Targeted Gene Sequencing Research Articles

Evaluation and Sequencing of 16S rRNA Gene for Molecular Identification of Prevalent <i>Salmonella</i> Strains Isolated from Food and Water

This Study is to identify prevalent strains using 16S rRNA, a standard molecular marker that reveals the phylogenetic. Salmonella contains the virulence gene Invasive-A (Inv-A), which increases the pathogenicity of the relationships between bacterial species. It is identified using PCR, targeted gene sequencing, and phylogenetic grouping. A total of 110 food and water samples collected from Karachi were examined to ascertain the prevalence. 16S rRNA gene is for smaller subunits and its function hasn't altered over time, making it one of the standard genes to identify bacteria and helps it connect to the host's epithelial cells, facilitating the infection. Combining the amplification of these two genes with gene sequencing and phylogenetic analysis can help determine where these species actually originated.16S rRNA and the Inv-A gene were amplified in the positive samples. MEGA-X is used to build a phylogenetic Maximum Likelihood tree while using existing and newly discovered research sequences. The examination of this tree reveals the relative proximity or divergence of other Salmonella species from these isolates as well as the geographic origin of these species.

Robust Copy Number Alteration Detection (RCNA): A Novel Algorithm for Detecting Copy-Number Alterations for Targeted Gene Sequencing Experiments

Robust Copy Number Alteration Detection (RCNA): A Novel Algorithm for Detecting Copy-Number Alterations for Targeted Gene Sequencing Experiments

Clinical and Molecular Profile of Neurofibromatosis Type 1 Patients Using Revised Diagnostic Criteria - A Retrospective Cohort Study.

To catalog and correlate the clinical features and mutational spectrum of neurofibromatosis type 1 (NF1) patients attending a tertiary care center in India. NF1 patients with confirmed molecular diagnosis from 2014 to 2021 were included in the study. The molecular tests used for the diagnosis were exome sequencing, targeted gene sequencing, and Multiple Ligation Probe Assay. Forty-two clinically diagnosed NF1 patients who had confirmed molecular diagnosis, which is now part of the revised diagnostic criteria, were included in the analysis. Nonsense variants were the most frequently observed (35.71%), followed by frameshift (23.8%), splice site (14.29%), deletion (11.9%), missense (9.5%), and in-frame deletions (4.76%) in our case series. Three variants (c. 5269-1G > C, c. 1541_1542del AG, and c. 6853_6854insA) were identified in more than one patient, suggesting that the variants are widely distributed in the gene and lack any mutational hotspot. This study supports the previous findings that patients with the variant c. 2970_2972delAAT do not develop neurofibromas; however, it was not necessary for those with whole gene deletion to have dysmorphic features as reported by other studies. The study could not establish any correlation between the type of variants and specific clinical features. Around 28% of mutations could be identified by screening exons 14, 28, 37, 46 and intron 37 in this population. This study will contribute to a better understanding of the phenotypic variability of neurofibromatosis patients. The variable expressivity seen in NF1 suggests that modifying genes may be involved in the development of particular clinical features in addition to NF1 mutations.

Characterization of knock-in zebrafish models with a novel SLC4A3 variant identified in a short QT syndrome family

