Amino Acid Mutations Research Articles

Smad1 Promotes Tumorigenicity and Chemoresistance of Glioblastoma by Sequestering p300 From p53.

Acetylation is critically required for p53 activation, though it remains poorly understood how p53 acetylation is regulated in glioblastoma (GBM). This study reveals that p53 acetylation is a favorable prognostic marker for GBM, regardless of p53 status, and that Smad1, a key negative regulator of p53 acetylation, is involved in this process. Smad1 forms a complex with p53 and p300, inhibiting p300's interaction with p53 and leading to reduced p53 acetylation and increased Smad1 acetylation in GBM. This results in enhanced tumor growth and resistance to chemotherapy, particularly in tumors with missense mutant p53. Acetylation of K373 is found to be essential for Smad1's oncogenic function but does not confer chemoresistance in the absence of p53. Through molecular docking, it is discovered that Smad1 and p53 both interact with the acetyltransferase domain of p300, but at different amino acid sites. Disturbing the interface of Smad1 through amino acid mutations abolishes the Smad1-p300 complex and promotes p53 acetylation. Therefore, a small molecule is identified through virtual screening that specifically disrupts the Smad1-p300 interaction, offering a promising strategy for inhibiting GBM and increasing chemosensitivity by inhibiting Smad1 acetylation and restoring p53 acetylation.

Genomic characterization and tissue tropism variations of two porcine delta coronavirus strains isolated in China

The porcine delta coronavirus (PDCoV) is a member of the Delta coronavirus genus, which can lead to diarrhea, vomiting, and mortality in piglets. First detected in Hong Kong in 2012, PDCoV has since spread globally. In January 2024, two strains, CHN-ANHZ-2024 and CHN-JSSQ-2024, were isolated from diarrheal piglets in Anhui and Jiangsu provinces. Immunofluorescence assays, electron microscopy, and genome sequencing were performed. Genome analysis revealed that both PDCoV strains belonged to the Chinese lineage, exhibiting amino acid mutations in the S1 region compared to other strains within the lineage. Amino acid mutation at position 530L is uniquely associated with the Thai strain. Notably, CHN-JSSQ-2024 was identified as a recombinant strain of DH1 and CHN-AHHN-2024, with the recombination occurring in the S2 subunit. CHN-ANHZ-2024 caused severe diarrhea with an 80% mortality rate, whereas CHN-JSSQ-2024 resulted in mild diarrhea without mortality. Viral load analysis showed CHN-ANHZ-2024 primarily infecting the brain and kidneys, while CHN-JSSQ-2024 targeted the lungs, revealing notable differences in tissue tropism. We designed the RNA scope Probe-PDCoV-N to visualize viral RNA in the positively detected organs, viral RNA was detected in the brain, cerebellum, kidneys, and lungs of the infected piglets. This study highlights significant differences in the pathogenicity and organ tropism of two PDCoV strains. The CHN-ANHZ-2024 strain caused severe diarrhea and high mortality in piglets, while the CHN-JSSQ-2024 strain exhibited much milder symptoms. Additionally, the study elucidated notable differences in organ tropism between the strains, offering valuable insights into the epidemiological characteristics and pathogenic mechanisms of PDCoV. These findings provide a foundation for the development of targeted prevention and treatment strategies tailored to specific strains in the future.

Molecular characterization of circulating infectious bursal disease viruses in chickens from different Egyptian governorates during 2023

