Generation Sequencing Techniques Research Articles

Practical considerations for pathological diagnosis and molecular profiling of cholangiocarcinoma: an expert review for best practices.

Advances in precision medicine have expanded access to targeted therapies and demand for molecular profiling of cholangiocarcinoma (CCA) patients in routine clinical practice. However, pathologists face challenges in establishing a definitive intrahepatic CCA (iCCA) diagnosis while preserving sufficient tissue for molecular profiling. Additionally, they frequently face challenges in optimal tissue handling to preserve nucleic acid integrity. This article first identifies the challenges in establishing a definitive diagnosis of iCCA in a lesional liver biopsy while preserving sufficient tissue for molecular profiling. Then, the authors explore the clinical value of molecular profiling, the basic principles of single gene and next-generation sequencing (NGS) techniques, and the challenges in tissue sampling for genomic testing. They also propose an algorithm for best practice in tissue management for molecular profiling of CCA. Several practical challenges face pathologists during tissue sampling and processing for molecular profiling. Optimized tissue processing, careful tissue handling, and selection of appropriate approaches to molecular testing are essential to ensure that the highest possible quality of diagnostic information is provided in the greatest proportion of cases.

Increased Diagnostic Yield by Reanalysis of Whole Exome Sequencing Data in Mitochondrial Disease.

The genetic diagnosis of mitochondrial disorders is complicated by its genetic and phenotypic complexity. Next generation sequencing techniques have much improved the diagnostic yield for these conditions. A cohort of individuals with multiple respiratory chain deficiencies, reported in the literature 10 years ago, had a diagnostic rate of 60% by whole exome sequencing (WES) but 40% remained undiagnosed. We aimed to identify a genetic diagnosis by reanalysis of the WES data for the undiagnosed arm of this 10-year-old cohort of patients with suspected mitochondrial disorders. The WES data was transferred and processed by the RD-Connect Genome-Phenome Analysis Platform (GPAP) using their standardized pipeline. Variant prioritisation was carried out on the RD-Connect GPAP. Singleton WES data from 14 individuals was reanalysed. We identified a possible or likely genetic diagnosis in 8 patients (8/14, 57%). The variants identified were in a combination of mitochondrial DNA (n = 1, MT-TN), nuclear encoded mitochondrial genes (n = 2, PDHA1, and SUCLA2) and nuclear genes associated with nonmitochondrial disorders (n = 5, PNPLA2, CDC40, NBAS and SLC7A7). Variants in both the NBAS and CDC40 genes were established as disease causing after the original cohort was published. We increased the diagnostic yield for the original cohort by 15% without generating any further genomic data. In the era of multiomics we highlight that reanalysis of existing WES data is a valid tool for generating additional diagnosis in patients with suspected mitochondrial disease, particularly when more time has passed to allow for new bioinformatic pipelines to emerge, for the development of new tools in variant interpretation aiding in reclassification of variants and the expansion of scientific knowledge on additional genes.

Molecular Characterization and Tolerance Potential of Culturable Crude Oil-Degrading Microbes in Santa-Barbara River, Bayelsa State, Nigeria

There has been chronic contamination of the surrounding aquatic and terrestrial environments in the Niger Delta region of Nigeria due to the enormous activities of crude oil exploration. This study aimed to characterize and assess the crude oil tolerance potential of indigenous microbes from crude oil spills in Santa Barbara River, Bayelsa State, Nigeria, that could serve as potential consortia for bioremediation of the crude oil spills. Total crude oil hydrocarbon-utilizing bacteria count (THUBC) and total crude oil hydrocarbon-utilizing fungi count (THUFC) in the samples were determined with a culture-dependent spread plate technique. Bacterial and fungal isolates were characterised using standard phenotypic and 16S/Internal Transcribed Spacer region rRNA gene sequencing techniques, respectively. The tolerance of autochthonous bacterial isolates to different concentrations of crude oil was subsequently determined. THUBC and THUFC in crude oil-polluted water samples ranged from 1.88 log10 CFU/ml to 2.74 log10 CFU/ml and from 0.00 log10 CFU/ml to 1.70 log10 CFU/ml, respectively. Representative strains of Proteus mirabilis, Bacillus subtilis, Pseudomonas aeruginosa, Micrococcus luteus and Aspergillus flavus isolates obtained from crude oil-polluted water samples were deposited in the GenBank (NCBI) under accession numbers OQ969924, OQ969951, OQ969987, OQ970009 and OQ975908. Proteus mirabilis, Pseudomonas aeruginosa and Aspergillus flavus demonstrated the most significant tolerance to crude oil pollutants (minimum crude oil inhibitory concentrations (MIC) = 80%) followed by Bacillus subtilis and Micrococcus luteus (MIC= 40%). The findings from this study are pointers to the potential role of the microbial isolates as bioremediation consortia to remediate the polluted Santa Barbara River.

