Enhancers of the PAIR4 regulatory module promote distal VH gene recombination at the Igh locus.

While extended loop extrusion across the entire Igh locus controls VH -DJH recombination, local regulatory sequences, such as the PAIR elements, may also activate VH gene recombination in pro-B-cells. Here, we show that PAIR-associated VH 8 genes contain a conserved putative regulatory element (V8E) in their downstream sequences. To investigate the function of PAIR4 and its V8.7E, we deleted 890 kb containing all 14 PAIRs in the Igh 5' region, which reduced distal VH gene recombination over a 100-kb distance on either side of the deletion. Reconstitution by insertion of PAIR4-V8.7E strongly activated distal VH gene recombination. PAIR4 alone resulted in lower induction of recombination, indicating that PAIR4 and V8.7E function as one regulatory unit. The pro-B-cell-specific activity of PAIR4 depends on CTCF, as mutation of its CTCF-binding site led to sustained PAIR4 activity in pre-B and immature B-cells and to PAIR4 activation in T-cells. Notably, insertion of V8.8E was sufficient to activate VH gene recombination. Hence, enhancers of the PAIR4-V8.7E module and V8.8E element activate distal VH gene recombination and thus contribute to the diversification of the BCR repertoire in the context of loop extrusion.

Spliceosome component PHD finger 5A is essential for early B lymphopoiesis.

The spliceosome, a multi-megadalton ribonucleoprotein complex, is essential for pre-mRNA splicing in the nucleus and ensuring genomic stability. Its precise and dynamic assembly is pivotal for its function. Spliceosome malfunctions can lead to developmental abnormalities and potentially contribute to tumorigenesis. The specific role of the spliceosome in B cell development is poorly understood. Here, we reveal that the spliceosomal U2 snRNP component PHD finger protein 5A (Phf5a) is vital for early B cell development. Loss of Phf5a results in pronounced defects in B cell development, causing an arrest at the transition from pre-pro-B to early pro-B cell stage in the bone marrow of mutant mice. Phf5a-deficient B cells exhibit impaired immunoglobulin heavy (IgH) chain expression due to defective V-to-DJ gene rearrangement. Mechanistically, our findings suggest that Phf5a facilitates IgH gene rearrangement by regulating the activity of recombination-activating gene endonuclease and influencing chromatin interactions at the Igh locus.

Microbial diversity and ecological complexity emerging from environmental variation and horizontal gene transfer in a simple mathematical model.

BACKGROUND: Microbiomes are generally characterized by high diversity of coexisting microbial species and strains, and microbiome composition typically remains stable across a broad range of conditions. However, under fixed conditions, microbial ecology conforms with the exclusion principle under which two populations competing for the same resource within the same niche cannot coexist because the less fit population inevitably goes extinct. Therefore, the long-term persistence of microbiome diversity calls for an explanation. RESULTS: To explore the conditions for stabilization of microbial diversity, we developed a simple mathematical model consisting of two competing populations that could exchange a single gene allele via horizontal gene transfer (HGT). We found that, although in a fixed environment, with unbiased HGT, the system obeyed the exclusion principle, in an oscillating environment, within large regions of the phase space bounded by the rates of reproduction and HGT, the two populations coexist. Moreover, depending on the parameter combination, all three major types of symbiosis were obtained, namely, pure competition, host-parasite relationship, and mutualism. In each of these regimes, certain parameter combinations provided for synergy, that is, a greater total abundance of both populations compared to the abundance of the winning population in the fixed environment. CONCLUSIONS: The results of this modeling study show that basic phenomena that are universal in microbial communities, namely, environmental variation and HGT, provide for stabilization and persistence of microbial diversity, and emergence of ecological complexity.

Construction of knockout mutants in Mycobacterium intracellulare ATCC13950 strain using a thermosensitive plasmid containing negative selection marker rpsL.

