Fabricating Multiphasic Angiogenic Scaffolds Using Amyloid/Roxadustat-Assisted High-Temperature Protein Printing.

Repairing multiphasic defects is cumbersome. This study presents new soft and hard scaffold designs aimed at facilitating the regeneration of multiphasic defects by enhancing angiogenesis and improving cell attachment. Here, the nonimmunogenic, nontoxic, and cost-effective human serum albumin (HSA) fibril (HSA-F) was used to fabricate thermostable (up to 90 °C) and hard printable polymers. Additionally, using a 10.0 mg/mL HSA-F, an innovative hydrogel was synthesized in a mixture with 2.0% chitosan-conjugated arginine, which can gel in a cell-friendly and pH physiological environment (pH 7.4). The presence of HSA-F in both hard and soft scaffolds led to an increase in significant attachment of the scaffolds to the human periodontal ligament fibroblast (PDLF), human umbilical vein endothelial cell (HUVEC), and human osteoblast. Further studies showed that migration (up to 157%), proliferation (up to 400%), and metabolism (up to 210%) of these cells have also improved in the direction of tissue repair. By examining different in vitro and ex ovo experiments, we observed that the final multiphasic scaffold can increase blood vessel density in the process of per-vascularization as well as angiogenesis. By providing a coculture environment including PDLF and HUVEC, important cross-talk between these two cells prevails in the presence of roxadustat drug, a proangiogenic in this study. In vitro and ex ovo results demonstrated significant enhancements in the angiogenic response and cell attachment, indicating the effectiveness of the proposed design. This approach holds promise for the regeneration of complex tissue defects by providing a conducive environment for vascularization and cellular integration, thus promoting tissue healing.

PPInterface: A Comprehensive Dataset of 3D Protein-Protein Interface Structures.

The PPInterface dataset contains 815,082 interface structures, providing the most comprehensive structural information on protein-protein interfaces. This resource is extracted from over 215,000 three-dimensional protein structures stored in the Protein Data Bank (PDB). The dataset contains a wide range of protein complexes, providing a wealth of information for researchers investigating the structural properties of protein-protein interactions. The accompanying web server has a user-friendly interface that allows for efficient search and download functions. Researchers can access detailed information on protein interface structures, visualize them, and explore a variety of features, increasing the dataset's utility and accessibility. The dataset and web server can be found at https://3dpath.ku.edu.tr/PPInt/.

GRP78 recognizes EV-F 3D protein and activates NF-κB to repress virus replication by interacting with CHUK/IKBKB.

Enteroviruses are the causative agents associated with several human and animal diseases, posing a significant threat to human and animal health. As one of the host immune defense strategies, innate immunity plays a crucial role in defending against invading pathogens, where the host utilizes a variety of mechanisms to inhibit or eliminate the pathogen. Here, we report a new strategy for the host to repress enterovirus replication by the 78 kDa glucose-regulated protein (GRP78), also known as heat shock protein family A member 5 (HSPA5). The GRP78 recognizes the EV-encoded RNA-dependent RNA polymerases (RdRPs) 3D protein and interacts with the nuclear factor kappa B kinase complex (CHUK) and subunit beta gene (IKBKB) to facilitate the phosphorylation and nuclear translocation of NF-κB, which induces the production of inflammatory factors and leads to a broad inhibition of enterovirus replication. These findings demonstrate a new role of GRP78 in regulating host innate immunity in response to viral infection and provide new insights into the mechanism underlying enterovirus replication and NF-κB activation.IMPORTANCEGRP78 is known as a molecular chaperone for protein folding and plays a critical role in maintaining protein folding and participating in cell proliferation, cell survival, apoptosis, and metabolism. However, the functions of GRP78 to participate in enterovirus genome replication and innate immune responses are rarely documented. In this study, we explored the functions of the EV-3D-interacting protein GRP78 and found that GRP78 inhibits enterovirus replication by activating NF-κB through binding to EV-F 3D and interacting with the NF-κB signaling molecules CHUK/IKBKB. This is the first report that GRP78 interacts with CHUK/IKBKB to activate the NF-κB signaling pathway, which leads to the expression of the proinflammatory cytokines and inhibition of enterovirus replication. These results demonstrate a unique mechanism of virus replication regulation by GRP78 and provide insights into the prevention and treatment of viral infections.

