidities of COVID-19 and a comparative study was done to identify the preserved module of genes in both. Enrichment analysis showed that the members of this module are significantly involved in translation and ribosome synthesis.
ARTICLE | doi:10.20944/preprints202112.0290.v3
Subject: Life Sciences, Molecular Biology Keywords: crescentic glomerulonephritis; BET proteins; NOTCH; GREMLIN; Chronic kidney disease; Bromodomain
Online: 23 December 2021 (14:40:45 CET)
Crescentic glomerulonephritis is a devastating autoimmune disease that without early and properly treat-ment may rapidly progress to end-stage renal disease and death. Current immunosuppressive treatment provided limited efficacy and an important burden of adverse events. Epigenetic drugs are a source of novel therapeutic tools. Among them, bromodomain and extraterminal domain (BET) inhibitors (iBETs) block the interaction between bromodomains and acetylated proteins, including histones and transcription factors. iBETs have demonstrated protective effects on malignancy, inflammatory conditions and experi-mental kidney disease. Recently, Gremlin-1 was proposed as a urinary biomarker of disease progression in human anti-neutrophil cytoplasmic antibody (ANCA)-associated crescentic glomerulonephritis. We have now evaluated whether iBETs regulate Gremlin-1 in experimental anti-glomerular basement membrane nephritis induced by nephrotoxic serum (NTS) in mice, a model of human crescentic glomerulonephritis. In NTS-injected mice, the iBET JQ1 inhibited renal Gremlin-1 overexpression and diminished glomerular damage, including podocyte loss. Chromatin immunoprecipitation assay demonstrated BRD4 enrichment of the Grem-1 gene promoter in injured kidneys, consistent with Gremlin-1 epigenetic regulation. Moreover, JQ1 blocked BRD4 binding and inhibited Grem-1 gene transcription. The beneficial effect of iBETs was also mediated by targeting NOTCH signaling pathway. JQ1 inhibited the gene expression of the NOTCH effec-tors Hes-1 and Hey-1 in NTS-injured kidneys. Our results further support the role for epigenetic drugs, such as iBETs, in the treatment of rapidly progressive crescentic glomerulonephritis.
ARTICLE | doi:10.20944/preprints202112.0330.v1
Subject: Life Sciences, Other Keywords: biostimulant; carotenoids; chlorophyll; IBA; leaf senescence; NAA; phenology; soluble proteins
Online: 21 December 2021 (12:40:44 CET)
Some biostimulants, including plant origin preparations, act similarly to plant hormones. Moreover, the supplementation of known and unknown rooting cofactors can stimulate rhizogenesis in cuttings. The aim of this research was to assess the response of difficult-to-root and long-rooting stem cuttings of the once-blooming old variety Rosa ‘Hurdal’ to preparations of plant origin. The hypothesis was that plant origin preparations could enhance rooting processes by inhibiting chlorophyll a/b degradation in leaves and postponing leaf senescence, simultaneously increasing the quality of cuttings. The one-bud stem cuttings were made in four phenological stages: (H1) flower buds closed, (H2) open flowers, (H3) just after petal fall, (H4) 7-14 days after petal shedding. They were treated with either standard commercial powder preparations containing 0.4% indolebutyric acid (IBA) or 0.2% naphthalene acetic acid (NAA) as well as with commercial plant origin preparations that this work will henceforth refer to as: Algae Extract, Organic Preparation, and Plant Extract. The cuttings were evaluated after 12 weeks of rooting them in two substrates: peat-perlite and peat-sand (v:v; 1:1). Mean root percentages for both substrates were noted after preparation from stage H1 (74.5 %), H2 (59.5 %), H3 (50.8 %) shoots. The H4 cuttings didn’t root at all and were not considered further. The means for all phenology stages together were the highest by the use of 0.6 % Algae Extract, 0.012 % and 0.02 % Organic Preparation, 0.2 % and 0.4 % Plant Extract. The lowest means were reported for the control cuttings as well as NAA and IBA treatment. Plant origin preparations encouraged growth parameters but did not unequivocally inhibit the decrease of chlorophyll content in the cuttings’ leaves. Rooting percentage depended on the quality of cuttings as well as chlorophyll a/b and soluble protein content in leaves in both rooting substrates.
ARTICLE | doi:10.20944/preprints202112.0254.v1
Subject: Materials Science, Polymers & Plastics Keywords: Peptides and proteins; Counterions; Ions; Carboxyls; Molecular dynamics; Force fields
Online: 15 December 2021 (11:14:28 CET)
Electrostatic interactions have a determining role in conformational and dynamic behavior of polyelectrolyte molecules . In this study, anionic polyelectrolyte molecules, poly(glutamic acid) (PGA) and poly(aspartic acid) (PASA), in water solution with the most commonly used K+ or Na+ counterions were investigated using atomistic molecular dynamics (MD) simulations. Seven common force fields, AMBER99SB-ILDN, AMBER14SB, AMBER-FB15, CHARMM22*, CHARMM27, CHARMM36m and OPLS-AA/L, both with their native parameters and with the non-bonded fix (NBFIX) and electronic continuum corrections (ECC) to were studied. These corrections have bene introduced to correct for the problem of overbinding of ions to the charged groups of polyelectrolytes. Physical properties, such as molecular sizes, local structure and dynamics, were studied using two types of common counterions, potassium and sodium. The results show that in some cases, the macroion size and dynamics depend strongly on the models (parameters) for the counterions due to strong overbinding of ions and charged side chain groups. The local structures and dynamics are more sensitive on dihedral angle parameterization resulting in a preference for defined monomer conformations amd the type of correction used.
ARTICLE | doi:10.20944/preprints202111.0376.v1
Subject: Life Sciences, Microbiology Keywords: Mast cells; Leishmania mexicana; sand fly salivary proteins; sexual hormones
Online: 22 November 2021 (10:51:24 CET)
Mast cells (MCs) play a crucial role during infections with Leishmania, that is transmitted through the bite of an infected sand fly that injects saliva together with the parasite. Sand fly saliva is a complex fluid that modulates the host immune response. In addition, hormonal factors modulate the host immune response, impacting the susceptibility to infections. Thus, to assess the impact of androgens and salivary proteins of sand fly vectors on the mast cell (MC) response to Leishmania infections, we infected orchiectomized male mice with the parasite in the presence or absence of sand fly salivary proteins and analyzed the inflammatory response of MCs. Our results showed a differential MC response to the parasite and to vector salivary proteins in mice deprived of gonadal hormones, as compared to sham-operated mice. Orchidectomy induced a different pattern of activation in MC of animals infected with Leishmania and vector-salivary proteins. Our results show that during Leishmania infection, androgens modulate the innate immunity response against the parasite and salivary proteins of the sand fly vector.
Subject: Life Sciences, Biochemistry Keywords: circular RNAs (circRNAs); biogenesis; trans-acting proteins; cis-regulatory elements
Online: 16 July 2021 (14:48:13 CEST)
Circular RNAs (circRNAs), which are a class of non-coding RNA with covalently closed loops, play important roles in epigenetics regulation of gene expression at both the transcriptional and post-transcriptional level. Accumulating evidence demonstrated that numerous circRNAs were abnormally expressed in tumors and their dysregulation was involved in the tumorigenesis and metastasis of cancer. Although the functional mechanisms of many circRNAs have been revealed, why circRNAs are dysregulated in cancer remains elusive. CircRNAs are generated by a “backsplicing” process, which is regulated by different cis-regulatory elements and trans-acting proteins. Therefore, how these cis- and trans-elements change during tumorigenesis and how they regulate the biogenesis of circRNAs in cancer are two questions that interest us. In this review, we summarized the pathways for the biogenesis of circRNAs; and then illustrated why circRNAs dysregulated in cancer by discussing the changes of cis-regulatory elements and trans-acting proteins that related to circRNA splicing and maturation in cancer.
ARTICLE | doi:10.20944/preprints202005.0150.v2
Subject: Biology, Other Keywords: chaperone function; heat-shock proteins; lipid binding; phosphatidylserine; protein refolding
Online: 29 July 2020 (12:18:02 CEST)
HspA1A, a molecular chaperone, translocates to the plasma membrane (PM) of stressed and cancer cells. This translocation results in HspA1A’s cell-surface presentation, which renders tumors radiation insensitive. To specifically inhibit the lipid-driven HspA1A’s PM translocation and devise new therapeutics it is imperative to characterize the unknown HspA1A’s lipid-binding regions and determine the relationship between the chaperone and lipid-binding functions. To elucidate this relationship, we determined the effect of phosphatidylserine (PS)-binding on the secondary structure and chaperone functions of HspA1A. Circular dichroism revealed that binding to PS resulted in minimal modification on HspA1A’s secondary structure. Measuring the release of inorganic phosphate revealed that PS-binding had no effect on HspA1A’s ATPase activity. In contrast, PS-binding showed subtle but consistent increases in HspA1A’s refolding activities. Furthermore, using a Lysine-71-Arginine mutation (K71A; a null-ATPase mutant) of HspA1A we show that although K71A binds to PS with affinities similar to the WT, the kinetics of the binding are significantly different, probably because of the mutant’s inability to achieve specific conformations. These observations suggest a two-step binding model that includes conformational changes and strongly support the notion that the chaperone and lipid-binding activities of HspA1A are dependent but the regions mediating these functions do not overlap. These findings provide the basis for future interventions to inhibit HspA1A’s PM-translocation in tumor cells, making them sensitive to radiation therapy.
ARTICLE | doi:10.20944/preprints202004.0008.v1
Subject: Life Sciences, Biophysics Keywords: bottom-up synthetic biology; motor proteins; pattern formation; self-organization
Online: 2 April 2020 (04:02:53 CEST)
Cortical actomyosin flows, among other mechanisms, scale up spontaneous symmetry breaking and thus play pivotal roles in cell differentiation, division, and motility. According to many model systems, myosin motor-induced local contractions of initially isotropic actomyosin cortices are nucleation points for generating cortical flows. However, the positive feedback mechanisms by which spontaneous contractions can be amplified towards large-scale directed flows remain mostly speculative. To investigate such a process on spherical surfaces, we reconstituted and confined initially isotropic minimal actomyosin cortices to the interfaces of emulsion droplets. The presence of ATP leads to myosin-induced local contractions that self-organize and amplify into directed, large-scale actomyosin flows. By combining our experiments with theory, we found that the feedback mechanism leading to a coordinated, directional motion of actomyosin clusters can be described as asymmetric cluster vibrations, caused by intrinsic non-isotropic ATP consumption, in conjunction with spatial confinement. By tracking individual actomyosin clusters, we identified fingerprints of vibrational states as the basis of directed motions. These vibrations may represent a generic key driver of directed actomyosin flows under spatial confinement in vitro and in living systems.
ARTICLE | doi:10.20944/preprints201905.0103.v2
Subject: Life Sciences, Molecular Biology Keywords: L-arginine, embryonic development, intracytoplasmic vacuoles, immunoglobulin, heat shock proteins
Online: 10 May 2019 (14:03:49 CEST)
The objective of this study was to evaluate the effect of in ovo injection of L-arginine (L-Arg) into Ross broiler eggs at different embryonic developmental stages on their survival, hatchability, and body weight (BW). Additionally, we have analyzed the levels of serum glutamic-oxaloacetic transaminase (SGOT) and serum glutamic-pyruvic transaminase (SGPT), protein expression of heat shock proteins (HSPs), also we have the determined micronuclei (MN) and nuclear abnormality (NA). Results showed that survival and hatching rates as well as body weight were increased on the 14th day incubation compared to 8th and 18th day incubation at lower concentration of L-Arg. Moreover, the levels of SGOT and SGPT were also significantly (P < 0.05) increased at 14th day incubation at the same concentration (100μg/μl/egg) of injection. In addition, IgM levels were increased on the 14th day incubation compared to other days. The protein expressions of HSP-47, HSP-60, and HSP-70 in the liver were significantly down-regulated whereas the expression of myogenin and MyoD were significantly up-regulated on the 14th day after incubation in treated with all different doses such as 100μg, 1000μg and 2500μg/μl/egg namely 3T1, 3T2 and 3T3 respectively. However, the treatment with low dose of L-Arg down-regulated expression levels of those proteins on the 14th day incubation. Histopathology of liver by hematoxylin and eosin (H&E) straining showed that the majority of liver damage, specifically intracytoplasmic vacuoles, were observed in 3T1, 3T2, and 3T3. The minimum dose of 100 μg/ml/egg on the 14th day of incubation significantly prevented intracytoplasmic vacuole damages. These results demonstrate that in ovo administration of L-Arg at (100μg/μl/egg) may be an effective method to increase chick BW, hatch rate, increasing muscle growth related proteins and promote the immune response through increasing IgM on the 14th day of incubation period.