BACKGROUND: Nontuberculous mycobacterial disease has emerged worldwide over the past 20 years. However, there are currently few reports on the established technique for constructing knockout mutants of nontuberculous mycobacteria. Therefore, gene recombination techniques for nontuberculous mycobacteria require further research. RESULTS: We constructed vector pPR23LHR that harbors the ribosomal protein S12 gene (rpsL+) as a dominant negative selection marker and the hygromycin (Hyg) and lacZ cassettes as positive selection markers. We constructed knockout mutants of proteasomal genes, which we found to be required for hypoxic pellicle formation in Mycobacterium intracellulare by functional genomic analysis. The knockout mutants showed impaired hypoxic pellicle formation, consistent with previous data using epoxomicin, a proteasomal inhibitor. CONCLUSIONS: Our findings demonstrate that rpsL+ is an efficient dominant negative selection marker for gene recombination in nontuberculous mycobacteria. Our temperature-sensitive rpsL+ method for the construction of knockout mutants will facilitate functional assays to validate the virulence factors of nontuberculous mycobacteria and the pathogenesis of nontuberculous mycobacterial disease.

Altered cell and RNA isoform diversity in aging Down syndrome brains.

Down syndrome (DS), trisomy of human chromosome 21 (HSA21), is characterized by lifelong cognitive impairments and the development of the neuropathological hallmarks of Alzheimer's disease (AD). The cellular and molecular modifications responsible for these effects are not understood. Here we performed single-nucleus RNA sequencing (snRNA-seq) employing both short- (Illumina) and long-read (Pacific Biosciences) sequencing technologies on a total of 29 DS and non-DS control prefrontal cortex samples. In DS, the ratio of inhibitory-to-excitatory neurons was significantly increased, which was not observed in previous reports examining sporadic AD. DS microglial transcriptomes displayed AD-related aging and activation signatures in advance of AD neuropathology, with increased microglial expression of C1q complement genes (associated with dendritic pruning) and the HSA21 transcription factor gene RUNX1 Long-read sequencing detected vast RNA isoform diversity within and among specific cell types, including numerous sequences that differed between DS and control brains. Notably, over 8,000 genes produced RNAs containing intra-exonic junctions, including amyloid precursor protein (APP) that had previously been associated with somatic gene recombination. These and related results illuminate large-scale cellular and transcriptomic alterations as features of the aging DS brain.

Characterization of a novel papillomavirus identified from a whale (Delphinapterus leucas) pharyngeal metagenomic library.

Here, using viral metagenomic method, a novel whale papillomavirus (temporarily named wPV, GenBank accession number OP856597) was discovered in a whale (Delphinapterus leucas) pharyngeal metagenomic library. The complete genome size of wPV is 7179 bp, with GC content of 54.4% and a nucleotide composition of 23.4% A, 22.3% T, 28.4% G, and 25.9% C. The viral genome has a typical papillomavirus organization pattern, and five ORFs were predicted, including two late genes encoding L1 and L2, and three early genes encoding E1, E2, and E6. Pairwise sequence comparison and phylogenetic analysis based on the L1 gene sequence indicated that wPV may be a novel species within genus Dyodeltapapillomavirus. In addition, the E2 region of wPV was predicted to have a potential recombination event. The discovery of this novel papillomavirus increases our understanding of the viral ecology of marine mammals, providing insights into possible future infectious diseases.

Establishment of a gene recombination method for a major human skin commensal fungus, Malassezia restricta, using Agrobacterium tumefaciens-mediated gene transfer system.

