Gene Copy Number Research Articles

Exploring genome-transcriptome correlations in cancer.

We examine the complex relationship between genomic copy number variation (CNV) and gene expression, highlighting the relevance to cancer biology and other biological contexts. By tracing the history of genometranscriptome correlations, we emphasize the complexity and challenges in understanding these interactions, particularly within the heterogeneous landscape of human cancers. Recent advances in computational algorithms and high-throughput single-cell multi-omic sequencing technologies are discussed, demonstrating their potential to refine our understanding of cancer biology and their limitations. The integration of genomic and transcriptomic analyses, which offers novel insights into tumor evolution and heterogeneity as well as therapeutic strategies, is presented as a crucial approach for advancing cancer research.

Detecting gene copy number alterations by Oncomine Comprehensive genomic profiling in a comparative study on FFPE tumor samples

Copy number alterations (CNAs) play a fundamental role in cancer development and constitute a potential tool for tailored treatments. The CNAs recognition in formalin fixed paraffin embedded (FFPE) material for diagnostic purposes has relied for years mainly on fluorescence in situ hybridization. The introduction of other procedures, such as Next-Generation Sequencing has dramatically improved CNAs discovery at genome-wide level. The detection of CNAs by NGS in FFPE material is, nonetheless, a complex issue, which still requires validation studies. Herein, the CNAs detection by a widely used NGS assay (Oncomine Comprehensive Assay plus®, OCA+) were evaluated in 14 FFPE samples mirroring diagnostic daily practice and compared to a whole-genome assay. OCA+, a targeted DNA panel, showed lower CNAs detection sensitivity and equal specificity for gains and losses. According to proprietary software pipeline, OCA+ accurately identified gains characterized by CN ≥ 5,2. No significant threshold maximizing the difference between true and false positive losses was found. Orthogonal FISH tests validated seven CNAs characterized by CN gain ≥ 6 or complete loss. Considering the CNAs growing significance in precision medicine, our findings further prompt towards a robust validation of NGS detection in FFPE materials.

Convergent Evolution and Predictability of Gene Copy Numbers Associated with Diets in Mammals.

Convergent evolution, the evolution of the same or similar phenotypes in phylogenetically independent lineages, is a widespread phenomenon in nature. If the genetic basis for convergent evolution is predictable to some extent, it may be possible to infer organismic phenotypes and the capability of organisms to utilize new ecological resources based on genome sequence data. While repeated amino acid changes have been studied in association with convergent evolution, relatively little is known about the potential contribution of repeated gene copy number changes. In this study, we explore whether gene copy number changes of particular gene families are linked to diet shifts in mammals and assess whether trophic ecology can be inferred from the copy numbers of a specific set of gene families. Using 86 mammalian genome sequences, we identified 24 gene families with a trend toward higher copy numbers in herbivores, carnivores, and omnivores, even after phylogenetic corrections. We were able to confirm previous findings on genes such as amylase, olfactory receptors, and xenobiotic metabolism genes, and identify novel gene families whose copy numbers correlate with dietary patterns. For example, omnivores exhibited higher copy numbers of genes encoding regulators of translation. We also established a discriminant function based on the copy numbers of 13 gene families that can help predict trophic ecology to some extent. These findings highlight a possible association between convergent evolution and repeated copy number changes in specific gene families, suggesting the potential to develop a method for predicting animal ecology from genome sequence data.

A barley pan-transcriptome reveals layers of genotype-dependent transcriptional complexity.

A pan-transcriptome describes the transcriptional and post-transcriptional consequences of genome diversity from multiple individuals within a species. We developed a barley pan-transcriptome using 20 inbred genotypes representing domesticated barley diversity by generating and analyzing short- and long-read RNA-sequencing datasets from multiple tissues. To overcome single reference bias in transcript quantification, we constructed genotype-specific reference transcript datasets (RTDs) and integrated these into a linear pan-genome framework to create a pan-RTD, allowing transcript categorization as core, shell or cloud. Focusing on the core (expressed in all genotypes), we observed significant transcript abundance variation among tissues and between genotypes driven partly by RNA processing, gene copy number, structural rearrangements and conservation of promotor motifs. Network analyses revealed conserved co-expression module::tissue correlations and frequent functional diversification. To complement the pan-transcriptome, we constructed a comprehensive cultivar (cv.) Morex gene-expression atlas and illustrate how these combined datasets can be used to guide biological inquiry.