Abstract Background Short QT syndrome (SQTS) is one of the lethal inherited arrhythmia syndromes leading to ventricular fibrillation (VF) and cardiac death. It is mainly caused by pathogenic variants in ion channel related genes, especially KCNH2 which produces IKr. Recently, SLC4A3 encoding a membrane-localized anion exchanger 3 (AE3) that regulates intracellular pH (pHi), has been reported as a causative gene for SQTS. However, the molecular mechanism of SLC4A3 variants which cause SQTS remains unclear. Purpose Functional characterization of a novel SLC4A3 variant identified in a SQTS family. Methods We performed target gene and whole exome sequencing to detect pathogenic variants in a family with SQTS. Patch-clamp analyses were performed to determine the electrophysiological properties using HEK293 cells. We constructed stable cell lines expressing wild-type (WT) or the SLC4A3 variant based on HEK293 cells. To evaluate the intracellular pH (pHi) level, we measured pHi using BCECF-AM via time-lapse microscopy. To clarify the pathogenesis of the SLC4A3 variant in vivo, we injected CRISPR-Cas9 and a repair template into one-cell stage zebrafish embryos and established a SLC4A3 knock-in (KI) zebrafish model (hereafter referred to as slc4a3 zebrafish). Action potential durations (APD) were recorded in slc4a3 zebrafish at 3 days post-fertilization (dpf). Results We identified two novel variants in KCNH2 (c.280 c>t, p.H70Y) and SLC4A3 (c.1059 c>a, p.N353K) in a SQTS patient. Both variants were co-segregated in the patient’s family with short QT intervals (Figure A). By electrophysiological analysis, KCNH2-H70Y did not increase IKr. We over-expressed KCNH2 (WT and H70Y), KCNQ1, and KCNJ2 in the SLC4A3 stable cell lines (WT and N353K) though no increase of IKr, IKs, and IK1 was observed. In the analysis of pHi, cells expressing SLC4A3-N353K showed significantly slower pHi alternations than those with WT, indicating the loss-of-function effect of transport kinetics in the regulation of pHi. In the electrophysiological analysis of the slc4a3 zebrafish, which harbors the paralogous variant of SLC4A3-N353K, APD20, 50, and 70 were significantly shortened in the slc4a3 KI zebrafish (HOMO) compared to the control (WT), providing evidence for the shortened QT interval with the SLC4A3 variant in vivo (Figure B). Furthermore, slc4a3 zebrafish developed severe cardiac edema (HET and HOMO: Figure C, arrows) with no gross morphological defects in other organs, which may indicate the specific role of SLC4A3-N353K in cardiac function. Conclusion We identified a novel SLC4A3 variant, p.N353K, in a family with SQTS and revealed its contributions to the pHi control and the shortened APD. Further analysis will be necessary to clarify the mechanism by which SLC4A3-N353K causes QT shortening.

Targeted gene sequencing and hearing follow-up in 7501 newborns reveals an improved strategy for newborn hearing screening.

Hearing loss is a common congenital condition. Concurrent newborn hearing and limited genetic screening has been implemented in China for the last decade. However, the role of gene sequencing screening has not been evaluated. In this study, we enrolled 7501 newborns (52.7% male, 47.3% female) in our Newborn Screening with Targeted Sequencing (NESTS) program, and 90 common deafness genes were sequenced for them. Hearing status assessments were conducted via telephone from February 2021 to August 2022, for children aged 3 to 48 months. Of the universal newborn hearing screening, 126 (1.7%) newborns did not pass. Targeted sequencing identified 150 genetically positive newborns (2.0%), with 25 exhibiting dual-positive results in both screening. Following diagnostic audiometry revealed 18 hearing loss newborns and half of them had abnormal results in both screening. The positive predictive value for universal newborn hearing screening alone was merely 14.3% (18/126). However, when combined with targeted sequencing, this rate increased to 36.0% (9/25). Furthermore, limited genetic screening identified 316 carriers of hot-spot variants, but none exhibited biallelic variants. All 15 hot-spot carriers who failed physical screening demonstrated normal hearing during follow-up. In this cohort study of 7501 Newborns, Combining targeted sequencing with universal newborn hearing screening demonstrated technical feasibility and clinical utility of identifying individuals with hearing loss, especially when coupled with genetic counseling and closed-loop management. It is suggested to use this integrated method as an improved strategy instead of the current limited genetic screening program in some regions of China.

A Python program to merge Sanger sequences: an update.

Gene cloning is an important step in investigating gene structure and function. To verify gene sequence, Sanger sequencing is used, which may produce several overlapping sequencing files that need to be merged before alignment to the target gene sequence is performed. Previously, we reported the Python program to Merge Sanger sequences (https://peerj.com/articles/11354/), which ran in command line and relied heavily on EMBOSS suite. In this updated version of the program, we have made several remarkable improvements. It provides a graphical user interface (GUI) written with tkinter, which is convenient and stable. It does not require users to rename the input sequences before performing merging. With regard to the implementation, the updated version utilizes Python function (Align.PairwiseAligner) to align adjacent sequences, which is more flexible (can adjust program parameter i.e., the number of first-time consecutive matching bases). The new version of the program makes merging Sanger sequences much more convenient and facilitates gene study.

The clinical and genetic aspects of six individuals with GH1 variants and isolated growth hormone deficiency type II.

