Search Results (109)

This study presents an in silico proof of concept exploring whether deep learning models can perform conditional mitochondrial DNA (mtDNA) sequence prediction across species boundaries. A CNN–BiLSTM model was trained under a leave-one-species-out (LOSO) scheme on complete mitochondrial genomes from 21 vertebrate species, primarily archosaurs. Model behavior was evaluated through multiple complementary tests. Under context-conditioned settings, the model performed next-nucleotide prediction using overlapping 200 bp windows to assemble contiguous 2000 bp fragments for held-out species; the resulting high token-level accuracy (>99%) under teacher forcing is reported as a diagnostic of conditional modeling capacity. To assess leakage-free performance, a two-flank masked-span imputation task was conducted as the primary evaluation, requiring free-running reconstruction of 500 bp interior spans using only distal flanking context; in this setting, the model consistently outperformed nearest-neighbor and demonstrated competitive performance relative to flank-copy baselines. Additional robustness analyses examined sensitivity to window placement, genomic region (coding versus D-loop), and random initialization. Biological plausibility was further assessed by comparing predicted fragments to reconstructed ancestral sequences and against composition-matched null models, where observed identities significantly exceeded null expectations. Using the National Center for Biotechnology Information (NCBI) BLAST web interface, BLASTn species identification was performed solely as a biological plausibility check, recovering the correct species as the top hit in all cases. Although limited by dataset size and the absence of ancient DNA damage modeling, these results demonstrate the feasibility of conditional mtDNA sequence prediction as an initial step toward more advanced generative and evolutionary modeling frameworks. Full article

Importance: The coronavirus disease 2019 (COVID-19) was the third leading cause of mortality in the United States for three years in a row. The genetic contributions to disease severity remain unclear and many previously identified single nucleotide polymorphisms (SNPs) have not been replicated nor linked with functional significance. Objective: To identify SNPs associated with mortality among hospitalized COVID-19 patients supplemented by expression quantitative trait loci (eQTL) evidence to infer plausible functional mechanisms related to COVID-19 severity. Design: A quality-controlled genome-wide association study (GWAS) supported by robust gene-level omnibus kernel association tests (SKAT-O), functional prediction, and eQTL analyses of the top GWAS signal. Setting: Massachusetts General Hospital (MGH). Participants: 370 adult ICU patients with SARS-CoV-2 infection and acute hypoxemic respiratory failure and floor patients with mild hypoxemia managed with supplemental oxygen consecutively admitted to MGH between March and June 2020 (Surge 1), and January and March 2021 (Surge 2) with baseline clinical characteristics and demographics collected. Exposures: Low-pass genotyped SNPs from whole blood and aggregated SNP-sets of potential disease susceptibility loci with $\pm$500 kb flanking regions. Main Outcomes & Measures: Genome-wide individual SNP associations and SNP-set associations with mortality outcomes from 370 severe COVID-19 cases. Results: After LD pruning (<0.8) and false discovery rate adjustment (<0.05), we identified rs7420371 G>A of the receptor transporter protein 5 (RTP5) gene as the top independent signal significantly associated with 30- and 60-day mortality among severe COVID-19 patients (OR, 2.32; 95% CI, 1.59–3.39; p = 4.92 × 10⁻⁹ and OR, 2.06; 95% CI, 1.43–2.97; p = 5.43 × 10⁻⁸, respectively). SKAT-O analyses on the RTP5 SNP-set showed associations with both mortality outcomes (p = 5.90 × 10⁻⁵ and 6.17 × 10⁻⁵, respectively). eQTL analysis showed rs7420371 A allele significantly upregulated the mRNA expression of RTP5 in 266 cerebellum tissues, in 277 cerebellar hemisphere tissues, and in 270 cerebral cortex samples. Conclusions & Relevance: We discovered a novel, independent, and potentially functional SNP RTP5 rs7420371 G>A to be significantly associated with COVID-19 mortality. The A allele is significantly associated with elevated mRNA expression of RTP5 in the brain, an important protein coding gene that modulates olfactory binding and taste perceptions in response to SARS-CoV-2 infection. Full article

