Region Of Gene Research Articles

Presence of the Anopheles culicifacies complex species A in southeast Iran

BackgroundThe Anopheles culicifacies complex is one of the most important malaria vectors in Southeast Asia and Southeastern Iran. Although the sibling species within this complex are morphologically indistinguishable, they differ significantly in their disease transmission potential, blood-feeding behaviour, and other biological traits. Cytogenetic and chromosomal studies have identified five sibling species within this complex: A, B, C, D, and E. Understanding the species composition and distribution of this complex is crucial for malaria control strategies.ObjectivesThis study aimed to identify the sibling species of the An. culicifacies complex in Qaleh Ganj County, Kerman Province, Southeastern Iran. Specifically, the study sought to determine the presence of species A, which is known to be a primary vector of malaria in the region.MethodsWe employed allele-specific PCR and sequencing PCR techniques to identify the sibling species. DNA was extracted from mosquito specimens, and the D3 region of the 28S rDNA gene and a segment of the COII gene from the mitochondrial genome (mtDNA) were targeted for amplification and sequencing.ResultsData analysis revealed a positive correlation between An. culicifacies s.l. specimens and altitude, with most specimens collected from mountainous areas. Both allele-specific PCR and sequencing PCR confirmed the presence of species A in the study areas of Kerman Province.ConclusionsGiven that species A is a primary malaria vector, the findings of this study provide valuable insights for guiding malaria control strategies in Southeastern Iran. Further studies are recommended to assess the vector competence and ecological dynamics of other species within the An. culicifacies complex in the region.

Changes in RNA Splicing: A New Paradigm of Transcriptional Responses to Probiotic Action in the Mammalian Brain

Elucidating the gene regulatory mechanisms underlying the gut–brain axis is critical for uncovering novel gut–brain interaction pathways and developing therapeutic strategies for gut bacteria-associated neurological disorders. Most studies have primarily investigated how gut bacteria modulate host epigenetics and gene expression; their impact on host alternative splicing, particularly in the brain, remains largely unexplored. Here, we investigated the effects of the gut-associated probiotic Lacidofil® on alternative splicing across 10 regions of the rat brain using published RNA-sequencing data. The Lacidofil® altogether altered 2941 differential splicing events, predominantly, skipped exon (SE) and mutually exclusive exon (MXE) events. Protein–protein interactions and a KEGG analysis of differentially spliced genes (DSGs) revealed consistent enrichment in the spliceosome and vesicle transport complexes, as well as in pathways related to neurodegenerative diseases, synaptic function and plasticity, and substance addiction across brain regions. Using the PsyGeNET platform, we found that DSGs from the locus coeruleus (LConly), medial preoptic area (mPOA), and ventral dentate gyrus (venDG) were enriched in depression-associated or schizophrenia-associated genes. Notably, we highlight the App gene, where Lacidofil® precisely regulated the splicing of two exons causally involved in amyloid β protein-based neurodegenerative diseases. Although the splicing factors exhibited both splicing plasticity and expression plasticity in response to Lacidofil®, the overlap between DSGs and differentially expressed genes (DEGs) in most brain regions was rather low. Our study provides novel mechanistic insight into how gut probiotics might influence brain function through the modulation of RNA splicing.

First Report of Anthracnoseon on Annona squamosa L. Caused by Colletotrichum fructicola in China.

