The World Health Organization has estimated an annual occurrence of approximately 392 million Dengue virus (DENV) infections in more than 100 countries where the virus is endemic, and this represents a serious threat to humanity. DENV is a serologic group with four distinct serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) belonging to the genus Flavivirus, in the family Flaviviridae. Dengue is the most widespread mosquito-borne disease in the world. The ~10.7 kb DENV genome encodes three structural proteins (capsid [C], pre-membrane [prM], and envelope [E]) and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The NS1 protein is a membrane-associated dimer and a secreted, lipid-associated hexamer. Dimeric NS1 is found on membranes both in cellular compartments and cell surfaces. Secreted NS1 (sNS1) is often present in patient serum at very high levels, which correlates with severe dengue symptoms. This study was conducted to discover how NS1 protein, microRNAs-15/16 (miRNAs-15/16), and apoptosis are related during DENV-4 infection in human liver cell lines. Huh 7.5 and HepG2 cells were infected with DENV-4, and miRNAs-15/16, viral load, NS1 protein, and caspases-3/7 were quantified after different times of infection. This study demonstrated that miRNAs-15/16 are overexpressed during infection of HepG2 and Huh 7.5 cells by DENV-4 and have a relationship with NS1 protein expression, viral load, and activity of caspases-3/7, thus making these miRNAs potential injury markers during DENV infection in human hepatocytes.

Short Linear Motifs (SLiMs) are short linear sequences that can mediate protein-protein interaction. Mimicking eukaryotic SLiMs to compete with extra or intracellular binding partners or to sequester host proteins is the crucial strategy of viruses to pervert the host system. The evolved proteins in viruses facilitate minimal protein-protein interactions that significantly affect intracellular signaling networks. Unfortunately, very little information about the SARS-CoV-2 SLiMs is known, especially across the SARS-CoV-2 variants. Through ELM database-based sequence analysis of spike protein from all the major SARS-CoV-2 variants, we identified four overriding SLiMs in the SARS-CoV-2 Omicron variant including LIG_TRFH_1, LIG_REV1ctd_RIR_1, LIG_CaM_NSCaTE_8, and MOD_LATS_1. These SLiMs are highly likely to interfere with various immune functions, interact with host intracellular proteins, regulate cellular pathways, and lubricate viral infection and transmission. These cellular interactions possibly serve as potential therapeutic targets for these variants, and this approach can be further exploited to combat emerging SARS-CoV-2 variants.

Photothrombotic stroke (PTS) stimulates the level of N- and C-terminal fragments of Amyloid precursor protein (APP) growth in the cytoplasm of ischemic penumbra cells not earlier but at 24 hours. Here we have shown that APP fragments are visualized in thin unmyelinated fibers of neurons, in containing mitochondria large fibers and in synapses but absent in the nuclei. At 24 hours after PTS, some elements of the destroyed tissue accumulated a significant amount of APP protein. The level of ADAM10 α-secretase decreased on the first day after PTS in the rat brain cortex and ADAM-10 co-localized with the lipid raft marker caveolin-1. PTS caused no changes in the level of β-secretase BACE1 either on the first day after PTS or in the early recovery period. The expression of proteins of the γ-secretase complex: presenilin-1 and nicastrin increased in astrocytes, but not in penumbra neurons after PTS. The β-secretase inhibitor LY2886721 did not affect the infarct size of the mouse cerebral cortex and the level of apoptosis of cells in the perifocal region after PTS. Whereas the inhibitor of γ-secretase DAPT reduced the expression of glial fibrillary acidic protein (GFAP) in astrocytes, prevented the growth of apoptosis of mouse cerebral cortex cells reducing the infarct volume on the 7th and 14th days after PTS. DAPT may be considered as a drug for stroke therapy.

The study compared the influence of chitosan sources on rumen fermentation, methane emission and milk production in lactating dairy cows fed a glycerin-based diet. Six, lactating Holstein-Frisian crossbreeds (410 ± 5.0 kg BW, 120 ± 21 day-in-milk), were arranged in a 3 x 3 replicated Latin square design. In addition to control, a 2% chitosan extract supplement and a 2% commercial chitosan supplement of dry matter intake were the treatments. The results denoted that no significant differences on daily dry matter, nutrients or estimated energy intake were noted when cows received different sources of chitosan. Nutrient digestibility was not influenced differently by extraction based or commercial chitosan supplements. The pH, temperature, ammonia nitrogen, blood urea and microbial count were similar among treatments. The different sources of chitosan supplements did not change the totals of volatile fatty acids, acetate and butyrate; in contrast, different chitosan sources influenced (P<0.05) propionate content. The ruminal acetate to propionate ratio was markedly (P<0.05) reduced with chitosan supplement, but no change appeared between sources of chitosan. At 4 hours after feeding, the methane estimation significantly decreased with the addition of chitosan supplementation (P<0.05) compared to the control group. The purine derivatives and microbial protein synthesis were not altered by the treatments. No significant differences existed on milk yield, milk composition or milk urea nitrogen when cows received different sources of chitosan (P>0.05). In sum, supplementing extracted chitosan showed more potential than did commercial chitosan for enhancing economic efficiency and recycling shrimp residues, therefore, reducing environmental waste.

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.

Crimean Congo hemorrhagic fever virus (CCHFV) is the causative agent of a globally-spread tick-borne zoonotic infection with an eminent risk of fatal human disease. Imminent public health threat posed by disseminated virus activity and lack of an approved therapeutic make CCHFV an urgent target for vaccine development. We described the construction of a DNA vector expressing nucleocapsid protein (N) of CCHFV (pV-N13) and investigated its potential to stimulate cytokine and total/specific antibody responses in BALB/c and challenge experiment in IFNAR-/- mice. Due to lack of sufficient antibody stimulation towards N protein, we have selected CD24 protein as a potential adjuvant which has proliferative effect on B and T cells. Overall, our N expressing construct when administered solely or in combination with pCD24 vector elicited significant cellular and humoral responses in BALB/c, despite variations in particular cytokines and total antibodies. However, the stimulated antibodies produced due to expression of N protein have shown no neutralizing ability in VNA. Furthermore, challenge experiments were revealed protection potential of N expressing construct in IFNAR -/- mice model. In conclusion, we have shown that CD24 has prominent effect as a genetic adjuvant when co-delivers with a synergic foreign gene expressing vector. Besides, targeting of S segment of CCHFV can be considered as a practical way in developing vaccine against this virus due to its ability to induce immune response which leads to protection in challenge assays in IFN-gamma defective mice models.

The Late Heavy Bombardment Period (4.1 to 3.8 billion years ago) of heightened impact cratering activity on young Earth is likely the driving force for the origin of life. During the Eoarchean, asteroids such as carbonaceous chondrites delivered the building blocks of life and water to early Earth. Asteroid collisions created innumerable hydrothermal crater lakes in the Eoarchean crust which inadvertently became the perfect cradle for prebiotic chemistry. These hydrothermal crater lakes were filled with cosmic water and the building blocks of life. forming a thick prebiotic soup. The unique combination of exogenous delivery of extraterrestrial building blocks of life, and the endogenous biosynthesis in hydrothermal impact crater lakes very likely gave rise to life. A new symbiotic model for the origin of life within the hydrothermal crater lakes is here proposed. In this scenario, life arose around four billion years ago through five hierarchical stages of increasing molecular complexity: cosmic, geologic, chemical, information, and biological. During the prebiotic synthesis, membranes first appeared in the hydrothermal crater lakes, followed by the simultaneous origin of RNA and protein molecules, creating the RNA/protein world. These proteins were noncoded protein enzymes that facilitated chemical reactions. RNA molecules formed in the hydrothermal crater basin by polymerization of the nucleotides on the montmorillonite mineral substrate. Similarly, the initial synthesis of abiotic protein enzymes was mediated by the condensation of amino acids on pyrite surfaces. The regular wet-dry cycles within the crater lakes assisted further concentration, condensation, and polymerization of the RNAs and proteins. Lipid membranes randomly encapsulated amino acids, RNA, and protein molecules from the prebiotic soup to initiate a molecular symbiosis inside the protocells, this led to the hierarchical emergence of several cell components. As the role of protein enzymes became essential for catalytic process in the RNA/protein world, Darwinian selection from noncoded to coded protein synthesis led to translation systems and the genetic code, heralding the information stage. In this stage, the biochemical pathways suggest the successive emergence of translation machineries such as tRNAs, aaRS, mRNAs, and of ribosomes for protein synthesis. The molecular attraction between tRNA and amino acid led to the emergence of translation machinery and the genetic code. tRNA is an ancient molecule that created mRNA for the purpose of storing amino acid information like a digital strip. Each mRNA strand became the storage device for genetic information that encoded the amino acid sequences in triplet nucleotides. As information became available in the digital languages of the codon within mRNA, biosynthesis became less random and more organized and directional. The original translation machinery was simpler before the emergence of the ribosome than that of today. We review three main concepts on the origin and evolution of the genetic code: the stereochemical theory, the coevolution theory, and adaptive theory. We believe that these three theories are not mutually exclusive, but are compatible with our coevolution model of translations machines and the genetic code. We suggest biosynthetic pathways as the origin of the translation machine that provided the framework for the origin of the genetic code. During translation, the genetic code developed in three stages coincident with the refinement of the translation machinery: GNC code with four codons and four amino acids during interactions of pre-tRNA/pre-aaRS /pre-mRNA, SNS code consisting of 16 codons and 10 amino acids appeared during the tRNA/aaRS/mRNA interaction, and finally the universal genetic code evolved with the emergence of the tRNA/aaRS/mRNA/ribosome machine. The universal code consists of 64 codons and 20 amino acids, with a redundancy that minimizes errors in translation. To address the question of the origin of the biological information system in the RNA/protein world, we converted letter codons into numerical codons in the Universal Genetic Code Table. We developed a software called CATI (Codon-Amino Acid-Translator-Imitator) to translate randomly chosen numerical codons into corresponding amino acids and vice versa, gaining insight into how translation might have worked in the RNA/protein world. We simulated the likely biochemical pathways for the origin of translation and the genetic code using the Model-View-Controller (MVC) software framework, and the translation machinery step-by-step. We used AnyLogic software to simulate and visualize the evolution of the translation machines and the genetic code. We conclude that the emergence of the information age from the RNA/protein world was a watershed event in the origin of life about four billion years ago.

Nucleocytoplasmic large DNA viruses (NCLDVs) have massive genomes and particle sizes compared to other known viruses. NCLDVs, including poxviruses, encode ATPases of the FtsK/HerA superfamily to facilitate genome encapsidation. However, their biochemical and structural characteristics are yet to be discerned. In this study, we demonstrate that the viral ATPases are significantly shorter than their bacterial homologs, representing the minimal ATPase core of the FtsK/HerA superfamily. We analyzed the sequence and structural features of the vaccinia virus A32 protein and determined their roles in the stability of the A32 protein and its ATPase activity. We sought to purify A32 by various techniques and noted that recombinant A32 expressed in E. coli is highly insoluble and unstable in solution, possibly because of the disordered C-terminus. N-terminal fusion with the thioredoxin solubility tag could alleviate this issue to some extent, but subsequent tag cleavage results in increased susceptibility to precipitation and degradation. We also predicted a conserved coiled-coil motif (CCM) towards the C-terminus of VV A32. ATPase activity of A32 is known to increase in the presence of DNA. Our comparative analysis of wildtype A32 versus CCM mutants suggests that this DNA dependence of A32’s ATPase activity is likely regulated by the CCM. Since CCM is also known to facilitate protein oligomerization, these findings provide new opportunities for further detailed characterization of A32.

Due to its devastating consequences, late life depression is an important public health problem. The aim of the study was the analysis of variables which may potentially influence risk of depression (GDS-SF). Furthermore, the aim was to study possible mediating effect of given variables on the relationship between the total protein concentration and risk of depression in older-adults with chronic diseases, and physical function impairment. The research sample included a total of 132 older adults with chronic conditions and physical function impairments, remaining under a long-term care in residential environment. Negative linear correlation was observed between patients’ physical functionality, total protein concentration, concentration of HDL cholesterol, arm circumference, and the risk of depression. Considerably stronger relationship was observed between total protein concentration, and GDS-SF, in elderly suffering from sensory dysfunction (b = −6.42, 95% CI = −11.27; −1.58). The effect of the mediation between depression risk is correlated to total protein concentration in blood serum, and the mediators are probably low function impairment and low levels of 25 (OH)D vitamin. Cohort control research is suggested to confirm the hypothesis.

The SARS-CoV-2 nucleocapsid protein (N protein) is critical to viral replication by undergoing liquid-liquid phase separation to seed the formation of a ribonucleoprotein (RNP) complex to drive viral genomic RNA (gRNA) translation and in also suppressing both stress granules and processing bodies which is postulated to increase uncoated gRNA availability. The N protein can also form biomolecular condensates with a broad range of host endogenous proteins including RNA binding proteins (RBPs). Amongst these RBPs are proteins that are associated with pathological neuronal and glial cytoplasmic inclusions across several adult-onset neurodegenerative disorders, including TAR DNA binding protein 43 kDa (TDP-43) which forms pathological inclusions in over 95% of amyotrophic lateral sclerosis cases. In this study, we demonstrate that the N protein can form biomolecular condensates with TDP-43 and that this is dependant on the N protein C-terminus domain (N-CTD) and the intrinsically disordered C-terminus domain of TDP-43. This process is markedly accelerated in the presence of RNA. In silico modelling suggests that the biomolecular condensate that formed in the presence of RNA is composed of a N protein quadriplex in which the intrinsically disordered TDP-43 C terminus domain is incorporated.