REVIEW | doi:10.20944/preprints201807.0492.v1
Subject: Physical Sciences, Other Keywords: membranes; vesicles; lipids; proteins; mesophase separation; domains; lipid rafts; clusters
Online: 25 July 2018 (15:50:38 CEST)
Cell plasma membranes display a dramatically rich structural complexity characterized by functional sub-wavelength domains with specific lipid and protein composition. Under favorable experimental conditions, patterned morphologies can also be observed in vitro on model systems such as supported membranes or lipid vesicles. Lipid mixtures separating in liquid-ordered and liquid-disordered phases below a demixing temperature play a pivotal role in this context. Protein-protein and protein-lipid interactions also contribute to membrane shaping by promoting small domains or clusters. Such phase separations displaying characteristic length-scales falling in-between the nanoscopic, molecular scale on the one hand and the macroscopic scale on the other hand, are named mesophases in soft condensed matter physics. In this Review, we propose a classification of the diverse mechanisms leading to mesophase separation in biomembranes. We distinguish between mechanisms relying upon equilibrium thermodynamics and those involving out-of-equilibrium mechanisms, notably active membrane recycling. In equilibrium, we show that the mechanisms generically dwell on an up-down symmetry breaking between the upper and lower bilayer leaflets. Symmetry breaking is an ubiquitous mechanism in condensed matter physics at the heart of several important phenomena. In the present case, it can be either spontaneous (domain buckling) or explicit, i.e. due to an external cause (global or local vesicle bending properties). Whenever possible, theoretical predictions and simulation results are confronted to experiments on model systems or living cells, which enables us to identify the most realistic mechanisms from a biological perspective.
REVIEW | doi:10.20944/preprints201702.0048.v2
Subject: Medicine & Pharmacology, Pharmacology & Toxicology Keywords: ostreolysin A/pleurotolysin B; equinatoxins; pore-forming proteins; biological effects
Online: 5 April 2017 (15:36:57 CEST)
Acidic ostreolysin A/pleurotolysin B (OlyA/PlyB, formerly known as ostreolysin (Oly), and basic 20 kDa equinatoxins (EqTs) are cytolytic proteins isolated from the edible mushroom Pleurotus ostreatus and the sea anemone Actinia equina, respectively. Both toxins, although from different sources, share many similar biological activities: (i) colloid-osmotic shock by forming pores in cellular and artificial membranes enriched in cholesterol and sphingomyelin; (ii) increased vascular endothelial wall permeability in vivo and perivascular oedema; (iii) dose-dependent contraction of coronary vessels; (iv) haemolysis with pronounced hyperkalaemia in vivo; (v) bradycardia, myocardial ischemia and ventricular extrasystoles accompanied by progressive fall of arterial blood pressure and respiratory arrest in rodents. Both types of toxins are haemolytic within nanomolar range concentrations, and it seems that hyperkalaemia plays an important role in toxin cardiotoxicity. However, it was observed that the haemolytically more active EqT III is less toxic than EqT I, the most toxic and least haemolytic EqT. In mice, EqT II is more than 30 times more toxic than OlyA/PlyB when applied intravenously. These observations imply that haemolysis with hyperkalaemia is not the sole cause of the lethal activity of both toxins. Additional mechanisms responsible for lethal action of the two toxins are direct effects on heart, coronary vasoconstriction and related myocardial hypoxia. In this review, we appraise the pathophysiological mechanisms related to the chemical structure of OlyA/PlyB and EqTs, as well as their toxicity.
ARTICLE | doi:10.20944/preprints202211.0188.v1
Subject: Medicine & Pharmacology, Pharmacology & Toxicology Keywords: radionuclide molecular imaging; HER2; scaffold proteins; DARPin; ADAPT6; technetium-99m; preclinical
Online: 10 November 2022 (03:31:20 CET)
Non-invasive radionuclide molecular visualization of human epidermal growth factor receptor type 2 (HER2) can provide stratifcation of patients for HER2-targeting therapy. This method can also enable monitoring of the response to such therapies and thereby the treatment will be more personalized and efficient. Phase I clinical evaluation of two scaffold protein-based imaging probes, [99mTc]Tc-(HE)3-G3 and [99mTc]Tc-ADAPT6, demonstrated that their injections are safe, well-tolerated and cause low level of absorbed and equivalent doses. The goal of this preclinical study was to select the best probe for patients’ stratification and for response monitoring. Biodis-tribution of both tracers was compared in mice bearing SKOV-3 xenografts with high HER2 ex-pression and MDA-MB-468 xenografts with very low expression. Changes in accumulation of both probes in SKOV-3 tumors 24 h after injection of trastuzumab were evaluated. Both [99mTc]Tc-ADAPT6 and [99mTc]Tc-(HE)3-G3 permitted high contrast imaging of HER2-expressing tumors and clear discrimination between tumors with high and low HER2 expression. However, [99mTc]Tc-ADAPT6 has better preconditions for higher sensitivity and specificity of stratification. On the other hand, [99mTc]Tc-(HE)3-G3 is capable to sense the decrease of HER2 expression on re-sponse to trastuzumab therapy only 24 h after injection of loading dose. This indicates that this tracer would be better for monitoring early response to such treatment. Results of this study should be considered in planning of further clinical development of HER2 imaging probes.
ARTICLE | doi:10.20944/preprints202210.0087.v1
Subject: Life Sciences, Biochemistry Keywords: Echinoderm; Heat shock proteins; Ubiquitin; Regeneration; RNA-seq; Spinal cord injury
Online: 8 October 2022 (03:02:32 CEST)
Injury to the central nervous system (CNS), in most vertebrate animals, results in permanent damage and lack of function, due to their limited regenerative capacities. In contrast, echinoderms can fully regenerate their radial nerve cord (RNC) following transection, with little or no scarring. Investigators have associated the regenerative capacity of some organisms with the stress response and inflammation produced by the injury. Here we explore the gene activation profile of the stressed holothurian CNS. To do this, we performed RNA sequencing on isolated RNC explants submitted to the stress of transection and enzyme dissection and compared them to explants kept in culture for 3 days following dissection. We describe stress-associated genes, including members of heat-shock families, ubiquitin-related pathways, transposons, and apoptosis that were differentially expressed. Surprisingly, the stress response does not induce apoptosis in this system. Other genes associated with stress in other animal models, such as hero proteins and those associated with the integrated stress response, were not found to be differentially expressed either. Our results provide a new viewpoint on the stress response in the nervous system of an organism with an amazing regenerative capacity. This is the first step to deciphering the molecular processes that allow echinoderms to undergo fully functional CNS regeneration while also providing a comparative view for students of the stress response in other organisms.
ARTICLE | doi:10.20944/preprints202108.0137.v1
Subject: Life Sciences, Biochemistry Keywords: RNA binding proteins; SF1; Hrb87F; Bru1; Drosophila; flight muscle; RNAi; splicing
Online: 5 August 2021 (10:42:09 CEST)
The proper regulation of RNA processing is critical for muscle development and the fine-tuning of contractile ability between muscle fiber-types. RNA binding proteins (RBPs) regulate the diverse steps in RNA processing including alternative splicing, which generates fiber-type specific isoforms of structural proteins that confer contractile sarcomeres with distinct biomechanical properties. Alternative splicing is disrupted in muscle diseases such as myotonic dystrophy and dilated cardiomyopathy, and is altered after intense exercise as well as with aging. It is therefore important to understand splicing and RBP function, but currently only a small fraction of the hundreds of annotated RBPs expressed in muscle have been characterized. Here we demonstrate the utility of Drosophila as a genetic model system to investigate basic developmental mechanisms of RBP function in myogenesis. We find that RBPs exhibit dynamic temporal and fiber-type specific expression patterns in mRNA-Seq data and display muscle-specific phenotypes. We performed knockdown with 105 RNAi hairpins targeting 35 RBPs and report associated lethality, flight, myofiber and sarcomere defects, including flight muscle phenotypes for Doa, Rm62, mub, mbl, sbr, and clu. Interestingly, knockdown phenotypes of spliceosome components, as highlighted by phenotypes for A-complex components SF1 and Hrb87F (hnRNPA1), revealed level- and temporal-dependent myofibril defects. We further show that splicing mediated by SF1 and Hrb87F is necessary for Z-disc stability and proper myofibril development, and strong knockdown of either gene results in impaired localization of Kettin to the Z-disc. Our results expand the number of RBPs with a described phenotype in muscle and underscore the diversity in myofibril and transcriptomic phenotypes associated with splicing defects. Drosophila is thus a useful model to gain disease-relevant insight into cellular and molecular phenotypes observed when expression levels of splicing factors, spliceosome components and splicing dynamics are altered.
REVIEW | doi:10.20944/preprints202103.0066.v1
Subject: Biology, Anatomy & Morphology Keywords: GRAS protein, DELLA, Intrinsically Disordered Proteins, Arbuscular Mycorrhizal association, abiotic stress
Online: 2 March 2021 (10:01:42 CET)
The GAI‐RGA ‐ and ‐SCR (GRAS) proteins belong to the plant-specific transcription factor gene family and involved in several developmental processes, phytohormone and phytochrome signaling, symbiosis, stress responses etc. GRAS proteins have a conserved GRAS domain at C-terminal and hypervariable N-terminal. The C-terminal conserved domain directly affects the function of the GRAS proteins. For instance, in Arabidopsis, mutations in this domain in Slender rice 1 (SLR1) and Repressor of GA (RGA) proteins cause significant phenotypic changes. GRAS proteins have been reported in more than 30 plant species and till now it has been divided into 17 subfamilies. This review highlighted GRAS protein's importance during several biological processes in plants, structural features of GRAS proteins, their expansion and diversification in the plants, GRAS-interacting proteins complexes and their role in biological processes. We also summarized available recent research that utilized CRISPR-Cas9 technology to manipulate GRAS genes in a plant for different traits. Further, the exploitation of GRAS genes in crop improvement programs has also been discussed
REVIEW | doi:10.20944/preprints202006.0211.v1
Subject: Life Sciences, Immunology Keywords: COVID-19; diagnosis; ELISA; RDT; point-of-care test; antibody; proteins
Online: 17 June 2020 (08:36:25 CEST)
The ongoing pandemic of COVID-19 has not only commenced a global health emergency but agitated various aspects of humanity. During this period of crisis researchers over the world have ramped their efforts to constrain the disease in all possible ways whether it is vaccination, therapy, or diagnosis. Since the spread of the disease has not yet elapsed sharing the ongoing research findings could be the key to disease control and management. An early and efficient diagnosis could leverage the outcome until a successful vaccine is developed. Molecular tests both in-house and commercial kits are preferably being used worldwide in the COVID-19 diagnosis. However, the limitation of high prices and lengthy procedures impede their use for mass testing. Keeping the constant rise of infection in mind search for an alternative test that should be cost-effective, simple, and suitable for large scale testing and surveillance is a need of an hour. One such alternative could be the immunological tests. Therefore, in the last few months deluge of immunological rapid tests has been developed and validated across the globe. The objective of the present review is to share the diagnostic performance of various immunological assays reported so far in SARS-CoV-2 case detection. The article consolidated the studies (published and preprints) related to the serological tests such as chemiluminescence, enzyme-linked and lateral flow-based point-of-care tests in COVID-19 diagnosis and updated the current scenario. This review will hopefully be an add-on in COVID-19 research and will contribute to congregate the evidence for decision-making.
