ARTICLE | doi:10.20944/preprints202102.0332.v1
Subject: Life Sciences, Molecular Biology Keywords: Angiogenesis; Pulmonary hypertension; Endothelium; PHD2 Deficiency; Hypoxia
Online: 16 February 2021 (13:43:56 CET)
Endothelial autocrine signaling is essential to maintain vascular hemostasis. There is limited in-formation about the role of endothelial autocrine signaling in regulating severe pulmonary vas-cular remodeling during the onset of pulmonary arterial hypertension (PAH). In this study, we employed the first severe PAH mouse model, Egln1Tie2Cre (Tie2Cre-mediated disruption of Egln1) mice, to identify the novel autocrine signaling mediating the pulmonary vascular endothelial cells (PVECs) hyperproliferation and the pathogenesis of PAH. PVECs isolated from Egln1Tie2Cre lung expressed upregulation of many growth factors or angiocrine factors such as CXCL12, and exhib-ited hyperproliferative phenotype in coincident with upregulation of proliferation specific tran-scriptional factor FoxM1. Treatment of CXCL12 on PVECs increased FoxM1 expression, which was blocked by CXCL12 receptor CXCR4 antagonist AMD3100 in culture human PVECs. Endo-thelial specific deletion of Cxcl12 (Egln1/Cxcl12Tie2 Cre) or AMD3100 treatment in Egln1Tie2Cre mice downregulated FoxM1 expression in vivo. We then generated and characterized a novel mouse model with endothelial specific FoxM1 deletion in Egln1Tie2Cre mice (Egln1/Foxm1Tie2Cre), and found that endothelial FoxM1 deletion reduced pulmonary vascular remodeling and right ventricular systolic pressure. Together, our study identified a novel mechanism of endothelial autocrine sig-naling in regulating PVECs hyperproliferation and pulmonary vascular remodeling in PAH.
Mon, 25 January 2021
REVIEW | doi:10.20944/preprints202101.0521.v1
Subject: Life Sciences, Molecular Biology Keywords: Data integration; multi-omics; integration strategies; genomics
Online: 25 January 2021 (16:19:31 CET)
Metabolomics deals with multiple and complex chemical reactions within living organisms and how these are influenced by external or internal perturbations. It lies at the heart of omics profiling technologies not only as the underlying biochemical layer that reflects information expressed by the genome, the transcriptome and the proteome, but also as the closest layer to the phenome. The combination of metabolomics data with the information available from genomics, transcriptomics, and proteomics offers unprecedented possibilities to enhance current understanding of biological functions, elucidate their underlying mechanisms and uncover hidden associations between omics variables. As a result, a vast array of computational tools have been developed to assist with integrative analysis of metabolomics data with different omics. Here, we review and propose five criteria – hypothesis, data types, strategies, study design and study focus – to classify statistical multi-omics data integration approaches into state-of-the-art classes under which all existing statistical methods fall. The purpose of this review is to look at various aspects that lead the choice of the statistical integrative analysis pipeline in terms of the different classes. We will draw a particular attention to metabolomics and genomics data to assist those new to this field in the choice of the integrative analysis pipeline.
Fri, 25 December 2020
REVIEW | doi:10.20944/preprints202012.0659.v1
Online: 25 December 2020 (13:00:28 CET)
Pirin is an oxidative stress (OS) sensor belonging to the functionally diverse cupin superfamily of proteins. Pirin is a suggested quercetinase and transcriptional activator of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. Its biological role in cancer development remains as a novel area of study. This review shows accumulating evidence on the contribution of Pirin in epithelial cancers, signaling pathways involved, and as a suggested therapeutic target. Finally, we propose a model in which Pirin is upregulated by physical, chemical or biological factors involved in OS and cancer development.
TECHNICAL NOTE | doi:10.20944/preprints202012.0641.v1
Subject: Life Sciences, Molecular Biology Keywords: Neurodegeneration; dopamine; L-DOPA; dopaminergic; DBA; Western; IR; TH; Parkinson disease
Online: 25 December 2020 (07:10:33 CET)
We present a robust, fresh-frozen approach to immunohistochemistry (IHC), without committing the tissue to IHC via fixation and cryopreservation while maintaining long-term storage, using LiCor-based infrared (IR) quantification for sensitive assessment of TH in immunoreacted mid-brain sections for quantitative comparison across studies. In fresh-frozen tissue stored up to 1 year prior to IHC reaction, we found our method to be highly sensitive to rotenone treatment in 3-month-old Sprague-Dawley rats, and correlated with a significant decline in rotarod laten-cy-to-fall measurement by approximately 2.5 fold. The measured midbrain region revealed a 31% lower TH signal when compared to control (p<0.01 by t test, n=5). Bivariate analysis of in-tegrated TH counts versus rotarod latency-to-fall indicates a positive slope and modest but sig-nificant correlation of R2=0.68 (p<0.05, n=10). These results indicate this rapid, instrument-based quantification method by IR detection successfully quantifies TH levels in rat brain tissue, while taking only 5 days from euthanasia to data output. This approach also allows for the identifica-tion of multiple targets by IHC with the simultaneous performance of downstream molecular analysis within the same animal tissue, allowing for the use of fewer animals per study.
Tue, 15 December 2020
REVIEW | doi:10.20944/preprints202012.0377.v1
Subject: Life Sciences, Molecular Biology Keywords: Feature Selection, Feature Ranking, Grouping, Clustering, Biological Knowledge.
Online: 15 December 2020 (12:10:44 CET)
In the last two decades, there have been massive advancements in high throughput technologies, which resulted in the exponential growth of public repositories of gene expression datasets for various phenotypes. It is possible to unravel biomarkers by comparing the gene expression levels under different conditions, such as disease vs. control, treated vs. not treated, drug A vs. drug B, etc. This problem refers to a well-studied problem in the machine learning domain, i.e., the feature selection problem. In biological data analysis, most of the computational feature selection methodologies were taken from other fields, without considering the nature of the biological data. For gene expression data analysis, most of the existing feature selection methods rely on expression values alone to select the genes; and biological knowledge is integrated at the end of the analysis in order to gain biological insights or to support the initial findings. Thus, integrative approaches that utilize the biological knowledge while performing feature selection are necessary for this kind of data. The main idea behind the integrative gene selection process is to generate a ranked list of genes considering both the statistical metrics that are applied to the gene expression data, and the biological background information which is provided as external datasets. Since the integrative approach attracted attention in the gene expression domain, lately the gene selection process shifted from being purely data-centric to more incorporative analysis with additional biological knowledge.
Fri, 4 December 2020
REVIEW | doi:10.20944/preprints202012.0097.v1
Subject: Life Sciences, Molecular Biology Keywords: acute respiratory distress syndrome; mesenchymal stem/stromal cells,; exosome; COVID-19; Clinical trials
Online: 4 December 2020 (10:10:11 CET)
Coronavirus disease 2019 (COVID-19) caused by novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reached a global epidemic across the world after first reported in Wuhan, China’s Hubei province in December 2019. The pandemic is also associated with acute respiratory distress syndrome (ARDS) characterized by excess inflammation, progressive arterial hypoxemia and dyspnea. Mesenchymal stem/ stromal cells (MSCs) have been investigated as treatment for ARDS due to immunomodulatory property. Exosomes derived from MSCs play an important role in paracrine signaling of MSCs, thereby contributed to immunomodulation of the immune microenvironment. Exosomes are emerged as potential alternative to MSC cell therapy with superiority of safety. In this review, we will introduce MSC-derived exosomes and briefly discuss current progress on MSCs and exosomes in ARDS, which may have clinical implications in pathogenesis and treatment of COVID-19.
Fri, 16 October 2020
ARTICLE | doi:10.20944/preprints202010.0339.v1
Subject: Life Sciences, Molecular Biology Keywords: aging; paternal exposure; sperm; semen; epigenetics; sncRNA; piRNA; miRNA; 2,2′,4,4′-tetrabromodiphenyl ether; PBDE; BDE-47; perinatal; environment.
Online: 16 October 2020 (08:10:16 CEST)
Advanced paternal age at fertilization is a risk factor for multiple disorders in offspring and may be linked with age-related epigenetic changes in fathers sperm. Understanding of aging-related epigenetic changes in sperm and environmental factors that modify such changes is needed. Here we characterize changes in sperm sncRNA between young pubertal and mature rats. We also analyze modification of these changes by exposure to environmental xenobiotic 2,2’,4,4’-tetrabromodiphenyl ether (BDE-47). SncRNA libraries prepared from epididymal spermatozoa were sequenced and analyzed using DESeq 2. Distribution of small RNA fractions changed with age, with fractions mapping to rRNA and lncRNA decreasing and fractions mapping to tRNA and miRNA increasing. 249 miRNA, 908 piRNA and 227 tRNA-derived RNA were differentially expressed (2-fold change, FDR p ≤ 0.05) between age groups in control animals. Differentially expressed miRNA and piRNA were enriched for protein-coding targets involved in development and metabolism, piRNA were enriched for LTR targets. BDE-47 accelerated age dependent changes in sncRNA in younger animals, decelerated these changes in older animals and increased the variance in expression of all sncRNA. Our results indicate that the natural aging process has profound effects on sperm sncRNA profiles and this effect may be modified by environmental exposures.
Thu, 24 September 2020
Subject: Life Sciences, Molecular Biology Keywords: COVID-19; SARS-CoV-2; RT-PCR; control strategy; Morocco
Online: 24 September 2020 (10:45:21 CEST)
Since March 2nd, 2020, date of the first SARS-COV-2 detected case in Morocco; multiples activities were adopted as COVID-19 control strategies. If the first period of COVID-19 noticed a few numbers of cases and deaths, the second half from July until today is marked with an exponential increase of the number of cases and a spread in almost all provinces with more intensive care needs and more deaths. The fatality rate of this disease is mainly compared with the highest developed countries in Europe and America. Instead of comparing the strategy and the deaths, with similar Arabic, African or Asiatic Middle-Income countries like Tunisia, Jordan, Cote d’Ivoire, Uzbekistan; that socio-demographic situation, behaviours, population density, and individual vulnerabilities create less confounding factors to make fair comparisons. Thus, this report has the aim to present how the COVID-19 pandemic was dealt in Morocco during this 200 days, by highlighting some discrepancies with corrective advice to get better future control results against COVID-19 and afford a possible comparison with other countries.The policy analysis approach was followed as a method to defines the pitfalls themes and to compare with the updated available international information about was it work and what is not needed to do. In conclusion, molecular biology represented by q-RT-PCR is the immediate action to do to enhance the diagnostic and the overall control strategy by knowing how to interpret its results following the time progress of cycle quantification values.
Tue, 22 September 2020
REVIEW | doi:10.20944/preprints202009.0513.v1
Subject: Life Sciences, Molecular Biology Keywords: PIP2; membrane dynamics; disease; actin dynamics; phosphoinositides; signaling
Online: 22 September 2020 (09:04:28 CEST)
In this review we summarize the recent development in understanding the role of PIP2 in cellular function and signaling. We first discuss the effect of PIP2 on actin binding proteins addressing the mechanism of the actin cytoskeletal dynamics such as polymerization or depolymerization of the filamentous network or the coupling to membrane to generate forces. Next, we outline the role of PIP2 in membrane dynamics. We summarized how the membrane organization depends upon PIP2 in the presence of ions or transmembrane proteins that are sensitive to membrane curvature. We discuss how clathrin coated pits interact with adaptor proteins during the endocytosis process, which is facilitated by PIP2. Finally, we discuss the role of PIP2 in cell signaling and diseases.
Sun, 20 September 2020
ARTICLE | doi:10.20944/preprints202009.0473.v1
Subject: Life Sciences, Molecular Biology Keywords: antioxidant enzymes; DNA methylation; epigenetics; plant resistance; root-knot nematodes; ROS; tomato
Online: 20 September 2020 (14:35:29 CEST)
Two wild-type field populations of root-knot nematodes (Mi-Vfield, Mj-TunC2field), and two isolates selected for virulence in laboratory on resistant tomato cultivars (SM2V, SM11C2), were used to induce a resistance reaction in tomato to the soil-borne parasites. Epigenetic and metabolic mechanisms of resistance were detected and compared with those occurring in partially or fully successful infections. The activated epigenetic mechanisms in plant resistance, as opposed to those activated in infected plants, were detected by analysing the methylated status of total DNA, by ELISA methods, and the expression level of key genes involved in the methylation pathway, by qRT-PCR. DNA hypo-methylation and down-regulation of two methyl-transferase genes (CMT2, DRM5), characterized the only true resistant reaction obtained by inoculating the Mi-1.2-carrying resistant tomato cv Rossol with the avirulent field population Mi-Vfield. On the contrary, in the roots into which nematodes were allowed to develop and reproduce, total DNA was generally found to be hyper-methylated and methyl-transferase genes up-loaded. DNA hypo-methylation was considered to be the upstream mechanism that triggers the general gene over-expression observed in plant resistance. Gene silencing induced by nematodes may be obtained through DNA hyper-methylation and methyl-transferase gene activation. Plant resistance is also characterized by an inhibition of the anti-oxidant enzyme system and activation of the defence enzyme chitinase, as opposed to the activation of such a system and inhibition of the defence enzyme glucanase in roots infested by nematodes.
Sat, 19 September 2020
ARTICLE | doi:10.20944/preprints202009.0450.v1
Subject: Life Sciences, Molecular Biology Keywords: COVID-19; rRT-PCR; dilution; viral diagnosis; RNA extraction
Online: 19 September 2020 (08:27:13 CEST)
Although rRT-PCR is the gold standard method for SARS-CoV-2 detection, some factors, such as amplification inhibitors presence, lead to false-negative results. Here we describe differences between rRT-PCR results for SARS-CoV-2 infection in normal and diluted samples, simulating the need for dilution due to amplification inhibitors presence. Viral RNA extraction of nasopharyngeal swabs samples from 20 patients previously detected as 'Negative' and 21 patients detected as 'Positive' for SARS-CoV-2 was realized with the EasyExtract DNA-RNA (Interprise®) for extraction. rRT-PCR was realized with OneStep/COVID-19 (IBMP) kit with normal and diluted (80µl of H₂O RNAse free) samples, totaling 82 tests. The results indicate that there is an average variation (ɑ < 0.05) delaying Ct between the amplification results of internal control (IC), N Gene (NG), and ORF-1ab (OF) of 1.811Ct, 3.840Ct, and 3.842Ct, respectively. The extraction kit does not completely purify the inhibitor compounds, therefore non-amplification by inhibitors may occur. In this study, we obtained a 19.04% false-negative diagnosis after sample dilution, and this process reduces the efficiency of rRT-PCR to 29.8% for detecting SARS-CoV-2. Knowing the rRT-PCR standards of diluted samples can help in the identification of false-negative cases, and consequently avoid a wrong diagnosis.
Thu, 17 September 2020
ARTICLE | doi:10.20944/preprints202009.0383.v1
Subject: Life Sciences, Molecular Biology Keywords: Methylglyoxal; Glyoxalase system; AGEs; glutathione; hyperglycemia
Online: 17 September 2020 (07:45:11 CEST)
Chronic high glucose (HG) exposure increases methylglyoxal (MG)-derived AGEs and is involved in the onset of pathological conditions, such as diabetes, atherosclerosis and chronic‐degenerative diseases. Under physiologic condition the harmful effects of MG are contrasted by glyoxalase system that is involved in the detoxification of Reactive Carbonyl Species (RCS) and maintain the homeostasis of the redox environment of the cell. Polyphenols are the most abundant antioxidants in the diet and present various health benefits. The study aimed at investigating the role of polyphenols extracted from an apple high in polyphenols (Calville White Winter), on glyco-oxidative stress induced by chronic HG-exposure. Intestinal Caco-2 cells were treated in physiological glucose condition (25mM) as a control and in HG condition (50mM) with or without apple extract for one week. Our data demonstrated that HG-treatment triggers glyco-oxidation stress with a significantly increase in ROS, lipid peroxidation, AGEs and Glyoxalase I (GlxI) activity with a significant decrease in total antioxidant intracellular defense. Treatment with polyphenols under HG condition restores to the control levels GlxI activity, decreases Glyoxalase II (GlxII) in relation to the control and induces a drop of glyco-oxidative damage. This paper seeks to highlight the roles of polyphenols in glyco-oxidative stress.
Sun, 13 September 2020
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.
Fri, 11 September 2020
HYPOTHESIS | doi:10.20944/preprints202003.0419.v9
Subject: Life Sciences, Molecular Biology Keywords: ATP hypothesis; origin of genetic code; life’s building block; probiotic “soup”; coevolution; biochemical system; missing “matchmaker”; energy transformation; informatization; structuralization; precellular selection; photo- chemical origin of life; virus; anti-life form; 2019-nCoV
Online: 11 September 2020 (08:39:39 CEST)
A plenty of theories on the origin of genetic codes have been proposed so far, yet all ignored the energetic driving force, its relation to the biochemical system, and most importantly, the missing “matchmaker” between proteins and nucleic acids. Here, a new hypothesis is proposed, according to which ATP is at the origin of the primordial genetic code by driving the coevolution of the genetic code with the pristine biochemical system. This hypothesis aims to show how the genetic code was produced e.g. by photochemical reactions in a protocell that derived from a lipid vesicle enclosing various life’s building blocks (e.g. nucleotides and peptides). At extant cell, ATP is the only energetic product of photosynthesis, and is at the energetic heart of the biochemical systems. ATP could energetically form and elongate chains of both polynucleotides and polypeptides, thus acting a “matchmaker” between these two bio-polymers and eventually mediating precellular biochemical innovation from energy transformation to informatization. ATP was not the only one that could drive the formation of polynucleotides and polypeptides, but favored by precellular selection. The protocell innovated a photosynthesis system to produce ATP efficiently and regularly with the aids of proteins and RNA/DNA. The completion of permanently recording the genetic information by DNA marked the dawn of cellular life operated by Darwinian evolution. The ATP hypothesis assumes or supports the photochemical origin of life, shedding light on the origins of both photosynthetic and biochemical systems, which remains largely unknown thus far. Based on ATP hypothesis, virus (like the new coronavirus) could not be the earliest life on Earth, as it has neither biochemical systems nor lipid bilayer membrane that provided relatively isolated environment for the development of protobiochemical reactions, although it owns the genetic code of a cellular life. Virus could not be a bridge between life and non-life, but is an anti-life substance, as it depletes cellular material for its own replication, and then spreads by destroying the host cells. It can be imagined that if cellular life are completely wiped out by the virus, the complete destruction of life on Earth would be inevitable.