EV71 5'UTR interacts with 3D protein affecting replication through the AKT-mTOR pathway.

BACKGROUND: EV71 is one of the important pathogens of Hand-foot-and-mouth disease (HFMD), which causes serious neurological symptoms. Several studies have speculated that there will be interaction between 5'UTR and 3D protein. However, whether 5'UTR interacts with the 3D protein in regulating virus replication has not been clarified. METHODS: Four 5'UTR mutation sites (nt88C/T, nt90-102-3C, nt157G/A and nt574T/A) and two 3D protein mutation sites (S37N and R142K) were mutated or co-mutated using virulent strains as templates. The replication of these mutant viruses and their effect on autophagy were determined. RESULTS: 5'UTR single-point mutant strains, except for EGFP-EV71(nt90-102-3C), triggered replication attenuation. The replication ability of them was weaker than that of the parent strain the virulent strain SDLY107 which is the fatal strain that can cause severe neurological complications. While the replication level of the co-mutant strains showed different characteristics. 5 co-mutant strains with interaction were screened: EGFP-EV71(S37N-nt88C/T), EGFP-EV71(S37N-nt574T/A), EGFP-EV71(R142K-nt574T/A), EGFP-EV71(R142K-nt88C/T), and EGFP-EV71(R142K-nt157G/A). The results showed that the high replicative strains significantly promoted the accumulation of autophagosomes in host cells and hindered the degradation of autolysosomes. The low replicative strains had a low ability to regulate the autophagy of host cells. In addition, the high replicative strains also significantly inhibited the phosphorylation of AKT and mTOR. CONCLUSIONS: EV71 5'UTR interacted with the 3D protein during virus replication. The co-mutation of S37N and nt88C/T, S37N and nt574T/ A, R142K and nt574T/A induced incomplete autophagy of host cells and promoted virus replication by inhibiting the autophagy pathway AKT-mTOR. The co-mutation of R142K and nt88C/T, and R142K and nt157G/A significantly reduced the inhibitory effect of EV71 on the AKT-mTOR pathway and reduced the replication ability of the virus.

Genomic profile of eGFP-tagged senecavirus A subjected to serial plaque-to-plaque transfers.

Senecavirus A (SVA) belongs to the genus Senecavirus in the family Picornaviridae. This virus possesses a positive-sense, single-stranded RNA genome, approximately 7200 nt in length, composed of a single 5' untranslated region, encoding region and 3' untranslated region. In this study, a recombinant SVA tagged with enhanced green fluorescent protein (eGFP) sequence, rSVA-eGFP, was rescued from its cDNA clone using reverse genetics. The passage-5 (P5) rSVA-eGFP was totally subjected to 55 rounds of consecutive fluorescent plaque-to-fluorescent plaque (FP-FP) transfers, and one extra common passaging in vitro. The P61 viral stock was analyzed by next-generation sequencing. The result showed ten single-nucleotide mutations (SNMs) in the rSVA-eGFP genome, including nine transitions and only one transversion. The P61 progeny still showed a complete eGFP sequence, indicating no occurrence of copy-choice recombination within the eGFP region during serial FP-FP transfers. In other words, this progeny was genetically deficient in the recombination of eGFP sequence (RES), namely, an RES-deficient strain. Out of ten SNMs, three were missense mutations, leading to single-amino acid mutations (SAAMs): F15V in L protein, A74T in VP2, and E53R in 3D protein. The E53R was predicted to be spatially adjacent to the RNA channel of 3D protein, perhaps involved in the emergence of RES-deficient strain. In conclusion, this study uncovered a global landscape of rSVA-eGFP genome after serial FP-FP transfers, and moreover shed light on a putative SAAM possibly related to the RES-deficient mechanism.