Annona squamosa L. is a tropical fruit, cultivated in various provinces of China, such as Guangxi, Taiwan, and Yunnan. This fruit has good edible and medicinal value. In November 2021, anthracnose on A. squamosa L. fruits was observed in a plantation in Longzhou (22°11´N, 106°51´E), Guangxi, China. The incidence rate of the plantation was 13.6%. The symptoms included gray-black to black spots, initially small and blackish brown, which expanded into large irregular spots(Fig.1A-B). To isolate the pathogen, symptomatic fruits were collected and small pieces (5 mm2) were cut from the lesions, surface sterilized in 75% ethanol for 15 seconds, followed by 2% AI sodium hypochlorite solution for 3 minutes, rinsed three times with sterile water, and incubated on PDA at 28°C. A total of 23 isolates with identical morphology were obtained and 3 representative isolates (FLZ-1 to FLZ-3) were selected for further characterization. The fungal colonies presented concentric rings on PDA plates, initially white, later turning gray green and producing light orange conidia(Fig.1C). The mature conidia were cylindrical, unicellular, with dimensions of 11.09-20.02 μm × 4.06-6.54 μm, usually containing 2 oil droplets with a size of 3.21-8.36 μm × 2.50-4.55 μm, and L/W ratio of 1.11-2.09(n=100) (Fig.1D). These morphological features were aligned with those of Colletotrichum spp. (Weir et al. 2012). To confirm the identity of the isolates, DNA was extracted and specific gene regions were amplified and sequenced using the following primer sets: ITS (ITS1 and ITS4), GAPDH (GPD1 and GPD2), ACT (ACT-512F and ACT-783R), TUB (T1 and Bt2b), CHS-1 (CHS-79F and CHS-354R), and ApMat (AM-F and AM-R) (Wang et al. 2024). Sequences were submitted to GenBank (Accession nos. PP968176 to PP968178 for ITS, PQ122814 to PQ122816 for GAPDH, PQ122808 to PQ122810 for ACT, PQ169643 to PQ169645 for TUB2, PQ122811 to PQ122813 for CHS-1, and PQ152017 to PQ152019 for ApMat). Maximum likelihood phylogenetic analysis using combined sequences of ITS, GAPDH, ACT, TUB2, CHS-1 and ApMat in MEGA X confirmed the isolates as Colletotrichum fructicola (Fig.2), marking the first report of this pathogen on A. squamosa L. in China. Pathogenicity tests were conducted on wound and nonwound healthy A. squamosa L. fruits to confirm the pathogenicity of the isolated C. fructicola. The fruits were wounded using a sterile needle. Each wounded and nonwounded fruit was inoculated with a 10 µL conidial suspension (1×106 conidia/mL) drop at corresponding sites. Control fruits were inoculated with sterile water. Five fruits per treatment were used and the experiment repeated three times. The fruits were placed in an incubator at 28°C and 85% RH. Anthracnose symptoms developed on the inoculated fruits within 5 days (Fig.1E-F), while control fruits remained symptom-free (Fig.1G-H). C. fructicola was successfully reisolated from the symptomatic fruits, fulfilling Koch's postulates and confirming its role as the causal agent of the disease. According to previous reports, C. fructicola also causes anthracnose on Psidium guajava (William R. O et al. 2024), leaf spots on apple (Moreira Rafaele R et al. 2019), and Kadsura coccinea (Jiang et al.2021). This study identifies C. fructicola as the pathogen causing anthracnose on A. squamosa L. and no cases have yet been discovered in the field in China. This report will raise awareness among growers about the anthracnose disease of A. squamosa L. and develop effective disease control strategies.

Genetic Diversity of Tobacco mosaic virus (TMV) isolates from tobacco growing fields of Western Anatolia, Türkiye

Tobacco mosaic virus (TMV) is an important plant virus that is common in agriculture. It is the first evidence of the existence of viruses in history. Studies on the genetic diversity of the CP gene of TMV, which plays a leading role in host interaction, are limited both in our country and around the world. Genetic diversity analyses were conducted on ten isolates of the full CP gene region of TMV obtained from the most intensive tobacco cultivation areas in our country, and compared with global isolates. TMV infection was detected in 32 out of 300 plants collected from the Aegean and Marmara regions (Çanakkale, Balıkesir, İzmir, Manisa, Uşak, Aydın and Denizli) between 2019 and 2020 using conventional molecular techniques. To genetically characterize the virus, 10 samples were selected from each region, and the complete CP gene region sequences were determined. The aligned CP gene region sequences of TMV from Türkiye and its global isolates exhibited nucleotide homology ratios ranging from 87.7% to100%, with amino acid ratios ranging from 88.7% to 100%. The Türkiye isolates displayed similarity rates of 98.5% to 100% at the nucleotide level and 98.7% and 100% at the amino acid level. In phylogenetic analysis, the 196 known isolates of TMV registered in GenBank, belong to the CP gene region, were divided into two main clades (I and II) and two subclades (Ia and Ib). Turkish isolates were clustered in the major branch with the main clade I and subclade Ia isolates. Therefore, genetic analyses were performed on the CP gene region isolates obtained from different parts of the world and a wide range of hosts, including the isolates obtained from Türkiye. The results showed high genetic stability, similar to many tobamoviruses.

Localization and Molecular Cloning of the ASMT Gene for Melatonin Synthesis in Pigs

Melatonin is synthesized in multiple tissues and organs of pigs, and existing studies have shown the presence of the melatonin-synthesizing enzyme ASMT protein. However, the genomic information for the ASMT gene has been lacking. The aim of this study was to locate the genomic information of the ASMT gene in pigs using comparative genomics analysis and then obtain the coding region information through molecular cloning. First, using the NCBI Genome Data Viewer, we found that in most animals, the AKAP17A gene is often located next to the ASMT gene, with both genes arranged in the same direction. Similarly, the P2RY8 gene is commonly adjacent to the ASMTL gene, also in the same orientation. We also discovered that the ASMTL gene is frequently adjacent to the ASMT gene and arranged in the opposite direction. Using the “three-point localization” principle, we inferred the position of the ASMT gene based on the coordinates of AKAP17A and ASMTL in pigs. Our results revealed that on the pig X chromosome, a gene called LOC110258194 is located next to the AKAP17A and ASMTL genes, and its arrangement aligns with the ASMT gene in other species. Additionally, Ensembl contains a gene, ENSSSCG00000032659, at the same position, with completely overlapping exons, though it is not annotated as ASMT. Further analysis using the TreeFam tool from EMBL-EBI and the CDD tool from NCBI revealed that LOC110258194 and ENSSSCG00000032659 do not contain the typical Maf domain of ASMTL and, thus, should not be annotated as ASMTL, but rather as the ASMT gene. Using a slow-down PCR method for high-GC content genes, we successfully cloned the full CDS region of the pig ASMT gene and identified a new transcript missing Exon 6 and Exon 7. This transcript was submitted to NCBI and assigned the GenBank accession number MW847601. Our results represent the first successful localization of the ASMT gene in pigs, the first cloning of the ASMT gene’s coding region, and the first discovery of a new transcript of the pig ASMT gene.

