Full-length Gene Research Articles

Evaluation of fipronil efficacy and first molecular report of gamma-aminobutyric acid (GABA) gated chloride channel gene of Rhipicephalus microplus ticks in China and Pakistan.

Southern cattle tick, Rhipicephalus (R) microplus, is a major challenge in transmitting Babesia spp., Theileria spp., and Anaplasma spp., worldwide. Mitigation of this tick and associated pathogens is crucial for animal and human-health. In present study, laboratory-based larval packet tests (LPTs) were performed to assess fipronil efficacy against R. microplus ticks collected from four Chinese localities, Nanning, Guangshui, Macheng, and Tengchong. Additionally, we reported first molecular characterization of the full-length gamma-aminobutyric acid (GABA)-gated chloride channel gene of R. microplus ticks from China and Pakistan (Khyber Pakhtunkhwa and Punjab). For LPTs bioassay, fipronil's concentrations (5-ppm, 25-ppm, 50-ppm, 75-ppm, 100-ppm) in acetone and DNase-free water were prepared. The highest average mortality rate of larvae was observed in Nanning (4.34-98.13 %) while lowest in Tengchong (5.70-82.29 %) via triplicate LPTs. The recorded LC50 values were 33.2 ppm, 35.7 ppm, 49.7 ppm, and 55.4 ppm, respectively, for the four localities (Guangshui, Macheng, Nanning, and Tengchong) while the LC99 values were 722 ppm, 827 ppm, 949 ppm, and 1342.3 ppm, respectively. The resistance factors (RR50) were 13.83, 14.88, 20.71, and 23.08, indicating the development of level-II resistance in the tick populations from these regions respectively. Molecularly, consensus sequences of R. microplus GABA-gene from China and Pakistan resulted 99.24-99.52 % and 99.10-99.39 % identities with dieldrin-susceptible NRFS (GQ398111.1) and dieldrin-resistant (GQ398112.1) R. microplus ticks, respectively. Findings revealed 21 single nucleotide polymorphisms (SNPs) [8-non-synonymous (NS) and 13-synonymous (S)], including specifically 10-SNPs from Nanning, 9-SNPs from Macheng, 7-SNPs from Guangshui, 10-SNPs from Tengchong, and 9-SNPs from Pakistani R. microplus full-length GABA gene. Explicitly, we obtained one NS-SNP in TM3 (T-871-G; F-291-V) and two NS-SNPs in TM4 (A-1438-G; N-480-D and A-1439-G; N-480-G) regions of Chinese and Pakistani, while one NS-SNP in TM2 (A-763-G; T-255-A) region of Pakistani R. microplus ticks was documented. Coexistence among SNPs in TM3 (T-871-G; F-291-V), TM4 (A-1439-G; N-480-G) and one linker SNP (A-1378-G; S-460-G) were also recorded. Presence of SNPs and their coexistence suggest the possible tri-dimensional structural modification in GABA-Cl channels that might interfere with binding of fipronil, resulting in development of resistance. This comprehensive research will provide a basis for governmental and pharmaceutical industries for development of effective tick control-strategies as well as to manage the effectiveness and functionality of the available acaricides.

Cost-Effective Detection of SNPs and Structural Variations in Full-Length Genes of Wheat and Sunflower Using Multiplex PCR and Rapid Nanopore Kit

The rapid identification of allele variants in target genes is crucial for accelerating marker-assisted selection (MAS) in plant breeding. Although current high-throughput genotyping methods are efficient in detecting known polymorphisms, they are limited when multiple variant sites are scattered along the gene. This study presents a target amplicon sequencing approach using Oxford Nanopore Technologies (ONT-TAS) to rapidly sequence full-length genes and identify allele variants in sunflower and wheat collections. This procedure combines multiplex PCR and a rapid sequencing kit, significantly reducing the time and cost compared to previous methods. The efficiency of the approach was demonstrated by sequencing four genes (Ahasl1, Ahasl2, Ahasl3, and FAD2) in 40 sunflower genotypes and three genes (Ppo, Wx, and Lox) in 30 wheat genotypes. The ONT-TAS revealed a complete picture of SNPs and InDels distributed over the individual alleles, enabling rapid (4.5 h for PCR and sequencing) characterization of the genetic diversity of the target genes in the germplasm collections. The results showed a significant diversity of the Ahasl1/Ahasl3 and Wx-A/Lox-B genes in the sunflower and wheat collections, respectively. This method offers a high-throughput, cost-effective (USD 3.4 per gene) solution for genotyping and identifying novel allele variants in plant breeding programs.