Withania somnifera mitochondrial atp4 gene editing alters the ATP synthase b subunit, independent of salt stress.

Numerous studies have shown that stress in plant cells and organelles with transport electron chains is related to RNA editing. The ATP synthase complex present in mitochondria plays a crucial role in cellular respiration and consists of several subunits. Among them is the b subunit, which is encoded by the mitochondrial atp4 gene. Computing-based analysis of the effects of RNA editing of the Withania somnifera atp4 gene in mitochondria leading to alterations in the b subunit of ATP synthase. Using the CLC Genomic Workbench 3, RNA editing analysis between the control and salt stress conditions was not significantly different. Depending on RNA editing, the tertiary structure model revealed a change in the states of the b subunit, reflecting differences in the central stalk and F1-catalytic domain. The study found that polar edits in the N-terminus of the b subunit allow for efficient H + ion selectivity and introduce a new coiled-coil alpha-helical structure that may help stabilize the complex. The most noteworthy finding of this study was the strong impact of these editing events on the tertiary structure of the b subunit, which has the potential to affect the ATPase activity and indicate that the editing in this subunit aimed to restore the original active protein and not as a response to salt stress.

High pressure freezing and cryo-sectioning can be used for protein structure determination by electron diffraction.

Electron diffraction of three-dimensional nanometer sized crystals has emerged since 2013 as an efficient technique to solve the structure of both small organic molecules and model proteins. However, the major bottleneck of the technique when applied to protein samples is to produce nano-crystals that do not exceed 200 to 300 nm in at least one dimension and to deposit them on a grid while keeping the minimum amount of solvent around them. Since the presence of amorphous solvent around the crystal, necessary to preserve its integrity, increases the amount of diffuse scattering, thus degrading the signal-to noise ratio of the diffraction signal, other sample preparation strategies have been developed. One of them is the milling of thin crystal lamella using focused ion beam (FIB), which was successfully applied to several protein crystals. Here, we present a new approach that uses cryo-sectioning after high pressure freezing of dextran embedded protein crystals. 150 to 200 nm thick cryo-sections of hen egg white lysozyme tetragonal crystals where used for electron diffraction experiments. Complete diffraction data up to 2.9 Å resolution have been collected and the lysozyme structure has been solved by molecular replacement and refined against these data. Our data demonstrate that cryo-sectioning can preserve protein structure at high resolution and can be used as a new sample preparation technique for 3D electron diffraction experiments of protein crystals. The different orientations found in the crystal chips and their large number resulting from the cryo-sectioning make the latter an attractive approach as it combines advantages from both blotting approaches (number of crystals) and FIB-milling (controlled thickness and absence of solvent around the crystal).

In silico analysis of the possible crosstalk between O-linked ß-GlcNAcylation and phosphorylation sites of Disabled 1 adaptor protein in vertebrates.

Disabled 1 (Dab1) is an adaptor protein with essential functions regulated by reelin signaling and affects many biological processes in the nervous system, including cell motility, adhesion, cortical development, maturation, and synaptic plasticity. Posttranslational modifications directly guide the fates of cytoplasmic proteins to complete their functions correctly. Reciprocal crosstalk between O-GlcNAcylation and phosphorylation is a dynamic modification in cytoplasmic proteins. It modulates the functions of the proteins by regulating their interactions with other molecules in response to the continuously changeable cell microenvironment. Although Dab1 contains conserved recognition sites for phosphorylation in their N-terminal protein interaction domain, the O-ß-GlcNAcylation and phosphorylation sites of human Dab1 sequence, their reciprocal crosstalk, and potential kinases catalyzing the phosphorylation remain unknown. In this study, we determined potential thirty-seven O-ß-GlcNAcylation and sixty-seven phosphorylation sites. Conserved twenty-one residues of these glycosylated sites were also phosphorylated with various kinases, including ATM, CKI, DNAPK, GSK3, PKC, PKG, RSK, cdc2, cdk5, and p38MAPK. In addition, we analyzed these conserved sites at our constructed two- and three-dimensional structures of human Dab1 protein. Dab1 protein models were frequently composed of coil structures as well as α-helix and ß-strands. Many of these conserved crosstalk sites between O-ß-GlcNAcylation and phosphorylation were localized at the coil region of the protein model. These findings may guide biochemical, genetic, and glyco-biology based on further experiments about the Dab1 signaling process. Understanding these modifications might change the point of view of the Dab1 signaling process and treatment for pathological conditions in neurodegenerative diseases such as Alzheimer's disease.