Genome-Wide Analysis and Expression Profiling of Watermelon VQ Motif-Containing Genes Under Abiotic and Biotic Stresses

Valine-glutamine (VQ) motif-containing proteins play important roles in diverse plant developmental processes and signal transduction in response to biotic and abiotic stresses. However, no systematic investigation has been conducted on VQ genes in watermelon. In this study, we identified 31 watermelon VQ genes, which were classified into six subfamilies (I–VI). All of the deduced proteins contained a conserved FxxxVQxL/F/VTG motif. Eleven ClVQs were involved in segment duplication, which was the main factor in the expansion of the VQ family in watermelon. Numerous stress- and hormone-responsive cis-elements were detected in the putative promoter region of the ClVQ genes. Green fluorescent protein fusion proteins for ten selected ClVQs were localized in the nucleus, but three ClVQs also showed signals in cell membranes and the cell wall, thus confirming their predicted divergent functionality. Quantitative real-time PCR (qRT-PCR) analysis indicated that the majority of ClVQ genes were specifically or preferentially expressed in certain tissues or organs, especially in the male flower. Analyses of RNA-sequencing data under osmotic, cold, and drought stresses and Cucumber green mottle mosaic virus (CGMMV) infection revealed that the majority of ClVQ genes, especially those from subfamily IV, were responsive to these stresses. The results provide useful information for the functional characterization of watermelon ClVQ genes to unravel their biological roles.

Loss of Myostatin Affects m6A Modification but Not Semen Characteristics in Bull Spermatozoa

N6-methyladenosine (m6A) modification is a key methylation modification involved in reproductive processes. Myostatin gene editing (MT) in cattle is known to enhance muscle mass and productivity. However, the changes in m6A modification in MT bull sperm remain poorly understood. In the MT and wild-type (WT) groups, we identified 25,542 and 22,253 m6A peaks, respectively, mainly concentrated in the coding sequence (CDS) and 3′ untranslated region (UTR) of genes. The MT group showed an increase in gene transcription, but there was no significant difference in the overall m6A peaks pattern. There was also no significant difference in m6A motif and chromosome distribution between MT and WT groups. Most genes had less m6A modification sites. A total of 1120 m6A peaks were significantly different, corresponding to 1053 differentially m6A-methylated genes (DMMGs). These DMMGs are mainly associated with G protein-coupled receptor signaling pathways and the overall composition of the cell membrane. Furthermore, an MCL clustering analysis of 111 differentially m6A-methylated and expressed genes identified seven key genes (RHOA, DAAM1, EXOC4, GNA12, PRICKLE1, SCN1A, and STXBP5L), with the cytoskeleton and migration-related gene, RHOA, being the most important gene located at the center of the gene network. However, the analysis of sperm morphology and motility indicated no significant changes in semen volume, sperm count, sperm viability, plasma membrane integrity, acrosome membrane integrity, or mitochondrial membrane integrity. This study provides a map of m6A methylation in spermatozoa from MT and WT bulls, identifies key differential m6A genes that are affected by the myostatin gene but do not affect sperm morphology and viability in MT bulls, and provides a theoretical basis for the breeding quality of MT bulls.

Comprehensive Analysis of TSPAN32 Regulatory Networks and Their Role in Immune Cell Biology

Tetraspanin 32 (TSPAN32), a member of the tetraspanin superfamily, is one of several tumor-suppressing subtransferable fragments located in the imprinted gene domain of chromosome 11p15.5, a critical tumor-suppressor gene region. Although the biology of TSPAN32 remains largely unexplored, accumulating evidence suggests its involvement in hematopoietic functions. In this study, we performed a comprehensive analysis of the expression patterns and regulatory roles of TSPAN32. Notably, TSPAN32 is highly expressed in immune cells, particularly in natural killer (NK) cells and CD8+ T cells. The observed downregulation of TSPAN32 during immune cell activation highlights its potential role as a regulator of immune cell activation and metabolic adaptations, which are crucial for effective immune responses against pathogens and tumors. Moreover, the modulation of biological processes following TSPAN32 knockout further supports its critical role in regulating immune cell physiology and responses. These findings not only shed light on the biology of TSPAN32 but also provide the basis for exploring its diagnostic, prognostic, and therapeutic potential in autoimmune and inflammatory disorders, as well as in hematopoietic cancers.