Quantitative amplification ratio for established and emerging biomarkers in gastroesophageal (GE) cancer.

470 Background: Gene copy number (CN) amplifications (amps) and/or protein overexpression in ERBB2 , EGFR , FGFR2 , and MET represent established and emerging clinical targets in GE. Various methodologies are used to report gene amp or overexpression with some data suggesting FISH ratio may predict therapy response. Quantitative methods for NGS CN and relationship to ISH/IHC remains largely unresolved. We studied the landscape of targetable CN amps in GE to understand correlations of quantitative CN amp ratio and outcomes. Methods: GE tissue (N = 20,468) and liquid (N = 1,760) samples underwent NGS-based comprehensive genomic profiling (CGP). A genome-wide CN model for each sample was generated, segmented, and combined with variant allele frequencies of heterozygous SNPs to estimate the sample purity, ploidy, and CN for each segment. Genes were considered amplified at CN ≥ ploidy + 3 for ERBB2 and + 4 for other genes. Sub-analysis using amp ratio (ratio of the gene CN to the sample ploidy) ≥ 3 was performed. ctDNA tumor fraction (TF) was quantified using a composite algorithm (PMID: 38990098). The Flatiron Health-Foundation Medicine clinico genomic database, a nationwide (US-based) de-identified EHR-derived database linked to CGP data, was queried to assess external HER2 FISH or IHC and describe real-world (rw) progression-free survival (rwPFS) and overall survival (rwOS) of GE patients (pts) with ERBB2 amp on trastuzumab-based regimens. Results: ≥1 gene amps were detected in 15,341 (75%) GE tissue cases, and in 67% of cases using an amp ratio ≥3 threshold. The rates of amps in current clinical targets using each threshold were ERBB2 : 17%/12%, KRAS : 13%/12%, EGFR: 8.7%/7.2%, MET: 5.2%/4.0% , FGFR2: 3.1%/2.7% of tissue samples. The median amp ratio and percent of amps with ratio ≥ 3 were ERBB2 : 7.1/73%, KRAS : 14/92%, EGFR : 13/82%, MET : 6.3/76%, and FGFR2 : 29/88%. Amp ratio ≥ 3 was associated with focal amplicons vs amp ratio < 3 (median amplicon size 0.95 vs. 3.8 Mbp, p < 0.001). Of 1,382 pts with HER2 NGS and IHC results, concordance with IHC3+ was 68% and increasing ERBB2 amp ratio was associated with IHC 3+ status. Of 604 pts with HER2 NGS and FISH, concordance was 80% (118 positive, 366 negative). Higher ERBB2 amp ratio was also associated with longer rwOS and rwPFS as a continuous variable on first-line trastuzumab-based regimens. ≥1 gene amps were detected in 597 (34%) liquid samples (median TF 18%) and 32% with the amp ratio ≥3 threshold, increasing to 79% and 73% with TF ≥ 20% (n=355). Conclusions: Gene amps are common established or emerging GE targets and ERBB2 amp ratio is associated with outcomes for pts on trastuzumab-based regimens. Amp detection in liquid correlates with higher TF and amp ratio. As the number of therapies targeting gene amps and overexpression grows, quantification of the degree of amplification and harmonization across methodologies may be an important tool to select pts for optimal outcomes.