Wed, 9 September 2020
ARTICLE | doi:10.20944/preprints202009.0194.v1
Subject: Life Sciences, Molecular Biology Keywords: Oenothera biennis; Evening primrose; Oxidative stress; Cell death; Nrf2/HO-1
Online: 9 September 2020 (03:29:52 CEST)
Background: Oenothera biennis (evening primrose) produces bioactive substances with a diverse range of pharmacological functions. However, it is currently unknown whether extract prepared from the aerial parts of O. biennis (APOB) can protect the skin against oxidative stress. To investigate the protective effects of APOB against oxidative stress-induced damage in human skin keratinocytes (HaCaT) and elucidate the underlying mechanisms. Methods: We pretreated HaCaT cells with various concentrations of APOB or the antioxidant N-acetyl-L-cysteine before applying H2O2. We then compared the cell viability, intracellular reactive oxygen species (ROS) production, and DNA and mitochondrial damage between pretreated and untreated control cells using a range of assays, flow cytometry, and Western blot analysis and also examined the reducing power and DPPH free radical-scavenging activity of APOB. Results: APOB pretreatment significantly increased cell viability, effectively attenuated H2O2-induced comet tail formation, and inhibited H2O2-induced phosphorylation of the histone γH2AX, as well as the number of apoptotic bodies and Annexin V-positive cells. APOB was found to have a high reducing power and DPPH radical-scavenging activity and also exhibited scavenging activity against intracellular ROS accumulation and restored the loss of mitochondrial membrane potential caused by H2O2. APOB pretreatment almost totally reversed the enhanced cleavage of caspase-3, the degradation of poly (ADP-ribose)-polymerase (PARP), DNA fragmentation that usually occurs in the presence of H2O2 and increased the levels of heme oxygenase-1 (HO-1), a potent antioxidant enzyme that is associated with the induction of nuclear factor-erythroid 2-related factor 2 (Nrf2). Conclusions: APOB can protect HaCaT cells from H2O2-induced DNA damage and cell death by blocking cellular damage related to oxidative stress via a mechanism that affects ROS elimination and by activating the Nrf2/HO-1 signaling pathway.
Sat, 5 September 2020
ARTICLE | doi:10.20944/preprints202009.0108.v1
Subject: Life Sciences, Molecular Biology Keywords: Synuclein; CPT1A; MMP14; PYGL; Tfrc; Ireb2; Pgrmc1; Hmox1; Cyp46a1; Slc11a2; Slc25a37
Online: 5 September 2020 (03:54:37 CEST)
Iron deprivation activates mitophagy and extends lifespan in nematodes. In patients suffering from Parkinson’s disease (PD), PINK1-PRKN mutations via deficient mitophagy trigger iron accumulation and reduce lifespan. To evaluate molecular effects of iron chelator drugs as a potential PD therapy, we assessed fibroblasts by global proteome profiles and targeted transcript analyses. In mouse cells, iron shortage decreased protein abundance for iron-binding nucleotide metabolism enzymes (prominently XDH and ferritin homolog RRM2). It also decreased the expression of factors with a role for nucleotide surveillance, which associate with iron-sulfur-clusters (ISC), and are important for growth and survival. This widespread effect included prominently Nthl1-Ppat-Bdh2, but also mitochondrial Glrx5-Nfu1-Bola1, cytosolic Aco1-Abce1-Tyw5, and nuclear Dna2-Elp3-Pold1-Prim2. Incidentally, upregulated Pink1-Prkn levels explained mitophagy induction, the downregulated expression of Slc25a28 suggested it to function in iron export. The impact of PINK1 mutations in mouse and patient cells was pronounced only after iron overload, causing hyperreactive expression of ribosomal surveillance factor Abce1 and of ferritin, despite ferritin translation being repressed by IRP1. This misregulation might be explained by the deficiency of the ISC-biogenesis factor GLRX5. Our systematic survey suggests mitochondrial ISC-biogenesis and post-transcriptional iron regulation to be important in the decision, whether organisms undergo PD pathogenesis or healthy aging.
Wed, 2 September 2020
REVIEW | doi:10.20944/preprints202009.0040.v1
Subject: Life Sciences, Molecular Biology Keywords: Coronaviruses; COVID-19; Pakistan; current situation; human physiology; diagnosis; treatment
Online: 2 September 2020 (09:59:45 CEST)
Coronavirus that is also known as COVID-19 disease is produced by SARSCoV-2. This causative agent is highly contagious and can cause potentially fatal pneumonia worldwide with serious public health concerns. In the beginning among infected individuals, most of them were those who were mainly shown to the wet animal market in a big city of China known as Wuhan. So, it was suggested that this was almost certainly the zoonotic source of COVID-19illness. The transitional source of origin and their mode of transmission to humans were not known obviously. Conversely, from human to human rapidly transformation have been confirmed generally. Currently, there is no availability of FDA approved clinically antiviral drugs and/or vaccines to be used against the COVID-19. Afterward, SARS-CoV and MERS-CoV, the occurrence of SARS-CoV-2 has been manifested as the third sketch of an enormously pathogenic coronavirus into human population globally. In this review, we provide a brief overview of the history of COVID-19 in Pakistan up-to 18th weeks after beginning, current situation, epidemiology, and its impact on the human population. Moreover, we focused on physiological variation during the incubation period, genome analysis of SARS-CoV2, supportive treatment approaches, and safety measures in the Pakistani population, which may be supportive for combating the risk of COVID-19 epidemic. We also reviewed the future approaches for the development of therapeutic interventions and vaccines to cope with the COVID-19 epidemic.
Tue, 1 September 2020
ARTICLE | doi:10.20944/preprints202007.0438.v2
Subject: Life Sciences, Molecular Biology Keywords: structure-function relationships; enrichment analysis; antifungal activities; knottin; two-layer sandwich architecture
Online: 1 September 2020 (12:03:47 CEST)
Whether there is any inclination between structures and functions of antimicrobial peptides (AMPs) is a mystery yet to be unraveled. AMPs have various structures associated with many different antimicrobial functions, including antibacterial, anticancer, antifungal, antiparasitic and antiviral activities. However, none has yet reported any antimicrobial functional tendency within a specific category of protein/peptide structures nor any structural tendency of a specific antimicrobial function with respect to AMPs. Here we examine the relationships between structures categorized by three structural classification methods (CATH, SCOP and TM) and seven antimicrobial functions with respect to AMPs using an enrichment analysis. The results show that antifungal activities of AMPs were tightly related to two-layer sandwich structure of CATH, knottin fold of SCOP, and the first structural cluster of TM. The associations with knottin and TM cluster 1 even sustained through the AMPs with a low sequence identity. Besides, another significant mutual enrichment was observed between the third cluster of TM and anti-gram-positive-bacterial/anti-gram-negative-bacterial activities. The findings of the structure-function inclination further our understanding of AMPs and could help us design or discover new therapeutic potential AMPs.
Mon, 31 August 2020
HYPOTHESIS | doi:10.20944/preprints202003.0419.v8
Subject: Life Sciences, Molecular Biology Keywords: ATP hypothesis; origin of genetic code; life’s building block; probiotic “soup”; coevolution; biochemical system; missing “matchmaker”; energy transformation; informatization; structuralization; precellular selection; photochemical origin of life
Online: 31 August 2020 (08:05:13 CEST)
A plenty of theories on the origin of genetic codes have been proposed so far, yet all ignored the energetic driving force, its relation to the biochemical system, and most importantly, the missing “matchmaker” between proteins and nucleic acids. Here, a new hypothesis is proposed, according to which ATP is at the origin of the primordial genetic code by driving the coevolution of the genetic code with the pristine biochemical system. This hypothesis aims to show how the genetic code was produced by photochemical reactions in a protocell that derived from a lipid vesicle enclosing various life’s building blocks (e.g. nucleotides and peptides). At extant cell, ATP is the only energetic product of photosynthesis, and is at the energetic heart of the biochemical systems. ATP could energetically form and elongate chains of both polynucleotides and polypeptides, thus acting a “matchmaker” between these two bio-polymers and eventually mediating precellular biochemical innovation from energy transformation to informatization. ATP was not the only one that could drive the formation of polynucleotides and polypeptides, but favored by precellular selection. The protocell innovated a photosynthesis system to produce ATP efficiently and regularly with the aids of proteins and RNA/DNA. The completion of permanently recording the genetic information by DNA marked the dawn of cellular life operated by Darwinian evolution. The ATP hypothesis supports the photochemical origin of life, shedding light on the origins of both photosynthetic and biochemical systems, which remains largely unknown thus far.
Fri, 28 August 2020
Subject: Life Sciences, Molecular Biology Keywords: sleeping Beauty transposon; bidirectional promoters; gene expression; gene therapy; synthetic biology; RPBSA; EF-1; LMP2/TAP1
Online: 28 August 2020 (11:35:53 CEST)
Promoter choice is an essential consideration for transgene expression in gene therapy. The expression of multiple genes requires ribosomal entry or skip sites, or the use of multiple promoters. Promoters systems comprised of two separate, divergent promoters may significantly increase the size of genetic cassettes intended for use in gene therapy. However, an alternative approach is to use a single, compact bidirectional promoter. We identified strong and stable bidirectional activity of the RPBSA synthetic promoter comprised of a fragment of the human Rpl13a promoter, together with additional intron / exon structures. The Rpl13a-based promoter drove long-term bidirectional activity of fluorescent proteins. Similar results were obtained for the EF1-α and LMP2/TAP1 promoters. However, in a lentiviral vector, the divergent bidirectional systems failed to produce sufficient titres to translate into an expression system for dual chimeric antigen receptors (CAR) expression. Although bidirectional promoters show excellent applicability to drive short RNA in Sleeping Beauty transposon systems, their possible use in the lentiviral applications requiring longer and more complex RNA, such as dual CAR cassettes, is limited.
Thu, 27 August 2020
BRIEF REPORT | doi:10.20944/preprints202008.0595.v1
Subject: Life Sciences, Molecular Biology Keywords: COVID-19; de novo assembly; metagenomics; data quality
Online: 27 August 2020 (07:56:01 CEST)
An intense scientific debate is ongoing as to the origin of SARS-CoV-2. An oft-cited piece of information in this debate is the genome sequence of a bat coronavirus strain referred to as RaTG13 1 mentioned in a recent Nature paper 2 showing 96.2% genome homology with SARS-CoV-2. This is discussed as a fossil record of a strain whose current existence is unknown. The said strain is conjectured by many to have been part of the ancestral pool from which SARS-CoV-2 may have evolved 7, 8, 9. Multiple groups have been discussing the features of the genome sequence of the said strain. In this paper, we report that the currently specified level of details are grossly insufficient to draw inferences about the origin of SARS-CoV-2. De-novo assembly, KRONA analysis for metagenomic and re-examining data quality highlights the key issues with the RaTG13 genome and the need for a dispassionate review of this data. This work is a call to action for the scientific community to better collate scientific evidence about the origins of SARS-CoV-2 so that future incidence of such pandemics may be effectively mitigated.
Wed, 26 August 2020
ARTICLE | doi:10.20944/preprints202008.0587.v1
Subject: Life Sciences, Molecular Biology Keywords: RF/Microwaves; mobile phone; HEK293; APP; α-synuclein; oxidative stress; non-ionizing EMR
Online: 26 August 2020 (14:46:14 CEST)
The expanding use of devices emitting Pulsed Telecommunication Signals (PTS) has launched a serious debate over the possible effects of electromagnetic radiation (EMR) on living organisms. Our previous work has indicated that PTS exposure alters Amyloid Precursor Protein (APP) and alpha-synuclein (α-syn) metabolism in human cells of neural origin, providing a possible connection between exposure and neurodegeneration. This investigation aimed to reveal, in vitro in human non-neural cells (HEK293), the aftermath of the same exposure on the processing of APP and α-syn. Data presented here, indicate changes in APP metabolism, acquisition of different cellular topologies of the newly generated APP fragments, changes in monomeric α-syn accumulation and multimerization, indicating that APP and α-syn processing is possibly altered in the periphery by EMR. These effects are accompanied by a substantial increase in the levels of Reactive Oxygen Species (ROS). Further investigation is required in order to provide insights into the interaction of PTS with non-neural cells affecting the peripheral systemic functional stability. This is necessary because nowadays whole body human exposure from various EMR sources is a fact in normal life with the valid estimation that they may be increased in view of the forthcoming 5G telecommunications network implementation.
ARTICLE | doi:10.20944/preprints202008.0569.v1
Subject: Life Sciences, Molecular Biology Keywords: Recombinase polymerase amplification; Treponema pallidum; Haemophilus ducreyi; Molecular diagnostics; Point-of-care test
Online: 26 August 2020 (09:01:07 CEST)
Yaws is a skin debilitating disease caused by Treponema pallidum subspecies pertenue with most cases reported in children. World Health Organization (WHO) aims at total eradication of this disease through mass treatment of suspected cases followed by an intensive follow-up program. However, effective diagnosis is pivotal in the successful implementation of this control program. Recombinase polymerase amplification (RPA), an isothermal nucleic acid amplification technique offers a wider range of differentiation of pathogens including those isolated from chronic skin ulcers with similar characteristics such as Haemophilus ducreyi (H. ducreyi). We have developed a duplex RPA assay for the simultaneous detection of Treponema pallidum (T. pallidum) and H. ducreyi (TPHD-RPA). TPHD-RPA assay demonstrated no cross-reaction with other pathogens and enable detection of T. pallidum and H. ducreyi within 15 minutes at 42 oC. The duplex RPA assay was validated with 49 clinical samples from individuals confirmed to have yaws by serological tests. Compared with commercial multiplex real-time PCR, the TPHD-RPA assay demonstrated 94-95% sensitivity for T. pallidum and H. ducreyi confirmed samples, respectively and 100% specificity. This simple novel TPHD-RPA assay enables the rapid detection of both T. pallidum and H. ducreyi in yaws-like lesions. This test could support the yaws eradication programs by ensuring effective diagnosis as well as enable monitoring of eradication efforts success or failure and planning of follow-up interventions at the community level.
Mon, 24 August 2020
REVIEW | doi:10.20944/preprints202008.0527.v1
Subject: Life Sciences, Molecular Biology Keywords: transcriptional regulation; reaction theory; prediction of promoters; one-dimensional diffusion; rate equation; detailed balance; antenna effect; physicochemical techniques
Online: 24 August 2020 (10:09:12 CEST)
Transcriptional regulations have been widely studied as one of the main bridges between biology and other basic sciences as well as medicine. The traffic across it has been mostly unidirectional: chemistry and physics provided a lot of tools for biology, although the supply is now saturating. The traffic in opposite direction, the supply of subjects to develop chemistry and physics, has been only a little. However, if there are any, the supply will be at least from transcription, because the notion of chemical reaction is the strongest. This topic is aimed to increase the opposite traffic by introducing the forefront of physicochemical studies of transcription.
Fri, 21 August 2020
ARTICLE | doi:10.20944/preprints202008.0470.v1
Subject: Life Sciences, Molecular Biology Keywords: Amyotrophic Lateral Sclerosis (ALS); Fronto-Temporal-Lobar-Dementia; Tauopathies; synaptic plasticity; long-term potentiation; spatial learning; inositol signaling; neurexin; K-homology RNA-binding domain; Fragile-X-associated Tremor-Ataxia syndrome
Online: 21 August 2020 (04:39:19 CEST)
Spinocerebellar ataxia type 2 (SCA2) is caused by polyglutamine expansion in Ataxin-2 (ATXN2). This factor binds RNA/proteins to modify metabolism after stress, and to control calcium (Ca2+) homeostasis after stimuli, thus exerting crucial neuroprotection for cerebellar ataxias and corticospinal motor neuron degeneration. Our Atxn2-CAG100-Knock-In mouse faithfully models features observed in patients at pre-onset, early and terminal stages. Here, its cerebellar global RNA profiling revealed downregulation of signaling cascades to precede motor deficits. Validation work at mRNA/protein level defined alterations that were independent of constant physiological ATXN2 functions, but specific for RNA/aggregation toxicity, and progressive across the short lifespan. Earliest changes were detected at 3 months among Ca2+ channels/transporters (Itpr1, Ryr3, Atp2a2, Atp2a3, Trpc3), IP3 metabolism (Plcg1, Inpp5a, Itpka), and Ca2+-Calmodulin dependent kinases (Camk2a, Camk4). CaMKIV–Sam68 control over alternative splicing of Nrxn1, an adhesion component of glutamatergic synapses between granule and Purkinje neurons, was found affected. Systematic screening of pre/post-synapse components, with dendrite morphology assessment, suggested early impairment of CamKIIα abundance together with weakening of parallel fiber connectivity. These data reveal molecular changes due to ATXN2 pathology, impacting communication and excitability of cerebellar neurons. Discovery of such risk versus progression markers improves the assessment of pre-symptomatic treatments in SCA2 and related disorders.
