Sequence Variants Research Articles

The rhizosphere bacteriome is modified by wheat genotype and growth stage under increased imidazolinone herbicide residues

Abstract When imidazolinone herbicides persist longer than intended and remain active in the soil, they can have unknown impacts on soil health. This study investigated the impact of simulated soil residues of an imidazolinone herbicide on shoot dry matter and bacterial communities in the bulk and rhizosphere soil in tolerant and susceptible wheat genotypes, at two different crop growth stages. Four levels of gradient increased herbicide residues were applied, and rhizosphere bacterial diversity and community composition were analysed using 16S rRNA gene amplicon sequencing. Our results highlight that the shift in wheat rhizosphere bacteriome is driven more by the crop growth stage and wheat genotype than the presence and level of imidazolinone residues. Results showed a linear trend of increasing alpha diversity with increasing herbicide residues during the early crop growth stage, and a decrease in alpha diversity with increasing herbicide residues during the late crop growth stage, only for the tolerant genotype. The order Betaproteobacteriales in the rhizosphere was increased by herbicide residues to a greater extent than the other taxonomic groups. During the early growth stage, there were more ASV (amplicon sequence variant) enriched by imidazolinone herbicide residues in the rhizosphere of the tolerant genotype compared with the susceptible genotype. Future research work should consider studies with soils that have different physicochemical properties, and focus on other soil microbes of known significance to nutrient cycling and crop growth.

Nucleotide sequence variants, gene expression and serum profile of immune and antioxidant markers associated with brucellosis resistance/susceptibility in Shami goat

Brucellosis is a highly contagious zoonotic bacterial disease. It has considerable negative consequences on the animal production industry worldwide. The objective of this study was to investigate the genetic and molecular variations in Shami goat susceptible to Brucella infection. Blood samples were collected from fifty mature Shami goats (30 Brucella-infected does and 20 non-infection). DNA was extracted and selected parts the immunity; solute carrier family 11 member 1 (SLC11A1), toll-like receptor 1 (TLR1), toll-like receptor 9 (TLR9), SP110 nuclear body protein (SP110), the adenosine A3 receptor (ADORA3), caspase activating recruitment domain 15 (CARD15) and interferon regulatory factor 3 (IRF3), antioxidant glutathione peroxidase 1 (GPX1), nitric oxide synthase (NOS), NAD(P)H dehydrogenase [quinone] 1 (NQO1) and transcription factor NF-E2-related factor 2 (Nrf2) and erythritol related transketolase (TKT), ribose 5-phosphate isomerase (RPIA) and Adenosine monophosphate deaminase (AMPD) genes were sequenced. Likewise, the levels of gene expressions were investigated. The results identified polymorphic variants between healthy and infected does. Levels of gene expression of SLC11A1, TLR1, TLR9, SP110, ADORA3, CARD15, IRF3, HMOX1, TKT, RPIA and AMPD were significantly (P < 0.05) up regulated in the infected compared to the non-infected ones. On the other hand, GPX1, NOS, NQO1 and Nrf2 genes were significantly (P < 0.05) downregulated in the infected compared to the non-infected does. The results of serum profile indicated that there is a significant (P < 0.05) increase in the activities of AST, ALT, GGT, LDH, ALP as well as serum level of globulin, triglycerides, cholesterol, MDA, NO, IL-1β, TNF-α, IgM, IgG, haptoglobin and amyloid A. On the other hand, there were significant reductions in the glucose, total protein albumin, urea, calcium, inorganic phosphorus, sodium, copper, zinc, iron, TAC, GSH, SOD, GPx, IL-10 and fibrinogen in the infected compared to the non-infected does. Our results provide valuable information about the serum profile variations and putative genetic markers for Brucella infection in goats. This could be utilized in controlling goat brucellosis through selective breeding of natural resistant animals.

Influences of plant maternal effects, chemotype, and environment on the leaf bacterial community.