Biocontrol efficacy of Bacillus licheniformis and Bacillus amyloliquefaciens against rice pathogens.

Biocontrol is a cost-effective and eco-friendly approach to control plant pathogens using natural enemies. Antagonistic microorganisms or their derivatives specifically target the plant pathogens while minimizing the harm to non-target organisms. Bacterial blight and brown spot are the major rice diseases caused by Xanthomonas oryzae pv. oryzae (Xoo) and Bipolaris oryzae (Bo), respectively. This study was conducted to assess the plant growth-promoting potential and biocontrol activity of root-associated bacteria against the rice pathogens, Xoo and Bo. A total of 98 bacteria were isolated from rice roots and characterized for plant growth-promoting properties including phosphate solubilization, indole-3-acetic acid production, nitrogen fixation and biofilm formation. Based on these properties, 36 bacteria were selected and tested for biocontrol potential against rice pathogens via co-culturing antagonism assay. LE7 exhibited the maximum inhibition of 79%, while FR8, PE2, LE7, LR22 and LR28 also significantly reduced the growth of Xoo. Likewise, FR2, LR22, LR35 and LE7 significantly inhibited the growth of Bo, in which LR22 exhibited the maximum inhibition of 81%. Under controlled-conditions, LE7 and LR22 significantly reduced the disease incidence of Xoo and Bo, respectively, and improved the growth of rice. Full-length 16S rRNA gene sequencing of most potential bacterial isolates, LE7 and LR22, revealed their maximum identity with Bacillus amyloliquefaciens and Bacillus licheniformis, respectively. Application of Bacillus spp. as biocontrol agent represents enormous potential in rice farming. The most promising bacterial isolates could be used as bioinoculants for rice disease management and improved production in a sustainable manner.

Alginate-polylysine-alginate (APA) microencapsulated transgenic human amniotic epithelial cells ameliorate fibrosis in hypertrophic scars.

Hypertrophic scar (HS) is a severe skin fibrosis. Transplanting stem cells carrying anti-fibrotic cytokine genes, like interferon-gamma (IFN-γ), is a novel therapeutic strategy. Human amniotic epithelial cells (hAECs) are ideal seed cells and gene vectors. Microencapsulation creates a favorable environment for transplanted cells. This study investigates the effect of alginate-polylysine-alginate (APA)-microencapsulated hAECs modified with IFN-γ on HS fibrosis. hAECs were isolated from human placentas and characterized. The full-length IFN-γ gene was cloned into the pcDNA3.1 vector to create the recombinant plasmid IFN-γ-pcDNA3.1. This plasmid was then transfected into hAECs, resulting in the generation of IFN-γ-modified hAECs (IFN-γ-hAECs). Subsequently, these IFN-γ-hAECs were microencapsulated with APA to produce APA-IFN-γ-hAECs. In vitro, the release of IFN-γ, as well as the cellular and metabolic activities, growth, proliferation, migration, apoptosis, and trans-differentiation were assessed using HS-derived fibroblasts. In vivo, the weight loss of HS xenografts, collagen fiber arrangement, tissue oxidative stress, and inflammatory response were evaluated using a nude mouse model that had been transplanted with human HS tissues. In vitro, APA-IFN-γ-hAECs exhibited significantly sustained and enhanced IFN-γ release, increased cellular vitality, and inhibited fibroblast growth, proliferation, migration, and trans-differentiation into myofibroblasts. APA-IFN-γ-hAECs also remarkably downregulated extracellular matrix (ECM) components and promoted apoptosis. In vivo, they significantly accelerated the weight reduction of HS xenografts, improved collagen fiber arrangement, and mitigated oxidative stress and inflammation. This study suggests that APA-microencapsulated IFN-γ-hAECs may have potential in alleviating HS fibrosis, offering a new direction for exploring effective clinical HS management strategies.