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.

Transcription factors (TFs) regulate gene expression by recognizing specific target enhancers in the genome. The DNA-binding and regulatory activity of TFs depend on the presence of additional protein partners, leading to the formation of versatile and dynamic multimeric protein complexes. Visualizing these protein-protein interactions (PPIs) in the nucleus is key for decrypting the molecular cues underlying TF specificity in vivo. Over the last years, Bimolecular Fluorescence Complementation (BiFC) has been developed in several model systems and applied for the analysis of different types of PPIs. In particular, BiFC has been applied for analyzing PPIs with hundreds of TFs in the nucleus of live Drosophila embryos. However, the visualization of PPIs at the level of specific target enhancers or genomic regions of interest awaits the advent of DNA-labelling methods that could be coupled with BiFC. Here, we present a novel experimental strategy that we called BiFOR and that is based on the coupling of BiFC with the bacterial ANCHOR DNA-labelling system. We demonstrate that BiFOR enables to precisely quantify the enrichment of specific dimeric protein complexes on target enhancers in Drosophila salivary gland nuclei. Given its versatility and sensitivity, BiFOR could be applied more largely in other tissues during Drosophila development. Our work set up the experimental basis for these future applications.

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.

The aim of this study was to investigate the biochemical properties and gel-forming capacity of duck myofibrillar proteins under the effects of 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH)-mediated oxidation. Duck myofibrillar proteins were extracted and treated with different concentrations of AAPH solutions (0, 1, 3, 5, 10 mmol/L) and then analysed for carbonyl content, dynamic rheology, protein profiles and gel-forming properties (colour, water holding capacity, hardness, protein profile, microstructure). The results showed that with increasing AAPH concentration, the carbonyl content of protein showed an increasing trend (p &lt; 0.05); SDS-PAGE pattern results indicated that moderate oxidation (3 mmol/L AAPH) induced myosin aggregation via covalent bonds including disulfide, enhanced protein-protein, and thus improved protein-water interactions and gel strength of DMPs heat-induced gels. However, high oxidation (5 and 10 mmol/L AAPH) led to partial degradation of myosin heavy chain (MHC), as evidenced by lower storage modulus and irregular microstructure, which significantly reduced gelation ability. These results suggest that the internal relationship between alkanoperoxy radical-induced oxidation should be taken into account in the processing of duck meat, as mild protein oxidation is conducive to improving gel quality.

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

Background: Chagas disease is still a neglected disease considered a public health problem. Early diagnosis of cases is important to improve the prognosis of infected patients and prevent transmission. Serological tests are the method of choice for diagnosis. However, two serological tests are currently recommended to confirm positive cases. In this sense, more sensitive and specific serological tests need to be developed to overcome the problems currently faced. This study aimed to develop a new recombinant multiepitope protein for the diagnosis of Chagas disease, hereafter named rTC. Methods: The rTC was constructed based on amino acid sequences from different combinations of Trypanossoma cruzi antigens in the same polypeptide and tested in ELISA for detecting different Chagas disease. Results: rTC1 was able to discriminate between indeterminate (IND) and cardiac (CARD) cases and cross-reactive diseases, and healthy samples, with 96.6% sensitivity and 97.96% specificity, respectively. Conclusion: These data suggest a preliminary study that rTC1 has the po-tential to be tested in future studies against a larger serological panel for the diagnosis of Chagas disease.

Proteins, as gifts from nature, provide structure, sequence, and function templates for designing biomaterials. Here, we reported an engineered toolkit derived from a natural block copolymer, RfA1. RfA1 is composed of positively charged polyelectrolyte linker regions interspersed with highly conserved polyampholyte motifs. These linkers and motifs are constructional fragments and ready-to-use building blocks for synthetic design and construction. One functional and editable feature of RfA1 derivatives is their preferential distribution to cytoplasm or nucleoplasm, in a fragment-replication-determined manner. Based on this property, a prices spatiotemporal Tet-on demo was established, which effectively transports cargo peptides into nuclei at selective time points. Moreover, the functional homogeneities of either motifs or linkers were also verified, making them standardized building blocks for synthetic biology. In summary, this study provides a modularized, orthotropic and well-characterized toolkit for precise and spatiotemporal regulation of protein nucleocytoplasmic localization.

Members of the DUF34 (domain of unknown function 34) family, also known as the NIF3 protein superfamily, are ubiquitous across superkingdoms. Proteins of this family have been widely annotated as “GTP cyclohydrolase I type 2” through electronic propagation based on one study. Here, the annotation status of this protein family was examined through comprehensive literature review and integrative bioinformatic analyses that revealed varied pleiotropic associations and phenotypes. This analysis combined with functional complementation studies strongly challenges the current annotation and suggests that DUF34 family members may serve as metal ion insertases, chaperones, or metallocofactor maturases. This general molecular function could explain how DUF34 subgroups participate in highly diversified pathways such as cell differentiation, metal ion homeostasis, pathogen virulence, redox and universal stress responses.

Erlotinib inhibits epithelial growth factor receptor (EGFR) kinase activity and is used to treat non-small cell lung cancer (NSCLC). Despite its high efficacy, recurrence can occur in patients who become resistant to the drug. To address the underlying mechanism of Erlotinib resistance, we investigated additional mechanisms related to mode-of-drug-action, by multiple protein-binding interactions, besides EGFR by using drug affinity responsive target stability (DARTS) and liquid chromatography-mass spectrometry (LC-MS/MS) methods with non-labeled Erlotinib. DNA polymerase alpha subunit B (POLA2) was identified as a new Erlotinib binding protein that was validated by the DARTS platform, complemented with cellular thermal shift assays. Genetic knock-down of POLA2 promoted the anti-proliferative effect of the drug in the Erlotinib-resistant cell line H1299 with high POLA2 expression, whereas overexpression of POLA2 restored anti-proliferative effects in the Erlotinib-sensitive cell line HCC827 with low POLA2 expression. Importantly, POLA2 expression levels in four NSCLC cell lines were positively correlated with anti-proliferative Erlotinib efficacy, verified by bio-statistical analysis (Pearson correlation coefficient, R=0.9886). These results suggest that POLA2 is a novel complementary target protein of Erlotinib, and could clinically provide validity as a surrogate marker for drug resistance in patients with NSCLC.

Phylogenomics, the use of large datasets to examine phylogeny, has revolutionized the study of evolutionary relationships. However, genome-scale data have not been able to resolve all relationships in the tree of life; this could reflect, at least in part, the poor-fit of the models used to analyze heterogeneous datasets. Some of the heterogeneity may reflect the different patterns of selection on proteins based on their structures. To test that hypothesis, we developed a pipeline to divide phylogenomic protein datasets into subsets based on secondary structure and relative solvent accessibility. We then tested whether amino acids in different structural environments had distinct signals for the topology of the deepest branches in the metazoan tree. The most striking difference in phylogenetic signal reflected relative solvent accessibility; analyses of exposed sites (on the surface of proteins) yielded a tree that placed ctenophores sister to all other animals whereas sites buried inside proteins yielded a tree with a sponge-ctenophore clade. These differences in phylogenetic signal were not ameliorated when we repeated our analyses using the CAT model, a mixture model that is often used for analyses of protein datasets. In fact, the heterogeneous CAT model resulted in several rearrangements that are unlikely to represent evolutionary history. However, analyses conducted after recoding amino acids to limit the impact of deviations from compositional stationarity increased the congruence in the estimates of phylogeny for exposed and buried sites; after recoding amino acids both trees supported placement of ctenophores sister to all other animals. These results provide striking evidence that it is necessary to achieve a better understanding of the constraints due to protein structure to improve phylogenetic estimation.

This work describes practical immunization of European sea bass (Dicentrarchus labrax) juveniles against viral nervous necrosis virus (VNNV), a betanodavirus causing worldwide mortalities in many fish species. Protection was obtained with the so called spinycterin vehicles consisting in irreversibly DNA-damaged DNA-repair-less E.coli displaying at their surface a downsized antigen. In this work we, i) maximized bacterial expression levels by downsizing the C protein to a fragment (frgC91-220) containing most of its antigenicity, ii) developed an scalable autoinduction bacterial media based in soy-bean increasing membrane display and reproducibility, iii) enriched surface expression by screening different anchors from several prokaryotic origins (anchor+frgC91-220), iv) preserved frgC91-220 antigenicity by inactivating bacteria by irreversible DNA-damage by means of Ciprofloxacin, and v) increased safety using a repair-less E.coli strain as spinycterin chassis. These second generation of spinycterins protected fish against VNNV challenge with partial (Nmistic+frgC91-220) or 100 % (YBEL+frgC91-220 ) protection, in contrast to those fish immunized with frgC91-220 spinycterins. The proposed spinycterin platform has high levels of environmental safety and cost effectiveness, thus providing potential for small fish vaccines for sustainable aquaculture.

Drug repurposing is a valuable tool for combating the slowing rates of novel therapeutic discovery. The Computational Analysis of Novel Drug Opportunities (CANDO) platform performs shotgun repurposing of 2030 indications/diseases using 3733 drugs/compounds to predict interactions with 46,784 proteins and relating them via proteomic interaction signatures. An accuracy is calculated by comparing interaction similarities of drugs approved for the same indications. We performed a unique subset analysis by breaking down the full protein library into smaller subsets and then recombining the best performing subsets into larger supersets. Up to 14% improvement in accuracy is seen upon benchmarking the supersets, representing a 100–1000 fold reduction in the number of proteins considered relative to the full library. Further analysis revealed that libraries comprised of proteins with more equitably diverse ligand interactions are important for describing compound behavior. Using one of these libraries to generate putative drug candidates against malaria results in more drugs that could be validated in the biomedical literature than the list suggested by the full protein library. Our work elucidates the role of particular protein subsets and corresponding ligand interactions that play a role in drug repurposing, with implications for drug design and machine learning approaches to improve the CANDO platform.

Beginning in 2012, our understanding of human papillomavirus (HPV) subcellular trafficking has undergone a drastic paradigm shift. Work from multiple laboratories has revealed that HPV has evolved a unique means to deliver its viral genome (vDNA) to the cell nucleus, relying on a myriad of host cell proteins and processes. The major breakthrough finding from these recent endeavors was the realization of L2-dependent utilization of cellular sorting factors for the retrograde transport of vDNA away from degradative endo/lysosomal compartments to the Golgi, prior to mitosis-dependent nuclear accumulation of L2/vDNA. An overview of current models of HPV entry, subcellular trafficking, and the role of L2 during initial infection is provided below, highlighting unresolved questions and gaps in knowledge.

In tissue engineering scaffolds take the place of the natural extra cellular matrix (ECM). The natural ECM is the extracellular part of animal tissue that usually provides structural support to the animal cells in addition to performing various other important functions. The design aspect along with the choice of the material for the artificial scaffold is very crucial to cell differentiation, adhesion, proliferation, and the transport of the growth factors or other bio molecular signals. In addition to the material and design of the scaffolds, it is necessary to replicate the normal physiological situation if the scaffold has to function as an implant. The cells have to be located in the porous scaffold to form a three dimensional assembly. The article discusses the important factors to be considered while designing a scaffold for tissue engineering and regenerative medicine.

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.

The liver health of Gilthead sea bream and European sea bass fed fishmeal-free diets including various proportions of plant proteins, insect and poultry by-product meals, was investigated through biochemical and histological analyses using a new Liver Index (LI) formula. Four isoproteic (45% DM) and isolipidic (20% DM) diets were compared: a control, plant-based diet (CV) and other three test ones where 40% of a plant protein-rich ingredient mixture was replaced by Hermetia illucens (H40) or poultry by-product (P40) meals alone or in combination (H10P30). The trials lasted 12 and 18 weeks for sea bream and sea bass, respectively. The results so far ob-tained highlighted species-specific differences in the physiological response to dietary changes. In sea bream, the biochemical and histological responses suggest favorable physiological status and liver health, with higher serum cholesterol (CHO) and triglyceride (TAG) levels and moderate hepatocyte lipid accumulation with the H10P30 diet compared to CV (p<0.05). In sea bass, all diets resulted in elevated serum TAG levels and liver lipid accumulation particularly in fish fed the P40 one (p<0.05) which resulted in the highest LI, coupled with higher frequency of severe lipid accumulation, hypertrophy, cord loss, peripheral nuclei displacement and pyknosis. In conclusion sea bream well adapted to the test diets, while sea bass exhibited altered hepatic lipid metabolism leading to incipient liver steatosis, likely due to the high lipid content of the diets including the insect and poultry meals. The LI developed in this study proved to be a reliable tool for assessing the effects of dietary changes on liver health of sea bream and sea bass, consistent with biochemical and histological findings.

