Background: Inflammation is an important mechanism in atherosclerosis and plaque formation. C-reactive protein (CRP) is a common inflammatory biomarker associated with the risk of coro-nary heart disease. We investigated the relationship of CRP with findings from myocardial per-fusion imaging (MPI). Methods: In this retrospective study, 102 consecutive patients (mean age 71 years, 68% males) that underwent MPI (for diagnostic reasons or quantification of myocardial ischemia) and CRP determination (upper limit: 6mg/L) within 1 month from MPI, were included. Patients had no infection or recent acute coronary syndrome. Results: the median CRP level was 4mg/L (2,10) among the study population. Patients with raised CRP had higher summed stress score (SSS) (P = 0.006) and summed rest score (SRS) (P = 0.001) and higher risk for SSS > 3 (OR 9.25, 95% CI 2.03-42.13, P = 0.001) compared to those with low CRP. The association of SSS and SRS with CRP levels was more evident in patients over 70 years (P = 0.027 and P = 0.005 respectively). No significant difference in summed difference score was shown. The two groups had no difference in other risk factors (p > 0.05 for all comparisons). Conclusion: A high level of CRP was associated with the presence and extent of stress-induced myocardial ischemia in MPI.

Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides to modulate multiple signaling events in cells. PDEs are recognized to actively associate with cyclic nucleotide receptors (Protein Kinases, PK) in larger macromolecular assemblies referred to as signalosomes. Complexation of PDEs with PK generates an expanded active site which enhances PDE activity. This facilitates signalosome-associated PDEs to preferentially catalyze active hydrolysis of cyclic nucleotides bound to PK, and aid in signal termination. PDEs are important drug targets and current strategies for inhibitor discovery are based entirely on targeting conserved PDE catalytic domains. This often results in inhibitors with cross-reactivity amongst closely related PDEs and attendant unwanted side effects. Here, our approach targets PDE-PK complexes as they would occur in signalosomes, thereby offering greater specificity. Our developed fluorescence polarization assay has been adapted to identify inhibitors that block cyclic nucleotide pockets in PDE-PK complexes in one mode, and disrupt protein-protein interactions between PDEs and cyclic nucleotide activating protein kinases in a second mode. We tested this approach with three different systems: cAMP-specific PDE8-PKAR, cGMP-specific PDE5-PKG and dual-specificity RegA-RD complexes and ranked inhibitors according to their inhibition potency. Targeting PDE-PK complexes offers biochemical tools for describing the exquisite specificity of cyclic nucleotide signaling networks in cells.

Herpesviruses usurp cellular stress responses to avoid immune detection while simultaneously promoting viral replication and spread. The unfolded protein response (UPR) is an evolutionarily conserved stress response that is activated when the protein load in the ER saturates its chaperone folding capacity causing an accrual of misfolded proteins. Through translational and transcriptional reprogramming, the UPR aims to restore protein homeostasis; however, if this fails the cell undergoes apoptosis. It is commonly thought that many enveloped viruses, including herpesviruses, may activate the UPR due to saturation of the ER with nascent glycoproteins and thus these viruses may have evolved mechanisms to evade the potentially negative effects of UPR signaling. Over the past fifteen years there has been considerable effort to provide evidence that different viruses may reprogram the UPR to promote viral replication. Here we provide an overview of the molecular events of UPR activation, signaling and transcriptional outputs, and highlight key findings that demonstrate that the UPR is an important cellular stress response that herpesviruses have hijacked to facilitate persistent infection.

Metal-catalyzed lipid oxidation is a major factor in food waste, as it reduces shelf life. Addressing this issue, our study investigates the potential of hydrolysates derived from potato protein, a by-product of potato starch production, as metal chelating antioxidants. Through sequential enzymatic hydrolysis using Alcalase or Trypsin combined with Flavourzyme, we produced various hydrolysates, which were then fractionated via ultrafiltration. Using a combination of peptidomics and bioinformatics, we predicted the presence of metal-chelating and free radical scavenging peptides across all hydrolysate fractions, with a trend indicating a higher content of antioxidant peptides in lower molecular weight fractions. To validate these predictions, we utilized Surface Plasmon Resonance (SPR) and a 9-day emulsion storage experiment. While SPR demonstrated potential in identifying antioxidant activity, it faced challenges in differentiating between hydrolysate fractions due to significant standard errors. In the storage experiment, all hydrolysates showed lipid oxidation inhibition, though not as effectively as EDTA. Remarkably, one fraction (AF13) was not significantly different (p < 0.05) from EDTA in suppressing hexanal formation. These results highlight SPR and peptidomics/bioinformatics as promising yet limited methods for antioxidant screening. Importantly, this study reveals the potential of potato protein hydrolysates as antioxidants in food products, warranting further research.

In this study, the potential of whey protein hydrolysate (WPH) containing LDIQK peptide and treadmill exercise to prevent cognitive decline was investigated along with the neuroprotective mechanisms. Cognitive dysfunction was induced in mice by 1 mg/kg scopolamine, followed by administration of WPH at 100 and 200 mg/kg and/or treadmill exercise at 15 m/min for 30 min five days per week. Both WPH administration and treadmill exercise significantly improved memory in mice with scopolamine-induced cognitive impairment, which was attributed to several key mechanisms, including a reduction in oxidative stress based on decreased levels of reactive oxygen species and malondialdehyde in the brain tissue and an increase in acetylcholine by increasing choline acyltransferase and decreasing acetylcholine esterase levels. Exercise and WPH also exerted neuroprotective effects by inhibiting the hyperphosphorylation of tau protein, enhancing the expression of brain-derived neurotrophic factor, and inhibiting apoptosis by reducing the Bax/Bcl2 ratio in conjunction with downregulation of the mitogen-activated protein kinase pathway. Moreover, the impact of WPH and treadmill exercise extended to the gut microbiome, suggesting a potential link with cognitive improvement. These findings suggest that both WPH intake and treadmill exercise are effective strategies for mitigating cognitive impairment, providing promising avenues for treating neurodegenerative diseases.

Abstract: Over the years, several distinct pathogenic coronaviruses have emerged, including the pandemic SARS-CoV-2 which is difficult to curtail despite the availability of licensed vaccines. The difficulty in managing SARS-CoV-2 is linked to changes in the variants’ proteins, especially in the spike protein (S) used for viral entry. These mutations, especially in the S, enable the virus to evade the immune responses induced by natural infection or vaccination. However, some parts of the SP in the S1 subunit and the S2 subunit are considered conserved among coronaviruses. In this review, we will discuss the epitopes in the SARS-CoV-2 S1 and S2 subunit proteins that have been demonstrated by various studies to be conserved among coronaviruses and may be immunogenic for the development of vaccine. Considering the higher conservancy of the S2, we will further discuss the likely challenges that could limit the S2 subunit from inducing robust immune responses and the promising approaches to increase their immunogenicity.

The intricate mechanisms of the immune response elicited by vaccines and the interplay between the immune system and the central nervous system (CNS) are poorly understood. This review explores these mechanisms, emphasizing the interplay between the immune system and the central nervous system (CNS). A critical aspect is the examination of how vaccine-induced immune responses can interact with the CNS, influencing neuroinflammation and neuroimmune interactions. Furthermore, the link between vaccination and persistent symptoms observed in long-COVID patients is discussed. Vaccination initiates a complex cascade of events, starting with the production of specific proteins, such as S1/S2 spike proteins, leading to robust immune activation in the axillary lymph nodes. The role of macrophages and antigen-presenting cells (APCs) in cytokine production, antigen presentation, and the stimulation of B and T cells highlights the sophistication of the adaptive immune response. The migration of immune cell-derived exosomes to the brain plays a dynamic role in CNS inflammation, neurodegenerative processes and long COVID. On the other hand, the prolonged presence of viral and vaccine-derived spike proteins may contribute to the complex pathophysiology of Long-COVID, shedding light on the delicate balance between beneficial immune responses and possible adverse neurological outcomes of vaccination. It is worth rigorous monitoring and further research to understand the mechanisms of neuroinflammation and the persistence of spike proteins and their long-term effects on the brain, emphasising the importance of a nuanced approach to vaccine safety and efficacy in the context of COVID-19 and beyond.Principio del formulario

The JC polyomavirus virus (JCPyV) affects more than 80% of the human population in their early life stage. It mainly affects immunocompromised individuals where virus replication in oligodendrocytes and astrocytes may lead to fatal progressive multifocal encephalopathy (PML). Virus protein 1 (VP1) is one of the major structural proteins of the viral capsid, responsible for keeping the virus alive in the gastrointestinal and urinary tracts. VP1 is often targeted for antiviral drug and vaccine development. Similarly, this study implied immunoinformatics and molecular dynamics simulation-based approaches to design a multi-epitope subunit vaccine targeting JCPyV. The VP1 protein epitopic sequences, which are highly conserved, were used to build the vaccine. This designed vaccine includes two adjuvants, five HTL epitopes, six CTL epitopes, and two linear BCL epitopes to stimulate cellular, humoral, and innate immune responses against JCPyV. Furthermore, molecular docking and molecular dynamics simulation (100ns) studies were used to examine the interaction and stability of the vaccine protein with TLR4. Trajectory analysis showed that the vaccine and TLR4 receptor form a stable complex with the vaccine protein. Overall, this study contributed to the path of vaccine development against JCPyV and may prevent PML from happening. However, this study needs experimental validation to conclude our findings.

qNMR is a valuable technique for metrological studies due to the uniformity of its signal response for all chemical species of an isotope of interest, which enables compound-independent calibration. However, protein quantification remained challenging as large molecules produce wide, low-intensity signals that reduce the already low sensitivity. Combining qNMR with the hydrolysis of protein samples into amino acids circumvents many of these issues and facilitates the use of NMR spectroscopy for absolute protein and peptide quantification.In this work, different conditions have been tested for quantifying aromatic amino acids and proteins. First, we examined the pH-based signal shifts in the aromatic region. The preferable pH depends on the selection of the amino acids for quantification and which internal standard substance should be used to avoid peak overlap. Several aromatic compounds, such as terephthalic acid, sulfoisophthalic acid, and benzene tricarboxylic acid, have been applied as internal standards. The quantification of amino acids from an amino acid standard, as well as from a certified reference material (bovine serum albumin), was performed. Using the first two suggested internal standards, recovery was ~ 97 % for histidine, phenylalanine, and tyrosine at a concentration of approximately 1 mM in solution. Acidic hydrolysis of a certified reference material (CRM) of bovine serum albumin (BSA) and subsequent quantification of Phe and Tyr yielded recoveries of 98 ± 2 and 88 ± 4 %, respectively, at a protein concentration of 16 g/L or 250 µM.

The RNA-binding protein Human antigen R (HuR) regulates stability, translation, and nucleus-to-cytoplasm shuttling of its target mRNAs. The protein has been progressively recognized as a relevant therapeutic target for several pathologies like cancer, neurodegeneration, as well as inflammation. Inhibitors of mRNA binding to HuR might thus be beneficial against a variety of diseases. Here we present the rational identification of structurally novel HuR inhibitors. In particular, by combining chemoinformatics approaches, high throughput virtual screening and RNA–protein pull-down assays, we show that the 4-(2-(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)hydrazineyl)benzoate ligand exhibits dose-dependent HuR inhibition in binding experiments. Importantly, the chemical scaffold is new with respect to the so-far known HuR inhibitors, opening up a new avenue for the design of pharmaceutical agents targeting this important protein.

Background. Increasing evidence demonstrate that concentration of protein in infant formula >1.9g/100Kcal with high levels of insulinogenic aminoacids is associated with accelerated weight gain, increased fat mass accumulation and risk of adiposity. Purpose of this study was to conduct a systematic review to determine the metabolic effects in infants feed with infant formula optimized in protein. Methods. Systematic review was conducted according PRISMA Statement. RCTs with one intervention group (infant formula with 1.6-1.9gr of protein/100Kcal) and at least one comparative control group (infant formula with >1.9gr of protein/100Kcal) were included. Standardized mean differences (SMD), through random model were calculated. Results. 15 RCT were included. Optimized protein in infant formula was associated with less gain of BMI at 24 months of follow-up (SMD -0.25, IC95% -0.36 to -0.13, p 0.01) and less fat mass accumulation (SMD -0.68, IC95% -0.98 to -0.37, p 0.01). Optimized protein was also associated to less gain of weight, weight/age Z-score, weight/length Z-score, BUN (mmol/dL) and IGF1 (ng/ml). No effect on length/age Z-score was observed. Conclusions. Robust evidence showed optimized protein (1.6gr/100Kcal to 1.9gr/100Kcal) in infant formula is associated with metabolic benefits in infants with less weight gain, BMI and fat mass accumulation.

Yeast two-hybrid approaches, which are based on fusion proteins that must co-localise to the nucleus to reconstitute the transcriptional activity of GAL4 have greatly contributed to understand the nitrogen interaction network of cyanobacteria, whose main hubs are the trimeric PII and the monomeric PipX regulators. The bacterial two-hybrid system, based on the reconstitution in the E. coli cytoplasm of the adenylate cyclase of Bordetella pertussis, should provide a relatively faster and presumably more physiological assay for cyanobacterial proteins than the yeast system. Here we use the bacterial two-hybrid system to gain additional insights into the cyanobacterial PipX interaction network while simultaneously assessing the advantages and limitations of the two most popular two-hybrid systems. Comprehensive mutational analysis of PipX and bacterial two-hybrid assays were performed to compare outcomes between yeast and bacterial systems. We detected interactions previously recorded in the yeast two-hybrid system as negative as well as a “false positive”, the self-interaction of PipX, which is rather an indirect interaction dependent on PII homologs from the E. coli host, a result confirmed by Western analysis with relevant PipX variants. This is, to our knowledge, the first report of the molecular basis of a false positive in the bacterial two-hybrid system.

Microglia are the resident immune cells of the central nervous system. Upon stimulus presentation microglia polarize from a resting to an activated state. Microglial translocator protein 18 kDa (TSPO) is considered as a marker of inflammation. Here, we characterized the role of TSPO by investigating the impact of TSPO deficiency on human microglia. We used TSPO knockout (TSPO-/-) variants of the human C20 microglia cell line. We found a significant reduction in the TSPO-associated protein VDAC1 in TSPO-/- compared to control cells. Moreover, we assessed the impact of TSPO deficiency on calcium levels and the mitochondrial membrane potential. Cytosolic and mitochondrial calcium concentrations were increased in TSPO-/- cell lines, whereas the mitochondrial membrane potential tended to be lower. Assessment of the mitochondrial DNA copy number via RT-PCR revealed a decreased amount of mtDNA in the TSPO-/- cells when compared to controls. Moreover, metabolic profiles of C20 cells were strongly dependent on the glycolytic pathway. However, TSPO depletion did not affect the cellular metabolic profile. Measurement of the mRNA expression levels of the pro-inflammatory mediators revealed an attenuated response to proinflammatory stimuli in TSPO-depleted cells, implying a role of the TSPO protein in the process of microglial polarization.