Identifying bacterial fixation pathway of mediating soil carbon stock changes along tropical forest restoration

The mechanism mediating carbon accumulation changes along ropical forest restoration remains unclear. Here, we identified how functional bacteria, litter input, and abiotic variables control soil organic carbon stock changes along an age-chronosequence of tropical forest restoration. Over 51-yr recovery, significant increases in total organic carbon stocks (∼1.7 fold) were strongly associated with increases in copy number of carbon fixation bacterial genes (cbbL) (∼2.6 fold). The direct pathways of cbbL abundance, microbial and mineral-associated organic carbon explained 76 % of carbon stock variation. In contrast, litter carbon, soil water, and bulk density indirectly regulated carbon stocks through affecting cbbL abundance (68 %) and microbial carbon level (29 %). Furthermore, cbbL abundance had a higher contribution (71 %) to carbon fraction transformation than microbial carbon level (19 %). We suggest that tropical forest restoration controls carbon stocks primarily via direct bacterial fixation pathway mediated by litter carbon and physical soil variables. Our results are helpful to further understand the mechanism of tropical forest restoration regulating carbon transformation and accumulation.

Spinal Muscular Atrophy Update in Best Practices: Recommendations for Treatment Considerations.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder caused by biallelic variants of the Survival Motor Neuron 1 gene (SMN1) that affects approximately 1 in 15,000 live births. Availability of 3 SMN-enhancing treatments for SMA has led to urgency to review how clinicians and patients use these treatments for SMA, while additional research and real-world data and experience are being collected. This work describes important factors to assist with decision-making for SMN-enhancing treatments. A systematic literature review was conducted on SMN-enhancing treatments for SMA and related studies. A working group of American and European health care providers with expertise in SMA care identified barriers and developed recommendations through a modified Delphi technique with serial surveys and feedback through virtual meetings to fill gaps for information where evidence is limited. A community working group of an individual living with SMA and caregivers provided insight and perspective on SMA treatments and support through a virtual meeting to guide recommendations. The health care provider working group and the community working group agreed that when determining whether to start, change, add, or discontinue a treatment, essential considerations include patient and family/caregiver perspective, and treatment safety and side effects. When initiating treatment for patients newly diagnosed with SMA, important patient characteristics are age and Survival Motor Neuron 2 gene (SMN2) copy number. Furthermore, when initiating, changing, or adding treatment, current clinical status and comorbidities drive decision-making. When considering a medication or treatment plan change, unless there is an urgent indication, a treatment and associated patient outcomes should be monitored for a minimum of 6-12 months. When determining a treatment plan with an adolescent or adult with SMA, consider factors such as quality of life, burden vs benefit of treatment, and reproductive issues. Access to care coordination and interdisciplinary/multidisciplinary care are essential to treatment success. Sharing information about current knowledge of treatments and shared decision-making between health care providers and patients living with SMA and caregivers are essential to overcoming barriers to providing SMN-enhancing treatments.

MDMNI-DGD: a novel graph neural network approach for druggable gene discovery based on the integration of multi-omics data and the multi-view network

Accurately predicting druggable genes is of paramount importance for enhancing the efficacy of targeted therapies, reducing drug-related toxicities and improving patients' survival rates. Nevertheless, accurately predicting candidate cancer-druggable genes remains a critical challenge in translational medicine due to the high heterogeneity and complexity of cancer data. In this study, we proposed a novel graph neural approach called Druggable Gene Discovery based on the Integration of Multi-omics Data and the Multi-view Network (MDMNI-DGD), aiming to predict and evaluate cancer-druggable genes. MDMNI-DGD integrated a comprehensive set of multi-omics data, including copy number variations, DNA methylation, somatic mutations, and gene expression profiles. Simultaneously, it constructed the multi-view gene association network based on protein-protein interactions (PPI), protein structural domains, gene co-expression, pathway co-occurrence, gene sequence and gene ontology. Compared to other state-of-the-art approaches, MDMNI-DGD exhibits excellent performance in key evaluation metrics such as AUROC and AUPR. Moreover, the case study has also demonstrated the efficacy of our approach in discovering potentially druggable genes. Among more than 20,000 protein-coding genes, MDMNI-DGD successfully identified 872 potentially druggable genes. The findings from this investigation may serve to bolster the assessment of pan-cancer druggable genes, potentially catalyzing the development of more personalized and efficacious therapeutic interventions.