Production of recombinant proteins including the B-cell epitopes of autolysin A of Staphylococcus aureus isolated from clinical sheep mastitis and their potential for vaccine development.

Staphylococcus aureus is the most common clinical mastitis-associated pathogen in sheep which contributes to reduced welfare of affected animals and, therefore, compromises the quality and quantity of milk production. To prevent mastitis and its spread, it is essential to guarantee adequate breeding conditions and animal health, through the adoption of good farm management practices and the application of suitable biosecurity measures. Vaccination can play a strategic role in prevention, control, and eradication of diseases. The identification of secreted and cellular antigens of the predominant sheep-CC130/ST700/t1773 lineage would assist in the design of effective vaccine against mammary infections caused by S. aureus. In the current study, we carried out a 3D structural prediction analysis with the identification of the best B cell epitopes of the whole and secreted portion of S. aureus AtlA. Fragments of atlA, containing the main predicted epitopes, were amplified, cloned, and expressed in Escherichia coli for recombinant protein production. Two selected clones produced recombinant proteins (rAtl4 and rAtl8) showing strong reactivity with a hyperimmune serum against the native AtlA and with blood sera collected from sheep with clinical S. aureus mastitis. These may represent potential candidate protein-based vaccines able to elicit a protective immune response to be evaluated by vaccination and subsequent challenge of the vaccinated sheep.

Determine genetic variations in heat shock factor gene family (HSFs) and study their effect on the functional and structural characterization of protein in Tali goat.

In this study, the effect of genetic variations of four heat shock transcription factor genes (HSF1, HSF2, HSF4, and HSF5) on the 3 D protein structure and function were studied. We defined the breed-specific genetic variations of pooled DNA of Tali goat that differed from the goat reference sequence (CHI2.0). Disordered regions of HSF proteins were predicted using PONDR. Post-translation changes were studied by several predicted online servers. Then, the structure of the order region of proteins was anticipated by using the Swiss model. Tali goat HSF genes contain a total number of 181, 679, 91, and 301 SNPs for HSF1, 2, 4, and 5, respectively. Also, 5 and 3 variants were identified as nsSNPs in the coding region of HSF4 and HSF5, respectively. (r.145A/S), (r.322P/Y), (r.379T/C) in HSF4 and (r.300Q/P), (r.573E/Q) in HSF5 obtained the tolerant and high confidence (SIFT score) for nsSNPs. More than half of these proteins are predicted to be disordered (56, 50, 52, and 50%, respectively for HSF1, 2, 4, and 5). Phosphorylation, acetylation, glycosylation, and Sumoylation sites of HSFs were compared between Tali goat and reference goat. Three residues S145, S263, and S322 of HSF4 in Tali goat were phosphorylation sites, and in HSF5, the reference goat has a phosphorylation site in S593.

Bioinformatics Predicted Linear Epitopes of the Major Coat Protein of the Beet Yellows Virus for Detection of the Virus in the Cell Extract of the Infected Plant.