Alternaria alternata JTF001 Metabolites Recruit Beneficial Microorganisms to Reduce the Parasitism of Orobanche aegyptiaca in Tomato

Orobanche aegyptiaca is a holoparasitic weed that extracts water, nutrients, and growth regulators from host plants, leading to significant yield and quality losses. Biocontrol microbial metabolites have been shown to enhance plant resistance against parasitic plants, yet the underlying microbial mechanisms remain poorly understood. In this study, we investigated the role of Alternaria alternata JTF001 (J1) microbial metabolites in recruiting beneficial microbes to the tomato rhizosphere and promoting the establishment of a disease-suppressive microbiome. Pot experiments revealed that J1 metabolite application significantly reduced O. aegyptiaca parasitism. High-throughput sequencing of full-length 16S rRNA genes and ITS regions, along with in vitro culture assays, demonstrated an increase in the abundance of plant-beneficial bacteria, particularly Pseudomonas spp. The three candidate beneficial strains (zOTU_388, zOTU_533, and zOTU_2335) showed an increase of 5.7-fold, 5.4-fold, and 4.7-fold, respectively. These results indicate that J1 metabolites induce the recruitment of a disease-suppressive microbiome in tomato seedlings, effectively inhibiting O. aegyptiaca parasitism. Our findings suggest that microbial metabolites represent a promising strategy for managing parasitic plant infestations through microbial community modulation, offering significant implications for sustainable agricultural practices.

DOP022 Shared and Distinct Features of the Gut Microbiome in Immune-mediated Inflammatory Diseases: Initial Analysis from the INTEGRATE Cohort Study

Abstract Background Inflammatory bowel disease (IBD) and ankylosing spondylitis (AS) share overlapping pathophysiological mechanisms as immune-mediated inflammatory diseases. While gut microbial dysbiosis has been implicated in both conditions, the shared and distinct features of the oral-gut microbiome axis remain poorly understood. This study aims to characterize the oral and gut microbial signatures and their relationship with environmental factors in IBD and AS to identify common pathogenic pathways and disease-specific patterns. Methods The INTEGRATE study is a nationwide prospective observational study in Korea targeting recruitment of 1,100 IBD patients, 700 AS patients, and 2,244 healthy controls over five years (Table 1). Systematic collection includes clinical data and biological samples (saliva, stool, blood, intestinal tissues) for multi-omics analyses. Microbial characterization employs full-length 16S rRNA gene sequencing and shotgun metagenomics for saliva and stool samples, complemented by metabolomic analyses of saliva, stool, and plasma. This initial analysis includes gut microbiome data from 254 IBD patients, 156 AS patients, and 194 healthy controls. Results Alpha diversity analysis revealed a gradient pattern, with IBD patients showing lowest gut microbiome diversity, AS patients intermediate, and healthy controls highest (p<1.0e-12 for all indices, Fig. 1). In IBD patients, alpha diversity significantly correlated with disease activity indices (Harvey-Bradshaw Index score in Crohn's disease, total Mayo score in ulcerative colitis) and inflammatory markers (CRP, r=-0.13, p=0.042). Blood urea nitrogen levels showed positive correlation with alpha diversity in both IBD (r=0.21, p=0.00062) and AS cohorts (r=0.20, p=0.015). Beta diversity analysis demonstrated distinct clustering patterns (UniFrac distance), with AS patients positioned intermediately between IBD patients and healthy controls (Healthy vs IBD: R²=0.0502, Healthy vs AS: R²=0.0148, IBD vs AS: R²=0.0198; all p=0.001). Species-level analysis through full-length 16S rRNA sequencing identified specific taxa (Clostridium bolteae, Blautia hansenii) significantly enriched in both IBD and AS patients compared to controls. Conclusion Our initial analysis demonstrates distinct gut microbiome signatures across IMIDs, with a clear diversity gradient and shared taxonomic features between IBD and AS. These findings, coupled with specific clinical correlations, provide a foundation for understanding common pathogenic pathways. Ongoing multi-omics analyses and quantitative microbiome profiling will further elucidate the role of the oral-gut microbiome axis in these IMIDs, potentially identifying novel microbial biomarkers for precision medicine.

Characterization of the second type of tubuliform spidroin (TuSp1 variant 2) elucidates the essential role of cysteine within the repetitive domain in liquid-liquid phase separation-mediated silk formation and the mechanical properties of silk fibers.