Assessment of Nucleic Acid Extraction Kits for SARS-CoV-2 Surveillance in Wastewater Samples

Objective: The aim of this study is to evaluate the effectiveness of three commercial nucleic acid extraction kits (kit A, B and C) in isolating SARS-CoV-2 viral RNA from wastewater samples. Method: In this study, water samples were collected in March 2021 from three wastewater treatment plants located in different parts of Istanbul, and it was confirmed that they were negative for SARS-CoV-2. Different concentrations of the SARS-CoV-2 virus, previously inactivated at the BSL-3 laboratory of the Pendik Veterinary Control Institute, were added to the wastewater samples. RNA extraction and quantification were performed using commercial nucleic acid extraction kits and and RT-qPCR kit specific to SARS-CoV-2. Results: At the end of the study, it was determined that kit C yielded the highest total RNA and produced more consistent results, significantly outperforming the other two kits in terms of RNA yield and purity. Statistical analysis revealed significant differences in RNA concentrations (p < 0.05) and gene copy numbers (p < 0.01) between the kits, and kit C demonstrated superior linearity and reproducibility. Conclusion: According to the findings, although all three evaluated kits are suitable for detecting SARS-CoV-2 RNA in wastewater samples, kit C provides the most efficient and reliable performance, especially for high-throughput studies. Additionally, this study highlights the importance of selecting appropriate nucleic acid extraction methods for wastewater surveillance, which serves as an early warning system for outbreaks that threaten public health.

Prognostic Stratification of Epithelioid Pleural Mesothelioma Based on the Hippo-TEADs Network

Background. The Hippo pathway is the most frequently altered signaling in pleural mesothelioma (PM). Epithelioid PM (ePM) is associated with better outcome than non-epithelioid subtypes, but its prognosis can be heterogeneous. Here, we tried to stratify ePM using the expression levels of the Hippo-TEAD network. Methods. Thirty patients with ePM were included in this study. Tumors were stratified using the expression levels of 74 genes belonging to the Hippo-TEAD network and using the non-negative matrix factorization algorithm. Results were validated using ePM cases from the TCGA cohort. Alterations associated with the molecular subgroups were investigated using mutation and copy number alteration data from TCGA. Results. Two groups of ePM (i.e., HP1 and HP2) were identified and validated using TCGA data. HP2 comprises about one-third of tumors. These tumors are frequently high-grade (73% vs. 35%), have higher levels of downstream Hippo effectors (i.e., YAP1, WWTR1 and TEADs), lower levels of VSIR—which encodes for VISTA—and poorer PFS and OS. HP2 tumors commonly harbor homodeletions in Hippo core suppressors (25% vs. 3%), while no specific gene mutation or copy number alterations of Hippo genes was associated with the two groups. Conclusions. ePM can be stratified in prognostic subtypes based on the expression levels of the Hippo-TEAD network. Higher levels of downstream Hippo effectors are associated with poor response to platinum-pemetrexed doublet and worse OS. The stratification of ePM based on the activation of the YAP/TAZ-TEAD axis is an intriguing approach in the light of the inhibitors of this signaling that are currently under investigation.

Evolution and diversification of the momilactone biosynthetic gene cluster in the genus Oryza.

Plants are master chemists and collectively are able to produce hundreds of thousands of different organic compounds. The genes underlying the biosynthesis of many specialized metabolites are organized in biosynthetic gene clusters (BGCs), which is hypothesized to ensure their faithful coinheritance and to facilitate their coordinated expression. In rice (Oryza sativa), momilactones are diterpenoids that act in plant defence and various organismic interactions. Many of the genes essential for momilactone biosynthesis are grouped in a BGC. We applied comparative genomics of diploid and allotetraploid Oryza species to reconstruct the species-specific architecture, evolutionary trajectory, and sub-functionalisation of the momilactone biosynthetic gene cluster (MBGC) in the Oryza genus. Our data show that the evolution of the MBGC is marked by lineage-specific rearrangements and gene copy number variation, as well as by occasional cluster loss. We identified a distinct cluster architecture in Oryza coarctata, which represents the first instance of an alternative architecture of the MBGC in Oryza and strengthens the idea of a common origin of the cluster in Oryza and the distantly related genus Echinochloa. Our research illustrates the evolutionary and functional dynamics of a biosynthetic gene cluster within a plant genus.