Infectious bursal disease virus (IBDV) induces severe immunosuppression in chickens, leading to significant economic losses in the global poultry industry. This study investigated 52 chicken flocks, including commercial broilers, layers, and baladi, from various Egyptian governorates in 2023. These flocks exhibited symptoms of depression, along with kidney and bursa lesions, indicative of IBDV infection. Pooled Bursal homogenates were tested using RT-PCR with VP2-specific primers, revealing that 20 flocks tested positive for IBDV. Six representative samples were selected from 20 positive flocks for isolation in embryonated chicken eggs. The embryonic lesions observed included haemorrhage, skull swelling, and liver necrosis with a pale-yellow appearance, in addition to congestion and thickening in the chorioallantoic membrane (CAM). Partial amplification of the VP2 gene from the harvested embryo suspensions of the six IBDV isolates was performed for sequencing. Phylogenetic analysis of the sequences revealed that five IBDV isolates (VV4, VV5, VV6, VV10, and VV16) belonged to the very virulent strain group A3 cluster, whereas one isolate (VV2) clustered with Chinese Variant strains in the A2d group. Sequence analysis of the hypervariable region (HVR) of VP2 compared to that of Egypt-USC-IBD-1-2019 and vvIBDV/Beh21/Egypt/18 highly virulent IBDV strains revealed several amino acid mutations. The VP2 HVR of all isolates maintained the serine-rich heptapeptide sequence SWSASGS, which is adjacent to the major hydrophilic peak B and serves as a virulence marker. Histopathological examination revealed that bursae from chickens infected with vvIBDV exhibited marked interlobular oedema and lymphoid depletion. In contrast, bursae from chickens infected with Variant IBDV showed massive lymphoid depletion, with hyperplasia of the bursal capsule. These findings highlight the circulation of both virulent and Variant IBDV strains in Egyptian chicken flocks, complicating disease control. Consequently, there is a need to update vaccination programs and vaccine strains for IBDV in Egypt.

Creation of catalytic activity-improved hyperthermophilic PQQ-dependent aldose sugar dehydrogenase and its efficient use for high performance electro-device

PQQ-dependent aldose sugar dehydrogenase (PQQ-ASD) from the hyperthermophilic archaeon Pyrobaculum aerophilum (PaeASD) has great potential as an element for durable bioelectrodevices owing to its exceptional stability against high temperatures and across a broad pH spectrum. However, its application is constrained by low electric current output of the enzyme-immobilized electrodes, which is attributable to its low catalytic activity. A directed evolutionary approach was performed on PaeASD to improve enzyme activity, resulting in the identification of a PaeASD s24 mutant containing six amino acid substitutions, which exhibited a 16-fold higher specific activity than that of wild type. Although each single amino acid mutant among these substitutions exhibited lower enzyme activity than PaeASD s24, the double mutant R64Q/D350N showed enzyme activity comparable to that of PaeASD s24. These amino acids located in the vicinity of coenzyme PQQ within the PaeASD molecule are also highly conserved with those of PQQ-ASDs reported to date. Thus, these amino acids play crucial roles in the catalytic activity of PQQ-ASD. Furthermore, the Km value for d-glucose of PaeASD s24-immobilized electrode decreased to approximately 1/3 that of the wild-type-immobilized electrode. These results indicate that the PaeASD s24 mutant is an excellent catalyst for potential bioelectrodevice applications.

Prevalence and genetic diversity of porcine epidemic diarrhea virus in Southwest China during 2020–2022

Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), has been frequently occurring in the southwestern region of China over the past few years, continuously affecting the development of the swine industry. However, the genetic diversity and prevalence of PEDV strains circulating in the swine population in southwestern China in recent years have not been well studied. To address this gap, a total of 478 clinical samples were collected from 125 pig farms experiencing piglet diarrhea in 18 cities in southwestern China. The detection results revealed that 227 out of 478 samples tested positive for PEDV nucleic acid, with a positivity rate of 47.49%. Complete S gene sequences of 28 PEDV strains were obtained and classified into four subgroups, G1-a subgroup (classical strain), G1-b subgroup (S-INDEL), and two G2 subgroups (G2-a and G2-b), accounting for 17.86% (5/28), 3.57% (1/28), 35.71% (10/28), 42.86% (12/28) of the total sequenced strains, respectively. The coexistence of multiple genotypes indicates the complex genetic background and prevalence of PEDV in southwest China. Amino acid comparisons of the S proteins showed that the 28 PEDV strains sequenced in the study showed different patterns of variation in the epitope domains compared to vaccine strains belonging to different genotypes and contained many unique amino acid mutations compared to the reference strains, which might lead to immune escape of PEDV. The complex epidemiology of PEDV with multiple subgroups co-circulating in Southwest China underscores the importance of selecting appropriate vaccine strains based on locally prevalent strains and the ongoing need for epidemiological surveillance of PEDV. The emergence of new variant strains also highlights the urgency of developing updated vaccines, and effective management practices remain crucial for controlling PED outbreaks in pig farms.