Thu, 20 August 2020
REVIEW | doi:10.20944/preprints202008.0455.v1
Subject: Life Sciences, Molecular Biology Keywords: Hedgehog signaling pathway; GLI family; protein arginine methyl transferase; signal transduction
Online: 20 August 2020 (09:58:05 CEST)
The glioma-associated oncogene (GLI) family consists of GLI1, GLI2, and GLI3 in mammals, and is the effector in the Hedgehog signaling pathway. This family has important roles in the development and homeostasis of various tissues. To achieve these roles, the GLI family has widespread outputs. GLI activity is therefore strictly regulated at multiple levels, including via post-translational modifications for context-dependent GLI target gene expression. Conversely, dysregulated GLI activation has strong links with a variety of cancers. The protein arginine methyl transferase (PRMT) family is also associated with embryogenesis, homeostasis, and cancer via epigenetic modifications and signal transduction. In the PRMT family, PRMT1, PRMT5, and PRMT7 reportedly regulate GLI1 and GLI2 activity. PRMT1 methylates GLI1 to upregulate its activity and target gene expression. Cytoplasmic PRMT5 methylates GLI1 and is involved in GLI1 protein stabilization. In contrast, nucleic PRMT5 interacts with MENIN to suppress growth arrest-specific protein 1 expression, which assists Hedgehog ligand binding to Patched, indirectly resulting in downregulated GLI1 activity. PRMT7-mediated GLI2 methylation upregulates its activity through the dissociation of GLI2 and Suppressor of Fused. Therefore, PRMT1, PRMT5, and PRMT7 regulate GLI activity at multiple levels, and PRMT-mediated GLI dysregulation may be involved in cancer formation.
REVIEW | doi:10.20944/preprints202007.0485.v2
Subject: Life Sciences, Molecular Biology Keywords: extracellular vesicles; exosomes; microvesicles; differential ultracentrifugation; poly-ethylene glycol; immunoaffinity capture; microfluidics; size exclusion chromatography
Online: 20 August 2020 (09:44:09 CEST)
Extracellular vesicles (EVs) are membranous vesicles secreted by both prokaryotic and eukaryotic cells and play a vital role in intercellular communication. EVs are classified into several subtypes based on their origin, physical characteristics, and biomolecular makeup. Exosomes, a subtype of EVs, are released by the fusion of multivesicular bodies (MVB) with the plasma membrane of the cell. Several methods have been described in literature to isolate exosomes from biofluids including blood, urine, milk, and cell culture media among others. While differential ultracentrifugation (dUC), has been widely used to isolate exosomes, other techniques including ultrafiltration, precipitating agents such as poly-ethylene glycol (PEG), immunoaffinity capture, microfluidics and size exclusion chromatography (SEC) have emerged as credible alternatives with pros and cons associated with each. In this review, we provide a summary of commonly used exosomal isolation techniques with a focus on SEC as an ideal methodology. We evaluate the efficacy of SEC to isolate exosomes from an array of biological fluids, with a particular focus on its application to adipose tissue-derived exosomes. We argue that exosomes isolated via SEC are relatively pure and functional, and that this methodology is reproducible, scalable, inexpensive, and does not require specialized equipment or user expertise.
ARTICLE | doi:10.20944/preprints202005.0273.v2
Subject: Life Sciences, Molecular Biology Keywords: COVID-19; SARS-CoV-2; pandemic; IFITM3; rs12252; rs34481144; ethnic groups; bame; cytokine storm
Online: 20 August 2020 (04:29:43 CEST)
Evidence was brought forward in England and the USA that Black, Asian, Latino and Minority Ethnic people exhibit higher mortality risk from COVID-19 than White people. While socioeconomic factors were suggested to contribute to this trend, they arguably do not explain the range of the differences observed, allowing for possible genetic implications. Almost concurrently, the analysis of a cohort in Chinese COVID-19 patients proposed an association between the severity of the disease and the presence of the minor allele of rs12252 of the Interferon-induced transmembrane protein 3 (IFITM3) gene. This SNP, together with rs34481144, are the two most studied polymorphisms of IFITM3 and have been associated in the past with increased severity in Influenza, Dengue, Ebola, and HIV viruses. IFITM3 is an immune effector protein that is pivotal for the restriction of viral replication, but also for the regulation of cytokine production. Following up to these two developments in the ongoing SARS-CoV-2 pandemic, the present study investigates a possible association between the differences in mortality of ethnic groups in England and the combined haplotypes of rs12252 and rs34481144. The respective allele frequencies were collected for 26 populations from 1000 Genomes Project and subgroups were pooled wherever possible to create correspondences with ethnic groups in England. A significant correlation (r=0.9687, p= 0.0003) was observed between the reported Standardized Mortality Ratios and the frequency of the combined haplotype of both reference alleles, suggesting that the combination of reference alleles of the specific SNPs may be implicated in more severe outcomes of COVID-19. This study calls for further focus on the role of IFITM3 variants in the mechanism of cellular invasion of SARS-CoV-2, their impact in COVID-19 severity and their possible implications in vaccination efficacy.
Wed, 12 August 2020
ARTICLE | doi:10.20944/preprints202008.0281.v1
Subject: Life Sciences, Molecular Biology Keywords: Leishmania infantum; proteome; post-translational modifications (PTMs); proteogenomics; mass spectrometry
Online: 12 August 2020 (10:10:45 CEST)
Leishmania infantum is causative of visceral leishmaniasis (kala-azar), the most severe form of leishmaniasis, lethal if untreated. Few years ago, re-sequencing and de novo assembling of the L. infantum (strain JPCM5) genome was accomplished, and now we aimed to describe and characterize the experimental proteome of this species. In this work, we have performed a proteomic analysis from axenic cultured promastigotes and carried out a detailed comparison with other Leishmania experimental proteomes published to date. We identified 2,352 proteins based on the search of mass spectrometry data against a database built from the six-frame translated genome sequence of L. infantum. We have detected many proteins belonging to organelles such as glycosomes, mitochondria or flagellum, as well as many metabolic enzymes, and a large number of putative RNA binding proteins and molecular chaperones. Moreover, we listed the proteins presenting post-translational modifications, such as phosphorylations, acetylations and methylations, among others. On the other hand, the identification of peptides mapping to genomic regions previously annotated as non-coding has allowed to correct annotations, leading to N-terminal extension of protein sequences, and the uncovering of eight novel protein-coding genes. The alliance of proteomics, genomics and transcriptomics has resulted in a powerful combination for improving the L. infantum reference genome annotation.
Tue, 11 August 2020
COMMUNICATION | doi:10.20944/preprints202008.0253.v1
Subject: Life Sciences, Molecular Biology Keywords: microRNA; SARS-CoV-2; coronavirus; lung epithelia; cellular antiviral defence
Online: 11 August 2020 (06:05:40 CEST)
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an RNA virus, is responsible for coronavirus disease 2019 (COVID-19) pandemic of 2020. Experimental evidence suggests that microRNA can mediate an intracellular defence mechanism against some RNA viruses. The purpose of this study was to identify microRNA with predicted binding sites in the SARS-CoV-2 genome, compare these to their microRNA expression profiles in lung epithelial tissue and make inference towards possible roles for microRNA in mitigating coronavirus infection. We hypothesize that high expression of specific coronavirus-targeting microRNA in lung epithelia may protect against infection and viral propagation, conversely low expression may confer susceptibility to infection. We have identified 128 human microRNA with potential to target the SARS-CoV-2 genome, most of which have very low expression in lung epithelia. Six of these 128 microRNA are differentially expressed upon in vitro infection of SARS-CoV-2. Twenty-eight and 23 microRNA also target the SARS-CoV and MERS-CoV, respectively. In addition, 48 and 32 microRNA are commonly identified in two other studies. Further research into identifying bona fide coronavirus targeting microRNA will be useful in understanding the importance of microRNA as cellular defence mechanism against pathogenic coronavirus infections.
Tue, 4 August 2020
ARTICLE | doi:10.20944/preprints202008.0096.v1
Online: 4 August 2020 (11:34:58 CEST)
The promoter hypermethylation of the methylguanine-DNA methyltransferase (MGMT) gene is a frequently used biomarker in daily clinical practice as it is associated with a favorable prognosis in glioblastoma patients treated with temozolamide. In this study we carried out a clinical validation of a quantitative assay for MGMT methylation detection by comparing a novel MSP custom assay using double-probe characteristics (dp_qMSP) with the conventional MSP in 100 FFPE glioblastoma samples collected from a prospective study in La Paz University Hospital. We realized both determinations and established the best cutoff for the identification of positive-methylated samples using the quantitative data obtained from dp_qMSP. Kaplan-Meier curves and ROC time dependent or ROC(t) curves were employed for the comparison of both methodologies. Our results indicate that the optimal cutoff to categorize the MGMTm positive samples by using dp_qMSP is 3.75% methylation value. We obtained similar results using both assays in the same cohort of patients, in terms of progression free survival (PFS) and overall survival (OS) when analyzing the Kaplan-Meier curves. The results of ROC(t) curves showed that dp_qMSP increases the AUC (t) in comparison with MSP for predicting PFS and OS over time. We conclude that dp_qMSP is an alternative methodology compatible with the results obtained with the conventional MSP. This easy-to-use, objective and reliable methodology provides quantitative results and improves the diagnostic precision of patients with glioblastoma in terms of PFS and OS, making it a more competitive assay, suitable for clinical practice.
Fri, 31 July 2020
ARTICLE | doi:10.20944/preprints202007.0739.v1
Subject: Life Sciences, Molecular Biology Keywords: Lung-molGPA; exon 19 deletion; Leu858Arg; rare mutation; prognostic implication
Online: 31 July 2020 (09:30:53 CEST)
EGFR mutations are heterogenous but all carry the same weighting in the Lung-molGPA. The aim of this study was to elucidate the different prognostic implications of molecular subtypes and frontline TKIs in EGFR-mutated lung adenocarcinoma with synchronous brain metastases (BM) using the Lung-molGPA. Medical records were searched in hospital databases from 2011 to 2015. Patients with EGFR-mutated adenocarcinoma and brain metastases who received TKIs were included. The Kaplan-Meier method was used to estimate survival, and multivariate Cox proportional hazard models were used to estimate adjusted hazard ratios (HRs) and 95% confidence intervals (CIs). A total of 256 patients were included with a median overall survival (OS) of 17.2 months. In multivariate analysis of OS, only age (70 versus <70 years, HR:1.71, 95% CI:1.25-2.35, p<0.001), KPS (<70 versus 70, HR:1.71, 95% CI:1.26-2.31, p<0.001), and rare mutations (other versus exon 19 deletions, HR:1.78, 95% CI:1.04-3.05, p=0.037) remained statistically significant. In patients with a Lung-molGPA score 2.5, EGFR molecular subtypes had different median OS (exon 19 deletions versus Leu858Arg versus other, 18.8 vs 12.4 vs 12.1 months, p=0.021). In conclusion, different molecular subtypes treated with frontline TKIs have different prognostic implications in the Lung-molGPA. Further prospective studies are warranted to validate these findings.
Thu, 30 July 2020
ARTICLE | doi:10.20944/preprints202007.0711.v1
Subject: Life Sciences, Molecular Biology Keywords: co-expression network; residual feed intake; RNA-Seq
Online: 30 July 2020 (09:39:36 CEST)
Long non-coding RNA (lncRNA) can regulate several aspects of gene expression, being associated with complex phenotypes in humans and livestock species. In taurine beef cattle, recent evidence points to the involvement of lncRNA in feed efficiency (FE), a proxy for increased productivity and sustainability. Here, we hypothesized specific regulatory roles of lncRNA in FE of indicine cattle. Using RNA-Seq data from liver, muscle, hypothalamus, pituitary and adrenal gland from Nellore bulls with divergent FE, we submitted new transcripts to a series of filters to confidently predict lncRNA. Then, we identified lncRNA that were differentially expressed (DE) and/or key regulators of FE. Finally, we explored lncRNA genomic location and interactions with miRNA and mRNA to infer potential function. We were able to identify 126 relevant lncRNA for FE in Bos indicus, some with high homology to previously identified lncRNA in Bos taurus and some possible specific regulators of FE in indicine cattle. Moreover, lncRNA identified here were linked to previously described mechanisms related to FE in hypothalamus-pituitary-adrenal axis and are expected to help elucidate this complex phenotype. This study contributes to expanding the catalogue of lncRNA, particularly in indicine cattle, and identifies candidates for further studies in animal selection and management.
Sat, 25 July 2020
REVIEW | doi:10.20944/preprints202007.0478.v2
Subject: Life Sciences, Molecular Biology Keywords: Bioinformatics; Drug Design; Small Organic Molecule; Target identification; Web-based Server
Online: 25 July 2020 (17:50:30 CEST)
Drug design is used for different applications of bioinformatics tools analyze DNA, genome, and sequence target region of a small organic molecule in order to understand the molecules of disease. Bioinformatics tools are identified a newly wide research field and minimize future risks through web servers and data mining. Clinical sample test performed with the bioinformatics tools as the biomedical detective. A particular structure and configuration of protein obliging in Drug design concluded Bioinformatics. This review bioinformatics tools and webserver will discuss functions of small organic molecules according to clinical pharmacology.
Tue, 21 July 2020
REVIEW | doi:10.20944/preprints202007.0485.v1
Subject: Life Sciences, Molecular Biology Keywords: extracellular vesicles; exosomes; differential ultracentrifugation; poly-ethylene glycol; immunoaffinity capture; microfluidics; size exclusion chromatography
Online: 21 July 2020 (12:40:15 CEST)
Extracellular vesicles (EVs) are membranous vesicles secreted by both prokaryotic and eukaryotic cells and play a vital role in intercellular communication. EVs are classified into several subtypes based on their origin, physical characteristics, and biomolecular makeup. Exosomes, a subtype of EVs, are released by the fusion of multivesicular bodies (MVB) with the plasma membrane of the cell. Several methods have been described in literature to isolate exosomes from biofluids including blood, urine, milk, and cell culture media among others. While differential ultracentrifugation (dUC), has been widely used to isolate exosomes, other techniques including ultrafiltration, precipitating agents such as poly-ethylene glycol (PEG), immunoaffinity capture, microfluidics and size exclusion chromatography (SEC) have emerged as credible alternatives with pros and cons associated with each. In this review, we provide a summary of commonly used exosomal isolation methodologies with a focus on SEC as an ideal methodology. We argue that exosomes isolated via SEC are relatively pure and functional, and that this methodology is reproducible and scalable, of low-cost, and does not require specialized equipment or user expertise.
REVIEW | doi:10.20944/preprints202007.0478.v1
Subject: Life Sciences, Molecular Biology Keywords: Bioinformatics, Drug Design, Small Organic Molecule, Target identification, Web-based Server
Online: 21 July 2020 (11:10:02 CEST)
Drug design use application bioinformatics tools analyze DNA, genome, and sequence target region of a small organic molecule in order to understand the molecular of disease. Bioinformatics tools precise a newly wide research field and minimize future risks through web servers and data mining. Clinical sample test performed with the bioinformatics tools as the biomedical detective. A particular structure and configuration of protein obliging in Drug Design concluded Bioinformatics. This review bioinformatics tools and webserver discus according to clinical pharmacology
Mon, 20 July 2020
ARTICLE | doi:10.20944/preprints202007.0452.v1
Subject: Life Sciences, Molecular Biology Keywords: COVID-19; kidney disease; comorbidity; enrichment analysis; biological processes and pathways
Online: 20 July 2020 (02:50:36 CEST)
Background: Kidney damage is considered to be one of the risk factors for severity and mortality among COVID-19 patients. However, molecular nature of such observations remains unknown. Hypothesis: Altered gene expressions due to infection and in chronic kidney disease could explain severity in COVID-19 with kidney defects. Methods: We collected gene expression data from publicly available resources Gene Expression Omnibus CKD, Enrichr for deregulated genes in SARS-CoV infected cells in vitro, DisGeNET and others and carried out enrichment analysis using Enrichr. Result: Number of common genes altered in chronic kidney disease (CKD) and SARS-CoV infected cells was 2834. Enrichment analysis revealed that biological processes related viral life cycle and growth, cytokines, immunity, interferon, inflammation, apoptosis, autophagy, oxidative stress and others were significantly enriched with common deregulated genes. Similarly, significantly enriched pathways related to viral and bacterial infections, immunity and inflammation, cell cycle, ubiquitin mediated proteolysis, signaling pathways like Relaxin signaling pathway, mTOR signaling pathway, IL-17 signaling pathway, NF-kappa B signaling pathway were enriched with the common deregulated genes. These processes and pathways are known to be related to kidney damage. DisGeNET terms enriched include and related to Dengue fever, chronic Hepatitis, measles, retroviridae infections, respiratory syncytial virus Infections and many others. Kidney dysfunction related terms ischemia of kidney, renal fibrosis and diabetic nephropathy. Conclusion: Common deregulated genes in SARS-CoV infected cells and chronic kidney disease, as well as their enrichment with molecular processes and pathways relevant for viral pathogenesis and renal dysfunctions, could explain the severity of COVID-19 with kidney disease. This observation not only provides molecular relation of severity in COVID-19 with renal dysfunctions but might also help in the management and treatment targets for these cases.