96. Candida albicans bloodstream infections are comprised of diverse populations of strains, including antifungal tolerant strains that emerge during treatment failure

Abstract Background The longstanding model is that most bloodstream infections (BSIs) are caused by a single strain that passes through a bottleneck. C. albicans exhibits genomic and phenotypic plasticity, but it is unknown whether within-patient (pt) strain diversity is common during BSIs.Figure 1.C. albicans SNP phylogenyStrains within each patient's blood cultures differed by SNPs and indels Methods We determined short-read whole genome sequences (Illumina NextSeq) of 10 C. albicans strains from blood cultures (BCs) in each of 4 pts at baseline (n=4) and during persistent BSIs (3 of 4). In depth phenotypic assays were performed on strains from 1 pt.Figure 2.C. albicans large scale genomic variantsStrains within 3/4 pts (MN, QR, G) were distinct by large scale variants, including aneuploidies, copy number variations and LOH. Results Strains clustered by pt by single nucleotide polymorphism (SNP) phylogeny. BCs from each pt contained mixed populations of strains that differed by SNPs and indels (n=4 pts; 0-42 SNPs, 0-9 indels/pt), gene copy numbers (n=2), chromosome 5 or 7 aneuploidy (n=2), and loss of heterozygosity (n=1) [Fig 1, 2]. Chromosome 7 trisomy (Tri7) and euploid strains from 1 pt (MN) exhibited identical antifungal minimum inhibitory concentrations. However, euploid strains significantly outcompeted Tri7 strains in presence of micafungin in vitro, consistent with echinocandin tolerance [Fig 3]. In absence of micafungin, Tri7 strains significantly outcompeted euploid strains within human blood ex vivo and during mouse gastrointestinal colonization, colon invasion and BSIs, despite attenuated hyphal formation in vitro and in vivo [Fig 4]. Over-expression of chromosome 7 gene NRG1 (transcriptional repressor) in euploid strains recapitulated Tri7 phenotypes, including repressed hyphal and biofilm formation. Tri7 strains represented 76% of the baseline BC population in pt MN. After 2 days of micafungin treatment, euploid strains were ≥ 95% of the population. In control experiments, changes in ploidy were not evident following incubation of index strain M1 in BC bottles in vitro.Figure 3.Phenotypic differences between Tri7 and euploid strains from pt MNA) M2 (euploid) outcompetes M1 (Tri7) in presence of micafungin in vitro in absence of differences in MIC. B) Tri7 strains are attenuated in hyphal formation. C) NRG1 overexpression in euploid M2 background represses biofilm and hyphal formation. Conclusion Positive BCs are comprised of diverse C. albicans populations rather than single, genetically identical strains. Within-pt strains may demonstrate differences in responsiveness to antifungal agents and virulence that are not recognized by the clinical micro lab. In some cases, antifungal treatment failures are due to selection of pre-existing resistant or tolerant strains. Results challenge standard clinical and microbiology practices that focus on single Candida strains.Figure 4.Tri7 strain M1 outcompetes euploid strain M2 during GI colonization and disseminated candidiasis.A) GI colonization mouse model. B) Competitive indices within kidneys during disseminated candidiasis Disclosures Cornelius J. Clancy, MD, Cidara: Grant/Research Support|Gilead: Honoraria|Merck: Grant/Research Support|Scynexis: Advisor/Consultant|Shionogi: Advisor/Consultant|Venatorx: Advisor/Consultant

Novel Fermented Plant-Based Functional Beverage: Biological Potential and Impact on the Human Gut Microbiota