Chicken ANP32A-independent replication of highly pathogenic avian influenza viruses potentially leads to mammalian adaptation-related amino acid substitutions in viral PB2 and PA proteins.

Acidic nuclear phosphoprotein 32 family member A (ANP32A) is an important host factor that supports the efficient replication of avian influenza viruses (AIVs). To develop an antiviral strategy against Gs/Gd-lineage H5 highly pathogenic avian influenza (HPAI) viruses in chickens, we established chicken ANP32-knockout (chANP32A-KO) DF-1 cells and evaluated their antiviral efficacy through in vitro validation. The replication of all HPAI viruses tested in chANP32A-KO cells was significantly lower compared to that of wild-type DF-1 cells. However, when HPAI strains A/mountain hawk-eagle/Kumamoto/1/2007 (H5N1; MHE) and A/chicken/Aichi/2/2011 (H5N1; H5Aichi) were passed in chANP32A-KO cells, mutant viruses were generated, which exhibited comparable replication levels in both chANP32A-KO and wild-type DF-1 cells. Sequence analysis revealed that mammalian-adaptive amino acid mutations PB2_D256G and PA_T97I were present in the MHE mutant virus, and the PB2_E627K mutation was identified in the H5Aichi mutant virus. These mutations have also been reported to enhance the polymerase activity of AIVs in mammalian cells; however, the minigenome assay in the present study showed that the polymerase activity of mutant viruses in chANP32A-KO cells was not restored to levels comparable to those in wild-type DF-1 cells. These findings suggest that ANP32A-independent viral replication may induce amino acid substitutions associated with mammalian adaptation in AIVs. They also imply that the high efficiency of viral replication mediated by these amino acid mutations may not result from enhanced polymerase activity but rather involve other undefined mechanisms.IMPORTANCEDuring the host-switching of avian influenza viruses (AIVs) to mammalian hosts, introducing adaptive mutations into viral proteins is essential to ensure optimal functionality through virus-host protein interactions in mammalian cells. However, the mechanisms leading to adaptive mutations in viral proteins remain unclear. Among several host proteins that promote viral growth, acidic nuclear phosphoprotein 32 family member A (ANP32A) is known to be an important factor for efficient viral replication. Here, we generated mutant highly pathogenic avian influenza viruses capable of ANP32A-independent replication in a chicken-derived cell line. We demonstrated that several amino acid mutations found in the mutant viruses correspond to those associated with the mammalian adaptation of AIVs. These results suggest that ANP32A-independent viral replication is one of the mechanisms for introducing amino acid mutations that are reportedly involved in the mammalian adaptation of AIVs.

Optimized Second-Generation IL-4Rα Inhibition: Structural and Molecular Dynamics Properties of Rademikibart Fab-IL-4Rα Complex

Introduction: Global Burden of Disease studies showed atopic dermatitis (AD) and asthma are the top two immune-mediated inflammatory diseases at younger age [1]. Dupilumab was the initial first-generation interleukin-4 receptor alpha (IL-4Rα) inhibitor for treating both type I and type II IL-4Rα-dependent inflammatory disorders. Following recent clinical trials, rademikibart (previously, CBP-201) emerges as an optimized next-generation human monoclonal antibody with higher binding affinity to IL-4Rα compared to dupilumab [2]. It demonstrated better effect in inhibiting STAT6 intracellular signaling in vitro and provided similar potency inhibiting both IL-4 induced TARC release and IL-4 induced B cell activation [2]. Materials & Methods: X-ray crystallography was used to determine the atomic resolution 3D structure of rademikibart fragment antigen binding (Fab) bound to IL-4Rα. This structure was analyzed and compared computationally with the 2.82 Å resolution crystal structure of dupilumab Fab bound to IL-4Rα (Protein Data Bank Code 6WGL). Molecular dynamics studies on rademikibart and dupilumab bound to IL-4Rα examined the stability of the complexes and effects of amino acid mutations on complex formation. Results: The x-ray crystal structure of rademikibart Fab bound to IL-4Rα was determined at 2.71Å and compared to the complex of dupilumab Fab and IL-4Rα. The rotation angle between dupilumab and rademikibart bound to IL- 4Rα is 59.17°. This rotation enables the epitope of rademikibart, but not dupilumab, on IL-4Rα to overlap more closely with the conserved binding interface utilized by IL-4 and IL-13 cytokines. Molecular dynamics simulations of rademikibart Fab and dupilumab Fab complexed with IL-4Rα showed the third interface loop (residues 148 to 152 in domain 2) of IL-4Rα interacts directly with rademikibart, which is absent in dupilumab/IL- 4Rα complex. This finding is confirmed by analysis of the hydrogen bond interactions at the interface between the antibodies and IL-4Rα, demonstrating superior binding energy for rademikibart. Through single amino acid mutation analysis on rademikibart, we identified residue Y50 on rademikibart as the key residue interacting with IL- 4Rα’s third interface loop. Conclusion: Our data provide a molecular and structural rationale for the enhanced IL-4Rα inhibition by rademikibart over dupilumab, confirming rademikibart as an optimized second-generation IL-4Rα inhibitor.