Orb-weaver spiders utilize morphologically differentiated abdominal glands to produce up to seven types of silks throughout their life cycles. Tubuliform silk is unique as it serves to protect developing embryos and hatchlings. However, our current understanding of the relationship between structure and function of tubuliform silk protein remains limited. Here, we present the full-length gene sequence of the second type of tubuliform spidroin (TuSp1 variant 2) from the orb-weaver spider Leucauge blanda. The L. blanda TuSp1 variant 2 (TuSp1-v2) contains 18 tandemly arrayed repeats, with each repeat having a cysteine residue. We demonstrate that the cysteine in L. blanda TuSp1-v2 repeats can form intermolecular disulfide bond and promote the liquid-liquid phase separation (LLPS) for silk formation. Moreover, the presence of cysteine partially enhances the thermostability of soluble spidroins and the mechanical properties of fibers, as demonstrated by comparative analyses of miniature TuSp1-v2 and its mutants. The integration of mechanical and structural data indicates that the recombinant TuSp1-v2 fiber exhibits high UV-A stability in both its mechanical and structural properties. This study provides new insights into the functions of cysteine in repetitive region and implies promising potentials for development new spidroin-based biomaterials.

Mining and Expression Pattern Analysis of Genes Related to the Regulation of Flowering in Korean Pine (Pinus koraiensis)

Korean pine (Pinus koraiensis Siebold & Zucc.) is an important timber and economic tree species in northeast China. Its seeds serve as both a primary means of propagation and a significant economic product. In this study, we identified 12 full-length MADS-box genes based on the Korean pine flower-induced transcriptome data available in our laboratory. These genes were identified through multiple sequence alignment and screening for conserved structural domains. We analyzed the genetic relationships of these genes and predicted their physicochemical properties. Additionally, we examined the expression patterns of three SHORT VEGETATIVE PHASE (SVP) genes across different tissues and developmental stages of Korean pine. The results indicate that the amino acid composition, molecular weight, isoelectric point, and other physicochemical properties of the MADS-box gene family in Pinus koraiensis are generally similar, though some individual variations are observed. A total of 12 MADS-box family genes were identified from the Korean pine transcriptome, distributed across five subfamilies. Conserved motif analysis revealed that these genes share similar conserved sequences. Structural and physicochemical analyses showed that genes with similar sequences exhibited comparable characteristics. Expression levels of the SVP genes varied significantly across different developmental stages and tissues, with the expression of the three SVP genes in leaves being markedly higher than in buds (approximately 200-fold). The expression levels of these genes in leaves were not only higher than in buds but also exceeded those in other tissues. Based on these findings, we conclude that these three SVP genes primarily play a suppressive role in the process of flower bud formation, helping Korean pine maintain a juvenile state under certain conditions, and are also involved in the growth and development of its leaves. This research provides a basis for future studies on the flowering induction mechanism in Korean pine.

Titin fragment is a sensitive biomarker in Duchenne muscular dystrophy model mice carrying full-length human dystrophin gene on human artificial chromosome

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations of the dystrophin gene, which spans 2.4 Mb on the X chromosome. Creatine kinase (CK) activity in blood and titin fragment levels in urine have been identified as biomarkers in DMD to monitor disease progression and evaluate therapeutic intervention. However, the difference in the sensitivity of these biomarkers in DMD remains unclear. Previously, we generated transchromosomic mice carrying the full-length human dystrophin gene on a human artificial chromosome (DYS-HAC1) vector. The human dystrophin derived from DYS-HAC1 improved pathological phenotypes observed in DMD-null mice, which lack the entire 2.4 Mb of the dystrophin gene. In this study, we compared the values of plasma CK activity and urine/plasma titin fragment levels in wild-type (WT), DYS-HAC1, DMD-null, and DYS-HAC1; DMD-null mice. Plasma CK activity and urine/plasma titin fragment levels in DMD-null mice were significantly higher than those in WT mice. Although plasma CK activity showed no significant difference between WT and DYS-HAC1; DMD-null mice, urine/plasma titin fragment levels in DYS-HAC1; DMD-null mice were higher than those in WT mice. Human dystrophin in DYS-HAC1; DMD-null mice drastically improved muscular dystrophy phenotypes seen in DMD-null mice; however, the proportion of myofibers with central nuclei in DYS-HAC1; DMD-null mice had a tendency to be slightly higher than that in WT mice. These results suggest that urine/plasma titin fragment levels could be a more sensitive biomarker than plasma CK activity.

Brassica rapa receptor-like cytoplasmic kinase BrRLCK1 negatively regulates freezing tolerance in transgenic Arabidopsis via the CBF pathway.