Plant individuals within a species can differ markedly in their leaf chemical composition, forming so-called chemotypes. Little is known about whether such differences impact the microbial communities associated with leaves and how different environmental conditions may shape these relationships. We used Tanacetum vulgare as a model plant to study the impacts of maternal effects, leaf terpenoid chemotype, and the environment on the leaf bacterial community by growing plant clones in the field and a greenhouse. We hypothesized that all three factors affect the bacterial community of the leaves and that terpenoid and bacterial profiles as well as chemodiversity and microbial diversity are correlated. The results revealed that the leaf microbial community was significantly influenced by plant maternal effects and environmental conditions (field vs. greenhouse), but not by the leaf terpenoid profile. There was also no evidence for a correlation between terpenoid profiles and bacterial community composition and diversity. Overall, a higher number of unique amplicon sequence variants were found in the leaves of clones grown under field conditions than in those grown in the greenhouse. We also identified interactions between individual terpenoids and specific members of the leaf bacterial community. Our study suggests that terpenoid chemodiversity has, overall, little effect on the leaf bacterial community, but some terpenoids might affect specific beneficial species. While more studies are needed to investigate the relationship between plant chemodiversity and plant microbiomes, our results highlight the importance of integrating plant maternal effects, chemodiversity, and environment in understanding plant-microbiome interactions.

LevSeq: Rapid Generation of Sequence-Function Data for Directed Evolution and Machine Learning.

Sequence-function data provides valuable information about the protein functional landscape but is rarely obtained during directed evolution campaigns. Here, we present Long-read every variant Sequencing (LevSeq), a pipeline that combines a dual barcoding strategy with nanopore sequencing to rapidly generate sequence-function data for entire protein-coding genes. LevSeq integrates into existing protein engineering workflows and comes with open-source software for data analysis and visualization. The pipeline facilitates data-driven protein engineering by consolidating sequence-function data to inform directed evolution and provide the requisite data for machine learning-guided protein engineering (MLPE). LevSeq enables quality control of mutagenesis libraries prior to screening, which reduces time and resource costs. Simulation studies demonstrate LevSeq's ability to accurately detect variants under various experimental conditions. Finally, we show LevSeq's utility in engineering protoglobins for new-to-nature chemistry. Widespread adoption of LevSeq and sharing of the data will enhance our understanding of protein sequence-function landscapes and empower data-driven directed evolution.

Shewanella is a putative producer of polyunsaturated fatty acids in the gut soil of the composting earthworm Eisenia fetida.

Polyunsaturated fatty acids (PUFAs) play a crucial role in aiding bacteria to adapt to extreme and stressful environments. While there is a well-established understanding of their production, accrual, and transfer within marine ecosystems, knowledge about terrestrial environments remains limited. Investigation of the intestinal microbiome of earthworms has illuminated the presence of PUFAs presumably of microbial origin, which contrasts with the surrounding soil. To comprehensively study this phenomenon, a multi-faceted approach was employed, combining fatty acid analysis with amplicon sequencing of the PfaA-KS domain of the anaerobic fatty acid synthase gene (pfa), as well as the 16S rRNA and 18S rRNA genes. This methodology was applied to scrutinize the gut microbiome of Eisenia fetida, its compost-based dietary source, and the resultant castings. This study unveiled a distinct gut soil ecosystem from input compost and output castings in fatty acid profile as well as type and abundance of organisms. 16S sequencing provided insights into the microbial composition, showing increased relative abundance of certain Pseudomonadota, including Shewanellaceae, and Planctomycetota, including Gemmataceae within the gut microbiome compared to input bulk soil compost, while Actinomycetota and Bacillota were relatively enriched compared to the casted feces. Sequencing of the PfaA-KS domain revealed amplicon sequence variants (ASVs) belonging primarily to Shewanella. Intriguingly, the 20C PUFAs were identified only in gut soil samples, though PfaA-KS sequence abundance was highest in output castings, indicating a unique metabolism occurring only in the gut. Overall, the results indicate that Shewanella can explain PUFA enrichment in the gut environment because of the pfa gene presence detected via PfaA-KS sequence data.IMPORTANCEPrior research has demonstrated that earthworm microbiomes can potentially harbor polyunsaturated fatty acids (PUFAs) that are not found within their residing soil environment. Moreover, distinct indicator species have been pinpointed for various microbial genera in earthworm microbiomes. Nevertheless, none of these studies have integrated metataxonomic and fatty acid analyses to explore the origin of PUFA synthesis in any earthworm species, with the objective of identifying the specific organisms and locations responsible for this production. This study suggests that earthworms accumulate PUFAs produced from bacteria, especially Shewanella, activated through the gut ecosystem.