Influenza A virus shedding and reinfection during the post-weaning period in swine: longitudinal study of two nurseries.

Influenza A virus in swine (IAV-S) is common in the United States commercial swine population and has the potential for zoonotic transmission. To elucidate influenza shedding the domestic pig population, we evaluated two commercial swine farms in Illinois, United States, for 7 weeks. Farm 1 had a recent IAV-S outbreak. Farm 2 has had IAV-S circulating for several years. Forty post-weaning pigs on Farm 1 and 51 pigs from Farm 2 were individually monitored and sampled by nasal swabs for 7 weeks. RT-PCR results over time showed most piglets shed in the first 2 weeks post weaning, with 91.2% shedding in week one, and 36.3% in week two. No difference in the number of pigs shedding was found between the two nurseries. Reinfection events did differ between the farms, with 30% of piglets on Farm 1 becoming reinfected, compared to 7.8% on Farm 2. In addition, whole genome sequencing of nasal swab samples from each farm showed identical viruses circulating between the initial infection and the reinfection periods. Sequencing also allowed for nucleic and amino acid mutation analysis in the circulating viruses, as well the identification of a potential reverse zoonosis event. We saw antigenic site mutations arising in some pigs and MxA resistance genes in almost all samples. This study provided information on IAV-S circulation in nurseries to aid producers and veterinarians to screen appropriately for IAV-S, determine the duration of IAV-S shedding, and predict the occurrence of reinfection in the nursery period.

A nosocomial outbreak of colistin and carbapenem-resistant hypervirulent Klebsiella pneumoniae in a large teaching hospital

Recently, colistin and carbapenem-resistant hypervirulent Klebsiella pneumoniae (CCR-hvKP) has been observed sporadically. The aim of this study was to report a nosocomial outbreak due to CCR-hvKP, so as to control the transmission of CCR-hvKP and prevent future outbreaks. The clinical characteristics of five involved cases were analyzed and infection prevention and control measures were documented. Five CCR-hvKP isolates were discovered from the five involved cases. Molecular features of the isolates including sequence type, capsule locus, antimicrobial resistance genes, virulence factors and phylogenetic relationship were analyzed by whole-genome sequencing. Validation of the role of the deleterious amino acid mutations to colistin resistance was examined by complementation assays. PCR was performed to identify insertion sequences within the mgrB gene. Mouse intraperitoneal infection models were used to assess virulence phenotype. Five cases infected with CCR-hvKP were identified with a high attributable mortality rate of 60% in the patients. The five outbreak isolates belonged to the high-risk ST11-KL64 clone and were closely clustered. They were highly resistant to commonly used antibiotics and showed hypervirulent in vivo. WGS revealed multiple antimicrobial resistance genes such as blaKPC-2 and blaCTX-M-65 and important virulence factors. Concerning colistin resistance, amino acid mutations G53S in pmrA gene, and T157P, T246A and R256G in pmrB gene were indentified. Among them, the deleterious mutation T157P in pmrB gene was validated to be responsible for the resistance phenotype of isolate KP01, KP03 and KP05. In addition, disruption of mgrB gene by insertion sequences of ISKpn26 and IS903B was indentified in isolate KP02 and KP04, respectively. This is the first report of an outbreak caused by CCR-hvKP. The study highlights infection prevention and control measures are key to successfully fight against CCR-hvKP dissemination and nosocomial infections. Continuous surveillance should be performed to limit the spread of these isolates.