CONCEPT PAPER | doi:10.20944/preprints202006.0206.v1
Subject: Life Sciences, Biotechnology Keywords: serine protease; interferon; ribonucleoproteins; RNA binding proteins; SERPIN; A1AT; hemostasis rebalancing
Online: 17 June 2020 (03:20:54 CEST)
Emerging paradigms in interferon (IFN) biology suggest a dynamic INF induced interactome that extends through broader Interferon Stimulated Gene (ISG)- induction, which implicates interferon- ISG coordinated cross-talk with mRNA processing, post-translational modification and metabolic processes that underlie pathological (viral, autoimmune and tumor biology) and physiological (stem cell regenerative pathways) processes. INF immune responses can also be triggered by endogenous host-derived molecules that are generated in response to cellular stress or hemostasis imbalance to establish tissue repair and regeneration in first place, however, overactivation or lack of countermeasures can result in host tissue damage. The proteases are integral to viral and tumor pathology, and importantly serine proteases TMPRSS2 and trypsin have been identified as important molecular determinants underlying COVID-19 pathology, and emergence of coronaviruses cultured in vitro, respectively. We propose that pathogen associated proteases can act as novel stress-inducers to facilitate viral- competent immunomodulation. We term it as Protease Induced Transcriptomic/ epi-Transcriptomic Reshaping (PITTR) of host cells to counter cellular stress. We present a novel experimental model and our preliminary findings of trypsin- primed Caco-2 cells (CPT) that result in translational halt comparable to cells grown under serum-starvation conditions (CSS). CPT at escalating trypsin concentration (CPT- EC) induce upregulation of selective proteins that majorly map to ribosomal, RNA transport, and spliceosome ribonucleoproteins (RNPs). The inclusion of proinflammatory IL1-b to CPT (CPT- IL) resulted in global overexpression of proteins comparable to Caco-2 cells cultured in growth-factor rich serum conditions (CFBS), indicating a likely de-repression of trypsin- induced translational halt. Caco-2 cells display abortive interferon proteome under differential trypsin conditions (CPT, CPT-EC and CPT-IL), which is marked by complete lack of INF generation despite induction of intermediate ISGs, suggestive of protease (trypsin)- dependent regulation of INF response. Viruses regulate the proteome of stress granules (SGs) that are induced to cope transient translational halt as a central adaptive response to pathogen induced cellular stress. The integral components of SGs include non-translating mRNA, ribonucleoproteins (RNPs) and RNA binding proteins (RBPs), which together form biological condensates through a biophysical process involving weak electrostatic interactions through intrinsically disordered regions in RBPs resulting in liquid- liquid phase separation. We compared the CPT- EC proteome to the Mammalian Stress Granules Proteome (MSGP) database to explore potential RBPs that could possibly regulate INF response (and could act as potential anti-viral targets). Notably, differentially upregulated RNPs and potential RBPs from ISG family including ADAR and PRKRA, and RNA helicases implicated in viral pathogenesis were found to be upregulated in the CPT- EC proteome further strengthening the role of proteases (trypsin) in regulating INF pathways independent of the pathogen. We propose that the supplementation of viable SARS-CoV-2 viral loads to trypsin- primed host cells could recapitulate an infectious disease model, which may closely phenocopy pathogen- driven inflammation and signaling events. Based on the global downregulation of seven SERPINS (serine protease inhibitors) linked to thromboinflammation in our LCMS profiling data, we support the candidature of serine protease inhibitors for protease mediated viral pathologies. COVID-19 is increasingly linked to coagulopathy and resemblance to Neutrophil Extracellular Trap (NET) related thromboinflammatory features; SERPIN A1AT (alpha 1 antitrypsin) being a potent neutrophil- elastase inhibitor and a negative regulator of coagulation complement pathway may be a promising candidate for establishing hemostasis rebalancing in COVID-19 pathology.
ARTICLE | doi:10.20944/preprints201810.0521.v1
Subject: Medicine & Pharmacology, Gastroenterology Keywords: alcohol-induced Golgi disorganization; Golgi recovery; giantin; hepatic proteins; ethanol withdrawal
Online: 23 October 2018 (06:10:04 CEST)
Background: In hepatocytes and alcohol-metabolizing cultured cells, Golgi undergoes ethanol (EtOH)-induced disorganization. Periniclear and organized Golgi is important in liver homeostasis, but how the Golgi remains intact is unknown. Work from our laboratories showed that EtOH-altered cellular function could be reversed after alcohol removal; we wanted to determine whether this recovery would apply to Golgi. Methods: We used alcohol-metabolizing HepG2 (VA-13) cells (cultured with or without EtOH for 72 h) and rat hepatocytes (control and EtOH-fed (Lieber-DeCarli diet). For recovery, EtOH was removed and replenished with control medium (48 hours for VA-13 cells) or control diet (10 days for rats). Results: EtOH-induced Golgi disassembly was associated with de-dimerization of the largest Golgi matrix protein giantin, along with impaired transport of selected hepatic proteins. After recovery from EtOH, Golgi regained their compact structure, and alterations in giantin and protein transport were restored. In VA-13 cells, when we knocked down giantin, Rab6a GTPase or non-muscle Myosin IIB, minimal changes were observed in control conditions, but post-EtOH recovery was impaired. Conclusions: These data provide a link between Golgi organization and plasma membrane protein expression and identify several proteins whose expression is important to maintain Golgi structure during the recovery phase after EtOH administration.
CONCEPT PAPER | doi:10.20944/preprints202005.0182.v1
Subject: Life Sciences, Virology Keywords: Coronavirus Nsp proteins; ribosomal proteins homology; inhibition of ribosome turn over; rRNA methyltransferase; protein synthesis inhibition; low ATP formation; blood clotting; low blood pressure and coma
Online: 10 May 2020 (18:14:55 CEST)
Multi-Alignment method coupled with phylogenetic analysis we disclosed the Nsp9 and Nsp10 non-structural proteins of Corona Virus as rRNA RlmH/K methyltransferases with similarities with bin recombinase and int-core integrase fold. Further, Nsp9 has similarities to S8 ribosomal protein and Nap10 has similarity to S10 ribosomal protein. Previously, we showed Nsp13, Nsp14, Nsp15 and Nsp16 are also different types of rRNA RlmE/N and Cfr-like methyltransferases-ribonuclease with RNA helicase domains. Two domains of Nsp13 astonishingly have similarities to ribosomal proteins L6 and L9. Taken together, Nsp9/10 and Nsp13-16 proteins could mimic host ribosome assembly and also could methylate rRNA of mitobibosome preventing mitochondrial protein synthesis and oxidative phosphorylation. Low ATP synthesis causes lowering blood pressure following coma but very ATP concentration (1-10nM) surely induces platelets aggregation through vWA, collagen and GpIIb/IIIa proteins followed by fibrin formation and blood clotting as recently have seen in the lung of many Corona virus infected patients. We have also postulated that two polyproteins itself resemble like 28S and 38S mitoribosome subunits and compete with rRNAs inhibiting the ribosome turnover and new protein synthesis due to their similarities with many ribosomal proteins. Such finding may be valuable in computer-based novel drug design against Corona virus.
ARTICLE | doi:10.20944/preprints202301.0247.v1
Subject: Life Sciences, Biochemistry Keywords: statin; natural compounds; Bcl2 family proteins; intrinsic apoptosis pathway; caspase dependent apoptosis
Online: 13 January 2023 (09:29:40 CET)
Glioblastoma multiforme (GBM) is one of the deadliest cancers. Temozolomide (TMZ) is the most common chemotherapy used for GBM patients. Recently, combination chemotherapy strategies have more effective antitumor effects and focus on slowing down the development of chemotherapy resistance. A combination of TMZ and cholesterol lowering medications (statins) is currently under investigation in in vivo and clinical trials. In our current investigation, we have used a triple combination therapy of TMZ, Simvastatin (Simva), and Acetylshikonin (ASH) and investigated its apoptotic mechanism in GBM cell lines (U87 and U251). We used viability, apoptosis, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), caspase-3/-7, acridine orange (AO) and immunoblotting autophagy assays. Our results showed that TMZ/Simva/ASH combination therapy significantly induced more apoptosis compared to TMZ, Simva, ASH, and TMZ/Simva treatments in GBM cells. Apoptosis via TMZ/Simva/ASH treatment induced mitochondrial damage (increase of ROS, decrease of MMP) and induced caspase-3/7 activation in both GBM cell lines. Compared to all single treatments and the TMZ/Simva treatment, TMZ/Simva/ASH significantly increased positive acidic vacuole organelles. We further confirmed that the increase of AVOs during the TMZ/Simva/ASH treatment was due to partial inhibition of autophagy flux (accumulation of LC3β-II and decrease in p62 degradation) in GBM cells. Our investigation also showed that TMZ/Simva/ASH-induced cell death was depended on autophagy flux as further inhibition of autophagy flux increased TMZ/Simva/ASH-induced cell death in GBM cells. Finally, our results showed that TMZ/Simva/ASH treatment potentially depends on an increase of Bax expression in GBM cells. Our current investigation might open new avenues for more effective treatment of GBM but further investigations are required for better identification of the mechanisms.
ARTICLE | doi:10.20944/preprints202212.0331.v1
Subject: Life Sciences, Molecular Biology Keywords: SMN; RNA-binding proteins; head and neck cancers; squamous cell carcinoma; EGFR
Online: 19 December 2022 (09:02:58 CET)
Head and neck squamous cell carcinoma (HNSCC) arise from the mucosal epithelium in the oral cavity, pharynx, sino-nasal region, and larynx. Laryngeal squamous cell carcinoma (LSCC) represents one-third of all head and neck cancers. Dysregulated RNA-related pathways define an important molecular signature in this aggressive carcinoma. The Survival Motor Neuron (SMN) protein regulates fundamental aspects of the RNA metabolism but, curiously, its role in cancer is virtually unknown. For the first time, here we focus on SMN in cancer context. We conducted a pilot study in a total of 20 patients with LSCC where SMN was found overexpressed at both the protein and transcript levels. By a cellular model of human laryngeal carcinoma, we demonstrated that SMN impacts cancer-relevant behaviors and perturbs key players of cell migration, invasion, and adhesion. Furthermore, in LSCC we showed a physical interaction between SMN and the epidermal growth factor receptor (EGFR), whose overexpression is an important feature in these tumours. This study candidates SMN as novel therapeutic target in LSSC, and likely in the whole spectrum of HNSCC. Overall, we provide the first analysis of SMN in human cancer.
ARTICLE | doi:10.20944/preprints202210.0099.v1
Subject: Life Sciences, Microbiology Keywords: Leptospira spp., secretome; virulent-associated secreted proteins; in-vivo mimic mammalian condition
Online: 9 October 2022 (02:07:23 CEST)
Leptospirosis remains an important worldwide zoonotic disease caused by Leptospira spp affecting human and animals. This research aims to study the virulent-associated secreted proteins (protein secretome) of pathogenic Leptospira interrogans serovar Icterohemorrhagiae strain RGA (Leptospira RGA) transition from the environment to mammalian physiological osmolarity, temperature (37 °C) and carbon dioxide concentration (5% CO2) conditions for 24 h. Mass Spectrometry and bioinformatics approaches, we identified 69 potential secreted proteins from the culture supernatant of the Leptospira RGA isolate. We discovered transporters and porins such as phosphate porin, outer membrane efflux, ompA family protein, and polymer-forming cytoskeletal family protein under hyperosmotic condition. Under heat stress, degradation enzymes included zinc metallopeptidase, M23 family (LA3456, LA0709), Rhs family protein (LA1765), thermolysin metallopeptidase; / hydrolase family (LA1345, LA2501). Oxidative stress response proteins induced by osmolarity and temperature shifts included chaperon GrpE, DnaK (LA3705), antioxidants, i.e., thiol-specific redoxin, and peroxiredoxin (LA2809). In response to the in vivo transition, metabolic and other enzymes involved in energy production (COG:C), amino acid metabolism and transport (COG:E), and lipid metabolism and transport (COG:I), as well as moonlighting proteins functionally binding to plasminogen and fibronectin and regulating transcription, were also discovered. An overview of secreted proteins will supplement our understanding of Leptospira biology and pathogenesis during infection and also in response to environmental stimuli and their potential virulent determinants have the potential for developing leptospirosis vaccines and diagnosis.