Case Report: Prenatal diagnosis of novel compound heterozygous variants in WDR35 gene causing short-rib thoracic dysplasia 7 with or without polydactyly

BackgroundWhole exome sequencing (WES) technology has been increasingly used for the etiological diagnosis of fetuses with ultrasound anomalies. In this article, we report a novel deletion compound combined with a causative variant in WDR35 gene leading to short-rib thoracic dysplasia 7 (SRTD7) with or without polydactyly using WES.MethodsThis study involved a Chinese fetus with clinical features of skeletal dysplasia on ultrasound imaging, in whom chromosome abnormalities and copy number variants (CNVs) were detected by chromosomal microarray analysis (CMA), and sequence variants were detected by WES. The obtained results were further verified by Sanger sequencing or real time quantitative PCR (qPCR).ResultsNo chromosomal abnormality or CNVs were identified in the fetus by CMA. However, WES result revealed a 14.38-kb large novel deletion compound covering exon 7 to exon 12 combined with a missense variant NM_001006657.2:c.932G&amp;gt;T(p.W311l) in WDR35. Both variants were thought of as pathogenic, which was further confirmed by Sanger sequencing and qPCR. In addition, two compound heterozygous variants NM_015102.5:c.[1196A&amp;gt;G(p.E399G)];[1972C&amp;gt;T(p.R658*)] in NPHP4 gene were also identified in the fetus, which may be partially responsible for fetal kidney hyperechogenicity and oligohydramnios.ConclusionThis is the first study reporting a novel deletion compound combined with a causative missense variant in WDR35 leading to SRTD7. This finding may broaden the spectrum of variants of WDR35 gene and provide a valuable reference for clinical counseling of related abnormalities in pregnancies.

Plastome data provides new insights into population differentiation and evolution of Ginkgo in the Sichuan Basin of China

BackgroundGinkgo biloba L., an iconic living fossil, challenges traditional views of evolutionary stasis. While nuclear genomic studies have revealed population structure across China, the evolutionary patterns reflected in maternally inherited plastomes remain unclear, particularly in the Sichuan Basin - a potential glacial refugium that may have played a crucial role in Ginkgo’s persistence.ResultsAnalysis of 227 complete plastomes, including 81 newly sampled individuals from the Sichuan Basin, revealed three distinct maternal lineages differing from known nuclear genome patterns. We identified 170 sequence variants and extensive RNA editing (235 sites) with a bias toward hydrophobic amino acid conversions, suggesting active molecular evolution. A previously undocumented haplotype (IIA2), predominant in western Sichuan Basin populations, showed close genetic affinity with rare refugial haplotypes. Western populations exhibited higher haplotypic diversity and distinctive genetic structure, supporting the basin’s role as both glacial refugium and corridor for population expansion. Ancient trees (314–784 years) provided evidence for interaction between natural processes and historical human dispersal in shaping current genetic patterns.ConclusionsOur findings demonstrate substantial genetic diversity within Sichuan Basin Ginkgo populations and reveal dynamic molecular evolution through plastome variation and RNA editing patterns, challenging the notion of evolutionary stasis in this living fossil. This study provides crucial genomic resources for understanding Ginkgo’s evolution and informs conservation strategies for this endangered species.