Controlled fermentation carried out by selected starters might enhance the safety, nutritional, and biological profiles of non-dairy fermented products. This research aims to study the biological potential and impact on the human gut microbiota of a novel fermented finger millet-based product. Finger millet (Eleusine coracana), suspended in an aqueous sucrose-based solution, was fermented by Weissella confusa 2LABPT05 and Lactiplantibacillus plantarum 299v (1%, 1:1 ratio (v/v)), at 30 °C/200 rpm in an orbital incubator until pH ≈ 4.5–5.0. Microbial growth, phenolic compounds, antioxidant, and antidiabetic activities were evaluated. In vitro digestion followed by in vitro faecal fermentation were used to study the impact of the fermented plant-based functional beverage (PBFB) on the human gut microbiota. Antidiabetic activity (21% vs. 14%) and total phenolics (244 vs. 181 mg of gallic acid equivalents/kg PBFB) increased with fermentation. The digested fermented PBFB contributed to the increase, over the first 6 h, of the Bifidobacterium’s 16S rRNA gene copy numbers, concomitant with significant release of the acetic, propionic, and butyric short chain fatty acids, and also lactic acid. The novel PBFB has been shown to have antidiabetic potential and bifidogenic effects, and consequently its consumption might positively impact blood glucose levels and the human gut microbiota.

Nano-Selenium Elevating Leaf Quality and Growth Via Microbial-Regulating Nitrogen Availability Under Ammonium and Nitrate Spraying in Tea Plants.

Nano-selenium fertilizers can promote plant growth and nitrogen availability. However, little information is available on the effects of nano-selenium on tea leaf quality, soil nutrient availability and associated microbe-driven mechanisms. This study examined the effects of nano-selenium on the tea leaf quality and soil nitrogen cycling in 20-year-old tea plantations when the leaves were sprayed with ammonium or nitrate. Leaf selenium and amino acid contents increased ninefold and 9%, respectively, with nano-selenium in "Zhongcha108" and "Longjing43." Rhizosphere bacterial and fungal community compositions were more sensitive to selenium and nitrogen applications in "Longjing43" than in "Zhongcha108." "Zhongcha108" enriched more taxa related to microbial growth, while more taxa related to cellular maintenance and nutrient acquisition enriched in "Longjing43." Nano-selenium application decreased the copy number of AOA and AOB genes, and nosZ and nirK genes by 59%, 53%, 37% and 46% under ammonium, and by 77%, 43%, 38% and 65%, respectively, under nitrate spraying, in "Longjing43." However, the expression of these genes increased by nano-selenium in "Zhongcha108" with ammonium spraying. It is concluded that a nano-selenium application increases tea leaf quality, and this effect on nitrogen cycling and ecological functioning largely depends on the tea cultivar-specific bacterial and fungal composition and function.

Increase in IAA levels by EPSPS copy number variation relates to fitness advantage in Eleusine indica.

Long-term use of chemical weed control has led to some weedy species evolving herbicide resistance traits with fitness advantage. Our previous studies revealed glyphosate resistance in an Eleusine indica population due to copy number variation of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) comes with fitness advantage under non-competitive conditions. Here, transcriptomics and targeted metabolomics were used to investigate physiological basis associated with the fitness advantage. Relative copy number of EPSPS gene and plant dry weight of the glyphosate-resistant (R) population was 88.3- and 1.2- times, respectively, higher than that in the wild type (WT) plants that were isolated from within the R population. Seven genes were screened to be relevant to fitness growth trait by RNA-seq. The level of aromatic amino acids Tryptophan (Trp), Phenylalanine (Phe) and Tyrosine (Tyr), products in the shikimate pathway catalyzed by EPSPS, was 1.2-times higher in R compared to the WT plants. The metabolites associated with Trp metabolism indole-3-acetic acid (IAA), 3-indolepropionic acid (IPA), indole-3-acetamide (IAM) in the R plants were 2.0-, 1.8- and 1.4- times higher than that in the WT plants, respectively. All the results indicate that fitness advantage in the studied R E. indica population may be caused by higher IAA production due to over-expression of the EPSPS gene and pleiotropically by elevated carbon metabolism. The findings in this research can provide reference information for control strategies to the glyphosate-resistant E. indica. © 2025 Society of Chemical Industry.