Antisense inhibition of gene expression is usually achieved using nuclease-resistant oligonucleotide analogs that promote mRNA degradation through RNase H or RNase P, or by steric hindrance of translation. Bridge nucleic acids (BNAs) are nucleotide analogs available in a few chemical variants. We evaluated gapmers composed of an oligodeoxynucleotide flanked by BNA residues in a BNA₅-DNA₈-BNA₄ configuration, using the available variants: the original locked nucleic acid (LNA; 2′-O,4′-methylene bridge), cET (2′-O,4′-ethyl bridge), cMOE (2′-O,4′-methoxyethyl bridge), and BNANC (2′-O,4′-aminomethylene bridge). These gapmers were tested in vitro for their ability to induce cleavage of the model aac(6′)-Ib mRNA. All gapmers complementary to a previously identified region suitable for interaction with antisense oligomers induced RNase H-mediated degradation. Instead, only the LNA-containing gapmer also elicited RNase P-dependent cleavage, demonstrating dual RNA- and DNA-mimicking capability. In vitro coupled transcription–translation assays using cell lysates or reconstituted systems confirmed inhibition of expression and ruled out steric hindrance as the mechanism. In contrast, gapmers targeting the ribosome-binding site strongly inhibited expression by steric hindrance. These findings demonstrate that LNA-containing gapmers can exert their effects through multiple mechanisms, depending on the targeted mRNA region, thereby supporting their potential for synergistic inhibition of gene expression. Full article

Gene expression plays a fundamental role in defining the characteristics of living organisms. To deepen our understanding of tissue-specific gene expression, we analyzed transcript variant enrichment across different tissues in human and Drosophila melanogaster. Datasets are widely accessible for both of these organisms. Given the substantial volume of available information, we have focused our interest on three fundamentally distinct tissues: the brain, where both neuronal and glial cells exhibit a relatively high cellular surface area, thus requiring a large amount of lipids; the adipose tissue, which is well-known for lipid storage; and the testis, which contains a massive number of developing spermatids with high membrane requirement. These three organs have fundamental differences in their structure and function yet share some common features; they all have lipid-rich cells and have special metabolic pathways. Most studies focus on gene expression, and transcript level analyses are less common; therefore, we aimed to characterize the transcript profiles of these tissues and examine evolutionarily conserved pathways between humans and Drosophila. Additionally, we analyzed the flanking sequences of transcriptional start sites of tissue-enriched transcripts. Our findings suggest that Drosophila tissues exhibit more distinct regulation of gene expression in individual tissues (weaker correlation in expression and variable nucleotide content in core promoter), whereas human gene expression is more generalized, likely relying more heavily on distal regulatory elements for tissue-specific expression. Through network analysis, summarizing tissue specificity, physical interactions, and orthologue data, we identified shared central pathways among these tissues. A relatively large network was observable in the testis, where the ubiquitin proteasome system, various kinases and transcription factors showed central position in both organisms. Additionally, we highlighted the evolutionary potential of highly enriched testis-specific transcripts. This work provides valuable insights into the mechanisms underlying tissue-specific gene expression and evolutionary conservation. Full article

Different ginseng species, such as Panax ginseng and Panax quinquefolius, have various medicinal and economic values. Industrially and commercially, it is important to differentiate them. We have adopted the DNA hybridization method based on a single-nucleotide polymorphism (SNP) found in the ginseng DNA. The ginseng DNA samples were placed on a nitrocellulose membrane, and hybridization of the target sample with the probes immobilized on the membrane occurred, resulting in red spots for unaided eye visualization. We managed to demonstrate a spot test and then a lateral flow assay. Genomic DNAs were extracted from ginseng root samples and DNA amplification was used to generate the PCR products that flank the SNP site. We conclude that ginseng DNA can be differentiated based on a DNA lateral flow assay, detecting ~3 ng (in 1 μL) of PCR amplicons. Full article

Background: Next-generation sequencing (NGS) analysis of viral samples generates results dispersed across multiple files—genome assembly, variant calling, and functional annotations—making integrated interpretation challenging. Variants often yield numerous low-frequency or non-significant variants, yet only a small fraction are biologically relevant. Virologists must manually sift through extensive data to identify meaningful mutations, a time-consuming and error-prone process. To address these practical challenges, we developed vvv2_display, a dedicated summarization and visualization tool, integrated within comprehensive Galaxy workflows. Results: vvv2_display streamlines variant interpretation by consolidating key results into two concise and interoperable outputs. The first output is a PNG image showing alignment coverage depth and genomic annotations, with significant variants displayed along the genome as symbols whose height reflects frequency and shape indicates the affected protein. At a glance, this enables virologists to identify all deviations from a reference viral genome. Each significant variant is assigned a unique identifier that directly links to the second output: a tab-separated (TSV) text file listing only high-confidence variants, with frequencies, flanking nucleotides, and impacted genes and proteins. This cross-referenced design supports rapid, accurate, and intuitive data exploration. Availability: vvv2_display is open source, available on Github and installable via Mamba. Full article