Differences in gut microbial diversity and composition between growth phenotypes of farmed juvenile sandfish, Holothuria scabra

BackgroundThe observed growth variability of different aquaculture species in captivity hinders its large-scale production. For the sandfish Holothuria scabra, a tropical sea cucumber species, there is a scarcity of information on its intestinal microbiota in relation to host growth, which could provide insights into the processes that affect growth and identify microorganisms with probiotic or biochemical potential that could improve current production strategies. To address this gap, this study used 16 S rRNA amplicon sequencing to characterize differences in gut and fecal microbiota among large and small juveniles reared in floating ocean nurseries.ResultsWe recovered 5915 amplicon sequence variants and diversity indices revealed significant differences between large and small juveniles (p < 0.05). Gut microbiota of large juveniles had lower bacterial diversity than its smaller counterparts. The genus cluster Burkholderia-Caballeronia-Paraburkholderia (BCP) is the most common and abundant taxa found in the gut for both size categories but less abundant in fecal samples. Small juveniles had a higher abundance of members from the Roseobacter clade (Rhodobacteriaceae) such as Ruegeria, Shimia, Psuedoruegeria and Marivita among others while the genus Schlegelella (Caldimonas) and Bosea were primarily found in larger juveniles. Predicted physiological functions identified signatures of metabolism, biosynthesis, and biodegradation pathways unique for each size category. Significant differences in diversity and composition were also exhibited between the pooled fecal and gut sample types.ConclusionsThe bacterial composition in the intestinal tract of the sandfish H. scabra is an important factor in the observed growth variability in aquaculture. The results show differences in diversity, composition and predicted physiological functions between the size groups, despite being from the same cohort and environment. It was also evident that the fecal microbiota differs from the gut and does not correspond to size category, warranting caution in using the fecal matter as a proxy to infer microbial composition and interactions in the gastrointestinal tract. Understanding the roles that these microorganisms play in sandfish growth could support the development of strategies to manage size variation in captive-bred sea cucumbers, or for the promotion and selection for faster-growing individuals.

Coastal eutrophication transforms shallow micro-benthic reef communities.

Coral reefs are impacted worldwide by coastal eutrophication, which is often translated by a decrease in coral cover and an increase in potentially harmful invertebrates and algal blooms. Additionally to corals and other macro-benthos, micro-benthic communities are affected tremendously, however few studies reported the specific effect of eutrophication on those communities. This study addresses how micro-benthic communities are impacted by turbidity and associated eutrophication. To answer this question, we compared three groups that have been previously suggested as bioindicators of reef environmental conditions: foraminifera, diatoms and bacteria, from 12 islands (and 600 samples) in the Spermonde Archipelago (Indonesia) along a near- to offshore turbidity gradient. Insights from environmental DNA metabarcoding and satellite images highlighted differences between the reef slope (deep) and the reef flat (shallow) communities, a distinction that has been omitted in most previous studies. The reef flat communities were 1.5- to 2-fold more affected by turbidity parameters compared to the reef slope, which we argue is related to eutrophication. Based on previous work, we expected that prokaryotes would be the most impacted group by water quality. However, we found that large benthic foraminifera and diatom communities were highly impacted by turbidity, whereas the reef flat prokaryotic communities were primarily shaped by the substrate type. Additionally, a total of 112 exact sequence variants (ESVs) were identified as indicators of different turbidity levels, of which 87 foraminifera and diatom ESVs were associated with high-moderate turbid waters. Our study revealed fundamental knowledge and provides key information of the specific effect of turbidity and associated eutrophication and habitat variables (substrate type and reef area) on foraminifera, diatoms and prokaryotic communities. We argue that local and regional eutrophication participates in shaping reef flat micro-benthic communities, which may therefore play an important role as early warning signals for local degradation in coral reefs.

Return of Clinically Actionable Pharmacogenetic Results From Molecular Tumor Board DNA Sequencing Data: Workflow and Estimated Costs.