Some winter rapeseed (Brassica rapa) varieties can endure extremely low temperatures (-20°C to -32°C). However, because of a lack of mutant resources, the molecular mechanisms underlying cold tolerance in B. rapa remain unclear. In this study, we identified a low-temperature-sensitive mutant receptor-like cytoplasmic kinase (RLCK), BrRLCK1, using the B. rapa--Arabidopsis (Arabidopsis thaliana) full-length cDNA-overexpressing gene hunting system mutant library. BrRLCK1, localized to the plasma membrane and retained its localization under low temperatures. Phylogenetic analysis showed that BrRLCK1 is highly conserved across six widely cultivated Brassica species, but exhibits complexity due to genome hybridization and polyploidization. Notably, β-glucuronidase activity and qRT-PCR analysis showed that B. rapa BrRLCK1 and its homologous gene BrRLCK2 were mainly expressed in the main root, shoot, and leaves, with their expression being activated by low temperatures. Transgenic Arabodipsis expressing BrRLCK1 and BrRLCK2 reduced freezing tolerance and promoted root elongation. These combined results indicated that low temperatures can activate the expression of BrRLCK1 and BrRLCK2, negatively regulating freezing tolerance via the C-repeat-binding factor (CBF) pathway.

Evaluation of 16S rRNA genes sequences and genome-based analysis for identification of non-pathogenic Yersinia.

16S rRNA genes sequencing has been used for routine species identification and phylogenetic studies of bacteria. However, the high sequence similarity between some species and heterogeneity within copies at the intragenomic level could be a limiting factor of discriminatory ability. In this study, we aimed to compare 16S rRNA genes sequences and genome-based analysis (core SNPs and ANI) for identification of non-pathogenic Yersinia. We used complete and draft genomes of 373 Yersinia strains from the NCBI Genome database. The taxonomic affiliations of 34 genomes based on core SNPs and the ANI results did not match those specified in the GenBank database (NCBI). The intragenic homology of the 16S rRNA gene copies exceeded 99.5% in complete genomes, but above 50% of genomes have four or more variants of the 16S rRNA gene. Among 327 draft genomes of non-pathogenic Yersinia, 11% did not have a full-length 16S rRNA gene. Most of draft genomes has one copy of gene and it is not possible to define the intragenomic heterogenicity. The average homology of 16S rRNA gene was 98.76%, and the maximum variability was 2.85%. The low degree of genetic heterogenicity of the gene (0.36%) was determined in group Y. pekkanenii/Y. proxima/Y. aldovae/Y. intermedia/Y. kristensenii/Y. rochesterensis. The identical gene sequences were found in the genomes of the Y. intermedia and Y. rochesterensis strains identified using ANI and core SNPs analyses. The phylogenetic tree based on 16S rRNA genes differed from the tree based on core SNPs of the genomes and did not represent phylogenetic relationship between the Yersinia species. These findings will help to fill the data gaps in genome characteristics of deficiently studied non-pathogenic Yersinia.

Clair3-RNA: A deep learning-based small variant caller for long-read RNA sequencing data.

Variant calling using long-read RNA sequencing (lrRNA-seq) can be applied to diverse tasks, such as capturing full-length isoforms and gene expression profiling. It poses challenges, however, due to higher error rates than DNA data, the complexities of transcript diversity, RNA editing events, etc. In this paper, we propose Clair3-RNA, the first deep learning-based variant caller tailored for lrRNA-seq data. Clair3-RNA leverages the strengths of the Clair series' pipelines and incorporates several techniques optimized for lrRNA-seq data, such as uneven coverage normalization, refinement of training materials, editing site discovery, and the incorporation of phasing haplotype to enhance variant-calling performance. Clair3-RNA is available for various platforms, including PacBio and ONT complementary DNA sequencing (cDNA), and ONT direct RNA sequencing (dRNA). Our results demonstrated that Clair3-RNA achieved a ~91% SNP F1-score on the ONT platform using the latest ONT SQK-RNA004 kit (dRNA004) and a ~92% SNP F1-score in PacBio Iso-Seq and MAS-Seq for variants supported by at least four reads. The performance reached a ~95% and ~96% F1-score for ONT and PacBio, respectively, with at least ten supporting reads and disregarding the zygosity. With read phased, the performance reached ~97% for ONT and ~98% for PacBio. Extensive evaluation of various GIAB samples demonstrated that Clair3-RNA consistently outperformed existing callers and is capable of distinguishing RNA high-quality editing sites from variants accurately. Clair3-RNA is open-source and available at (https://github.com/HKU-BAL/Clair3-RNA).