Malassezia restricta is the most predominant fungus in the microbiome of human skin. This microorganism can cause or exacerbate Malassezia-associated skin dermatitis, seborrheic dermatitis, atopic dermatitis, and pityriasis versicolor. The virulence factors of M. restricta have not been analyzed because a gene recombination system has not been developed. In this study, we established an Agrobacterium tumefaciens-mediated gene transfer (ATMT) system, optimized for generating gene-deficient mutants of M. restricta. A mutant of FKB1 gene, which encodes the FKBP12 protein that binds to the calcineurin inhibitor tacrolimus, was generated using the ATMT system. Subsequently, the FKB1 gene was reintroduced into the FKB1 gene-deficient mutant for obtaining a gene-complemented strain. The wild-type strain of M. restricta was sensitive to tacrolimus, whereas the FKB1 gene-deficient mutant was resistant to tacrolimus; the phenotypic drug susceptibility in the mutant was restored by reintroducing the FKB1 gene. Contrastingly, the FKB1 gene-deficient mutant was not resistant to cyclosporine A, which also inhibits calcineurin by binding to cyclophilin A. The gene recombination system for M. restricta will facilitate in elucidating the molecular mechanisms causing Malassezia-associated dermatitis.

Brain cell somatic gene recombination and its phylogenetic foundations.

A new form of somatic gene recombination (SGR) has been identified in the human brain that affects the Alzheimer's disease gene, amyloid precursor protein (APP). SGR occurs when a gene sequence is cut and recombined within a single cell's genomic DNA, generally independent of DNA replication and the cell cycle. The newly identified brain SGR produces genomic complementary DNAs (gencDNAs) lacking introns, which integrate into locations distinct from germline loci. This brief review will present an overview of likely related recombination mechanisms and genomic cDNA-like sequences that implicate evolutionary origins for brain SGR. Similarities and differences exist between brain SGR and VDJ recombination in the immune system, the first identified SGR form that now has a well-defined enzymatic machinery. Both require gene transcription, but brain SGR uses an RNA intermediate and reverse transcriptase (RT) activity, which are characteristics shared with endogenous retrotransposons. The identified gencDNAs have similarities to other cDNA-like sequences existing throughout phylogeny, including intron-less genes and inactive germline processed pseudogenes, with likely overlapping biosynthetic processes. gencDNAs arise somatically in an individual to produce multiple copies; can be functional; appear most frequently within postmitotic cells; have diverse sequences; change with age; and can change with disease state. Normally occurring brain SGR may represent a mechanism for gene optimization and long-term cellular memory, whereas its dysregulation could underlie multiple brain disorders and, potentially, other diseases like cancer. The involvement of RT activity implicates already Food and Drug Administration-approved RT inhibitors as possible near-term interventions for managing SGR-associated diseases and suggest next-generation therapeutics targeting SGR elements.

Integration of large heterologous DNA fragments into the genome of Thermococcus kodakarensis.

In this study, a transformation system enabling large-scale gene recombination was developed for the hyperthermophilic archaeon Thermococcus kodakarensis. Using the uracil auxotroph T. kodakarensis KU216 (∆pyrF) as a parent strain, we constructed multiple host strains harboring two 1-kbp DNA regions from the genomes of either the hyperthermophilic archaeon Pyrococcus furiosus or Methanocaldococcus jannaschii. The two regions were selected so that the regions between them on the respective genomes would include pyrF genes, which can potentially be used for selection. Transformation using these host strains and genomic DNA from P. furiosus or M. jannaschii were carried out. Transformants with exogenous pyrF were obtained only using host strains with regions from P. furiosus, and only when the distances between the two regions were relatively short (2-5 kbp) on the P. furiosus genome. To insert longer DNA fragments, we examined the possibilities of using P. furiosus cells to provide intact genomic DNA. A cell pellet of P. furiosus was overlaid with that of T. kodakarensis so that cells were in direct contact. As a result, we were able to isolate T. kodakarensis strains harboring DNA fragments from P. furiosus with lengths of up to 75 kbp in a single transformation step.

[Genomic recombination of the vibrio cholerae serogroup O1 El Tor pandemic strains].