Spinocerebellar ataxia 3 (SCA3) is a neurodegenerative condition caused by an expansion of a polyglutamine tract within the ATXN3 gene. Normal alleles range from 12 to 44 repeats, while pathogenic alleles have 52 repeats or more. The canonical ATXN3 repeat tract sequence includes three interruptions at positions 3 (CAA), 4 (AAG), and 6 (CAA). The intragenic rs7158733 single-nucleotide polymorphism (SNP) flanks the ATXN3 repeat region and substitutes a TAC¹¹¹⁸ tyrosine codon with a TAA¹¹¹⁸ stop codon, resulting in a shorter ataxin-3aS isoform. We examined the distribution of SCA3 allele repeat sizes in a UK-based cohort presenting with an ataxic phenotype. The 6596 alleles showed a clear gap between normal and expanded alleles, with no intermediate alleles containing 41 to 57 repeats. We used clone sequencing to characterize the structure of the ATXN3 repeat region in a sub-cohort of 44 SCA3 patients. We observed that the three canonical interruptions were typically preserved. There was no association of the interruptions with age at onset detected in this cohort, given the limited power of this sub-cohort. We genotyped the rs7158733 SNP in a sub-cohort of 79 SCA3 patients and found that 74.7% of expanded alleles carried the A¹¹¹⁸ variant, which was associated with earlier disease onset. This study highlights the importance of rs7158733 genotyping alongside ATXN3 repeat sizing for patient evaluation, as this SNP modifies the effect of repeat size on age at onset in SCA3 for pathogenic alleles up to 69 repeats. Full article

The skin color of radish taproots is an important commodity character that directly affects the choice behavior of consumers. Here, we identified a skin color gene carried by a red-skinned inbred line, SXAU-R2. Genetic population was constructed by the crossing of SXAU-R2 and a white-skinned inbred line, SXAU-W2, and the taproots of F₁ plants exhibited intermediate color. In the F₂ population, the separation ratio of taproot skin color indicated that the phenotype was controlled by one major locus, named RST1 (Red-Skinned Taproot 1). Combined with bulked segregant analysis and RNA sequencing (BSA-seq), 2640 single nucleotide polymorphisms (SNPs) were detected between the annotated genes of the red skin bulk and white skin bulk. Molecular markers were developed in the SNP-enriched 27~32 Mbp region of chromosome 7, and then RST1 was mapped in the genetic interval between flanking markers SSR-14 and SSR-22. Using F_2:3 lines derived from a key F₂ heterozygote, RST1 was narrowed down into a 530 Kbp interval. There were 46 expressed annotated genes in the fine-mapping region, and a gene encoding MYB was selected as the candidate of RST1. Finally, based on Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and RT-qPCR, we identified the potential interacting genes RsbHLH and RsWD, as well as the latent target genes RsDFR and RsANS of RST1 in the anthocyanin synthesis pathway. These results provide an understanding of the genetic mechanisms regulating anthocyanin synthesis and offer an efficient molecular marker for the radish breeding of skin color. Full article

Enhancing the specificity and applicability of PCR-based genome-walking methods is highly desirable. A new and universal genome-walking tool, called center degenerated walking-primer PCR (CDWP-PCR), is presented in this study. CDWP-PCR involves adopting a center degenerated walking primer (cdWP) in the secondary/tertiary round of amplification. This cdWP is generated by degenerating the seven central nucleotides of the normal walking primer (nWP) used in primary PCR to NNNNNNN (where N includes the bases A, T, C, and G). Clearly, a partially complementary structure is formed between the two primers. Accordingly, the primary CDWP-PCR non-target products defined by the nWP are diluted in secondary/tertiary CDWP-PCR, as these non-targets have difficulty in annealing with the cdWP; conversely, the primary target product can still be efficiently amplified. The working performance of the proposed CDWP-PCR is verified through cloning of the unknown flanks of three known genes. All the clear DNA bands in the tertiary CDWP-PCRs are confirmed to be correct, and the largest DNA band is 8.0 kb. Overall, CDWP-PCR can be considered as a reliable supplement to existing genome-walking methods. Full article