Branched-chain amino acid (BCAA) levels are associated with skeletal muscle cross-sectional area (CSA). Serum BCAA levels are enhanced by whey protein supplementation (WPS), and evidence in clinical populations suggests an association of single nucleotide polymorphisms (SNPs) with BCAA metabolite levels. It is not known whether the same SNPs are associated with the ability to catabolise BCAAs from exogenous sources, such as WPS. The present study investigated whether possessing a higher number of alleles associated with increased BCAA metabolites correlates with muscle fiber CSA of m. vastus lateralis in physically active participants, and whether any relationship is enhanced by WPS. Endurance-trained participants (n=75) were grouped by self-reported habitual WPS consumption and genotyped for 5 SNPs (PPM1K rss1440580, APOA5 rs2072560, CBLN1 rs1420601, DDX19B rs12325419, TRMT61A rs58101275). Body mass, BMI and fat percentage were significantly lower and muscle mass higher in the WPS group (n=22) compared to Non-WPS (n=53). The number of BCAA-increasing alleles was correlated with fiber CSA in the WPS group (r=0.75, p&lt;0.0001) and was stronger for fast-twitch fibers (p=0.001) than slow-twitch fibers (p=0.048). Similar results remained when corrected for multiple covariates (age, physical activity, meat and dairy intake). No correlation was found in the Non-WPS group. This study presents novel evidence of a positive relationship between BCAA-increasing alleles and muscle fiber CSA in athletes habitually consuming WPS. We suggest that a high number of BCAA-increasing alleles improves the efficiency of WPS by stimulation of muscle protein synthesis, and contributes to greater fiber CSA.

Soy protein isolate (SPI) based materials are abundant, biocompatible, renewable, and biodegradable. In order to improve the tensile strength (TS) of SPI films, we prepared a novel composite film modified with microcrystalline cellulose (MCC) and metal nanoclusters (NCs) in this research. The effects of the modification of MCC on the properties of SPI-Cu NCs and Zn NCs films were investigated. Attenuated total reflectance-Fourier transformed infrared spectroscopy analyses and X-ray diffraction patterns characterized the strong interactions and reduction of the crystalline structure of the composite films. Scanning electron microscope showed the enhanced cross-linked and entangled structure of modified films. Compared with untreated SPI film, the tensile strength of the SPI-MCC-Cu and SPI-MCC-Zn films increased from 2.91 MPa to 13.95 and 6.52 MPa, respectively. Moreover, the results also indicated their favorable water resistance with higher water contact angle. Meanwhile, the composite films exhibited increased initial degradation temperatures, demonstrating their higher thermostability. The results suggested that MCC could effectively improve the performance of SPI-NCs films, which would provide a novel preparation method for environmentally friendly SPI-based films in the applications of packaging materials.

In Southeast Asia, the incidence of Leukemia, a malignant blood cancer originating from hema-topoietic progenitor cells, is on the rise, marked by a concerning 54% mortality rate. This study focuses on enhancing early-stage prediction to improve patient recovery prospects significantly. Leveraging Machine Learning and Data Science, we employ protein sequential data from frequently mutated genes such as BCL2, HSP90, PARP, and RB to predict Chronic Myeloid Leukemia (CML). Our approach relies on robust feature extraction techniques, namely Di-peptide Composition (DPC), Amino Acid Composition (AAC), and Pseudo amino acid composition (Pse-AAC), with prior attention to addressing outliers and validating feature selection through the Pearson Corre-lation Coefficient. Data augmentation ensures a well-rounded dataset for analysis. Employing a range of Machine Learning models, including Support Vector Machine (SVM), XGBoost, Random Forest (RF), K Nearest Neighbor (KNN), Decision Tree (DT), and Logistic Regression (LR), we achieve accuracy rates spanning from 66% to 94%. These classifiers undergo comprehensive as-sessment using performance metrics such as accuracy, sensitivity, specificity, F1-score, and the confusion matrix. Our proposed solution, encompassing a user-friendly web application dashboard, presents an invaluable tool for early CML diagnosis with profound implications for practitioners, offering a deploy-able asset within healthcare institutions and hospitals.

It has not been reasonably explained still now, how the “chicken and egg relationship” between gene and protein was formed, although RNA world hypothesis was proposed to aim at explaining formation process of the relationship about 40 years ago. On the other hand, GADV hypothesis advocates that life emerged after five members of the fundamental life system; cell structure, metabolism, tRNA, genetic code and gene, were piled up one by one onto immature [GADV]-proteins, which were produced by random joining of [GADV]-amino acids on the primitive Earth. Furthermore, formation process of the first (GNC)n RNA gene encoding a mature [GADV]-protein can be also reasonably explained according to the hypothesis as follows. Double-stranded (ds)-(GNC)n RNA could be produced by complementary strand synthesis of a single-stranded (ss)-(GNC)n RNA, which was formed by random joining of GNC anticodons carried by four types of AntiC-SL tRNAs. Successively, the first (GNC)n RNA gene was generated upon maturation of an immature [GADV]-protein, which was produced through expression of codon sequence on one strand of ds-(GNC)n RNA, as accumulating necessary base substitutions and raising a weak catalytic activity of the immature protein to a more active state. It is considered that, consequently, a gene encoding a mature protein, which has the “chicken and egg relationship”, was generated for the first time. Therefore, it can be concluded that the “chicken and egg relationship” was built not by formation of either one, gene or protein, but by maturation of an immature gene, which was lead by improvement of catalytic activity of an immature protein. That is, it can be concluded that the reason, why it was unable to give an answer to the question on the formation process of the “chicken-egg relationship” thus far, is because it was an unanswered question.

Alzheimer’s disease (AD) is the most common cause of dementia worldwide. Pathological deposits of neurotoxin proteins within the brain, such as amyloid-Beta and hyperphosphorylated tau tangles, are prominent features in AD. The prion protein (PrP) is involved in neurodegeneration via its conversion from the normal cellular form PrPc, to the infection form PrP Sc. Some studies indicated that posttranslationally modified PrPc isoforms plays a fundamental role in AD pathological progression. Several studies have shown that interaction of Aβ oligomers with N-terminal residues of the PrPc protein region appears critical for neuronal toxicity. The PrPc-Aβ binding always occur in AD brains and is never detected in nondemented controls and the binding of Aβ aggregates to PrPc is restricted to the N-terminus of PrPc.

Protein is the proteios building block of life. Evolutionarily, its sequence is not as conserved as its structure, making it more reasonable for protein structure, instead of protein sequence, to be the descriptor of protein function. Yet, in the National Center for Biotechnology Information (NCBI) database, the number of experimentally identified protein sequences is in great excess of that of experimentally determined protein structures inside the almost-half-a-century old Protein Data Bank (PDB). For instance, GPR151 is an proton-sensing G-protein coupled receptor (GPCR) originally identified as homologous to galanin receptors. As of March 19, 2020, GPR151’s structure has not been experimentally determined and deposited in PDB yet. Thus, an ab initio modelling approach was employed here to build a three-dimensional structure of GPR151. Overall, the ab initio GPR151 model presented herein constitutes the first structural hypothesis of GPR151 to be experimentally tested in future with previously published, currently ongoing and future GPR151 studies.

ShK domain-containing proteins are peptides found in different parasitic and venomous organisms. From a previous transcriptomic dataset from Bursaphelenchus xylophilus, a plant-endoparasitic nematode that infects forest tree species, we identified 96 transcripts potentially as ShK domain-containing proteins with unknown function in the nematode genome. This study aimed to characterize and explore the functional role of genes encoding ShK domain-containing proteins in B. xylophilus biology. We selected and functionally analyzed nine candidate genes specific from B. xylophilus. In situ hybridization revealed expression of one B. xylophilus ShK in the pharyngeal gland cells, suggesting their delivery into host cells. Most of the transcripts are highly expressed during infection and showed a significant upregulation in response to peroxide products compared to the nematode catalase enzymes. We reported for the first time, the potential involvement of ShK domain genes in oxidative stress, suggesting that these proteins may have an important role protecting or modulating the reactive oxygen species (ROS) activity of the host plant during parasitism.

Most neurodegenerative diseases have the characteristics of proteinopathies, i.e. they cause lesions to appear in vulnerable regions of the nervous system, corresponding to protein aggregates that progressively spread through the neuronal network as the symptoms progress. Alzheimer's disease is one of these proteinopathies. It is characterized by two lesions, neurofibrillary tangles (NFTs) and senile plaques, formed essentially of amyloid peptides (Aβ). A combination of factors ranging from genetic mutations to age-related changes in the cellular context converge in this disease to accelerate Aβ deposition. Over the last two decades, numerous studies have attempted to elucidate how structural determinants of its precursor (APP) modify Aβ production, and to understand the processes leading to the formation of different Aβ aggregates; e.g. fibrils and oligomers. The synthesis proposed in this review indicates that the same motifs can control APP function and Aβ production essentially by regulating membrane dimerization, and subsequently Aβ aggregation processes. The distinct properties of these motifs and the cellular context regulate the APP conformation to trigger the transition to the amyloid pathology. This concept can be transposed to the study of other proteinopathies, providing a framework for improving our understanding of these mechanisms that devastate neuronal functions.

The function of the endoplasmic reticulum (ER) can be impaired by the alternation of the extra- and intracellular environment such as disruption of calcium homeostasis, expression of mutated proteins and oxidative stress. In response to disruptions to ER homeostasis, eukaryotic cells activate canonical branches of signal transduction cascades, collectively termed the unfolded protein response (UPR). The UPR attempts to recover the protein folding capacity and avoid irreversible cellular damage. Additionally, the UPR has been shown to play unique physiological roles in the regulation of diverse cellular events, including cell differentiation and development and lipid biosynthesis. Recent studies have indicated that these important cellular events are also regulated by contact and communication among organelles. These reports suggest strong involvement among the UPR, organelle communication and regulation of cellular homeostasis. However, the precise mechanisms for the formation of contact sites and the regulation of its dynamics by UPR remain unresolved. In this review, we summarized the current understanding of how the UPR regulates morphological changes to the ER and the formation of contact sites between the ER and other organelles. We also review how UPR-dependent connections between the ER and other organelles affect cellular and physiological functions.

The function of the endoplasmic reticulum (ER) can be impaired by the alternation of the extra- and intracellular environment such as disruption of calcium homeostasis, expression of mutated proteins and oxidative stress. In response to disruptions to ER homeostasis, eukaryotic cells activate canonical branches of signal transduction cascades, collectively termed the unfolded protein response (UPR). The UPR attempts to recover the protein folding capacity and avoid irreversible cellular damage. Additionally, the UPR has been shown to play unique physiological roles in the regulation of diverse cellular events, including cell differentiation and development and lipid biosynthesis. Recent studies have indicated that these important cellular events are also regulated by contact and communication among organelles. These reports suggest strong involvement among the UPR, organelle communication and regulation of cellular homeostasis. However, the precise mechanisms for the formation of contact sites and the regulation of its dynamics by UPR remain unresolved. In this review, we summarized the current understanding of how the UPR regulates morphological changes to the ER and the formation of contact sites between the ER and other organelles. We also review how UPR-dependent connections between the ER and other organelles affect cellular and physiological functions.

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.

Exposure to ionizing radiation, accidental or intentional, may lead to delayed effects of acute radiation exposure (DEARE) that manifest as injury to organ systems including kidney, heart, and brain. This study examines the role of activated protein C (APC), a known mitigator of radiation-induced early toxicity, in long-term plasma metabolite and lipid panels that may be associated with DEARE. Male and female cohorts of C57BL/6N wild-type and APCHi transgenic mice were exposed to 9.5 Gy γ-rays with their hind-legs shielded to allow long-term survival that is necessary to monitor DEARE, and plasma was collected at 6 months for LC-MS based metabolomics and lipidomics. We observed significant dyslipidemia, indicative of inflammatory phenotype, upon radiation exposure. Additionally, observance of several other metabolic dysregulations was suggestive of gut damage, perturbations in TCA and urea cycles, and arginine metabolism. We also observed gender and genotype modulated metabolic perturbations post radiation exposure. The APCHi mice showed near normal abundance for several lipids. Moreover, restoration of plasma levels of some metabolites including amino acids, citric acid, and hypoxanthine in APCHi mice are indicative of APC-mediated protection from radiation injuries. With the help of these findings the role of APC in plasma molecular events after acute -radiation exposure in a gender-specific manner can be established for the first time.

Oncostatin M (OSM), an inflammatory cytokine belonging to interleukin-6 superfamily, is recognized as an essential factor secreted by osteal macrophages to orchestrate bone remodelling. Osteoprotegerin (OPG) produced by osteoblasts regulates osteoclastogenesis. We previously demonstrated that bone morphogenetic protein-4 (BMP-4) stimulates OPG synthesis in osteoblast-like MC3T3-E1 cells, and that SMAD1/5/8(9), p38 mitogen-activated protein kinase (MAPK), and p70 S6 kinase positively regulates the OPG synthesis. The present study is aimed to investigate the effect of OSM on BMP-4-stimulated OPG synthesis in osteoblasts. OSM suppressed the release and the mRNA expression of OPG upregulated by BMP-4 in MC3T3-E1 cells.  Neither the BMP-4-induced phosphorylation of SMAD1/5/9 nor that of p38 MAPK affected by OSM. On the other hand, the BMP-4-stimulated phosphorylation of p70 S6 kinase was considerably suppressed by OSM. These results strongly suggest that OSM suppresses the BMP-4-stimulated OPG synthesis via inhibition of the p70 S6 kinase-mediated pathway in osteoblasts.

It has been well known for a long time that inert gases, such as xenon (Xe), have significant biological effects. As these atoms are extremely unlikely to partake in direct chemical reactions with bio-molecules, such as proteins, lipids and nucleic acids, there must be some other mode of action to account for the effects reported. It has been shown that the topology of proteins allows for cavities and hydrophobic pockets, and it is through an interaction with such protein structures that inert gases are thought to have their action. Recently, it has been mooted that the relatively inert gas molecular hydrogen (H2), also may have its effects through such a mechanism, and so influencing protein structures and actions. H2 is thought to also act through interaction with redox active compounds, particularly the hydroxyl radical (·OH) and peroxynitrite (ONOO-), but not nitric oxide (NO·), superoxide anions (O2·-) or hydrogen peroxide (H2O2). However, instead of having a direct interaction with H2, is there any evidence that these redox compounds can also interact with Xe pockets and cavities in proteins, so either having an independent effect on proteins or interfering with the action of inert gases. This suggestion with be explored here.

Every protein consists of a linear sequence over an alphabet of $20$ letters/amino acids. The sequence unfolds in the $3$-dimensional space through secondary (local foldings), tertiary (bonds) and quaternary (disjoint multiple) structures. The mere existence of the genetic code for the $20$ letters of the linear chain could be predicted with the (informationally complete) irreducible characters of the finite group $G_n:=\mathbb{Z}_n \rtimes 2O$ (with $n=5$ or $7$ and $2O$ the binary octahedral group) in our previous two papers. It turns out that some quaternary structures of protein complexes display $n$-fold symmetries. We propose an approach of secondary structures based on free group theory. Our results are compared to other approaches of predicting secondary structures of proteins in terms of $\alpha$ helices, $\beta$ sheets and coils, or more refined techniques. It is shown that the secondary structure of proteins shows similarities to the structure of some hyperbolic $3$-manifolds. The hyperbolic $3$-manifold of smallest volume --Gieseking manifold--, some other $3$ manifolds and the oriented hypercartographic group are singled out as tentative models of such secondary structures. For the quaternary structure, there are links to the Kummer surface.