A leucine responsive small RNA AbcR200 regulates expression of the lactate utilization (lut) operon in Acinetobacter baumannii DS002.

Noncoding small RNAs are essential for modulating bacterial gene expression, especially under carbon and nutrient-limited conditions. In this study, by employing both in silico and molecular hybridization tools, we identified a carbon source responsive small RNA in A. baumannii DS002. Expression of corresponding gene, abcR200, located at the intergenic region of omt (O-methyltransferase) and orf72 genes, is under the transcriptional control of a global transcriptional factor, Leucine Responsive Regulatory Protein (Lrp). A sequence motif that serves as a target for Lrp was found overlapping the abcR200 promoter (PabcR200). Chromatin immunoprecipitation (ChIP) demonstrated that Lrp oligomers, formed under low leucine conditions, strongly interacted to the PabcR200. However, the observed interactions were disrupted in the presence of leucine, as leucine promoted dissociation of Lrp to monomers and dimers, the conformation unfavourable to interact with PabcR200. The abcR200 promoter activity increased with increase of exogenous leucine concentrations, and at 2 mM maximum promoter activity was observed. The AbcR200 target mRNAs were identified by analysing the transcriptome of abcR200 negative strain of A. baumannii. Intriguingly, in abcR200 negative background, expression of lut (lactate utilization) mRNA has increased, suggesting that lut mRNA as one of the mRNA targets for AbcR200. Consistent of this observation, there existed extensive sequence complementarity between AbcR200 and lut mRNA, especially in the regions coding LutP, LutE and LutR. In support of the observed sequence complementarity, the levels of lut mRNA encoded proteins got elevated in abcR200 negative HS002 strains suggesting a role for AbcR200 in translational inhibition of lut mRNA.

Tandem mass spectrometry screening and genetic analysis of neonates with Urea cycle disorders

To explore the results of four types of Urea cycle disorders (UCDs) in newborns from the Xuzhou region, assess the efficacy of newborn screening by tandem mass spectrometry (MS/MS), and analyze their genetic characteristics. A retrospective analysis was performed using tandem mass spectrometry to screen for inherited metabolic disorders in 691 712 newborns at the Maternal and Child Health Care Hospital of Xuzhou from November 2015 to December 2023. Ten children (cases 1-10) were diagnosed with Ornithine transcarbamylase deficiency (OTCD), Carbamoylphosphate synthase 1 deficiency (CPS1D), Arginase deficiency (ARGD), and Argininosuccinate synthase deficiency (ASSD) based on MS/MS and genetic testing. This study was approved by the Medical Ethics Committee of Xuzhou Maternity and Child Health Care Hospital (Ethics No.XZFY2024-051K-01J). A total of 691 712 neonates were screened for UCDs using MS/MS, which identified 1 237, 1 237, 510, and 1 009 initial positive cases for OTCD, CPS1D, ASSD, and ARGD, respectively. After genetic testing, 1 case of OTCD, 1 case of CPS1D, 1 case of ASSD, and 7 cases of ARGD were confirmed. The overall positive predictive value for these four UCDs was 0.362%. Among the 10 diagnosed UCD cases, four novel variants were identified, which included OTC: c.1024C>A (p.L342M) and ASS1: c.826A>G (p.M276V), c.695C>T (p.P232L) and c.694C>T (p.P232S). Bioinformatic analysis has rated these as variants of uncertain clinical significance or likely pathogenic based on guidelines from the American College of Medical Genetics and Genomics (ACMG). The incidence of four UCDs in neonates from the Xuzhou area is relatively low, and there is a correlation between genetic variants and clinical phenotypes. For novel variants with uncertain clinical significance or suspected pathogenicity, their pathogenicity should be clarified in conjunction with clinical and biochemical indicators. The four novel pathogenic variants of UCDs identified in this study have enriched the mutational spectrum of UCDs-associated genes in the Xuzhou region.

First Report of Agroathelia delphinii Causing Rhizome Rot of Polygonatum cyrtonema Hua (Duohua Huangjing) in China.