Pharmacogenetic testing can prevent severe toxicities from several oncology drug therapies; it also has the potential to improve the outcomes from supportive care drugs. Paired tumor and germline sequencing is increasingly common in oncology practice; these include sequencing of pharmacogenes, but the germline pharmacogenetic variants are rarely included in the clinical reports, despite many being clinically actionable. We established an informatics workflow to evaluate the clinical sequencing results for pharmacogenetic variants. We used the Aldy computational tool, which we have previously shown to determine the variant alleles in 14 pharmacogenes in clinical sequencing data with >99% accuracy, to identify pharmacogenetic variants in the clinical whole exome sequencing from our molecular tumor board. Patients with genetic variants that are clinically actionable for their individual therapy programs, including both treatment and supportive care, are referred to a clinical pharmacogenetics testing laboratory for confirmation. Through an evaluation of our weekly informatics workflow, we determined it took approximately 3.25 hours to complete the analysis of the sequencing data from approximately 20 patients. Using a United States pharmacist's median salary, we estimated the incremental added cost of the process to be only ~$15 per patient. This adds only a minor increase to the patient's cost of testing and has the potential to improve the safety and efficacy of their treatment.

Biodiversity of the Bacterial and Fungal Microbiome and Associated Inflammatory Cytokine Profile in Chronic Rhinosinusitis.

Dysbiosis of the bacterial and fungal microbiome has been increasingly implicated in the pathogenesis of chronic rhinosinusitis (CRS). This study explores the relationship between microbiome and mycobiome biodiversity and type 2 (T2) versus non-type 2 (NT2) inflammation. Mucosal tissues from the ethmoid sinus were collected during endoscopic sinus (CRS) and skull base (controls) surgery between January 2020 and July 2021. Specimens underwent 16S rRNA (bacterial) and internal transcribed spacer (fungal) gene sequencing, along with cytokine analysis using the Luminex assay. Based on cytokine (IL-4, IL-5, IL-13) concentrations and the presence of eosinophils, CRS cases were classified into T2 or NT2 inflammatory profiles. The relationships between CRS endotype and the biodiversity of the microbiome and mycobiome were assessed. Specimens from 92 patients (30 control, 31 CRSwNP, 31 CRSsNP) were included in the analyses. Among 62 CRS cases, 20 exhibited T2 inflammation and 42 exhibited NT2 inflammation. Compared with control specimens, NT2 specimens exhibited significantly lower amplicon sequence variants (mean difference -149, 95% CI [-261, -37], p=0.007), Shannon index (-0.48 [-0.79, -0.16], p=0.002), and Simpson index (-0.003 [-0.005, -0.001], p=0.002) for bacterial alpha diversity. However, no significant differences in bacterial alpha diversity were observed between T2 specimens and controls, or between T2 and NT2 specimens. Fungal biodiversity did not differ significantly across endotype and control groups. Dysbiosis of the sinus bacterial microbiome is more strongly associated with a NT2-mediated inflammatory profile than with a T2-mediated inflammatory profile.

Aeromonas isolation reveals this genus's contribution to antimicrobial resistance fluxes across the wastewater-treated water-river interface.

Aeromonas spp. are common members of water and wastewater microbiomes, but some are listed as opportunistic pathogens and are often reported to carry antimicrobial resistance (AMR) genes. We aimed to assess the performance of isolation media for capturing their distribution and their role in AMR dissemination into aquatic environments. We investigated the abundance, diversity, and AMR profile of Aeromonas isolates from wastewater and receiving water bodies at five municipal wastewater treatment plants in Denmark using three isolation media. This was then compared with the diversity estimated from community-wide 16S rRNA gene amplicon sequencing and resistance patterns inferred from high-throughput qPCR of resistance genes. Isolates from ampicillin sheep blood agar were the most phylogenetically diverse, but the overall Aeromonas recovery on the three media was similarly good and matched the dominant amplicon sequence variants. While the dominant phylotypes were ubiquitous, some types were only detected in treated wastewater and the receiving rivers. The resistance prevalence was moderate and mostly to beta-lactams and tetracyclines. Isolates resistant to piperacilin-tazobactam, cefepime, and tetracycline downstream of the plants were linked to wastewater origin. Overall, our work demonstrates Aeromonas and Aeromonas-mediated AMR fluxes at the wastewater/environment interfaces and provides methodological bases for monitoring aeromonads in wastewater and surface waters.