Background: Chronic Hepatitis B Virus (HBV) infection is a global health concern, associated with severe liver diseases, necessitating ongoing research on novel drug candidates. This study aims to identify potential drug candidates targeting HBV core protein (HBcAg) and disrupting capsid assembly, a critical step in the virus's life cycle. Methods: HBcAg in complex with HBV inhibitors were obtained from the Protein Data Bank (PDB). CavityPlus server was used for analysis of druggable cavity. Structure-based pharmacophores were extracted from identified cavities, and potential allosteric ligand binding sites were assessed using CavPharmer, CorrSite, and CovCys. LigandScout was employed for ligand-based pharmacophore screening against an FDA-approved library. The ZINC database was screened with features extracted from CavPharmer. Molecular docking studies were conducted using Autodock Vina. Lead compounds were selected based on docking scores, binding modes, and interactions within the druggable cavity. Results: Strong druggable pockets were found for Ciclopirox, while Compound 24, NVR10-001E2, and others showed medium to weak pockets. Ligand-based pharmacophores varied in size and complexity. Screening revealed potential hits matching these pharmacophores, including Ciclopirox olamine, Voriconazole, Enasidenib, and Statins. A large compound database search yielded additional hits like ZINC86859997 and ZINC63280172. Docking analyses confirmed these hits' potential, highlighting their interactions with critical HBc protein residues, offering promising leads for hepatitis B drug development. Conclusions: Voriconazole, Enasidenib, and Lovastatin have shown promises. These hits displayed favorable interactions with crucial HBc protein residues, indicating their potential as lead compounds The mechanism of action of statins with anti-HBV activities also highlighted. This comprehensive approach offers valuable insights into targeting HBc protein for antiviral drug discovery.

Flaviviruses replicate in membrane factories associated with the endoplasmic reticulum (ER). Significant levels of flavivirus viral protein accumulation contribute to ER stress. As a consequence, the host cell exhibits an Unfolded Protein Response (UPR), subsequently stimulating appropriate cellular responses such as adaptation, autophagy or apoptosis. The correct redox conditions of this compartment are essential to form native disulfide bonds in proteins. ZIKA virus (ZIKV) has ability to induce persistent ER stress leading to activation of UPR pathways. In this study, we wondered whether ZIKV affects the redox balance and consequently the oxidative protein folding in the ER. We found that ZIKV replication influences redox state leading to aggregation of viral envelope protein as amyloid-like structures in the infected cells.

The devastating impact of the ongoing coronavirus disease 2019 (COVID-19) on public health, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has made fighting of the COVID-19 pandemic is a top priority in medical research and pharmaceutical development. Surveillance of SARS-CoV-2 mutations is essential for the comprehension of SARS-CoV-2 variant diversity and their impact on virulence and pathogenicity. The SARS-CoV-2 open reading frame 10 (ORF10) protein interacts with multiple human proteins CUL2, ELOB, ELOC, MAP7D1, PPT1, RBX1, THTPA, TIMM8B, and ZYG11B expressed in the lung tissues. Mutations and co-mutations in the emerging SARS-CoV-2 ORF10 variants are expected to impact the severity of the virus and its associated consequences. In this article, We highlight 128 single mutations and 35 co-mutations in the unique SARS-CoV-2 ORF10 variants in this article. The possible predicted effects of these mutations and co-mutations on the secondary structure of ORF10 variants and host protein interactomes are presented. The findings highlight the possible effects of mutations and co-mutations on the emerging 140 ORF10 unique variants from secondary structure and intrinsic protein disorder perspectives.

Accurate determination of protein localization, levels, or protein-protein interactions is pivotal for the study of their function, and in situ protein labeling via homologous recombination has emerged as a critical tool in many organisms. While this approach has been refined in various model fungi, the study of protein function in most plant pathogens has predominantly relied on ex-situ or overexpression manipulations. To dissect the molecular mechanisms of development and infection for Verticillium dahliae, a formidable plant pathogen responsible for vascular wilt diseases, we have established a robust, homologous recombination-based in situ protein labeling strategy in this organism. Utilizing Agrobacterium tumefaciens-mediated transformation (ATMT), this methodology facilitates the precise tagging of specific proteins at their C-termini with epitopes, such as GFP and Flag, within the native context of V. dahliae. We demonstrate the efficacy of our approach through the in situ labeling of VdCf2 and VdDMM2, followed by subsequent confirmation via subcellular localization and protein-level analyses. Our findings confirm the applicability of homologous recombination for in situ protein labeling in V. dahliae and suggest its potential utility across a broad spectrum of filamentous fungi. This labeling method stands to significantly advance the field of functional genomics in plant pathogenic fungi, offering a versatile and powerful tool for the elucidation of protein function.

The receptor-type protein tyrosine phosphatases (RPTPs) are involved in a wide variety of physiological functions which are mediated via their diverse extracellular regions. They play key roles in cell-cell contacts, bind various ligands and are regulated by dimerization and other processes. Depending on the subgroup they have been described as everything from ‘rigid rods’ to ‘floppy tentacles’. Here we review current experimental structural knowledge on the extracellular region of RPTPs and draw on AlphaFold structural predictions to provide further insights into structure and function of these cellular signalling molecules.

(1) Background: In recent years, the increasing demand for alternative food shifted research toward new types of food sources. It is well known that many diseases are caused by oxidative stress, then supplementation of antioxidants can be proposed to reduce it. Cannabis Sativa (CS) is an interesting alternative source of antioxidant species. The use of CS for the extraction of these molecules could be optimized to maximize the antioxidant yield extracts. (2) Methods: This work aimed to investigate extraction methods of CBD oil from CS inflorescences and stalks to obtain antioxidant-enriched compounds for use in food by mixing with a carrier oil like hemp seed oil. Soxhlet (SE), dynamic maceration (DME) and supercritical fluid (SFE) extraction techniques were performed, using CS inflorescences and stalks; by High-Pressure Liquid Chromatography (HPLC) analysis the antioxidants were individuated and quantified; moreover, the antioxidant power was investigated by 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays and finally doughs enriched with hemp oil were studied by rheological measurements (3) Results: The best results in terms of CBD yield were obtained by dynamic maceration from only flowers and it also shows a good antioxidant power. (4) Conclusions: Formulation of protein bars with hemp oil enriched in CBD could be an excellent means for the consumption of products enriched with antioxidants because it preserves the CBD anti-inflammatory activity.

Cytochrome P450 oxidoreductase (POR) is the redox partner of steroid and drug-metabolizing cytochromes P450 located in the endoplasmic reticulum. Mutations in POR cause a broad range of metabolic disorders. The POR variant rs17853284 (P228L) identified by genome sequencing has been linked to lower testosterone levels and reduced P450 activities. We expressed POR wild type and the P228L variant in bacteria, purified the proteins, and performed protein stability and catalytic functional studies. Variant P228L affected the stability of the protein as evidenced by lower unfolding temperatures and higher sensitivity to urea denaturation. A significant reduction of small molecule metabolism was observed with POR P228L while activities of CYP3A4 were reduced by 25%, and activities of CYP3A5, and CYP2C9 were reduced by more than 40% compared to WT POR. The 17,20 lyase activity of CYP17A1 responsible for production of main androgen precursor dehydroepiandrosterone, was reduced to 27% of WT in presence of P228L variant of POR. Based on in silico and in vitro studies we predict that the change of proline to leucine may change the rigidity of the protein, causing conformational changes in POR, leading to altered electron transfer to redox partners. A single amino acid change can affect protein stability and cause a severe reduction in POR activity. Molecular characterization of individual POR mutations is crucial for a better understanding of the impact on different redox partners of POR.

Abstract: Due to their rapid evolution and their impact on health care, be-ta-lactamases, protein degrading beta-lactam antibiotics, are used as generic model of protein evolution. Therefore, we investigated the mutation effects in two distant beta-lactamases TEM-1 and CTX-M-15. Interestingly we found a site with a complex pattern of genetic interactions. Mutation G251W in TEM-1 is in-activating the protein’s function just as the reciprocal mutation W251G in CTXM-15. Phylogenetic analysis revealed that mutation G has been entrenched in TEM-1 background: while rarely observed throughout the phylogeny it is es-sential in TEM-1. Using a rescue experiment in TEM-1 G251W mutant, we could identify sites that alleviates the deviation from G to W. While few of these muta-tions could potentially involve local interactions, most of them were found on distant residues in the 3D structure. Many well-known mutations having an impact on protein stability, such as M182T, were recovered. Our results there-fore suggest that entrenchment of an amino acid may rely on diffuse interactions among multiple sites with a major impact on protein stability.

The recent outbreak of the new virus in Wuhan city, China from the sea food market has led to the identification of a new strain called the corona virus and named as novel corona virus (2019-nCoV) belonging to Coronaviridae family. This has created major havoc and concern due to the mortality of 250 persons and affecting more than 10,000 people. This virus causes sudden fever, pneumonia and also kidney failure. In this study a computational approach is proposed for drug and vaccine design. The spike protein sequences were collected from a protein database and analysed with various bioinformatics tools to identify suitable natural inhibitors for the N-terminal receptor binding domain of spike protein. Also, it is attempted to identify suitable vaccine candidates by identifying B-Cell and T-cell epitopes. In the drug design, the tanshinone Iia and methyl Tanshinonate were identified as natural inhibitors based on the docking score. In the vaccine design, B-cell epitope VLLPLVSSQCVNLTTRTQLPPAYTN was found to have the highest antigenicity. FVFLVLLPL of MHC class-I allele and FVFLVLLPL of MHC class-II allele were identified as best peptides based on a number of alleles and antigencity scores. The present study identifies natural inhibitors and putative antigenic epitopes which may be useful as effective drug and vaccine candidates for the eradication of novel corona virus.

HspA1A, a molecular chaperone, translocates to the plasma membrane (PM) of stressed and cancer cells. This translocation results in HspA1A’s cell-surface presentation, which renders tumors radiation insensitive. To specifically inhibit the lipid-driven HspA1A’s PM translocation and devise new therapeutics it is imperative to characterize the unknown HspA1A’s lipid-binding regions and determine the relationship between the chaperone and lipid-binding functions. To elucidate this relationship, we determined the effect of phosphatidylserine (PS)-binding on the secondary structure and chaperone functions of HspA1A. Circular dichroism revealed that binding to PS resulted in minimal modification on HspA1A’s secondary structure. Measuring the release of inorganic phosphate revealed that PS-binding had no effect on HspA1A’s ATPase activity. In contrast, PS-binding showed subtle but consistent increases in HspA1A’s refolding activities. Furthermore, using a Lysine-71-Arginine mutation (K71A; a null-ATPase mutant) of HspA1A we show that although K71A binds to PS with affinities similar to the WT, the kinetics of the binding are significantly different, probably because of the mutant’s inability to achieve specific conformations. These observations suggest a two-step binding model that includes conformational changes and strongly support the notion that the chaperone and lipid-binding activities of HspA1A are dependent but the regions mediating these functions do not overlap. These findings provide the basis for future interventions to inhibit HspA1A’s PM-translocation in tumor cells, making them sensitive to radiation therapy.

Background: The fulminant course of COVID-19, triggered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents with a high mortality rate and still lacks a causative treatment. C-reactive protein (CRP) has been shown to increase dramatically during the disease progression and correlates with deleterious outcomes. Selective CRP apheresis can reduce circulating CRP levels fast and effective. Methods: Seven hospitalized patients with documented severe COVID-19 progression, elevated CRP plasma levels (> 100 mg/L) and signs of respiratory failure were treated with CRP apheresis. 2-12 CRP apheresis sessions were performed generally in 24 h time intervals and depending on CRP plasma levels. Results: CRP apheresis reduced CRP plasma levels by up to 84% within a few hours, without exhibiting side effects in any patient. Despite signs of severe lung infiltration in all patients, only one patient died. The other patients showed improvements within the chest x-ray after CRP apheresis and were able to recover regardless of intubation and/or ECMO (4 patients). All remaining six patients were discharged from the hospital in good clinical condition. All patients had comorbidities. Conclusions: This case series presents a mortality rate of only 14%, which is dramatically lower than expected from the presented CRP levels as well as comorbidities and ventilation require-ments. Our clinical observations regarding the here presented seven patients support the hypoth-esis that CRP is a candidate to be therapeutically targeted in the early stage of severe COVID-19.

Three isoenergetic diets differing on their fishmeal/ soy protein concentrate (SPC) ratio were assessed on tissue growth and energy budget of juvenile crabs Scylla serrata in postmolt stages (PMolt) and in intermolt stages (IMolt). The average growth rate on dry matter basis, were 2.064 ± 0.324% and 0.492 ± 0.08% initial BW.day-1 during PMolt and IMolt stages respectively. The efficiency in feed conversion (FCE, %), protein retention (PRE, %) and energy retention (ERE, %) were similar for the 3 experimental diets. However, FCE, PRE and ERE in PMolt stages were 4 to 5 times higher than in IMolt stages. Feed intake, energy and protein required for growth in PMolt stages were obviously higher than in IMolt stages. The energy budgets (%total energy intake) were marginally affected by diet but were significantly affected by the molt stage. Maintenance energy was lower in PMolt stages (49.84 ± 4.9%) than in IMolt stages (83.33 ± 2.45%). The excess of maintenance energy in IMolt stages represents the portion set aside for next molt: shell energy content (4.97 ± 0.31%) and energy for ecdysis (± 28%). Conversely, recovery energy was significantly higher in PMolt stages (34.39 ± 0.99%) than in IMolt stages (8.33 ± 1.7%). In conclusion SPC sustained good tissue growth and good feed utilization and can be used as a main source of dietary protein for crab juveniles in captivity.

The study was done to determine the effect of supplementing different forms of L-methionine (L-Met) and acetate on protein synthesis in immortalized bovine mammary epithelial cell line (MAC-T Cell): Control, L-Met, conjugated L-Met and acetate (CMA), and non-conjugated L-Met and Acetate (NMA). Protein synthesis mechanism was determined by omics method. NMA group had the highest protein content in the media and β-casein mRNA expression levels (P < 0.05). The number of upregulated and downregulated proteins observed were 77 and 62 in CMA group and 50 and 81 in NMA group from 448 proteins, respectively (P < 0.05). NMA and CMA treatments stimulated pathways related to protein and energy metabolism (P < 0.05). Metabolomic analysis also revealed CMA and NMA treatments resulted in increases of several metabolites (P < 0.05). In conclusion, NMA treatment increased protein concentration and expression level of β-casein mRNA in MAC-T cells compared to CMA.