ARTICLE | doi:10.20944/preprints202208.0126.v1
Subject: Chemistry, Applied Chemistry Keywords: Eugenol; Essential oils; Nanoencapsulation; Biopesticides; Insecticides; Odorant binding proteins; Inverted virtual screening
Online: 5 August 2022 (14:43:22 CEST)
The eugenol derivative, ethyl 4-(2-methoxy-4-(oxiran-2-ylmethyl)phenoxy)butanoate 1, with promising insecticidal capability was encapsulated in liposomal formulations of egg-phosphatidylcholine/cholesterol (Egg-PC:Ch) 70:30 and of 100% dioleoylphosphatidylglycerol (DOPG). Compound-loaded Egg-PC:Ch liposomes exhibit small hydrodynamic diameters (below 100 nm), high encapsulation efficiency (88.8% ± 2.7%), higher stability and a more efficient compound release, being chosen for assays in Sf9 insect cells. Compound 1 elicited a loss of cell viability up to 80% after 72h of incubation. Relevantly, encapsulation maintained the toxicity of compound 1 towards insect cells, while it lowered toxicity towards human cells, thus showing the selectivity of the system. Structure based inverted virtual screening was used to predict the most likely targets and molecular dynamics simulations and free energy calculations were used to demonstrate that this molecule can form a stable complex with insect odorant binding proteins and/or acetylcholinesterase.
ARTICLE | doi:10.20944/preprints202206.0376.v1
Subject: Biology, Other Keywords: effector proteins; genome-wide analysis; Ganoderma boninense; basal stem rot; genome architecture
Online: 28 June 2022 (04:59:14 CEST)
Ganoderma boninense is the major causal agent for the basal stem rot (BSR) disease in oil palm, causing the progressive rot of the basal part of the stem. Despite its prominence, key pathogenicity determinants for the aggressive nature of hemibiotrophic infection remain unknown. In this study, genome sequencing and annotation of G. boninense T10 were carried out using the Illumina sequencing platform and comparative genome analysis was performed with previously reported G. boninense strains (NJ3 and G3). The pan-secretome of G. boninense was constructed and comprised of 937 core orthogroups, 243 accessory orthogroups, and 84 strain-specific orthogroups. A set of core candidate effector proteins (CEPs) were found to be enriched with catalytic protein classified as the carbohydrate-active enzymes, hydrolases as well as non-catalytic proteins. Differential expression analysis revealed an upregulation of CEP genes which was linked to the suppression of PTI signaling cascade while the downregulation of CEP genes was linked to the inhibition of PTI by preventing host defense elicitation. Genome architecture analysis revealed the one-speed architecture of the G. boninense genome and the lack of preferential association of CEP genes to the transposable elements. The findings obtained from this study would aid in the characterization of pathogenicity determinants and molecular biomarkers of BSR disease.
ARTICLE | doi:10.20944/preprints202008.0621.v2
Subject: Biology, Anatomy & Morphology Keywords: Clustering; Mutation; Amino acid substitution; Structural proteins; Biochemical properties; Functional sub-domains
Online: 4 March 2021 (10:17:15 CET)
SARS-CoV-2 is mutating and creating divergent variants across the world. An in-depth investigation of the amino acid substitution in the genomic signature of SARS-CoV-2 proteins is highly essential for understanding its host adaptation and infection biology. A total of 9587 SARS-CoV-2 structural protein sequences collected from 49 different countries are used to characterize protein-wise variants, substitution pattern (type and location), and major substitution changes. The majority of the substitutions are distinct, occurred mostly in a particular location, and leads to a change in amino acid's biochemical properties. In terms of mutational changes, Envelope (E) and Membrane (M) proteins are relatively stable than Nucleocapsid (N) and Spike (S) proteins. Several co-occurrence substitutions are observed, particularly in S and N proteins. Substitution specific to active sub-domains reveals that Heptapeptide Repeat, Fusion peptides, Transmembrane in S protein, and N-terminal and C-terminal domains in N protein are remarkably mutated, and also found few deleterious mutations in these domains.
REVIEW | doi:10.20944/preprints202012.0751.v1
Subject: Chemistry, Analytical Chemistry Keywords: Maillard reaction; protein-polysaccharide conjugate; plant proteins; technofunctionality; application; Amadori products; AGEs
Online: 30 December 2020 (11:21:19 CET)
Plant proteins being considered to become the most important protein source of the future, they must be able to replace the animal-derived proteins currently in use as technofunctional food ingredients. This poses challenges because plant proteins are oftentimes storage proteins with a high molecular weight and low water solubility. One promising approach to overcome these limitations is the glycation of plant proteins. The covalent bonding between the proteins and different carbohydrates created via the initial stage of the Maillard reaction can improve the technofunctional characteristics of these proteins without the involvement of potentially toxic chemicals. However, compared to studies with animal-derived proteins, glycation studies on plant proteins are currently still underrepresented in literature. This review provides an overview of the existing studies on the glycation of the major groups of plant proteins with different carbohydrates using different preparation methods. Emphasis is put on the reaction conditions used for glycation as well as the modifications to physicochemical properties and technofunctionality. Different applications of these glycated plant proteins in emulsions, foams, films, and encapsulation systems are introduced. Another focus lies on the reaction chemistry of the Maillard reaction and ways to harness it for controlled glycation and to limit the formation of undesired advanced glycation products. Finally, challenges related to the controlled glycation of plant proteins to improve their properties are discussed.
COMMUNICATION | doi:10.20944/preprints202011.0441.v1
Subject: Biology, Anatomy & Morphology Keywords: Sars-Cov-2; Proteomic,; Ghost proteome,; Alternative proteins,; AltOrf.; interactomic; drug repurposing
Online: 16 November 2020 (17:50:19 CET)
Conventionally, eukaryotic mRNAs were thought to be monocistronic, leading to the translation of a single protein. However, large-scale proteomics has led to a massive identification of proteins translated from mRNAs of alternative ORF (AltORFs), in addition to the predicted proteins issued from the reference ORF or from ncRNAs. These alternative proteins (AltProts) are not represented in the conventional protein databases and this “Ghost proteome” was not considered until recently. Some of these proteins are functional and there is growing evidence that they are involved in central functions in physiological and physiopathological context. Based on our experience with AltProts we have got interested in finding out their involvement in development of the SARS-CoV-2 virus, responsible for the 2020 Covid-19 outbreak. Thus, we have scrutinized the recently published data by Krogan and coworkers (2020) on the SARS-CoV-2 interactome with host cells by co-IP in the perspective of drug repurposing. The initial work has revealed the interaction between 332 human cellular RefProts with the 27 viral proteins. Re-interrogation of this data using 23 viral targets and including AltProts, followed by enrichment of the interaction networks, leads to identify 218 RefProts (in common to initial study) plus 56 AltProts involved in 93 interactions. This demonstrates the necessity to take into account the Ghost proteome for discovering new therapeutic targets and establish new therapeutic strategies. Missing the ghost proteome in the drug metabolism and pharmacokinetic (DMPK) drug development pipeline will certainly be a major limitation to the establishment of efficient therapies.
ARTICLE | doi:10.20944/preprints202003.0024.v1
Subject: Medicine & Pharmacology, Allergology Keywords: SARS-CoV-2; nonstructural proteins (NSP); NSP12; NSP7; NSP8; virtual screening; inhibitor
Online: 2 March 2020 (03:11:33 CET)
A novel coronavirus (SARS-CoV-2) that is initially found to trigger human severe respiratory illness in Wuhan City of China in 2019, has killed 2,718 people in China by February 26, 2020, and which has been recognized as a public health emergency of international concern as well. And the virus has spread to more than 38 countries around the world. However, the drug has not yet been officially licensed or approved to treat SARS-Cov-2 infection. NSP12-NSP7-NSP8 complex of SARS-CoV-2, essential for viral replication and transcription, is generally regarded as a potential target to fight against the virus. According to the NSP12-NSP7-NSP8 complex (PDB ID: 6NUR) structure of SARS, two homologous models were established for virtual screening in the present study, namely NSP12-NSP7 interface model and NSP12-NSP8 interface model. Seven compounds (Saquinavir, Tipranavir, Lonafarnib, Tegobuvir, Olysio, Filibuvir, and Cepharanthine) were selected for binding free energy calculations based on virtual screening and docking scores. All seven compounds can combine well with NSP12-NSP7-NSP8 in the homologous model, providing drug candidates for the treatment and prevention of SARS-CoV-2.
ARTICLE | doi:10.20944/preprints201807.0194.v2
Subject: Medicine & Pharmacology, Clinical Neurology Keywords: brain-derived neurotrophic factor; Frailty; Inflammasome proteins; Interleukin-1β; Peri-infarct area
Online: 23 August 2018 (07:59:58 CEST)
A risk of ischemic stroke increases exponentially after menopause. Even a mild-ischemic stroke can result in increased frailty. Frailty is a state of increased vulnerability to adverse outcomes, which subsequently increases risk of cerebrovascular events and severe cognitive decline, particularly after menopause. Several interventions to reduce frailty and subsequent risk of stroke and cognitive decline have been proposed in laboratory animals and patients. One of them is whole body vibration (WBV). WBV recuperates cerebral function and cognitive ability that deteriorates with increased frailty. The goal of the current study is to test the efficacy of WBV in reducing post-ischemic stroke frailty and brain damage in reproductively senescent female rats. Reproductively senescent Sprague–Dawley female rats were exposed to transient middle cerebral artery occlusion (tMCAO) and randomly assigned to either WBV or control groups. Animals placed in the WBV group underwent 30 days of WBV (40 Hz) treatment performed twice daily for 15 min each session, 5 days each week. The motor functions of animals belonging to both groups were tested intermittently and at the end of treatment period. Brains were then harvested for inflammatory markers and histopathological analysis. The results demonstrate a significant reduction in inflammatory markers and infarct volume with significant increases in brain-derived neurotrophic factor and improvement in functional activity after tMCAO in middle-aged female rats that were treated with WBV as compared to the control group. Our results may facilitate a faster translation of the WBV intervention for improved outcome after stroke, particularly among frail women.
ARTICLE | doi:10.20944/preprints202210.0302.v1
Subject: Life Sciences, Virology Keywords: poxviruses; monkeypox virus; envelope proteins; drug repurposing; bioactive phytochemicals; molecular docking, molecular simulation
Online: 20 October 2022 (09:32:02 CEST)
The monkeypox virus (MPXV) has become a major threat due to the increasing global caseload and the ongoing multi-country outbreak in non-endemic territories. Due to limited research in this avenue and the lack of intervention strategies, the present study was aimed to virtually screen bioactive phytochemicals against envelope proteins of MPXV via rigorous computational approaches. Molecular docking and molecular dynamic (MD) simulations were used to investigate the binding affinity of 12 phytochemicals against three envelope proteins of MPXV, viz., D13, A26, and H3. Silibinin, oleanolic acid, and ursolic acid were computationally identified as potential phytochemicals that showed strong binding affinity towards all the tested structural proteins of MPXV through molecular docking. The stability of the docked complexes was also confirmed by MD simulations. ADME analysis also computationally confirmed the drug-like properties of the phytochemicals, thereby asserting their suitability for consumption. Hence, this study envisions the candidature of bioactive phytochemicals as promising inhibitors against the MPXV, serving as template molecules that could further be experimentally evaluated for their efficacy against monkeypox.
REVIEW | doi:10.20944/preprints202208.0366.v1
Subject: Physical Sciences, General & Theoretical Physics Keywords: Curvature proteins; Membrane Remodelling; Protein-protein interactions; Mesoscopic Modeling; Backmapping and molecular reconstruction
Online: 22 August 2022 (03:20:01 CEST)
Specialized classes of proteins, working together in a tightly orchestrated manner, induce and maintain highly curved cellular and organelles membrane morphology. Due to the various ex- perimental constraints, including the resolution limits of imaging techniques, it is non-trivial to accurately elucidate interactions among the various components involved in membrane deformation. The spatial and temporal scales of the systems also make it formidable to investigate them using simulations with molecular details. Interestingly, mechanics-based mesoscopic models have been used with great success in recapitulating the membrane defor- mations observed in experiments. In this review, we collate together and discuss the various mechanics based mesoscopic models for protein-mediated membrane deformation studies. In particular, we provide an elaborate description of a mesoscopic model where the membrane is modeled as a triangulated sheet and proteins are represented as either nematics or fila- ments. This representation allows us to explore the various aspects of protein-protein and protein-membrane interactions as well as examine the underlying mechanistic pathways for emergent behavior such as curvature-mediated protein localization and membrane deforma- tion. We also put forward current efforts in the field towards back-mapping these mesoscopic models to finer-grained particle based models - a framework that could be used to explore how molecular interactions propagate to physical scales and vice-versa. We end the review with an integrative-modeling based road map where experimental imaging micrograph and biochemical data are combined with mesoscopic and molecular simulations methods in a theoretically consistent manner to faithfully recapitulate the multiple length and time scales in the membrane remodeling processes.