Calmodulins (CaMs) and calmodulin-like-proteins (CMLs) belong to families of calcium-sensors that act as calcium-ion (Ca2+) signal-decoding proteins and regulate downstream target proteins. As a tropical halophyte, Canavalia rosea shows great resistance to multiple abiotic stresses, including high salinity-alkaline, extreme drought, heat, and intense sunlight. However, investigations of calcium ion signal transduction involved in the stress responses of C. rosea are limited. The CaM and CML gene families have been identified and characterized in many species. Nevertheless, there is limited available information about these genes in C. rosea. In this study, a bioinformatic analyses, including the gene structures, conserved domains, phylogenetic relationships, chromosome distribution, and gene synteny, was comprehensively performed to identify and characterize the CrCaM and CrCML gene members. A spatio-temporal expression analysis in different organs and environmental conditions were then assayed using the RNA sequencing technique. Additionally, several CrCaM and CrCML members were then cloned and functionally characterized using the yeast heterogeneous expression system, and some of them were found to change the tolerance of yeast to heat, salt, alkaline, and high osmotic stresses. Furthermore, this indicated that the CrCaMs and CrCMLs play important roles in stress resistance. The results of this study provide a foundation for understanding the possible roles of plant CaM and CML genes, especially for halophyte C. rosea natural ecological adaptability. This study also provides a theoretical basis for further study of the physiological functions of plant CrCaMs and CrCMLs that are involved in multiple abiotic stresses.

The Ramazzottius varieornatus tardigrade is an extremotolerant terrestrial invertebrate belonging to the phylum of Tardigrada. At a length of 0.1-1.0 mm, tardigrades are small animals with an exceptional tolerance to extreme conditions such as high pressure, chemicals and irradia-tion. These properties have been attributed to the recently-discovered Dsup protein. Dsup is a nucleosome-binding protein that prevents DNA damage against X-ray and oxidative stress without impairing cell life, also in Dsup-transfected animal and plant cells. However, the precise “protective” role of this protein is still under study. We performed experiments on human cells and shows that, as compared to control cells, Dsup+ cells are more resistant to UV-C exposure and H2O2. Real-time PCR identified different expression patterns of endogenous genes involved in apoptosis, cell survival and DNA damage repair in Dsup+ cells in response to H2O2 and UV-C. While H2O2 treatment in Dsup+ cells only marginally involved the activation of pathways responsible for DNA repair reinforcing the idea of a direct protective effect of the protein on DNA, in UV-C exposed cells, Dsup efficiently upregulates DNA damage repair genes. In conclusion, our data may help to delineate the different mechanisms by which the Dsup protein operates in response to different insults.

Blocking the interaction of the SARS-CoV-2 spike protein with the human angiotensin-converting enzyme II (hACE2) protein serves as a therapeutic strategy for treating COVID-19. Traditional Chinese medicine (TCM) treatments containing bioactive products could alleviate the symptoms of severe COVID-19. However, the emergence of SARS-CoV-2 variants has complicated the process of developing broad-spectrum drugs. As such, the aim in this study was to explore the efficacy of TCM treatments for SARS-CoV-2 variants through targeting the interaction of the viral spike protein with the hACE2 receptor. The antiviral activity was systematically evaluated using a pseudovirus system. Scutellaria baicalensis was found to be effective against SARS-CoV-2 infection through affecting the interaction of viral spike protein with the hACE2 protein. The active molecules of S. baicalensis were identified and analyzed. Baicalein and baicalin, a flavone and a flavone glycoside, respectively, found in S. baicalensis, exhibited strong inhibitory activities, targeting the viral spike protein or hACE2 protein, respectively. Under optimized conditions, virus infection was inhibited by 98% via baicalein-treated pseudovirus and baicalin-treated hACE2. In summary, we identified the potential SARS-CoV-2 inhibitors of the interaction of the Omicron spike protein and hACE2 receptor from S. baicalensis. Future studies on the therapeutic application of baicalein and baicalin against SARS-CoV-2 variants are needed.

Background: COVID-19 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a recurrent endemic disease affecting the whole world. Since November 2021, Omicron and its subvariants are dominating. In order to prevent severe courses of disease, vaccines are needed to boost and maintain antibody levels capable of neutralizing Omicron. Recently we produced and characterized a SARS-CoV-2 vaccine based on a recombinant fusion protein consisting of hepatitis B virus (HBV)-derived PreS and two SARS-CoV-2 wild-type RBDs. Objectives: To develop a PreS-RBD vaccine which induces high levels of Omicron-specific neutralizing antibodies. Methods: We designed, produced, characterized and compared strain-specific (wild-type: W-PreS-W; Omicron: O-PreS-O), bivalent (mix of W-PreS-W and O-PreS-O) and chimeric (i.e., W-PreS-O) SARS-CoV-2 protein subunit vaccines. Immunogens were characterized in vitro by protein chemical methods, mass-spectrometry, circular dichroism in combination with thermal denaturation and immunological methods. In addition, BALB/c mice were immunized with aluminum hydroxide-adsorbed proteins and aluminum hydroxide alone (i.e., placebo) to study specific antibody and cytokine responses, safety and Omicron neutralization. Results: Defined and pure immunogens could be produced in large amounts as secreted and folded proteins in mammalian cells. Antibodies induced after vaccination with different doses of strain-specific, bivalent and chimeric PreS-RBD fusion proteins reacted with wild-type and Omicron RBD in a dose-dependent manner and resulted in a mixed Th1/Th2 immune response. Interestingly, RBD-specific IgG levels induced by the different vaccines were comparable but the W-PreS-O-induced virus neutralization titers against Omicron (median VNT50: 5000) were 7- and 2-fold higher than the W-PreS-W- and O-PreS-O-specific ones, respectively and 6-fold higher than those of the bivalent vaccine. Conclusion: Among the tested immunogens, the chimeric PreS-RBD subunit vaccine, W-PreS-O, induced the highest neutralizing antibody titers against Omicron. Thus, W-PreS-O seems to be a highly promising COVID-19 vaccine candidate for further preclinical and clinical evaluation.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) utilizes Angiotensin Converting Enzyme 2 (ACE2) as its main receptor for cell entry. We have bioengineered a soluble ACE2 protein termed ACE2 618-DDC-ABD that has increased binding to SARS-CoV-2 and prolonged duration of action. Here we investigated the protective effect of this protein when administered intranasally to k18-hACE2 mice infected with the aggressive SARS-CoV-2 delta variant. k18-hACE2 mice were infected with the SARS-CoV-2 delta variant by inoculation of a lethal dose (2x104 PFU) and followed for up to 14 days. ACE2-618-DDC-ABD (10mg/kg) or PBS was administered intranasally six hours prior and 24 and 48 hours post viral inoculation. All animals in the PBS-control group had succumbed to the disease on day 7 post infection (0% survival) whereas, by contrast, there was only one casualty in the group that received ACE2-618-DDC-ABD (90% survival). Mice in the ACE2-618-DDC-ABD group had minimal disease as assessed by a clinical score and stable weight and both brain and lung viral titers were markedly reduced. These findings demonstrate the efficacy of a bioengineered soluble ACE2 decoy with extended duration of action in protecting against the aggressive delta SARS-CoV-2 variant. Together with previous work these findings demonstrate the universal protective potential against current and future emerging SARS-CoV-2 variants.

AML is a highly aggressive and heterogeneous form of hematological cancer. Proteomics-based stratification of patients into more refined subgroups may contribute to a more precise characterization of the patient-derived AML cells. Here, we reanalyzed liquid chromatography-tandem mass spectrometry (LC-MS/MS) generated proteomic and phosphoproteomic data from 26 chemoresistant/relapse (RELAPSE) and 15 relapse-free (REL_FREE) AML patients. We considered not only the RELAPSE and REL_FREE characteristics but also integrated the French-American-British (FAB) classification, along with considering the presence of nucleophosmin 1 (NPM1) mutation and cytogenetically normal AML. We found a significant number of differentially enriched proteins (911) and phosphoproteins (257) between the various FAB subtypes in RELAPSE patients. Patients with the myeloblastic M1/M2 subtype showed higher levels of RNA processing-related routes and lower levels of signaling related to terms like translation and degranulation, when compared to the M4/M5 subtype. Moreover, we found that high abundance of proteins associated to mitochondrial translation and oxidative phosphorylation, particularly observed in the RELAPSE M4/M5 NPM1 mutated subgroup, distinguishes relapsing from non-relapsing AML patient cells with the FAB subtype M4/M5. Thus, the discovery of subtype-specific biomarkers through proteomic profiling may complement the existing classification system for AML and potentially aid in selecting personalized treatment strategies for individual patients.

Encapsulation is a valuable strategy to protect and deliver anthocyanins (ACNs), phenolic com-pounds with outstanding antioxidant capacity but limited stability. In this study, coacervation was used to encapsulate an ACNs-rich red cabbage extract (RCE). Two agri-food by-products polymers, whey protein isolate (WPI) and apple high-methoxyl pectin (HMP), were blended at pH 4.0 in a specific ratio to induce the formation of nanoparticles (NPs). The process optimisation yielded a monodispersed population (PDI < 0.200) of negatively charged (-17 mV) NPs with an average diameter of 380 nm. RCE concentration influenced size, charge, and antioxidant capacity in a dose-dependent manner. NPs were also sensitive to pH increase from 4 to 7, showing a progressive breakdown. The encapsulation efficiency was 30%, with a retention of ACNs within the polymeric matrix being influenced by their chemical structure: diacylated and/or C3-triglucoside forms were more efficiently encapsulated than monoacylated C3-diglucosides. In conclusion, we report a promising, simple, and sustainable method to produce monodispersed NPs for ACNs encapsu-lation and delivery. Evidence of differential binding of ACNs to NPs, dependent on specific ac-ylation/glycosylation patterns, indicate that care must be taken in the choice of the appropriate NPs formulation for the encapsulation of phenolic compounds.

Lasiodiplodia theobromae, as one of the causing agents associated with Chinese hickory trunk cankers, has caused huge economic losses to the Chinese hickory industry due to its extremely strong pathogenicity. Though the biological characteristics of this pathogen and the occurrence pattern of this disease have been reported, few studies have focused on investigating the mechanisms responsible for L. theobromae survival strategies and pathogenicity. The high-quality genome data and the efficient transformation system are the basis for researching above mechanism events. In this study we sequenced and assembled L. theobromae strain LTTK16-3, and established the first protoplasmic preparation method and polyethylene glycol (PEG) -mediated genetic transformation system for L. theobromae. These genetic information and transformation methods established the foundation for the future mechanisms study of L. theobromae and set up the possibility of targeted molecular improvements for Chinese hickory canker control.

Keywords: Red meat; Processed meat; poultry; dairy; mortality

Fibroblast activation protein (FAP) is a membrane-tethered serine protease overexpressed in the reactive stromal fibroblasts of &gt; 90% human carcinomas, which makes it a promising target for developing radiopharmaceuticals for imaging and therapy of carcinomas. Here, we synthesized two novel (R)-pyrrolidin-2-yl-boronic acid-based FAP-targeted ligands; SB02055 (DOTA-conjugated (R)-(1-((6-(3-(piperazin-1-yl)propoxy)quinoline-4-carbonyl)glycyl)pyrrolidin-2-yl)boronic acid) and SB04028 (DOTA-conjugated ((R)-1-((6-(3-(piperazin-1-yl)propoxy)quinoline-4-carbonyl)-D-alanyl)pyrrolidin-2-yl)boronic acid). natGa- and 68Ga-complexes of both ligands were evaluated in preclinical studies and compared to previously reported natGa/68Ga-complexed PNT6555. Enzymatic assays showed that FAP binding affinities (IC50) of natGa-SB02055, natGa-SB04028 and natGa-PNT6555 were 0.41±0.06, 13.9±1.29 and 78.1±4.59 nM, respectively. PET imaging and biodistribution studies in HEK293T:hFAP tumor-bearing mice showed that while [68Ga]Ga-SB02055 presented with a nominal tumor uptake (1.08±0.37 %ID/g), [68Ga]Ga-SB04028 demonstrated clear tumor visualization with ~1.5-fold higher tumor uptake (10.1±0.42 %ID/g) compared to [68Ga]Ga-PNT6555 (6.38±0.45 %ID/g). High accumulation in the bladder indicated renal excretion of all three tracers. [68Ga]Ga-SB04028 displayed low background level uptake in most normal organs, and comparable to [68Ga]Ga-PNT6555. However, since its tumor uptake was considerably higher than [68Ga]Ga-PNT6555, the corresponding tumor-to-organ uptake ratios for [68Ga]Ga-SB04028 were also significantly greater than [68Ga]Ga-PNT6555. Our data demonstrate that (R)-(((quinoline-4-carbonyl)-D-alanyl)pyrrolidin-2-yl)boronic acid is a promising pharmacophore for the design of FAP-targeted radiopharmaceuticals for cancer imaging and radioligand therapy.