Beet yellows virus, which belongs to the genus Closterovirus, family Closteroviridae and has a significant negative economic impact, has proven to be challenging to detect and diagnose. To obtain antibodies against BYV, we propose an easier bioinformatics approach than the isolation and purification of the wild virus as an antigen. We used the SWISS-MODEL Workspace (Biozentrum Basel) protein 3D prediction program to discover epitopes of major coat protein p22 lying on the surface of the BYV capsid. Sequences coding these epitopes were cloned into plasmid pQE-40 (Qiagen) in frame with mouse dihydrofolate reductase gene. Fused epitopes were expressed in Escherichia coli and isolated by the Ni-NTA affinity chromatography. Murine antibodies were raised against each epitope and in a combination of both and characterized by dot-ELISA and indirect ELISA. We successively used these antibodies for diagnosis of virus disease in systemically infected Tetragonia tetragonioides. We believe the approach described above can be used for diagnostics of difficult-to-obtain and hazardous-to-health viral infections.

A Missense Mutation (c.1037 G > C, p. R346P) in PAPSS2 Gene Results in Autosomal Recessive form of Brachyolmia Type 1 (Hobaek Form) in A Consanguineous Family.

BACKGROUND: Brachyolmia is a skeletal disorder with an autosomal mode of inheritance (both dominant and recessive) in which the patients have a short height, scoliosis and a reduced trunk size. METHODS: From the Muzaffargarh District in Pakistan, a consanguineous family with multiple Brachyolmia-affected subjects were enrolled in the present study. Basic epidemiological data and radiographs were collected for the subjects. Whole exome sequencing (WES) which was followed by Sanger sequencing was applied to report the geneticbasic of Brachyolmia. RESULTS: The WES identified a missense mutation (c.1037 G > C, p. R346P) in exon 9 of the PAPSS2 gene that was confirmed by the Sanger sequencing in the enrolled subjects. The mutation followed a Mendalian pattern with an autosomal recessive inheritance mode. Multiple sequence alignment by Clustal Omega indicated that the PAPSS2 mutation-containing domain is highly conserved. The HEK293T whole-cell extract that was transfected with the Myc-tagged PCMV6-PAPSS2 of both the wild and mutant constructs were resolved by SDS-PAGE as well as by a Western blot, which confirmed that there are different PAPSS2 protein expression patterns when they were compared between the control and Brachyolmia patients. This difference between the normal and mutated protein was not evident when the three-dimensional computational structures were generated using homology modeling. CONCLUSION: We report a missense mutation (c.1037 G > C, p. R346P) in the PAPSS2 gene that caused Brachyolmia in a consanguineous Pakistani family.

PDBsum1: A standalone program for generating PDBsum analyses.

PDBsum1 is a standalone set of programs to perform the same structural analyses as provided by the PDBsum web server (https://www.ebi.ac.uk/pdbsum). The server has pages for every entry in the Protein Data Bank (PDB) and can also process user-uploaded PDB files, returning a password-protected set of pages that are retained for around 3 months. The standalone version described here allows for in-house processing and indefinite retention of the results. All data files and images are pre-generated, rather than on-the-fly as in the web version, so can be easily accessed. The program runs on Linux, Windows, and mac operating systems and is freely available for academic use at https://www.ebi.ac.uk/thornton-srv/software/PDBsum1.

Fuzzy spherical truncation-based multi-linear protein descriptors: From their definition to application in structural-related predictions.