Objective: To analyze the genomic recombination of the vibrio cholerae serogroup O1 El Tor pandemic strains. Methods: A total of 292 complete or draft genome sequences of Vibrio cholerae O1 serogroup El Tor strains isolated from 1937 to 2015 were selected from National Biotechnology Information Center database. The genome alignment of strains was computed by snippy software by using N16961 as reference sequence. Then ClonalFrameML software was used to do the recombinant analysis. The wilcox.test function in agricolae package was used to compare the number recombinant segments and the total length of recombinant regions between small and large chromosomes. The kruskal function was used to compare the number recombinant segments and the total length of recombinant regions among different isolation continents. The KOBAS tool was used to do the gene ontology enrichment analysis of recombinant hotspot genes. Results: Of all 292 strains of Vibrio cholerae, 163 strains (55.8%) were recombined. The median of normalized recombinant segment number of small chromosome was 4.7×10(-6) (9.3×10(-7), 2.0×10(-5)), which was significantly larger than that of large chromosome [2.4×10(-6) (3.4×10(-7), 5.7×10(-6))] (P<0.001). The median (P(25),P(75)) of recombinant segment number of strains isolated from Africa, Asia, Europe, North America and South America were 23(1.0,33.0), 1.0(0.0,34.0), 6.0(2.0,13.0), 0.0(0.0,1.0) and 29.5(6.8,56.8), respectively, and the difference was statistically significant (P<0.001). The median (P(25),P(75)) of total length of recombinant regions of strains isolated from Africa, Asia, Europe, North America and South America were 233.0(4.0, 461.0), 11.0(0.0, 695.5), 56.0(4.0,111.0), 0.0(0.0,9.0) and 347.5(132.8,1 323.5) bp, respectively, and the difference was statistically significant (P<0.001). Gene ontology Enrichment analysis showed that the functions of 62 recombinant hotspot genes were mainly enrichment in chemotaxis, taxis, response to external stimulus, receptor activity and molecular transducer activity. Conclustion: In this study, we found that there were significant differences in the number of recombinant fragments and the length of recombinant regions between large and small chromosomes of Vibrio cholerae El Tor. We also found significant differences in the number of recombinant fragments and the total length of recombinant regions among different continents.

First detection of bovine coronavirus in Yak (Bos grunniens) and a bovine coronavirus genome with a recombinant HE gene.

The yak (Bosgrunniens) is a unique domestic bovine species that plays an indispensable role for herdsmen in the Qinghai-Tibet Plateau. Here, 336 diarrhoeic samples were collected from yaks on 29 farms in the Qinghai-Tibet Plateau from 2015 to 2017. Approximately 69.05 % (232/336) of the diarrhoeic samples were assessed as bovine coronavirus (BCoV)-positive by RT-PCR assay, and most of the detected strains showed a unique evolution based on 40 spike (S), nucleocapsid (N) and haemagglutinin-esterase (HE) gene fragments. Notably, the 12 complete S genes detected shared 1 identical amino acid mutation (E121V) in the S1 subunit compared with the other 150 complete S genes in the GenBank database. Furthermore, a BCoV strain (designated YAK/HY24/CH/2017) was isolated from one diarrhoeic sample (virus titre : 108.17TCID50 ml-1), and a phylogenetic analysis based on complete genome sequences revealed that strain YAK/HY24/CH/2017 has the closest genetic relationship with the BCoV prototype strain Mebus. Interestingly, 2 significant characteristics were observed in the genome of strain YAK/HY24/CH/2017 :  (1) the strain had 26 unique amino acid variations in the S gene compared with the other 150 BCoV S genes in the GenBank database and (2) a recombination event was identified between the esterase and lectin domains of the HE gene. In conclusion, this study revealed the high prevalence of BCoV in yaks in the Qinghai-Tibet Plateau. To the best of our knowledge, this is the first description of the molecular prevalence of BCoV in yaks and of a BCoV genome with an HE gene recombination.

Identification of Rice Large Grain Gene GW2 by Whole-Genome Sequencing of a Large Grain-Isogenic Line Integrated with Japonica Native Gene and Its Linkage Relationship with the Co-integrated Semidwarf Gene d60 on Chromosome 2.