ARTICLE | doi:10.20944/preprints202010.0351.v1
Subject: Life Sciences, Biochemistry Keywords: Streptococcus mutans; adhesion proteins; Antigen I/II; Structure-based virtual screening; molecular dynamics
Online: 16 October 2020 (13:48:43 CEST)
Streptococcus mutans is well known for having virulence factors associated with its cariogenic role, such as glucosyltransferases, which have been used as targets for the virtual screening of molecules with inhibitory capacity. The Antigen I/II of S. mutans is involved in the adhesion to the surface of the tooth and the bacterial co-aggregation in the biofilm formation, despite that, this protein has not been used as a target in a virtual strategy search for inhibitors. In this study we identified in silico and evaluated in vitro molecules with adhesion inhibitory potential on S. mutans Ag I/II. A virtual screening of 883,551 molecules was conducted, cytotoxicity analysis on fibroblast cells, S. mutans adhesion studies, scanning electron microscopy analysis for bacterial integrity, and molecular dynamics simulation were also performed. We have found three molecules (ZI-187, ZI-939, ZI-906) that were not cytotoxic and inhibited the adhesion of S. mutans to polystyrene microplates. Molecular dynamic simulation by 300 nanoseconds showed stability of the interaction between ZI-187 and Ag I/II (PDB: 3IPK). This work provides three new molecules that targets Ag I/II and have the capacity to inhibit in vitro the S. mutans adhesion on polystyrene microplates and provides a new computational line for the search and selection of safe inhibitory molecules against different pathogens.
REVIEW | doi:10.20944/preprints202010.0066.v1
Subject: Keywords: Vitis vinifera; Plasmopara viticola; plant-pathogen interaction; proteomics; defence-related proteins, ETS, ETI
Online: 5 October 2020 (10:58:33 CEST)
Grapevine is one of the most relevant crops in the world being used for economically important products such as wine. Yet, relevant grapevine cultivars are heavily affected by diseases such as the downy mildew disease caused by Plasmopara viticola. Improvements on grapevine resistance are made mainly by breeding techniques where resistance traits are introgressed into cultivars with desired grape characteristics. However, there is still a lack of knowledge on how resistant or tolerant cultivars tackle the P. viticola pathogen. In this study, using a shotgun proteomics LC-MS/MS approach, we unravel the protein modulation of a highly tolerant grapevine cultivar, V. vinifera ‘Regent’, in the first hours post inoculation (hpi) with P. viticola. At 6 hpi, proteins related to defence and to response to stimuli are negatively modulated while at 12 hpi there is an accumulation of proteins belonging to both categories. The co-occurrence of effector-triggered susceptibility (ETS) and effector-triggered immunity (ETI) is detected at both time-points, showing that these defence processes present high plasticity. The results obtained in this study unravel the tolerant grapevine defence strategy towards P. viticola and may provide valuable insights on resistance associated candidates and mechanisms, which may play an important role in the definition of new strategies on breeding approaches.
ARTICLE | doi:10.20944/preprints202009.0283.v1
Subject: Life Sciences, Molecular Biology Keywords: Decapod Crustaceans; dsRNA transport; Gene silencing mechanism; Serum dsRNA binding proteins; Systemic RNAi
Online: 13 September 2020 (12:09:03 CEST)
RNA interference (RNAi) has become a widely utilised method to study gene function, yet despite this, many of the mechanisms surrounding RNAi remain elusive. The core RNAi machinery is relatively well understood, however many of the systemic mechanisms, particularly double stranded RNA (dsRNA) transport, are not. Here, we demonstrate that dsRNA binding proteins in the serum contribute to systemic RNAi, and may be the limiting factor in RNAi capacity for species such as spiny lobsters where gene silencing is not functional. Incubating serum from a variety of species across phyla with dsRNA led to a gel mobility shift in species where systemic RNAi has been observed, with this response being absent in species where systemic RNAi has never been observed. Proteomic analysis suggested lipoproteins may be responsible for this phenomenon, and may transport dsRNA to spread the RNAi signal systemically. Following this, we identified the same gel shift in the slipper lobster Thenus australiensis and subsequently silenced the insulin androgenic gland hormone, marking the first time RNAi has been performed in any lobster species. These results pave the way for inducing RNAi in spiny lobsters, and better understanding the mechanisms of systemic RNAi in Crustacea, as well as across phyla.
ARTICLE | doi:10.20944/preprints201804.0017.v1
Subject: Life Sciences, Cell & Developmental Biology Keywords: minicircle; induced pluripotent stem cells; chondrogenesis; chondrocyte; bone morphogenetic proteins; transforming growth factors
Online: 2 April 2018 (09:59:50 CEST)
The human degenerative cartilage has low regenerative potential. Chondrocyte transplantation offers a promising strategy for cartilage treatment and regeneration. Currently chondrogenesis using human pluripotent stem cells are accomplished using human recombinant growth factors. Here, we differentiated human induced pluripotent stem cells (hiPSCs) into chondrocytes and cartilage pellet using minicircle vectors. Minicircles are used as a non-viral gene delivery system for gene therapy in various diseases. Non-viral gene delivery can produce growth factors without integrating into the host genome. Minicircle vectors containing bone morphogenetic protein 2 (BMP2) and transforming growth factor, beta 3 (TGFβ3) were successfully generated and delivered to hiPSC-derived outgrowth (OG) cells. Cell pellets generated using minicircle-transfected OG cells successfully differentiated into chondrogenic lineage. Chondrogenic pellets transfected with growth factor-encoding minicircles effectively recovered osteochondral defect in rat models. Taken together, this work shows the potential application of minicircles in cartilage regeneration using hiPSCs.
REVIEW | doi:10.20944/preprints201706.0068.v1
Subject: Life Sciences, Other Keywords: human cytolytic fusion proteins; immunotherapy; microtubule associated protein tau (MAP); cancer; inflammatory diseases
Online: 15 June 2017 (12:02:31 CEST)
Some of the most promising small molecule toxins used to generate antibody drug conjugates (ADCs) include anti-mitotic agents (e.g auristatin and its derivatives) which are designed to attack cancerous cells at their most vulnerable state during mitosis. We were interested to identify a human cystostatic protein eventually showing comparable activities and allowing the generation of corresponding targeted fully human cytolytic fusion proteins. Recently, we identified the human microtubule associated protein tau (MAP tau) which binds specifically to tubulin and modulates the stability of microtubules thereby blocking mitosis and presumably vesicular transport. By binding and stabilizing polymerized microtubule filaments, MAP tau-based fusion proteins skew microtubule dynamics towards cell cycle arrest and apoptosis. This biological activity makes rapidly proliferating cells (e.g cancer and inflammatory cells) an excellent target for MAP tau-based targeted treatments. Their superior selectivity for proliferating cells confers additional selectivity towards upregulated tumor-associated antigens at their surface, thereby preventing off-target related toxicity against normal cells bearing tumor-associated antigens at physiologically normal to low levels. In this review, we highlight recent findings on MAP tau-based targeted cytolytic fusion proteins reported in preclinical immunotherapeutic studies.
ARTICLE | doi:10.20944/preprints201612.0145.v1
Subject: Life Sciences, Biochemistry Keywords: ATP-dependent proteolysis, Non-native membrane proteins, Periplasmic domain, Crystal structure, Photosystem II.
Online: 29 December 2016 (17:16:52 CET)
Prompt removal of misfolded membrane proteins and misassembled membrane protein complexes is essential for membrane homeostasis. However, the elimination of these toxic proteins from the hydrophobic membrane environment has high energetic barriers. Transmembrane FtsH is the only known ATP-dependent protease responsible for this task, unlike other well-studied soluble ATP-dependent proteases. The mechanisms by which FtsH recognizes, unfolds, translocates, and proteolyzes its substrates remain unclear. Here, we report the crystal structures of the Thermotoga maritima FtsH periplasmic domain (PD) in an associative trimeric state at a 1.5-1.95 Å resolution. We also describe the pH-dependent oligomerization states of the isolated PD using dynamic light scattering. These observations help us understand how FtsH recognizes membrane-anchored misfolded proteins.
ARTICLE | doi:10.20944/preprints202207.0065.v1
Subject: Biology, Other Keywords: SARS-CoV-2; VSV replicon; PDC-109; Bovine seminal plasma; Fn-type 2 proteins
Online: 5 July 2022 (07:49:32 CEST)
Mammalian seminal plasma contains a multitude of bioactive components, including lipids, glucose, mineral elements, metabolites, proteins, cytokines and growth factors, with various functions during insemination and fertilization. The seminal plasma protein PDC-109 is one of the major soluble components of the bovine ejaculate and is crucially important for sperm motility, capacitation and acrosome reaction. A hitherto underappreciated function of seminal plasma is its anti-microbial and anti-viral activity, which may limit sexual transmission of infectious diseases during intercourse. We have recently discovered that PDC-109 inhibits the membrane fusion activity of influenza virus particles and significantly impairs viral infections at micromolar concentrations. Here we investigated whether the antiviral activity of PDC-109 is restricted to Influenza or if other mammalian viruses are similarly affected. We focused on Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), the etiological agent of the Coronavirus Disease 19 (COVID-19), thoroughly assessing PDC-109 inhibition with SARS-CoV-2 Spike (S)-pseudotyped reporter virus particles, but also live-virus infections. Consistent with our previous publications we found significant virus inhibition, albeit accompanied by substantial cytotoxicity. Using time-of-addition experiments however, we discovered treatment regimen that enable virus suppression without affecting cell viability. We furthermore demonstrated that PDC-109 is also able to impair infections mediated by the VSV glycoprotein (VSVg) thus indicating a broad pan-antiviral activity against multiple virus species and families.
ARTICLE | doi:10.20944/preprints202204.0312.v1
Subject: Engineering, Biomedical & Chemical Engineering Keywords: Tensional homeostasis; Traction microscopy; Gastric cancer cells; E-cadherin mutations; Extra-cellular matrix proteins
Online: 30 April 2022 (05:18:13 CEST)
In epithelia, breakdown of tensional homeostasis is closely associated with E-cadherin dysfunction and disruption of tissue function and integrity. In this study, we investigated the effect of E-cadherin mutations affecting distinct protein domains on tensional homeostasis of gastric cancer cells. We used micropattern traction microscopy to measure temporal fluctuations of cellular traction forces in AGS cells transfected with the wild-type E-cadherin or with variants affecting the extracellular, the juxtamembrane, and the intracellular domains of the protein. We focused on the dynamic aspect of tensional homeostasis, namely the ability of cells to maintain a consistent level of tension, with low temporal variability around a set point. Cells were cultured on hydrogels micropatterned with different extracellular matrix (ECM) proteins to test whether the ECM adhesion impacts cell behavior. A combination of Fibronectin and Vitronectin was used as a substrate that promotes the adhesive ability of E-cadherin dysfunctional cells, whereas Collagen VI was used to test an unfavorable ECM condition. Our results showed that mutations affecting distinct E-cadherin domains influenced differently cell tensional homeostasis, and pinpointed the juxtamembrane and intracellular regions of E-cadherin as the key players in this process. Furthermore, Fibronectin and Vitronectin might modulate cancer cell behavior towards tensional homeostasis.
Subject: Life Sciences, Biochemistry Keywords: heat shock proteins; Diabetes mellitus; beta-cells; endoplasmic reticulum stress; proteostasis; HSPB1; cytoprotection; apoptosis.