Analytical Validation of the Labcorp Plasma Complete Test, a Cell-free DNA Comprehensive Genomic Profiling Tool for Precision Oncology.

To help guide treatment decisions and clinical trial matching, tumor genomic profiling is an essential precision oncology tool. Liquid biopsy, a complementary approach to tissue testing, can assess tumor-specific DNA alterations circulating in the blood. Labcorp Plasma Complete is a next-generation sequencing, cell-free DNA comprehensive genomic profiling test that identifies clinically relevant somatic variants across 521 genes in advanced and metastatic solid cancers. Over 800 unique sequencing libraries across 27 cancer types were evaluated to establish analytical sensitivity, specificity, accuracy, and precision, reproducibility, and repeatability (PRR). Sensitivity was verified for each variant type, with a median variant allele frequency (VAF) of 1.25% and 1.27% for panel-wide SNVs and indels (sequence variants), respectively, with <1% VAF sensitivity observed for clinically actionable variants, 1.72-fold for copy number amplifications (CNAs), 0.48% fusion read fraction for translocations, and 0.47% sequence mutation VAF for MSI-High. Analytical specificity was 99.9999% for SNVs and 100% for all other variant types. PRR resulted in 94.9% average positive agreement (APA) and 99.9% average negative agreement (ANA) for sequence variants and 100% APA and ANA for CNAs, translocations, and MSI. Orthogonal assays were utilized to assess accuracy demonstrating an aggregate analytical concordance of 97.4% positive percent agreement and >99.99997% negative percent agreement for all variants. Overall, the test demonstrates high sensitivity, specificity, accuracy, and robustness to enable informed clinical decision-making.

Development and validation of a one-step SMN assay for genetic testing in spinal muscular atrophy via MALDI-TOF MS.

Spinal muscular atrophy (SMA) is a fatal neuromuscular disorder primarily attributed to the homozygous deletion of the survival motor neuron 1 (SMN1) gene, with disease severity closely correlated to the copy number variations (CNV) of SMN2. Conventional methodologies, however, fail to provide a comprehensive gene overview of SMN and are often both time-intensive and costly. In this study, we present a novel one-step MALDI-TOF MS assay for SMA gene testing. To accurately quantify CNV, we incorporated RPPH1 as an internal control alongside normal samples and competing templates targeting SMN1, SMN2, and RPPH1 for multiple corrections. The CNV assay enables precise quantification of exons 7/8 in both SMN1 and SMN2 genes, achieving a kappa value of 0.935 (P < 0.001) when compared with multiple ligation-dependent probe amplification (MLPA) during its development phase. This accuracy was further corroborated in a cohort comprising 78 individuals. To identify patients harboring compound heterozygous mutations or silent carriers, prevalent pathogenic variants along with sequence variants of SMN1 were integrated into our analysis framework; plasmids were constructed for methodological validation purposes. Utilizing these combinatorial assays for SMN detection, we identified one patient exhibiting a compound heterozygous mutation characterized by genotype [0 + 1d] and another subject presenting genotype [2 + 1], who harbored simultaneous variants of g.27134T > G and g.27706_27707delAT. The CNV assessment combined with pathogenic variants analysis developed through MALDI-TOF MS provides a comprehensive gene profile of SMN within a single analytical run. Given its unparalleled cost-effectiveness and time efficiency, this approach holds significant promise for further application in clinical diagnosis as well as newborn screening for SMA.

Characteristics of fecal mycobiota and bacteriota in laying hens during different laying periods.