Trauma to the spinal cord causes extensive neuronal death contributing to the loss of sensory-motor and autonomic functions below the injury level. Apoptosis affects neurons after spinal cord injury (SCI) and is associated with increased caspase activity. Cleavage of X-linked inhibitor of apoptosis protein (XIAP) after SCI may contribute to this rise of caspase activity. Accordingly, we have shown that the elevation of XIAP resulted in increased neuronal survival after SCI and improved functional recovery. Therefore, we hypothesize that neuronal overexpression of XIAP can be neuroprotective after SCI with improved functional recovery. In line with this, studies of a transgenic mouse with overexpression of XIAP in neurons revealed that higher levels of XIAP after spinal cord trauma favours neuronal survival, tissue preservation, and motor recovery after the spinal cord trauma. Using the human SH-SY5Y cells overexpressing XIAP we show further that XIAP reduced caspase activity and apoptotic cell death after pro-apoptotic stimuli. In conclusion, this study shows that the levels of XIAP expression are an important factor for the outcome after spinal cord trauma and identifies XIAP as an important therapeutic target for alleviating the deleterious effects of SCI.

Herein, we are proposing two chalcone molecules, (E)-1-(4-methoxyphenyl)-3-(p-tolyl) prop-2-en-1-one and (E)-3-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl) prop-2-en-1-one, based on the anticancer bioactive molecule Xanthohumol, which are suitable for further in vitro and in vivo studies. Their ability to create stable complexes with the antiapoptotic X-linked IAP (XIAP) protein makes them promising anticancer agents. The calculations were based on ligand-based and structure-based virtual screening combined for the pharmacophore built. Additionally, the structures passed Lipinski's rule for drug use, and their reactivity was confirmed using density functional theory studies. The candidates were chosen between 10639400 compounds, and the docking protocols were evaluated using molecular dynamics simulations.

The enterohemorrhagic Escherichia coli (EHEC) O157: H7 EspF is known to be a multifunctional effector that triggers several damage processes in the host cells. However, in the process of EHEC O157: H7 infection, the interactions between EspF, its N- or C-terminus, and host proteins are still unclear. In this work, we use co-immunoprecipitation combined with mass spectrometry (CoIP-MS) to screen the interactions between EspF/EspF-N/EspF-C terminus and host proteins. A total of 311 host proteins are analyzed. The N-terminus of EspF is found to interact with 192 proteins, whereas 205 proteins interact with the C-terminus. These proteins are mainly involved in RNA splicing, endoplasmic reticulum stress, and a variety of metabolic signaling pathways. Surprisingly, MS results reveal that EspF can also phosphorylate H2AX, suggesting that EspF may directly mediate DNA damage. Here, by western blot and immunofluorescence (IF), we verified that EspF can cause phosphorylation of H2AX and mediates cell multi-nuclearation and cell hypertrophy. Furthermore, we verify here for the first time that SMC1 interacts with EspF -C-terminus, and provide evidence that EspF increases p-SMC1 levels. p-SMC1 is known to influence S-phase cell cycle arrest and usually increases during periods of DNA damage. Our work revealed a novel interaction between EspF and the host protein SMC1 and lays a foundation for further research on EspF-mediated host DNA damage, apoptosis, and even colorectal carcinogenesis.

The importance of antibodies in health care and the biotechnology research and development demands not only knowledge of their experimental structures at high resolution, but also practical implementation of this knowledge for both effective and efficient design and production of antibody for its use in both medical and research applications. While the experimental wet-lab approach is usually costly, laborious and time-consuming, computational (dry-lab) approaches, in spite of their intrinsic limitations in comparison with its experimental (wet-lab) counterpart, provide a cheaper and faster alternative option. For the first time, this article reports a comprehensive set of structural electrostatic features extracted from experimentally determined antigen-antibody-related structures, including especially those structural electrostatic features at the interfaces of all experimentally determined antigen-antibody complex structures as of February 29, 2020, to facilitate effective and efficient machine learning-based computational antibody design using currently available experimental structures inside Protein Data Bank.

Phospholipase Dα1 (PLDα1) belongs to phospholipases, a large phospholipid hydrolyzing protein family. PLDα1 has a substrate preference for phosphatidylcholine leading to enzymatic production of phosphatidic acid, a lipid second messenger with multiple cellular functions. PLDα1 itself is implicated in biotic and abiotic stress responses. We present here a shot-gun differential proteomic analysis on roots of two pldα1 mutants compared to the Col-0 wild type. Our data suggest new roles of PLDα1 in endomembrane transport, mitochondrial protein import and protein quality control and glucosinolate biosynthesis. Thus, we identified proteins involved in endocytosis, endoplasmic reticulum-Golgi transport and attachment sites of endoplasmic reticulum and plasma membrane (V-type proton ATPases, protein transport protein SEC13 homolog A, vesicle-associated protein 1-2, vacuolar protein sorting-associated protein 29, syntaxin-32, all upregulated in the mutants), mitochondrial import and electron transport chain (mitochondrial import inner membrane translocase subunits TIM23-2 and TIM13, mitochondrial NADH dehydrogenases, ATP synthases, cytochrome c oxidase subunit 6b-1, ADP,ATP carrier protein 2, downregulated in the mutants) and glucosinolate biosynthesis (3-isopropylmalate dehydrogenases 1, 2 and 3, methylthioalkylmalate synthase 1, cytochrome P450 83B1, Glutathione S-transferase F9, indole glucosinolate O-methyltransferase 1, adenylyl-sulfate kinase 1, all upregulated in mutants). Our results suggest broader biological roles of PLDα1 as anticipated so far.

This study investigated how the type and homogenisation parameters of the feed mixture affect the efficiency of the encapsulation process and the microcapsule properties of phenol-rich grape pomace extract (GPE) during the spray drying process with goat whey protein (GW) as wall material. GPE was obtained by solid-liquid extraction of grapes with 50% ethanol. Effects of ultrasonic homogenisation parameters (UB): temperature: 30-50 °C; time: 5-30 min; stabilisation time after homogenisation: 15-45 min, and homogenisation parameters on a magnetic stirrer (MS): temperature (25-50 °C), time (10-60 min) and homogenisation speed (600-1200 rpm) were tested using response surface methodology (RSM) and Box-Behnken design. After selecting the optimal homogenisation conditions, the spray drying process parameters were optimised in the same way, namely: inlet air temperature (160-190 °C), GW fraction (0.5-3.5) and feed flow (6-10 mL/ min). The results showed that GW has a high potential as a wall material in the spray drying process, especially for the purpose of protecting polyphenolic compounds from GPE.

Clofazimine and Arbidol have both been reported to be effective in vitro SARS-CoV-2 fusion inhibitors. Both are promising drugs repurposed for treatment of COVID-19 and have been used in several previous and ongoing clinical trials. Small-molecule binding to expressed constructs of the trimeric S2 segment of Spike and the full-length SARS-CoV-2 spike protein were measured using a Surface Plasmon Resonance (SPR) binding assay. We demonstrate that Clofazimine, Toremifene, Arbidol and other Arbidol derivatives bind to the S2 segment of the Spike protein. Clofazimine provided the most reliable and highest quality SPR binding data to S2 over the conditions explored. A molecular docking approach was used to identify the most favorable binding sites on the S2 segment in the prefusion conformation, highlighting two possible small-molecule binding sites for fusion inhibitors. Results from molecular docking and modeling the structure-activity-relationship (SAR) of a newly reported series of Clofazimine derivatives supports the proposed Clofazimine binding site on the S2 segment. When the proposed Clofazimine binding site is superimposed with other experimentally determined coronavirus structures in structure-sequence alignments, the changes in sequence and structure may rationalize the broad-spectrum antiviral activity of Clofazimine in closely related coronavirus such as (SARS-CoV, MERS, hCoV-229E, hCoV-OC43).

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.

Parkinson's disease is a neurodegenerative disorder resulting from progressive damage to specific brain regions. Key symptoms encompass involuntary movements (tremor), slowness of movements (bradykinesia), and muscle rigidity. Although the precise cause remains uncertain, a combination of genetic and environmental factors is believed to contribute to its development. This study focuses on the interaction of the E3 ubiquitin-protein ligase parkin with natural compounds. Docking investigations revealed that Amentoflavone exhibited an excellent binding energy score of approximately -10 kcal/mol. The E3 ubiquitin-protein ligase parkin displayed a robust binding affinity with docked amentoflavone, evident from a high binding energy of -10 kcal/mol. Nonetheless, additional biological studies are crucial not only to validate its potential efficacy against Parkinson's and assess safety in terms of toxicity but also to perform further computational analyses confirming these preliminary assessments.

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.

The Omicron EG.5 lineage of SARS-CoV-2 is currently on a trajectory to become a dominant strain. This phase 2 study aims to evaluate the immunogenicity of SCTV01E-2, a tetravalent protein vaccine, with a specific emphasis on its effectiveness against Omicron EG.5, comparing it with its progenitor vaccine, SCTV01E (NCT05933512). As of September 12, 2023, 429 participants aged ≥18 years were randomized to SCTV01E (N=215) or SCTV01E-2 (N=214) groups. Both vaccines showed increases in neutralizing antibody (nAb) against Omicron EG.5, with a 5.7-fold increase and a 9.0-fold increase in SCTV01E and SCTV01E-2 groups 14 days post-vaccination, respectively. The predetermined statistical endpoints were achieved, showing that the geometric mean titer (GMT) of nAb and the seroresponse rate (SRR) against Omicron EG.5 were significantly higher in the SCTV01E-2 group compared to SCTV01E. Additionally, SCTV01E and SCTV01E-2 induced a 5.5-fold and a 5.9-fold increase of nAb against XBB.1, respectively. Reactogenicity was generally mild and transient. No vaccine related SAEs, AESIs or deaths were reported. In summary, SCTV01E-2 elicited robust neutralizing responses against Omicron EG.5 and XBB.1 without safety concerns, underlining its potential as a versatile COVID-19 vaccine against SARS-CoV-2 variants.

Kinases play an important role in regulating various intracellular signaling pathways that control cell proliferation, differentiation, survival and other cellular processes and their deregulation causes more than 400 diseases. Consequently, macrocyclization can be considered a noteworthy approach to develop new therapeutic agents for human diseases. Macrocyclization has emerged as an effective drug discovery strategy over the past decade to improve target selectivity and potency of small molecules. Small compounds with linear structures upon macrocyclization can lead to changes in their physicochemical and biological properties by firmly reducing conformational flexibility. A number of distinct protein kinases exhibit similar binding sites. Comparison of protein binding site provides crucial insights for the drug discovery and development. Binding site similarities are helpful to understand polypharmacology, identifying potential off-targets and repurposing of known drugs. In this review, we focused on comparing binding sites of those kinases for which macrocyclic inhibitors are available/studied so far. Furthermore, we calculated the volume of binding site pocket for each targeted kinase and then compared it with the binding site pocket of the kinase for which only acyclic inhibitors were designed till date. Our review and analysis of several explored kinases might be useful to target new protein kinases for macrocyclic drug discovery.

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.

Structure–function relationships in proteins have been one of the crucial scientific topics. Heme proteins have diverse and pivotal biological functions. Therefore, clarifying their structure–function correlation is significant to understand their functional mechanism and is informative for various fields of science. In this study, we constructed convolutional neural network models for predicting protein functions from the tertiary structures of heme-binding sites (active sites) of heme proteins to examine the structure–function correlation. As a result, we succeeded in the classification of oxygen-binding protein (OB), oxidoreductase (OR), proteins with both functions (OB–OR), and electron transport protein (ET) with high accuracy. Although the misclassification rate for OR and ET was high, the rates between OB and ET and between OB and OR were almost zero, indicating that the prediction model works well between protein groups with very different functions. However, predicting the function of proteins modified with amino acid mutation(s) remains a challenge. Our findings indicate a structure–function correlation in the active site of heme proteins. This study is expected to be applied to the prediction of more detailed protein functions such as catalytic reactions.

The development of antibody therapies against SARS-CoV-2 remains a challenging task during the ongoing COVID-19 pandemic. All approved therapeutic antibodies are directed against the receptor binding domain (RBD) of Spike and lost neutralization efficacy against continuously emerging SARS-CoV-2 variants, which especially mutate in the RBD region. Previously, phage display has been used to identify epitopes of antibody responses against several diseases. Such epitopes have been applied to design vaccines or neutralizing antibodies. Here, we constructed an ORFeome phage display library for the SARS-CoV-2 genome. Open reading frames (ORFs) representing the SARS-CoV-2 genome were displayed on the surface of phage particles in order to identify enriched immunogenic epitopes from COVID-19 patients. Library quality was assessed by both, NGS and epitope mapping of a monoclonal antibody with known binding site. The most prominent epitope captured represented parts of Spike´s fusion peptide (FP). It is associated with the cell entry mechanism of SARS-CoV-2 into the host cell and the serine protease TMPRSS2 cleaves Spike within this sequence. Blocking of this mechanism could be a potential target for non-RBD binding therapeutic anti-SARS-CoV-2 antibodies. As mutations within the FP amino acid sequence were rather rare among SARS-CoV-2 variants so far, this may be an advantage in the fight against future virus variants.