Background/Objectives: Some G-quadruplex (G4)-forming nucleic acid sequences bind a hemin cofactor to enhance its peroxidase-like activity. This has been implemented in a variety of bioanalytical assays benefiting from analyte-dependent peroxidation of a chromogenic organic substrate (e.g., ABTS) to produce a color change. Adenine and cytosine nucleotides in the vicinity of the G4 hemin-binding site promote the peroxidation reaction. In this work, the effect of G4 loop and flanking nucleotides on the colorimetric signal of split hybridization probes utilizing hemin-G4 signal reporters was tested. Methods: G4s varying by loop sequences and flanking nucleotides were tested with hemin for ABTS peroxidation (A₄₂₀), and the signal was compared with that produced by the most catalytically efficient complexes reported in the literature using one-way ANOVA and post hoc pairwise comparison with Tukey’s HSD test. The best G4s were used as signal transducers in the split peroxidase deoxyribozyme (sPDz) probes for sensing two model nucleic acid analytes, as well as in a cascade system, where the analyte-dependent assembly of an RNA-cleaving deoxyribozyme 10–23 results in G4 release. Results: Intramolecular G4s (G₃T)₃G₃TC or G₃T₃G₃ATTG₃T₃G₃ were found to be the most efficient hemin PDzs. When splitting intramolecular G4 for the purpose of sPDz probe design, the addition of a flanking d(TC) sequence at one of the G4 halves or d(ATT) in a loop connecting the second and third G-tracts helps boost analyte-dependent signal intensity. However, for the cascade system, the effect of d(TC) or d(ATT) in the released G4 was not fully consistent with the data reported for intramolecular G4-hemin complexes. Conclusions: Our findings offer guidance on the design of split hybridization probes utilizing the peroxidase-like activity of G4-hemin complexes as a signal transducer. Full article

CRISPR-Cas is an adaptive immune system found in bacteria and archaea that provides resistance against invading nucleic acids. Elements of this natural system have been harnessed to develop several genome editing tools, including CRISPR-Cas9. This technology relies on the ability of the nuclease Cas9 to cut DNA at specific locations directed by a guide RNA. In addition, the nuclease activity of Cas9 requires the presence of a short nucleotide motif (5′-NGG-3′ for Cas9 from Streptococcus pyogenes) called PAM, flanking the targeted region. As the reliance on this PAM is typically strict, diverse Cas9 variants recognising different PAM motifs have been studied to target a broader range of genomic sites. In this study, we assessed the potential of Cas9 from Streptococcus mutans strain P42S (SmutCas9) in gene editing. SmutCas9 recognises the rarely targeted 5′-NAA-3′ and 5′-NGAA-3′ PAMs. To test its efficacy, two genes of the virulent lactococcal phage p2 were edited, thereby demonstrating the potential of SmutCas9 for gene editing purposes, particularly in AT-rich genomes. Sequencing of total RNA also revealed the RNA components of this system, allowing further molecular characterisation of the type II-A CRISPR-Cas system of S. mutans. Full article

Background/Objectives: Cowpea is a major source of dietary protein and plays a key role in sustainable agriculture across sub-Saharan Africa (SSA), Asia, and Latin America. Research efforts have focused mainly on enhancing productivity through higher yield and resistance to biotic and abiotic stresses in cowpea. Understanding the genetic basis of yield and associated agronomic traits is crucial for improving crop productivity. This study aims to identify quantitative trait loci (QTL) associated with grain yield and related traits in cowpea under regular rainfed conditions. Methods: We developed a set of 316 F6:7 recombinant inbred lines (RILs) mapping populations derived from a cross between RP270 and CB27 using a single-seed descent breeding method. The RILs and their two parental lines were evaluated in the field for two years, 2022 and 2023, at the International Institute of Tropical Agriculture (IITA) in Ibadan, Nigeria. The cowpea mid-density genotyping panel consisting of 2602 quality DArTag single nucleotide polymorphisms (SNPs) was used to genotype the RIL population. Results: Seven major QTLs, each explaining ≥10% of phenotypic variance, were detected for 100-seed weight, number of days to flower, number of pods per plant, number of branches per plant, and number of peduncles per plant. Putative genes associated with yield and related traits were identified within significant flanking markers. Further efforts to validate these loci will help to better understand their roles in yield and associated traits in cowpea. Full article