Transcription near arrested DNA replication forks triggers ribosomal DNA copy number changes.

DNA copy number changes via chromosomal rearrangements or the production of extrachromosomal circular DNA. Here, we demonstrate that the histone deacetylase Sir2 maintains the copy number of budding yeast ribosomal RNA gene [ribosomal DNA (rDNA)] by suppressing end resection of DNA double-strand breaks (DSBs) formed upon DNA replication fork arrest in the rDNA and their subsequent homologous recombination (HR)-mediated rDNA copy number changes during DSB repair. Sir2 represses transcription from the regulatory promoter E-pro located near the fork arresting site. When Sir2 is absent, this transcription is stimulated but terminated by arrested replication forks. This transcription-replication collision induces DSB formation, DSB end resection and the Mre11-Rad50-Xrs2 complex-dependent DSB repair that is prone to chromosomal rDNA copy number changes and the production of extrachromosomal rDNA circles. Therefore, repression of transcription near arrested replication forks is critical for the maintenance of rDNA stability by directing DSB repair into the HR-independent, rearrangement-free pathway.

Unelongated Stems are an Active Nitrogen-Fixing Site in Rice Stems Supported by Both Sugar and Methane Under Low Nitrogen Conditions

Enhancing nitrogen (N) fixation in rice plants can reduce N fertilizer application and contribute to sustainable rice production, particularly under low-N conditions. However, detailed microbial and metabolic characterization of N fixation in rice stems, unlike in the well-studied roots, has not been investigated. Therefore, the aim of this study was to determine the active N-fixing sites, their diazotroph communities, and the usability of possible carbon sources in stems compared with roots. The N-fixing activity and copy number of the nitrogenase gene in the rice stem were high in the outer part of the unelongated stem (basal node), especially in the epidermis. N fixation, estimated using the acetylene reduction assay, was also higher in the leaf sheath and root than in the inner part of the unelongated stem and culm. Amplicon sequence variants (ASVs) close to sugar-utilizing heterotrophic diazotrophs belonging to Betaproteobacteria and type II methanotrophic diazotrophs belonging to Alphaproteobacteria were abundant in the outer part of the unelongated stems. Media containing crushed unelongated stems exhibited N-fixing activity when sucrose, glucose, and methane were added as the sole carbon sources. This suggested that N fixation in the unelongated stems was at least partly supported by sugars (sucrose and glucose) and methane as carbon sources. ASVs close to sugar-utilizing heterotrophs belonging to Actinobacteria were also highly abundant in the unelongated stem; however, their functions need to be further elucidated. The present finding that diazotrophs in rice stems can use sugars such as sucrose and glucose synthesized by rice plants provides new insights into enhancing N fixation in rice stems.

Application of Minimally Invasive Oral Swab Samples for qPCR-Based Sexing in Neognathae Birds.

Birds are inherently social creatures that rely on pairing to enhance their well-being. Since many bird species lack obvious physical differences between females and males, sex identification is essential for ensuring their welfare. Additionally, early determination of the sexes of birds is crucial for their breeders, especially considering that most companion birds do not display clear sexual characteristics. Molecular genetic sexing has been demonstrated to be the most reliable method for determining the sexes of monomorphic birds. The objective of the present study was to demonstrate rapid, effective, and precise identification of sex in birds through quantitative real-time PCR (qPCR) using samples obtained via a minimally invasive technique (oral swabs). This qPCR method assesses variations in gene copy numbers within conserved Z-specific genes such as CHRNA6, DDX4, VPS13A, LPAR1, and TMEM161B, which are absent from the W chromosome. A total of 34 samples were included in this study from the following 17 bird species: domestic pigeon (Columba livia domestica), domestic chicken (Gallus gallus domesticus), domestic goose (Anser anser f domesticus), domestic duck (Anas platyrhynchos domesticus), Mute swan (Cygnus olor), Budgerigar (Melopsittacus undulatus), Lovebird (Agapornis roseicollis), Cockatiel (Nymphicus hollandicus), Red-rumped parrot (Psephotus haematonotus), Rose-ringed parakeet (Psittacula krameri), African grey parrot (Psittacus erithacus), domestic Canary (Serinus canaria forma domestica), Goldfinch (Carduelis carduelis major), Gouldian Finch (Chloebia gouldiae), Red Siskin (Carduelis cucullata), Australian Zebra Finch (Taeniopygia castanotis), and Common buzzard (Buteo buteo). The results proved that the CHRNA6, DDX4, VPS13A, LPAR1, and TMEM161B genes can reveal the sexes in the Neognath birds tested.