The aim of this work was to test polyamines as potential natural substrates of the Acinetobacter baumannii chlorhexidine efflux protein AceI using near-UV synchrotron radiation circular dichroism (SRCD) spectroscopy. The Gram-negative bacterium A. Baumannii is a leading cause of hospital-acquired infections and an important foodborne pathogen. A. Baumannii strains are becoming increasingly resistant to antimicrobial agents, including the synthetic antiseptic chlorhexidine. AceI was the founding member of the recently recognised PACE family of bacterial multidrug efflux proteins. Using the plasmid construct pTTQ18-aceI(His₆) containing the A. Baumannii aceI gene directly upstream from a His₆-tag coding sequence, expression of AceI(His₆) was amplified in E. coli BL21(DE3) cells. Near-UV (250-340 nm) SRCD measurements were performed on detergent-solubilised and purified AceI(His₆) at 20 °C. Sample and SRCD experimental conditions were identified that detected binding of the triamine spermidine to AceI(His₆). In a titration with spermidine (0-10 mM) this binding was saturable and fitting of the curve for the change in signal intensity produced an apparent binding affinity (K_D) of 3.97 +/- 0.45 mM. These SRCD results were the first experimental evidence obtained for polyamines as natural substrates of PACE proteins.

Featured Application: Bio-electronic devices take advantages of some specific duties of biological matter. The specific ability of some proteins to use sunlight is considered for the realization of photo-electronic devices . Here the focus is on the role of the pH, whose variations seem to affect the protein conductance

Till date there are three full length COVID 19 virus genome sequences available from India. The earlier two were reported from Kerala, Southern India and the more recent one has been reported from Gujarat, western part of India. In this paper we report two novel mutations in the Spike protein sequence of Gujarat’s isolate. These mutations are based on comparison with the original Wuhan sequence. The two mutations have been found to be located just upstream and downstream of the receptor binding domain (RBD). Out of these two one has also been found to affect the secondary structure of the S1 domain. Since both of these mutations lie near the receptor binding domain, they might influence the spike receptor interactions by changing the conformation of the spike protein S1 domain. These mutations are uniquely placed and might be important in the context of vaccine engineering and therapeutic interventions.

The emergence of proteins in the prebiotic world was a watershed event at the origin of life. With their astonishing versatility, the protein enzymes catalyzed crucial biochemical reactions within protocells into more complex biomolecules in diverse metabolic pathways, whereas structural proteins provided strength and permeability in the cell membrane. Five major biochemical innovations followed in succession after availability of various kinds of protein molecules during decoding and translation of mRNAs. These are: (1) the modification of the phospholipid membrane into the plasma membrane; (2) the origin of primitive cytoplasm; (3) primitive gene regulation; (4) the beginnings of the virus world; and (5) the advent of DNA. The creative role of viruses during prebiotic synthesis led to the origin of the DNA world, when DNA replaced mRNA as the major genome of the protocells. With the advent of DNA, replication of information was entirely dissociated from its expression. Because DNA is much more stable than mRNA with more storage capacity, it is a superb archive for information systems in the form of base sequences. DNA progressively took over the replicative storage function of mRNA, leaving the latter for protein synthesis. Genetic information began to flow from DNA to mRNA to protein in a two-step process involving transcription and translation. In the biological stage, DNA replication was central to the binary fission of the first cell, orchestrated by the duplication of genomes and then the division of the parent cell into two identical daughter cells. With the onset of binary fission, the population of primitive cells grew rapidly in the hydrothermal vent environment, undergoing Darwinian evolution and diversification by mutation. The habitat of the earliest fossil record (≥ 3.5 Ga) from the Archean sedimentary rocks of Canada, Greenland, Australia, South Africa, and India offers a new window on the early radiation of microbial life. The development of anoxygenic and then oxygenic photosynthesis from early hyperthermophiles would have allowed life to escape the hydrothermal setting to the mesophilic global ocean.

Background: Although lower extremity revascularization is commonly performed in hemodialysis patients, poor prognosis remains a major problem. Protein-energy wasting is reportedly associated with chronic inflammation and advanced atherosclerosis in hemodialysis patients. We investigated the association between the geriatric nutritional risk index (GNRI) as a surrogate marker of protein-energy wasting, C-reactive protein (CRP), and their joint roles in the prediction of amputation and/or mortality. Methods: We enrolled 800 patients successfully underwent lower extremity revascularization. Patients were divided into low, middle and high tertile (T1, T2 and T3) according to GNRI and CRP levels, respectively. Results: Amputation-free survival rates for 8 years were 47.0%, 56.9%, and 69.5% in T1, T2, and T3 of the GNRI, and 65.8%, 58.7%, and 33.2% for T1, T2, and T3 of CRP, respectively (p<0.0001 for both). Declined GNRI [adjusted hazard ratio (aHR) 1.78, 95% confidence interval (CI) 1.24-2.59, p=0.0016 for T1 vs. T3] and elevated CRP (aHR 1.86, 95%CI 1.30-2.70, p=0.0007 for T3 vs. T1) were independent predictors of amputation and/or mortality. In the combined setting of both variables, the risk was 3.77-fold higher (95% CI 1.97-7.69, p<0.0001) in the T1 of GNRI with T3 of CRP than in the T3 of GNRI with T1 of CRP. Conclusions: Patients with pre-procedural decreased GNRI and elevated CRP levels frequently experienced amputation and/or mortality, and a combination of both variables could more accurately stratify the risk.

Background: C-reactive protein (CRP) is a predictive biomarker of colorectal surgery (CRS) complications but has not been evaluated by comparing patients undergoing CRS with intracorporeal anastomosis (IA) or extracorporeal anastomosis (EA). Therefore, we aimed to describe postoperative CRP values between two groups of patients who underwent laparoscopic right hemicolectomy for right-side colon cancer with IA or EA. Methods: We retrospectively reviewed the data of patients affected by right-side colon cancers who underwent laparoscopic right-hemicolectomy with ileo-colic IA or EA between January 2016 and December 2022. Results: A total of 340 patients underwent laparoscopic right hemicolectomy. EA was performed in 238 (70%) and IA in 102 (30%) cases. Patients with IA showed significantly higher values of CRP in the second, third and fourth postoperative days (15.2 mg/dL vs 9.5 mg/dL p=

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.

: 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.

The non-structural protein (NS) and nucleoprotein (NP) of the severe fever with thrombocytopenia syndrome virus (SFTSV) encoded by the S segment are crucial for viral pathogenesis. They reside in viroplasm-like structures (VLS), but their interaction and their significance in viral propagation remain unclear. Here, we investigated the significance of the association between NS and NP during viral infection through in-silico and in-vitro analyses. Through in-silico analysis, three possible binding sites were predicted, at NSs1-32, NSs56-82, and NSs207-237. Rationally, three mutant NSs were developed by site-directed mutagenesis and tested for NP interaction by co-immunoprecipitation. NSsW61Y failed to interact with the nucleoprotein, which was substantiated by the conformational changes observed in the structural analyses. Additionally, molecular docking analysis corroborated that the NSW61Y mutant protein does not interact well compared to wild-type NS. Over-expression of wild-type NS in HeLa cells increased the SFTSV replication by five folds, but NSsW61Y exhibited 1.9-folds less viral replication than wild-type. We demonstrated that the W61Y alteration was implicated in the reduction of NS-NP interaction and viral replication. Thus, the present study identified a critical NS site, which could be targeted for development of therapeutic regimens against SFTSV.

This research investigated the antihypertensive effects of tamarind products and compared their potentials based on their animal model’s data verified by molecular docking, multitarget interactions in network pharmacology, and dynamic simulation assays. The GC-MS characterized tamarind products were administered to the cholesterol-induced hypertensive Wistar albino rat models. Two-week-intervened animals were dissected to collect serum and organs which were respectively subjected to the analyses of hypertension-linked markers and tissue architectures. Lead biometabolites of tamarinds interacted with eight target receptors in molecular docking and dynamic simulation studies and they were tested for multitarget interactions using network pharmacological analyses. Results showed that serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), C-reactive protein (CRP), Troponin I, and lipid profiles were maximally reinstated by the phenolic-enriched ripened sour over the sweet tamarind flesh extracts, but seed extracts had a smaller influence. Among the tamarind’s biometabolites, Gamma-sitosterol (ϒ-sitosterol) was found to be the best ligand in managing hypertension via the possible ligand-receptor interactions with the guanylate cyclase which displayed the best drug-likeliness through the highest binding energy -9.3 Kcal. Multitargeted interactions-based degree algorithm and phylogenetic tree of pathways showed 14 targeted genes, of which NR3C1, REN, PPARG, and CYP11B1 hub genes, were consistently modulated by the ϒ-sitosterol to reduce hypertension and related risk factors. The dynamic simulation study showed that the P-RMSD values of ϒ-sitosterol-guanylate cyclase were stable between 75.00 to 100.00 ns at the binding pocket. The findings demonstrate that ripened sour tamarind extract may be supplemented to be a more prospective antihypertensive target or nutraceuticals affirming through advanced preclinical and clinical studies.

Introduction: Neuroinflammation following lipopolysaccharide (LPS) administration induces locomotor deficit and anxiety-like behavior. In this study, minocycline was compared to memantine, the NMDA receptor antagonist, for its effects on LPS-induced locomotor deficit and anxiety-like behavior in rats. Methodology: Adult male Sprague Dawley rats were administered either two different doses of minocycline (25 or 50 mg/kg/day, i.p.) or 10 mg/kg/day of memantine (i.p.) for 14 days four days prior to LPS (5 mg/kg, i.p.) injection. The locomotor activity and anxiety-like behavior were assessed using the open field test (OFT). The phosphorylated tau protein level was measured using ELISA while the expression and density of brain-derived neurotrophic factor (BDNF) and cAMP response element-binding protein (CREB) protein in the medial prefrontal cortex (mPFC) were measured using immunohistochemistry and western blot, respectively. Results: In the mPFC, minocycline treatment reduced the locomotor deficit and anxiety-like behavior, reduced phosphorylated tau protein level, and upregulated BDNF/CREB protein expression comparable to memantine, with the higher dose of minocycline having better benefits. Conclusion: Minocycline treatment attenuated LPS-induced locomotor deficit and anxiety-like behavior in rats, possibly via a decrease in phosphorylated tau protein levels and an increase in the expression of the BDNF/CREB proteins.

Prophylactic vaccines are important in the control of visceral leishmaniasis (VL) and as economic measures for public health. Despite the efforts to develop a vaccine against human VL caused by Leishmania infantum, none is available, and the focus has shifted to developing vaccines against canine visceral leishmaniasis (CVL). Currently, commercially available vaccines are targeted at CVL but are not effective. Different strategies have been applied in developing and improving vaccines, such as using chimeric proteins to expand vaccine coverage. The search for patents can be a way of tracking vaccines that have the potential to be marketed. In this context, the present work presents a summary of immunological aspects relevant to VL vaccine development with a focus on the composition of chimeric protein vaccines for CVL deposited in patent banks as an important approach for biotechnological development. The resulting data could facilitate the screening and selection of antigens to compose vaccine candidates with high performance against VL.

Transcription of glycine-rich RNA-binding protein 2 (PeGRP2) transiently increased in root and shoot of Populus euphratica (salt-resistant poplar) upon initial salt exposure and tended to decrease after long-term NaCl stress (100 mM, 12 days). PeGRP2 overexpression in the hybrid Populus tremula × P. alba ‘717-1B4’ (P. × canescens) increased salt sensitivity, which was reflected in plant growth and photosynthesis. PeGRP2 contains a conserved RNA recognition motif domain at the N-terminus, and RNA affinity purification (RAP) sequencing was developed to enrich target mRNAs that physically interact with PeGRP2 in P. × canescens. RAP sequencing combined with RT-qPCR revealed that NaCl decreased the transcripts of PeGRP2-interacting mRNAs encoding photosynthetic proteins, antioxidative enzymes, ATPases, and Na+/H+ antiporters in transgenic poplar. Particularly, PeGRP2 negatively affected the stability of target mRNAs encoding photosynthetic proteins (PETC and RBCMT), antioxidant enzymes (SOD[Mn], CDSP32 and CYB1-2), ATPases (AHA11, ACA8 and ACA9), and Na+/H+ antiporter (NHA1). This resulted in (i) a greater reduction in Fv/Fm, YII, ETR and Pn, (ii) a less pronounced salt activation of antioxidative enzymes, and (iii) a reduced ability to maintain Na+ homeostasis in transgenic poplars during long-term salt stress, leading to a lowered ability to tolerate salinity stress.

Background: We previously reported that human keratinocytes express protease-activated receptor (PAR)-2 and play an important role in activated protein C (APC)-induced cutaneous wound healing. This study investigated the involvement of PAR-2 in the production of gelatinolytic matrix metalloproteinases (MMP)-2 and -9 by APC during cutaneous wound healing. Methods: Full-thickness excisional wounds were made on the dorsum of male C57BL/6 mice. Wounds were treated with APC on days 1, 2, and 3 post-wounding. Cultured neonatal foreskin keratinocytes were treated with APC with or without intact PAR-2 signalling to examine the effects on MMP-2 and MMP-9 production. Murine dermal fibroblasts from PAR2 knock-out (KO) mice were also assessed. MMP-2 and -9 were measured by gelatin zymography, fluorometric assay, and immunohistochemistry. Results: APC accelerated wound healing in WT mice, but had negligible effect in PAR-2 KO mice. APC stimulated murine cutaneous wound healing was associated with differential 0temporal production of MMP-2 and MMP-9, with the latter peaking on Day 1 and the former on Day 6. In-hibition of PAR-2 in human keratinocytes reduced APC-induced MMP-2 activity 25~50, but had little effect on MMP-9. Similarly, APC-induced MMP-2 activation was reduced by 40% in cultured dermal fibroblasts derived from PAR-2 KO mice. Conclusion: This study shows for the first time that PAR-2 is essential for APC-induced MMP-2 production. Considering the important role of MMP-2 in wound healing, this work helps explain the underlying mechanisms of action of APC to promote wound healing through PAR2.