Isolated growth hormone deficiency type II (IGHD II) is an autosomal dominant disorder characterized by a GH1 gene variant resulting in a significant reduction in growth hormone (GH) secretion and a subsequent decrease of plasma insulin-like growth factor 1 (IGF-1) levels and eventual growth impairment. This study aimed to identify causative variants in six Chinese families with IGHD II, exploring both clinical and genetic characteristics. Detailed clinical data, including clinical presentations, physical charateristics, medical and family histories, as well as genetic test results, were systematically examined. Six children, comprising four males and two females, with a mean age of 4.64 ± 1.15 years, exhibited short stature with a mean height of -3.95 ± 1.41 SDS. Four of them had a family history of short stature, while one patient presented with pulmonary hypertension. All children demonstrated GH deficiency in growth hormone stimulation tests (mean peak GH value: 2.83 ± 2.46 ng/mL). Exome sequencing for the six patients and targeted gene sequencing for their family members revealed heterozygous variants in the GH1 gene, including Exon2-5del, c.334T>C, c.291 + 1G>A, c.291 + 2T>A, 1.5 kb deletion, and 1.7 kb deletion, with four variants being novel. Four patients underwent human recombinant growth hormone (rhGH) replacement therapy, initiating treatment at a mean age of 4.6 ± 0.7 years. The mean height increase in patients was 1.21 ± 0.3 SDS in the first six months of treatment and 1.79 ± 0.15 SDS in the first year. Our findings contribute to expanding the genotypic and phenotypic spectra of individuals with IGHD II.

Activity-Based DNA-Encoded Library Screening for Selective Inhibitors of Eukaryotic Translation.

Small molecule probes exist for only ∼2% of human proteins because most lack functional binding pockets or cannot be assayed for high-throughput screening. Selective translation modulation circumvents canonical druggability and assay development constraints by using in vitro transcription-translation (IVTT) as a universal biochemical screening assay. We developed an IVTT activity assay by fusing a GFP reporter to various target gene sequences and screened the target sequences for inhibitors in microfluidic picoliter-scale droplets using a 5,348-member translation inhibitor DNA-encoded library (DEL). Screening a proof-of-concept PCSK9-GFP reporter yielded many hits; 6/7 hits inhibited PCSK9-GFP IVTT (IC50 1-20 μM), and the lead hit reduced PCSK9 levels in HepG2 cells. Preliminary selectivity was informed by counterscreening the DEL against a frameshift mutant PCSK9-GFP reporter. A plug-and-play approach to assay development and screening was demonstrated by scouting the DEL for activity using reporter genes of targets with difficult-to-assay or even unknown function (RPL27, KRASG12D, MST1, USO1). This microfluidic IVTT DEL screening platform could scale probe discovery to the human proteome and perhaps more broadly across the tree of life.

Targeted gene sequencing and transcriptome sequencing reveal characteristics of NUP98 rearrangement in pediatric acute myeloid leukemia

BackgroundNUP98 rearrangements (NUP98-r) are rare but overrepresented mutations in pediatric acute myeloid leukemia (AML) patients. NUP98-r is often associated with chemotherapy resistance and a particularly poor prognosis. Therefore, characterizing pediatric AML with NUP98-r to identify aberrations is critically important.MethodsHere, we retrospectively analyzed the clinicopathological features, genomic and transcriptomic landscapes, treatments, and outcomes of pediatric patients with AML.ResultsNine patients with NUP98-r mutations were identified in our cohort of 142 patients. Ten mutated genes were detected in patients with NUP98-r. The frequency of FLT3-ITD mutations differed significantly between the groups harboring NUP98-r and those without NUP98-r (P = 0.035). Unsupervised hierarchical clustering via RNA sequencing data from 21 AML patients revealed that NUP98-r samples clustered together, strongly suggesting a distinct subtype. Compared with that in the non-NUP98-r fusion and no fusion groups, CMAHP expression was significantly upregulated in the NUP98-r samples (P < 0.001 and P = 0.001, respectively). Multivariate Cox regression analyses demonstrated that patients harboring NUP98-r (P < 0.001) and WT1 mutations (P = 0.030) had worse relapse-free survival, and patients harboring NUP98-r (P < 0.008) presented lower overall survival.ConclusionsThese investigations contribute to the understanding of the molecular characteristics, risk stratification, and prognostic evaluation of pediatric AML patients.

Harnessing Virtual Insights: A Computational Study on the Molecular Characterization of a Drought‐Inducible RNA‐Binding Protein for Enhancing Drought Tolerance in Mungbean (Vigna radiata L.)