Development of a Recombinant Infectious Bronchitis Virus Vaccine Expressing Infectious Laryngotracheitis Virus Multiple Epitopes

Infectious laryngotracheitis (ILT) is a highly contagious disease, usually controlled by vaccination with live attenuated vaccines. However, the latent infection and adverse reactions caused by the live attenuated vaccines against infectious laryngotracheitis virus (ILTV) have limited its use in poultry. Infectious bronchitis virus (IBV) is considered a potential vector for vaccine development, but the issue of poor stability in recombinant IBV expressing foreign genes has not yet been resolved. In this study, we designed a multi-epitope cassette (gD-T/B) containing multiple T and B cell epitopes of ILTV gD protein. The genetic stability of the full-length gD gene and the gD-T/B multi-epitope cassette replacing non-essential genes in IBV was systematically analyzed. We found that, at the same insertion site, the stability of inserting gD-T/B multi-epitope cassette was consistently higher compared to the full-length gD gene. This difference may be related to the presence of more signals affecting virus replication or transcription in larger heterologous genes. In addition, the stability of recombinant IBV varied depending on the genome region being replaced. When the gene 5 was replaced, rH120-Δ5ab-gD-T/B was maintained up to at least passage 20 (P20). Compared with the parental virus H120 strain, rH120-Δ5ab-gD-T/B showed similar growth kinetics. Clinical observations and scoring of clinical signs in the vaccination-challenge experiment showed that rH120-Δ5ab-gD-T/B provided 90% protection against virulent ILTV, effectively alleviating clinical signs caused by infection with a virulent strain of ILTV. Furthermore, rH120-Δ5ab-gD-T/B significantly reduced the replication and shedding of ILTV in the trachea. Overall, this study suggests that rH120-Δ5ab-gD-T/B is a promising candidate vaccine against ILTV.

Exploring the genomic landscape of Chlamydiifrater species: novel features include multiple truncated major outer membrane proteins, unique genes and chlamydial plasticity zone orthologs

Recently discovered obligate intracellular bacteria belonging to the genus Chlamydiifrater with the species of Chlamydiifrater phoenicopteri and Chlamydiifrater volucris were studied to explore the composition of their genomes and their relatedness to Chlamydia, the other genus of the family Chlamydiaceae. We investigated 4 isolates of Cf. volucris, 2 of them newly sequenced, and one of Cf. phoenicopteri alongside 12 representatives of the Chlamydia species. Our study uncovers previously unrecognized genomic structures within Chlamydiifrater using a hybrid sequencing approach and advanced annotation pipelines, providing insights into species-specific adaptations and evolutionary dynamics. The integration of long-read sequencing data, comprehensive re-annotation strategies and pan-genomics enabled the localization of the unique plasticity zone and the identification of novel gene clusters in Chlamydiifrater strains, which improves our understanding of chlamydial genome architecture and plasticity in the family Chlamydiaceae. Our analysis revealed that 761 CDS (~80%) are shared among members of both genera. We further identified 158 unique genes of Chlamydiifrater species, but their annotation remains challenging because of the absence of functionally annotated orthologs in public databases. A full-length ompA gene encoding the major outer membrane porin was seen in all Chlamydiifrater strains. We also describe the localization and structure of multiple truncated CDS of ompA family members, representing one of this study’s most interesting findings. While genome analysis of Chlamydiifrater spp. confirmed numerous common features shared with representatives of the genus Chlamydia, many unique genomic elements were identified that underpin the distinct phenotype and separate genetic position of these new microorganisms.

Targeted correction of megabase-scale CNTN6 duplication in induced pluripotent stem cells and impacts on gene expression.

Copy number variations of the human CNTN6 gene, resulting from megabase-scale microdeletions or microduplications in the 3p26.3 region, are frequently implicated in neurodevelopmental disorders such as intellectual disability and developmental delay. However, duplication of the full-length human CNTN6 gene presents with variable penetrance, resulting in phenotypes that range from neurodevelopmental disorders to no visible pathologies, even within the same family. Previously, we obtained a set of induced pluripotent stem cell lines derived from a patient with a CNTN6 gene duplication and from two healthy donors. Our findings demonstrated that CNTN6 expression in neurons carrying the duplication was significantly reduced. Additionally, the expression from the CNTN6 duplicated allele was markedly lower compared to the wild-type allele. Here, we first introduce a system for correcting megabase-scale duplications in induced pluripotent stem cells and secondly analyze the impact of this correction on CNTN6 gene expression. We showed that the deletion of one copy of the CNTN6 duplication did not affect the expression levels of the remaining allele in the neuronal cells.