This study introduces a set of fuzzy spherically truncated three-dimensional (3D) multi-linear descriptors for proteins. These indices codify geometric structural information from kth spherically truncated spatial-(dis)similarity two-tuple and three-tuple tensors. The coefficients of these truncated tensors are calculated by applying a smoothing value to the 3D structural encoding based on the relationships between two and three amino acids of a protein embedded into a sphere. At considering, the geometrical center of the protein matches with center of the sphere, the distance between each amino acid involved in any specific interaction and the geometrical center of the protein can be computed. Then, the fuzzy membership degree of each amino acid from an spherical region of interest is computed by fuzzy membership functions (FMFs). The truncation value is finally a combination of the membership degrees from interacting amino acids, by applying the arithmetic mean as fusion rule. Several fuzzy membership functions with diverse biases on the calculation of amino acids memberships (e.g., Z-shaped (close to the center), PI-shaped (middle region), and A-Gaussian (far from the center)) were considered as well as traditional truncation functions (e.g., Switching). Such truncation functions were comparatively evaluated by exploring: 1) the frequency of membership degrees, 2) the variability and orthogonality analyses among them based on the Shannon Entropy's and Principal Component's methods, respectively, and 3) the prediction performance of alignment-free prediction of protein folding rates and structural classes. These analyses unraveled the singularity of the proposed fuzzy spherically truncated MDs with respect to the classical (non-truncated) ones and respect to the MDs truncated with traditional functions. They also showed an improved prediction power by attaining an external correlation coefficient of 95.82% in the folding rate modelling and an accuracy of 100% in distinguishing structural protein classes. These outcomes are better than the ones attained by existing approaches, justifying the theoretical contribution of this report. Thus, the fuzzy spherically truncated-based protein descriptors from MuLiMs-MCoMPAs (http://tomocomd.com/mulims-mcompas) are promising alignment-free predictors for modeling protein functions and properties.

Phylogenetic analysis and characterization of arsenic (As) transforming bacterial marker proteins following isolation of As-tolerant indigenous bacteria.

Marker proteins play a significant role in bacterial arsenic (As) transformation. Phylogenetic analysis and three-dimensional (3D) characteristics of As transforming bacterial marker proteins guide the evolutionary origin and As transforming potential of the species. Indeed, As-tolerant bacteria also show a significant level of As transformation. Hence, characterization of As transforming bacterial marker proteins, isolation of As transforming bacteria, and proper integration of the findings may guide to elucidate how bacteria transform As. Therefore, phylogenetic analysis and 3D characterization of As transforming bacterial marker protein following isolation of potential indigenous As-tolerant indigenous bacteria were done to explore the mechanism of bacterial As transformation. Phylogenetic analysis of ten As transforming marker proteins (arsA, arsB, arsC, arsD, arsR, aioA, arrA, aioB, acr1, and acr3) in 20 potential bacterial genomes (except 19 for the acr3) were studied. Some bacterial genomes featured up to five marker proteins, and therefore, 3D characteristics of the marker proteins were analyzed in those genomes having three-to-five marker proteins. In phylogeny, species in close clades represent their phylogenetic resemblances and may have similar functions. P. aeruginosa, E. coli, and K. pneumonia were found to be more effective due to having the highest number (five) of marker proteins. In 3D protein modeling, most of the marker proteins were found to be active. Among 19 indigenous bacterial isolates, multiple isolates showed tolerance up to 50 mM As(III) and 250 mM As(V), which may potentially transform a significant quantities of As. Hence, integration of the results of phylogenetic analysis, 3D protein characteristics, and As tolerance in the bacterial isolates could guide to explore the mechanism of how bacteria transform As at cellular and molecular levels.

Antimicrobial Proteins and Peptides in Avian Eggshell: Structural Diversity and Potential Roles in Biomineralization.

The calcitic avian eggshell provides physical protection for the embryo during its development, but also regulates water and gaseous exchange, and is a calcium source for bone mineralization. The calcified eggshell has been extensively investigated in the chicken. It is characterized by an inventory of more than 900 matrix proteins. In addition to proteins involved in shell mineralization and regulation of its microstructure, the shell also contains numerous antimicrobial proteins and peptides (AMPPs) including lectin-like proteins, Bacterial Permeability Increasing/Lipopolysaccharide Binding Protein/PLUNC family proteins, defensins, antiproteases, and chelators, which contribute to the innate immune protection of the egg. In parallel, some of these proteins are thought to be crucial determinants of the eggshell texture and its resulting mechanical properties. During the progressive solubilization of the inner mineralized eggshell during embryonic development (to provide calcium to the embryo), some antimicrobials may be released simultaneously to reinforce egg defense and protect the egg from contamination by external pathogens, through a weakened eggshell. This review provides a comprehensive overview of the diversity of avian eggshell AMPPs, their three-dimensional structures and their mechanism of antimicrobial activity. The published chicken eggshell proteome databases are integrated for a comprehensive inventory of its AMPPs. Their biochemical features, potential dual function as antimicrobials and as regulators of eggshell biomineralization, and their phylogenetic evolution will be described and discussed with regard to their three-dimensional structural characteristics. Finally, the repertoire of chicken eggshell AMPPs are compared to orthologs identified in other avian and non-avian eggshells. This approach sheds light on the similarities and differences exhibited by AMPPs, depending on bird species, and leads to a better understanding of their sequential or dual role in biomineralization and innate immunity.