Genetic analysis of "InochinoIchi," an exceptionally large grain rice variety, was conducted through five continuous backcrosses with Koshihikari as a recurrent parent using the large grain F3 plant in Koshihikari × Inochinoichi as a nonrecurrent parent. Thorough the F2 and all BCnF2 generations, large, medium, and small grain segregated in a 1:2:1 ratio, indicating that the large grain is controlled by a single allele. Mapping by using simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers with small grain homozygous segregants in the F2 of Nipponbare × Inochinoichi, revealed linkage with around 7.7 Mb markers from the distal end of the short arm of chromosome 2. Whole-genome sequencing on a large grain isogenic Koshihikari (BC4F2) using next-generation sequencing (NGS) identified a single nucleotide deletion in GW2 gene, which is located 8.1 Mb from the end of chromosome 2, encoding a RING protein with E3 ubiquitin ligase activity. The GW2-integrated isogenic Koshihikari showed a 34% increase in thousand kernel weight compared to Koshihikari, while retaining a taste score of 80. We further developed a large grain/semi-dwarf isogenic Koshihikari integrated with GW2 and the semidwarfing gene d60, which was found to be localized on chromosome 2. The combined genotype secured high yielding while providing robustness to withstand climate change, which can contribute to the New Green Revolution.

Full genome-based genotyping system for rotavirus H and detection of potential gene recombination in nonstructural protein 3 between porcine rotavirus H and rotavirus C.

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

Modified neonicotinoid insecticide with bi-directional selective toxicity and drug resistance.

A three-dimensional quantitative structure-activity relationship (3D-QSAR) model was established based on the molecular structures and the negative logarithm of experimental lethal concentration 50 values (pLC50) of neonicotinoid insecticides. Then, the mechanisms of bi-directional selective toxic effects and drug resistance were determined using homology modeling and molecular docking analyses. The results of the model showed that the 1-, 2-, 4-, and 12- positions of neonicotinoid insecticides strongly affected their toxicity, and that the introduction of bulky or electropositive groups at these positions could increase the pLC50 values. Using Compound 19 as a template, we designed 37 derivatives with greater toxicity (increased by 0.04-11.45%). Among them, 20 derivatives had bioconcentrations lower than that of Compound 19 (reduced by 0.38-147.88%). Further screening of Compound 19 and the 20 derivatives mentioned above by homology modeling and acetylcholine receptors (AChRs) molecular docking analyses showed that 10 derivatives had bi-directional selective toxic effects against pests and bees. Further docking analyses of Compound 19 and these 10 derivatives identified that Derivative-33 showed decreased docking with superoxide dismutase (SOD) and glutathione S transferase (GST) in pests and enhanced docking with these enzymes in bees, indicating bi-directional selective resistance for pests and bees. Accordingly, Derivative-33 was selected as a new insecticide with high toxicity to pests and low toxicity to bees (bi-directional selective toxicity), low resistance in pest populations, and high resistance in bee populations. This study provides valuable reference data and will be useful for the development of strategies to produce new environmentally friendly pesticides.

Enhanced production of tanshinone IIA in endophytic fungi Emericella foeniculicola by genome shuffling.

CONTEXT: Tanshinone IIA, commercially produced from Salvia miltiorrhiza Bunge (C.Y.Wu) (Labiatae), has various biological benefits. Currently, this compound is mainly extracted from plants. However, because of the long growth cycle and the unstable quality of plants, the market demands can barely be satisfied. OBJECTIVE: The genomic shuffling technology is applied to screen the high-yield tanshinone IIA strain, which could be used to replace the plant S. miltiorrhiza for the production of tanshinone IIA. The change in the production of tanshinone IIA is clarified by comparing it with the original strain. MATERIALS AND METHODS: Tanshinone IIA was extracted from Strains cells, which was prepared through 0.5 mL protoplast samples by using hypertonic solution I from two different strains. Then, it was analyzed by high-performance liquid chromatography at 30 °C and UV 270 nm. Total DNA from the strains was extracted for RAPD amplification and electrophoresis to isolate the product. RESULTS: In this study, a high-yield tanshinone IIA strain F-3.4 was screened and the yield of tanshinone IIA was increased by 387.56 ± 0.02 mg/g, 11.07 times higher than that of the original strain TR21. DISCUSSION: This study shows that the genetic basis of high-yield strains is achieved through genome shuffling, which proves that genome shuffling can shorten the breeding cycle and improve the mutagenesis efficiency in obtaining the strains with good traits and it is a useful method for the molecular breeding of industrial strains.