Protocol to Retrieve Unknown Flanking DNA Using Fork PCR for Genome Walking.

PCR-based genome walking is one of the prevalent techniques implemented to acquire unknown flanking genomic DNAs. The worth of genome walking includes but is not limited to cloning full-length genes, mining new genes, and discovering regulatory regions of genes. Therefore, this technique has advanced molecular biology and related fields. However, the PCR amplification specificity of this technique needs to be further improved. Here, a practical protocol based on fork PCR is proposed for genome walking. This PCR uses a fork primer set of three arbitrary primers to execute walking amplification task, where the primary fork primer mediates walking by partially annealing to an unknown flank, and the fork-like structure formed between the three primers participates in inhibiting non-target amplification. In primary fork PCR, the low-annealing temperature (25 °C) cycle allows the primary fork primer to anneal to many sites of the genome, synthesizing a cluster of single-stranded DNAs; the subsequent 65 °C cycle processes the target single-strand into double-strand via the site-specific primer; then, the remaining 65 °C cycles selectively enrich this target DNA. However, any non-target single-stranded DNA formed in the 25 °C cycle cannot be further processed in the following 65 °C cycles because it lacks an exact binding site for any primer. Secondary, or even tertiary nested fork PCR further selectively enriches the target DNA. The practicability of fork PCR was validated by walking three genes in Levilactobacillus brevis CD0817 and one gene in Oryza sativa. The results indicated that the proposed protocol can serve as a supplement to the existing genome walking protocols. Key features • This protocol builds upon the method developed by Pan et al. [1], which is applicable to genome-walking for any species. • The developed protocol is a random priming PCR-based genome-walking scheme. • Two rounds of nested fork PCR amplifications suffice to release a positive walking result.

Maternal Allergic Disease Phenotype and Infant Birth Season Influence the Human Milk Microbiome.

Early infancy is a critical period for immune development. In addition to being the primary food source during early infancy, human milk also provides multiple bioactive components that shape the infant gut microbiome and immune system and provides a constant source of exposure to maternal microbiota. Given the potential interplay between allergic diseases and the human microbiome, this study aimed to characterise the milk microbiome of allergic mothers. Full-length 16S rRNA gene sequencing was performed on milk samples collected at 3 and 6 months postpartum from 196 women with allergic disease. Multivariate linear mixed models were constructed to identify the maternal, infant, and environmental determinants of the milk microbiome. Human milk microbiome composition and beta diversity varied over time (PERMANOVA R2 = 0.011, p = 0.011). The season of infant birth emerged as the strongest determinant of the microbiome community structure (PERMANOVA R2 = 0.014, p = 0.011) with impacts on five of the most abundant taxa. The milk microbiome also varied according to the type of maternal allergic disease (allergic rhinitis, asthma, atopic dermatitis, and food allergy). Additionally, infant formula exposure reduced the relative abundance of several typical oral taxa in milk. In conclusion, the milk microbiome of allergic mothers was strongly shaped by the season of infant birth, maternal allergic disease phenotype, and infant feeding mode. Maternal allergic disease history and infant season of birth should therefore be considered in future studies of infant and maternal microbiota. Trial Registration: ClinicalTrials.gov identifier: ACTRN12606000281594.

Comparative analysis of the human microbiome from four different regions of China and machine learning-based geographical inference.