Short-term exposure to antibiotics begets long-term disturbance in gut microbial metabolism and molecular ecological networks

BackgroundAntibiotic exposure can occur in medical settings and from environmental sources. Long-term effects of brief antibiotic exposure in early life are largely unknown.ResultsPost a short-term treatment by ceftriaxone to C57BL/6 mice in early life, a 14-month observation was performed using 16S rRNA gene-sequencing technique, metabolomics analysis, and metagenomics analysis on the effects of ceftriaxone exposure. Firstly, the results showed that antibiotic pre-treatment significantly disturbed gut microbial α and β diversities (P < 0.05). Both Chao1 indices and Shannon indices manifested recovery trends over time, but they didn’t entirely recover to the baseline of control throughout the experiment. Secondly, antibiotic pre-treatment reduced the complexity of gut molecular ecological networks (MENs). Various network parameters were affected and manifested recovery trends over time with different degrees, such as nodes (P < 0.001, R2 = 0.6563), links (P < 0.01, R2 = 0.4543), number of modules (P = 0.0672, R2 = 0.2523), relative modularity (P = 0.6714, R2 = 0.0155), number of keystones (P = 0.1003, R2 = 0.2090), robustness_random (P = 0.79, R2 = 0.0063), and vulnerability (P = 0.0528, R2 = 0.28). The network parameters didn't entirely recover. Antibiotic exposure obviously reduced the number of key species in gut MENs. Interestingly, new keystones appeared during the recovery process of network complexity. Changes in network stability might be caused by variations in network complexity, which supports the ecological theory that complexity begets stability. Besides, the metabolism profiles of the antibiotic group and control were significantly different. Correlation analysis showed that antibiotic-induced differences in gut microbial metabolism were related to MEN changes. Antibiotic exposure also caused long-term effects on gut microbial functional networks in mice.ConclusionsThese results suggest that short-term antibiotic exposure in early life will cause long-term negative impacts on gut microbial diversity, MENs, and microbial metabolism. Therefore, great concern should be raised about children’s brief exposure to antibiotics if the results observed in mice are applicable to humans.D3-AZHri_jesG1CHKmDZa1Video

Haplotype determination of the Bombyx mori nucleopolyhedrovirus by Nanopore sequencing and linkage of single nucleotide variants.

Naturally occurring isolates of baculoviruses, such as the Bombyx mori nucleopolyhedrovirus (BmNPV), usually consist of numerous genetically different haplotypes. Deciphering the different haplotypes of such isolates is hampered by the large size of the dsDNA genome, as well as the short read length of next generation sequencing (NGS) techniques that are widely applied for baculovirus isolate characterization. In this study, we addressed this challenge by combining the accuracy of NGS to determine single nucleotide variants (SNVs) as genetic markers with the long read length of Nanopore sequencing technique. This hybrid approach allowed the comprehensive analysis of genetically homogeneous and heterogeneous isolates of BmNPV. Specifically, this allowed the identification of two putative major haplotypes in the heterogeneous isolate BmNPV-Ja by SNV position linkage. SNV positions, which were determined based on NGS data, were linked by the long Nanopore reads in a Position Weight Matrix. Using a modified Expectation-Maximization algorithm, the Nanopore reads were assigned according to the occurrence of variable SNV positions by machine learning. The cohorts of reads were de novo assembled, which led to the identification of BmNPV haplotypes. The method demonstrated the strength of the combined approach of short- and long-read sequencing techniques to decipher the genetic diversity of baculovirus isolates.