Sun, 19 July 2020
ARTICLE | doi:10.20944/preprints202007.0438.v1
Subject: Life Sciences, Molecular Biology Keywords: structure-function relationships; enrichment analysis; antifungal activities; knottin; two-layer sandwich architecture
Online: 19 July 2020 (20:40:29 CEST)
Whether there is any inclination between structures and functions of antimicrobial peptides (AMPs) is a mystery yet to be unraveled. AMPs have various structures associated with many different antimicrobial functions, including antibacterial, anticancer, antifungal, antiparasitic and antiviral activities. However, none has yet reported any antimicrobial functional tendency within a specific category of protein/peptide structures nor any structural tendency of a specific antimicrobial function with respect to AMPs. Here we examine the relationships between structures categorized by three structural classification methods (CATH, SCOP and TM) and seven antimicrobial functions with respect to AMPs using an enrichment analysis. The results show that antifungal activities of AMPs were tightly related to two-layer sandwich structure of CATH, knottin fold of SCOP, and the first structural cluster of TM, especially to knottin, whose robust association sustained through the AMPs with a low sequence identity. In addition, another significant mutual enrichment was observed between the third cluster of TM and anti-gram-positive-bacterial/anti-gram-negative-bacterial activities. The findings of the structure-function inclination further our understanding of AMPs and could help us design or discover new therapeutic potential AMPs.
Thu, 16 July 2020
REVIEW | doi:10.20944/preprints202007.0349.v1
Subject: Life Sciences, Molecular Biology Keywords: systematic review; extracellular vesicles; EVs; asthma; therapy; inflammation; respiratory disease; airway hyperresponsiveness; BALF
Online: 16 July 2020 (12:52:03 CEST)
Asthma is the most common chronic disease in children. It is characterized by difficulty in breathing and chronic airway inflammation associated with narrowing of the airways, and airway hyperresponsiveness. If left untreated, asthma can lead to respiratory distress and even death. A number of medications are available and prescribed to manage asthma. Yet despite that, only half of the asthmatic patients are able to control their condition. Extracellular vesicles (EVs) play an important role in transporting contents such as nucleic acids, proteins, and lipids to other cells. While EVs have been extensively studied as biomarkers of various pathological states, evidence indicates that they can play protective and therapeutic roles in mitigating diseases such as cancer, cardiovascular disease and asthma. Here we propose to conduct a systematic review that provides a detailed analysis of the therapeutic effect of EVs in mitigating the primary (inflammation, airway hyperresponsiveness) and secondary outcomes (airway remodelling, molecular indices of cellular signalling, and inflammatory mediators in serum) associated with asthma in preclinical studies.
Mon, 13 July 2020
COMMUNICATION | doi:10.20944/preprints202007.0279.v1
Subject: Life Sciences, Molecular Biology Keywords: COVID19; sub-Sahara Africa; PCR testing; Capacity building
Online: 13 July 2020 (06:32:48 CEST)
The novel COVID-19 pandemic prompted an unprecedented Institutional reaction to aggregate existing capacity from silos of research laboratories to establish a multidisciplinary research laboratory for COVID19 testing. In less than two weeks, resources were mobilized from the community to strengthen public health response and epidemic control. Such strengthening of institutional research capacity to support public health response contributes to a natural knowledge transfer, facilitates collaboration, and generates new frontiers for knowledge production that should ultimately lead to professional development and retention of skilled human resources. This report describes the pre-established mechanisms and involvement of the authors that made it possible to set-up a multidisciplinary laboratory in a remarkably short period of time. We also discuss the opportunities and sustainability of multidisciplinary laboratory research post-COVID19. Existing institutional capacity can be repurposed to establish multidisciplinary research laboratories to support the strengthening of basic and clinical translational research capacity in resource limited settings and impact on public health and scientific knowledge for socioeconomic development.
Sun, 12 July 2020
ARTICLE | doi:10.20944/preprints202007.0251.v1
Subject: Life Sciences, Molecular Biology Keywords: SARS-CoV-2; COVID-19; Spike protein; Mutant; Genome
Online: 12 July 2020 (12:03:16 CEST)
The severity of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), greatly varies from patient to patient. In the present study, we explored and compared mutation profiles of SARS-CoV-2 isolated from mildly affected and severely affected COVID-19 patients in order to explore any relationship between mutation profile and disease severity. Genomic sequences of SARS-CoV-2 were downloaded from GISAID database. With the help of Genome Detective Coronavirus Typing Tool, genomic sequences were aligned with the Wuhan seafood market pneumonia virus reference sequence and all the mutations were identified. Distribution of mutant variants was then compared between mildly and severely affected groups. Among the numerous mutations detected, 14,408C>T and 23,403A>G mutations resulting in RNA-dependent RNA polymerase (RdRp) P323L and spike protein D614G mutations, respectively, were found predominantly in severely affected group (>82%) compared with mildly affected group (<46%, p<0.001). The 241C>T mutation in the non-coding region of the genome was also found predominantly in severely affected group. The 3,037C>T, a silent mutation, also appeared in relatively high frequency in severely affected group. We concluded that RdRp P323L and spike protein D614G mutations predominate in severely affected COVID-19 patients. Further studies will be required to explore whether these mutations have any impact on the severity of COVID-19.
Fri, 10 July 2020
BRIEF REPORT | doi:10.20944/preprints202007.0201.v1
Online: 10 July 2020 (01:57:16 CEST)
The outbreak of coronavirus disease-19 (COVID-19) has infected more than 11 million people and has claimed more than 530.000 deaths world-wide. In July 2020, still, there is no specific treatment for disease caused by the novel coronavirus. In the search to curb the global pandemic COVID-19, some eastern and developing countries have approved various treatment with controversial efficacy, among that the use of the antimalarial Hydroxychloroquine (HCQ), so far with inconclusive clinical evidence of effectiveness. On the other hand, computer-based screening suggest that HCQs analog are promising molecules, to impair viral replication in vitro. Therefore, what is emerging from this complex background, is the need to understand molecular mechanism beyond drugs that can be helpful against viral infection for this and future pandemic. The intent of this Brief Report is to highlight: i) the involvement of the Mitogen Activated Protein Kinase (MAPK) cascade in viral infection and ii) the urgent need to have molecular data on the effectiveness of the combination of MAPK inhibitors together with HCQ and HCQs analogs in curbing viral infection. We are convinced that a better understanding of the patterns of elicited molecular mechanisms will be critical for new molecular approaches to this severe disease
Thu, 9 July 2020
COMMUNICATION | doi:10.20944/preprints202007.0193.v1
Online: 9 July 2020 (12:30:11 CEST)
Glioblastoma multiforme (GBM) is classified as WHO grade IV Astrocytoma & is the most common highly aggressive form of primary brain tumor. Garde IV tumor are highly recurrent even after treatment, with patient survival rate is less than two years from the time of diagnosis. This might be due to overexpression of one of the factors such as ATP-binding cassette transporters (ABC transporters) responsible for drug resistance. ABCC transporter family a member of ABC transporter was found to mostly responsible for multi drug resistance (MDR) in cancer cells. On the other hand, heterogeneous nuclear ribonucleoprotein (hnRNP's) an alternative splicing factor play different role in various cellular process such as nucleic acid metabolism, transcription and translation regulation, among them hnRNPA1 is best studied and its aberrant deregulation favor development of cancer. This study was focused on to study the function of hnRNPA1 in the expression analysis of ABCC transporter (responsible for MDR) in glioma cell lines. The expression of ABCC transporter (ABCC4 and ABCC6) gene was examined in two glioma cell line i.e. U87MG and T98G in normal and knockdown two alternative variants of hnRNPA1 by Quantitative Realtime PCR and Reverse Transcription PCR. We found that ABCC4 was significantly overexpressed in hnRNPA1 Variant 2 knockdown cells (si hnRNPA1 V2) in U87 (3-fold) and in T98G (18.34-fold), While hnRNPA1 Variant 1 knockdown cells (hnRNPA1 V1i) does not shows any significant effect. Further, the expression of ABCC6 was decreased in both hnRNPA1 V1i (0.40-fold) and hnRNPA1 V2i (0.48-fold) in U87. Reverse transcription-based result was complemented with normal PCR based detection strategy after running in agarose gel for U87MG and T98G glioma cell line. Finally, this result indicates that hnRNPA1 an alternative splicing factor regulate the expression of ABCC4 and ABCC6 transporter which are responsible for multiple drug resistance in cancer. This information will help in future for the development of an alternative method for the treatment of drug resistance cases in brain tumor and other tumors by targeting hnRNPA1 splicing factor.
Sun, 5 July 2020
CONCEPT PAPER | doi:10.20944/preprints202007.0080.v1
Subject: Life Sciences, Molecular Biology Keywords: plant parasitic nematodes; xenobiotic metabolism; plant resistance; gene silencing
Online: 5 July 2020 (15:12:51 CEST)
The molecular interaction between the nematode and the host plant cells is complex and sophisticated. Initial contact with the plant parasitic nematodes (PPNs) triggers immune response in the host plant system which includes the release of toxic molecules. To put a bridle on this immune response, PPNs trigger pivotal cytoprotective mechanisms, such as antioxidant and detoxification pathways. Mechanisms of these pathways have been studied in PPNs and the specific genes involved have been targeted for gene silencing research in view of developing novel control measures. However, one of the important group of proteins involved in detoxification pathways known as ABC-transporters are not being studied until recently in PPNs. This opinion article focusses on the current knowledge and future prospects of ABC transporters in PPNs.
Sun, 21 June 2020
REVIEW | doi:10.20944/preprints202006.0263.v1
Subject: Life Sciences, Molecular Biology Keywords: biomarker; blood/plasma; cancer; ctDNA; liquid biopsy
Online: 21 June 2020 (11:22:36 CEST)
Circulating tumor DNA (ctDNA) in fluids has gained attention because ctDNA seems to identify tumor-specific abnormalities, which could be used for diagnosis, follow-up of treatment, and prognosis: the so-called liquid biopsy. Liquid biopsy is a minimally invasive approach and presents the sum of ctDNA from primary and secondary tumor sites. It has been possible not only to quantify the amount of ctDNA but also to identify (epi)genetic changes. Specific mutations in genes have been identified in the plasma of patients with several types of cancer, which highlights ctDNA as a possible cancer biomarker. However, achieving detectable concentrations of ctDNA in body fluids is not an easy task. ctDNA fragments present a short half-life, and there are no cut-off values to discriminate high and low ctDNA concentrations. Here, we discuss the use of ctDNA as a cancer biomarker, the main methodologies, the inherent difficulties, and the clinical predictive value of ctDNA.
ARTICLE | doi:10.20944/preprints202006.0261.v1
Subject: Life Sciences, Molecular Biology Keywords: adverse outcome pathway; toxicity pathway; computational toxicology
Online: 21 June 2020 (11:10:34 CEST)
Background: Targeted methods that dominated toxicological research until recently did not allow for screening of all molecular changes involved in toxic response. Therefore, it is difficult to infer if all major mechanisms of toxicity have already been discovered, or if some of them are still overlooked. Objectives: To identify molecular mechanisms sensitive to chemical exposures in an unbiased manner. Methods: We used data on 641,516 unique chemical-gene interactions from the Comparative Toxicogenomic Database. Only data from high-throughput gene expression experiments with human, rat or mouse cells/tissues were extracted. The total number of chemical-gene interactions was calculated for every gene, and used as a measure of gene sensitivity to chemical exposures. These values were further used in enrichment analyses to identify molecular mechanisms sensitive to chemical exposures. Results: Remarkably, use of different input subsets with non-overlapping lists of chemical compounds identified largely the same genes and molecular pathways as most sensitive to chemical exposures, indicative of an unbiased nature of our analysis. One of the most important findings of this study is that almost every known molecular mechanism may be affected by chemical exposures. Predictably, xenobiotic metabolism pathways and mechanisms of cellular response to stress and damage were among the most sensitive. Additionally, our analysis identified a range of highly sensitive molecular pathways, which are not widely recognized by modern toxicology as major targets of toxicants, including lipid metabolism pathways, longevity regulation cascade and cytokine mediated signaling. Discussion: Molecular mechanisms identified as the most sensitive to chemical exposures are relevant for significant public health problems, such as aging, cancer, metabolic and autoimmune disease. Thus, public health system will likely benefit from future research focus on these sensitive molecular mechanisms. Additionally, approach used in this study may guide identification of priority adverse outcome pathways (AOP) for in-vitro and in-silico toxicity testing methods.
BRIEF REPORT | doi:10.20944/preprints202006.0255.v1
Online: 21 June 2020 (10:45:09 CEST)
CD44 glycoprotein is ubiqutously present and situated at the extracellular surface of cell. Its role as an adhesion recptor was modified depending upon the state of post addition of glycomoeties under varied microenvironment. Many alternative spliced variants of CD44 were expressed by human cells depending upon the attainment during mutifunctional signal disposition. The function of splice factors, and hnRNPA family (Heterogenous ribonucleoproteins A) were dicussed in context to CD44 alternative variants. Glioma cell cline U87 MG were used in this study to search more about the affect of hnRNPA1 and hnRNPA2/B1 knockdown in CD44 V6 and CD44V10 expression. We found in preliminary study that splice factor knock down reduces the alternative splice variants of CD44 such as V6 & V10. Therefore, potential of this study to elaborate further the mechanism in vivo model, to validate the alternative spliced variant in context to interaction with extracellular microenvironment.
Wed, 17 June 2020
ARTICLE | doi:10.20944/preprints202006.0222.v1
Subject: Life Sciences, Molecular Biology Keywords: astaxanthin; muscle atrophy; mitochondria; oxidative stress
Online: 17 June 2020 (13:29:15 CEST)
Astaxanthin (AX) is a carotenoid that exerts potent antioxidant activity and acts in the lipid bilayer. This study aimed to investigate the effects of AX on muscle atrophy-mediated disturbance of mitochondria that have a lipid bilayer. Tail suspension was used to establish muscle- atrophied mouse models. AX diet fed to tail-suspension mice prevented loss of muscle weight and decreased myofiber size in the soleus muscle. Additionally, AX improved down-regulation of mitochondrial respiratory chain complexes II and III in the soleus muscle after tail suspension. To confirm the AX phenotype in the soleus muscle, we examined its effects on mitochondria using Sol8 myotubes derived from the soleus muscle. We found that AX was preferentially detected in the mitochondrial fraction; it significantly suppressed mitochondrial complex III-driven production of reactive oxygen species in Sol8 myotubes. Moreover, AX inhibited the activation of caspase 3 via inhibiting the release of cytochrome c into the cytosol in antimycin A-treated Sol8 myotubes. These results suggested that AX inhibited mitochondrial oxidative stress through a mitochondria-mediated apoptosis pathway and thus prevented muscle atrophy.
Sun, 14 June 2020
ARTICLE | doi:10.20944/preprints202006.0184.v1
Online: 14 June 2020 (16:00:35 CEST)
Spike protein is the surface glycoprotein of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) necessary for the entry of the virus via the transmembrane receptors of the human endothelial cells of the respiratoty system for the virus to be engulfed causing COVID-19 disease after priming by type II transmembrane protease TMPRSS2 and then binding with the angiotensin-converting enzyme 2 (ACE2). Therefore, mutations and amino acid variants analysis are essential in understanding the mechanism of binding of spike protein with its receptor to have an insights on possibilities to design a peptide or nucleotide-based vaccine for COVID-19. Here, we employed Iterative Threading Assembly Refinement (I-TASSER) and Multiple Alignment using Fast Fourier Transform (MAFFT) to predict the three-dimensional monomer structure of spike protein of SARS-CoV-2 and to analyze the amino acid variants for protein sequences from GISAID database for samples collected from Jordan in a try to find an explanation for the low confirmed number of COVID-19 in Jordan. Our Protein Homology/analogY Recognition Engine V 2.0 (Phyre2) findings showed four single amino acid variants (SAV) found in 20 samples of SARS-CoV-2. What is equal to 5% of samples showed tyrosine deletion at Y144 located in the SARS-CoV-like_Spike_S1_NTD (N terminal domain), 62% showed aspartate substitution to glycine at D614G located in the SARS-CoV-2_Spike_S1_RBD (spike recognition binding site), 5% showed aspartate substitution to tyrosine at D1139Y and 5% showed glycine substitution to serine at G1167S both located in the Corona_S2 domain. The findings have shown lower mutational sensitivity in all variants that might not affect the function of spike glycoprotein except for D614G, which has the highest mutational sensitivity score (5 out of 9) indicating a higher likelihood to affect the function of the spike protein. This might suggest, in general, a reduced transmitability of SARS-CoV-2 in Jordan.
Tue, 9 June 2020
REVIEW | doi:10.20944/preprints202006.0123.v1
Subject: Life Sciences, Molecular Biology Keywords: Lignocellulosic biomass crops; biofuels; plant miRNAs; miR156; miR156/SPL-system; plant biotechnology; abiotic and biotic stresses; bio-confinement
Online: 9 June 2020 (11:52:23 CEST)
Currently, energy security and environmental degradation are the two biggest challenges before humanity that can be surmounted with the use of green and sustainable biofuels produced from lignocellulosic crops. In the future, to ensure adequate and cost-effective supply of biofuels, it requires a sufficient amount of amenable and quality lignocellulosic feedstocks. Therefore, agricultural yields of lignocellulosic biomass crops should be substantially increased by intense genetic maneuvering of key gene regulatory mechanisms and signaling pathways that control plant biomass yield. Recently, numerous miRNAs families are identified, characterized, and validated across the plant kingdom. Plant microRNAs (miRNAs) are 21 to 24 nucleotides long, non-coding small RNAs, act as regulators of their target genes via inducing modifications in transcription, translation, and epigenome. MiRNAs represent many hallmark characteristics like sequence-specific regulation, tissue, and species-specific expression, evolutionary conservation, and functional diversity. They coordinate well physiological and life cycle processes in plants under adverse environmental conditions. Hence, miRNAs offer accurate, precise, and efficient regulatory switches in the miRNA-targeted genetic networks. It is evident from the study of the miR156 family and its target SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes network that controls highly significant agronomic traits in crop plants. The miR156/SPL module acts as a master circuit that synchronizes many intricate complex biological functions such as growth and development, and metabolic processes by sensing internal and external environmental signals in plants. Therefore, miR156 can prove a potential target for miRNAs based plant biotechnology to harmonize complex biofuel traits and improve biomass yield in lignocellulosic biomass crops.