RNA editing is a significant mechanism underlying genetic variation and protein molecule alteration; C-to-U RNA editing, specifically, is important in the regulation of mammalian genetic diversity. The ability to define and limit accesses of enzymatic machinery to avoid the modification of unintended targets is key to the success of RNA editing. Identification of the core component of the apoB RNA editing holoenzyme, APOBEC, and investigation into new candidate genes encoding other elements of the complex could reveal further details regarding APOBEC-mediated mRNA editing. Menkes disease is a recessive X-chromosome-linked hereditary syndrome in humans, caused by defective copper metabolism due to mutations in the ATP7A gene, which encodes a copper transport protein. Here, we generated plasmids encoding the MS2 system and the APOBEC1 deaminase domain and used a guide RNA with flanking MS2 sites to restore mutated Atp7a in fibroblasts from a macular mouse model of Menkes disease withs T>C mutation. Around 35% of the mutated C nucleotide (nt) was restored to U, demonstrating that our RNA editing system is reliable and has potential for therapeutic clinical application. RNA base editing via human RNA-guided cytidine deaminases is a potentially attractive approach for in vivo therapeutic application and provides opportunities for new developments in this field. Full article

Our previous study identified the apolipoprotein M (APOM) and cytochrome P450 family 7 subfamily A polypeptide 1 (CYP7A1) genes as candidates for milk traits in dairy cattle, which were significantly up-regulated in liver tissue of Holstein cows between the dry and lactation periods. The two genes play critical roles in the peroxisome proliferator-activated receptor (PPAR) pathway. In this study, we further confirmed whether the APOM and CYP7A1 genes had significant genetic impacts on milk production traits in a Chinese Holstein population. By dual-direction sequencing of the polymerase chain reaction (PCR) products of the complete coding sequences and 2000 bp of the 5′ and 3′ flanking regions on pooled DNA sample, seven and three single nucleotide polymorphisms (SNPs) were identified in APOM and CYP7A1, respectively. With SAS 9.2, phenotype-genotype association analysis revealed such SNPs were significantly associated with at least one of the milk production traits, including 305-day milk yield, milk fat yield, milk fat percentage, milk protein yield, and milk protein percentage in the first and second lactations (p = <0.01~0.04). With Haploview 4.2, we further found that six SNPs in APOM and thee SNPs in CYP7A1 formed one haplotype, respectively. The haplotypes were significantly associated with at least one of milk production traits as well (p = <0.01~0.02). Of note, we found the SNPs in the 5′ regulatory region, rs209293266 and rs110721287 in APOM and rs42765359 in CYP7A1, significantly impacted the gene transcriptional activity after mutation (p < 0.01) through changing the transcription factor binding sites by using luciferase assay experiments. Additionally, with RNAfold Web Server, rs110098953 and rs378530166 changed the mRNA secondary structures of APOM and CYP7A1 genes, respectively. In summary, our research is the first to demonstrate that APOM and CYP7A1 genes have significantly genetic effects on milk yield and composition traits, and the identified SNPs may serve as available genetic markers for genomic selection program in dairy cattle. Full article

In warm environments, thermoregulation in poultry is controlled by heat shock protein 70 (HSP70), whose expression is controlled by heat shock factor 3 (HSF3). Although the association between genetic polymorphisms in these genes and thermotolerance as well as reproductive traits has been extensively studied in mammals, the association has not yet been studied in poultry. This study aimed to explore the relationship between single-nucleotide polymorphisms (SNPs) in these genes and the egg production traits of Bangladeshi hilly chickens. Sequencing and allele-specific PCR (AS-PCR) were used to detect new SNPs and perform genotyping. We identified two novel SNPs (G-399A and A-68G) in the 5′-flanking regions of HSP70 that were significantly associated with egg numbers (ENs) at 161–190 days and increased egg weight (EW) at 40 weeks of age. Furthermore, three SNPs in HSP70 (A258G, C276G and C1431A) and one SNP in HSF3 (A-1388G) were associated with EN at different ages. The haplotype and combined genotypic effects of these two genes were found to be associated with age at sexual maturity (ASM), EN, EW, and body weight at ASM. The identified SNPs and their corresponding haplotypes may be useful in selective breeding to enhance the productivity of chickens in warm environments. Full article