AbstractAbiotic stresses, particularly drought and salinity, significantly impact agricultural crop yields, with mung bean (Vigna radiata L.) being no exception. This annual green legume, vital to agricultural systems in Asia, is generally resilient to limited water supply; however, severe drought conditions at critical growth stages can drastically diminish both its quality and yield. Drought stress negatively affects various morpho‐physicochemical properties, impeding plant growth and highlighting the need for sustainable approaches to enhance mung bean's drought tolerance through improved farming practices. In this study, we focused on identifying and understanding the molecular mechanisms underlying drought tolerance in mung beans. We retrieved the target gene sequence from the NCBI database, but found no direct sequence similarity, which led to the use of homology modeling. The structure of a known protein (PDB ID: 5T9P) was identified as the most suitable template. Motif prediction using the Motif Search tool highlighted specific motifs relevant to drought stress responses. To investigate potential drought‐tolerance mechanisms, we screened ABA agonists using Density Functional Theory (DFT) analysis. Molecular docking and MD simulations revealed the interactions between these ligands and the modeled protein, shedding light on how they might influence drought tolerance. We performed molecular dynamics (MD) simulations to assess the stability and conformational dynamics of the modeled protein across different time scales. Our findings underscore the potential of ABA agonists to modulate physiological responses and enhance drought resistance. This research offers valuable insights into the conformational dynamics of drought‐related proteins in mung beans, elucidating the molecular underpinnings of drought response mechanisms. By advancing our understanding of these processes, our findings provide novel strategies for improving breeding approaches aimed at enhancing crop resilience against water scarcity and other abiotic stresses. This work promises to mitigate the detrimental effects of drought on mung bean yields and supports sustainable agricultural practices, thereby contributing to global food security.

Clonal hematopoiesis in LGI1-antibody encephalitis.

Leucine-rich glioma-inactivated 1 (LGI1)-antibody encephalitis (LGI1e), the major form of autoimmune encephalitis (AE) presented with memory loss and faciobrachial dystonic seizure, commonly develops in aged population. Hematologic aging is often accompanied by clonal hematopoiesis (CH), a phenomenon in which specific mutations accumulate, potentially leading to autoimmune disorders or malignancies. Our research aimed to investigate the connection between clonal hematopoiesis of indeterminate potential (CHIP) and LGI1e. Peripheral blood samples from consecutive LGI1e patients were collected and analyzed for 24 clonal CHIP using targeted gene sequencing. The results were compared to a control dataset from an ethnically matched health care cohort. Patient characteristics were analyzed based on their CHIP status. A total of 52 LGI1e patients were enrolled for this study. Among them, three patients (5.8%) exhibited functional mutations in the ASXL1 gene, one of the CHIP-associated genes analyzed by targeted sequencing. This frequency was significantly higher compared to that of the control cohort (1%, p = 0.015). Nevertheless, the patients showed no difference in the clinical characteristics, laboratory results, and immunotherapy outcomes. LGI1e showed high frequency of ASXL1 functional mutation in the CHIP analysis, which may contribute to the underlying pathogenesis. Further research is needed to determine its direct role in the development of autoimmunity and disease progression.

Diversifying selection identified in immune epitopes of bovine coronavirus isolates from Irish cattle.

Bovine betacoronavirus (BoCoV) is a pneumoenteric pathogen of cattle that is closely related to human coronavirus OC43. Vaccines are administered to protect against diseases caused by BoCoV, but knowledge gaps exist with regard to correlates of protection and the effect of immune evasion on driving evolution. In this study, immune epitopes were mapped onto BoCoV structural proteins, including spike and haemagglutinin esterase (HE), and then supported with targeted gene sequencing of Irish clinical isolates and selective pressure analysis. Increased prevalence of diversifying selection and amino acid changes in some mapped immune epitopes suggests that immune escape is selecting for non-synonymous mutations arising in these regions. Selection analysis and sequencing provided increased support for neutralising antibody (nAb) epitopes compared to others, suggesting that nAbs are an important arm of the immune response to BoCoV. Phylogenetic analysis of spike and HE sequences showed that Irish isolates from this study were in the European clade, except for one HE sequence that sat in the Asian/American clade, while the spike gene of this sample was in the European clade. Recombination between a European and an Asian/American isolate would give rise to such a sequence. This study has gathered evidence suggesting that pressure to evade the nAb response is contributing to BoCoV evolution.