Although ionizing radiation (IR) is widely used for therapeutic and research purposes, studies on low-dose ionizing radiation (LDIR) are limited compared with those on other IR approaches, such as high-dose gamma irradiation and ultraviolet irradiation. High-dose IR affects DNA damage response and nucleotide-protein crosslinking, among other processes; however, the molecular consequences of LDIR have been poorly investigated. Here, we developed a method to profile RNA species crosslinked to an RNA-binding protein, human antigen R (HuR), using LDIR and high-throughput RNA sequencing. The RNA fragments isolated via LDIR-crosslinking and immunoprecipitation sequencing were crosslinked to HuR and protected from RNase-mediated digestion. Upon crosslinking HuR to target mRNAs, such as PAX6, ZFP91, NR2F6, and CAND2, the transcripts degraded rapidly in human cell lines. Additionally, PAX6 and NR2F6 downregulation mediated the beneficial effects of LDIR on cell viability. Thus, our approach provides a method for investigating post-transcriptional gene regulation using LDIR.

In the crustacean immune system, leucine-rich repeat (LRR) is one of the major structures for recognizing pathogen-associated molecular patterns (PAMPs). LRR domain-containing proteins belong to the LRR family, which is a large group of proteins with more than 6000 genes in the database. They are involved in very diverse physiological functions mainly by interacting with other proteins. In a previous study, the LvLRRm, a transmembrane protein containing only LRR domain, was identified in the white leg shrimp, Litopenaeus vannamei. Its versatile role in performing multiple immunomodulation activities has been reported. However, there is still a lack of research on its efficient function at the protein level. To investigate its interactions with other proteins, we applied a convenient method called ‘Hybrid LRR technique’ to produce a recombinant LvLRRm. The LvLRRm and hagfish’s variable lymphocyte receptors (VLRs) fragments were fused to the conserved LxxLxLxxN motif while retaining the β-strand. In addition, we established interactions between hybrid-proteins and flagellin of Salmonella by performing surface plasmon resonance (SPR) analysis. The designed hybrid proteins showed potential for universal applications without losing protein functions.

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.

Dysfunction of the WW domain-containing adaptor with coiled-coil, WAC, gene underlies a rare autosomal dominant disorder, DeSanto-Shinawi syndrome (DESSH). DESSH is associated with facial dysmorphia, hypotonia, and cognitive alterations, including attention deficit hyperactivity disorder and autism. How the WAC protein localizes and functions in neural cells is critical to understanding its role during development. To understand the genotype-phenotype role of WAC, we developed a knowledgebase of WAC expression, evolution, human genomics, and structural/motif analysis combined with human protein domain deletions to assess how conserved domains guide cellular distribution. Then assessed in a cell type implicated in DESSH, cortical GABAergic neurons. WAC contains conserved charged amino acids, phosphorylation signals, and enriched nuclear motifs, suggesting a role in cellular signaling and gene transcription. Human DESSH variants are found within these regions. We also discovered and tested a nuclear localaization domain that impacts the cellular distribution of the protein. These data provide new insights into the potential roles of this critical developmental gene, establishing a platform to assess further translational studies, including the screening of missense genetic variants in WAC. Moreover, these studies are essential for understanding the role of human WAC variants in more diverse neurological phenotypes, including autism spectrum disorder.

Background: According to newly published studies, patients with Coronavirus Disease 2019 (COVID-19) have significantly lower serum 25-hydroxyvitamin D 25(OH)D levels than those without the SARS-CoV-2 infection. Vitamin D insufficiency may be a predictor of poor prognosis in COVID-19 patients. Vitamin D supplementation is related to improved clinical outcomes in terms of intensive care unit (ICU) admission and death, particularly in individuals with moderate-to-severe forms of COVID-19 requiring hospitalization. Materials and methods: This placebo-controlled five-day study was performed on 100 hospitalized COVID-19 patients with vitamin D insufficiency randomized into two groups. Vitamin D in the form of a sublingual sprayable microemulsion (LYL love your life® sunD3 LYLmicro™) was given three times daily after breakfast, lunch, and dinner (daily dose 12,000 IU) to 52 patients with blood vitamin D levels below 30 ng/ml. 48 patients in the control group received a placebo spray in the same daily regiment. Results: We observed a statistically significant increase by 8.6 ng/ml in median vitamin D level after five days of high-dose vitamin D3 spray therapy. 90.2% of subjects reached normal serum levels of vitamin D. There was a strong correlation between changes in vitamin D and C-reactive protein (CRP) levels in individuals with moderately severe disease (p<0.05), while mild and severe cases showed no statistical significance comparing case and control groups. There were no statistically significant changes in ferritin and interleukin-6 (IL-6) levels. Conclusions: In this study, we demonstrated the possibility to rapidly increase circulating levels of vitamin D just in 5 days of high-dose intervention and found a statistically significant decrease of CRP levels in patients with moderately severe COVID-19. We were not able to detect statistically significant changes in ferritin and IL-6 levels during the 5-day vitamin D intervention.

Novel cultivation technologies demand the adaptation of existing analytical concepts. Metabolic flux analysis (MFA) requires stable-isotope labeling of biomass-bound protein as the primary information source. Obtaining the required protein in cultivation set-ups where biomass is scarce or inaccessible due to low cell densities and cell immobilization is difficult to date. We developed a non-disruptive analytical concept for 13C-based metabolic flux analysis based on secreted protein as an information carrier for isotope mapping in the protein-bound amino acids. This "metabolic flux probe" (MFP) concept was investigated in different cultivation set-ups with a recombinant, protein-secreting yeast strain. The obtained results grant insight into intracellular protein turnover dynamics. Experiments under metabolic but isotopically nonstationary conditions in continuous glucose-limited chemostats at high dilution rates demonstrated faster incorporation of isotope information from labeled glucose into the recombinant reporter protein than in biomass-bound protein. Our results suggest that the reporter protein was polymerized from intracellular amino acid pools with higher turnover rates than biomass-bound protein. The latter aspect might be vital for 13C-flux analyses under isotopically nonstationary conditions for analyzing fast metabolic dynamics.

Rhes is one of the most interesting proteins regulated by thyroid hormones that, through the inhibition of the striatal cAMP/PKA pathway, acts as a modulator of dopamine neurotransmission. It is expressed at high levels in the dorsal striatum, with a medial-to-lateral expression gradient reflecting that of both dopamine D2 and adenosine A2A receptors. Rhes is also present in the hippocampus, cerebral cortex, olfactory tubercle and bulb, substantia nigra pars compacta (SNc) and ventral tegmental area of the rodent brain. In line with Rhes-dependent regulation of dopaminergic transmission, several data showed that lack of Rhes enhanced cocaine and amphetamine-induced motor stimulation in mice. Previous studies showed that pharmacological depletion of dopamine significantly reduces Rhes mRNA levels in rodents, non-human primates and Parkinson’s disease (PD) patients, suggesting a link between dopaminergic innervation and physiological Rhes mRNA expression. Rhes protein binds to and activates striatal mTORC1, and modulates L-DOPA-induced dyskinesia in PD rodent models. Finally, Rhes is involved in the survival of mouse midbrain dopaminergic neurons of SNc, thus pointing towards a Rhes-dependent modulation of autophagy and mitophagy processes, and encouraging further investigations about mechanisms underlying dysfunctions of the nigrostriatal system.

p53 and p73 are critical tumor suppressors often inactivated in human cancers through various mechanisms. Owing to high structural homology, the proteins have many common functions and recognize the same set of genes involved in apoptosis and cell cycle regulation. p53 is known as the ‘guardian of the genome’ and together with p73 form a barrier against cancer development and progression. The TP53 is mutated in more than 50% of all human cancers and the germline mutations in TP53 predispose to the early onset of multiple tumors in Li-Fraumeni Syndrome (LFS), the inherited cancer predisposition. In cancers where TP53 gene is intact, p53 is degraded. Despite the ongoing efforts, the treatment of cancers remains challenging. This is due to late diagnoses, the toxicity of current standard of care and marginal benefit of newly approved therapies. Presently, the endeavours focus on reactivating p53 exclusively, neglecting the potential of the restoration of p73 protein for cancer eradication. Taken that several small molecules reactivating p53 failed in clinical trials, there is a need to develop new treatments targeting p53 proteins in cancer. This review outlines the most advanced strategies to reactivate p53 and p73 and describes drug repurposing approaches for the efficient reinstatement of the p53 proteins for cancer therapy.

Since the Coronavirus disease (COVID-19) outbreak at the end of 2019, the past two month has seen an acceleration both in and outside China in the R&D of the diagnostics, vaccines and therapeutics for this novel coronavirus. As one of the molecular forces that determine protein structure, electrostatic effects dominate many aspects of protein behaviour and biological function. Thus, incorporating currently available experimental structures related to COVID-19, this article reports a simple python-based analysis tool and a LaTeX-based editing tool to extract and summarize the electrostatic features from experimentally determined structures, to strengthen our understanding of COVID-19's structure and function and to facilitate machine-learning and structure-based computational design of its neutralizing antibodies and/or small molecule(s) as potential therapeutic candidates. Finally, this article puts forward a brief update of the structurally observed electrostatic features of the COVID-19 coronavirus.

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.

Pseudomonas aeruginosa is an emerging opportunistic pathogen responsible for cystic fibrosis and nosocomial infections. In addition, empirical treatments are become inefficient due to their multiple-antibiotic resistance and extensive colonizing ability. Quorum sensing (QS) plays a vital role in the regulation of virulence factors in P. aeruginosa. Attenuation of virulence by QS inhibition could be an alternative and effective approach to control infections. Therefore, we sought to discover new QS inhibitors (QSIs) against LasR receptor in P. aeruginosa using chemoinformatics. Initially, a structure-based high-throughput virtual screening was performed using the LasR active site that identified 61404 relevant molecules. E-pharmacophore (ADAHH) screening of these molecules rendered 72 QSI candidates. In standard-precision docking, only 7 compounds were found as potential QSIs due to their higher binding affinity to LasR receptor (-7.53 to -10.32 kcal/mol compared to -7.43 kcal/mol of native ligands). The ADMET properties of these compounds were suitable to be QSIs. Later, extra-precision docking and binding energy calculation suggested ZINC19765885 and ZINC72387263 as the most promising QSIs. The dynamic simulation of the docked complexes showed good binding stability and molecular interactions. The current study suggested that these two compounds could be used in P. aeruginosa QS inhibition to combat bacterial infections.

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.

Background: Early diagnosis of postoperative complications is an urgent task, allowing timely prescribing of appropriate therapy and reducing the cost of patient treatment. Prognostic models based on various data could help in the early diagnosis of postoperative complications after cardiac surgery. Objectives: The purpose of the study was to determine whether an integrated approach based on clinical data along with metabolites and biomarkers had greater predictive value than the models built on less data. Methods: The study included patients (n=62) admitted for planned cardiac surgery (coronary artery bypass grafting with cardiopulmonary bypass), who were retrospectively divided into two groups depending on the presence (n=26) or absence (n=36) of postoperative complications of all types. Clinical and laboratory data on the first day after surgery were analyzed. Additionally, patients' blood samples were collected before and on the first day after surgery to determine biomarkers (interleukin-6, procalcitonin, NT-proBNP, high-sensitivity troponin T) and metabolites (8 aromatic and 2 dicarboxylic acids were detected using gas chromatography-mass spectrometry). Results: Patients before surgery, compared with healthy donors, differed in the concentration of metabolites (p &lt; 0.05), and had concentrations of a number of biomarkers exceeding the corresponding reference values. Multivariate PLS-DA models, predicting the presence or absence of postoperative complications, were built using clinical data (ROC-AUC=0.71), concentrations of metabolites and biomarkers (ROC-AUC=0.60), and the entire data set (ROC-AUC=0.75). For comparison we built univariate models using the EuroScore2 (ROC-AUC=0.59) and SOFA (ROC-AUC=0.76) scales; concentrations of lactate (ROC-AUC=0.71), the dynamic changes of 4-hydroxyphenyllactic acid (ROC-AUC=0.69), and the sum of three sepsis-associated metabolites (ROC-AUC=0.70). The proposed complex model using entire data set was comparable to the SOFA scale, which confirms the expediency of searching for new predictive models based on a variety of factors.