Online: 8 July 2021 (14:03:59 CEST)
During type 1 diabetes mellitus (T1DM) development, beta-cells undergo intense endoplasmic reticulum (ER) stress that could result in apoptosis through the failure of adaptation to the unfolded protein response (UPR). Islet transplantation is considered an attractive alternative among beta-cell replacement therapies for T1DM. To avoid the loss of beta-cells that will jeopardize the transplant´s outcome, several strategies are being studied. We have previously shown that prolactin induces protection against pro-inflammatory cytokines and redox imbalance-induced beta-cell death by increasing heat shock protein B1 (HSPB1) levels. Since the role of HSPB1 in beta cells has not been deeply studied, we investigated the mechanisms involved in unbalanced protein homeostasis caused by intense ER stress and overload of the proteasomal protein degradation pathway. We tested whether HSPB1-mediated cytoprotective effects involved UPR modulation and improvement of protein degradation via the ubiquitin-proteasome system. We demonstrated that increased levels of HSPB1: attenuated levels of pro-apoptotic proteins like CHOP and BIM, increased protein ubiquitination and the speed of proteasomal protein degradation. Our data showed that HSPB1 induced resistance to proteotoxic stress and thus enhanced cell survival via an increase in beta-cell proteolytic capacity. These results could contribute to generate strategies aiming at optimization of beta-cell replacement therapies.
ARTICLE | doi:10.20944/preprints202105.0752.v1
Subject: Life Sciences, Biochemistry Keywords: Candida albicans; invasive candidiasis; secretome; secreted proteins; hypha; diagnosis; biomarkers; immunoproteomics; SERPA; serologic response.
Online: 31 May 2021 (11:40:35 CEST)
Invasive candidiasis (IC) is associated with high morbidity and mortality in hospitalized patients if not diagnosed early. Long-term use of central venous catheters is a predisposing factor for IC. Hyphal forms of Candida albicans (the major etiological agent of IC) are related to invasion of host tissues. The secreted proteins of hyphae are involved in virulence, host interaction, immune response, and immune evasion. To identify IC diagnostic biomarker candidates, we characterized the C. albicans hyphal secretome by gel-free proteomic analysis, and further assessed the antibody-reactivity patterns to this subproteome in serum pools from 12 patients with non-catheter-associated IC (ncIC), 11 patients with catheter-associated IC (cIC), and 11 non-IC patients. We identified 301 secreted hyphal proteins stratified to stem from the extracellular region, cell wall, cell surface, or intracellular compartments. ncIC and cIC patients had higher antibody levels to the hyphal secretome than non-IC patients. Seven secreted hyphal proteins were identified to be immunogenic (Bgl2, Eno1, Pgk1, Glx3, Sap5, Pra1 and Tdh3). Antibody-reactivity patterns to Bgl2, Eno1, Pgk1 and Glx3 discriminated IC patients from non-IC patients, while those to Sap5, Pra1 and Tdh3 differentiated between cIC and non-IC patients. These proteins may be useful for development of future IC diagnostic tests.
Subject: Life Sciences, Biochemistry Keywords: Rainbow trout; intestinal epithelium; intestinal stem cells; self-renewal; fish nutrition; brush border proteins
Online: 5 November 2020 (09:28:19 CET)
We previously showed that, based on the frequency and distribution of specific cell types, rainbow trout (RT) intestinal mucosa is divided in two regions that form a complex nonlinear 3D pattern and have a different renewal rate. This work had two aims. First, to investigate whether the unusual distribution of cell populations reflects a similar distribution of functional activities. To this end, we determined the protein expression pattern of three well defined enterocytes functional markers: Peptide Transporter 1 (PepT1), Sodium-Glucose/Galactose Transporter 1 (SGLT-1) and fatty acid-binding protein 2 (Fabp2). Second, to characterize the structure of RT intestinal stem cells (ISC) niche and to determine whether the different proliferative rates correlate with a different organization and/or extension of the stem cells population. We studied the expression and localization of well-characterized mammal ISC markers: LGR5, HOPX, SOX9, NOTCH1, DLL1 and WNT3A. Our results indicate that morphological similarity is associated with similar function only between the first portion of the mid-intestine and the apical part of the complex folds in the second portion. Mammal ISC markers are all expressed in RT but their localization is completely different suggesting also substantial functional differences. Finally, higher renewal rates are supported by a more abundant ISC population.
ARTICLE | doi:10.20944/preprints201901.0013.v1
Subject: Life Sciences, Molecular Biology Keywords: Aedes aegypti, Leucine-Rich Repeat Proteins, Zika virus, Chikungunya virus, immune responses, gene expression
Online: 3 January 2019 (12:12:05 CET)
Aedes aegypti (L.) is the primary vector of chikungunya, dengue, yellow fever and Zika viruses. The leucine-rich repeats (LRR)-containing domain is evolutionarily conserved in many proteins associated with innate immunity in invertebrates and vertebrates, as well as plants. We focused on the AaeLRIM1 and AaeAPL1 gene expressions in response to Zika virus (ZIKV) and Chikungunya virus (CHIKV) infection using a time course study, as well as the developmental expressions in the eggs, larvae, pupae, and adults. RNA-seq analysis data provided 60 leucine-rich repeat related transcriptions in Ae. aegypti in response to Zika virus (Accession number: GSE118858, https://www.ncbi.nlm.nih.gov/gds/?term=GSE118858). RNA-seq analysis data showed that AaeLRIM1 (AAEL012086-RA) and AaeAPL1 (AAEL009520-RA) were significantly upregulated 2.5 and 3-fold during infection by ZIKV 7-days post infection (dpi) of an Ae. aegypti Key West strain compared to an Orlando strain. The qPCR data showed that LRR-containing proteins AaeLRIM1, AaeAPL1 and five paralogues were expressed 100-fold lower than other nuclear genes, such as defensin, during all developmental stages examined. Together, these data provide insights into transcription profiles of LRR proteins of Ae. aegypti during its development and in response to infection with emergent arboviruses.
REVIEW | doi:10.20944/preprints201705.0129.v1
Subject: Life Sciences, Biophysics Keywords: cell fate decision; cancer attractors; gene network dynamics; EMT; drug resistance; intrinsically disordered proteins
Online: 17 May 2017 (06:25:17 CEST)
Waddington’s epigenetic landscape, a famous metaphor in developmental biology, depicts how a stem cell progresses from an undifferentiated phenotype to a differentiated one. The concept of “landscape” in the context of dynamical system theory represents a high-dimensional cell state space, in which each cell phenotype is considered as an “attractor” that is determined by interactions among multiple variables (molecular players), and is buffered against environmental fluctuations. Further, biological noise is thought to play an important role during these cell-fate decisions and in fact controls transitions between different phenotypes. Here, we discuss these phenotypic transitions in cancer from a dynamical systems perspective and invoke the concept of “cancer attractors” – hidden stable states of the underlying regulatory network that are not occupied by normal cells. Using epithelial-to-mesenchymal transition (EMT), cancer stem-like properties, metabolic reprogramming and the emergence of drug/hormone resistance as examples, we illustrate how phenotypic plasticity in cancer cells enables them to acquire hybrid phenotypes (such as hybrid epithelial/mesenchymal and hybrid metabolic phenotypes) that tend to be more aggressive and notoriously resilient to drug/hormone treatment. Furthermore, we highlight multiple factors that may give rise to phenotypic plasticity in cancer cells, such as (a) multi-stability or oscillatory behaviors governed by underlying regulatory networks involved in cell-fate decisions in cancer cells, and (b) network rewiring due to conformational dynamics of intrinsically disordered proteins (IDPs) that are highly enriched in cancer cells. We conclude by discussing why a therapeutic approach that promotes ‘recanalization’, i.e. the exit from “cancer attractors” and re-entry into “normal attractors”, is more likely to succeed rather than a conventional approach that targets individual molecules/pathways.
REVIEW | doi:10.20944/preprints202110.0168.v1
Subject: Life Sciences, Cell & Developmental Biology Keywords: heat shock proteins; co-chaperones; protein quality control; protein folding; protein degradation; cardioprotection; neuroprotection; cancer
Online: 11 October 2021 (14:38:49 CEST)
Heat shock proteins (HSPs) are a family of molecular chaperones that regulate essential protein refolding and triage decisions to maintaining protein homeostasis. Numerous co-chaperone proteins directly interact and modify the function of HSPs, and these interactions impact the outcome of protein triage, impacting everything from structural proteins to cell signaling mediators. The chaperone/co-chaperone machinery protects against various stressors to ensuring cellular function in the face of stress. However, coding mutations, expression changes, and post-translational modifications of the chaperone/co-chaperone machinery can alter the cellular stress response. Importantly, these dysfunctions appear to contribute to numerous human diseases. Therapeutic targeting of chaperones is an attractive but challenging approach due to the vast functions of HSPs, likely contributing to the off-target effects of these therapies. Current efforts focus on targeting co-chaperones to develop precise treatments for numerous diseases caused by defects in protein quality control. This review focuses on the recent developments regarding selected HSP70/HSP90 co-chaperones, focusing on cardioprotection, neuroprotection, and cancer. We also discuss therapeutic approaches that highlight both the utility and challenges of targeting co-chaperones.
ARTICLE | doi:10.20944/preprints202107.0531.v1
Subject: Biology, Anatomy & Morphology Keywords: A.thaliana; HaloTag; RNA-binding proteins; RNA pulldown assay; RNA-protein complexes; cold shock domain protein
Online: 23 July 2021 (09:32:28 CEST)
Study of RNA-protein interactions and identification of RNA targets are among the key aspects of understanding the RNA biology. Currently, various methods are available to investigate these interactions, in particular, RNA pulldown assay. In the present paper, a method based on the HaloTag technology is presented that is called Halo-RPD (HaloTag RNA PullDown). The proposed protocol uses plants with stable fusion protein expression and the MagneBeads magnetic beads to capture RNA-protein complexes directly from the cytoplasmic lysate of transgenic A. thaliana plants. The key stages described in the paper are as follows: 1) preparation of the magnetic beads 2) tissue homogenization and collection of control samples 3) precipitation and wash of RNA-protein complexes; 4) evaluation of protein binding efficacy; 5) RNA isolation; 6) analysis of the obtained RNA. Recommendations for better NGS assay designs are provided.
ARTICLE | doi:10.20944/preprints202104.0464.v1
Subject: Medicine & Pharmacology, Allergology Keywords: tumor-associated macrophage; exosomes; extracellular vesicles; heat shock proteins; oral cancer; fluorescent labeling of exosomes
Online: 19 April 2021 (11:50:52 CEST)
Tumor-associated macrophages are a key component in the tumor microenvironment, secreting extracellular vesicles (EVs) such as exosomes and other various factors for intercellular communication. However, macrophage-derived EVs heterogeneity and their cytotoxicity to cancer cells has not been well understood. Here, we aimed to separately isolate various types of macro-phage-EVs by size exclusion chromatography (SEC) method and investigate EV transmission and cytotoxicity to oral cancer cells. For fluorescence-labeling of cellular and EV membranes, palmitoylation signal-fused GFP and tdTomato were expressed in THP-1 monocytic cells and HSC-3 oral cancer cells, respectively. We found that fluorescence-labeled EVs secreted by macrophages were highly transmissive to oral cancer cells than those from parental monocytic cells. In a co-culture system and conditioned medium (CM), a macrophage-secreted unidentified factor was cytotoxic to oral cancer cells. We fractionated macrophage-derived EVs by the SEC method and performed western blotting to characterize various EV types. Three fractions were characterized: small exosomes (EXO-S: < 50 nm) fraction containing HSP90α, HSP90β, CD63 (EV marker) and β-actin; large exosomes (EXO-L: 50-200 nm) fraction containing CD9 (EV marker) and HSP90β; large EVs (100-500 nm) fraction. Notably, the macrophage-derived small exosomes fraction was cytotoxic to oral cancer cells, while large exosomes and large EVs were not. There-fore, it was implicated that macrophage-derived small exosomes are cytotoxic with high trans-mission potential to cancer cells.