Multiplex Approach of Metabolomic and Transcriptomic Reveals the Biosynthetic Mechanism of Light-induced Flavonoids and CGA in Chrysanthemum

Light, as an important environmental signal for plant growth and development, has been reported to be involved in the metabolism of phenylpropane. However, the light-induced phenylpropane biosynthesis mechanism in Chrysanthemum morifolium Ramat., especially flavonoid and chlorogenic acid (CGA) metabolism, is not clear. In this study, we found the flower phenotype of chrysanthemum 'HangBaiJu' became lighter after bagging. Besides, the content of main active ingredients, such as Luteolin-7-O-glucoside (Lu-7-O-G), Diosmetin-7-O-glucoside (Di-7-O-G), Apigenin-7-O-glucoside, Apigenin and CGA, was significantly decreased. Comparative transcriptome analysis (pairwise comparison, WGCNA, and k-means clustering) revealed that the expression of structural genes, such as CmPAL, CmCHS1/2, CmF3’H, CmFNS and CmHQT, was notably declined under dark conditions. Meanwhile, ‘bridge proteins’ CmMYB3/6/16 and signal transduction factors CmBBX20/22.2/22.3 were identified as key regulatory factors in the light-mediated synthesis of flavonoids and CGA in chrysanthemum. Among that, CmBBX20 could promote the accumulation of flavonoids and CGA by directly binding to the G-box core sequences of downstream target genes based on transient overexpression and Y1H assays. Overall, the preliminary molecular mechanism of BBXs and MYBs coordinately regulating the accumulation of flavonoids and CGA in chrysanthemum under different light inductions has been clarified, which provided a theoretical basis for the molecular breeding and quality improvement of chrysanthemum.

Targeted gene sequencing reveals disparate genomic mutations between young and older adults in renal cell carcinoma

BackgroundRenal cell carcinoma (RCC) is a type of cancer that can develop at any point in adulthood, spanning the range of age-related changes that occur in the body. However, the specific molecular mechanisms underlying the connections between age and genetic mutations in RCC have not been extensively investigated.MethodsClinical and genetic data from patients diagnosed with RCC were collected from two prominent medical centers in China as well as the TCGA dataset. The patients were categorized into two groups based on their prognosticated age: young adults (YAs) and older adults (OAs). Univariate and multivariate analysis were employed to evaluate the relationships between age and genetic mutations. Furthermore, a mediation analysis was conducted to assess the association between age and overall survival, with genetic disparities serving as a mediator.ResultsOur analysis revealed significant differences in clinical presentation between YAs and OAs with RCC, including histopathological types, histopathological tumor stage, and sarcomatoid differentiation. YAs were found to have lower mutation burden and significantly mutated genes (SMGs) of RCC. However, we did not observe any significant differences between the two groups in terms of 10 canonical oncogenic signaling pathways-related genes mutation, telomerase-related genes (TRGs) mutation, copy number changes, and genetic mutations associated with clinically actionable targeted drugs. Importantly, we demonstrate superior survival outcomes in YAs, and we confirmed the mediating effect of genetic disparities on these survival outcome differences between YAs and OAs.ConclusionOur findings reveal previously unrecognized associations between age and the molecular underpinnings of RCC. These associations may serve as valuable insights to guide precision diagnostics and treatments for RCC.

A Systematic Study on Mirror Repeats in CTLA4 and KCNJ11 Genes of Diabetes

All cellular functions are controlled by the regulatory material which is constituted majorly by variety type of repeat elements. Among that types a unique pattern of bases form a Mirror repeat (MR) which thought to plays variety of roles at the genomic level that include regulation of gene expression, genomic integrity, triplex structure formation etc. The present research focused on identification of Mirror repeats in different genes types involved in both diabetes types. Two different genes CTLA4 (T1D) &amp; KCNJ11 (T2D) were targeted to identify these sequence type. A manual method refers as FPCB is utilized to extract out MR sequences. The targeted gene sequences were divided into short regions so that it can be handled out easily while bioinformatics based analysis. Among the both types it was reported that both genes have different pattern of distribution of MR sequences. CTLA4 have 346 MR sequences in total in which MR235 have highest length of 50bps. In case of KCNJ11 a total no of 235 MR sequences were reported in which MR105 having highest length of 58bps. The present study will be helpful to elucidate the roles of MR sequences in diseases development &amp; progression. It will be helpful for sequence based protein finding studies using bioinformatics tools as well as wet lab experiments.