Sun, 31 May 2020
REVIEW | doi:10.20944/preprints202005.0519.v1
Subject: Life Sciences, Molecular Biology Keywords: Angiotensin-Converting Enzyme 2; Spike glycoprotein; TMPRESS2; Furin; Malayan pangolin
Online: 31 May 2020 (21:41:44 CEST)
The outbreak of a novel coronavirus (SARS-CoV2) associated with acute respiratory disease called COVID-19 marked the introduction of the third spillover of an animal CoV to humans in the last 2 decades. The SARS-CoV2 genome analysis with various bioinformatics tools revealed that it belongs to beta CoVs genera, with highly similar genome as bat coronavirus and receptor binding domain (RBD) of spike glycoprotein as Malayan pangolin coronavirus. Based on its genetic proximity, SARS-CoV2 is likely to be originated from bat derived CoV and transmitted to humans via an unknown intermediate mammalian host, probably Malayan pangolin. Further spike protein S1/S2 cleavage site of SARS-CoV2 has acquired polybasic furin cleavage site which is absent in bat and pangolin suggesting natural selection either in an animal host before zoonotic transfer or in humans following zoonotic transfer. In the current review, we recapitulate a preliminary opinion about the disease, origin and life cycle of SARS-CoV2, roles of virus proteins in pathogenesis, commonalities and differences between different corona viruses. We have also highlighted the evidences regarding the potential drugs and vaccine candidates with their modes of action to cope with this viral outbreak. Our review provides comprehensive up-dated information on molecular aspects of the SARS-CoV2.
Sun, 24 May 2020
Subject: Life Sciences, Molecular Biology Keywords: body shape index; body roundness index; metabolic risk factors; Southern China adults
Online: 24 May 2020 (16:44:12 CEST)
This purpose was to compare the ability of body shape index (ABSI) and body roundness index (BRI) with waist circumference (WC), body mass index (BMI), waist-to-hip ratio (WHR), waist-to-height ratio (WHtR) and body adiposity index (BAI) to predict metabolic risk. The cross-sectional study was conducted in 17,360 Chinese subjects (were aged 18-95 years old) who escaped cardiovascular disease (CVD) or diabetes. Biochemical and anthropometric variables were measured by trained staff. Receiver operating characteristic curve (ROC) and optimal cutoff values of obesity indices were recruited to compare the predictive ability for metabolic risk factors. The mean age of subjects was 53.7(13.1) years, 41.6 % were males. Within young group (<60 years), the areas under the curve (AUC) demonstrated that WC, BMI, WHR, WHtR and BRI were able to similarly predict high metabolic risk in males (0.74 vs. 0.74 vs. 0.73 vs. 0.73 vs. 0.73) and in females (0.73 vs. 0.73 vs. 0.71 vs. 0.73 vs. 0.73), while the approximate predictive ability were only acquired in males (0.73 vs. 0.73 vs. 0.70 vs. 0.73 vs. 0.73) within elder group (≥60 years). The optimal cut-off values of BRI for high metabolic risk were calculated in males (<60 y: 3.49 vs. ≥60 y: 3.46) and females (<60 y: 3.47 vs. ≥60 y: 3.60). Meanwhile, BRI displayed a strong prediction to elevated BP and elevated TG in males (AUC = 0.64; AUC = 0.70) and to elevated BP, elevated TG and elevated SUA in females (AUC = 0.67; AUC = 0.69; AUC =0.70). BRI was able to similarly predict high metabolic risk compared to WC, BMI, WHR, WHtR and BAI, while ABSI was not. Moreover, BRI revealed specific predictive ability for elevated BP, elevated TG and elevated SUA.
Sun, 17 May 2020
Subject: Life Sciences, Molecular Biology Keywords: COVID-19; SARS-CoV-2; companion animals; cross-infection
Online: 17 May 2020 (15:27:24 CEST)
Since the COVID-19 caused by SARS-CoV-2 break out in Wuhan China from Dec. 2019, it has spread to hundreds of countries up to now. Scientists from all over the world have paid tremendous efforts to research and try to control the disease. Previous studies suggested that some of the wild animals could be intermediate hosts between humans and origination of SARS-CoV-2, and some companion animals of humans can be infected by SARS-CoV-2, which raised our curiosity about cross-infection of SARS-CoV-2 between animals and humans. Thus, we select some kinds of animals that might have contact with humans to estimate the susceptibility to SARS-CoV-2 in different animals by evolutionary analysis of their receptors for SARS-CoV-2. The results show that some companion animals of the Felidae family like the cat has a higher infection possibility while the species of the Rodent family like the rat and mouse having close contact with humans show an opposite result, which consist with recent animal experiments and researches. These should raise concerns about cross-infection between human and companion animals or animals having close contact with humans which might turn animals into depositaries of the coronavirus even after control of SARS-CoV-2 spreading and cause second or more waves of infections after social reopening. Another side of our results stands by the opinion that bioinformatic analysis can be consistent with experiments in some respects so that we can prevent unnecessary sacrifice of laboratory animals in future experiments.
Sat, 16 May 2020
ARTICLE | doi:10.20944/preprints202005.0273.v1
Subject: Life Sciences, Molecular Biology Keywords: COVID-19; SARS-CoV-2; pandemic; IFITM3; rs12252; rs34481144; ethnic groups; bame; cytokine storm
Online: 16 May 2020 (17:38:28 CEST)
Evidence was recently brought forward in England and the USA that Black, Asian, Latinos and Minority Ethnic people present higher mortality risk from COVID-19 than White people. While socioeconomic factors were suggested to contribute to this trend, they arguably do not explain the range of the differences observed, allowing for possible genetic implications. Almost concurrently, the analysis of a cohort in Chinese COVID-19 patients proposed an association between the severity of the disease and the presence of the minor allele of rs12252 of the IFITM3 gene. This SNP, together with rs34481144, are the two most studied polymorphisms of IFITM3 and have been associated in the past with increased severity in Influenza, Dengue, Ebola, and HIV viruses. Interferon-induced transmembrane protein 3 is an immune effector protein that is pivotal for the restriction of viral replication, but also for the regulation of cytokine production. Following up to these two developments in the SARS-CoV-2 pandemic, the present study investigates a possible connection between differences in mortality of ethnic groups in England and the haplotypes of rs12252 and rs34481144. The respective allele frequencies were collected for all 1000 Genomes Project’s populations and subgroups were pooled wherever possible to create correspondences with ethnic groups in England. A strong correlation was observed between the reported Standardized Mortality Ratios and the frequency of the combined haplotype of both reference alleles. If confirmed clinically, this finding could be pointing at possible hijacking of IFITM3 by SARS-CoV-2 virus and is expected to impact our understanding of the disease mechanisms behind COVID-19.
Thu, 14 May 2020
Subject: Life Sciences, Molecular Biology Keywords: COVID-19; SARS-CoV-2; companion animal; cross-infection
Online: 14 May 2020 (11:54:31 CEST)
Since the COVID-19 caused by SARS-CoV-2 break out in Wuhan China from Dec. 2019, it has spread to hundreds of countries up to now. Scientists from all over the world have paid tremendous efforts to research and try to control the disease. Previous studies suggested that some of the wild animals could be intermediate hosts between humans and origination of SARS-CoV-2, and some companion animals of humans can be infected by SARS-CoV-2, which raised our curiosity about cross-infection of SARS-CoV-2 between animals and humans. Thus, we select some kinds of animals that might have contact with humans to estimate the susceptibility to SARS-CoV-2 in different animals by evolutionary analysis of their receptors for SARS-CoV-2. The results show that some companion animals of the Felidae family like the cat has a higher infection possibility while the species of the Rodent family like the rat and mouse having close contact with humans show an opposite result, which consist with recent animal experiments and researches. These should raise concerns about cross-infection between human and companion animals or animals to have close contact with humans which might grow into depositaries of the virus after control of SARS-CoV-2 spreading and cause second or more infection wave after social reopening. Another side of our results stands by the opinion that bioinformatic analysis can be consistent with practical experiments in some respects so that we can prevent unnecessary sacrifice of laboratory animals in future experiments.
Sat, 9 May 2020
REVIEW | doi:10.20944/preprints202005.0158.v1
Subject: Life Sciences, Molecular Biology Keywords: apoptosis; cancer; phosphorylation; kinases; Bcl-2
Online: 9 May 2020 (08:40:40 CEST)
The regulation of apoptosis depends upon the Bcl-2 protein family. The process of cell death and survival is highly complicated and regulated by various types of extrinsic as well as intrinsic network of biological system. Several enzymes and regulators play crucial role in cell death and survival cycle not only in healthy but also in pathological state particularly in cancer. In cancerous cells, various proto-oncogenes and anti-apoptotic proteins are activated and responsible for the cell survival and longevity. The mechanism of activation and inactivation of various proteins in cell survival is regulated by the process of phosphorylation (kinases) and dephosphorylation (phosphatases). The current review will summarize the dynamics of Bcl-2 phosphorylation and its role in apoptosis and cell survival.
Thu, 7 May 2020
ARTICLE | doi:10.20944/preprints202005.0134.v1
Subject: Life Sciences, Molecular Biology Keywords: Crispr-Cas9; ACE2 gene; SARS-CoV-2; new coronavirus; COVID-19
Online: 7 May 2020 (15:26:06 CEST)
The human angiotensin-converting enzyme 2 (ACE2) has a crucial role on blood pressure control; however, ACE2 is also the primary SARS-CoV-2 (S domain) virus receptor. Inhibiting or even reducing the expression of the native ACE2 might diminish the viral entry into the cells, but may cause a failure of ACE2 biological activity, primarily in patients with comorbidities, including diabetes mellitus or hypertension. Since the ACE2 catalytic site and the SARS-Cov-2 receptor are distinct, we designed a Crispr-Cas9 model system, predicting the respective sequences for a guide RNA (gRNA) and a single-stranded oligo dideoxy nucleotide (ssODN), to introduce point mutations into the exon 1 of the human ACE2 gene, which encodes the alpha-helix, implicated on the binding of the SARS-CoV-2 envelope S protein. Protein modeling predicted that the specific substitutions of residues Phe28, Lys31, and Tyr41 for Ala at the ACE2 alpha-helix do not significantly alter ACE2 native conformation. The analysis of the impact of these mutations on ACE2 receptor function predicted a weakening of the binding of the SARS-CoV-2 protein S. An experimental genome editing of cells based on these Crispr-Cas9 elements might reduce the SARS-CoV-2 ability to enter the epithelial cell, preserving the biological activity of ACE2 enzyme.
Tue, 5 May 2020
ARTICLE | doi:10.20944/preprints202005.0065.v1
Subject: Life Sciences, Molecular Biology Keywords: memory formation; moonlighting protein; protein-protein interaction; astrocyte-neuron lactate shuttle
Online: 5 May 2020 (06:09:47 CEST)
Long-term potentiation (LTP) is a molecular basis of memory formation. Here, we demonstrate that LTP critically depends on muscle fructose 1,6-bisphosphatase 2 (Fbp2) – a glyconeogenic enzyme and moonlighting protein protecting mitochondria against stress. We show that LTP induction regulates Fbp2 association with neuronal mitochondria and Camk2, and that the Fbp2-Camk2 interaction correlates with Camk2 autophosphorylation. Silencing of Fbp2 expression or simultaneous inhibition and tetramerization of the enzyme with a synthetic effector mimicking the action of physiological inhibitors (NAD+ and AMP) abolishes Camk2 autoactivation and blocks formation of the early phase of LTP and expression of the late phase LTP markers. Astrocyte-derived lactate reduces NAD+/NADH ratio in neurons and thus, diminishes the pool of tetrameric and increases the fraction of dimeric Fbp2. We therefore hypothesize that this NAD+-level-dependent increase of the Fbp2 dimer/tetramer ratio might be a crucial mechanism in which astrocyte-neuron lactate shuttle stimulates LTP formation.
ARTICLE | doi:10.20944/preprints202005.0063.v1
Subject: Life Sciences, Molecular Biology Keywords: p53-Mdm2; mutant p53; oncogene; stress; regulatory network; cancer dynamics
Online: 5 May 2020 (05:55:27 CEST)
We study a minimal model of the stress-driven p53 regulatory network that includes competition between active and mutant forms of the tumor-suppressor gene p53. Depending on the nature of the external stress signal, four distinct dynamical states are observed. These states can be distinguished by dierent dynamical properties and correspond to active, apoptotic, pre-malignant and cancer states. Transitions between any two of these states are found to be unidirectional and irreversible if the stress signal is either oscillatory or constant. When the signal decays exponentially, the apoptotic state vanishes, and for low stress the pre-malignant state is bounded by two critical points, allowing the system to transition reversibly from the active to the pre-malignant state. For signicantly large stress, the range of the pre-malignant state expands and the system moves to the cancerous state which is a stable attractor. This suggests that identification of the pre-malignant state may be important both for therapeutic intervention as well as for drug discovery.
Sun, 3 May 2020
ARTICLE | doi:10.20944/preprints202005.0040.v1
Online: 3 May 2020 (08:49:19 CEST)
To address the expression pattern of the SARS-CoV-2 receptor ACE2 and the viral priming protease, TMPRSS2, in the respiratory tract, this study investigated RNA sequencing transcriptome profiling of samples of airway and oral mucosa. As shown, ACE2 has medium levels of expression in both small airway epithelium and masticatory mucosa, and high levels of expression in nasal epithelium. The expression of ACE2 is low in mucosal associated invariant T (MAIT) cells, and can’t be detected in alveolar macrophages. TMPRSS2 is highly expressed in small airway epithelium and nasal epithelium, and has lower expression in masticatory mucosa. Our results highlights that the nasal mucosa is the most susceptible locus in the respiratory tract for SARS-CoV-2 infection and consequently for subsequent droplet transmission and should be the focus for protection against SARS-CoV-2 infection.
Thu, 30 April 2020
COMMUNICATION | doi:10.20944/preprints202004.0533.v1
Online: 30 April 2020 (14:04:40 CEST)
The Standard Genetic Code (SGC) exists in every organism known on Earth. SGC evolution via early unique codon assignment, then later wobble, yields coding resembling the near-universal code. Below, later wobble also creates an optimal route to accurate codon assignment. This assignment time matches a previous mean time for ordered codes, exhibiting ≥ 90% of SGC order. Accurate evolution is also accessible, sufficiently frequent to appear in populations of 103 to 104 codes. SGC-like coding capacity, code order and assignments therefore arise together, in one attainable evolutionary intermediate. Examples, which plausibly resemble coding at evolutionary domain separation, are characterized.
Fri, 24 April 2020
ARTICLE | doi:10.20944/preprints202004.0425.v1
Subject: Life Sciences, Molecular Biology Keywords: SARS-CoV-2; COVID-19; 5’UTR; miRNAs; RNAi; GapmeRs
Online: 24 April 2020 (04:16:20 CEST)
After the increasing number of SARS-CoV-2 infections all over the world, researchers and clinicians are struggling to find a vaccine or innovative therapeutic strategies to treat this viral infection. The SARS-CoV infection that occurred in 2002, MERS and other more common infectious diseases such as HCV, led to the discovery of many RNA-based drugs. Among them, siRNAs and antisense LNAs have been demonstrated to have effective antiviral effects both in animal models and humans. Owing to the high genomic homology of SARS-CoV-2 and SARS-CoV (80-82%) the use of these molecules could be employed successfully also to target this emerging coronavirus. Trying to translate this approach to treat COVID-19, we analyzed the common structural features of viral 5’UTR regions that can be targeted by non-coding RNAs and we also identified miRNAs binding sites suitable for designing RNA-based drugs to be employed successfully against SARS-CoV-2.
Sun, 19 April 2020
Subject: Life Sciences, Molecular Biology Keywords: SARS-CoV-2; 4521 lines; mutations; MAF (minor allele frequency); deamination
Online: 19 April 2020 (07:05:58 CEST)
Aims: The sequencing data of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) are rapidly emerging. The mutation profile across SARS-CoV-2 populations is an important inference of the evolution of coronaviruses. Materials & Methods: With 4,521 lines of SARS-CoV-2, we obtained 3,169 unique point mutation sites in the SARS-CoV-2 genome. We counted the numbers and calculated the MAF (minor allele frequency) of each mutation type. Results: Nearly half of the point mutations are C-T mismatches and 20% are A-G mismatches. The MAF of C-T and A-G mismatches is significantly higher than MAF of other mutation types. Conclusions: The excessive C-T mismatches do not resemble the random mutation profile, and are likely to be explained by the cytosine-to-uridine deamination system in hosts. Not only the population analyses in previous studies are questionable, but also the 17% divergence between SARS-CoV-2 and RaTG13 could be erroneous due to the deamination.