Polygonatum cyrtonema Hua (Duohua Huangjing, Asparagaceae in angiosperms) is a traditional medicinal and edible plant in China. Its rhizomes can potentially enhance immunity, reduce tumor growth and the effects of aging, improve memory, and even reduce blood sugar levels (Zhao et al. 2020). Many Polygonatum-derived health-care products have been developed, and annual demand in China for processed Polygonatum is up to 4000 tons (Su et al. 2018). However, in May 2024 there was an outbreak of rhizome rot in the P. cyrtonema Hua planting fields (N31°01'50″, E121°26'31″) located on the Shanghai Jiao Tong University campus, China. Approximately 40 % of the plants were affected. The surfaces of the rhizomes appeared rotten and were covered with white and turquoise hyphae. We also observed brown, basal stem necrosis, as well as leaf yellowing and wilting. To isolate the causal organism, affected rhizome tissues were disinfected with 75% ethanol for 1 min, rinsed 5 times with sterile water, placed on potato dextrose agar (PDA) medium containing 50 µg/ml of Chlortetracycline, and then incubated at 28 ℃. Hyphal tips of the resulting fungal colonies were sub-cultured after 3 days of incubation. The suspect pathogen had white, fluffy aerial mycelium, and grew radially at an average rate of 13.75 ± 0.55 mm/day. White sclerotia werebserved after 5 days of incubation, which subsequently turned yellow-brown, and ultimately reddish brown after 2 weeks. Mature sclerotia were of the globular or ellipsoidal type (0.6-2.1 mm in diameter with a mean of 1.23 mm; n = 100). Septal hyphae and clamp connections were observed with the Olympus microscope. These morphological features were similar to the description of Agroathelia delphinii (anamorph Sclerotium delphinii), a known pathogen of delphinium (Punja and Damiani 1996). Our isolated strain was denoted as HYW-1. For molecular identification, HYW-1 genomic DNA was extracted using the CTAB method (Mahadevakumar et al. 2018). The internal transcribed spacer region (ITS) and large subunit rRNA (LSU) gene were subsequently PCR-amplified with the primer pairs ITS1/ITS4 and LR5/LROR (White et al. 1990) and then sequenced at Sangon Biotech (Shanghai, China). The resulting HYW-1 ITS and LSU sequences were submitted to the NCBI nr database for BlastN analysis (GenBank accessions PP976559 and PP907792, respectively), and they were 99.69%, and 99.46% identical to the corresponding to the type strain of Agroathelia delphinii (Welch) Redhead (Amylocorticiaceae, Agaricomycotina) sequences (GenBank accessions JN241576 and MH866785, respectively). Based on the ITS and LSU sequences, a phylogenetic tree was generated using the the maximum likelihood method of MEGA 7.0. Strains HYW-1 and CBS 305.32 were members of the same clade. To complete Koch's Postulates, two HYW-1 hyphal discs (5 mm in diameter) were placed on each of the rhizomes of six healthy P. cyrtonema Hua seedlings. As a control, two uncolonized agar discs were placed on the rhizomes of six additional healthy seedlings. All seedlings were moved to a greenhouse with conditions of 90% RH and 28 °C. White hyphae were observed on the surface of inoculated plants after 3 days, and leaf wilt was observed after 10 days. The control seedlings showed no symptomatology. Infected tissues were collected for pathogen re-isolation, and subsequent ITS sequence analysis revealed that the causal agent was consistent with the original isolate HYW-1. To the best of our knowledge, this is the first report of A. delphinii causing rhizome rot on P. cyrtonema Hua in China.

Characteristics and Clinical Significance of Gut Microbiota in Patients With Epstein-Barr Virus-Associated Liver Dysfunction.

Infectious mononucleosis (IM) is mainly triggered by Epstein-Barr virus (EBV) infection. There are few studies on the role of the gut microbiota in IM and EBV-associated liver dysfunction. The aim of this study was to investigate the characteristics of the gut microbiota in the EBV-associated liver dysfunction and to evaluate the relationship between the severity of gut microbiota dysbiosis and cytokine levels. A case-control study was performed. Individuals meeting the inclusion and exclusion criteria for EBV-induced IM were enrolled and their fecal and blood samples were collected. The V3-V4 region of the 16s rDNA gene of fecal microbiota was sequenced; bioinformatics analysis including α-diversity, β-diversity, and linear discriminant analysis (LDA) effect size (LEfSe) was performed; and the correlation between bacteria and clinical indices was analysed. A total of 48 participants completed fecal and blood tests, including 18 IM, 11 EBV-associated liver dysfunction, 12 healthy children and 7 EBV-negative liver dysfunction. The α-diversity and β-diversity of the gut microbiota in the EBV-associated liver dysfunction was more than that in IM. The abundance of Granulicatella, Enterococcus, Atopobium and Acinetobacter increased, while the abundance of Prevotella, Sutterella, Collinsella, Desulfovibrio decreased in the EBV-associated liver dysfunction compared with the IM. The abundance of Enterococcus, Atopobium and Acinetobacter correlated positively with the levels of IL-1β, IL-6, TNF-α and CD8+ cytotoxic T lymphocytes%. Gut microbiota of EBV-associated liver dysfunction was significantly disturbed and associated with systemic immune response.