The genus Flavivirus contains pathogenic vertebrate-infecting flaviviruses (VIFs) and in-sect-specific flaviviruses (ISF). ISF transmission to vertebrates is inhibited at multiple stages of the cellular infection cycle, via yet to be elucidated specific antiviral responses. The Zinc-finger an-tiviral protein (ZAP) in vertebrate cells can bind CpG dinucleotides in viral RNA, limiting virus replication. Interestingly, the genomes of ISFs contain more CpG dinucleotides compared to VIFs. In this study, we investigated whether ZAP prevents two recently discovered lineage II ISFs, Binjari (BinJV) and Hidden Valley viruses (HVV) from replicating in vertebrate cells. BinJV protein and dsRNA replication intermediates were readily observed in human ZAP knockout cells when cultured at 34 ˚C. In ZAP expressing cells, inhibition of the interferon response via interferon response factors 3/7 did not improve BinJV protein expression, whereas treatment with kinase inhibitor C16, known to reduce ZAP’s antiviral function, did. Importantly, at 34 ˚C both BinJV and HVV successfully completed the infection cycle in human ZAP knockout cells evident from infectious progeny virus in the cell culture supernatant. Therefore, we identify vertebrate ZAP as an important barrier that protects vertebrate cells from ISF infection This provides new insights into flavivirus evolution and the mechanisms associated with host switching.

Hepatitis B virus (HBV) is the dominant risk factor for hepatocellular carcinoma (HCC). HBV X protein (HBx) plays crucial roles in HCC carcinogenesis. HBx interferes with several signaling pathways including Notch1 pathway in HCC. In our study, we found that Notch1 was highly expressed in HCC especially in large HCC. Notch1 and HBx co-localized in HCC and their levels were positively correlated with each other. Notch1 expression was more elevated in HepG2.2.15 than that in HepG2. HBx activated Notch1 pathway in HepG2.2.15. Repression of HBx and Notch1 pathway attenuated the growth of HepG2.2.15. Notch1, ERK and AKT pathways were inhibited after a γ-secretase inhibitor treatment. Dual-specificity phosphatase 1 (DUSP1) and phosphatase and tensin homolog (PTEN) were up-regulated after the γ-secretase inhibitor treatment and Hes1 inhibition. Luciferase reporter assays showed that Hes1 repressed the promoters of DUSP1 and PTEN and this was reverted by γ-secretase inhibitor treatment. Western blotting demonstrated that DUSP1 dephosphorylated pERK and PTEN dephosphorylated pAKT. Collectively, we reported a link among HBx, Notch1 pathway, DUSP1/PTEN, and ERK/AKT pathways, which influenced HCC cell survival and could be a therapeutic target for HCC.

This study was conducted to investigate the effect of supplementing different types of methionine (L and D-type) and its precursor (HMBi) on milk protein synthesis using immortalized bovine mammary epithelial cell line (MAC-T Cell); D-methionine (D-Met), L-methionine (L-met) and 2-hydroxy-4-methylthiobutanoic acid I (HMBi), an isopropyl ester of the hydroxy analogue precursor of methionine. The underlying mechanism of milk protein synthesis by adding D- and L-type amino acid as well as HMBi was elucidated through omics analysis to verify the metabolism pathway. Results showed that HMBi group showed the highest beta casein mRNA expression levels compared to D- and L-Met groups and highest medium protein although not different with the L-Met treatment. The observed upregulated (>2 protein expression vs. control) and downregulated (<0.5 protein expression vs. control) proteins in L-Met, D-Met and HMBi treated groups were: 39, 77; 46, 68; and 40, 78, respectively. Interestingly, based on protein pathway analysis, L-Met treated group stimulated the ATP synthesis, PI3 kinase and pyruvate metabolism. On the other hand, the D-Met group stimulated fructose-galactose metabolism, glycolysis pathway, PI3 kinase and pyruvate metabolism. And lastly HMBi-treated group stimulated pentose phosphate pathway and glycolysis pathway. Metabolite analysis revealed that L-Met treated group resulted in the increase in 11 metabolites. On the other hand, D-Met treated group showed increase in 7 metabolites and decreased of uridine monophosphate (UMP). HMBi supplementation caused increases of 3 metabolites and decreased of UMP and N-acetyl-L-glutamate Addition of different isoforms of Met stimulated the production of intermediate metabolites for energy production. Addition of L-Met stimulated the production of energy metabolites such as pyruvate, malate, and fumarate, well-known as intermediates of Krebs cycle. On the other hand, HMBi supplementation resulted in increases of energy metabolite glucose 1-phosphate and 6-phosphogluconate. Results showed that HMBi-treated group exhibited highest expression of β-casein mRNA expression by stimulating proteins and metabolites as well as protein and metabolic pathways involved in protein and energy synthesis. As a result, HMBi-treated group resulted in highest protein concentration but not significantly different with L-Met. Both the D- and L-isoforms has considerably the same medium protein concentration and β-casein mRNA expression higher than the control. So, D- and L-Met isoforms can be used alternatively without any significant change in protein synthesis efficiency in bovine mammary epithelial cells.

Keywords: Marine sulfated glycans; SARS-CoV-2; Omicron XBB.1.5, Spike protein; Heparin

Apoptosis, also known as the programmed death of cells, is responsible for maintaining the homeostasis of tissues and this function is carried out by caspases. The process of apoptosis is carried out via two distinct pathways: the extrinsic pathway, which is governed by death receptors, and the intrinsic pathway, also known as the mitochondrial pathway. The BCL-2 protein family encoded by the BCL-2 gene, located at the 18q21.33 chromosomal location, is in charge of regulating the intrinsic pathway, which is responsible for inducing cell death via the permeabilization of the mitochondrial membrane and the release of apoptosis - inducing components. The BCL-2 homology (BH1, BH2, BH3, BH4) domains of this family proteins are crucial for their functioning and their common BH domains allow interactions between members of the same family and can also serve as indications of pro- or anti-apoptotic activity. A direct correlation may be shown between the overexpression of BCL-2 and the postponement of cell death. It has been determined that a change in the expression of BCL-2 is the root cause of a variety of malignancies, including lung, breast, melanoma, and chronic lymphocytic leukemia, Multiple Sclerosis, Diabetes. In this review, we discuss the therapeutic potential of regulating BCL-2 family connections and their relevance to health and disease.

One of the major problems caused by repeated subcutaneous insulin injections in diabetic patients is insulin amyloidosis. Understanding the molecular mechanism of amyloid fibril formation of insulin and finding effective compounds to its inhibit or eliminate is very important and extensive research has been done on it. In this study, the anti-amyloidogenic and destabilizing effects of the pyrogallol, as a phenolic compound, on human insulin protein were investigated by CR absorbance, ThT and ANS fluorescence, FTIR spectroscopy and atomic force microscopy. According to the obtained results, the formation of amyloid fibrils at pH 2.0 and 50 °C was confirmed by CR, ThT, ANS, FTIR assays. Microscopic images also showed the twisted and long structures of amyloid fibrils. Simultaneous incubation of the protein with pyrogallol at different concentrations reduced the intensities of CR, ThT and ANS in a dose-dependent manner and no trace of fibrillar structures was observed in the microscopic images. FTIR spectroscopy also showed that the position of amide I band in the spectrum of samples containing pyrogallol was shifted. Based on the findings in this study, it can be concluded that pyrogallol can be effective in preventing and suppressing human insulin amyloid fibrils.

A separate group consists of compounds that are able to simultaneously interact with both orthosteric and allosteric sites, which are classified as bitopic GPCR ligands [74, 76-81]. They have two pharmacophores, one of which binds with high affinity to the orthosteric site, while the other, with lower affinity, binds to the allosteric site. If these sites are spatially separated in the receptor, then the pharmacophores in the bitopic ligand must be connected with a flexible linker, the length of which exactly corresponds to the distance between the orthosteric and allosteric sites. At the same time, it is important that the linker does not significantly affect the conformational rearrangements in the receptor caused by its activation by orthosteric and (or) allosteric agonists [74, 79].

The presence of a non-structural protein 1 (NS1) in tick-borne encephalitis (TBE) vaccines and the possible induction of an NS1-specific immune response in vaccinated individuals remains a somewhat controversial topic. Previously, we detected the presence of NS1 in Encepur TBE vaccine by mass spectrometry and found the induction of NS1-specific IgG antibodies in mice vaccinated with FSME-Immun TBE vaccine. Here, in this follow-up study, we examined the dynamics and extent of the NS1-specific IgG response in mice vaccinated with these two vaccines in more detail and compared it with the IgG response to the whole virus (WV). Mice were vaccinated at two-week intervals with a total of six doses of each vaccine, and levels of IgG antibodies to TBE virus WV and NS1 were measured by ELISA after each dose. Both vaccines elicited a robust anti-WV IgG response after two doses. The Encepur vaccine did not elicit NS1-specific IgG even after all six doses. In contrast, FSME-Immun vaccine triggered production of NS1-specific IgG after four doses. The results indicate that FSME-Immun is the only vaccine that elicits an NS1-specific antibody response in mice. However, compared to WV-specific IgG, the NS1-specific response is weaker, and a higher number of doses is required to induce detectable levels of NS1-specific IgG antibodies.

N-Acetyl-L-glutamate kinase (NAGK) catalyzes the rate-limiting step in the ornithine/arginine biosynthesis pathway in eukaryotic and bacterial oxygenic phototrophs. NAGK is the most highly conserved target of the PII signal transduction protein in Cyanobacteria and Archaeplastida (red algae and Chlorophyta). However, yet there is still much to be learnt on how NAGK is regulated in vivo. The use of unicellular green Chlamydomonas reinhardtii as a model system has already been instrumental in identifying several key regulation mechanisms that control nitrogen (N) metabolism. With a combination of molecular-genetic and biochemical approaches, we show the existence of the complex CrNAGK control at transcriptional level, which is dependent on N source and N availability. In growing cells, CrNAGK requires CrPII to properly sense the feedback inhibitor arginine. Moreover, we provided primary evidence that CrPII is only partly responsible for regulating CrNAGK activity to adapt to changing nutritional conditions. Collectively, our results suggest that in vivo CrNAGK is tuned at transcriptional and post-translational levels, and CrPII and additional as yet unknown factor(s) are integral parts of this regulation.

Multidrug resistance protein 4 (MRP4) is an energy-dependent membrane transporter that is responsible for cellular efflux of a broad range of xenobiotics and physiological substrates. In this trial, we aimed to investigate the co-effects of aging and MRP4 deficiency using gene expression microarray and morphological and electrophysiological analyses of the mouse retina. Mrp4-knockout (null) mice and wild-type (WT) mice were reared in the same condition to 8–12 wk (young) or 45–55 wk (aged). Microarray analysis identified 186 differently expressed genes from the retinas of aged Mrp4-null mice as compared to that from aged WT mice, and subsequence gene ontology and KEGG pathway analyses showed that differently expressed genes were related to lens, eye development, vision, and transcellular barrier function that are involved in metabolic pathways or viral infection pathways. No significant change in thickness was observed for each retinal layer among young/aged WT mice and young/aged Mrp4-null mice. Moreover, immunohistochemical analyses of retinal cell type did not exhibit an overt change in the cellular morphology or distribution among the 4 age/genotype groups, and the electroretinogram responses showed no significant differences in the amplitude or the latency between aged WT mice and aged Mrp4-null mice. Aging would be an insufficient stress to cause some damage to the retina in the presence of MRP4 deficiency.

In addition to nucleic acids, various viral proteins are involved in the assembly of virion. If it is possible to create a biologically active agent that leads to the degradation of one of the key proteins for virus assembly and/or destroys the viral factory, then this agent will effectively cope with virus replication. One of these key proteins for the SARS-CoV-2 virus is the nucleocapsid protein (N-protein). As such a bioactive agent, we offer a modular nanotransporter (MNT) developed by us, which, in addition to an antibody mimetic to the N-protein, contains an amino acid sequence for the attraction of the Keap1 E3 ubiquitin ligase. This should lead to the subsequent degradation of the N-protein. We have shown that functional properties of modules within the MNT permit its internalization into target cells, endosome escape into the cytosol, and binding to the N-protein. Using flow cytometry and Western blot, it was demonstrated that significant degradation of N-protein is observed when A549 and A431 cells transformed with N-protein are incubated with the developed MNTs. The proposed MNTs open up a new approach for the treatment of viral diseases.

The PAX8/PPARγ rearrangement, producing the PAX8–PPARγ fusion protein (PPFP), is thought to play an essential role in the oncogenesis of thyroid follicular tumors. To identify PPFP-targeted drug candidates and establish an early standard of care for thyroid tumors, we performed ensemble docking-based compound screening. Specifically, we investigated which pocket structure should be adopted to search for a promising ligand compound for the PPFP; the position of the ligand binding pocket on the PPARγ side of the PPFP is common to that of PPARγ; however, the shape is slightly different between them due to environmental factors. We developed a method for selecting a PPFP structure with a relevant pocket and high prediction accuracy for ligand binding. This method was validated using PPARγ, whose structure and activity values are known for many compounds. Then, we performed docking calculations to PPFP for 97 drug or drug-like compounds with a thiazolidine backbone, which is one of the characteristics of ligands that bind well to PPARγ, registered in the DrugBank database. Furthermore, the binding affinities of promising ligand candidates were estimated more reliably using the molecular mechanics-Poisson–Boltzmann surface area method. Thus, we propose promising drug candidates for the PPFP with a thiazolidine backbone.

Achieving global goals on sustainable nutrition, health, and wellbeing will depend on delivering enhanced diets to humankind. This will require, among others, instantaneous access to information on food quality at key points within agri-food systems. Although stationary methods are usually used to quantify grain quality (wet-lab chemistry, benchtop NIR spectrometer); these do not suit many required user-cases, such as stakeholders in decentralized agri-food-chains that are typical for emerging economies. Therefore, we explored new technologies and models that might aid these particular user-cases. For this purpose, we generated the NIR spectra of 328 grain samples from multiple cereals (finger millet, foxtail millet, maize, pearl millet, sorghum) with a standard benchtop NIR Spectrometer (DS2500, FOSS) and a novel mobile NIR-based sensor (HL-EVT5, Hone). We explored a range of classical deterministic and novel machine learning (ML)-driven models to build calibrations out of the NIR spectra. We were able to build relevant calibrations out of both types of spectra. At the same time, ML-based methods enhanced the prediction capacity of calibration models compared to classical deterministic methods. We also documented that the prediction of grain protein content based on NIR spectra generated by a mobile sensor (HL-EVT5, Hone) was highly relevant for quantitative protein predictions (R2 = 0.91, RMSE = 0.97, RPD = 3.48). Thus, the findings of this study lay the foundations on which to expand the utilization of NIR spectroscopy applications for agricultural research and development.