An epidemiologic surveillance study based on wastewater and respiratory specimens reveals influenza a virus prevalence and mutations in Taiyuan, China during 2023–2024

BackgroundInfluenza is a major cause of morbidity and mortality. Influenza A virus (IAV) is one of the most important pathogens causing influenza and often causes global pandemics due to its tendency to mutate. We aim to use epidemiology based on wastewater and respiratory specimens to understand the occurrence of influenza A virus infections in Taiyuan City.MethodsA retrospective epidemiology surveillance was carried out at the First Hospital of Shanxi Medical University (FHSMU) and five wastewater treatment plants (WTPs) in Taiyuan city from 2023 to 2024. Reverse transcription real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect influenza A viruses in wastewater and respiratory specimens. High-throughput whole genome sequencing was performed on 17 strains obtained in this study, and subsequent analyses included characterization, phylogenetic construction, amino acid mutation analysis, and antigenic structural variability assessment.Results520 wastewater samples and 1,203 throat swab samples were collected. We detected RNA concentration from pH1N1 and H3N2 viruses in wastewater and got 17 genome sequences (5 of pH1N1 and 12 of H3N2) in respiratory specimens. Whole-genome sequencing showed co-prevalence of pH1N1 viruses in the branches of 6B.1 A.5a.2a.1 and H3N2 viruses in the branches of 3 C.2a1b.2a.2a.3a.a in Taiyuan from 2023 to 2024. Moreover, a HA mutation (N138D), predicted to be of high phenotypic consequence, was found in 8 Taiyuan H3N2 sequences.ConclusionThis study highlights the predominant presence of pH1N1 and H3N2 strains in Taiyuan. The analysis also identified amino acid site variations in the HA antigenic epitopes in H3N2 strains, which may contribute to immune escape.

Deep mutational scanning of H5 hemagglutinin to inform influenza virus surveillance.

H5 influenza is considered a potential pandemic threat. Recently, H5 viruses belonging to clade 2.3.4.4b have caused large outbreaks in avian and multiple nonhuman mammalian species. Previous studies have identified molecular phenotypes of the viral hemagglutinin (HA) protein that contribute to pandemic potential in humans, including cell entry, receptor preference, HA stability, and reduced neutralization by polyclonal sera. However, prior experimental work has only measured how these phenotypes are affected by a handful of the >10,000 different possible amino-acid mutations to HA. Here, we use pseudovirus deep mutational scanning to measure how all mutations to a 2.3.4.4b H5 HA affect each phenotype. We identify mutations that allow HA to better bind α2-6-linked sialic acids and show that some viruses already carry mutations that stabilize HA. We also measure how all HA mutations affect neutralization by sera from mice and ferrets vaccinated against or infected with 2.3.4.4b H5 viruses. These antigenic maps enable rapid assessment of when new viral strains have acquired mutations that may create mismatches with candidate vaccine virus, and we show that a mutation present in some recent H5 HAs causes a large antigenic change. Overall, the systematic nature of deep mutational scanning combined with the safety of pseudoviruses enables comprehensive measurements of the phenotypic effects of mutations that can inform real-time interpretation of viral variation observed during surveillance of H5 influenza.

Predicting binding events in very flexible, allosteric, multi-domain proteins.