A new pipeline SPICE identifies novel JUN-IKZF1 composite elements.

Transcription factor partners can cooperatively bind to DNA composite elements to augment gene transcription. Here, we report a novel protein-DNA binding screening pipeline, termed Spacing Preference Identification of Composite Elements (SPICE), that can systematically predict protein binding partners and DNA motif spacing preferences. Using SPICE, we successfully identified known composite elements, such as AP1-IRF composite elements (AICEs) and STAT5 tetramers, and also uncovered several novel binding partners, including JUN-IKZF1 composite elements. One such novel interaction was identified at CNS9, an upstream conserved noncoding region in the human IL10 gene, which harbors a non-canonical IKZF1 binding site. We confirmed the cooperative binding of JUN and IKZF1 and showed that the activity of an IL10-luciferase reporter construct in primary B and T cells depended on both this site and the AP1 binding site within this composite element. Overall, our findings reveal an unappreciated global association of IKZF1 and AP1 and establish SPICE as a valuable new pipeline for predicting novel transcription binding complexes.

Genetic Basis and Identification of Candidate Genes for Alkalinity Tolerance Trait in Spotted Sea Bass (Lateolabrax maculatus) by Genome-Wide Association Study (GWAS).

Given the challenges of overcrowded coastal aquaculture spaces and insufficient production, utilizing saline-alkaline water areas represents a vital strategy to alleviate these bottlenecks. Spotted sea bass (Lateolabrax maculatus), with its formidable osmoregulatory capabilities, is an ideal candidate to develop a saline-alkaline tolerant strain. In our study, genotypic and phenotypic data from 287 L. maculatus individuals exposed to carbonate alkaline conditions were collected, and a genome-wide association study (GWAS) conducted to elucidate genetic basis related to carbonate alkalinity tolerance trait. Results showed that 14 SNPs and 8 InDels were markedly related to carbonate alkalinity tolerance trait, and 404 candidate genes were pinpointed within a ± 300-kb region surrounding these variants. Notably, the most significant SNP (SNP_05_17240108), along with two adjacent SNPs (SNP_05_17240102 and SNP_05_17240340) and two InDels (InDel_05_17240228 and InDel_05_17240231), was situated in the intron region of trio gene that could play vital roles in cell remodeling, and cell junction and activity of aquaporins to deal with carbonate alkalinity stress. Furthermore, candidate genes were significantly involved in pathways associated with carbohydrate metabolism, cell remodeling, ion transport, and RNA degradation, which were consistent with RNA-Seq analysis results of gills and kidneys in response to alkalinity stress. Our study will contribute to elucidate the genetic basis of alkalinity tolerance and the identified SNPs and InDels could be used for marker-assisted selection (MAS) and genomic selection (GS) for alkalinity tolerance trait in the breeding programs of spotted sea bass.

Systematically developing a registry of splice-site creating variants utilizing massive publicly available transcriptome sequence data

Genomic variants causing abnormal splicing play important roles in genetic disorders and cancer development. Among them, variants that cause the formation of novel splice-sites (splice-site creating variants, SSCVs) are particularly difficult to identify and often overlooked in genomic studies. Additionally, these SSCVs are frequently considered promising candidates for treatment with splice-switching antisense oligonucleotides (ASOs). To leverage massive transcriptome sequence data such as those available from the Sequence Read Archive, we develop a novel framework to screen for SSCVs solely using transcriptome data. We apply it to 322,072 publicly available transcriptomes and identify 30,130 SSCVs. Among them, 5121 SSCVs affect disease-causing variants. By utilizing this extensive collection of SSCVs, we reveal the characteristics of Alu exonization via SSCVs, especially the hotspots of SSCVs within Alu sequences and their evolutionary relationships. We discover novel gain-of-function SSCVs in the deep intronic region of the NOTCH1 gene and demonstrate that their activation can be suppressed using splice-switching ASOs. Collectively, we provide a systematic approach for automatically acquiring a registry of SSCVs, which facilitates the elucidation of novel biological mechanisms underlying splicing and serves as a valuable resource for drug discovery. The catalogs of SSCVs identified in this study are accessible on the SSCV DB (https://sscvdb.io).

Optogenetic control of horizontally acquired genes prevent stuck fermentations in yeast.