REVIEW | doi:10.20944/preprints202101.0026.v1
Subject: Life Sciences, Biochemistry Keywords: 20S proteasome; protein degradation; intrinsically disordered proteins; enzyme functional cycle; peptides; peptidome; proteome; oxidative stress
Online: 4 January 2021 (12:16:42 CET)
Four decades of proteasome research have yielded extensive information on ubiquitin-dependent proteolysis. The archetype of proteasomes is a 20S barrel-shaped complex that does not rely on ubiquitin as a degradation signal but can degrade substrates with a considerable unstructured stretch. Since roughly half of all proteasomes in most eukaryotic cells are free 20S complexes, ubiquitin-independent protein degradation may coexist with ubiquitin-dependent degradation by the highly regulated 26S proteasome. This article reviews recent advances in our understanding of the biochemical and structural features that underlie the proteolytic mechanism of 20S proteasomes. The two outer α-rings of 20S proteasomes provide a number of potential docking sites for loosely folded polypeptides. The binding of a substrate can induce asymmetric conformational changes, trigger gate opening, and initiate its own degradation through a protease-driven translocation mechanism. Consequently, the substrate translocates through two additional narrow apertures augmented by the β-catalytic active sites. The overall pulling force through the two annuli results in a protease-like unfolding of the substrate and subsequent proteolysis in the catalytic chamber. Although both proteasomes contain identical β-catalytic active sites, the differential translocation mechanisms yield distinct peptide products. Non-overlapping substrate repertoires and product outcomes rationalize cohabitation of both proteasome complexes in cells.
ARTICLE | doi:10.20944/preprints202009.0607.v1
Subject: Chemistry, Analytical Chemistry Keywords: Milk Serum; whey proteins; RP-HPLC-UV; free amino acids; RP-HPLC-FLD; antimicrobial study
Online: 25 September 2020 (11:45:09 CEST)
The aim of this study was characterization of some dairy drinks based on Milk Serum regarding major whey proteins (WP) and free amino acids (FAAs) using reversed phase high performance liquid chromatographic (RP-HPLC) methods. The studied WP, -lactalbumin (-La), bovine serum albumin (BSA), -lactoglobulin A (-Lg A) and -lactoglobulin B (-Lg B) were separated on Aeris XB-C18 column at 214 nm detection. The RP-HPLC method was validated by selectivity, linearity (R2 ≥0.99), sensitivity (LOQ, 1.35–10.08 µg mL−1), accuracy (recovery 96.79-103.07%) and precision (% RSD ≤ 4.13%). The total studied WP in studied dairy drinks varied between 1.42 and 3.047 g·L-1. The chromatographic profile of FAAs (aspartic acid, glutamic acid, serine, histidine, arginine, glycine, threonine, alanine, tyrosine, cysteine, tryptophan, methionine, valine, phenylalanine, isoleucine, leucine and lysine) was determined in lyophilized concentrate of Milk Serum by RP-HPLC using pre-column derivatization reaction with orthophthalaldehyde (OPA). The total studied FAAs in studied samples varied between 1.103 and 1.119 mg·g-1. Moreover, the Milk Serum showed bacteriostatic activity against two bacterial strains Escherichia coli and Staphylococcus aureus. The obtained results confirm that dairy drinks based on the Milk Serum constitutes a valuable sources of bioactive components with benefits for human healthy nutrition.
ARTICLE | doi:10.20944/preprints202002.0041.v1
Subject: Medicine & Pharmacology, Pharmacology & Toxicology Keywords: geranylgeranyl acetone (GGA); heat shock proteins (Hsps); HT-22 (hippocampal neuronal) cells; mitochondrial membrane potentials
Online: 4 February 2020 (10:24:57 CET)
Geranylgeranyl acetone (GGA) protects against various types of cell damages by upregulating heat shock proteins. We investigated whether GGA protect neuronal cells from cell death induced by oxidative stress. Glutamate exposure was lethal to HT-22 cells which comprise a neuronal line derived from mouse hippocampus. This configuration is often used as a model for hippocampus neurodegeneration in vitro. In the present study, GGA protected HT-22 cells from glutamate-induced oxidative stress. GGA pretreatment did not induce Hsps. Moreover, reactive oxygen species increased to the same extent in both GGA-pretreated and untreated cells exposed to glutamate. In contrast, glutamate exposure and GGA pretreatment increased mitochondrial membrane potential. However, increases in intracellular Ca2+ concentration were inhibited by GGA pretreatment. In addition, the increase of phosphorylated ERKs by the glutamate exposure was inhibited by GGA pretreatment. These findings suggest that GGA protects HT-22 cells from glutamate-provoked cell death without Hsp induction and that the mitochondrial calcium buffering capacity plays an important role in this protective effect.
ARTICLE | doi:10.20944/preprints202301.0167.v1
Subject: Chemistry, Food Chemistry Keywords: Almond; Almond beverage; Almond skin; Antiradical activity; Green extraction; Hydrodynamic cavitation; Nutritional values; Polyphenols; Proteins; Vitamins
Online: 10 January 2023 (02:12:56 CET)
Almond beverages are gaining ever-increasing consumer preference in the growing market of non-alcoholic vegetable beverages, ranking in first place among oilseed-based drinks, mainly due to the perceived healthy benefits. However, the high cost of the raw material, time and energy consuming pre-treatments such as soaking, blanching and peeling, and post-treatments such as thermal sterilization, leading also to the loss of valuable macronutrients and micronutrients, hinder the sustainability, affordability and spread of almond beverages. Hydrodynamic cavitation processes were applied, as a single-unit operation, to the extraction in water of almond skinless kernels in the form of flour and fine grains, and to whole almond seeds in the form of coarse grains, up to high concentrations. The results showed full compliance with a high-end commercial product and with the expected levels based on the properties of the raw materials. The concentrated extract obtained from whole almond seeds showed a comparatively much higher antiradical activity, likely due to the contribution of the almond kernel skin. In conclusion, hydrodynamic cavitation could represent a convenient alternative processing route to the production of conventional and new integral almond beverages, avoiding multiple and costly technological steps, while affording fast production cycles of potentially healthier beverages.
REVIEW | doi:10.20944/preprints202203.0344.v1
Subject: Life Sciences, Biochemistry Keywords: carbonic anhydrase; carbonic anhydrase related proteins; acid-base balance; ion transport; pH regulation; motor coordination; zebrafish
Online: 25 March 2022 (09:22:10 CET)
During the last decades, zebrafish (Danio rerio) has become one of the most important model organisms to study different physiological and biological phenomena. Research field of carbonic anhydrases (CAs) and carbonic anhydrase related proteins (CARPs) is not an exception in this. The best-known known function of CAs is the regulation of acid-base balance. However, studies performed with zebrafish, among others, have revealed important roles for these proteins in many other physiological processes, some of which have been unpredicted in the light of the previous studies and suggestions. Examples are roles in zebrafish pigmentation as well as motor coordination. Disruption of the function of these proteins may generate even lethal outcomes. In this review, we summarize the current knowledge of CA-related studies performed in zebrafish.
REVIEW | doi:10.20944/preprints202111.0190.v1
Subject: Keywords: Malaria; proteases; Plasmodium rhomboids; dipeptidyl aminopeptidases; apical membrane antigen; subtilisin-like proteins; glucose transporters; schizogony; plasmepsins
Online: 9 November 2021 (15:50:12 CET)
There is an overarching need to find alternative treatment options for malaria and this quest is more pressing in current times due to the morbidity and mortality data arising from most endemic countries and partially owing to the fact that the SARS-Cov-2 pandemic has diverted much public health attention. Additionally, the therapeutic options available for malaria has been severely threatened with the emergence of resistance to almost all existing drugs by the human malaria parasite. The Artemisinin Combination Therapies (ACTs) which hitherto have been the mainstay for malaria have encountered resistance in South East Asia, a notorious ground zero for the emergence of antimalarial drug resistance. This review analyses few key druggable targets of the parasite and the potential to leverage strategic inhibitors to mitigate the scourge of malaria by providing a concise assessment of the essential proteins of the malaria parasite that could serve as targets. Furthermore, this work provides a summary of the advances made in malaria parasite biology and the potential to leverage such findings for antimalarial drug production.
Subject: Medicine & Pharmacology, Pharmacology & Toxicology Keywords: Covid-19; proteins; normal-modes; protein-drug interactions; chloroquine; ivermectin; remdesivir; sofosbuvir; boceprevir; α-difluoromethylornithine (DMFO)
Online: 11 August 2020 (04:09:43 CEST)
We report the results of our study of approved drugs as potential treatments for COVID 19, based on the application of various bioinformatics predictive methods. The drugs studied include chloroquine, ivermectin, remdesivir, sofosbuvir, boceprevir, and α-difluoromethylornithine (DMFO). Our results indicate that these small molecules selectively bind to stable, kinetically active residues and residues adjoining them on the surface of proteins and inside protein pockets, and that some prefer hydrophobic over other active sites. Our approach is not restricted to viruses and can facilitate rational drug design, as well as improve our understanding of molecular interactions, in general.
Subject: Biology, Physiology Keywords: calmodulin; calmodulin binding proteins; calmodulin binding motifs; calcium signalling; EF hands; heart arrythmia; neurodegeneration; Dictyostelium discoideum
Online: 15 December 2019 (15:44:35 CET)
Dictyostelium discoideum is gaining increasing attention as a model organism for the study of calcium binding and calmodulin function in basic biological events as well as human diseases. After a short overview of calcium-binding proteins, the structure of Dictyostelium calmodulin and the conformational changes effected by calcium ion binding to its four EF hands is compared to its human counterpart, emphasizing the highly conserved nature of this central regulatory protein. The calcium-dependent and -independent motifs involved in calmodulin binding to target proteins are discussed with examples of the diversity of calmodulin binding proteins that have been studied in this amoebozoan. The methods used to identify and characterize calmodulin binding proteins is covered followed by the ways Dictyostelium is currently being used as a system to study several neurodegenerative diseases and how it could serve as a model for studying calmodulinopathies such as those associated with specific types of heart arrythmia. Because of its rapid developmental cycles, its genetic tractability, and a richly endowed stock center, Dictyostelium is in a position to become a leader in the field of calmodulin research.
REVIEW | doi:10.20944/preprints201911.0252.v1
Subject: Biology, Ecology Keywords: data; sequence; information; entropy; genome; gene; proteins; time-series; modeling; meta-genomics; transcriptomics; proteomics; bioinformatics; DNA
Online: 22 November 2019 (02:28:15 CET)
Today massive amounts of sequenced metagenomic and -transcriptomic data from different ecological niches and environmental locations are available. Scientific progress depends critically on methods that allow extracting useful information from the various types of sequence data. Here, we will first discuss types of information contained in the various flavours of biological sequence data, and how this information can be interpreted to increase our scientific knowledge and understanding. We argue that a mechanistic understanding is required to consistently interpret experimental observations, and that this understanding is greatly facilitated by the generation and analysis of dynamic mathematical models. We conclude that, in order to construct mathematical models and to test mechanistic hypotheses, time-series data is of critical importance. We review diverse techniques to analyse time-series data and discuss various approaches by which time-series of biological sequence data was successfully used to derive and test mechanistic hypotheses. Analysing the bottlenecks of current strategies in the extraction of knowledge and understanding from data, we conclude that combined experimental and theoretical efforts should be implemented as early as possible during the planning phase of individual experiments and scientific research projects.
ARTICLE | doi:10.20944/preprints201706.0114.v1
Subject: Life Sciences, Biochemistry Keywords: proteins；time-varying； parallel； architectures；Conditional Random Fields (CRF)；Graphics Processing Units； Block Coordinate Descent algorithm
Online: 26 June 2017 (05:47:40 CEST)
Proteins are the workhorses of the cell that perform biological functions by interacting with other proteins. Many statistical methods for protein-protein interaction (PPI) have been studied without considering time-dependent changes in networks and the functionalities. I introduced a novel method that models PPI networks as being dynamic in nature and evolving time-varying multivariate distribution with Conditional Random Fields (CRF). This research is directed towards implementing this new combinatorial algorithm on massively parallel architectures such as Graphics Processing Units (GPUs) for efficient computations for large scale bioinformatics datasets. I compared Conditional Random Fields (CRF) and the proposed novel method using CRF combined with the Block Coordinate Descent algorithm for human protein-protein interaction data set. Both are implemented on GPU-Accelerated Computing Architecture and the proposed novel method showed the advantages in predicting protein-protein interaction sites. I also show that the proposed approach is more efficient in 6.13% than standalone CRF++ in predicting protein-protein interaction sites.