Subject: Life Sciences, Molecular Biology Keywords: lncRNA; breast cancer; alternative splicing; estrogen receptor; RNA-Seq
Online: 19 April 2020 (04:29:31 CEST)
Background: DSCAM-AS1 is a cancer-related long noncoding RNA with higher expression levels in Luminal A, B and HER2-positive Breast Cancer (BC), where its expression is strongly dependent on Estrogen Receptor Alpha (ERα). Methods: To decipher its function, DSCAM-AS1 expression was measured by qRT-PCR in tissue samples from 93 BC patients in addition to a meta-analysis of 30 gene expression datasets, together with the evaluation of its association with clinical data. By computational analyses of our RNA-Seq in MCF-7 cells, we investigated the DSCAM-AS1 knock-down effects at both gene and isoform levels. Results: We confirmed DSCAM-AS1 overexpression in high grade Luminal A, B and HER2+ BCs and found a significant correlation with disease relapse. 908 genes were regulated by DSCAM-AS1-silencing, primarily involved in cell cycle and inflammatory response. Noteworthy, the analysis of alternative splicing and isoform regulation revealed 2,085 splicing events regulated by DSCAM-AS1, enriched in differential polyadenylation sites and 3’UTR shortening events. Finally, the DSCAM-AS1-interacting splicing factor hnRNPL was predicted as the most enriched RBP for exon skipping and 3’UTR events. Conclusion: The relevance of DSCAM-AS1 overexpression in BC is confirmed by clinical data and further enhanced by its possible involvement in the regulation of RNA processing, which is emerging as one of the most important dysfunctions in cancer.
ARTICLE | doi:10.20944/preprints202004.0315.v1
Subject: Life Sciences, Molecular Biology Keywords: COVID-19; SARS-CoV2; ACE2 receptor; medical cannabis; CBD
Online: 19 April 2020 (02:45:50 CEST)
With the rapidly growing pandemic of COVID-19 caused by the new and challenging to treat zoonotic SARS-CoV2 coronavirus, there is an urgent need for new therapies and prevention strategies that can help curtail disease spread and reduce mortality. Inhibition of viral entry and thereby spread constitute plausible therapeutic avenues. Similar to other respiratory pathogens, SARS-CoV2 is transmitted through respiratory droplets, with potential for aerosol and contact spread. It uses receptor-mediated entry into the human host via angiotensin-converting enzyme II (ACE2) that is expressed in lung tissue, as well as oral and nasal mucosa, kidney, testes, and the gastrointestinal tract. Modulation of ACE2 levels in these gateway tissues may prove a plausible strategy for decreasing disease susceptibility. Cannabis sativa, especially one high in the anti-inflammatory cannabinoid cannabidiol (CBD), has been proposed to modulate gene expression and inflammation and harbour anti-cancer and anti-inflammatory properties. Working under the Health Canada research license, we have developed over 800 new Cannabis sativa lines and extracts and hypothesized that high-CBD C. sativa extracts may be used to modulate ACE2 expression in COVID-19 target tissues. Screening C. sativa extracts using artificial human 3D models of oral, airway, and intestinal tissues, we identified 13 high CBD C. sativa extracts that modulate ACE2 gene expression and ACE2 protein levels. Our initial data suggest that some C. sativa extract down-regulate serine protease TMPRSS2, another critical protein required for SARS-CoV2 entry into host cells. While our most effective extracts require further large-scale validation, our study is crucial for the future analysis of the effects of medical cannabis on COVID-19. The extracts of our most successful and novel high CBD C. sativa lines, pending further investigation, may become a useful and safe addition to the treatment of COVID-19 as an adjunct therapy. They can be used to develop easy-to-use preventative treatments in the form of mouthwash and throat gargle products for both clinical and at-home use. Such products ought to be tested for their potential to decrease viral entry via the oral mucosa. Given the current dire and rapidly evolving epidemiological situation, every possible therapeutic opportunity and avenue must be considered.
Wed, 15 April 2020
ARTICLE | doi:10.20944/preprints202004.0240.v1
Online: 15 April 2020 (10:00:52 CEST)
The novel coronavirus 2019 (nCov-2019/Covid-2019/2019-nCov) has become a pandemic in a very short span of time. It has caused significant loss to human lives, economy, daily life. The key development against the nCov-2019 remains apprehended when it comes to discovery of its vaccine or medicines for the treatment. Drugs used for the treatment of HIV ( inhibitors of HIV protease) are being largely used for the treatment of nCov-2019. Therefore, we conducted a study by docking a set of natural compounds with reported protease activity against HIV or SARS coronavirus against the protease of nCov-2019. The Bavachinin ranked the top among natural compounds with binding energy of -7.74±0.152 Kcal/mol, RMSD 0.823±0.024 Å, predicted pKd 5.59 and predicted dG of -7.56 Kcal/mol. The finding infers that these three compounds could have the potential to inhibit the nCov-2019 protease. The finding was supported with reputed research publications.
Thu, 9 April 2020
COMMUNICATION | doi:10.20944/preprints202004.0146.v1
Subject: Life Sciences, Molecular Biology Keywords: Coronavirus Disease 2019; SARS-CoV-2; main protease; molecular docking-based virtual screening
Online: 9 April 2020 (09:34:45 CEST)
Coronavirus Disease 2019 (Covid-19) was first described in December 2019 in Wuhan, Hubei Province, China; and produced by a novel coronavirus designed as the acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Covid-19 has become a pandemic reaching over 1.3 million confirmed cases and 73,000 deaths. Several efforts have been done to identify pharmacological agents that can be used to treat patients and protect healthcare professionals. The sequencing of the virus genome not only has offered the possibility to develop a vaccine, but also to identified and characterize the virus proteins. Among these proteins, main protease (Mpro) has been identified as a potential therapeutic target, since it is essential for the processing other viral proteins. Crystal structures of SARS-CoV-2 Mpro and inhibitors has been described during the last months. To describe additional compounds that can inhibit SARS-CoV-2 Mpro, in this study we performed a molecular docking-based virtual screening against a library of experimental and approved drugs. Top 10 hits included Pictilisib, Nimorazole, Ergoloid mesylates, Lumacaftor, Cefuroxime, Cepharanhine, and Nilotinib. These compounds were predicted to have higher binding affinity for SARS-CoV-2 Mpro than previously reported inhibitors for this protein, suggesting a higher potential to inhibit virus replication. Since the identified drugs have both pre-clinical and clinical information, we consider that these results may contribute to the identification of treatment alternative for Covid-19. Nevertheless, in vitro and in vivo confirmation should be performed before these compounds could be translated to the clinic.
Wed, 8 April 2020
ARTICLE | doi:10.20944/preprints201910.0146.v2
Subject: Life Sciences, Molecular Biology Keywords: NGS data analysis; bioinformatics pipelines; NGS pipelines
Online: 8 April 2020 (06:21:10 CEST)
Next-generation sequencing (NGS) has been a widely-used technology in biomedical research for understanding the role of molecular genetics of cells in health and disease. A variety of computational tools have been developed to analyse the vastly growing NGS data, which often require bioinformatics skills, tedious work and significant amount of time. To facilitate data processing steps minding the gap between biologists and bioinformaticians, we developed CSI NGS Portal, an online platform which gathers established bioinformatics pipelines to provide fully automated NGS data analysis and sharing in a user-friendly website. The portal currently provides 16 standard pipelines for analysing data from DNA, RNA, smallRNA, ChIP, RIP, 4C, SHAPE, circRNA, eCLIP, Bisulfite and scRNA sequencing, and is flexible to expand with new pipelines. The users can upload raw data in fastq format and submit jobs in a few clicks, and the results will be self-accessible via the portal to view/download/share in real-time. The output can be readily used as the final report or as input for other tools depending on the pipeline. Overall, CSI NGS Portal helps researchers rapidly analyse their NGS data and share results with colleagues without the aid of a bioinformatician. The portal is freely available at: https://csibioinfo.nus.edu.sg/csingsportal
Tue, 31 March 2020
BRIEF REPORT | doi:10.20944/preprints202003.0446.v1
Online: 31 March 2020 (05:08:29 CEST)
The COVID-19 pandemic caused by SARS-COV-2 has infected over 500,000 people causing over 25,000 deaths in the last 10 weeks. A key host cellular protein required for the virus entry is angiotensin-converting enzyme 2 (ACE2). Recent studies have reported that patients with hypertension and diabetes treated with ACE inhibitors or angiotensin receptor blockers might be at a higher risk of COVID-19 infection as these drugs have been reported to increase ACE2 expression. This has raised the need to systematically investigate the effect of different drugs including antihypertensives on modulating ACE2 expression. Here, we analyzed a publicly available CMAP dataset of pre/post transcriptomic profiles for drug treatment in cell lines for over 20,000 small molecules. We show that only one subclass of antihypertensives drugs - ACE inhibitors, are significantly enriched for drugs up-regulating ACE2 expression. Studying the effects of the 672 clinically approved drugs in CMAP, we chart the drug categories that affect ACE2 expression. Specifically, we find that panobinostat (an HDAC inhibitor) confers the highest up-regulation of ACE2 expression while isotretinoin (a vitamin A derivative) is its strongest down-regulator. Our results provide initial candidates guiding further in vitro and in vivo studies aimed at assessing drug effects on ACE2 expression.
Tue, 24 March 2020
ARTICLE | doi:10.20944/preprints202003.0357.v1
Subject: Life Sciences, Molecular Biology Keywords: Benzo[a]pyrene; Microtubule; Neuroblastoma; Cell cycle arrest, Neuropeptide Y
Online: 24 March 2020 (08:41:53 CET)
Benzo[a]pyrene (B[a]P), is a family member of polycyclic aromatic hydrocarbons and a widespread environmental pollutant and neurotoxicant that contribute to the development of cancer. Microtubules are polymers of tubulin that form part of the cytoskeleton and target for anticancer drugs. Furthermore, NPY significantly increased the percentage of cells in S and G2/M phases. However, little is known about the specific role of NPY in proliferation and the underlying protective mechanism remains unclear. Hence, the aim of this work was to investigate the effect of B[a]P on SH-SY5Y neuroblastoma cells and to explore the potential mechanism for alteration of tubulin-microtubule equilibrium causing mitotic arrest and NPY expression. The present findings showed B[a]P treatment significantly increase number of SH-SY5Y cells in S and G2/M phase as compared to G1 phase and provokes cell cycle arrest that correlated with significant decrease in G0/G1 cells. Immunofluorescence study showed significantly distorted tubulin arrangement from metaphasic plate in formation of bipolar mitotic spindle apparatus. Further, higher doses of B[a]P treatment lead to chromosomal abnormalities accompanied by DNA damage due ROS causing oxidative stress showing significant decrease in tubulin protein around spindle. The results of present study demonstrated that NPY exerts a proliferative and protective effect on B[a]P-induced oxidative stress in a dose-dependent manner in vitro and importantly, these effects may be mediated via mitotic arrest and involved in spindle arrangement during cell division. Our findings addresses a novel pathological outcomes of B[a]P-induced NPY expression by oxidative stress through spindle abnormalities leading to microtubule disruption.
Wed, 18 March 2020
REVIEW | doi:10.20944/preprints202003.0290.v1
Subject: Life Sciences, Molecular Biology Keywords: Histone PTM; RNA Polymerase II; ChIP-seq; chromatin; epigenetics; transcriptional interference; plant; Transcription Cycle; Transcription
Online: 18 March 2020 (17:14:28 CET)
Post-translational modifications (PTMs) of histone residues shape the landscape of gene expression by modulating the dynamic process of RNAPII transcription. The contribution of particular histone modifications to the definition of distinct RNAPII transcription stages remains poorly characterized in plants. Chromatin Immuno-precipitation combined with next-generation sequencing (ChIP-seq) resolves the genomic distribution of histone modifications. Here, we review histone PTM ChIP-seq data in Arabidopsis thaliana and find support for a Genomic Positioning System (GPS) that guides RNAPII transcription. We review the roles of histone PTM “readers”, “writers” and “erasers”, with a focus on the regulation of gene expression and biological functions in plants. The distinct functions of RNAPII transcription during the plant transcription cycle may in part rely on the characteristic histone PTMs profiles that distinguish transcription stages.
Fri, 13 March 2020
ARTICLE | doi:10.20944/preprints202003.0230.v1
Subject: Life Sciences, Molecular Biology Keywords: Ixeris dentate; lactobacillus, salivation; xerostomia
Online: 13 March 2020 (10:18:15 CET)
Dry mouth, hyposalivation, or xerostomia is a significant problem in diabetic patients; however, there was no way to relieve these symptoms. This study was aimed to evaluate the effects of Ixeris dentata (IXD) in combination with lactobacillus extract on the salivation rate in diabetes-induced dry mouth, and its mechanism was also investigated. In the streptozotocin-induced diabetes model, dry mouth condition was established as a model. Both control and diabetic rats were treated with a sublingual spray of either water or IXD and subsequently treated with or without a spray of lactobacillus extract. In diabetes condition, the salivary flow rate, amylase activity, and aquaporin-5 and Na+/H+ exchanger (NHE-1) expressions were markedly decreased, whereas they were more significantly recovered in the sequential treatment of IXD-lactobacillus extract than each single treatment. Furthermore, oxidative stress and its related ER stress response were especially regulated in the IXD/lactobacillus extract condition, where the following anti-oxidative enzymes; GSH:GSSG ratio, superoxide dismutase (SOD), and glutathione peroxidase (GPx) were involved. This study suggests that the combination of IXD and lactobacillus would be a potential alternative medicine against diabetes-induced hyposalivation and xerostomia.
Tue, 10 March 2020
ARTICLE | doi:10.20944/preprints202003.0168.v1
Subject: Life Sciences, Molecular Biology Keywords: NF-kappaB; HIF; ARNT; TRAF6; Transcription; ChIP; Drosophila
Online: 10 March 2020 (11:14:37 CET)
NF-B signalling is crucial for cellular responses to inflammation but has also been associated with the hypoxia response. NF-B and HIF transcription factors possess an intense molecular crosstalk. Although it is known that HIF-1beta modulates NF-kappaB transcriptional response, very little is understood regarding how HIF-1beta contributes to NF-kappaB signalling. Here, we demonstrate that HIF-1beta is required for full NF-kappaB activation in cells following canonical and non-canonical stimuli. We found that HIF-1beta specifically controls TRAF6 expression in human cells but also in Drosophila melanogaster. HIF-1beta binds to the TRAF6 gene and controls its expression independently of HIF-1alpha. Furthermore, exogenous TRAF6 expression is able to rescue all of the cellular phenotypes observed in the absence of HIF-1beta. These results indicate that HIF-1beta is an important regulator of NF-kappaB with consequences for homeostasis and human disease.
Sat, 7 March 2020
Online: 7 March 2020 (03:05:56 CET)
Heterosis or hybrid vigour is a phenomenon in which hybrid progeny exhibit superior yield and biomass to parental lines and has been used to breed F1 hybrid cultivars in many crops. A similar level of heterosis in all F1 individuals is expected as they are genetically identical. However, we found variation of rosette size in individual F1 plants from a cross between C24 and Columbia-0 accessions of Arabidopsis thaliana. Big sized F1 plants had 26.1% larger leaf area in the 1st and 2nd leaves than medium sized F1 plants at 14 days after sowing in spite of the identical genetic background. We identified differentially expressed genes between big and medium sized F1 plants by microarray; genes involved in the category of stress response were overrepresented. We made transgenic plants overexpressing 21 genes, which were differentially expressed between the two size classes, some lines had increased plant size at 14 or 21 days after sowing but not at all time points. Change of expression levels in stress responsive genes among individual F1 plants, implying epigenetic changes, could generate the variation in plant size of individual F1 plants in A. thaliana.
Fri, 6 March 2020
ARTICLE | doi:10.20944/preprints202003.0106.v1
Subject: Life Sciences, Molecular Biology Keywords: purinergic P2X4 receptor; TM1 and TM2 segments; ethanol and agonist action; mutagenesis; molecular model
Online: 6 March 2020 (04:45:49 CET)
Mouse models of alcohol use disorder (AUD) revealed a subtype of purinergic receptors (P2X4Rs) as a promising target for AUD drug development. We have previously demonstrated that residues at the transmembrane (TM)-ectodomain interface and within TM1 segment contribute to the formation of an ethanol action pocket in P2X4Rs. In the present study, we tested the hypothesis that there are more residues in TM segments, which are important for ethanol sensitivity of P2X4Rs. Using site-directed mutagenesis and two-electrode voltage-clamp electrophysiology in Xenopus oocytes, we found that arginine at position 33 (R33) in the TM1 segment plays a role in ethanol sensitivity of P2X4Rs. Molecular models in both closed and open states provided evidence for interactions between R33 and aspartic acid at position 354 (D354) of the neighboring TM2 segment. Further work with mixtures of wild-type (WT) and reciprocal single (R33D:WT, D354R:WT) and double (R33D-D354R:WT) mutants confirmed the importance of this interaction for ethanol sensitivity, ivermectin action and channel function. Additionally, our findings suggest that valine at TM1 position 49 plays a role in P2X4R function by providing flexibility during channel opening. Collectively, these findings identified new activity sites, and suggest the importance of TM1-TM2 interaction for channel function and ethanol sensitivity of P2X4Rs.
Fri, 28 February 2020
ARTICLE | doi:10.20944/preprints202002.0423.v1
Subject: Life Sciences, Molecular Biology Keywords: ITF2_TCF4; chemotheraoy resistance; NSCLC; HOXD9; Wnt pathway
Online: 28 February 2020 (11:58:28 CET)
Despite often leading to platinum resistance, platinum-based chemotherapy continues to be the standard treatment for many epithelial tumors. In this study we analyze and validated the cytogenetic alterations that arise after treatment in four lung and ovarian paired cisplatin-sensitive/resistant cell lines by 1-million array-CGH and qRT-PCR methodologies. RNA-sequencing, functional transfection assays and gene-pathway activity analysis were used to identify genes with a potential role in the development of this malignancy. Results were further explored in 55 lung and ovarian primary tumors and control samples and in two extensive in silico databases. Long-term cell exposure to platinum induces the frequent deletion of ITF2 gene. Its expression re-sensitizes tumor cells to platinum and recovers the levels of Wnt/β-catenin transcriptional activity. ITF2 expression was also frequently downregulated in epithelial tumors, predicting a worse overall survival. We also identified an inverse correlation between ITF2 and HOXD9 expression, revealing that NSCLC patients with lower expression of HOXD9 have a better overall survival rate. We define the implication of ITF2 as a molecular mechanism behind the development of cisplatin resistance probably through the activation of the Wnt-signaling pathway. This data highlights the possible role of ITF2 and HOXD9 as novel therapeutic targets for platinum resistant tumors.