The human microbiome, the community of microorganisms that reside on and inside the human body, is critically important for health and disease. However, it is influenced by various factors and may vary among individuals residing in distinct geographic regions. In this study, 220 samples, consisting of sterile swabs from palmar skin and oral and nasal cavities were collected from Chinese Han individuals living in Shanghai, Chifeng, Kunming, and Urumqi, representing the geographic regions of east, northeast, southwest, and northwest China. The full-length 16S rRNA gene of the microbiota in each sample was sequenced using the PacBio single-molecule real-time sequencing platform, followed by clustering the sequences into operational taxonomic units (OTUs). The analysis revealed significant differences in microbial communities among the four regions. Cutibacterium was the most abundant bacterium in palmar samples from Shanghai and Kunming, Psychrobacter in Chifeng samples, and Psychrobacillus in Urumqi samples. Additionally, Streptococcus and Staphylococcus were the dominant bacteria in the oral and nasal cavities. Individuals from the four regions could be distinguished and predicted based on a model constructed using the random forest algorithm, with the predictive effect of palmar microbiota being better than that of oral and nasal cavities. The prediction accuracy using hypervariable regions (V3-V4 and V4-V5) was comparable with that of using the entire 16S rRNA. Overall, our study highlights the distinctiveness of the human microbiome in individuals living in these four regions. Furthermore, the microbiome can serve as a biomarker for geographic origin inference, which has immense application value in forensic science.IMPORTANCEMicrobial communities in human hosts play a significant role in health and disease, varying in species, quantity, and composition due to factors such as gender, ethnicity, health status, lifestyle, and living environment. The characteristics of microbial composition at various body sites of individuals from different regions remain largely unexplored. This study utilized single-molecule real-time sequencing technology to detect the entire 16S rRNA gene of bacteria residing in the palmar skin, oral, and nasal cavities of Han individuals from four regions in China. The composition and structure of the bacteria at these three body sites were well characterized and found to differ regionally. The results elucidate the differences in bacterial communities colonizing these body sites across different regions and reveal the influence of geographical factors on human bacteria. These findings not only contribute to a deeper understanding of the diversity and geographical distribution of human bacteria but also enrich the microbiome data of the Asian population for further studies.

Integrated Analysis of Oral Microbiome and Metabolome in T2DM Patients With Varying Glycemic Status.

This study aimed to analyze the subgingival and salivary microbiome and metabolome in diabetic periodontitis patients with varying glycemic levels. Forty-two diabetic periodontitis patients were sampled of saliva, gingival crevicular fluid (GCF), and blood, and categorized into three groups based on systemic glycemic status. The microbiome was assessed using full-length 16S rRNA gene sequencing. Gas chromatography-mass spectrometry was performed for metabolome analysis. The similarity in the structure and function of the flora in saliva and GCF was evident under good blood glucose control. Conversely, inadequate blood glucose control demonstrated a more distinct separation from saliva flora. Both salivary and GCF microorganisms exhibited greater periodontal pathogenicity, with salivary metabolites showing stronger associations with inflammation when Hemoglobin A1c (HbA1c) > 6.5%. Some periodontal pathogens, such as Veillonella atypica, showed significantly positive correlations with proinflammatory metabolites, including lactic acid and putrescine, etc. Salivary microbes demonstrated more sensitive responses than GCF to changes in blood glucose levels among type 2 diabetes mellitus (T2DM) patients. Under active blood glucose control, it indicates lower periodontal pathogenicity and inflammatory correlation in the oral microecology of T2DM patients. Saliva appears to offer superior diagnostic and monitoring value compared to GCF in the context of systemic disease surveillance.

Coat-protein Gene Based Identification and Characterization of Yellow Mosaic Virus Infecting Blackgram in Northern Karnataka, India

Aims: Coat-protein (CP) based identification and characterization of yellow mosaic virus infecting blackgram. Place and Duration of Study: The study was carried out during Kharif 2023 at College of Agriculture, Vijayapur. Methodology: The whole genomic DNA was isolated from the leaf tissues of both healthy blackgram plants and plants infected with the yellow mosaic virus by using a modified CTAB method. Specific primers for YMV were tried to amplify coat protein region of blackgram YMV. Results: Sequence analysis revealed that the CP gene of the begomovirus under study shared 99.8 per cent similarity with MYMV Shivamogga isolate (OM106035.1) at the nucleotide level. When comparing the deduced amino acid sequences of individual proteins from YMV infecting blackgram at Vijayapur with those of other begomoviruses, the highest identity was found with the MYMV Shivamogga isolate (OM106035.1), showing 84.59 per cent similarity. A phylogenetic tree constructed based on the full-length coat protein gene sequence of MYMV, along with 37 other Geminivirus sequences, formed two major clusters in which MYMV-Vijayapur isolate appeared in Cluster I. Conclusion: The results of the current study, based on similarities in coat protein sequence at both the nucleotide and amino acid levels, as well as phylogenetic analysis, confirmed the prevalence of MYMV strain rather than MYMIV, HgYMV and DoYMV in blackgram in northern parts of Karnataka.