Diversification of defensins and NLRs in Arabidopsis species by different evolutionary mechanisms.

BACKGROUND: Genes encoding proteins underlying host-pathogen co-evolution and which are selected for new resistance specificities frequently are under positive selection, a process that maintains diversity. Here, we tested the contribution of natural selection, recombination and transcriptional divergence to the evolutionary diversification of the plant defensins superfamily in three Arabidopsis species. The intracellular NOD-like receptor (NLR) family was used for comparison because positive selection has been well documented in its members. Similar to defensins, NLRs are encoded by a large and polymorphic gene family and many of their members are involved in the immune response. RESULTS: Gene trees of Arabidopsis defensins (DEFLs) show a high prevalence of clades containing orthologs. This indicates that their diversity dates back to a common ancestor and species-specific duplications did not significantly contribute to gene family expansion. DEFLs are characterized by a pervasive pattern of neutral evolution with infrequent positive and negative selection as well as recombination. In comparison, most NLR alignment groups are characterized by frequent occurrence of positive selection and recombination in their leucine-rich repeat (LRR) domain as well negative selection in their nucleotide-binding (NB-ARC) domain. While major NLR subgroups are expressed in pistils and leaves both in presence or absence of pathogen infection, the members of DEFL alignment groups are predominantly transcribed in pistils. Furthermore, conserved groups of NLRs and DEFLs are differentially expressed in response to Fusarium graminearum regardless of whether these genes are under positive selection or not. CONCLUSIONS: The present analyses of NLRs expands previous studies in Arabidopsis thaliana and highlights contrasting patterns of purifying and diversifying selection affecting different gene regions. DEFL genes show a different evolutionary trend, with fewer recombination events and significantly fewer instances of natural selection. Their heterogeneous expression pattern suggests that transcriptional divergence probably made the major contribution to functional diversification. In comparison to smaller families encoding pathogenesis-related (PR) proteins under positive selection, DEFLs are involved in a wide variety of processes that altogether might pose structural and functional trade-offs to their family-wide pattern of evolution.

Characterization of a functional recombinant human creatine kinase-MB isoenzyme prepared by tandem affinity purification from Escherichia coli.

Creatine kinase isoform CK-MB has been widely applied as a biomarker of myocardial injury. While a variety of methods have been used to measure CK-MB activity or mass in clinical laboratories, a CK-MB standard is needed to eliminate between-method bias. Because the in vitro expression of human creatine kinase generates three isoenzymes, CK-MM, CK-MB, and CK-BB, it is important to establish an effective method to purify the isoform CK-MB from the mixture. In this study, we aimed at using tandem affinity purification (TAP) to purify recombinant CK-MB protein and evaluate its value in clinical laboratories. After the optimized sequence coding CK-M and CK-B were synthesized, they were combined with TAP tags (6His and SBP) and inserted into a pRSFDuet vector; then, the constructed 6His-CK-M-SBP-CK-B-pRSF plasmid was transformed into Escherichia coli BL21 (DE3) for expression. After TAP, we obtained purified CK-MB protein. We also did recovery testing using the engineered CK-MB and standard CK-MB (Randox) at different concentrations, and the results suggested that the engineered CK-MB could be used as the reference material. Moreover, the stability study of recombinant CK-MB showed high stability during long-term storage at -80 °C. In conclusion, the TAP-purified recombinant CK-MB protein may be a much better and cheaper standard or reference material for clinical laboratories.

Genetic variation and evolutionary characteristics of Echovirus 11: new variant within genotype D5 associated with neonatal death found in China.