Endometrial Cancer (EC) is the sixth most commonly occurring cancer in women with 380 000 cases in 2018. Sadly, EC morbidity and mortality are continuously increasing, therefore the medical society have a substantial need for an accurate and inexpensive diagnostic test for EC early detection and a prognostic tool for treatment planning and evaluation. Considering experience with different types of cancers C-reactive protein (CRP) appears to be a promising diagnostic and prognostic factor. Aiming to investigate its potential and in view of EC authors, this paper reviewed the following databases for metanalysis, randomized controlled trials and review articles published up to June 2020: Pubmed, Scopus, Google scholar and ClinicalKey. Studies indicate CRP >3.33 mg/l correlate with EC incidence with HR = 2.29 (p<0.05). Moreover, High-sensitivity CRP assay allows to detect CRP in very low concentrations and distinguish patients with endometriosis, soft tissue sarcomas and possibly EC. Preoperational and postoperational CRP, as well as its dynamic change are independent prognostic factors for EC and are more reliable if analyzed together. However, CRP-to-albumin ratio as well as Glasgow Prognostic Scale have greater prognostic value that CRP alone. Additionally, CRP is possibly a mediator of carcinogenesis and cancer progression through activation of inter alia FcgRs/MAPK/ERK, FcgRs/IL-6/AKT/STAT3 and FcgRs/NF-κB/NLRP3 pathways.

Interpretation of increased gain in the bovine animals is difficult to be validated due to inherent genetic variation to meet their requirement for energy and protein, and those may relate to the bull species, e.g., Thai swamp buffalo. Therein, the study aimed at investigating and predicting protein requirement systems, with providing abundant energy intake 2.20 Mcal/kg DM for maintenance and growth of Thai swamp buffaloes using the comparative prolonged feeding trial for 90 days. Sixteen bull Thai swamp buffaloes at the initial (Age: 18-24 months; BW: 233 ± 25.0 kg) were as-signed into four treatment groups, four buffaloes each, fed 5.42, 6.96, 8.94, and 10.71% DM crude protein (CP). CP intake, BW, and physiological fluid were determined. The net CP requirements for maintenance and growth of Thai swamp buffaloes were 5.41 g CP/kg W0.75 and 0.46 g CP/g average daily gain (ADG), respectively. Our results indicated that CP requirement increases, when BW increases. An increased dietary CP resulted in an increased number at blood urine nitrogen (N), N absorption, total volatile fatty acid, urinary purine derivative, and the microbial N. Notably, the net CP requirement for growth of Thai swamp buffalo was higher than it reported in NRC, but the maintenance was lower.

Substrate binding proteins (SBP) bind to specific ligands in the periplasmic region and bind to membrane proteins to participate in transport or signal transduction. Typical SBPs consist of two α/β domains and recognize the substrate by hinge motion between two domains. Conversely, short length Rhodothermus marinus SBP (named as RmSBP) exists around the methyl-accepting chemotaxis protein. We previously determined the crystal structure of RmSBP consisting of a single α/β domain, but the substrate recognition mechanism is still unclear. To better understand the short length RmSBP, we performed comparative structure analysis, computational substrate docking, and X-ray crystallographic study. RmSBP shares a high level of similarity in α/β domain with other SBP proteins, but it has a distinct topology in the C-term region. The substrate binding model suggested that conformational change in the peripheral region of RmSBP was required to recognize the substrate. We determined the crystal structures of RmSBP at pH 5.5, 6.0, and 7.5. RmSBP showed structural flexibility of the β1-α2 loop, β5-β6 loop, and extended C-term domain based on the electron density map and temperature B-factor analysis. These results provide information that will further the understanding of the function of the short length SBP.

Scrub typhus threatens one billion people in the Asia-Pacific area and cases have emerged outside this region. It is caused by infection with any of the multitude of strains of the bacterium, Orientia tsutsugamushi. A vaccine that affords heterologous protection and a commercially available molecular diagnostic assay are lacking. Herein, we determined that the nucleotide and translated amino acid sequences of outer membrane protein A (OmpA) are highly conserved among 51 O. tsutsugamushi isolates. Molecular modeling revealed the predicted tertiary structure of O. tsutsugamushi OmpA to be very similar to that of the phylogenetically related pathogen, Anaplasma phagocytophilum, including the location of a helix that contains residues functionally essential for A. phagocytophilum infection. PCR primers were developed that amplified ompA DNA from all O. tsutsugamushi strains, but not from negative control bacteria. Using these primers in quantitative real-time PCR enabled sensitive detection and quantitation of O. tsutsugamushi ompA DNA from organs of mice that had been experimentally infected with the Karp or Gilliam strains. The high degree of OmpA conservation among O. tsutsugamushi strains evidences its potential to serve as a molecular diagnostic target and justifies its consideration as a candidate for developing a broadly protective scrub typhus vaccine.

: F-box proteins are a large gene family in plants, and play crucial roles in plant growth, development, and stress response. However, a comprehensive investigation of F-box family genes in cultivated peanut (Arachis hypogaea L.), has not been undertaken. In this study, we used a bioinformatics approach to identify and analyze F-box protein family genes in the cultivated peanut genome, and examined their expression profiles. Herein, a total of 95 F-box protein family members on 18 chromosomes were identified in cultivated peanut, which were sequentially designed as AhFBX1-AhFBX95. Phylogenetic analysis classified these AhFBXs into four different groups (Group I - IV), and eight subfamilies based on the structural domains of the C-terminal. Structural analysis revealed the presence of ten unique motifs within these genes. Additionally, the investigation of non-synonymous and synonymous substitution rates (Ka/Ks), and synteny in the F-box proteins, provided valuable insights into the evolutionary and domestication processes of AhFBX genes in peanut. Furthermore, in silico expression profiling of 22 different tissues demonstrated that most genes were predominantly expressed in leaves, pistils, and the aerial gynophore tip, while some members showed tissue-specific expression at the apex of both vegetative and reproductive shoots. Subsequently, we validated the expression of 9 differentially expressed genes (DEGs) using transcriptomeic data of lateral branch development and qRT-PCR analysis. These DEGs may be involved in regulating the development of lateral branches and subsequently affect the architecture of peanut plants.

In this study, we reported that novel single-chain fusion-proteins linked thromboxane A2 (TXA2) receptor (TP) to a selected G-protein α-subunit q (SC-TP-Gαq), or s (SC-TP-Gαs) could be stably expressed in megakaryocytes (MK). We tested the MK-released platelet-linked particles (PLPs) to be used as a vehicle to deliver the over-expressed SC-TP-Gαq), or s (SC-TP-Gαs) to regulate human platelet function. To understand how the single-chain TP-Gα fusion proteins could regulate opposite platelet activities by an identical ligand TXA2, we used dual-binding domains linked to different signaling pathways within a single polypeptide chain using a 3D structural model. The immature MKs were cultured and transfected with cDNAs constructed from structural models of the individual SC-TP-Gαq and SC-TP-Gαs, respectively. After transient expression was identified, the immature MKs stably expressing SC-TP-Gαq or SC-TP-Gαs (stable cell lines) were selected. The stable cell lines were induced into mature MKs which released PLPs. Western blot analysis confirmed that the released PLPs were carrying the recombinant SC-TP-Gαq or SC-TP-Gαs. Flow cytometry analysis showed the PLPs carrying SC-TP-Gαq were able to perform the activity by promoting platelet aggregation. In contrast, PLPs carrying SC-TP-Gαs reversed Gq to Gs signaling to inhibit platelet aggregation. This is the first time demonstrating that SC-TP-Gαq and SC-TP-Gαs were successfully overexpressed in MK cells and released as PLPs with proper folding. This bio-engineering led to the formation of two sets of biologically active PLP forms, which mediate calcium and cAMP signaling, respectively. As a result, these PLPs are able to bind to identical endogenous TXA2 with opposite activities, inhibiting and promoting platelet aggregation as required for therapeutic process.The results also demonstrated the nucleus-free PLPs could be used to deliver recombinant membrane-bound GPCRs to regulate cellular activity in general.

Kiwifruit is a perennial horticultural crop species with high nutritional and economic value. However, various pathogens stress brings a serious threat to kiwifruit. Pentatricopeptide repeat proteins (PPRs) are characterized by tandem repetitions of 35 amino acid motifs, and have been found to function in plant RNA editing. However, the roles of PPRs in plant development and disease resistance remain unclear. In this study, we performed a genome-wide identification and characterization of PPR gene family in two kiwifruit species (Actinidia chinensis (Ach), and Actinidia eriantha (Ace)) with markedly different disease resistance. A total of 497 and 499 PPRs were identified in Ach and Ace, respectively. All the kiwifruit PPRs could be phylogenetically divided into four subfamilies. There were about 40.68% PPRs predicted to be localized to mitochondria or chloroplasts. Synteny analysis showed that the expansion of kiwifruit PPRs mainly came from segmental duplication. Based on RNA-seq data from the fruit during 12 periods of development and maturity, weighted correlation network analysis suggested that two PPRs Actinidia20495.t1 and Actinidia15159.t1 were involved in the fruit development and maturation. In addition, we observed different gene expression of PPRs and chloroplast RNA editing profiles between resistant and susceptible kiwifruits after pathogen infection, indicating the roles of PPRs in stress response by modulating the editing extend of mRNA. The differentially expressed upstream transcription factors of PPRs were further identified, they may regulate resistance adaption by modulating the PPRs expression. These results provide a solid foundation for further analyses of the functions and molecular evolution of PPRs, in particular, for clarifying the resistance mechanisms in kiwifruits and breeding new cultivars with high resistance.

Novel SARS-CoV-2, a bat based virus originated in Wuhan, China that caused a global pandemic in December, 2019 belongs to the Betacorona virus family and contains single stranded genome of ~29Kbp. The host cell invasion of SARS-CoV-2 is facilitated by interaction of C-Terminal Domain (CTD) of Spike (S) protein of virus and host ACE2 receptor in the presence of TMPRSS seine protease secreted by the host cell. In this study the mutation hotspots of S-protein will be identified and the impact of such mutation in the binding affinity will be studied. Additionally, the lead molecule which can bind to the mutated protein also will be identified. Multiple sequence alignment of the spike protein sequence of SARS-CoV-2 shows the number of single amino acid mutation hotspots such as L5F, R214L, R408I, G476S, V483A, H519Q, A520S, T572I, D614G and H655Y. Among these mutations D614G has 57.5% occurrence and G476S, V483A has 7.5% occurrence. The mutated proteins were modelled based on wild type homolog and docked to ACE2 receptor. When the mutated S protein is docked, the ∆G (binding free energy) value is very minimal in mutated protein showed the stability of variants. By the drug repurposing method, 1000 FDA approved drugs were virtually screened for its binding to RBD of S1 domain. Among these drugs Digitoxin, Gliquidone and Zorubicin Hcl binds to spike proteins with higher docking score (lesser than -8.5 Kcal/mol) to both wild type and mutants.

Pneumonia caused by a new coronavirus SARS-CoV-2 has caused serious harm to people's lives and health in Wuhan, China. By February 26, 2020, over 80,000 people were infected and 2,814 died from the infection. The initial route of infection is the binding of the spike protein (S protein) of the virus to the angiotensin-converting enzyme 2 (ACE2). From bioinformatics analysis, we found that the S protein of SARS-CoV-2 produced an evolutionary mutation of K403R compared with the S protein of SARS-CoV, forming an adjacent RGD motif at the interaction surface. As the RGD motif is considered as a ligand for many cell surface integrins, we proposed that the binding of S protein of SARS-CoV-2 with integrins may facilitate the infection process of the virus. Therefore, high-throughput virtual screening was performed by choosing the key residues of S protein interface of SARS-CoV-2 and the adjacent RGD motif as potential binding site, to search for the potential agents targeting interaction of S protein of SARS-CoV-2 with both ACE2 and integrins as potential therapeutic drugs. Various libraries including the FDA-approved drugs etc. were screened, and Nadide, Losartan, 9'''-Methyllithospermate B and Leonurine etc. were identified as representative potential drugs candidate for COVID-19.

Human RAD51 protein (HsRad51)-promoted DNA strand exchange, a crucial step in homologous recombination, is regulated by proteins and calcium ions. The activator protein Swi5-Sfr1 and Ca2+ ions stimulate different reaction steps and induce a perpendicular orientation of DNA bases in the presynaptic complex. To investigate the importance of base orientation in the strand exchange reaction, we examined the Ca2+ concentration dependence of strand exchange activities and structural changes in the presynaptic complex. Our results show that optimal D-loop formation (strand exchange with closed circular DNA) requires Ca2+ concentrations greater than 5 mM, while 1 mM is sufficient for strand exchange between two oligonucleotides. The structural change, which is evidenced by an increase in fluorescence intensity of poly(dεA) (a poly(dA) analog), reaches a plateau at 1 mM Ca2+. Meanwhile, the linear dichroism signal intensity at 260 nm, which is indicative of rigid perpendicular DNA base orientation, requires >2 mM Ca2+ for saturation and thus correlates with the stimulation of D-loop formation. Therefore, Ca2+ exerts two different effects. Thermal stability measurements suggest that HsRad51 binds two Ca2+ ions with KD values of 0.3 mM and 2.5 mM, implying that one step is stimulated by one Ca2+ bond and the other by two Ca2+ bonds. We further discuss the parallels between Mg2+ activation of RecA and Ca2+ activation of HsRad51.

The knowledge of protein function is essential for the study of biological processes, the understanding of disease mechanism and the exploration of novel therapeutic target. Apart from experimental methods, a number of in-silico approaches have been developed and extensively used for protein function prediction. Among these approaches, BLAST predicts functions based on protein sequence similarity, and machine learning predicts functional families from protein sequences irrespective of their similarity, which complements BLAST and other methods in predicting diverse classes of proteins including distantly related proteins and homologous proteins of different functions. However, their identification accuracies and the false discovery rate have not yet been assessed so far, which greatly limits the usage of these prediction algorithms. Herein, a comprehensive comparison of the performances among four popular functional prediction algorithms (BLAST, SVM, PNN and KNN) was conducted. In particular, the performance of these algorithms were systematically assessed by four metrics (sensitivity, specificity, accuracy and Matthews correlation coefficient) based on the independent test datasets generated from 93 protein families defined by UniProtKB Keywords. Moreover, the false discovery rates of these algorithms were evaluated by scanning the genomes of four representative model species (homo sapiens, arabidopsis thaliana, saccharomyces cerevisiae and mycobacterium tuberculosis). As a result, the substantially higher sensitivity and stability of BLAST and SVM were observed compared with that of PNN and KNN. But the machine learning algorithms (PNN, KNN and SVM) were found capable of significantly reducing the false discovery rate (SVM < PNN ≈ KNN). In summary, this study comprehensively assessed the performance of four popular algorithms applied to protein function prediction, which could facilitate the selection of the most appropriate method in the related biomedical research.