Knowledge of the structures formed by proteins and small molecules is key to understand the molecular principles of chemotherapy and for designing new and more effective drugs. During the early stage of a drug discovery program, it is customary to predict ligand-protein complexes in silico , particularly when screening large compound databases. While virtual screening based on molecular docking is widely used for this purpose, it generally fails in mimicking binding events associated with large conformational changes in the protein, particularly when the latter involve multiple domains. In this work, we describe a new methodology to generate bound-like conformations of very flexible and allosteric proteins bearing multiple binding sites by exploiting only information on the unbound structure and the putative binding sites. The protocol is validated on the paradigm enzyme adenylate kinase, for which we generated a significant fraction of bound-like structures. A fraction of these conformations, employed in ensemble-docking calculations, allowed to find native-like poses of substrates and inhibitors (binding to the active form of the enzyme), as well as catalytically incompetent analogs (binding the inactive form). Our protocol provides a general framework for the generation of bound-like conformations of challenging drug targets that are suitable to host different ligands, demonstrating high sensitivity to the fine chemical details that regulate protein's activity. We foresee applications in virtual screening, in the prediction of the impact of amino acid mutations on structure and dynamics, and in protein engineering.

Phylotranscriptomics reveals the phylogeny of Asparagales and the evolution of allium flavor biosynthesis

Asparagales, the largest monocot order, is renowned for its ecological, economic, and medicinal significance. Here, we leverage transcriptome data from 455 Asparagales species to explore the phylogeny of Asparagales. Moreover, we investigate the evolutionary patterns of the genes involved in allium flavor formation. We not only establish a robust bifurcating phylogeny of Asparagales but also explore their reticulate relationships. Notably, we find that eight genes involved in the biosynthesis of allium flavor compounds underwent expansion in Allium species. Furthermore, we observe Allium-specific mutations in one amino acid within alliinase and three within lachrymatory factor synthase. Overall, our findings highlight the role of gene expansion, increased expression, and amino acid mutations in driving the evolution of Allium-specific compounds. These insights not only deepen our understanding of the phylogeny of Asparagales but also illuminate the genetic mechanisms underpinning specialized compounds.

Low nucleotide diversity of the Plasmodium falciparum AP2-EXP2 gene among clinical samples from Ghana.

PfAP2-EXP2 is located within chromosome 6 of Plasmodium falciparum recently identified to be undergoing an extensive selective sweep in West African isolates. The gene encoding this transcription factor, PfAP2-EXP2, is essential and thus likely subject to purifying selection that limits variants in the parasite population despite its genomic location. 72 Plasmodium falciparum field samples and 801 clinical sequences from the Pf6 MalariaGEN dataset of Ghanaian origin, were integrated and analysed. A total of 14 single nucleotide variants of which 5 were missense variants, were identified after quality checks and filtering. Except for one, all identified variants were rare among the clinical samples obtained in this study (Minor allelic frequency < 0.01). Further results revealed a considerably low dN/dS value (0.208) suggesting the presence of purifying selection. Further, all the mutant amino acids were wildtype residues in AP2-EXP2 orthologous proteins-tentatively suggesting a genus-level conservation of amino acid residues. Computational analysis and predictions corroborated these findings. Despite the recent extensive selective sweep within chromosome 6 of West African isolates, PfAP2-EXP2 of Ghanaian origin exhibits low nucleotide diversity and very low dN/dS consistent with purifying selection acting to maintain the function of an essential gene. The conservation of AP2-EXP2 is an important factor that makes it a potential drug target.

Molecular dynamics simulations provide insights into ULK-101 potency and selectivity toward autophagic kinases ULK1/2

Kinase domains are highly conserved within proteins in both sequence and structure. Many factors, including phosphorylation, amino acid substitutions or mutations, and small molecule inhibitor binding, influence conformations of the kinase domain and enzymatic activity. ULK1 and ULK2 are serine/threonine kinases that serve important roles in autophagy, an intracellular recycling process capable of degrading proteins and organelles via fusion with lysosomes. ULK1/2 are emerging as therapeutic targets in human cancer, particularly KRAS-driven malignancies. Here, we performed molecular dynamics (MD) simulations to hypothesize bound poses for the ULK1/2 small molecule inhibitor, ULK-101. We observed stable bound states for ULK-101 to the adenosine triphosphate (ATP)-binding site of ULK2, coordinated by hydrogen bonding with the hinge backbone and the catalytic lysine sidechain. Notably, ULK-101 occupies a hydrophobic pocket associated with the N-terminus of the αC-helix. Large movements in the phosphate-binding loop (P-loop) are also associated with ULK-101 inhibitor binding and exit from ULK2. Together, our data support a model to explain ULK-101 potency toward ULK1/2.