A novel mutation Trp-2027-Gly in acetyl-CoA carboxylase confers resistance to cyhalofop-butyl in Chinese sprangletop (Leptochloa chinensis).

Chinese sprangletop [Leptochloa chinensis (L.) Nees] control is threatened by resistance to acetyl-CoA carboxylase (ACCase)-inhibiting herbicides. In this study, a L. chinensis population, HFLJ18, that survived cyhalofop-butyl [aryloxyphenoxypropionate (APP) herbicide, CyB] treatment was collected from a rice field in Lujiang County, Anhui Province, China. This study aimed to evaluate the susceptibility of HFLJ18 to herbicides with different modes-of-action and investigate the potential mechanisms of resistance to CyB. The HFLJ18 population exhibited high levels of resistance to CyB (10.92-fold) and showed resistance to the ACCase inhibitors metamifop (4.63-fold) and fenoxaprop-P-ethyl (8.39-fold), but was susceptible to clethodim, pinoxaden, florpyrauxifen-benzyl, oxadiazon and pretilachlor. Target gene sequencing revealed a novel Trp-to-Gly substitution at codon position 2027 of ACCase in the resistant plants. Molecular docking revealed that the spatial structure of ACCase changed significantly following the substitution, as indicated by reduced H-bonds. A newly derived cleaved amplified polymorphic sequence (dCAPS) marker was subsequently developed to detect the Trp-2027-Gly mutation in the ACCase of L. chinensis. Additionally, pretreatment with the cytochrome P450 (P450) inhibitor piperonyl butoxide (PBO) and the glutathione S-transferase (GST) inhibitor 4-chloro-7-nitrobenzoxadiazole (NBD-Cl) did not reverse resistance to CyB, suggesting that nontarget-site resistance mechanisms were not involved in CyB resistance in the HFLJ18 population. Overall, the resistance to CyB in the HFLJ18 population derived from the mutation of ACCase gene, and to the best of our knowledge, this is the first report of the ACCase Trp-2027-Gly mutation conferring resistance to ACCase-inhibiting herbicides in grass species. © 2024 Society of Chemical Industry.

Horizontal gaze palsy with progressive scoliosis: Further expanding the ROBO3 spectrum.

Horizontal gaze palsy with progressive scoliosis (HGPPS) is a rare, autosomal recessive disorder resulting from axonal midline crossing defect due to variants in ROBO3. We retrospectively evaluated demographics, clinical phenotype, course of spinal deformities, and neuroimaging findings of six Turkish patients with HGPPS. We performed targeted gene testing by next-generation sequencing. The median age at symptom onset and diagnosis was 1.5 years (0.5-4), and 11 years (2-16), respectively. Oculomotor signs were the most common presenting symptom (n = 4), followed by scoliosis (n = 2). The course of scoliosis was progressive and accompanied by kyphosis, showed intrafamilial variability, and was corrected surgically in three of the patients. Intellectual disability (n = 4), hypergonadotropic hypogonadism (n = 2), hearing loss (n = 2), and tranisent movement disorders (n = 1) were additional features. Targeted gene sequencing revealed five distinct homozygous variants. Of the four novel variants, two of them were located in the acceptor site of the noncoding region of the gene, remaining two were missense and frameshift variants, located in immunoglobulin-like domain-2, and cytoplasmic signaling motif 2, respectively. Structural magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI) showed the absence of decussation of superior cerebellar peduncle and dorsal transverse pontine fibers. Spectrum of HGPPS is further expanded with novel variants in the ROBO3 with clinical and radiological fingerprints. Spinal deformities require close orthopedic screening and individualized approach. Intellectual disability and hearing loss emerge as additional features. Hypogonadism and transient subtle movement disorders require further attention and confirmation from other series.

Melodious tuning of microbial dynamics in biofloc, cage, and pond culturing system: a study on Pangasius pangasius fish gut microbiome.