The Genetics of Human Sleep and Sleep Disorders.

Healthy sleep is vital for humans to achieve optimal health and longevity. Poor sleep and sleep disorders are strongly associated with increased morbidity and mortality. However, the importance of good sleep continues to be underrecognized. Mechanisms regulating sleep and its functions in humans remain mostly unclear even after decades of dedicated research. Advancements in gene sequencing techniques and computational methodologies have paved the way for various genetic analysis approaches, which have provided some insights into human sleep genetics. This review summarizes our current knowledge of the genetic basis underlying human sleep traits and sleep disorders. We also highlight the use of animal models to validate genetic findings from human sleep studies and discuss potential molecular mechanisms and signaling pathways involved in the regulation of human sleep.

Prostate cancer invasion is promoted by the miR-96-5p-induced NDRG1 deficiency through NF-κB regulation.

The N-myc downstream-regulated gene 1 (NDRG1) has been discovered as a significant gene in the progression of cancers. However, the regulatory mechanism of NDRG1 remained obscure in prostate cancer (PCa). The miR-96-5p and NDRG1 expression levels were evaluated in PCa cell lines, and prostate tissues, and validated in public databases by real-time polymerase chain reaction, western blot analysis, and immunohistochemistry. The function of miR-96-5p and NDRG1 were investigated by scratch assay and transwell assays in vitro, and mouse xenograft assay in vivo. The candidate pathway regulated by NDRG1 was conducted by the next-generation gene sequencing technique. Immunofluorescence and luciferase assays were used to detect the relation between miR-96-5p, NDRG1, and NF-kB pathway. Overexpressing NDRG1 suppresses the migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro, and inhibits metastasis in vivo. Moreover, miR-96-5p contributes to NDRG1 deficiency and promotes PCa cell migration and invasion. Furthermore, NDRG1 loss activates the NF-kB pathway, which stimulates p65 and IKBa phosphorylation and induces EMT in PCa. MiR-96-5p promotes the migration and invasion of PCa by targeting NDRG1 and regulating the NF-kB pathway.

Carnitine palmitoyltransferase II (CPT II) deficiency responsible for refractory cardiac arrhythmias, acute multiorgan failure and early fatal outcome