In order to study the effects of irradiation on the physicochemical properties and digestion behavior of chickpea protein, chickpea protein concentrate (CPC) was treated with electron beam irradiation (EBI) at doses of 5, 10, 15 and 20 kGy, respectively. After irradiation, protein solubility first increased at 10 kGy and 15 kGy, and then decreased at higher dose of 20 kGy. This was supported by SDS-PAGE where the intensity of major protein bands first increased and then decreased. Increased does of EBI generally led to greater oxidative modification of proteins in CPC, indicated by reduced sulfhydryls and increased carbonyls. In addition, protein structure was modified by EBI shown by Fourier transform infrared spectroscopy analysis where α-helix generally decreased and β-sheet increased. Although the protein digestibility was not significantly affected by EBI, the peptidomic analysis of the digests revealed significant difference among CPC irradiated with varying doses. A total of 337 peptides were identified from CPC irradiated with 0 kGy, 10 kGy and 20 kGy, respectively, with 18 overlapping peptides and 60, 29 and 40 peptides specific to the groups of 0, 10 and 20 kGy respectively. Theoretical calculation showed that the distribution of peptide length, hydrophobicity, net charge, and C-terminal residues were affected by irradiation. The 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity showed a marginal decrease with increasing dose of irradiation. In conclusion, EBI led to oxidative modification and structural changes of the chickpea protein, which subsequently affected the physicochemical properties of peptides obtained from in-vitro digestion of CPC despite of similar digestibility.

G protein-coupled receptors (GPCRs) are expressed essentially on all cells, facilitating cellular responses to external stimuli, and are involved in nearly every biological process. Several members of this family play significant roles in the regulation of adipogenesis and adipose me-tabolism. However, the expression and functional significance of a vast number of GPCRs in adipose tissue are unknown. We used a high-throughput RT-PCR panel to determine the expres-sion of the entire repertoire of non-sensory GPCRs in mouse white, and brown adipose tissue and assess changes in their expression during adipogenic differentiation of murine adipocyte cell line, 3T3-L1. In addition, the expression of GPCRs in subcutaneous adipose tissues from lean, obese, and diabetic human subjects was evaluated by re-analyzing RNA-sequencing data. We detected a total of 292 and 271 GPCRs in mouse white and brown adipose tissue, respectively. There is a significant overlap in the expression of GPCRs between the two adipose tissue depots but several GPCRs are specifically expressed in one of the two tissue types. Adipogenic differ-entiation of 3T3-L1 cells had a profound impact on the expression of several GPCRs. RNA se-quencing of subcutaneous adipose from healthy human subjects detected 255 GPCRs and obesity significantly changed the expression of several GPCRs in adipose tissue. Finally, we report sev-eral highly expressed GPCRs with no known role in adipose biology whose expression was sig-nificantly altered during adipogenic differentiation and/or in the diseased human subjects. These GPCRs could play an important role in adipose metabolism and serve as a valuable translational resource for obesity and metabolic research.

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.

Ustekinumab, a new anti-interleukin-12/23 antibody, is an effective treatment for ulcerative colitis; however, data regarding predictive factors of its efficacy are limited. This study aimed to identify biomarkers that can predict the long-term outcome of ustekinumab treatment. We retrospectively reviewed the records of patients with active ulcerative colitis treated with ustekinumab at our hospital from June 2020 to January 2023. We divided patients into non-remission and remission groups, and examined whether baseline biomarkers, including C-reactive protein-to-lymphocyte ratio, and early treatment response could predict clinical remission at week 48 of ustekinumab treatment. Of the 33 patients included in the study, 21 (63.6%) were in clinical remission at week 48 of ustekinumab treatment. The baseline C-reactive protein-to-lymphocyte ratio value was identified as an independent prognostic factor for clinical remission at week 48 (odds ratio: 10, 95% confidence interval: 1.6–62.4, p=0.014), with the cutoff value of 3.353 showing excellent prognostic performance (sensitivity: 71.4%, specificity: 83.3%). Furthermore, the clinical response at week 4 (p=0.009) and that at week 8 (p=0.005) were significantly associated with clinical remission at week 48. The baseline C-reactive protein-to-lymphocyte ratio value and early treatment response are useful biomarkers to predict the long-term efficacy of ustekinumab treatment.

The COVID-19 pandemic was declared in March 2020 and is still ongoing. The course of the epidemic remains uncertain, and mutations in the coronavirus, as well as the emergence of new variants of concern, make it possible that there will be periodic surges in the number of cases. Moreover, it is not clear how quickly and to what extent the effectiveness of vaccination will decline as the virus continues to mutate. One possible solution to combat the rapidly mutating coronavirus is the development of pan-coronavirus vaccines that stimulate the production of antibodies against conserved elements of the virus. Another approach could be the creation of safe vaccine platforms that can be rapidly adapted to deliver new, specific antigens in response to viral mutations. Recombinant probiotic microorganisms that can produce viral antigens by inserting specific viral DNA fragments into their genome show promise as a platform and vector for mucosal vaccine antigen delivery. With a well-established safety profile and the ability to transform into vaccine candidates, probiotic bacteria have significant research potential. The authors of this study have developed a convenient and universal technique for inserting the DNA sequences of pathogenic bacteria and viruses into the gene that encodes the pili protein of the probiotic strain E. faecium L3. The paper presents data on the immunogenic properties of two E. faecium L3 vaccine strains, which produce two different fragments of the coronavirus S1 protein, and provides an assessment of the protective efficacy of these oral vaccines against coronavirus infection in Syrian hamsters.

Arf-like protein 2 (ARL2) is a ubiquitously expressed small GTPase with multiple functions. In cell culture, ARL2 participates with tubulin cofactor D (TBCD) in the neogenesis of tubulin αβ-heterodimers, the building blocks of microtubules. To evaluate this function in retina, we conditionally deleted ARL2 in mouse retina at two distinct stages, either during embryonic development (retArl2-/-) or after ciliogenesis specifically in rods (rodArl2-/-). retArl2-/- retina sections displayed distorted nuclear layers and a disrupted microtubule cytoskeleton (MTC) as early as postnatal day 6 (P6). Rod and cone outer segments (OS) did not form. By contrast, rod ARL2 knockouts were stable at postnatal day 35 and revealed normal ERG responses. Cytoplasmic dynein is reduced in retArl2-/- inner segments (IS), suggesting that dynein may be unstable in the absence of a normal MTC. We investigated microtubular stability in the absence of either ARL2 (retARL2-/-) or DYNC1H1 (retDync1h1-/-), the dynein heavy chain, and found that both the retArl2-/- and retDync1h1-/- retinas exhibited reduced microtubules and nuclear layer distortion. The results suggest that ARL2 and dynein depend on each other to generate a functional MTC during early photoreceptor development.

Under the oxidative stress condition, the small RNA (sRNA) Oxys that acts as essential post-transcriptional regulators of gene expression is produced and plays a regulatory function with the assistance of the RNA chaperone Hfq protein. Interestingly, experimental studies found that the N48A mutation of Hfq protein could enhance the binding affinity with OxyS while resulting in defection of gene regulation. But, how the Hfq protein interacts with sRNA Oxys and the origin of the stronger affinity of N48A mutation are both unclear. In this paper, molecular dynamics (MD) simulations were performed on the complex structure of Hfq and OxyS to explore their binding mechanism. The molecular mechanics generalized Born surface area (MM/GBSA) and interaction entropy (IE) method were combined to calculate the binding free energy between Hfq and OxyS sRNA, and the computational result is in excellent correlation with the experimental result. Per-residue decomposition of the binding free energy revealed that the enhanced binding ability of the N48A mutation mainly comes from the increased van der Waals interactions (vdW). This research explores the binding mechanism between Oxys and chaperone protein Hfq, and revealed the origin of the strong binding affinity of N48A mutation. The results provided important insights on the mechanism of gene expression regulation affected by protein mutations.

Objective: Obstructive sleep apnea syndrome (OSAS) is a common sleep disorder that is caused by the reduction or cessation of airflow in the upper airway. Irisin, retinol-binding protein-4 (RBP-4), and adiponectin are the three significant factors in the metabolic process of the human body. The objective of this study was to investigate whether plasma irisin, RBP-4, and adiponectin levels are associated with the severity of OSAS. Methods: According to inclusion and exclusion criteria, 125 patients with OSAS and 46 healthy, gender matched controls were included to the study. The patients were classified according to the apnea hypopnea index (AHI) as 14 mild cases (5<AHI<15), 23 moderate OSAS cases (15<AHI<30) and 88 severe OSAS cases (AHI>30). The plasma irisin, RBP-4 and adiponectin levels were measured and compared between groups. Results: RBP-4 levels were higher in severe OSAS compared to moderate (p<0.05) and mild (p<0.001) OSAS groups, irisin levels were significantly lower compared to moderate (p<0.05) and mild (p<0.001) OSAS groups. There was a negative correlation between irisin and RBP-4 (r=-0.379; p<0.001) and AHI (r=-0.834; p<0.001) and a positive correlation with minimum oxygen saturation (MOS) (r=0.341; p<0.001) in the patient group. In the patient group, there was a negative correlation between adiponectin and BMI (r=-0.682; p<0.001), RBP-4 (r=-0.292; p<0.01) and AHI (r=-0.179; p<0.05) and a positive correlation with MOS (r=0.397; p<0.001). In the patient group, there was a positive correlation between RBP-4 and BMI (r=0.406; p<0.001) and AHI (r=0.473; p<0.001) and a negative correlation with MOS (r=-0.805; p<0.001). As a predictor of OSAS, adiponectin showed the highest specificity (84.8%) and RBP-4 the highest sensitivity (92.0%). Conclusion: The results of the present study demonstrated that the severity of OSAS causes more metabolic problems, including decreased plasma irisin and adiponectin levels and an increased plasma RBP-4 level. As a result, patients with an AHI score greater than thirty should be closely monitored for metabolic and cardiovascular abnormalities.

One of the most commonly known chronic neurodegenerative disorders, Alzheimer’s disease (AD), manifests the common type of dementia in 60–80% of cases. From a clinical standpoint, a patent cognitive decline and a severe change in personality, as caused by a loss of neurons, is~usually evident in AD with about 50 million people affected in 2016. The disease progression in patients is distinguished by a gradual plummet in cognitive functions, eliciting symptoms such as memory loss, and eventually requiring full-time medical care. From a histopathological standpoint, the~defining characteristics are intracellular aggregations of hyper-phosphorylated tau protein, known as neurofibrillary tangles (NFT), and depositions of amyloid β-peptides (Aβ) in the brain. The~abnormal phosphorylation of tau protein is attributed to a wide gamut of neurological disorders known as tauopathies. In addition to the hyperphosphorylated tau lesions, neuroinflammatory processes could occur in a sustained manner through astro-glial activation, resulting in the disease progression. Recent findings have suggested a strong interplay between the mechanism of Tau phosphorylation, disruption of microtubules, and synaptic loss and pathology of AD. The mechanisms underlying these interactions along with their respective consequences in Tau pathology are still ill-defined. Thus, in this review: (1) we highlight the interplays existing between Tau pathology and AD; and (2) take a closer look into its role while identifying some promising therapeutic advances including state of the art imaging~techniques.

Variable and sometimes contrasting effects of using artificial rhizobium inocula to increase grain legume performance have been reported. The objectives of this systematic review and meta-analysis were therefore to (i) estimate the overall effect of rhizobium inocula on grain yield and protein content and (ii) identify potential confounding factors that explain the observed variation. A web of Science database literature search was carried out in 2016. Data from all field experiments, greenhouse and pot experiments that compared grain yields and protein contents in Rhizobium/Bradyrhizobium-inoculated with non-inoculated control crops were extracted and analyzed using previously described unweighted and weighted meta-analysis protocols. GRADE assessments of the strength of evidence of results from the weighted meta-analyses were carried which included an assessment of the risk of publication bias. Results: For unweighted meta-analyses we identified eligible data in 236 and 36 studies for grain yield and protein content respectively. For weighted meta-analyses we identified eligible data in 92 and 8 studies for grain yield and protein content respectively. Rhizobium inoculation was found to (i) significantly increase grain yield in both the unweighted (mean percent difference: 41%, 95%CI 36,46, p&lt;0.0001) and weighted meta-analysis (Standardized mean difference: 1.56, 95%CI 1.26,1.87, p&lt;0.0001) and (ii) significantly increase protein content in the unweighted meta-analysis (mean percent difference: 11%, 95%CI 5,18, p&lt;0.0001), but not the weighed meta-analyses which only detected a trend (0.1&gt;p&gt;0.05) towards higher protein content in inoculated grain legume crops. Multi-level model-based weighted meta-analyses suggest that the (i) type of experiment, (ii) type of fertilizer used in experiments, (iii) grain legume type/species and (iv) the country in which experiments were carried out, were confounding factors. The main limitation of the evidence is that a large number of publications did not report measures of variation and could therefore not be included in the more scientifically sound weighted meta-analyses. However based on the large evidence-base available for grain yield it can be concluded that overall Bradyrhizobium/Rhizobium seed inoculation results can increase grain yield by ~40% compared with non-inoculated grain legume crops.

This study was first time designed to investigate the effect of oxidation on protein digestion and transport properties in boiled sea cucumber body wall (BSCBW) via simulated digestion combine with everted-rat-gut-sac models. Boiling heat treatments led to protein oxidation in SCBW, manifested by increases in free radical intensity, thiobarbituric acid reactive substances, carbonyl groups, disulfide bonds, dityrosine bonds, advanced glycation end products, protein hydrophobicity and aggregation, declines in free sulfhydryl group, and secondary structure transition from α-helix to β-sheet. Boiling for 2 h caused the anti-digestion collagen unfolding, provided the action site for protease, and improved the protein digestion and transport levels. On the contrary, excessive oxidative modification of 4 h-BSCBW resulted in decreased protein digestion and transport levels. From the perspective of texture, digestion and transport properties, boiling for 2 h can obtain sea cucumber products with better edible and digestible properties, which is considered to be a better processing condition.

Background: Cutaneous squamous skin cell carcinoma (SCC) is the second most frequent type of non- melanoma skin cancer and the second cause of death by skin cancer in caucasian population. However, at present it is difficult to predict patients with worst SCC prognosis. Objective: To identify proteins whose expression level could predict SCC infiltration in SCC arising from actinic keratosis (AC). Methods: A total of 20 biopsies of 20 different patients were studied, 10 were from SCC-AK samples and 10 from normal skin. Early infiltrated SCC-AK were selected on histological examination and to determine the expression of proteins fresh skin samples were processed by 2DE-electrophoresis Results: The expression levels of three proteins namely alpha-hemoglobin, heat shock protein (Hsp)-27 and 70 were significantly increased in SCC-AK samples with respect to normal control skin. However, only the expression level of Hsp70 protein positively correlated with the level of SCC-AK dermis infiltration. Immnunohistological examination suggested that the increased expression of Hsp70 proteins seems to mainly occur in the keratinocytes cytoplasm. The increased cytoplasmic Hsp70 expression in SCC-AK was confirmed by Western-blot experiments. Conclusion: Cytoplasmic expression of Hsp70 could be potential biomarker of early infiltration of SCC arising from an AK. Keywords: actinic keratosis, cutaneous squamous cell carcinoma; cytoplasm, skin cancer; heat shock protein.