Rapid production of recombinant rotaviruses by overexpression of NSP2 and NSP5 genes with modified nucleotide sequences.

Reverse genetics systems for rotaviruses (RV) facilitate the generation of genetically engineered RVs by transfection of 11 plasmids encoding 11 genomic viral RNA segments. In addition to viral genome expression, overexpression of NSP2 and NSP5 has been used to increase the rescue efficiency of recombinant RVs. Here, we showed that the overexpression of nucleotide sequence-modified NSP2 and NSP5 enabled the rapid and efficient production of recombinant RVs. Using improved reverse genetics, we established a reverse genetics system for human and bovine RV clinical isolates, as well as laboratory strains of bovine RV (NCDV and UK) and porcine RV (Gottfried). In addition, we rescued low-replicating recombinant RVs carrying a mutant NSP4 lacking the double-layered particle-binding domain, which was deficient in the efficient production of mature virions. These advancements in reverse genetics enabled the generation of molecular clones of RV clinical isolates and recombinant RVs harboring critical amino acid mutations, offering a versatile platform for investigating RV biology and pathogenesis.IMPORTANCERecombinant rotavirus (RV) synthesis via reverse genetics relies on both the viral propagation capacity and the efficiency of the experimental system. Since the establishment of our reverse genetics system, several enhancements have been implemented to augment the rescue efficiency. Nevertheless, challenges persist in generating RV clinical strains and recombinant viruses with low replication capacities. Notably, this improved reverse genetics system successfully facilitated the establishment of molecular clones of human and bovine RV clinical isolates. Fecal samples from patients with RV typically harbor quasi-species or, occasionally, multiple genotypes of RV. In the present study, we performed the genetic sequencing of clinical viral strains during the early propagation stages in cultured cells. Subsequently, infectious viruses were synthesized, allowing the characterization of circulating viruses in nature. This approach provides valuable insights into the genetic diversity and dynamics of RV populations and contributes to a more comprehensive understanding of viral pathogenesis and evolution.

Novel Intertypic Recombinant Coxsackievirus A2 Containing Specific Amino Acid Mutations in the RNA-Dependent RNA Polymerase Potentially Associated With Its Emergence.

Coxsackievirus A2 (CVA2), a member of enterovirus A species (EV-A), is associated with diverse human diseases and occasionally causes acute gastroenteritis (AGE). In Thailand, CVA2 emerged as the predominant genotype in 2019. The increasing incidence of CVA2, coupled with the limited availability of full-length genomes, highlights the need for more complete genome sequence analysis to facilitate molecular epidemiology study. This study aimed to investigate the molecular epidemiology, evolutionary dynamics, and recombination characteristics of CVA2 associated with AGE in Thailand from 2013 to 2022. A total of 19 full-genome sequences of CVA2 isolated from stool samples of AGE patients in Thailand were characterized and analyzed together with the reference sequences available in the GenBank database. A novel lineage of CVA2 (subgenotype C5) was detected with the potential recombination with CVA10 within the P2 and P3 regions. Specific consensus amino acid mutations, A61S in the VP3 gene and R136K in the 3D (RdRp) gene, were identified in all CVA2 recombinant strains. Additionally, the S45G mutation in the RdRp gene was found to be potentially associated with the emergence of CVA2 infection in 2019. In conclusion, this study reveals potential intertypic recombinant events and specific mutations in CVA2 strains isolated from AGE patients and provides a broader understanding of its evolutionary epidemiology.