BackgroundCarnitine palmitoyltransferase II (CPT II) deficiency is a rare inborn error of mitochondrial fatty acid metabolism with autosomal recessive pattern of inheritance. Its phenotype is highly variable (neonatal, infantile, and adult onset) on the base of mutations of the CPT II gene. In affected subjects, long-chain acylcarnitines cannot be subdivided into carnitine and acyl-CoA, leading to their toxic accumulation in different organs. Neonatal form is the most severe, and all the reported patients died within a few days to 6 months after birth. Hereby, we report on a male late-preterm newborn who presented refractory cardiac arrhythmias and acute multiorgan (hepatic, renal, muscular) injury, leading to cerebral hemorrhage, hydrocephalus, cardiovascular failure and early (day 5 of life) to death. Subsequently, extended metabolic screening and target next generation sequencing (NGS) analysis allowed the CPT II deficiency diagnosis.Case presentationThe male proband was born at 36+ 4 weeks of gestation by spontaneous vaginal delivery. Parents were healthy and nonconsanguineous, although both coming from Nigeria. Family history was unremarkable. Apgar score was 9/9. At birth, anthropometric measures were as follows: weight 2850 g (47th centile, -0.07 standard deviations, SD), length 50 cm (81st centile, + 0.89 SD) and occipitofrontal circumference (OFC) 35 cm (87th centile, + 1.14 SD). On day 2 of life our newborn showed bradycardia (heart rate around 80 bpm) and hypotonia, and was then transferred to the Neonatal Intensive Care Unit (NICU). There, he subsequently manifested many episodes of ventricular tachycardia, which were treated with pharmacological (magnesium sulfate) and electrical cardioversion. Due to the critical conditions of the baby (hepatic, renal and cardiac dysfunctions) and to guarantee optimal management of the arrythmias, he was transferred to the Pediatric Cardiology Reference Center of our region (Sicily, Italy), where he died 2 days later. Thereafter, the carnitines profile evidenced by the extended metabolic screening resulted compatible with a fatty acid oxidation defect (increased levels of acylcarnitines C16 and C18, and low of C2); afterwards, the targeted next generation sequencing (NGS) analysis revealed the known c.680 C > T p. (Pro227Leu) homozygous missense mutation of the CPTII gene, for diagnosis of CPT II deficiency. Genetic investigations have been, then, extended to the baby’s parents, who were identified as heterozygous carriers of the same variant. When we meet again the parents for genetic counseling, the mother was within the first trimester of her second pregnancy. Therefore, we offered to the couple and performed the prenatal target NGS analysis on chorionic villi sample, which did not detect any alterations, excluding thus the CPT II deficiency in their second child.ConclusionsCPTII deficiency may be suspected in newborns showing cardiac arrhythmias, associated or not with hypertrophic cardiomyopathy, polycystic kidneys, brain malformations, hepatomegaly. Its diagnosis should be even more suspected and investigated in cases of increased plasmatic levels of creatine phosphokinase and acylcarnitines in addition to kidney, heart and liver dysfunctions, as occurred in the present patient. Accurate family history, extended metabolic screening, and multidisciplinary approach are necessary for diagnosis and adequate management of affected subjects. Next generation sequencing (NGS) techniques allow the identification of the CPTII gene mutation, essential to confirm the diagnosis before or after birth, as well as to calculate the recurrence risk for family members. Our report broads the knowledge of the genetic and molecular bases of such rare disease, improving its clinical characterization, and provides useful indications for the treatment of patients.

Feruloylated Oligosaccharides Prevented Influenza-Induced Lung Inflammation via the RIG-I/MAVS/TRAF3 Pathway.

Uncontrolled inflammation contributes significantly to the mortality in acute respiratory infections. Our previous research has demonstrated that maize bran feruloylated oligosaccharides (FOs) possess notable anti-inflammatory properties linked to the NF-kB pathway regulation. In this study, we clarified that the oral administration of FOs moderately inhibited H1N1 virus infection and reduced lung inflammation in influenza-infected mice by decreasing a wide spectrum of cytokines (IFN-α, IFN-β, IL-6, IL-10, and IL-23) in the lungs. The mechanism involves FOs suppressing the transduction of the RIG-I/MAVS/TRAF3 signaling pathway, subsequently lowering the expression of NF-κB. In silico analysis suggests that FOs have a greater binding affinity for the RIG-I/MAVS signaling complex. This indicates that FOs have potential as promising targets for immune modulation. Moreover, in MAVS knockout mice, we confirmed that the anti-inflammatory function of FOs against influenza depends on MAVS. Comprehensive analysis using 16S rRNA gene sequencing and metabolite profiling techniques showed that FOs have the potential to restore immunity by modulating the gut microbiota. In conclusion, our study demonstrates that FOs are effective anti-inflammatory phytochemicals in inhibiting lung inflammation caused by influenza. This suggests that FOs could serve as a potential nutritional strategy for preventing the H1N1 virus infection and associated lung inflammation.

Functional diversity of apoptotic vesicle subpopulations from bone marrow mesenchymal stem cells in tissue regeneration.