Viruses frequently contain overlapping genes, which encode functionally unrelated proteins from the same DNA or RNA region but in different reading frames. Yet overlapping genes are often overlooked during genome annotation, in particular in DNA viruses. Here we looked for the presence of overlapping genes likely to encode a functional protein in human parvovirus B19 (genus erythroparvovirus), using an experimentally validated software, Synplot2. Synplot2 detected an open reading frame, X, conserved in all erythroparvoviruses, which overlaps the VP1 capsid gene, and is under highly significant selection pressure. In a related virus, human parvovirus (genus tetraparvovirus), Synplot2 also detected an open reading frame under highly significant selection pressure, ARF1, which overlaps the VP1 gene. X and ARF1 have exactly the same location (both overlap the region of VP1 encoding the phospholipase A2 domain), and encode proteins with similar predicted properties, such as a transmembrane region, strongly suggesting that they are homologous. These findings provide compelling evidence that the X protein must be expressed and functional. It is probably translated either from a polycistronic mRNA by a non-canonical mechanism, or from an unmapped monocistronic mRNA. Finally, we also discovered proteins predicted to be expressed from a frame overlapping VP1 in other species related to parvovirus B19: porcine parvovirus 2 (Z protein) and bovine parvovirus 3 (X-like protein).

Aptamers are versatile oligonucleotide ligands used for molecular recognition of diverse targets. However, application of aptamers to the field of amyloid β-protein (Aβ) has been limited so far. Aβ is an intrinsically disordered protein that exists in a dynamic conformational equilibrium, presenting time-dependent ensembles of short-lived, metastable structures and assemblies that have been generally difficult to isolate and characterize. Moreover, despite understanding of potential physiological roles of Aβ, this peptide has been linked to the pathogenesis of Alzheimer disease, and its pathogenic roles remain controversial. Accumulated scientific evidence thus far highlights undesirable or nonspecific interactions between selected aptamers and different Aβ assemblies likely due to metastable nature of Aβ or inherent affinity of RNA oligonucleotides to β-sheet-rich fibrillar structures of amyloidogenic proteins. Accordingly, lessons drawn from Aβ–aptamer studies emphasize that purity and uniformity of the protein target and rigorous characterization of aptamers’ specificity are important for realizing and garnering the full potential of aptamers selected for recongizing Aβ or other intrinsically disordered proteins. This review summarizes studies of aptamers selected for recognizing different Aβ assemblies and highlights controversies, difficulties, and limitations of such studies.

Increasing the availability of alternative protein from insects is important to solving food shortages. Not only are insects a rich source of protein, the use of insect ingredi-ents can reduce food waste. Insects are thus a potentially valuable ingredient for food industries and even food eaten on deep-space missions. The three-dimensional produc-tion of food on space missions has gained attention owing to its potential to reduce au-tonomous food production and produce sustainable food for long-duration space mis-sions. This study investigated the printability and rheological properties of a high-protein food system derived from mealworms and guar gum used as a stabilizer to im-prove printability. Stability and rheological properties were analyzed for various print-ing parameters. The results indicate that as the guar gum concentration was increased from 0 to 1.75%, the yield stress of the mealworm paste increased from 39 to 1096 Pa. Increasing the guar gum concentration thus resulted in a mealworm paste that was more viscous, exhibited shear thinning behavior, could support itself and was thus more stable. In summary, introducing guar gum resulted in a mealworm paste with rheological properties more suitable for printing in terms of printability and stability.

Type 2 diabetes mellitus may be complicated with chronic complications including diabetic neuropathy (DN). Timely diagnosis is crucial in treatment and maybe reversal of this complication. C reactive protein to albumin ratio (CAR) is considered as a marker of inflammation in various diseases that characterized with acute or chronic inflammation. Since diabetic neuropathy and type 2 diabetes mellitus are also associated with chronic, low grade inflammation, we aimed to study CAR levels of type 2 diabetic subjects with diabetic neuropathy and to compare to those in patients without diabetic neuropathy. Type 2 diabetic patients presented to our institutional outpatient clinics were divided into two according to the presence of DN. Subjects with DN were listed as DN group and others were as non-DN group. Characteristics and laboratory data, including CAR, in DN and non-DN groups were compared. Median CAR of the DN and non-DN groups were 2,19 (0,2-49)% and 0,56 (0,02-5,8)%, respectively (p&lt;0.001). CAR was significantly and positively correlated with age (r=0.15, p&lt;0.001), weight (r=0.19, p=0.01), BMI (r=0.11, p=0.03), waist circumference (r=0.10, p=0.046), fasting plasma glucose (r=0.14, p=0.004), serum creatinine (r=0.25, p&lt;0.001), triglyceride (r=0.17, p&lt;0.001), and LDL-cholesterol (r=0.13, p=0.001) levels. Moreover, CAR was inversely correlated with eGFR levels (r=-0.16, p&lt;0.001). The sensitivity and specificity of CAR (when higher than 1,02%) in predicting diabetic neuropathy were 78% and 73%, respectively (AUC: 0.84, p&lt;0.001, 95% CI: 0,82-0,87). Moreover, high CAR level was an independent risk factor of diabetic neuropathy (p&lt;0.001, OR: 1.34, 95% CI: 0.48-1.62). In conclusion, elevated CAR levels could be considered as a marker of diabetic neuropathy.

Flaviviruses replicate in membranous factories associated with the endoplasmic reticulum (ER). Significant levels of flavivirus polyprotein integration contribute to ER stress and the host cell may exhibit an Unfolded Protein Response (UPR) to this protein accumulation, stimulating appropriate cellular responses such as adaptation, autophagy or cell death. These different stress responses support other antiviral strategies initiated by infected cells and can help to overcome viral infection. In epithelial A549 cells, a model currently used to study the flavivirus infection cycle and the host cell responses, all three pathways leading to UPR are activated during infection by Dengue virus (DENV), Yellow Fever virus (YFV) or West Nile virus (WNV). In the present study, we investigated the capacity of ZIKA virus (ZIKV) to induce ER stress in A549 cells. We observed that the cells respond to ZIKV infection by implementing an UPR through activation of the IRE1 and PERK pathway without activation of the ATF6 branch. By modulating the ER stress response, we found that UPR inducers significantly inhibit ZIKV replication. Interestingly, our findings provide evidence that ZIKV could manipulate the UPR to escape this host cell defence system. Since incomplete UPR could lead to unresolved and persistent ER stress, we found that ZIKV infection was associated with an abnormal accumulation of viral envelope proteins, which were aggregated with non-native disulfide bridges. As the presence of these “amyloid like” protein polymers may be cytopathic, our observations provide new insights into specific neuropathologies associated with ZIKA virus infections.

Our previous studies demonstrated that quercetin (Q) could be ingested and metabolized by macrophages and exerted prophylactic immuno-stimulatory activity and therapeutic anti-inflammatory effects on lipopolysaccharide (LPS)-treated macrophages ex vivo. To further clarify its possible anti-inflammatory mechanism, Q was selected to treat mouse peritoneal macrophages that obtained from female BALB/c mice exposed to LPS i.p. for 12 h. Relative gene expression of pro-/anti-inflammatory (TNF-α/IL-10) cytokines and components of inflammation-related intracellular signaling pathways (TLR2, TLR4, NF-κB, JAK2 and STAT3) was analyzed using two-step reverse transcription (RT) and real-time quantitative polymerase chain reaction (qPCR). STAT3 protein phosphorylation was determined using an in-cell ELISA method. As a result, Q and its metabolite quercetin-3-O-β-D-glucuronide (Q3G) decreased TNF-α gene expression amounts and ratios of pro-/anti-inflammatory (TNF-α/IL-10) cytokine gene expressions, but increased IL-10 gene expression amounts in activated inflammatory macrophages, supporting a substantial anti-inflammatory potential of Q and Q3G treatments. However, Q3G had lower effects than those of Q. Importantly, Q inhibited TLR2 gene expression and phosphorylation of STAT3 protein in the inflamed cells. Our results are the first report to suggest that Q inhibits LPS-induced inflammation ex vivo through suppressing TLR2 gene expression and STAT3 protein phosphorylation in activated inflammatory macrophages. Q has potential to further apply for treating inflammation-associated diseases.

It is estimated that about 50% of geriatric rehabilitation patients suffer from sarcopenia. Thereby, malnutrition is frequently associated with sarcopenia and dietary intake is the main modifiable risk factor. During hospitalization, older adults are recommended to consume more dietary protein than the current recommended dietary allowance of 0.8 g/kg body weight per day to optimize the recovery of muscular strength and physical function. This prospective study examined the effect of a short-term protein supplementation with protein-enriched foods and drinks on hand-grip strength, nutritional status and physical function in older patients at risk of malnutrition during a three week inpatient orthopedic rehabilitation stay. The Mini Nutritional Assessment (MNA) was used to assess malnutrition. Patients with a MNA score ≤ 23.5 points were randomly assigned to an intervention group (goal: to consume 1.2–1.5 g protein/kg body weight per day) or a control group (standard care). Both groups carried out the same rehabilitation program. Physical recovery parameters were determined at admission and discharge. There was a trend for participants in the intervention group to consume more protein than the control group (P = 0.058): 95.3 (SD 13.2) g/day as compared to 77.2 (SD 24.2) g/day, which corresponds to a mean protein intake of 1.6 (SD 0.3) g/kg/day vs. 1.3 (SD 0.5) g/kg/day. Dietary protein supplementation increased body weight by an average of 0.9 (SD 1.1) kg and fat mass by an average of 0.9 (SD 1.2) kg as compared with baseline (P = 0.039 and P = 0.050, respectively). There was no significant change in hand-grip strength, body composition and physical function. In conclusion, a short-term intervention with protein-enriched foods and drinks enabled older patients at risk of malnutrition to increase their protein intake to levels that are higher than their required intake. In these older individuals with appropriate protein intake, dietary protein supplementation did not result in a greater improvement in physical recovery outcomes during short-term inpatient rehabilitation. The intervention improved dietary protein intake, but further research (e.g., a full-scale randomized controlled trial with sufficient power) is required to determine the effects on physical function outcomes.

The use of intelligent sensor-based packaging in food products provide quick evaluation of the quaility of the food. This study was done to develop bio-based color sensor from surimi-based color sensor incorporated with sappan wood extract (SWE) for smart food packaging. The SWE with different concentration (0.15%, 0.25%, and 0.35%) was incorporated to the myofibrillar protein-based edible film. The sappan wood-surimi edible film (SSEF) was subjected to physical properties analysis and the color changes at different pH values and soaking time at different condition were evaluated. Based on the results, different concentration of the SWE significantly (p&lt;0.05) affected the physical properties of the film. With the increasing of pH values, the darkness, redness, and blueness of the film was increased. Based on the evaluation of the SSEF with different soaking condition, the color changes of the film in acidic condition were more stable than in neutral and alkaline condition. The results from this study showed that SSEF have the possibilities to be used as a smart food packaging possessing the capabilities to act as color sensor due to its sensitivity to the changes in pH condition of the product.

The AT-HOOK MOTIF NUCLEAR LOCALIZED PROTEIN (AHL) gene family encodes embryophyte-specific nuclear proteins with DNA binding activity. They modulate gene expression and affect various developmental processes in plants. We identify AHL18 (At3G60870) as a developmental modulator of root system architecture and growth. AHL18 regulates the length of the proliferation domain and number of dividing cells in the root apical meristem and thereby, cell production. Both primary root growth and lateral root development respond according to AHL18 transcription level. The ahl18 knock-out plants show reduced root systems due to a shorter primary root and a lower number of lateral roots. This change results from a higher number of arrested and non-developing lateral root primordia (LRP) rather than from decreased initiation. Overexpression of AHL18 results in a more extensive root system, longer primary roots, and increased density of lateral root initiation events. Formation of lateral roots is affected during the initiation of LRP and later development. AHL18 regulate root apical meristem activity, lateral root initiation and emergence, which is in accord with localization of its expression.

Avian metapneumovirus subgroup C (aMPV/C) causes respiratory diseases and egg dropping in chickens and turkeys, resulting in severe economic losses to the poultry industry worldwide. Integrin β1 (ITGB1), a transmembrane cell adhesion molecule, is present in various cells and mediates numerous viral infections. Herein, we demonstrate that ITGB1 is essential for aMPV/C infection in cultured DF-1 cells, as evidenced by the inhibition of viral binding by EDTA blockade, Arg-Ser-Asp (RSD) peptide, monoclonal antibody against ITGB1, and ITGB1 short interfering (si) RNA knockdown in cultured DF-1 cells. Simulation of the binding process between the aMPV/C fusion (F) protein and avian-derived ITGB1 using molecular dynamics showed that ITGB1 may be a host-factor for benefiting aMPV/C attachment or internalization. Transient expression of avian ITGB1 rendered porcine and feline non-permissive cells (DQ cells and CRFK cells, respectively) susceptible to aMPV/C infection. Replication kinetic of aMPV/C in siRNA-knockdown cells revealed that ITGB1 plays an important role in aMPV/C infection at the early stage (attachment and internalization). aMPV/C was also able to infect efficiently human non-small cell lung cancer (A549) cells. This may be due to a consequence of the similar structures of both metapneumovirus F protein-specific motifs (RSD for aMPV/C and RGD for human metapneumovirus) recognized by ITGB1. Overexpression of avian-derived ITGB1 and human-derived ITGB1 in A549 cells enhanced aMPV/C infectivity. Taken together, this study demonstrated that ITGB1 acts as an essential receptor for aMPV/C attachment and internalization into host cells in which facilitates aMPV/C infection.

Medicinal plants possess a surplus of novel and biologically active secondary metabolites that are responsible for counteracting diseases. Traditionally, Gomphandra tetrandra (Wall.) Sleumer is used to treat mental disorders. The present research was designed to explore phytochemicals from the ethanol leaf extract of Gomphandra tetrandra (Wall.) Sleumer to identify the potential pharmacophore(s) in the treatment of neurological disorders. The chemical compounds of the experimental plant were identified through GC-MS analysis. Besides, in-vitro antioxidant activity was assessed using different methods. Furthermore, in-vivo neurological activity was assessed in Swiss-albino mice. Computer aided analysis was appraised to determine the best-fit phytoconstituent of a total of fifteen identified compounds in the experimental plant extract against beta-amyloid precursor protein. The experimental extract revealed fifteen compounds in GC-MS analysis and the highest content was 9, 12, 15-octadecatrienoic acid (Z, Z, Z). Also, the extract showed potent anti-oxidant activity in in-vitro assays. Furthermore, in in-vivo neurological assays, the extract disclosed significant (p<0.05) neurological activity. The most favorable phytochemicals as neurological agents were selected via ADMET profiling and molecular docking was studied with beta-amyloid precursor protein. Moreover, in the computer aided study, 1, 5-Diphenyl-2H-1, 2, 4-triazoline-3-thione (Pub Chem CID: 2802516) was more active than other identified compounds with strong binding affinity to beta-amyloid precursor protein. The present in vivo and in silico studies revealed neuropharmacological features of G. tetrandra leaves extract as a natural agent against neurological disorders, especially Alzheimer’s disease.