Mycobiota represents an important component of the gut microbiome in poultry and plays important roles in host nutrition and metabolism. However, the understanding of gut mycobiota in laying hens during the production cycle is limited. The present study aimed to characterize the structure and diversity of fecal mycobiota and bacteriota and examine the interplays between both microbial communities in laying hens during different laying periods. Sequencing of the internal transcribed spacer 1 (ITS1) and 16S rRNA gene amplicon was performed on 50 fecal samples of laying hens at 5 different time points during the laying cycle. The analysis yielded 1,314 and 3,840 amplicon sequence variants (ASVs), respectively. The results showed that Ascomycota and Basidiomycota were the most predominant. The statistical analysis of fecal flora composition succession in laying hens showed that different laying periods were one of the main factors affecting the fecal flora of laying hens. Mycobiota displayed greater variability across different laying periods compared to the bacterial community, in terms of taxonomic structure and community diversity. Co-occurrence analysis revealed varying degrees of interaction between the mycobiota and bacteriota during different laying periods. The present study aimed to improve the understanding of the fecal mycobiota and bacterial community of laying hens across different laying periods and has provided basic data support for further research into the complex fecal microbiota of laying hens.

Effect of specific sound frequency on production performance, egg quality and physiological characteristics of laying hens

The study investigated the effects of violin note B7 (Violin), double bass note E1 (Bass) and Mozart’s Sonata for Two Pianos in D Major, K. 448 (Mozart), on laying hens in terms of the production performance, egg quality and physiological characteristics. One hundred and sixty 600-day-old laying hens (ISA Brown) were randomly categorised into four groups with four replicates (10 hens/ replicate), and exposed to specific sound frequency for 12 weeks. The results indicated that Violin group presented significantly higher thick albumin height, Haugh unit and yolk index compared to the control group (p < .05). The treatment groups exhibited higher level of glutathione peroxidase in the jejunum–ileum and serum compared to the control group (p < .05). The Violin and Bass groups displayed lower levels of interleukin (IL)-1β in the jejunum–ileum, while the levels of IL-6 were lower in treatment groups compared to the control group (p < .05). The expression of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) gene was lower in the treatment groups than the control group (p < .05). Bass and Mozart groups exhibited lower messenger RNA (mRNA) level of inducible nitric oxide synthase (iNOS) and tumour necrosis factor-α (TNF-α) compared to those in the control group. The number of ASVs (amplicon sequence variants) in the jejunum–ileum of the Violin group was three times higher than that in the control group. These observations suggest that specific sound frequency stimulation can enhance the egg quality and also aid in regulating oxidative stress of laying hens.

A conifer metabolite corrects episodic ataxia type 1 by voltage sensor-mediated ligand activation of Kv1.1

Loss-of-function sequence variants in KCNA1 , which encodes the voltage-gated potassium channel Kv1.1, cause Episodic Ataxia Type 1 (EA1) and epilepsy. Due to a paucity of drugs that directly rescue mutant Kv1.1 channel function, current therapeutic strategies for KCNA1 -linked disorders involve indirect modulation of neuronal excitability. Native Americans have traditionally used conifer extracts to treat paralysis, weakness, and pain, all of which may involve altered electrical activity and/or Kv1.1 dysfunction specifically. Here, screening conifer extracts, we found that Chamaecyparis pisifera increases wild-type (WT) Kv1.1 activity, as does its prominent metabolite, the abietane diterpenoid pisiferic acid. Uniquely, pisiferic acid also restored function in 12/12 EA1-linked mutant Kv1.1 channels tested in vitro. Crucially, pisiferic acid (1 mg/kg) restored WT function in Kv1.1 E283K/+ mice, a model of human EA1. Experimentally validated all-atom molecular dynamics simulations in a neuron-like membrane revealed that the Kv1.1 voltage-sensing domain (VSD) also acts as a ligand-binding domain akin to those of classic ligand-gated channels; binding of pisiferic acid induces a conformational shift in the VSD that ligand-dependently opens the pore. Conifer metabolite pisiferic acid is a promising and versatile therapeutic lead for EA1 and other Kv1.1-linked disorders.

Comparing subsampling strategies for metagenomic analysis in microbial studies using amplicon sequence variants versus operational taxonomic units.