Aquaculture, a rapidly expanding sector, meets the global surging demand for aquatic food. Pangasius, a highly valued freshwater species, has seen a significant increase in demand due to its adaptability and potential for high yields, making it a promising candidate for aquaculture in India. This study investigates the gut microbiome composition of Pangasius pangasius fish cultured in three different systems (biofloc, cage, and pond). Metagenomic DNA extraction and 16S rRNA gene-targeted sequencing were performed. Outcomes revealed distinct microbial compositions across culture types, with significant differences in species richness and diversity, specifically in the biofloc system, compared to cages and ponds. Taxonomic analysis identified prevalent phyla such as Firmicutes and Fusobacteriota, with varying abundances among culture systems. The genus-level analysis highlighted dominant genera such as Cetobacterium and WWE3. Functional profiling indicated differences in enzymatic activity and metabolic pathways, emphasizing each culture sample type's unique microbial community structures. Notably, the microbiota from BF samples exhibited significant differences and unique metabolic pathways compared to the microbiota from C and P samples, which showed greater similarity and shared several common metabolic pathways. These findings highlight substantial differences in microbial diversity across the culturing systems, reflecting the microbiota's ability to adapt to specific environments and their potential role in promoting fish growth within those environments. Overall, this study provides insights into the gut microbiome diversity and functionality in Pangasius pangasius across different aquaculture environments, contributing to a better understanding of host-microbe interactions and aquaculture management strategies.

Effective target genes for RNA interference-based management of the cabbage stem flea beetle.

The cabbage stem flea beetle (CSFB, Psylliodes chrysocephala) is a key pest of oilseed rape. The ban on neonicotinoids in the European Union due to environmental concerns and the emergence of pyrethroid-resistant populations have made the control of CSFB extremely challenging. In search of a solution, we have recently shown that RNA interference (RNAi) has potential in the management of CSFB. However, the previously tested target genes for RNAi-mediated pest control (subsequently called target genes) exhibited moderate and slow-acting lethal effects. In this study, 27 double-stranded RNAs (dsRNAs) were orally delivered to identify highly effective target genes in CSFB adults by leveraging the findings of a genome-wide RNAi screen in Tribolium castaneum. Our screen using 500 ng of dsRNA identified 10 moderately effective (> 50% mortality) and 4 highly effective target genes (100% mortality in 8-13 days). The latter mainly included proteasome subunits. Gene expression measurements confirmed target gene silencing and dose-response studies revealed LD50 values as low as ~20 ng in 14 days following a single exposure to dsRNA. Four highly effective dsRNAs also inhibited leaf damage (up to ~75%) and one affected locomotion. The sequences of promising target genes were subjected to in silico target prediction in non-target organisms, for example, beneficials such as honeybees, to design environmentally friendly dsRNAs. Overall, the study provides valuable insights for the development of dsRNA-based insecticides against CSFB.

Prognostic value of genomic mutation signature associated with immune microenvironment in southern Chinese patients with esophageal squamous cell carcinoma

The genomic landscape of esophageal squamous cell cancer (ESCC), as well as its impact on the regulation of immune microenvironment, is not well understood. Thus, tumor samples from 92 patients were collected from two centers and subjected to targeted-gene sequencing. We identified frequently mutated genes, including TP53, KMT2C, KMT2D, LRP1B, and FAT1. The most frequent mutation sites were ALOX12B (c.1565C > T), SLX4 (c.2786C > T), LRIG1 (c.746A > G), and SPEN (c.6915_6917del) (6.5%). Pathway analysis revealed dysregulation of cell cycle regulation, epigenetic regulation, PI3K/AKT signaling, and NOTCH signaling. A 17-mutated gene-related risk model was constructed using random survival forest analysis and showed significant prognostic value in both our cohort and the validation cohort. Based on the Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression (ESTIMATE) algorithm, the Tumor Immune Dysfunction and Exclusion (TIDE) algorithm, and the MCPcounter algorithm, we found that the risk score calculated by the risk model was significantly correlated with stimulatory immune checkpoints (TNFSF4, ITGB2, CXCL10, CXCL9, and BTN3A1; p < 0.05). Additionally, it was significantly associated with markers that are important in predicting response to immunotherapy (CD274, IFNG, and TAMM2; p < 0.05). Furthermore, the results of immunofluorescence double staining showed that patients with high risk scores had a significantly higher level of M2 macrophage than those with low risk scores (p < 0.05). In conclusion, our study provides insights into the genomic landscape of ESCC and highlights the prognostic value of a genomic mutation signature associated with the immune microenvironment in southern Chinese patients with ESCC.