Apoptosis releases numerous apoptotic vesicles that regulate processes such as cell proliferation, immunity, and tissue regeneration and repair. Now, it has also emerged as an attractive candidate for biotherapeutics. However, apoptotic vesicles encompass a diverse range of subtypes, and it remains unclear which specific subtypes play a pivotal role. In this study, we successfully isolated different apoptotic vesicle subtypes based on their sizes and characterized them using NTA and TEM techniques, respectively. We compared the functional variances among the distinct subtypes of apoptotic vesicles in terms of stem cell proliferation, migration, and differentiation, as well as for endothelial cell and macrophage function, effectively identifying subtypes that exhibit discernible functional differences. ApoSEV (with diameter<1000nm) promoted stem cell proliferation, migration, and multi-potent differentiation, and accelerated skin wound healing of diabetes mouse model, while apoBD (with diameter>1000nm) played the opposite effect on cell function and tissue regeneration. Lastly, employing protein analysis and gene sequencing techniques, we elucidated the intrinsic mechanisms underlying these differences between different subtypes of apoEVs. Collectively, this study identified that apoptotic vesicle subtypes possessed distinct bio-functions in regulating stem cell function and behaviour and modulating tissue regeneration, which primarily attribute to the distinct profiling of protein and mRNA in different subtypes. This comprehensive analysis of specific subtypes of apoEVs would provide novel insights for potential therapeutic applications in cell biology and tissue regeneration.

Advances in pediatric gliomas: from molecular characterization to personalized treatments.

Pediatric gliomas, consisting of both pediatric low-grade (pLGG) and high-grade gliomas (pHGG), are the most frequently occurring brain tumors in children. Over the last decade, several milestone advancements in treatments have been achieved as a result of stronger understanding of the molecular biology behind these tumors. This review provides an overview of pLGG and pHGG highlighting their clinical presentation, molecular characteristics, and latest advancements in therapeutic treatments. Conclusion: The increasing understanding of the molecular biology characterizing pediatric low and high grade gliomas has revolutionized treatment options for these patients, especially in pLGG. The implementation of next generation sequencing techniques for these tumors is crucial in obtaining less toxic and more efficacious treatments. What is Known: • Pediatric Gliomas are the most common brain tumour in children. They are responsible for significant morbidity and mortality in this population. What is New: • Over the last two decades, there has been a significant increase in our global understanding of the molecular background of pediatric low and high grade gliomas. • The implementation of next generation sequencing techniques for these tumors is crucial in obtaining less toxic and more efficacious treatments, with the ultimate goal of improving both the survival and the quality of life of these patients.

Degradation of Dimethyl Phthalate and Diethyl Phthalate by Bacteria Isolated from Jazan, Saudi Arabia

P hthalate esters (PAEs) are commonly utilized in commercial, industrial, and medical applications. Various concentrations of PAEs have been detected in seawater samples and treated wastewater obtained from wastewater treatment plants in Saudi Arabia. Since these compounds pose adverse effects to human health, it is vital to develop efficient strategies to eliminate phthalate contamination from environments. In the present study, an enrichment technique was used to isolate phthalate-degrading bacteria from seawater and from activated sludge collected at Jazan Wastewater Treatment Plant, Saudi Arabia. Bacteria with the capability of degrading dimethyl phthalate (DMP) or diethyl phthalate (DEP) were isolated using minimum salt medium supplemented with these compounds as the sole carbon and energy source. High-performance liquid chromatography (HPLC) was utilized to evaluate the residual concentration of DMP and DEP. Using the 16S rRNA gene sequencing technique, the isolates with maximum DMP biodegradation activity were identified as Achromobacter sp. M1 and Pseudomonas sp. M2, while the isolates with maximum DEP biodegradation activity were identified as Pseudomonas sp. E2 and Pseudomonas sp. E3. The optimum bacterial growth was achieved at 30°C, pH 7.0, and 2% NaCl, except for Pseudomonas sp. E3, which demonstrated its maximum growth in the absence of NaCl. The highest biodegradation rate for all the bacterial strains (&gt; 99.0%) was achieved at 30°C, pH 7.0, and up to 2% NaCl within 4 days. Concurrently, Pseudomonas sp. M2 and Pseudomonas sp. E2 exhibited remarkable degradation rates (97.5% and 56.7%, respectively) at 4% NaCl whereas Pseudomonas sp. E3 showed its highest DEP degradation (82.4%) after 4 days in the absence of NaCl. This study offers potential candidates, including Achromobacter sp. M1, Pseudomonas sp. M2, Pseudomonas sp. E2, and Pseudomonas sp. E3, for the proficient biodegradation of DMP and DEP.