The microbiome is increasingly regarded as a key component of human health, and analysis of microbiome data can aid in the development of precision medicine. Due to the high cost of shotgun metagenomic sequencing (SM-seq), microbiome analyses can be done cost-effectively in two phases: Phase 1-sequencing of 16S ribosomal RNA, and Phase 2-SM-seq of an informative subsample. Existing research suggests strategies to select the subsample based on biological diversity and dissimilarity metrics calculated using operational taxonomic units (OTUs). However, the microbiome field has progressed towards amplicon sequencing variants (ASVs), as they provide more precise microbe identification and sample diversity information. The aim of this work is to compare the subsampling strategies for two-phase metagenomic studies when using ASVs instead of OTUs, and to propose data driven strategies for subsample selection through dimension reduction techniques. We used 199 samples of infant-gut microbiome data from the DIABIMMUNE project to generate ASVs and OTUs, then generated subsamples based on five existing biologically driven subsampling methods and two data driven methods. Linear discriminant analysis Effect Size (LEfSe) was used to assess differential representation of taxa between the subsamples and the overall sample. The use of ASVs showed a 50-93% agreement in the subsample selection with the use of OTUs for the subsampling methods evaluated, and showed a similar bacterial representation across all methods. Although sampling using ASVs and OTUs typically lead to similar results for each subsample, ASVs had more clades that differed in expression levels between allergic and non-allergic individuals across all sample sizes compared to OTUs, and led to more biomarkers discovered at Phase 2-SM-seq level.

Seed tuber microbiome can predict growth potential of potato varieties.

Potato vigour, the growth potential of seed potatoes, is a key agronomic trait that varies significantly across production fields due to factors such as genetic background and environmental conditions. Seed tuber microbiomes are thought to influence plant health and crop performance, yet the precise relationships between microbiome composition and potato vigour remain unclear. Here we conducted microbiome sequencing on seed tuber eyes and heel ends from 6 potato varieties grown in 240 fields. By using time-resolved drone imaging of three trial fields in the next season to track crop development, we were able to link microbiome composition with potato vigour. We used microbiome data at varying taxonomic resolutions to build random forest predictive models and found that amplicon sequence variants provided the highest predictive accuracy for potato vigour. The model revealed variety-specific relationships between the seed tuber microbiome and next season's crop vigour in independent trial fields. With a coefficient of determination value of 0.69 for the best-performing variety, the model accurately predicted vigour in seed tubers from fields not previously included in the analysis. Moreover, the model identified key microbial indicators of vigour from which a Streptomyces, an Acinetobacter and a Cellvibrio amplicon sequence variant stood out as the most important contributors to the model's accuracy. This study shows that seed potato vigour can be reliably predicted based on the microbiota associated with seed tuber eyes, potentially guiding future microbiome-informed breeding strategies.

Deciphering regulatory architectures of bacterial promoters from synthetic expression patterns.

For the vast majority of genes in sequenced genomes, there is limited understanding of how they are regulated. Without such knowledge, it is not possible to perform a quantitative theory-experiment dialogue on how such genes give rise to physiological and evolutionary adaptation. One category of high-throughput experiments used to understand the sequence-phenotype relationship of the transcriptome is massively parallel reporter assays (MPRAs). However, to improve the versatility and scalability of MPRAs, we need a "theory of the experiment" to help us better understand the impact of various biological and experimental parameters on the interpretation of experimental data. These parameters include binding site copy number, where a large number of specific binding sites may titrate away transcription factors, as well as the presence of overlapping binding sites, which may affect analysis of the degree of mutual dependence between mutations in the regulatory region and expression levels. To that end, in this paper we create tens of thousands of synthetic gene expression outputs for bacterial promoters using both equilibrium and out-of-equilibrium models. These models make it possible to imitate the summary statistics (information footprints and expression shift matrices) used to characterize the output of MPRAs and thus to infer the underlying regulatory architecture. Specifically, we use a more refined implementation of the so-called thermodynamic models in which the binding energies of each sequence variant are derived from energy matrices. Our simulations reveal important effects of the parameters on MPRA data and we demonstrate our ability to optimize MPRA experimental designs with the goal of generating thermodynamic models of the transcriptome with base-pair specificity. Further, this approach makes it possible to carefully examine the mapping between mutations in binding sites and their corresponding expression profiles, a tool useful not only for developing a theory of transcription, but also for exploring regulatory evolution.