Echovirus 11 (E11) has gained attention owing to its association with severe neonatal infections. From 2018 to 2023, a surge in severe neonatal cases and fatalities linked to a novel variant of genotype D5 was documented in China, France, and Italy. However, the prevention and control of E11 variants have been hampered by limited background data on the virus circulation and genetic variance. Therefore, the present study investigated the circulating dynamics of E11 and the genetic variation and molecular evolution of genotype D5 through the collection of strains from the national acute flaccid paralysis (AFP) and hand, foot, and mouth disease (HFMD) surveillance system in China during 2000-2022 and genetic sequences published in the GenBank database. The results of this study revealed a prevalent dynamic of E11 circulation, with D5 being the predominant genotype worldwide. Further phylogenetic analysis of genotype D5 indicated that it could be subdivided into three important geographic clusters (D5-CHN1: 2014-2019, D5-CHN2: 2016-2022, and D5-EUR: 2022-2023). Additionally, variant-specific (144) amino acid mutation sites and positive-selection pressure sites (132, 262) were identified in the VP1 region. Cluster-specific recombination patterns were also identified, with CVB5, E6, and CVB4 as the major recombinant viruses. These findings provide a preliminary landscape of E11 circulation worldwide and basic scientific data for further study of the pathogenicity of E11 variants.

[Preparation of recombinant mussel mucin Mfp-3P and its promotion of wound healing].

To investigate the role of recombinant mussel mucin in wound healing, we aimed to prepare this mucin using Pichia pastoris as the host microbe. Our method involved constructing a genetically engineered strain of P. pastoris that expressed a fusion protein consisting of Mfp-3 and preCol-P peptide segments of mussel. After fermentation and purification, we obtained a pure recombinant mussel mucin product. We then conducted experiments to evaluate its effect at both the cellular and animal levels. At the cellular level, we examined its impact on the proliferation and migration of mouse fibroblast L929. At the animal level, we assessed its ability to promote wound healing after full-layer skin resection in rats. Our results showed that the recombinant mussel mucin protein has a content of 90.28% and a purity of 96.49%. The content of 3,4-dihydroxyphenylalanine (DOPA) was 0.73 wt%, and the endotoxin content was less than 0.5 EU/mg. Importantly, the recombinant mussel mucin protein significantly promoted both the migration and proliferation of mouse fibroblast, as well as the wound healing in rat skin. In conclusion, our findings demonstrate that recombinant mussel mucin has the potential to promote wound healing and can be considered a promising medical biomaterial.

Nucleoside Reverse Transcriptase Inhibitor Exposure Is Associated with Lower Alzheimer's Disease Risk: A Retrospective Cohort Proof-of-Concept Study.

Brain somatic gene recombination (SGR) and the endogenous reverse transcriptases (RTs) that produce it have been implicated in the etiology of Alzheimer's disease (AD), suggesting RT inhibitors as novel prophylactics or therapeutics. This retrospective, proof-of-concept study evaluated the incidence of AD in people with human immunodeficiency virus (HIV) with or without exposure to nucleoside RT inhibitors (NRTIs) using de-identified medical claims data. Eligible participants were aged ≥60 years, without pre-existing AD diagnoses, and pursued medical services in the United States from October 2015 to September 2016. Cohorts 1 (N = 46,218) and 2 (N = 32,923) had HIV. Cohort 1 had prescription claims for at least one NRTI within the exposure period; Cohort 2 did not. Cohort 3 (N = 150,819) had medical claims for the common cold without evidence of HIV or antiretroviral therapy. The cumulative incidence of new AD cases over the ensuing 2.75-year observation period was lowest in patients with NRTI exposure and highest in controls. Age- and sex-adjusted hazard ratios showed a significantly decreased risk for AD in Cohort 1 compared with Cohorts 2 (HR 0.88, p < 0.05) and 3 (HR 0.84, p < 0.05). Sub-grouping identified a decreased AD risk in patients with NRTI exposure but without protease inhibitor (PI) exposure. Prospective clinical trials and the development of next-generation agents targeting brain RTs are warranted.