Features and evolutionary adaptations of the mitochondrial genome of Garuga forrestii W. W. Sm.

Garuga forrestii W. W. Sm. is a tree species of the Burseraceae family, endemic to China, found in hot/warm-dry valleys. This species plays a crucial role in maintaining biodiversity in these ecosystems. We performed de novo assembly of the Garuga forrestii mitochondrial genome using PMAT (v.1.5.4), resulting in a typical circular molecule of 606,853 bp. The genome consists of 31 tRNA genes, 3 rRNA genes, 35 protein-coding genes, and 1 pseudogene. The study also investigates RNA editing sites and evolutionary patterns. The mitochondrial genome exhibits a low proportion of repetitive sequences (3.30%), suggesting a highly conserved structure. A high copy number of the trnM-CAT gene (4 copies) is noted, which may contribute to genomic rearrangement and adaptive evolution. Among the 476 RNA editing sites, hydrophilic-hydrophobic and hydrophobic-hydrophobic editing events are most common, accounting for 77.10%. Negative selection predominates among most genes (Ka/Ks < 1), while a few genes (e.g., matR, nad3, rps1, rps12, and rps4) show signs of positive selection (Ka/Ks > 1), potentially conferring evolutionary advantages. Additionally, a significant A/T bias is observed at the third codon position. Phylogenomic analysis supports the APG IV classification, with no evidence of horizontal gene transfer. This mitochondrial genome offers valuable insights into the adaptive mechanisms and evolutionary processes of Garuga forrestii. It enhances our understanding of the species' biogeography in tropical Southeast Asia and Southwest China, providing key information on the evolutionary history of this genus.

Crustacean Zooplankton Ingestion of Potentially Toxic Microcystis: In Situ Estimation Using mcyE Gene Gut Content Detection in a Large Temperate Eutrophic Lake.

Grazing by zooplankton can regulate bloom-forming cyanobacteria but can also transfer toxin-producing cells, as well as toxic metabolites, to the food web. While laboratory investigations have provided extensive knowledge on zooplankton and toxic cyanobacteria interactions, information on zooplankton feeding on toxin-producing cyanobacteria in natural water bodies remains scarce. In this study, we quantified Microcystis-specific mcyE synthase genes from the gut contents of various cladoceran and copepod taxa to assess the in situ crustacean community and taxon-specific ingestion of potentially toxic Microcystis in Lake Peipsi, a large eutrophic lake in Estonia, Northern Europe. Microcystis cells with mcyE genes were found in all crustaceans examined. However, some species, such as the cyclopoid copepod Mesocyclops leuckarti, were more efficient in ingesting potentially toxic Microcystis than other co-occurring cladocerans (Daphnia spp., Bosmina spp., Chydorus sphaericus) and copepods (Eudiaptomus gracilis). The amount of toxigenic Microcystis cells grazed by crustacean population changed temporarily, and copepods were the predominant consumers of toxigenic Microcystis during several months of the 5-month study period. Crustacean ingestion of toxigenic Microcystis was not related to Microcystis biomass or mcyE gene copy numbers in the environment but was instead related to the abundance of major crustacean grazers. Our findings emphasize the close interaction between crustacean zooplankton and toxigenic Microcystis, indicating that some species may play a more significant role in linking toxic cells within the food web than others.