Milk serves as a crucial source of natural bioactive compounds essential for human nutrition and health. The gastrointestinal digestion of milk proteins releases various biologically active peptides that positively impact human health and enhance physiological performance. With the growing production of cheese, Greek yogurt, and other dairy products, the volume of unutilized acid whey is significantly increasing. Acid whey, containing almost all whey proteins and approximately one-quarter of casein, presents a valuable raw material for generating peptides with potential health benefits. These peptides exhibit properties such as antioxidant, antimicrobial, anti-inflammatory, anticarcinogenic, antihypertensive, antithrombotic, opioid, mineral-binding, and growth-stimulating activities, contributing to improved human immunity and the treatment of chronic diseases. Enzymatic hydrolysis of milk proteins has been developed as a method to produce these peptides, facilitating their use in food fortification. To incorporate whey into functional beverages, it undergoes fermentation by lactic acid bacteria with proteolytic activity. To specifically enrich these beverages with peptides exhibiting targeted bioactivities, it is essential to select lactic acid bacteria strains possessing the requisite protease profiles. Additionally, the sensory properties of the final product can be enhanced using starter cultures with diverse metabolic capabilities, even in the absence of added flavoring agents. Given the imperative to develop waste-free technologies and enhance the profitability of the dairy industry, there is a growing need to efficiently utilize and transform acid whey into a broad spectrum of valuable products and food additives. This approach is justified not only by the nutritional benefits of the bioactive components in whey but also by the commercial potential of safe and health-enhancing products.

Vaccines against SARS-CoV-2 have been pivotal in overcoming the Covid-19 pandemic yet understanding the subsequent outcomes and immunological effects remain crucial, especially for at-risk groups e.g. people living with human immunodeficiency virus (HIV) (PLWH). In this study we report the longitudinal IgA and IgG antibody titers, as well as antibody-mediated angiotensin converting enzyme 2 (ACE2) binding blockade, against the SARS-CoV-2 spike (S) proteins after 1 and 2 doses of the ChAdOx1 nCoV-19 vaccine in a population of Black PLWH. Here, we report that PLWH (N = 103) did not produce an anti-S IgA response after infection or vaccination, however, anti-S IgG was detected in response to vaccination and infection, with the highest level detected for infected vaccinated participants. The anti-IgG and ACE2 blockade assays revealed that both vaccination and infection resulted in IgG production, however, only vaccination resulted in a moderate increase in ACE2 binding blockade to the ancestral S protein. Vaccination with a previous infection results in the greatest anti-S IgG and ACE2 blockade for the ancestral S protein. In conclusion, PLWH produce an anti-S IgG response to the ChAdOx1 nCoV-19 vaccine and/or infection, and ChAdOx1 nCoV-19 vaccination with a previous infection produced more neutralizing antibodies than vaccination alone.

Bone morphogenetic protein (BMP) and platelet-derived growth factor (PDGF) are known to regulate and stimulate osteogenesis, both proteins playing an important role in bone homeostasis, constituting good candidates for osteoporosis´ treatment. Herein we aim to evaluate the effects of recombinant human BMP-7 (rhBMP7) and PDGF-BB (rhPDGF-BB) in oophorectomy-induced osteoporosis in rats. To this end, 40 Sprague-Dawley rats were submitted to oophorectomy surgery and treatments were started at the 100th day after the surgery, when all animals showed signs of osteoporosis. These peptide growth factors were applied intravenously once or twice a week and animals were followed for a total of five weeks. Two weeks after the end of the treatments, the animals were euthanized, the tissues were collected and Alkaline Phosphatase, X-ray, micro-CT and histology were assessed. The results indicate that the most promising treatments are: 20 µg/kg PDGF-BB + 30 µg/kg BMP-7 2x/week and 30 µg/kg BMP-7 2x/week, presenting an increase of 15% (p&lt;0.05) and 13% (p&lt;0.05) in bone volume fraction and 21% (p&lt;0.05) and 23% (p&lt;0.05) in trabecular number, respectively. In conclusion, rhPDGF-BB and rhBMP-7 are capable of inducing an increase in bone volume and density in this osteoporotic animal model, constituting interesting candidates for osteoporosis treatment.

Neuropsychiatric disorders are globally public-health concern, in which diagnosis might be based on symptoms that often vary across individuals. Schizophrenia is one of the major neuropsychiatric disorders, which may affect millions worldwide. Detection of predictive biomarkers is the most intensively developing approach for the disease. However, the biochemical alterations have not been comprehensively distinguished up to the present time. In addition, there is less confidence of finding a specific biomarker for neuropsychiatric disorders including schizophrenia, but rather a specific characteristic behavioral pattern. Maternal immune activation has been considered to be one of the important factors for the development of neuropsychiatric disorders. Here, the mouse model of neuropsychiatric disorders has been built, in which poly-I:C, sodium dextran sulfate (DSS) and κ-carrageenan (CGN) were used for the maternal immune activation during the pregnancy of individuals. Subsequently, we challenged to link some of biochemical changes of p62 and GLAST in the offspring mice brain to the alteration of several pathological behaviors. Consequently, it has been shown that autophagy might be involved in the development of neuropsychiatric disorders including schizophrenia. To our knowledge, this is the first report of the significant correlation between pathological behavior and the biochemical alterations in neuropsychiatric disorder model animals. The relative ease of conducting these evaluation-tasks would make them useful for testing novel therapeutics designed to ameliorate the symptoms of several psychiatric disorders.

Tissue programmed death ligand-1 (PD-L1) protein is the recognized predictive immune biomarker of immune checkpoint inhibitor (ICI) treatment benefit in metastatic non-small cell lung cancer (NSCLC). However, tissue PD-L1 protein testing can be limited by tumor heterogeneity and fraught with tissue acquisition difficulties. A plasma PD-L1 assay potentially overcomes these tissue limitations. Patients with metastatic NSCLC treated with first-line ICI-based treatment and available results of plasma cfRNA PD-L1 by real-time polymerase chain reaction (RT-PCR) and tissue PD-L1 protein PD-L1 with the Dako 22C3 monoclonal antibody were retrospectively assessed for median and landmark 3-year overall survival (OS). OS was identical whether positive plasma cfRNA PD-L1 expression or positive tissue PD-L1 protein expression (median OS 15 months; 3-year landmark OS 30%; hazard ratio (HR) 0.97; 95% CI, 0.44-2.10). Positive plasma cfRNA PD-L1 patients also demonstrated a numerically longer median and higher 3-year OS compared to patients lacking PD-L1 expression (median 15 months versus 8 months; 3-year landmark OS 30% versus 15%; HR 0.56; 95% CI, 0.27-1.17). Plasma cfRNA PD-L1 expression by RT-PCR was similarly predictive of ICI-based treatment benefit as tissue PD-L1 protein expression.

The aim of the study was to evaluate the effect of crude protein (CP) levels in concentrate and Sesbania grandiflora pod meal (SG) supplementations on feed intake, rumen fermentation, and methane (CH4) mitigation in Thai purebred beef cattle. Four cattle with 100 ± 5.0 kg body weight were used in this study. A 2 × 2 factorial experiment in a 4 × 4 Latin square design were conducted, in which factor A was the CP contents in concentrate of 14, and 16% of dry matter (DM) and factor B was the supplement contents of SG at 0.4% and 0.6% DM intake, respectively. The results showed that the CP contents in concentrate and SG had no interaction effect on intake, digestibility, ruminal ecologies, ruminal fermentation products, and nitrogen utilization. Increasing CP contents in concentrate did not influence DM intake and nutrients’ digestibility, and SG supplementation at 0.6% significantly (P<0.05) decreased CP digestibility. Increasing CP content to 16% increased significantly (P<0.05) the ruminal ammonia nitrogen (NH3-N) concentration while decreased significantly (P<0.05) the NH3-N concentration, protozoal number, and blood urea nitrogen (BUN) at 4 h post-feeding. The 0.6% supplementation of the SG increased significantly average total volatile fatty acids (VFAs) and propionate (C3) concentration while decreased significantly average acetate (C2), C2:C3 ratio, and CH4 production, which was 2.71% for C2, 13.17% for C2:C3 ratio, and 4.37% for CH4 production lower than 0.4% supplementation. Fecal nitrogen excretion was significantly decreased when supplemented with 0.6% of the SG. In conclusion, 0.6% of the SG supplementation showed a greater effect on intake, rumen manipulation, and CH4 mitigation and would recommend supplementation to a concentrate-based diet containing either 14% or 16% CP content.

The spike protein of SARS coronavirus (SARS-CoV) attaches the virus to its cellular receptor, angiotensin-converting enzyme 2 (ACE2), which is mediated by the receptor binding domain (RBD) of the spike protein. Recently, an analysis based on decade-long structural studies of SARS was reported to illustrate with atomic-level details receptor recognition by the novel coronavirus from Wuhan, i.e., 2019-nCoV. Here, this article reports a comprehensive set of structural electrostatic analysis of all SARS-CoV spike protein RBD-related structures as of February 13, 2020, aiming at identifying the electrostatic hot spots for SARS-CoV spike protein to be complexed with ACE2 and its neutralizing antibodies. First, this article identified a structural action mechanism of the F26G19 antibody (of SARS-CoV spike protein), where its Asp56 residue binds to the Arg426 of the SARS-CoV spike protein RBD against the formation of the interfacial Arg426-Glu329 salt bridges between ACE2 and the SARS-CoV spike protein RBD. Second, a hypothesis is reported that a pair of electrostatic clips exist at the interface between ACE2 and the SARS-CoV spike protein RBD, including both Arg426-Glu329 and His445-Glu23-Lys447 salt bridges. Last, this article reports a structurally identified interfacial Glu35-Arg479 salt bridge which helps stabilize the complex structure of ACE2 and the SARS-CoV spike protein RBD. Overall, the structurally identified electrostatic hot spots reported here may be useful for the design of SARS-CoV-neutralizing antibodies in future.

Molecular hydrogen (H2) has been shown to have antioxidant and anti-inflammatory activities that may reduce the development and progression of many diseases. In this study, Hydrogen-rich water (HRW) was obtained by reacting hybrid magnesium-carbon hydrogen storage materials with water. Then the effects of intake of HRW on the activities of xenobiotic-metabolizing enzymes, membrane transporters, and oxidative stress in rats were investigated. Rats were given HRW ad libitum for 4 weeks. Results showed that intake of HRW had no significant effect on the activities of various cytochrome P450 (CYP) enzymes (CYP1A1, 1A2, 2B, 2C, 2D, 2E1, 3A, 4A), glutathione-S-transferase and UDP-glucuronosyltransferase. Except for a slight lower plasma glucose concentration, intake of HRW had no effect on other plasma biochemical parameters in rats. P-Glycoprotein and multidrug resistance-associated protein (Mrp)2 protein expressions in liver were elevated after intake of HRW. However, HRW had no significant effects on glutathione, glutathione peroxidase, or lipid peroxidation in liver. Results from this study suggest that consumption of HRW may not affect xenobiotic metabolism or oxidative stress in liver. However, intake of HRW may increase the efflux of xenobiotics or toxic substances from the liver into bile by enhancing the expression of p-glycoprotein and Mrp2 protein.

The paper focuses on the application of a multilevel, integrative and territorial approach to the protection of cultural heritage through planning instruments in Serbia. It points out the problems of the sectoral approach and discusses international recommendations that can be integrated into the planning methodology. The case study is the area of Djerdap National Park, which has an already established international and national importance. The research suggests that the cultural heritage in the area, with the exception of the Roman limes, is still recognized at the level of individual localities. The study emphasizes the importance of aligning all levels of governance to safeguard the whole spatial entity rather than focusing solely on its particular areas. The interna-tional and national importance of the planning area contributes to the strengthening of awareness of heritage and its institutional protection. However, the vitality of the area, which is key to its sustainable development, comes from the local level. Planning documents contribute to the inte-gration of local values and local communities into development programs and projects.

The impact of interaction SS with MJO on rainfall along Java Island to East Nusa Tenggara has been studied using daily rainfall observation data from 2140 weather observation stations, which are also equipped with GSMaP, OLR, and ERA5 data. There were 61 SS events found, 17 during active MJO and 44 in inactive MJO. Rainfall increases more in the western study area when the active MJO and more in the eastern study area when the inactive MJO is due to SS. SS reduces rainfall in the Jakarta region, regardless of the activity or inactivity of the MJO. SS causes extreme rainfall to only occur in a small part of certain areas, so it tends to significantly reduce the possibility of extreme rainfall. In the southern part of the IMC, SS predominates over MJO in supporting increased water vapor transport. Large-scale synoptic circulations like the monsoon, SS, and MJO can interact with local land-sea wind circulation to produce spatiotemporal variability in rainfall on Java Island. Rainfall mostly increases in the afternoon and decreases in the morning when SS occurs, whether there is MJO or not. Convective instability analysis indicates that SS increases precipitation, most likely by lowering CIN and raising VIMFC.