Dietary Galacto-oligosaccharides Ameliorate Atopic Dermatitis-like Skin Inflammation and Behavioral Deficits by Modulating Gut Microbiota-Brain-Skin Axis.

Atopic dermatitis (AD), a chronic, highly pruritic, and inflammatory skin disorder, often coexists with psychiatric comorbidities including anxiety and depression, posing considerable challenges for treatment. The current research aims at evaluating the efficacy and potential therapeutic mechanism of galacto-oligosaccharides (GOS) on AD-like skin lesions and comorbid anxiety/depressive disorders. Macroscopical and histopathological examination showed that GOS could markedly relieve skin inflammation by decreasing the production of IgE, IL-4, IL-13, IFN-γ, and TNF-α and regulating the PPAR-γ/NF-κB signaling in DNFB-induced AD mice. Moreover, GOS significantly improved the anxiety- and depressive-like symptoms as mirrored by the behavior tests including FST, TST, OFT, and EZM through normalizing the neurotransmitter levels of 5-HT, DA, NE, and CORT in the brain. Mechanistically, by virtue of the high-throughput 16S rRNA gene sequencing and GC-MS techniques, GOS restructured the gut microbiota and specifically induced the proliferation of Lactobacillus and Alloprevotella, leading to an increase in the total content of fecal SCFAs, in particular acetate and butyrate. Pearson correlation analysis found a marked correlation among the altered gut microbiota/SCFAs, AD-associated skin manifestations, and comorbid behavioral phenotypes. Collectively, this work highlights that GOS is a promising strategy against both AD and associated depressive symptoms by modulating the gut microbiota-brain-skin axis.

Phylogenomics of the geometrid tribe Palyadini (Lepidoptera: Geometridae) reveals contrasting patterns of phylogenetic signal in wing colour characters.

Next generation sequencing techniques currently represent a practical and efficient way to infer robust evolutionary hypotheses. Palyadini is a small Neotropical tribe of geometrid moths composed of six genera that feature strikingly colourful wings. Here, we investigated patterns of evolution and amount of phylogenetic signal contained in various colour characters featured in the wings of members of this tribe by (i) inferring a robust phylogenetic hypothesis using ultraconserved elements (UCEs), and afterwards, (ii) mapping the morphological characters onto the molecular topology under a parsimonious ancestral character optimization. Our matrix, obtained with 60% completeness, includes 754 UCE loci and 73 taxa (64 ingroup, nine outgroup). Maximum likelihood and parsimony generated largely identical topologies with strongly supported nodes, except for one node inside the genus Opisthoxia. According to our topology, most wing colour characters are reconstructed as homoplastic, particularly at the tribe level, but five of the seven provide evidence supporting common ancestry at the genus level. Our results emphasize, once again, that no character system is infallible, and that more research is necessary to take our understanding of the evolution of wing colour in moths to a level comparable with the knowledge we have for butterflies.

NUP214 fusion genes in acute leukemias: genetic characterization of rare cases.

Alterations of the NUP214 gene (9q34) are recurrent in acute leukemias. Rearrangements of chromosomal band 9q34 targeting this locus can be karyotypically distinct, for example t(6;9)(p22;q34)/DEK::NUP214, or cryptic, in which case no visible change of 9q34 is seen by chromosome banding. We examined 9 cases of acute leukemia with NUP214 rearrangement by array Comparative Genomic Hybridization (aCGH), reverse-transcription polymerase chain reaction (RT-PCR), and cycle sequencing/Sanger sequencing to detect which fusion genes had been generated. The chimeras DEK::NUP214, SET::NUP214, and NUP214::ABL1 were found, only the first of which can be readily detected by karyotyping. The identification of a specific NUP214 rearrangement is fundamental in the management of these patients, i.e., AMLs with DEK::NUP214 are classified as an adverse risk group and might be considered for allogenic transplant. Genome- and/or transcriptome-based next generation sequencing (NGS) techniques can be used to screen for these fusions, but we hereby present an alternative, step-wise procedure to detect these rearrangements.