Nitrogen limitations in the grape must be the main cause of stuck fermentations during the winemaking process. In Saccharomyces cerevisiae, a genetic segment known as region A, which harbors 12 protein-coding genes, was acquired horizontally from a phylogenetically distant yeast species. This region is mainly present in the genome of wine yeast strains, carrying genes that have been associated with nitrogen utilization. Despite the putative importance of region A in yeast fermentation, its contribution to the fermentative process is largely unknown. In this work, we used a wine yeast strain to evaluate the contribution of region A to the fermentation process. To do this, we first sequenced the genome of the wine yeast strain using long-read sequencing and determined that region A is present in a single copy. We then implemented an optogenetic system in this wine yeast strain to precisely regulate the expression of each gene, generating a collection of 12 strains that allow for light-activated gene expression. To evaluate the role of these genes during fermentation, we assayed this collection using microculture and fermentation experiments in synthetic must with varying amounts of nitrogen concentration. Our results show that changes in gene expression for genes within this region can impact growth parameters and fermentation rate. We additionally found that the expression of various genes in region A is necessary to complete the fermentation process and prevent stuck fermentations under low nitrogen conditions. Altogether, our optogenetics-based approach demonstrates the importance of region A in completing fermentation under nitrogen-limited conditions.IMPORTANCEStuck fermentations due to limited nitrogen availability in grape must represent one of the main problems in the winemaking industry. Nitrogen limitation in grape must reduces yeast biomass and fermentation rate, resulting in incomplete fermentations with high levels of residual sugar, undesired by-products, and microbiological instability. Here, we used an optogenetic approach to demonstrate that expression of genes within region A is necessary to complete fermentations under low nitrogen availability. Overall, our results suggest that region A is a genetic signature for adaptation to low nitrogen conditions.

Emergence of ceftriaxone-resistant Neisseria gonorrhoeae through horizontal gene transfer among Neisseria spp.

It has been suggested that the emergence of ceftriaxone-resistant strains of Neisseria gonorrhoeae involves the incorporation of the penA gene from commensal Neisseria spp. that are resistant to ceftriaxone. However, the mechanism of this mosaic penA generation is unknown. We obtained 10 strains of commensal Neisseria spp. showing ceftriaxone MIC >0.5 mg/L. The similarity of the penA gene region of these commensal Neisseria spp. strains and some ceftriaxone-resistant N. gonorrhoeae strains was investigated. To obtain transformants, commensal Neisseria spp., Neisseria lactamica, gDNA was used as donor DNA and a N. gonorrhoeae strain as the recipient. The sequence similarity in certain regions of penA-murE between some of the commensal Neisseria spp. strains and the N. gonorrhoeae FC428 strain was very high. The sequence of these regions was very similar among some ceftriaxone-resistant strains of Neisseria spp. The PenA of the transformants matched the full PenA 60 of the original FC428 strain. Furthermore, our findings indicated that the source of resistance could have been a penA fragment derived from Neisseria spp. that originally carried the same sequence. We suggest that FC428 developed ceftriaxone resistance by acquiring part of the penA-murE gene region from N. lactamica through horizontal gene transfer. The ceftriaxone-resistant N. lactamica shown here may also have emerged by acquiring part of penA from other Neisseria spp. From this work, our data provide insights into the understanding of the mechanism underlying the evolution of drug-resistant gonorrhea-causing strains.

A genome-wide scan of non-coding RNAs and enhancers for refractive error and myopia.

Refractive error (RE) and myopia are complex polygenic conditions with the majority of genome-wide associated genetic variants in non-exonic regions. Given this, and the onset during childhood, gene-regulation is expected to play an important role in its pathogenesis. This prompted us to explore beyond traditional gene finding approaches. We performed a genetic association study between variants in non-coding RNAs and enhancers, and RE and myopia. We obtained single-nucleotide polymorphisms (SNPs) in microRNA (miRNA) genes, miRNA-binding sites, long non-coding RNAs genes (lncRNAs) and enhancers from publicly available databases: miRNASNPv2, PolymiRTS, VISTA Enhancer Browser, FANTOM5 and lncRNASNP2. We investigated whether SNPs overlapping these elements were associated with RE and myopia leveraged from a large GWAS meta-analysis (N = 160,420). With genetic risk scores (GRSs) per element, we investigated the joint effect of associated variants on RE, axial length (AL)/corneal radius (CR), and AL progression in an independent child cohort, the Generation R Study (N = 3638 children). We constructed a score for biological plausibility per SNP in highly confident miRNA-binding sites and enhancers in chromatin accessible regions. We found that SNPs in two miRNA genes, 14 enhancers and 81 lncRNA genes in chromatin accessible regions and 54 highly confident miRNA-binding sites, were in RE and myopia-associated loci. GRSs from SNPs in enhancers were significantly associated with RE, AL/CR and AL progression. GRSs from lncRNAs were significantly associated with all AL/CR and AL progression. GRSs from miRNAs were not associated with any ocular biometric measurement. GRSs from miRNA-binding sites showed suggestive but inconsistent significance. We prioritized candidate miRNA binding sites and candidate enhancers for future functional validation. Pathways of target and host genes of highly ranked variants included eye development (BMP4, MPPED2), neurogenesis (DDIT4, NTM), extracellular matrix (ANTXR2, BMP3), photoreceptor metabolism (DNAJB12), photoreceptor morphogenesis (CHDR1), neural signaling (VIPR2) and TGF-beta signaling (ANAPC16). This is the first large-scale study of non-coding RNAs and enhancers for RE and myopia. Enhancers and lncRNAs could be of large importance as they are associated with childhood myopia. We provide a confident blueprint for future functional validation by prioritizing candidate miRNA binding sites and candidate enhancers.