In order to shorten the detection time and improve the average precision on embedded devices, A lightweight and high accuracy model is proposed for passion fruit in complex environments (backlight, occlusion, overlap, sunny, cloudy, rainy). Firstly, replacing the backbone network of YOLOv5 with a lightweight GhostNet model reduces the number of parameters and computation while improving detection speed. Secondly, a new feature branch is added to the GhostNet network, and the feature fusion layer in the neck network is reconstructed to effectively combine the lower-level and higher-level features, which not only improves the accuracy of the model but also maintains its lightweight. Finally, the knowledge distillation methods are used to transfer the knowledge from the more capable teacher model to the less capable student model, which significantly improving the detection accuracy. The improved model is denoted as G-YOLO-NK. The average accuracy of the G-YOLO-NK network is 96.00%, which is 1.00% higher than the original YOLOv5s model. Furthermore, the improved model size is 7.14MB, reduced to half of the original model, and the real-time detection frame rate is 11.25 FPS on the Jetson Nano. Compared to the state-of-the-art model, the proposed model outperforms them in terms of average precision and detection performance. The present work provides an effective model for real-time detection of passion fruits in complex orchard scenes, which can provide valuable technical support for the development of orchard picking robots and greatly improve the intelligence level of orchards.

The Simultaneous Localization And Mapping (SLAM) scanner is an easy and portable Light Detection And Ranging (LiDAR) data acquisition device. Its main output is a 3D point cloud covering the scanned scene. Regarding the importance of accuracy in the survey domain, this paper aims to assess the accuracy of two SLAM scanners: the NavVis VLX and the BLK2GO Scanner. This assessment is conducted for both outdoor and indoor environments. In this context, two types of reference data are used: the Total Station (TS) and the static scanner Z+F Imager 5016. To carry out the assessment, four comparisons are tested: cloud to cloud, cloud to mesh, mesh to mesh, and edge detection board assessment. However, the results of the assessments confirm that the accuracy of indoor SLAM scanner measurements (5 mm) is greater than that of outdoor ones (between 10 mm and 60 mm). Moreover, the comparison of cloud to cloud provides the best accuracy regarding direct accuracy measurement without manipulations. Finally, based on the high accuracy, scanning speed, flexibility, and the accuracy differences between tested cases, it is confirmed that SLAM scanners are effective tools for data acquisition.

In recent years, electroencephalogram (EEG) emotion recognition has emerged as a research hotspot in the field of artificial intelligence (AI). Despite advancements in EEG-based emotion recognition, room for enhancing performance persists due to EEG signal redundancy and limitations in feature extraction, leading to inefficient models and emotional information loss. To fully utilize EEG's emotional information and improve recognition accuracy while reducing computational costs, this paper proposes a Convolutional-Recurrent Hybrid Network with a dual-stream adaptive approach and an attention mechanism (CSA-SA-CRTNN). Firstly, the model utilizes a CSAM module to assign corresponding weights to EEG channels. Then, an adaptive dual-stream convolutional-recurrent network (SA-CRNN and MHSA-CRNN) is applied to extract local spatial-temporal features. After that, the extracted local features are concatenated and fed into a temporal convolutional network with a multi-head self-attention mechanism (MHSA-TCN) to capture global information. Finally, the extracted EEG information is used for emotion classification. We conducted binary and ternary classification experiments on the DEAP dataset, achieving 99.26% and 99.15% accuracy for arousal and valence in binary classification, and 97.69% and 98.05% in ternary classification, surpassing relevant algorithms. Additionally, the model's efficiency is significantly higher than other models, achieving better accuracy with lower resource consumption.

Glycosidic linkage analysis was conducted on the unfractionated polysaccharides in the alcohol-insoluble residues (AIRs) prepared from six red seaweeds (Gracilariopsis sp., Prionitis sp., Mastocarpus papillatus, Callophyllis sp., Mazzaella splendens, and Palmaria palmata) using GC-MS/FID analysis of partially methylated alditol acetates (PMAAs). The cell walls of P. palmata primarily contained mixed-linkage xylans and small amounts of sulfated galactans and cellulose. In contrast, the unfractionated polysaccharides of the other five species were rich in galactans displaying diverse 3,6-anhydro-galactose and galactose linkages with varied sulfation patterns. Different levels of cellulose were also observed. This glycosidic linkage method offers advantages for cellulose analysis over traditional monosaccharide analysis that is known for underrepresenting glucose in crystalline cellulose. Relative linkage compositions calculated from GC-MS and GC-FID measurements showed that anhydro sugar linkages generated more response in the latter detection method. This improved linkage workflow presents a useful tool for studying polysaccharide structural variations across red seaweed species. Furthermore, for the first time, relative linkage compositions from GC-MS and GC-FID measurements, along with normalized FID and total ion current (TIC) chromatograms without peak assignments, were analyzed using principal component analysis (PCA) as a proof-of-concept demonstration of the technique's potential to differentiate various red seaweed species.

Background: Educating college students about the importance of good sleep habits could lead to the adoption of healthier sleep routines, including sufficient sleep duration and sleep quality. The current pre-post study examines the impact of completing a sleep education program called “Sleep 101” on (1) sleep hygiene practices, (2) sleep quality, and (3) weekday and weekend sleep behaviors among college students. “Sleep 101” is an interactive online course developed to educate college students about the importance of sleep by addressing basic sleep physiology, the impact of sleep on mood, academic and physical performance, the interactions between sleep and various substances, and common sleep disorders. Methods: 338 college students were recruited from a midsized public university in the US and invited to participate in “Sleep 101”, of which 25 participants completed the baseline and post-intervention questionnaire. The Sleep Hygiene Index (SHI) was utilized to evaluate sleep hygiene practices, while standard questions were employed to assess weekday and weekend sleep behaviors and overall sleep quality. Results: Those who completed the study demonstrated a significant increase in weekday sleep duration and were less likely to engage in negative prior bedtime activities. In addition, 40% of those who completed baseline and post-intervention assessments noticed improvements in their sleep quality. Conclusion: These findings suggest that a brief online program is beneficial in improving sleep behaviors among college and could be a scalable approach to addressing current sleep issues and preventing future sleep problems in college students.

The profile of secondary metabolites occurring in apple cuticular layer is not only characteristic for particular apple cultivar, but also dynamically reflects various external factors in growing environment. In this study, the possibility to authenticate apple samples based on analysis of their cuticular layer extracts was investigated. Ultra-high-performance liquid chromatography coupled to high resolution tandem mass spectrometry (UHPLC-HRMS/MS) was employed for obtaining metabolomic fingerprints. In total, 274 authentic apple samples, four cultivars harvested within the seasons 2020 to 2022 in the Czech Republic and Poland were analysed. The generated complex data processed by univariate and multivariate statistical methods enabled to build classification models for discrimination of apple cultivars as well as their geographical origin. The models showed very good performance in differentiation of Czech and Polish samples for three of four cultivars: ‘Gala’, ‘Golden Delicious’ and ‘Idared’, moreover, the validity of the models was verified over harvest seasons. The diagnostic markers, besides metabolites of triterpene biosynthetic pathway, mainly represented wax esters. ‘Jonagold’, which is known to be susceptible to mutations, was the only cultivar for which the unambiguous classification of geographical origin was not possible.

Lymphadenomegaly is well described in dogs with neoplastic diseases, but it can also reflect in-flammation. However, its role in assessments of inflammation is little reported in the veterinary literature. Lymph nodes are most easily assessed superficially through palpation, but computed tomography (CT) enables evaluation of deep lymph nodes. Accordingly, we investigated associ-ations between maximal lymph node diameter and plasma concentrations of C-reactive protein (CRP), a sensitive inflammatory marker, targeting three deep lymph nodes (sternal, cranial me-diastinal, and internal iliac). We evaluated data from small dogs with non-neoplastic diseases that underwent synchronous CT and blood biochemistry at our institution. We found that cranial mediastinal lymph node diameter was significantly greater in dogs with clinically elevated CRP than those with clinically unremarkable CRP (CRP 0.7-7.0< mg/dL vs.

Background: An aberrant cellular microenvironment characterized by pathological cells or inflammation represents an added risk factor across various cancer types. While the significance of chronic inflammation in the development of most diffuse tumors has been extensively studied, an exception to this analysis exists in the context of Chondrosarcomas. Chondrosarcomas represent 20–30% of all skeletal sarcomas and have an estimated incidence of 1 in 100,000 worldwide. The mean age of presentation is 50, with over 70% of patients over the age of 40 at the time of diagnosis. The aim of this retrospective study was to analyze the role of C-reactive protein (CRP) as a prognostic factor in relation to the histopathological findings for Chondrosarcoma. Methods: This retrospective study included 73 patients with confirmed Chondrosarcoma diagnosis treated between 2004 and 2019. Preoperative CRP determination was assessed in mg/dL with a non-pathological value below 0.5 mg/dL. Disease-free survival time was calculated as the time between the initial diagnosis and an event like local recurrence or metastasis. Follow-up status was described in death of disease, no evidence of disease and alive with disease. The exclusion criteria of this study included insufficient laboratory values, lack of information regarding the follow-up status and incomplete histopathological report. Results: The calculate risk estimation of a reduced follow-up time was 2,25 time higher in the patients with an CRP level &gt;0.5 mg/dL (HR 2,25 and 95% CI 1,13-4,45) and 3 times higher in patients with a tumour size &gt;pT2 (HR 3 and 95% CI 1,59-5,92). We can easily confirm that the most elevated risk for a reduce follow-up time relies in chondrosarcomas high grading, preoperative pathological CRP- level and a size &gt;8 cm. Conclusions: In Chondrosarcoma cases, we can consider CRP pretreatment value &gt;0.5 mg/dL a sensitive prognostic and sensitive risk factor for distant metastasis.

African swine fever virus (ASFV) is a large double stranded DNA arbovirus that is highly contagious and seriously endangers the lives of domestic and wild pigs. In the past decade, African swine fever (ASF) has spread in many countries in the Caucasus, Russian Federation, Eastern Europe and Asia, causing significant losses to the pig industry. At present, there is a lack of effective vaccine and treatment for ASF. Therefore, the rapid and accurate detection is crucial for ASF prevention and control. In this study, we have developed a portable lateral flow strip (LFS) detection method mediated by recombinase polymerase amplification (RPA) and DNA enzyme (LwCas13a), which is performed at 37 ℃ and visualized by eyes without the need for complex instruments. This RPA-LwCas13a-LFS is based on the ASFV structural protein p17 gene (D117L), with a detection sensitivity up to 2 gene copies. This method is highly specific and has no cross reactivity to 7 other pig viruses. In the detection of 33 clinical samples, the p17 (D117L) RPA-LwCas13a-LFS had 100% coincidence with conventional quantitative PCR (qPCR). These findings demonstrate the potential of this simple, rapid, sensitive, and specific ASFV detection method for on-site ASFV detection.

The pathogenesis of coronavirus disease 2019 (COVID-19) is associated with a hyperinflammatory response. The mechanisms of SARS-CoV-2-induced inflammation are scantly known. Methylglyoxal (MG) is a glycolysis-derived by-product, endowed with a potent glycating action, leading to the formation of advanced glycation end products (AGEs), the major of which is MG-H1. MG-H1 exerts strong pro-inflammatory effects, frequently mediated by the receptor for AGEs (RAGE). Here, we investigated the involvement of MG-H1/RAGE axis as a potential novel mechanism in SARS-CoV-2-induced inflammation by resorting to human bronchial (BEAS-2B) and alveolar (A549) epithelial cells, expressing different levels of ACE2 receptor (R), exposed to SARS-CoV-2 spike protein 1 (S1). Interestingly, we found in BEAS-2B cells that do not express ACE2-R, that S1 exerted a pro-inflammatory action through a novel MG-H1/RAGE-based pathway. RAGE expression levels in nasopharyngeal swabs from SARS-Cov-2 positive and negative individuals, as well as glyoxalase 1 expression, the major scavenging enzyme of MG, seem to support the results obtained in vitro. Altogether, our findings reveal a novel mechanism involved in the inflammation triggered by S1, paving the way to the study of MG-H1/RAGE inflammatory axis in SARS-CoV-2 infection as a potential therapeutic target to mitigate COVID-19 associated pathogenic inflammation.

Pregnancy associated plasma protein-A (PAPP-A) plays an integral role in breast cancer (BC), especially triple negative breast cancer (TNBC). This subtype accounts for the most aggressive BC, possesses high tumor heterogeneity, is least responsive to standard treatments and has the poorest clinical outcomes. There is a critical need to address the lack of effective targeted therapeutic options available. PAPP-A is a protein that is highly elevated during pregnancy. Frequently, higher PAPP-A expression is detected in tumors than in healthy tissues. The increase in expression coincides with increased rates of aggressive cancers. In BC, PAPP-A has been demonstrated to play a role in tumor initiation, progression, metastasis including epithelial-mesenchymal transition (EMT), as well as acting as a biomarker for predicting patient outcomes. In this review, we present the role of PAPP-A, with specific focus on TNBC. The structure and function of PAPP-A, belonging to the pappalysin subfamily, and its proteolytic activity are assessed. We highlight the link of BC and PAPP-A with respect to the IGFBP/IGF axis, EMT, the window of susceptibility and the impact of pregnancy. Importantly, the relevance of PAPP-A as a TNBC clinical marker is reviewed and its influence on immune-related pathways are explored. The relationship and mechanisms involving PAPP-A reveal the potential for more treatment options that can lead to successful immunotherapeutic targets and the ability to assist with better predicting clinical outcomes in TNBC.