Currently, entire world is crumbled due to COVID-19 caused by novel SARS-CoV-2. Globally, over 5 million people are infected by SARS-CoV-2 with 6% fatality rate. The surface spike (S) protein plays a key role in the pathogenesis of SARS-CoV-2 by mediating viral entry through human angiotensin converting enzyme 2 (hACE2) receptors on the host cell and there is a big global race to find viral neutralizing antibodies and vaccine against S protein of SARS-CoV-2. Since SARS-CoV-2 evolved into 10 different clades in a very short span, a study on sipke protein mutation is essential to have effective vaccine coverage globally. Based on the mutation analysis of S protein from 166 Indian SARS-CoV-2 genome, a total of 40 different SNPs comprising of 14 synonymous and 26 non-synonymous mutations were observed, and notably, Indian S protein diverged into two major clusters, D614 and G614, with 11 different types. Majority of Indian strains fall in A2a and O clusters. Alarmingly, we have observed six SNPs at RBD and notably two of them at RBM (S438F and S494P). S494P SNP, similar to Bat–SARS like-CoV, may indicate a low ACE2 binding affinity. Interestingly 38% of Indian strains harbor a characteristic D614G SNP which was found predominantly in A2a cluster, mostly comprising USA and European strains with high disease severity. The association of disease severity with D614G SNP is well-correlated in states with high death rate except Maharashtra. Notably, more than 50% of D614G mutation were observed in Northern part of India and 14% in Southern part but not in Kerala and Tamil Nadu strains. Highly conserved motif, D614 (608-VAVLYQDVNCT-618) in upstream and also few downstream, of S1/S2 furin cleavage site may indicate specific key role in efficient interaction with host proteases in pathogenesis. Further studies are warranted to clarify the impact of SD614G SNP association to disease severity . Interestingly, C2367T (Y789Y) synonymous SNP is observed in 37% of Indian strains and notably similar SNPs with degeneracy bases were observed which is a key indication for the possibility of misdiagnosis by Real-Time PCR and revised strategies are needed for the precise diagnosis. Circulation of high number of signature SNPs [D614G and C2367T (Y789Y)] in certain states may be an early indication of emergence of community transmission in India. Further large genome sequence data from India will aid in deep understanding on the diversity of circulating SASR-Cov-2 and its impact on disease severity, origin of imported cases to India, community spread, effect on diagnosis and vaccine coverage.

Fluctuations of protein three-dimensional structures and large-scale conformational transitions are crucial for the biological function of proteins and their complexes. Experimental studies of such phenomena remain very challenging and therefore molecular modeling can be a good alternative or a valuable supporting tool for the investigation of large molecular systems and long-time events. In this mini-review, we present two alternative approaches to the coarse-grained (CG) modeling of dynamic properties of protein systems. We discuss two CG representations of polypeptide chains used for Monte Carlo dynamics simulations of protein local dynamics and conformational transitions and, on other hand, highly simplified structure-based Elastic Network Models of protein flexibility. In contrast to classical Molecular Dynamics the modeling strategies discussed here allow quite accurate modeling of much larger systems and longer time dynamic phenomena. We briefly describe the main features of these models and outline some of their applications, including modeling of near-native structure fluctuations, sampling of large regions of the protein conformational space, or possible support for the structure prediction of large proteins and their complexes.

Rotavirus species H (RVH) has been detected in pigs, humans and bats. Moreover, porcine RVHs have been recently identified in several swine-producing countries. Despite their zoonotic impact, genome information of RVHs is still limited. This study aimed to establish a tentative complete genome-based genotyping system for RVHs, by appending genomic sequences from 12 porcine RVHs identified in Japan between 2013 and 2015 to those from human and other porcine RVHs reported in previous studies. Phylogenetic analysis of 11 RNA segments indicated that porcine RVHs could be classified into multiple genotypes. Consequently, the genotype classification for RVHs revealed the existence of genotypes 10G, 6P, 6I, 3R, 4C, 7M, 6A, 2N, 4T, 6E, 3H for the genes VP7, VP4, VP6, VP1, VP2, VP3, NSP1, NSP2, NSP3, NSP4 and NSP5, respectively. Surprisingly, two distinctive types in NSP1 and NSP3 genes were identified from among the twelve porcine RVHs. Our data suggest a potentially novel gene rearrangement event between porcine RVH and rotavirus species C in the NSP3 gene. These findings would provide a new insight in understanding for evolution of RV.

Cell-penetrating peptides (CPPs), a group of small peptides capable of promoting the transport of molecular cargo across the plasma membrane, have become important tools in promoting the cellular uptake of exogenously delivered macromolecules. GV1001, a peptide derived from a reverse-transcriptase subunit of telomerase (hTERT) and developed as a vaccine against various cancers, reportedly has unexpected CPP properties. Unlike typical CPPs, such as the HIV-1 TAT peptide, GV1001 enabled the cytosolic delivery of macromolecules such as proteins, DNA and siRNA via extracellular heat shock protein 90 (eHSP90) and 70 (eHSP70) complexes. The eHSP-GV1001 interaction may have biological effects in addition to its cytosolic delivery function. GV1001 was originally designed as a MHC class II-binding cancer epitope, but its CPP properties may contribute to its strong anti-cancer immune response relative to other telomerase peptide-based vaccines. Cell signaling via eHSP-GV1001 binding may lead to unexpected biological effects, such as direct anticancer or antiviral effects. In this review, we focus on the CPP effects of GV1001 bound to eHSP90 and ehsp70.

Structural and biochemical studies have recently revealed a range of rationally engineered nanobodies with efficient neutralizing capacity against SARS-CoV-2 virus and resilience against mutational escape. In this work, we combined atomistic simulations and conformational dynamics analysis with the ensemble-based mutational profiling of binding interactions for a diverse panel of SARS-CoV-2 spike complexes with nanobodies. Using this computational toolkit, we identified dynamic signatures and binding affinity fingerprints for the SARS-CoV-2 spike protein complexes with nanobodies Nb6 and Nb20, VHH E, a pair combination VHH E+U, a biparatopic nanobody VHH VE, and a combination of CC12.3 antibody and VHH V/W nanobodies. Through ensemble-based deep mutational profiling of stability and binding affinities, we identify critical hotspots and characterize molecular mechanisms of SARS-CoV-2 spike protein binding with single ultra-potent nanobodies, nanobody cocktails and biparatopic nanobodies. By quantifying dynamic and energetic determinants of the SARS-CoV-2 S binding with nanobodies, we also examine the effects of circulating variants and escaping mutations. We found that mutational escape mechanisms may be controlled through structurally and energetically adaptable binding hotspots located in the host receptor-accessible binding epitope that are dynamically coupled to the stability centers in the distant epitope targeted by VHH U/V/W nanobodies. The results of this study suggested a mechanism in which through cooperative dynamic changes, nanobody combinations and biparatopic nanobody can modulate the global protein response and induce the increased resilience to common escape mutants.

DDX3 is an ATP-dependent RNA helicase involved in multiple cellular activities, including RNA metabolism and innate immunity. DDX3 is known to assist the replication of some viruses while restricting some others through direct interaction with the viral proteins. However, the role of DDX3 in the replication of the hepatitis E virus (HEV) is unknown. In this study, DDX3 is shown to interact with the HEV capsid protein and provide an indispensable role in HEV replication. The DDX3 C-terminal domain was demonstrated to interact with the capsid protein, which was previously demonstrated to inhibit the production of type I interferons. Knockdown of DDX3 compromised the capsid protein-mediated blockage of interferon induction. Notably, DDX3 silencing led to a significant reduction in HEV replication. Also, the ATPase activity of DDX3 is required for the HEV replication as an ATPase-null mutant DDX3 failed to rescue the viral replication in the DDX3-silenced cells. These results demonstrate a pro-viral role of DDX3 in HEV replication, providing further insights into the virus-cell interactions.

Many of the wide-ranging symptoms of COVID-19 have not been associated previously with a respiratory illness. A similarity between these atypical symptoms and the symptoms of malnutrition was observed. To investigate, a comparison of COVID-19 symptomatology with recognised states of malnutrition showed a significant relationship that was further explored in a review of the literature. This hypothesis links the atypical symptoms to the effects of increasing protein malnutrition. The rapid appearance of malnutrition symptoms at the beginning of the COVID-19 infection is concurrent with the rapid cell proliferation of the immune system. Although previous work has, in general, assumed that the body is capable of providing for the needs of the immune system during infection, heightened immune activity from the commencement of COVID-19 could require an early increase in energy yielding sources. Should insufficient availability of protein resources occur, this could quickly lead to malnutrition. Any continuing insufficiency, during the active phase of the virus, might result in widespread depletions in the tissues and the consequential malfunctioning of many organs. A new appraisal of the immune system requirements needed to counteract the impact of the novel virus, COVID-19, could show the potential benefit for earlier protein intervention in the disease process. This hypothesis could also have relevance for other life situations where a rapid cell proliferation might occur.

Porcine epidemic diarrhea virus (PEDV) has affected the pork industry worldwide and during outbreaks the mortality of piglets has reached 100%. Lipid nanocarriers are commonly used in the development of immunostimulatory particles due to their biocompatibility and slow-release delivery properties. In this study, we developed a lipid nanoparticle (LNP) complex based on glycyrrhizinic acid (GA) and tested its efficacy as an adjuvant in mice immunized with the recombinant N-terminal domain (NTD) of porcine epidemic diarrhea virus (PEDV) spike (S) protein (rNTD-S). The dispersion stability analysis (Z-potential–27.6 mV) confirmed the size and charge stability of the LNP-GA, demonstrating that the particles were homogeneously dispersed and strongly anionic, that favors nanoparticles binding with the rNTD-S protein, which showed a slightly positive charge (2.11 mV) by in silico analysis. TEM image of LNP-GA revealed nanostructures with a spherical-bilayer lipid vesicle (⁓100 nm). The immunogenicity of the LNP-GA-rNTD-S complex induced an efficient humoral response 14 days after the first immunization (P &lt; 0.05) as well as an influence on the cellular immune response by decreasing serum TNF-α and IL-1β concentrations, which was associated with an anti-inflammatory effect.

Background: Food waste has been a major social problem in recent years. Annually, 1.3 billion tons of food is wasted worldwide. It is recommended that food waste should be reduced at every stage of production. By-products from food processing have high nutritional value; therefore their use in the design of new products is advisable. Methods: By-products from tomato processing (tomato waste-TW) and pepper (defatted pepper seeds-DPS, pepper placenta-PP) were used at a level of 10-30% to produce extruded pasta. The farinographic characteristics, chemical composition, cooking quality, and color of the pasta were studied. Results: The results showed a significant up to 27% increase in the protein content in the TW30 samples, compared to the control. The TDF content increased over five times in DPS30 and TW30 (27.99% and 25.44%). The amino acid composition of the pasta improved with the fortification, but failed to achieve the complete protein by FAO. The DPS30, PP20, PP30, and all TW samples can be considered high-protein products according to the EU definition (min. 20% energy from protein). Conclusion: Vegetable waste can be a valuable additive for improvement of the nutritional value of food. Their addition is part of the zero-waste trend, which has a positive impact on the environment.

Porcine circovirus type 2 (PCV2) is a small non-enveloped DNA virus that causes swine immunosuppression by inducing apoptosis in lymphocytes. The ORF3 protein plays a major role in PCV2-induced apoptosis in porcine kidney cells, but there is little information regarding this protein in PCV2-infected lymphocytes. In this study, hybridoma screening and epitope mapping were determined by using an indirect ELISA. The mAb 7D3 against ORF3 peptide (residues 35–65) of PCV2 were generated in this study. In vivo situation, the mAb 7D3 recognized ORF3 protein existed in PCV2-infected apoptotic porcine PBMCs. It is noteworthy that thimerosal interfered with the binding of mAb 7D3 to epitope and it was diminished by adding cysteine. Additionally, thimerosal interacting with cysteine-containing peptide was demonstrated by the PTI assay. Furthermore, thimerosal specifically interacted with the antigen-binding sites of mAb 7D3. This study suggested that thimerosal blockade the occlusion of the antigen-binding sites of mAb 7D3 to bind ORF3 peptide (residues 35–65) via thimerosal interacting with cysteine residues which should be located within the antigen-binding sites of mAb 7D3. Overall, the mAb 7D3 has been characterized and it will be a valuables tool in future studies of ORF3 function and the wider mechanism of cell apoptosis caused by PCV2 infection. Similarly, these techniques will be useful for applications in detecting thimerosal too.

Staphylococcus aureus is a common pathogen in human. Methicillin resistant Staphylococcus aureus (MRSA) infection poses a big and perplexing difficulty in terms of therapy. The acquisition of the non-native gene PBP2a, which has a decreased tolerance for β-lactam antibiotics, frequently confers resistance. PBP2a has a less attraction for methicillin and it helps bacteria to continue peptidoglycan biosynthesis, and it is cell wall’s core component in bacteria. So, even in the presence of methicillin or any other antibiotic, bacteria develop resistance. Due to resistance-causing genes, S. aureus becomes MRSA. The main premise of the resistance mechanism is well understood. The current demand for novel antibiotics is legitimate in the face of therapeutic concerns posed by resistant micro-organisms. The emphasis of this review is on PBP2a scaffolds and the different screening approaches used to find PBP2a inhibitors. Penicillin, Cephalosporins, Pyrazole-Benzimidazole based derivatives, Oxadiazole containing derivatives, non-β-lactam allosteric inhibitors, 4-(3H)-Quinazolinones, Pyrrolylated chalcone, Bis-2-Oxoazetidinyl macrocycle (β-lactam antibiotics with 1, 3-Bridges), Macrocycle-embedded β-lactams as novel inhibitors, Pyridine-Coupled Pyrimidinones, novel Naphthalimide corbelled aminothiazoximes, non-covalent inhibitors, Investigational-β-lactam antibiotics Carbapenem, novel Benzoxazole derivatives, Pyrazolylpyridine analogues, and other miscellaneous classes of scaffolds for PBP2a are also represented as well as with their biological activity is discussed. The penicillin-binding protein is also discussed, which is the crucial target for the cell wall of MRSA. Various aspects of PBP2a, the cell wall of bacteria, peptidoglycans, different crystal structures of PBP2a, synthetic routes for PBP2a inhibitors, and future perspectives of MRSA inhibitors are also enumerated.

Base on the sequence of S genes, which encode spike proteins, we previously identified three different types (North American, S INDEL and S large-DEL types) of porcine epidemic diarrhea virus (PEDV) that have re-emerged in Japan since 2013. Base on experimental infections with the North American and S large-DEL types, we also hypothesized that PEDV virulence may be linked to the S1 subunit of the S protein. To test this hypothesis, we have now assayed in gnotobiotic piglets various recombinant PEDVs generated by reverse genetics. Piglets inoculated with CV777 maintained in National Institute of Animal Health, along with piglets infected with a recombinant form of the same virus, developed subclinical to mild diarrhea. In contrast, severe watery diarrhea, dehydration, weight loss, astasia, and high mortality were observed in piglets inoculated with recombinant strains in which the S gene was partially or fully replaced with corresponding sequences from the highly virulent Japanese PEDV isolate OKN-1/JPN/2013. Indeed, symptoms resembled those in piglets inoculated with the OKN-1/JPN/2013, and were especially pronounced in younger piglets. Collectively, the data demonstrate that the S1 subunit of the S protein is an important determinant of PEDV virulence, and advance development of new vaccine candidate.