ARTICLE | doi:10.20944/preprints202010.0316.v1
Subject: Life Sciences, Biochemistry Keywords: Type 2 diabetes; cancer; shared pathways; shared genes and proteins; relationship between cancer and type 2 diabetes
Online: 15 October 2020 (09:47:35 CEST)
Obesity, type 2 diabetes, and different forms of cancers are among the leading human diseases and highly complex in terms of diagnostic and therapeutic approaches. Diabetes and cancer are among the most frequent and complex diseases and based on epidemiological evidence and study it can be concluded that the patients suffering from diabetes are considered to be significantly at higher risk for a number of cancer types. Both these diseases are among the highly complex and heterogeneous in nature. There are a number of evidences which support the hypothesis that these diseases interlinked and obesity may aggravate the risk(s) of both these diseases type 2 diabetes and different types of cancers. Multi-level unwanted alterations such as (epi-)genetic alterations, changes at the transcriptional level, and altered signaling pathways (receptor, cytoplasmic, and nuclear level) are the major source which promotes a number of complex diseases and such heterogeneous level of complexities are considered as the major barrier in the development of therapeutic. With so many known challenges, it is critical to understand the relationships and the common shared causes between type 2 diabetes and cancer which is difficult to unravel and understand. Furthermore, the real complexity arises during diagnosis from contended corroborations that specific drug(s) (individually or in combination) during diagnosis process of type 2 diabetes may increase or decrease the cancer risk or affect cancer prognosis. In this review article, we have presented the recent and most updated evidences from the studies where the origin, biological background, correlation between them have been presented or proved. Furthermore, we have summarized the methodological challenges and tasks that are frequently encountered. we have also outlined the physiological links between type 2 diabetes and cancers. Finally, we have presented and summarized the outline of the hallmarks for both these diseases diabetes and cancer.
ARTICLE | doi:10.20944/preprints201712.0106.v2
Subject: Life Sciences, Biochemistry Keywords: graphene; electrochemical biosensors; cancer; diagnosis; electrical detection; Alzheimer’s disease; dementia; neurodegenerative disorders; cardiovascular; blood biomarkers; antibodies; proteins
Online: 2 January 2018 (05:21:45 CET)
We report on the development of chemical vapour deposition (CVD) based graphene field effect transistor (GFET) immunosensors for the sensitive detection of Human Chorionic Gonadotropin (hCG), a glycoprotein risk biomarker of certain cancers. The GFET sensors were fabricated on Si/SiO2 substrate using photolithography with evaporated chromium and sputtered gold contacts. GFET channels were functionalized with a linker molecule to immobile anti-hCG antibody on the surface of graphene. Binding reaction of the antibody with varying concentration levels of hCG antigen demonstrated the limit of detection of the GFET sensors to be below 1 pg/mL using four-probe electrical measurements. We also show annealing can significantly improve the carrier transport properties of GFETs and shift the Dirac point (Fermi level) with reduced p-doping in back-gated measurements. The developed GFET biosensors are generic and could find applications in a broad range of medical diagnostics in addition to cancer, such as neurodegenerative (Alzheimer’s, Parkinson’s and Lewy body) and cardiovascular disorders.
REVIEW | doi:10.20944/preprints201705.0161.v1
Subject: Medicine & Pharmacology, Ophthalmology Keywords: dry eye; lacrimal gland; lipids and lipidomics; Meibomian gland; ocular surface disorders; proteins and proteomics; tear film
Online: 22 May 2017 (07:54:47 CEST)
The alteration of the delicate balance that regulates the secretion and distribution of the tear film determines the dry eye (DE) syndrome, because the tear film represents the interface between the eye and the environment. Despite having a multifactorial origin, the main risk factors for the emergence of the ocular disease are female gender and advanced age. Likewise, morphological changes in several glands and in chemical composition of their secretions such as proteins, mucins, lipidics, aqueous tears, and salinity, are highly relevant factors to maintain a condition of good health of the ocular anterior segment. Another key factor of recurrence and onset of the disease is the presence of local and/or systemic infiammation that reflex on the ocular surface. However, it is one of the most commonly encountered disease in clinical practice and many other causes related to daily life and to lengthen the average life will contribute to the beginning. This review will consider how and what disorders of the ocular surface are responsible for a widespread pathology so. In the end, the most appropriate and new therapies will be briefly exposed according to the specific pathology.
ARTICLE | doi:10.20944/preprints202208.0478.v1
Subject: Life Sciences, Microbiology Keywords: antibacterial proteins; encapsulating protein; high molecular-weight bacteriocins; insect patho-genic bacterium; phage tail-like protein; purification methods
Online: 29 August 2022 (09:00:10 CEST)
Brevibacillus laterosporus (Bl) is a Gram-positive and spore-forming bacterium belonging to the Brevibacillus brevis phylogenetic cluster. Globally, insect pathogenic strains of the bacterium have been isolated, characterised, and some activities patented. Two isolates, Bl 1821L and Bl 1951, exhibiting pathogenicity against the diamondback moth and mosquitoes, are under development as a biopesticide in New Zealand. However, due to the suspected activity of putative antibacterial proteins (ABPs), the endemic isolates often grow erratically. Various purification methods including size exclusion chromatography, sucrose density gradient centrifugation, polyethylene glycol precipitation, and ammonium sulphate precipitation employed in this study enabled the isolation of two putative antibacterial proteins of ~30 kD and ~48 kD from Bl 1821L and one putative antibacterial protein of ~30 kD from Bl 1951. Purification of the uninduced cultures of Bl 1821L and Bl 1951 also yielded the protein bands of ~30 kD and ~48 kD on SDS-PAGE which indicated their spontaneous induction. Disc diffusion assay was used to determine the antagonistic activities of the putative ABPs. Subsequent transmission electron microscope (TEM) examination of purified putative antibacterial protein-containing solution showed the presence of encapsulin (~30 kD) and polysheath (~48 kD) like structures. Although only the ~30 kD protein was purified from Bl 1951, both structures were seen in this strain under TEM. Furthermore, while assessing the antibacterial activity of some fractions of Bl 1951 against Bl 1821L in size exclusion chromatography method, population of Bl 1821L persister cells was noted. Overall, this work added a wealth of knowledge for the purification of the HMW proteins (bacteriocins) of the Gram-positive bacteria including Bl.
ARTICLE | doi:10.20944/preprints202208.0293.v1
Subject: Physical Sciences, Other Keywords: Golden ratio; Irrational numbers; Fibonacci sequence; Spiral galaxy; Planetary orbits; KAM Theorem; Ultimatum game; Proteins; Penrose tiling; Quasicrystals
Online: 16 August 2022 (14:29:27 CEST)
The Golden ratio is an irrational number that has a tendency to appear in many different scientific and artistic fields. It may be found in natural phenomena across a vast range of length scales; from galactic to atomic. In this review, the mathematical properties of the Golden ratio are discussed before exploring where in nature it has been found; beginning at astronomical scales and progressing to smaller lengths, until reaching those of atomic and quantum physics. In making such a tour across length scales, it is illustrated just how prevalent this single number is within the natural universe.
Subject: Biology, Physiology Keywords: vitamin A transporters; all-trans retinol; retinyl esters; LRAT; STRA6; RBPR2; RBP4; retinol-binding proteins; photoreceptors; visual function
Online: 11 October 2021 (14:15:27 CEST)
Vitamins are essential compounds obtained through diet that are necessary for normal devel-opment and function in an organism. One of the most important vitamins for human physiology is Vitamin A, a group of retinoid compounds and carotenoids which generally function as a mediator for cell growth, differentiation, immunity, and embryonic development, as well as serving as a key component in the phototransduction cycle in vertebrate retina. For humans, vitamin A is obtained through the diet, where provitamin A carotenoids like β-carotene, or preformed vitamin A such as retinyl esters are absorbed into the body via the small intestine and converted into all-trans retinol within the intestinal enterocytes. Specifically, once absorbed, carotenoids are cleaved by carote-noid cleavage oxygenases (CCOs), such as BCO1, to produce all-trans retinal that subsequently gets converted into all-trans retinol. CRBP2 bound retinol is then converted into retinyl esters (REs) by the enzyme lecithin retinol acyltransferase (LRAT) in the endoplasmic reticulum, which is then packaged into chylomicrons and sent into the bloodstream for storage in hepatic stellate cells in the liver or for functional use in peripheral tissues such as the retina. All-trans retinol also travels through the bloodstream bound to retinol binding protein 4 (RBP4), where it enters cells with the assistance of the transmembrane transporters, stimulated by retinoic acid 6 (STRA6) in peripheral tissues or retinol binding protein receptor 2 (RBPR2) in systemic tissues (e.g. in the retina and the liver respectively). Much is known about the intake, metabolism, storage, and function of vitamin A compounds, especially with regard to its impact on eye development and visual function in the retinoid cycle. However, there is much to learn about the role of vitamin A as a transcription factor in development and cell growth, as well as how peripheral cells signal hepatocytes to secrete all-trans retinol into the blood for peripheral cell use. This article aims to review literature re-garding the major known pathways of vitamin A intake from dietary sources into hepatocytes, vitamin A excretion by hepatocytes, as well as vitamin A usage within the retinoid cycle in the RPE and retina to provide insight on future directions of novel membrane transporters for vitamin A in retinal cell physiology and visual function.
ARTICLE | doi:10.20944/preprints202109.0503.v1
Subject: Biology, Animal Sciences & Zoology Keywords: mitogenome; transmembrane proteins; substitution matrix; JTT matrix; molecular evolution; partitioned models; mixture models; RY coding; cyto-nuclear discordance
Online: 29 September 2021 (16:57:38 CEST)
Phylogenomic analyses have revolutionized the study of biodiversity, but they have revealed that estimated tree topologies can depend, at least in part, on the subset of the genome that is analyzed. For example, estimates of trees for avian orders differ if protein coding or non-coding data are analyzed. The bird tree is a good study system because the historical signal for relationships among orders is very weak, which should permit subtle non-historical signals to be identified, while monophyly of orders is strongly corroborated, allowing identification of strong non-historical signals. Hydrophobic amino acids in mitochondrially-encoded proteins, which are expected to be found in transmembrane helices, have been hypothesized to be associated with non-historical signals. We tested this hypothesis by comparing the evolution of transmembrane helices and extramembrane segments of mitochondrial proteins from 420 bird species, sampled from most avian orders. We estimated amino acids exchangeabilities for both structural environments and assessed the performance of phylogenetic analysis using each data type. We compared those relative exchangeabilities with values calculated using a substitution dataset for transmembrane helices from a variety of sampled set of nuclear- and mitochondrially-encoded proteins, allowing us to compare the bird-specific mitochondrial models with a general model of transmembrane protein evolution. To complement our amino acid analyses, we examined the impact of protein structure on patterns of nucleotide evolution. Models of transmembrane and extramembrane sequence evolution for amino acids and nucleotides exhibited striking differences, but there was no evidence for strong topological data type effects. However, incorporating protein structure into analyses of mitochondrially-encoded proteins improved model fit. Thus, we believe that considering protein structure will improve analyses of mitogenomic data, both in birds and in other taxa.
ARTICLE | doi:10.20944/preprints202107.0526.v1
Subject: Life Sciences, Biochemistry Keywords: maternal pre-gestational obesity; placenta; lipid metabolism; fatty acid transporter proteins; isoprostanoids; neuroprostanes; isoprostanes; docosahexaenoic acid; arachidonic acid
Online: 23 July 2021 (08:04:47 CEST)
The rise in prevalence of obesity in women of reproductive age in both developed and developing countries might propagate intergenerational cycles of detrimental effects on metabolic health, contributing to substantial economic burden on society. Placental lipid metabolism might be disrupted by maternal obesity, which possibly affects the life-long health of the offspring. Here, we investigated placental lipid metabolism and handling from women with pre-gestational obesity as a sole pregnancy complication and compared to placental responses of lean women. Open profile and targeted lipidomics were used to assess placental lipids and oxidized products of docosahexahenoic acid (DHA), neuroprostanes, and arachidonic acid (AA), isoprostanes. Placental fatty acid transporters FABP1, FABP3 and endothelial lipase protein were measured. Despite no signs of overall alterations in lipid content, increased contents of DHA, AA, DHA-derived neuroprostanes and AA-derived isoprostanes and decreased content of FABP1 protein were found in placentas from obese women. Multivariate analyses suggested that these oxidised fatty acids are associated with maternal and placental inflammation and also with birth weight. These results might shed light on the molecular mechanisms associated with altered fatty acid metabolism and lipid handling in maternal pre-gestational obesity, placing these oxidized fatty acids as novel mediators of placental function.