Wed, 26 February 2020
ARTICLE | doi:10.20944/preprints202002.0391.v1
Subject: Life Sciences, Molecular Biology Keywords: Highly Pathogenic Avian Influenza Virus, Low Pathogenic Avian Influenza Virus, Evolution, Virulence Determinants, non-H5/H7, Cleavage Site, Chicken-to-Chicken Transmission, Virulence, Protease
Online: 26 February 2020 (11:13:35 CET)
Highly pathogenic (HP) avian influenza viruses (AIVs) are naturally restricted to H5 and H7 subtypes with a polybasic cleavage site (CS) in the hemagglutinin (HA) and any AIV with an intravenous pathogenicity index (IVPI) ≥1.2. Only few non-H5/H7 viruses fulfill the criteria of HPAIVs; nevertheless, it remains unknown why these viruses did not spread in domestic birds. In 2012, a unique H4N2 virus with a polybasic CS 322PEKRRTR/G329 was isolated from quails in California which, however, was avirulent in chickens. This is the only known non-H5/H7 virus with four basic amino acids in the HACS. Here, we investigated the virulence of this virus in chickens after expansion of the polybasic CS by substitution of T327R (322PEKRRRR/G329) or T327K (322PEKRRKR/G329) with or without reassortment with HPAIVs H5N1 and H7N7. The impact of single mutations or reassortment on virus fitness in vitro and in vivo was studied. Efficient cell culture replication of T327R/K carrying H4N2 viruses increased by trypsin, particularly in MDCK cells, and reassortment with HPAIV H5N1. Likewise, replication, virus excretion and bird-to-bird transmission of H4N2 was remarkably compromised by the CS mutations, but restored after reassortment with HPAIV H5N1, although not with HPAIV H7N7. Viruses carrying the H4-HA with or without R327 or K327 mutations and the other gene segments from HPAIV H5N1 exhibited high virulence and efficient transmission in chickens. Together, increasing the number of basic amino acids in the H4N2 HACS was detrimental for viral fitness particularly in vivo but compensated by reassortment with HPAIV H5N1. This may explain the absence of non-H5/H7 HPAIVs in poultry.
Mon, 24 February 2020
ARTICLE | doi:10.20944/preprints201910.0026.v2
Subject: Life Sciences, Molecular Biology Keywords: ticks; Ixodes scapularis; blood digestion; serine proteases; trypsin
Online: 24 February 2020 (04:45:09 CET)
Ixodes scapularis is the major vector of Lyme disease in the eastern United States. Each active life stage (larva, nymph, and adult) takes a blood meal either for developing and molting to the next stage (larvae and nymphs) or for oviposition (adult females). This protein-rich blood meal is the only food taken by Ixodes ticks and therefore blood digestion is very important for tick survival. Most studies on blood digestion in ticks have shown that the initial stages of digestion are carried out by cathepsin proteases within acidic digestive cells. However, most of these studies have focused on partially engorged ticks. In other hematophagous arthropods, the serine proteases play an important role in blood protein degradation. In this study, we determined transcript expression of four I. scapularis serine proteases with previously characterized roles in blood digestion. RNA interference was used for functional analysis and a trypsin-benzoyl-D, L-arginine 4-nitoanilide assay was used to measure active trypsin levels. An in vitro hemoglobinolytic assay was performed with or without serine protease inhibitor. Our data suggest that trypsin levels increase significantly after blood feeding and peaked in larvae, nymphs, and adults at 3, 1, and 1 day post host detachment, respectively. The knockdown of three previously identified serine proteases by RNAi negatively impacted blood intake, survival, fecundity, levels of active trypsin in the gut and resulted in lower hemoglobin degradation in vitro. A trypsin inhibitor, PMSF, blocked the action of trypsin in the gut extract resulting in 65% lower hemoglobin degradation. We provide evidence of the serine proteases as digestive enzymes in fully engorged, replete females. Our data also demonstrated that in addition to blood digestion, these serine proteases might have a role in blood feeding success in I. scapularis.
Sun, 23 February 2020
BRIEF REPORT | doi:10.20944/preprints202002.0332.v1
Subject: Life Sciences, Molecular Biology Keywords: nucleic acids; airborne microorganisms; far infrared radiation; RNA virus
Online: 23 February 2020 (15:51:12 CET)
Emergence of zoonotic-human pathogens is proven to be a lethal threat to public health, and RNA virus including influenza viruses, severe acute respiratory syndrome coronavirus, middle east respiratory syndrome coronavirus, Wuhan coronavirus (COVID-19), plays a pivotal role. As those viruses as airborne microorganisms spread mainly by tiny airborne particles, it is important to de-active those airborne particles before their entry into human bodies. In this study, we investigated the effect of far infrared (FIR) radiation on inhibition of airborne microorganisms. The result confirmed that double stand DNA from airborne microorganisms were stable under mild FIR radiation. However, single strand RNA from them was found to be sensitive to FIR radiation, indicating that RNA virus in airborne particles is instable under FIR radiation. Based on this observation, two models on usage of FIR radiation to prevent RNA virus transmission and cure RNA virus infection were proposed, implying that FIR radiation might be a cheap, convenient, and efficient method in clinic to treat RNA virus.
Mon, 17 February 2020
Online: 17 February 2020 (15:25:43 CET)
A novel coronavirus (2019-nCoV) was first identified in Wuhan, Hubei Province, and then spreads to the other Provinces of China. 2019-nCoV was reported to share the same receptor, Angiotensin-converting enzyme 2 (ACE2), with SARS-CoV. Previous studies have found ACE2 is abundantly present in humans in the epithelia of the lung and small intestines, and they found ACE2 expression in the basal layer of the non-keratinizing squamous epithelium in nasal and oral mucosa and the nasopharynx. Here based on the public single-cell RNA-Seq datasets, we analyzed the ACE2 expression in the nasal, mouth, lung, and colon tissues. We find that the number of ACE2-expressing cells in the nasal tissue and mouth is comparable to the number of ACE2-expressing cells in the lung tissue and colon. We also find that ACE2 tends to be co-expressed with HLA-DRB1, which plays a central role in the immune system by presenting peptides derived from extracellular proteins, in the nasal, mouth, lung, and colon tissues at single-cell resolution. We hope this provides valuable information for virus-prevention strategy and therapeutic strategy development.
Sun, 16 February 2020
REVIEW | doi:10.20944/preprints202002.0209.v1
Subject: Life Sciences, Molecular Biology Keywords: bone morphogenetic protein 4; molecular mechanism; delivery; clinical application; malignant glioma
Online: 16 February 2020 (04:19:22 CET)
Malignant gliomas are heterogeneous neoplasms. Glioma stem-like cells (GSCs) are undifferentiated and self-renewing cells that develop and maintain these tumors. These cells are the main population that resist current therapies. Genomic and epigenomic analyses has identified various molecular subtypes. Bone morphogenetic protein 4 (BMP4) reduces the number of GSCs through differentiation and induction of apoptosis, thus increasing therapeutic sensitivity. However, the short half-life of BMP4 impedes its clinical application. We have previously reviewed BMP4 signaling in central nervous system development and glioma tumorigenesis and its’ potential as a treatment target in human gliomas. Recent advances in understanding both adult and pediatric malignant gliomas highlight critical roles of BMP4 signaling pathways in the regulation of tumor biology, and indicate its’ potential as a therapeutic molecule. Furthermore, significant progress has been made on synthesizing BMP4 biocompatible delivery materials, which can bind to and markedly extend BMP4 half-life. Here, we review current research associated with BMP4 in brain tumors, especially in pediatric malignant gliomas. We also summarize BMP4 delivery strategies, with a focus on biocompatible BMP4 binding peptide amphiphile nanostructures as promising novel delivery platforms for treatment of these devastating tumors.
Tue, 4 February 2020
ARTICLE | doi:10.20944/preprints202002.0038.v1
Subject: Life Sciences, Molecular Biology Keywords: allele specific expression; 6-BA; DNA methylation; long noncoding RNA; siRNA; poplar
Online: 4 February 2020 (05:22:28 CET)
The cytokinins play important roles in plant growth and development by regulating gene expression at genome wide level. DNA methylation is responsive to the external environment, but whether DNA methylation changes in response to cytokinin treatment to regulate gene expression is still unclear. Here, we used bisulfite sequencing and RNA sequencing to examine genome-wide DNA methylation and gene expression patterns in poplar (Populus tomentosa) after treatment with the synthetic cytokinin 6-benzylaminopurine (6-BA). We identified 566 significantly differentially methylated regions (DMRs) in response to 6-BA treatment. Transcriptome analysis showed that 501 protein-coding genes, 262 long non-coding RNAs, and 15,793 24-nt small interfering RNAs were differentially expressed under 6-BA treatment. Among these, 79% were differentially expressed between alleles in P. tomentosa. Combined DNA methylation and gene expression analysis demonstrated that DNA methylation plays an important role in regulating allele-specific gene expression. To further investigate the relationship between these 6-BA-responsive genes and phenotypic variation, we performed SNP analysis of 507 6-BA-responsive DMRs via re-sequencing using a natural population of P. tomentosa and identified 206 SNPs that were significantly associated with growth and wood properties. Association analysis indicated that 53% of loci with allele-specific expression had primarily dominant effects on poplar traits. Our comprehensive analyses of P. tomentosa DNA methylation and the regulation of allele-specific gene expression suggest that DNA methylation is an important regulator of imbalanced expression between allelic loci.
REVIEW | doi:10.20944/preprints202002.0036.v1
Online: 4 February 2020 (04:51:50 CET)
Crohn's disease (CD) results from an aberrant immune response against commensal microbiota in genetically susceptible hosts. However, the nature of immune defects, the microflora involved, and genetic susceptibility remain incompletely defined and controversial. This review seeks to describe the present state of association between CD and renal disease; moreover, we highlight the convergence of CD with amyloidosis that can trigger sustained inflammation, producing the pathological alteration observed in both diseases. The following MESH terms were searched in PubMed, PubMed Central (PMC), and Web of Science: “Crohn´s disease” and “renal disease.” The R RISmed package was used for PubMed and PMC. The abnormal humoral immune response is described along with alterations in immune cell migration mechanisms in CD during inflammation.
Mon, 3 February 2020
ARTICLE | doi:10.20944/preprints202002.0029.v1
Subject: Life Sciences, Molecular Biology Keywords: Myalgic Encephalomyelitis; Chronic Fatigue Syndrome; mitochondria; Complex V; TORC1; Seahorse respirometry; biomarker; diagnosis; ME/CFS
Online: 3 February 2020 (10:36:05 CET)
Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a devastating illness whose biomedical basis is now beginning to be elucidated. We reported previously that, after recovery from frozen storage, lymphocytes (peripheral blood monocytic cells, PBMCs) from ME/CFS patients die faster in culture medium than those from healthy controls. We also found that lymphoblastoid cell lines (lymphoblasts) derived from these PBMCs exhibit multiple abnormalities in mitochondrial respiratory function and signalling activity by the cellular stress-sensing kinase TORC1. These differences were correlated with disease severity, as measured by the Richardson and Lidbury Weighted Standing Test. The clarity of the differences between these cells derived from ME/CFS patient blood and those from healthy controls suggested that they may provide useful biomarkers for ME/CFS. Here we report a preliminary investigation into that possibility using a variety of analytical classification tools, including linear discriminant analysis, logistic regression and Receiver Operating Characteristic (ROC) curve analysis. We found that results from three different tests, lymphocyte death rate, mitochondrial respiratory function and TORC1 activity could each individually serve as biomarker with better than 90% sensitivity but only modest specificity vís a vís healthy controls. However, in combination they provided a cell-based biomarker with sensitivity and specificity approaching 100% in our sample. This level of sensitivity and specificity was almost equalled by a suggested protocol in which the frozen lymphocyte death rate was used as a highly sensitive test to triage positive samples to the more time consuming and expensive tests measuring lymphoblast respiratory function and TORC1 activity. This protocol provides a promising biomarker that could assist in more rapid and accurate diagnosis of ME/CFS.
Tue, 28 January 2020
REVIEW | doi:10.20944/preprints202001.0330.v1
Subject: Life Sciences, Molecular Biology Keywords: fatty acyl desaturase; Δ6 - desaturase; long-chain polyunsaturated fatty acid; LC-PUFA; ω3; ω6; EPA; DHA; AA; essential fatty acid; health; fish; transgene
Online: 28 January 2020 (04:39:09 CET)
Fatty acid desaturase 2 (Fads2) is the key enzyme of long chain polyunsaturated fatty acid (LC-PUFA) biosynthesis. Endogenous production of these biomolecules in vertebrates, if present, is insufficient to meet demand. Hence, LC-PUFA are considered as conditionally-essential. At present however, LC-PUFA are globally-limited nutrients due to anthropogenic factors. Attention of research is given therefore to find ways to maximize endogenous LC-PUFA production especially in production species, whereby deeper knowledge on molecular mechanisms of enzymatic steps involved is being generated. This review first briefly informs about the milestones in the history of LC-PUFA essentiality exploration before it focuses on the main aim – to highlight the fascinating Fads2 potential to play roles fundamental to adaptation to novel environmental conditions. Investigations are summarized, which elucidate the evolutionary history of fish Fads2 providing an explanation for the remarkable plasticity of this enzyme in fish. Further, structural implications of Fads2 substrate specificity are discussed and some relevant studies performed on organisms other than fish are mentioned in cases when such studies have so far not been conducted on fish models. The importance of Fads2 in the context of growing aquaculture demand and dwindling LC-PUFA supply is depicted and a few remedies in the form of genetic engineering to improve endogenous production of these biomolecules are outlined.
Mon, 27 January 2020
REVIEW | doi:10.20944/preprints202001.0325.v1
Subject: Life Sciences, Molecular Biology Keywords: QSAR evolution; Multi-target QSAR; Monte Carlo method; Fuzzy sets
Online: 27 January 2020 (09:29:19 CET)
Ability of quantitative structure – property / activity relationships (QSPRs/QSARs) to serve for epistemological processes in natural sciences is discussed. Some weirdness of QSPR/QSAR state-of-art are listed. There are some contradictions in the research results in this area. Sometimes, these should be classified as paradoxes or weirdness. These points often are ignored. Here these are listed and briefly commented. In addition, hypothesises on the future evolution of the QSPR/QSAR theory and practice are suggested.
Tue, 21 January 2020
ARTICLE | doi:10.20944/preprints202001.0244.v1
Subject: Life Sciences, Molecular Biology Keywords: Stress; epigenetics; senescence; cognition; age-related cognitive decline; Alzheimer’s disease; SAMP8; SAMR1; oxidative stress; inflammation; autophagy
Online: 21 January 2020 (11:44:35 CET)
Cognitive and behavioural disturbances are growing public healthcare issue for the modern society, as stressful lifestyle is becoming more and more common. Besides, several pieces of evidence state that environment is crucial in the development of several diseases as well as compromising healthy aging. Therefore, it is important to study the effects of stress on cognition and its relationship with aging. To address these queries, Chronic Mild Stress (CMS) paradigm was used in the senescence-accelerated mouse prone 8 (SAMP8) and resistant 1 (SAMR1). On one hand, we determined the changes produced in the three main epigenetic marks after 4 weeks of CMS treatment, such as a reduction in histone posttranslational modifications and DNA methylation, and up-regulation or down-regulation of several miRNA involved in different cellular processes in mice. In addition, CMS treatment induced reactive oxygen species (ROS) accumulation and loss of antioxidant defence mechanisms, as well as inflammatory signalling activation through NF-κB pathway and astrogliosis markers, like Gfap. Remarkably, CMS altered mTORC1 signalling in both strains, decreasing autophagy only in SAMR1 mice. We found a decrease in glycogen synthase kinase 3 β (GSK-3β) inactivation, hyperphosphorylation of Tau and an increase in sAPPβ protein levels in mice under CMS. Moreover, reduction in the non-amyloidogenic secretase ADAM10 protein levels was found in SAMR1 CMS group. Consequently, detrimental effects on behaviour and cognitive performance were detected in CMS treated mice, affecting mainly SAMR1 mice, promoting a turning to SAMP8 phenotype. In conclusion, CMS is a feasible intervention to understand the influence of stress on epigenetic mechanisms underlying cognition and accelerating senescence.
Mon, 30 December 2019
Subject: Life Sciences, Molecular Biology Keywords: BE4; CRISPR-Cas9; Tyr; cytosine base editing; mouse model
Online: 30 December 2019 (11:16:30 CET)
Most human genetic disease arises from point mutations. These genetic diseases can theoretically be corrected by gene therapy but clinic practice is still far from mature. Nearly half of the pathogenic single-nucleotide polymorphisms (SNPs) are caused by G:C>A:T or T:A>C:G base changes. The best current methods to repair these changes are by base editing without footprint using recently developed CRISPR-Cas9 technology by David Liu’s lab. These base editing methods have been confirmed with precision and efficiency in cultured mammalian cells, but it is barely confirmed and the efficiency is still very low. Animal models are important in dissecting pathogenic mechanism for human genetic diseases and efficacy testing of base correction in vivo. Cytidine base editor BE4 is a newly developed version of cytidine base editing system that converts cytidine (C) to uridine (U) in cultured mammalian cells but has not been proven in vivo. In this study, we have tested this system in cells to inactivate GFP gene and in mice by introducing single-base substitution that leads to a stop codon in tyrosinase gene. High percentage albino coat-colored mice were obtained from black coat-colored donor zygotes after pronuclei microinjection. Sequencing results showed that expected base changes were obtained with high precision and efficiency (56.25%). There are no off-targeting events identified in predicted off-target sites. Results confirm BE4 system can work in vivo with high precision and efficacy, and has great potentials in clinic to repair human genetic mutations.