Exploring caffeine as a disruptor of membrane integrity and genomic stability in Staphylococcus aureus: functional and in silico analysis.

To explore the mechanistic underpinnings of caffeine as a potent antibacterial against Staphylococcus aureus ATCC 25923 via in vitro functional assays, whole-genome sequencing, and in silico docking studies. In vitro studies established that caffeine's minimum inhibitory concentration (MIC) against S. aureus ATCC 25923 is 0.01544mmol/mL. Functional assays along with Scanning Electron Microscopy confirmed that caffeine at 0.030089mmol/mL (2MIC) released nucleotide constituents (nucleotide leakage assay) and effluxed potassium ions (potassium efflux assay) thereby, further validating caffeine's role as a membrane-active antimicrobial agent. Whole genome sequencing of control versus caffeine treated samples revealed a significant drop in read mapping percentage from 99.96 to 23.68% and GC content from 30.69 to 6.93%. This massive reduction in the treated sample was a consequence of single nucleotide polymorphisms (SNPs, 50,303), along with insertions and deletions (InDels, 62). Several of these caffeine-induced mutations were found to be harbouring the coding regions of genes involved in processes such as cell membrane organization, bacterial virulence, and DNA repair processes. Thus, implying a caffeine-mediated genomic rearrangement and instability. In silico docking studies revealed a strong binding affinity of caffeine to key cell wall proteins ltaA (-6.9kcal/mol) and ltaS (-6.5kcal/mol) respectively. The dynamic simulation studies revealed caffeine's interaction with receptor ltaS remained stable, with low deviations and minimal fluctuations. Although caffeine has been widely investigated for its antibacterial properties, its specific mechanisms of action, notably its effects on the cell membrane and genomic integrity in S. aureus ATCC 25923, are little understood. This study thus offers a comprehensive functional genomic analysis of caffeine as an antibacterial against S. aureus.

Enhancement of FK520 production in Streptomyces hygroscopicus var. ascomyceticus ATCC 14891 by overexpressing the regulatory gene fkbR2.

Ascomycin (FK520) is a 23-membered macrolide antibiotic primarily produced by the Streptomyces hygroscopicus var. ascomyceticus. Structurally similar to tacrolimus and rapamycin, it serves as an effective immunosuppressant widely used in the treatment of rejection reactions after organ transplantation and certain autoimmune diseases. Currently, FK520 is mainly produced through microbial fermentation, but its yield remains low. Since the gene fkbR2 is a regulatory gene within the FK520 biosynthesis gene cluster that has not been studied, this paper focuses on the overexpression of the gene fkbR2 in Streptomyces hygroscopicus var. ascomyceticus ATCC 14891 (WT). By constructing a strain with overexpressed fkbR2 gene, we initially obtained a high-yield strain R2-17 through shake flask fermentation, with a 28% increase in yield compared to WT. In the process of further improving the stability of the high-yield strain, this paper defines two indices: high-yield index and stability index. After two consecutive rounds of natural breeding, strain R2-17 achieved a high-yield index of 100% and a stability index of 80%. Finally, the high-yield strain R2-17-3-10 was successfully screened, and the yield was increased by 34% compared with the strain WT, reaching 686.47mg/L. A comparative analysis between the high-yield strain R2-17-3-10 and the original strain WT revealed differences in fermentation process parameters such as FK520 synthesis rate, pH, bacterial growth, glycerol consumption rate, ammonia nitrogen level, and ammonium ion concentration. In addition, the transcription levels of genes involved in the synthesis of precursors 4,5-dihydroxycyclohex-1-enecarboxylic acid (fkbO), ethylmalonyl-CoA (fkbE, fkbU, fkbS), and pipecolic acid (fkbL), as well as pathway-specific regulatory genes (fkbN, fkbR1), were significantly increased at different time points in the high-yield strain R2-17-3-10. EMSAs analysis showed that the FkbR2 protein could not bind to the promoter region of above genes. This suggests that the gene fkbR2 may enhance the supply of FK520 synthetic precursors by indirectly regulating the transcription levels of these genes, thereby promoting an increase in FK520 production. These results demonstrate that modifying genes within the biosynthetic gene clusters of natural products can be successfully applied to increase the yields of industrially and clinically important compounds. However, it is found that fkbR2 gene is a regulatory gene that has not been fully studied, and it is worth further studying its regulatory mechanism.