Divergent modulation of activated protein C pleiotropic functions by antibodies that differ by a single amino acid

AbstractActivated protein C (APC) is a pleiotropic plasma protease with diverse functions derived from its anticoagulant, anti-inflammatory, and cytoprotective activities. The selective uncoupling and/or modulation of these APC activities by antibodies may have therapeutic benefit in diseases such as traumatic bleeding, hemophilia, sepsis, and ischemia. TPP-26870 is an antibody that targets a nonactive site of APC for the selective modulation of APC activities. To optimize the potency of TPP-26870, variants with single amino acid mutation in the complementarity-determining regions (CDRs) were screened, and 21 variants with improved KD were identified. Interestingly, the affinity maturation of TPP-26870 did not merely generate a panel of variants with higher potency in functional assays. Functional data demonstrated that the pleiotropic functions of APC were very sensitive to epitope-CDR interactions. Single amino acid mutations within the CDRs of TPP-26870 were sufficient to elicit divergent antagonistic and agonistic effects on the various APC functional activities. These include prolonged in vitro APC plasma half-life, increased inhibition of anticoagulant activity, and agonistic enhancement of histone H3 cleavage, while having less impact on protease-activated receptor 1 cleavage, compared with TPP-26870. This study illustrates that APC is highly sensitive to non–active site targeting that can lead to unpredictable changes in its activity profile of this pleiotropic enzyme. Furthermore, this study demonstrates the ability to modify APC functions to advance the potential development of APC-targeted antibodies as therapeutics for the treatment of diseases including trauma bleeding, hemophilia, ischemia, and sepsis.

Epidemiological and Genetic Insights of the Circulating Foot-and-Mouth Disease Virus Serotypes in Egypt

Foot-and-mouth disease virus (FMDV) remains a major threat to livestock in Egypt, with ongoing outbreaks involving serotypes A, O, and SAT2. This study aimed to improve the understanding of these circulating FMDV strains to improve control measures. Between 2022 and 2023, 134 cattle samples from across Egypt were analyzed, revealing a 67.9% positivity rate for Pan FMDV. Of these positive samples, 64 were identified as serotype A and 27 as serotype O. Genetic analysis indicated that serotype O strains clustered within the EA-3 topotype, suggesting endemic persistence and potential vaccine evasion, while serotype A strains were associated with the African topotype and linked to regions such as Ethiopia, Kenya, and Sudan. Notable amino acid mutations in the VP1 protein of both serotypes highlighted potential challenges to vaccine effectiveness. These findings underscore the need for enhanced surveillance, timely vaccine updates, and regional cooperation to effectively manage FMD outbreaks in Egypt and neighboring countries.

A Trp-574-Leu mutation in acetolactate synthase confers imazamox resistance in barnyardgrass (Echinochloa crus-galli) from China

Abstract Barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] is increasingly infesting imidazolinone-tolerant (IMI-T) rice fields in China, imazamox resistance of E. crus-galli has become the major concern for weed management in IMI-T rice fields. In this study, the susceptible population JLGY-3 (S) and the suspected resistant population JHXY-2 (R) collected from IMI-T rice fields were used as research subjects. When treated with imazamox, JHXY-2 (R) population showed a high level of herbicide resistance with a resistance index (RI) of 31.2. JHXY-2 (R) was cross-resistant to all five acetolactate synthase (ALS) inhibitors from different chemical families, but sensitive to herbicides inhibiting acetyl-CoA carboxylase (ACCase). In order to understand the reason why JHXY-2 (R) was resistant to imazamox, we performed experiments to characterize potential TSR and NTSR mechanisms. A trp-574-leu amino acid mutation in ALS and low imazamox ALS sensitivity were the main mechanism underlying imazamox resistance in this JHXY-2 (R) population. There was no significant difference in ALS gene expression and ALS protein abundance between R and S populations. High-performance liquid chromatography-tandem mass spectrometry analysis showed enhanced metabolism of imazamox in JHXY-2 (R), which was in contrast to the results of pretreatment with a metabolic enzyme inhibitor. Treatments with the P450/GST inhibitors did not alter the resistance level of JHXY-2 (R) against imazamox. To further clarify the NTSR mechanism of JHXY-2 (R), transcriptome sequencing showed that there was almost no significant difference in the expression of P450 and GST metabolic enzyme genes between R and S populations, and only GST-U1 showed a significant induction in R population. In conclusion, amino acid mutation and higher enzyme activity of ALS are the main causes of imazamox resistance in JHXY-2 (R). However, given the differences in imazamox residues in the leaves of the E. crus-galli, there may still be undetectable NTSR that are causing imazamox resistance in the R population.