In Silico Characterisation of the Late Embryogenesis Abundant (LEA) Protein Families and Their Role in Desiccation Tolerance in Ramonda serbica Panc.

Ramonda serbica Panc. is an ancient resurrection plant able to survive a long desiccation period and recover metabolic functions upon watering. The accumulation of protective late embryogenesis abundant proteins (LEAPs) is a desiccation tolerance hallmark. To propose their role in R. serbica desiccation tolerance, we structurally characterised LEAPs and evaluated LEA gene expression levels in hydrated and desiccated leaves. By integrating de novo transcriptomics and homologues LEAP domains, 318 R. serbica LEAPs were identified and classified according to their conserved motifs and phylogeny. The in silico analysis revealed that hydrophilic LEA4 proteins exhibited an exceptionally high tendency to form amphipathic α-helices. The most abundant, atypical LEA2 group contained more hydrophobic proteins predicted to fold into the defined globular domains. Within the desiccation-upregulated LEA genes, the majority encoded highly disordered DEH1, LEA1, LEA4.2, and LEA4.3 proteins, while the greatest portion of downregulated genes encoded LEA2.3 and LEA2.5 proteins. While dehydrins might chelate metals and bind DNA under water deficit, other intrinsically disordered LEAPs might participate in forming intracellular proteinaceous condensates or adopt amphipathic α-helical conformation, enabling them to stabilise desiccation-sensitive proteins and membranes. This comprehensive LEAPs structural characterisation is essential to understanding their function and regulation during desiccation aiming at crop drought tolerance improvement.

Senecavirus a 3D Interacts with NLRP3 to Induce IL-1ß Production by Activating NF-κB and Ion Channel Signals.

Senecavirus A (SVA) infection induces inflammation in animals, such as fever, diarrhea, vesicles and erosions, and even death. The inflammatory cytokine interleukin-1ß (IL-1ß) plays a pivotal role in inflammatory responses to combat microbes. Although SVA infection can produce inflammatory clinical symptoms, the modulation of IL-1ß production by SVA infection remains unknown at present. Here, both in vitro and in vivo, SVA robustly induced IL-1ß production in macrophages and pigs. Infection performed in NOD-, LRR-, and pyrin domain-containing three (NLRP3) knockdown cells indicated that NLRP3 is essential for SVA-induced IL-1ß secretion. Importantly, we identified that the 1 to 154 amino acid (aa) portion of SVA 3D binds to the NLRP3 NACHT domain to activate NLRP3 inflammasome assembly and IL-1ß secretion. In addition, the SVA 3D protein interacts with IKKα and IKKß to induce NF-κB activation, which facilitates pro-IL-1ß transcription. Meanwhile, 3D induces p65 nucleus entry. Moreover, SVA 3D induces calcium influx and potassium efflux, which triggers IL-1ß secretion. Ion channels might be related to 3D binding with NLRP3, resulting in NLRP3-ASC complex assembly. We found that 3D protein expression induced tissue hemorrhage and swelling in the mice model. Consistently, expression of 3D in mice caused IL-1ß maturation and secretion. In the natural host of pigs, we confirmed that 3D also induced IL-1ß production. Our data reveal a novel mechanism underlying the activation of the NLRP3 inflammasome after SVA 3D expression, which provides clues for controlling pig's inflammation during the SVA infection. IMPORTANCE Inflammation refers to the response of the immune system to viral, bacterial, and fungal infections or other foreign particles in the body, which can involve the production of a wide array of soluble inflammatory mediators. The NLRP3 inflammasome is one of the best-characterized inflammasome leading to IL-1ß production and maturation. Senecavirus A (SVA) is an oncolytic virus that can cause fever, vesicles and erosions, severe fatal diarrhea, and even the sudden death of piglets. In this study, we demonstrated that 1 to 154 aa of SVA polymerase protein 3D interacts with the NACHT domain of NLRP3 to induce IL-1ß production via the NF-κB signaling pathway and ion channel signal. Our study unveils the mechanism underlying the regulation of inflammasome assembly and production of IL-1ß in response to SVA infection that will help better understand the modulation of host inflammation in pathogens invasion and development of the vaccine.