On the limits of 16S rRNA gene-based metagenome prediction and functional profiling.

Molecular profiling techniques such as metagenomics, metatranscriptomics or metabolomics offer important insights into the functional diversity of the microbiome. In contrast, 16S rRNA gene sequencing, a widespread and cost-effective technique to measure microbial diversity, only allows for indirect estimation of microbial function. To mitigate this, tools such as PICRUSt2, Tax4Fun2, PanFP and MetGEM infer functional profiles from 16S rRNA gene sequencing data using different algorithms. Prior studies have cast doubts on the quality of these predictions, motivating us to systematically evaluate these tools using matched 16S rRNA gene sequencing, metagenomic datasets, and simulated data. Our contribution is threefold: (i) using simulated data, we investigate if technical biases could explain the discordance between inferred and expected results; (ii) considering human cohorts for type two diabetes, colorectal cancer and obesity, we test if health-related differential abundance measures of functional categories are concordant between 16S rRNA gene-inferred and metagenome-derived profiles and; (iii) since 16S rRNA gene copy number is an important confounder in functional profiles inference, we investigate if a customised copy number normalisation with the rrnDB database could improve the results. Our results show that 16S rRNA gene-based functional inference tools generally do not have the necessary sensitivity to delineate health-related functional changes in the microbiome and should thus be used with care. Furthermore, we outline important differences in the individual tools tested and offer recommendations for tool selection.

The Regulatory Effect of Huangshui Polysaccharides on Intestinal Microbiota and Metabolites during In Vitro Fermentation.

Huangshui polysaccharides (HSPs) have attracted extensive attention recently for their biological activity and physicochemical property. This research investigated the extraction, structural characterization, and prebiotic activity of three different HSPs (HSP40-0, HSP60-0, and HSP80-0) in vitro to reveal the scientific support for the high-value utilization of Huangshui. HSPs were heteropolysaccharide with diverse structures and surface morphologies. Comprehensive analysis was conducted through 16S rRNA gene sequencing and metabolite profiling techniques, and results showed that HSPs had different potentials to regulate the gut microbiota due to their different structures; for instance, both HSP40-0 and HSP80-0 could notably increase the relative abundance of Bacteroidota, whereas HSP60-0 could increase the relative abundance of Phascolarctobacterium. In addition, HSPs upregulated beneficial differential metabolites, especially short-chain fatty acids (SCFAs). Fermentation products containing these metabolites exhibited anti-inflammatory effects on LPS-treated Caco-2 cells. This study will provide reference for exploring the relationship between the natural polysaccharide structure and the prebiotic activity and widen the application of Huangshui.

Bacillus cereus: an effective bio-inoculant for promoting salt stress tolerance of rice seedlings under saline soil conditions