Sun, 29 December 2019
ARTICLE | doi:10.20944/preprints201912.0373.v1
Subject: Life Sciences, Molecular Biology Keywords: bacterial flagellar motility; flagellin; Salmonella; FljB; FliC; electron cryomicroscopy; viscosity; infection
Online: 29 December 2019 (07:47:57 CET)
The bacterial flagellum is a motility organelle, consisting of a long helical filament as a propeller and a rotary motor that drives rapid filament rotation to produce thrust. Salmonella enterica serovar Typhimurium has two genes of flagellin, fljB and fliC, for flagellar filament formation and autonomously switches their expression at a frequency of 10-3–10-4 per cell per generation. We report here differences in their structures and motility functions under high viscosity conditions. A Salmonella strain expressing FljB showed a higher motility than the one expressing FliC under high viscousity. To examine the reasons for this motility difference, we carried out structural analyses of the FljB filament by electron cryomicroscopy and found that the structure is nearly identical to that of the FliC filament except for the position and orientation of the outermost domain D3 of flagellin. The density of domain D3 was much lower in FljB than FliC, suggesting that domain D3 of FljB is more flexible and mobile than that of FliC. These differences suggest that domain D3 plays an important role not only in changing antigenicity of the filament but also in optimizing motility function of the filament as a propeller under different conditions.
Fri, 27 December 2019
ARTICLE | doi:10.20944/preprints201912.0358.v1
Subject: Life Sciences, Molecular Biology Keywords: fibromyalgia; Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS); HERV (Human Endogenous Retrovirus); transposable elements; epigenetics; DNA methylation; tsRNAs (transfer RNA small fragments); interferon; non-Hodgkin's lymphoma
Online: 27 December 2019 (03:30:53 CET)
The development of nucleic acid sequencing technology and the unprecedented availability of metadata has evidenced that 45% of human genome constituted by transposable elements (TEs) is not only transcriptionally active but also physiologically needed. Aberrant regulation of TEs, and of human retroviral endogenous sequences (HERVs) in particular, associates with several neurologic and autoimmune diseases, including the Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) frequently comorbid with fibromyalgia (FM). However, no study has yet addressed whether abnormal expression of these sequences correlates with FM. The work presented here shows, for the first time, that in fact HERVs of the H, K and W types are overexpressed in the cells of the immune system of FM patients with or without comorbid ME/CFS. The patients with increased HERV expression (N=14) presented increased levels of interferon (INF-β and INF-γ) but unchanged levels of TNF-α. In support of our proposal that TE activation is a contributor to FM, we find that the tRNA pools are decreased in comparison to matched healthy participants (N=14). The findings reported here could explain the flu-like symptoms FM patients present with in the absence of concomitant infections. Future work towards identifying specific genomic loci differentially affected in FM and ME/CFS is granted.
Sun, 15 December 2019
REVIEW | doi:10.20944/preprints201905.0140.v2
Subject: Life Sciences, Molecular Biology Keywords: protein crystallization; protein sample qualification; JAXA PCG; microgravity
Online: 15 December 2019 (13:43:29 CET)
We summarize how to obtain protein crystals from which better diffraction images can be obtained. In particular, we describe in detail the quality evaluation of the protein sample, the crystallization methods and crystallization conditions, the flash-cooling protection of the crystal, and the crystallization under a microgravity environment.
ARTICLE | doi:10.20944/preprints201912.0189.v1
Subject: Life Sciences, Molecular Biology Keywords: nanoparticles; silver; L-arginine; conjugation; muscle growth
Online: 15 December 2019 (13:37:00 CET)
The aim of the study was to analyze the in ovo injection of chemically and biologically synthesized silver nano-particles (Ag NPs) using Brassica oleracea L. var capitate f. Rubra, (BOL) conjugation with L-Arginine (L-Arg) on the immune, muscle growth, survivability and hatchability of the broiler chickens. L-Arg (100 μg) with 1000 µg (BOL-Ag NPs) and L-Arg (100 μg) with 100 µg (C-Ag NPs) were injected into fertile eggs at 8, 14 and 18 of incubation. Survival and hatched chicks were calculated. Survivability and hatchability were unaffected by the injected dose of L-Arg (100 μg) with 1000 µg (BOL-Ag NPs) and L-Arg (100 μg) with 100 µg (C-Ag NPs) but it significantly improved when the eggs were injected on day 14 of incubation compared with those injected on days 8 or 18. Moreover, the protein expression of muscle development markers such as myogenin and myoD were significantly up-related in 14 d incubation whereas the heat shock proteins (HSPs) such as HSP-60 and HSP-70 were significantly up-regulated in 18 d incubation. In addition, the in ovo injection on 18 d significantly increased the serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) as well the immunoglobulin (IgM) levels were increased in 14-day incubation period in serum at the same concentration. Collectively, these results indicate that injecting L-Arg (100 μg) with 1000 µg of BOL-Ag NPs or L-Arg (100 μg) with 100 µg of C-Ag NPs to eggs at day 14 after incubation could improve their hatchability, survival rate, and muscle growth marker.
Thu, 5 December 2019
REVIEW | doi:10.20944/preprints201912.0066.v1
Subject: Life Sciences, Molecular Biology Keywords: gdnf; microglia; astrocyte; neuroinflammation; ret; gfr α1; gene therapy; Parkinson’s disease
Online: 5 December 2019 (11:10:05 CET)
In healthy adult brain, glial cell line-derived neurotrophic factor (GDNF) is exclusively expressed by neurons and in some instances, it has furthermore been shown to derive from a single neuronal subpopulation. Secreted GDNF acts in a paracrine fashion by forming a complex with GDNF family receptor α1 (GFRα1) which is mainly expressed by neurons and can act in cis as a membrane-bound or in trans as a soluble factor. The GDNF/GFRα1 complex signals through interaction with RET (“rearranged during transfection”) or with a lower affinity with neural cell adhesion molecule (NCAM). GDNF can also signal independently from GFRα1 via interaction with syndecan-3. RET being expressed by neurons involved in several pathways: nigro-striatal dopaminergic neurons, motor neurons, enteric neurons, sensory neurons, etc. could be the main determinant of the specificity of GDNF pro-survival effect. In injured brain, de novo expression of GDNF occurs in glial cells. Neuroinflammation has been reported to induce GDNF expression in activated astrocytes and microglia, infiltrating macrophages, nestin-positive neural stem cells and neuron/glia (NG2) progenitors. This disease-related GDNF overexpression can be either beneficial or detrimental depending on the localization in the brain and the level and duration of glial cells activation. Some reports also describe upregulation of RET and GFRα1 in glial cells, suggesting that GDNF could modulate neuroinflammation.
Tue, 3 December 2019
REVIEW | doi:10.20944/preprints201912.0021.v1
Subject: Life Sciences, Molecular Biology Keywords: protein 0th-order structure; origin of gene; origin of protein; origin of genetic code; GNC primeval genetic code hypothesis; SNS primitive genetic code hypothesis
Online: 3 December 2019 (11:10:12 CET)
Understanding the mechanism, how entirely new (EntNew) gene/protein or the first ancestral gene/protein of a family was created, should be one of the most important issues in the biological sciences. However, the mechanism is totally unknown still now. On the other hand, it is well known that mature protein is generally rigid and one catalytic center exists on the protein. Creation of such a mature EntNew gene/protein should be, of course, carried out through random process, because it cannot be designed in advance. However, the EntNew gene/protein never be created by random polymerization of the respective monomeric units, because of the extraordinary large sequence diversities of ~10180 and ~10130, respectively. Protein 0th-order structure or a specific amino acid composition, in which immature but water-soluble protein can be produced even through random process, holds the key for solving the difficult problem. As it was fragmentally described in the previous papers how and where EntNew gene/protein was created, I describe in detail in this review three processes generating EntNew gene/protein with some flexibility under three genetic codes, the universal genetic code, SNS primitive code and GNC primeval code, and discuss why the mature gene/protein could be created through the processes.
Sat, 30 November 2019
ARTICLE | doi:10.20944/preprints201911.0399.v1
Subject: Life Sciences, Molecular Biology Keywords: olivo-ponto-cerebellar atrophy (OPCA); Purkinje cells; Calcium/calmodulin dependent protein kinase II (CAMK2); Plastin (Fimbrin); Hirano bodies; Huntington’s disease
Online: 30 November 2019 (14:47:30 CET)
Ataxin-2 (human gene symbol ATXN2, protein ATXN2) is the disease protein of Spinocerebellar Ataxia type 2 (SCA2). The large expansions of a polyglutamine (polyQ) stretch above a threshold of ~33 glutamines cause the multi-system nervous atrophy SCA2, while intermediate expansions of 29-32 glutamines contribute to the risk of the motor neuron diseases Amyotrophic Lateral Sclerosis (ALS) and Fronto-Temporal Lobar Dementia (FTLD). To elucidate the cellular function of ATXN2, we further characterized its direct interaction with alpha-Actinin-1 (symbol ACTN1), which had been observed in high-throughput yeast-two-hybrid surveys. An endogenous complex of ATXN2 and ACTN1 proteins was detected by co-immunoprecipitation. In vitro GST-tag pull-down experiments showed that the Calponin-Homology-domain at the N-terminus of ACTN1 binds to the N-terminus of ATXN2. Although an impact of the polyQ expansion on the interaction was not evident in pull-down experiments, a recent characterization of aged Atxn2-CAG100-KnockIn mice provides evidence. Both proteins associated in the cytosol and at the plasma membrane, as determined by sedimentation experiments in mouse brain, and by immunofluorescence microscopy of a transfected monkey cell line and of rat primary hippocampal neurons. In view of the roles of ACTN1 for spine plasticity and postsynaptic receptor control via reassembly of cortical actin, our data help to explain the impaired dendrite maintenance in SCA2.
Sun, 24 November 2019
Subject: Life Sciences, Molecular Biology Keywords: Kandelia obovata; mangrove; hydrogen sulfide; salt tolerance; comparative proteome
Online: 24 November 2019 (14:25:34 CET)
As a dominant mangrove species, Kandelia obovata is distributed in an intertidal marsh with an active H2S release. Whether H2S participates in the salt tolerance of mangrove plant is still ambiguous although increasing evidence have demonstrated that H2S functions in plant responses to multiple abiotic stresses. In this study, as an H2S donor, NaHS was used to investigate the regulatory mechanism of H2S on salt tolerance of K. obovata seedlings using a combined physiological and proteomic analysis. The results showed that the reduction in photosynthesis (Pn) caused by 400 mM NaCl was recovered by the addition of NaHS (200 μM). Furthermore, the application of H2S enhanced the quantum efficiency of PSII and the membrane lipid stability, implying that H2S is beneficial to the survival of K. obovata seedlings under high salinity. We further identified 37 differentially expressed proteins by proteomic approaches under salinity and NaHS treatment. Among them, the proteins related to photosynthesis, primary metabolism, stress response and hormone biosynthesis were primarily enriched. The physiological and proteomic results highlighted that exogenous H2S up-regulated photosynthesis and energy metabolism to help K. obovata to cope with high salinity. Specifically, H2S increased photosynthetic electron transfer, chlorophyll biosynthesis and carbon fixation in K. obovata leaves under salt stress. Furthermore, the abundances of other proteins related to metabolic pathway, such as antioxidation (APX, CSD2, PDX1), protein synthesis (HSP, Cpn 20), nitrogen metabolism (GS2, GS1:1), glycolysis (PGK, TPI), AsA-GSH cycle were increased by H2S under high salinity. These findings provide new insights into the roles of H2S in the adaptations of mangrove plant K. obovata to high salinity environment.
Fri, 15 November 2019
ARTICLE | doi:10.20944/preprints201911.0177.v1
Subject: Life Sciences, Molecular Biology Keywords: macrophage migration inhibitory factor; HNSCC cell lines; proliferation; cell cycle; apoptosis
Online: 15 November 2019 (08:48:50 CET)
Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine that contributes to the progression of several cancers. MIF overexpression has been reported in head and neck squamous cell carcinoma (HNSCC) patients. However, the exact role of MIF in HNSCC is not fully understood. Our aim was to evaluate the amount of secreted MIF and the role of MIF in the proliferation, cell cycle, and apoptosis in HNSCC cell lines. The MIF levels in conditioned media from human primary (HN18 and HN30) and metastatic (HN17 and HN31) HNSCC cell lines were evaluated using ELISA. The HNSCC cell lines were treated with recombinant MIF and its effect on proliferation, cell cycle, and apoptotic status was determined by MTT and flow cytometry, respectively. The HNSCC-secreted MIF concentration ranged from 49.33‒860 pg/ml. Exogenous MIF (25 ng/ml) significantly increased HN18, HN30, and HN31 cell proliferation. Moreover, MIF induced cell cycle progression and inhibited apoptosis in these cells. However, MIF did not affect growth or apoptosis in HN17 cell. In conclusion, the HNSCC cell lines were evaluated secrete MIF. Exogenous MIF promotes various effects on proliferation, cell cycle, and apoptosis in HNSCC cells.
Thu, 14 November 2019
Subject: Life Sciences, Molecular Biology Keywords: DNA lesions; 7,8-dihydro-8-oxoguanine (8-oxoG); mutagenic activity; method of detection; cell nuclear extracts; mice organs and embryos
Online: 14 November 2019 (09:00:19 CET)
We propose an improved earlier described “mirror” method  for detecting in cell nuclear extracts mutations that arise in DNA during its replication due to misincorporation of deoxyadenosine-5’-monophosphate (dAMP) opposite 7,8-dihydro-8-oxoguanine (8-oxoG). The method is based on the synthesis of a complementary chain (“mirror”) by nuclear extracts of different mice organs on a template containing 8-oxoG inside and dideoxycytidine residue (ddC) at the 3’-end. The “mirror”was amplified by PCR using primers part of which was non-complementary to the template. It allowed obtaining the “framed mirror” products. The misincorporation of dAMP in “framed mirror” products forms an EcoRI restriction site. The restriction analysis of double-stranded “framed mirror” products allows a quantification of the mutation frequency in nuclear extracts. The data obtained showed that the mutagenic potential of 8-oxoG markedly varied in different organs of adult mice and embryos.
Wed, 6 November 2019
ARTICLE | doi:10.20944/preprints201911.0061.v1
Subject: Life Sciences, Molecular Biology Keywords: rett syndrome; intrinsically disordered region; phylogenetic profile analysis; post-transcriptional modification; methyl-cpg-binding protein 2; cyclin-dependent kinase-like 5; forkhead box protein g1
Online: 6 November 2019 (10:58:54 CET)
Rett syndrome (RTT), a neurodevelopmental disorder, is mainly caused by mutations in methyl CpG-binding protein 2 (MECP2), which alter the functions of domains to either bind to methylated DNA or interact with a transcriptional co-repressor complex. It has been established that alterations in cyclin-dependent kinase-like 5 (CDKL5) or forkhead box protein G1 (FOXG1) correspond to distinct neurodevelopmental disorders, given that a series of studies have indicated that RTT is also caused by alterations in either one of these genes. We tried to elucidate RTT through evolution and structure assessment of MeCP2, CDKL5, and FOXG1, by focusing on their binding partners and disordered structures. Here, we provide insight into the similarities of the FOXG1 and MECP2 binding partners evolution and function. On the other hand, we suggest that CDKL5 could be a potential candidate for a classical RTT treatment, particularly based on its disordered structure that spans after the catalytic domain to the C-terminus, which shows abundant linear motifs that can bind to molecules with divergent structures of similar affinity. Additionally, we provide insight into the relationship between disordered structure and disease.
Mon, 4 November 2019
REVIEW | doi:10.20944/preprints201911.0031.v1
Subject: Life Sciences, Molecular Biology Keywords: lncRNA; translation; transcription; splicing; brain; cerebral cortex; neurogenesis; synaptic plasticity; neurons
Online: 4 November 2019 (03:06:01 CET)
Mammalian genomes encode tens of thousands of long-noncoding RNAs (lncRNAs), which are capable of interactions with DNA, RNA and protein molecules, thereby enabling a variety of transcriptional and post-transcriptional regulatory activities. Strikingly, about 40% of lncRNAs are expressed specifically in the brain in precisely regulated temporal and spatial expression patterns. In stark contrast to the highly conserved repertoire of protein-coding genes, thousands of new lncRNAs have appeared during primate nervous system evolution with hundreds of human-specific lncRNAs. Their evolvable nature and the myriad of potential functions make lncRNAs ideal candidates for drivers of human brain evolution. The human brain displays the largest relative volume of any animal species and the most remarkable cognitive abilities. In addition to brain size, structural reorganization and adaptive changes represent crucial hallmarks of human brain evolution. LncRNAs are increasingly reported to be involved in neurodevelopmental processes including proliferation, neurite outgrowth and synaptogenesis, as well as in neuroplasticity, suggested to underlie human brain evolution. Hence, evolutionary human brain adaptations are proposed to be essentially driven by lncRNAs, which will be discussed in this review.
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