Structural aspects of ß-glucosidase of Myceliophthora thermophila (MtBgl3c) by homology modelling and molecular docking.

Cellulases are the enzymes with diverse range of industrial applications. Cellulases degrade cellulose into monomeric glucose units by hydrolysing ß-1,4-glycosidic bonds. There are three components of cellulases: a) endoglucanase, b) exoglucanase and c) ß-glucosidase which act synergistically in cellulose bioconversion. The cellulases are the third largest industrial enzymes with a great potential in bioethanol production. In this investigation, a ß-glucosidase of a thermophilic fungus Myceliophthora thermophila (MtBgl3c) was analysed for its structural characterization using in silico approaches. The protein structure of MtBgl3c is unknown, therefore an attempt has been made to model 3D structure using Modeller 9.23 software. The MtBgl3c protein model generated was validated from Verify 3D and ERRAT scores of 89.37% and 71.25%, respectively derived from SAVES. Using RAMPAGE the Ramachandran plot was generated, which predicted the accuracy of the 3D model with 91.5% amino acid residues in the favored region. The ion binding and N-glycosylation sites were also predicted. The generated model was docked with cellobiose to predict the most favorable binding sites of MtBgl3c. The key amino acid residues involved in cellobiose bonding are Val88, Asp106, Asp287, Tyr255, Arg170, Glu514. The catalytic conserved amino residues of MtBgl3c were identified. The dock score of cellobiose with MtBgl3c is much lower (-6.46 kcal/mol) than that of glucose (-5.61 kcal/mol), suggesting its high affinity for cellobiose. The docking data of MtBgl3c with glucose illustrate its tolerance to glucose. The present study provides insight into structural characteristics of the MtBgl3c which can be further validated by experimental data. Highlights3D structure of ß-glucosidase (MtBgl3c) of Myceliophthora thermophila is being proposed based on computational analysesThe amino acid residues Asp106, Asp287, Tyr255, Arg170 and Glu514 have been identified to play catalytically important role in substrate bindingDocking and interaction of MtBgl3c with cellobiose and glucose has been confirmedDocking analysis of MtBgl3c with glucose suggested its glucose toleranceThe data would be useful in engineering enzymes for attaining higher catalytic efficiencyCommunicated by Ramaswamy H. Sarma.

PDBsum extras: SARS-CoV-2 and AlphaFold models.

The PDBsum web server provides structural analyses of the entries in the Protein Data Bank (PDB). Two recent additions are described here. The first is the detailed analysis of the SARS-CoV-2 virus protein structures in the PDB. These include the variants of concern, which are shown both on the sequences and 3D structures of the proteins. The second addition is the inclusion of the available AlphaFold models for human proteins. The pages allow a search of the protein against existing structures in the PDB via the Sequence Annotated by Structure (SAS) server, so one can easily compare the predicted model against experimentally determined structures. The server is freely accessible to all at http://www.ebi.ac.uk/pdbsum.