Plant growth-promoting rhizobacteria (PGPR) are a powerful tool to maintain sustainable agriculture and promote plant resistance to biotic and abiotic types of stress. Salinity, a major abiotic stress hampers plant growth, development, and yield. Salt-tolerant PGPR are effective agents for ameliorating salinity effects on rice plants. The present study endeavored to isolate, determine halotolerant ability, characterize Plant Growth Promoting (PGP) traits, and finally observe the effect of PGPR strain on plant growth promotion of rice plants under saline and non-saline conditions. Based on the 16S rRNA gene sequencing technique, the rhizobacterial strain DB2 was identified as Bacillus cereus ATCC 14579(T) from NCMR, NCCS Pune. To check the growth-promoting ability, the strain was inoculated with two rice genotypes named Chinsurah Nona I (salt tolerant-non aromatic) and Badshabhog (aromatic) under polyhouse conditions. Results showed a significant increment in relative water content (RWC), total chlorophyll content (TCC), root length (RL), and shoot length (SL) in both rice genotypes inoculated with DB2 under both saline and non-saline conditions. Under non-saline conditions enhancement of RWC, TCC, RL, and SL was better in DB2 inoculated Chinsurah Nona I than in Badshabhog inoculated with DB2. Whereas, DB2-inoculated Badshabhog showed more recovery of RWC, TCC, RL, and SL than DB2-inoculated Chinsurah Nona I under saline conditions. Under salt stress conditions, inoculation with the rhizobacterial strain showed a significant reduction in electrolytic leakage (EL) in rhizobacteria inoculated with both rice genotypes. Moreover, DB2 inoculation showed a significant reduction in Na+ content in the roots of Chinsurah Nona I (44.6%) and Badshabhog (24.5%) rice genotypes. The present study has indicated that the application of salt-tolerant PGPR may be an effective and sustainable solution for rice cultivation under salt-stress conditions.

A bacteriophage against Citrobacter braakii and its synergistic effect with antibiotics.

A bacteriophage BD49 specific for Citrobacter braakii was screened out and purified by double-layer plate method. It consists of a polyhedral head of 93.1 ± 1.2nm long and 72.9 ± 4.2nm wide, tail fibers, collar, sheath and baseplate. The bacteriophage was identified by morphology observed with transmission electron microscope (TEM), whole genome sequencing carried out by Illumina next generation sequencing (NGS) technique, and gene annotation based on Clusters of Orthologous Groups of proteins (COG) database. It was identified primarily as a member of Caudovirales by morphology and further determined as Caudovirales, Myoviridae, and Citrobacter bacteriophage by alignment of its whole genome sequence with the NCBI database and establishment of phylogenetic tree. The bacteriophage showed good environmental suitability with optimal multiplicity of infection (MOI) of 0.01, proliferation time of 80min, optimum living temperature of 30-40°C, and living pH of 5-10. In addition, it exhibited synergistic effect with ciprofloxacin against C. braakii in antibacterial tests.

Gene sequencing applications to combat oral-cavity related disorders: a systematic review with meta-analysis

Gene sequencing (GS) has numerous applications in combatting oral-cavity related disorders, including identifying genetic risk factors for diseases, developing targeted therapies, and improving diagnostic methods. It can help identify specific genetic mutations or variations that increase the risk of developing oral-cavity related disorders, such as oral cancer, periodontal disease, and cleft lip and palate. By the means of the following investigation, our primary objective was to assess the impact of GS technique in diagnosing and potentially treating diseases of the oral cavity by the means of a systematic review and meta-analysis. We commenced by defining the terms "gene sequencing," "oral cavity," and "disorders" as the important elements in our investigation's subject. Next, relevant databases like PubMed, Scopus, Embase, Web of Science, and Google Scholar were searched using keywords and synonyms for each concept, such as "genomic sequencing," "DNA sequencing," "oral health," "oral diseases," "dental caries," "periodontal disease," "oral cancer," and "salivary gland disorders." We combined several search terms, such as "gene sequencing AND oral disorders AND periodontal disease" or "oral cancer OR genomic sequencing," to further hone your search results using Boolean operators like "AND" and "OR." The oral cavity analysis obtained by CS in the selected articles revealed that most of the disorders were, in fact, a direct causal event influenced by the oral microbiome. Moreover, each sampled oral cavity evidenced a different microbial community, which predicted the precipitation of benign as well as malignant conditions, though not on a definitive basis. In the last ten years, genomic sequencing had advanced remarkably as majority of our selected studies observed, making it possible to diagnose and treat a variety of oral and maxillofacial disorders, including cancer. It was also used to ascertain a person's genetic make-up as well as to spot numerous genetic abnormalities that can predispose individuals to diseases. Understanding the different sequencing techniques and the resulting genetic anomalies may help with their clinical application and lead to an improvement in illness diagnosis and prognosis as a whole in the field of dentistry.