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Study Design.: A retrospective, observational study. Objective.: To evaluate the ability and value of metagenomic next-generation sequencing (mNGS) in detecting pathogens from spinal infections. Background.: The pathogenic diagnosis of primary spinal infection is challenging. The widespread application of mNGs in clinical practice makes it particularly useful in detecting rare, emerging, and atypical complex infectious diseases. Methods.: From January 2019 to December 2023, 120 samples were retrospectively collected from patients suspected of spinal infections and undergoing treatment. Pairwise comparisons between traditional laboratory tests and mNGS were conducted for all cases. Results.: Among the 120 cases, 95 were diagnosed as spinal infections, while 25 were classified as noninfectious. Microbiological evidence was found in 59 cases, while 36 cases were clinically diagnosed as spinal infections without definitive microbiological evidence. Rare microorganisms such as Aspergillus fumigatus , Taifanglania major , and Coxiella burnetii were detected by mNGS. The positive rate of mNGS was significantly higher at 88.42% compared with microbiological culture (43.16%), P <0.001. At the genus level, mNGS exhibited a consistency rate of 86.44% (51/59) with confirmed microorganisms. MNGS demonstrated very good agreement with clinically confirmed microorganisms at the genus level (κ=0.833). The sensitivity, specificity, positive predictive value, and negative predictive value of mNGS were 86.44%, 92.00%, 96.23%, and 74.19%, respectively. Conclusions.: The mNGS test exhibits rapidity, efficiency, and accuracy, rendering it of immense diagnostic and therapeutic value in the realm of spinal infection diseases. [ABSTRACT FROM AUTHOR]

The mitochondrial genome of Metazoa is predominantly maternally inherited, providing a valuable genetic marker in various fields such as phylogenetics and phylogeography. However, mitochondrial heteroplasmy, where multiple copies of mitochondrial DNA (mtDNA) coexist within an individual or even cell, presents a challenge to the use of mtDNA markers. This phenomenon may arise from replication errors, mutagenic processes, or paternal leakage. In this study, we investigated mitochondrial heteroplasmy in the freshwater bug Aphelocheirus aestivalis (Fabricius, 1794) using both Sanger and Illumina sequencing approaches. Our analyses confirmed the presence of diverse mtDNA variants within individual specimens collected from various locations across Europe. Signals of mitochondrial heteroplasmy were observed in chromatograms derived from both female and male individuals, indicating a prevalent phenomenon across A. aestivalis populations in general. Further comparative analyses of COI (partial cytochrome c oxidase subunit I) haplotypes indicated their high similarity and complex phylogenetic relationships. In turn, Illumina sequencing of a single male specimen revealed 102 heteroplasmic sites across the protein-coding sequences of mitogenome. Our results suggest the widespread and heritable nature of mitochondrial heteroplasmy in A. aestivalis, highlighting its importance for future phylogenetic and phylogeographic studies. [ABSTRACT FROM AUTHOR]

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Given the substantial chemical energy and harmful pollutants stored in refining wastewater (RW), energy recovery from RW has emerged as a promising strategy to address both energy scarcity and environmental degradation. In this study, we constructed a microbial fuel cell (MFC) facilitated by an electroactive microbial community to harness energy from RW. After the treatment of RW by MFC, high efficient removal rates of chemical oxygen demand (89.76%), NH 4 +-N (61.78%), total phosphorus (78.38%), chroma (81.53%), and turbidity (86.57%) were achieved. During the treatment of RW, the MFC system produced a maximum output voltage of 347 mV and a maximum power density of 337 mW m−2. High-throughput sequencing analysis revealed that the dominant phylum of domesticated microbial community was Proteobacteria (97.66%) and the dominant class was Gammaproteobacteria (70.10%). Furthermore, three dominant exoelectrogens (Paraburkholderia fungorum YS95, Pseudomonas sp. YS, and Stenotrophomonas maltophilia XS) capable of extracellular electron transfer were identified from the anodic samples. Notably, P. fungorum YS95 was identified as a novel exoelectrogen with significant naphthalene degradation capability. These results highlight the MFCs' efficiency in treating RW and recovering energy, with the discovery of exoelectrogens revealing the untapped potential for energy recovery from petroleum hydrocarbon pollutants. [Display omitted] • Refining wastewater treatment was achieved in MFC with 89.76% COD removal rate. • Energy recovery from refining wastewater was obtained with high power density. • Three dominant exoelectrogens were identified based on high-throughput sequencing. • A green alternative for energy recovery from refining wastewater was established. [ABSTRACT FROM AUTHOR]

This document discusses a rare case of aleukemic soft-tissue relapse in T-prolymphocytic leukemia post allogeneic hematopoietic cell transplantation. T-cell prolymphocytic leukemia is a rare T-cell malignancy with specific immunophenotypic characteristics. Treatment typically involves alemtuzumab followed by allogeneic hematopoietic cell transplant, but relapse rates remain high. The case study presented in the document highlights the challenges in managing this disease and the need for further research into targeted therapies. [Extracted from the article]

Wiskott-Aldrich syndrome (WAS) (MIM #301000) is a rare X-linked primary immunodeficiency due to mutations in the WAS gene, characterized by thrombocytopenia with small platelets, eczema, recurrent infections, and an increased incidence of autoimmunity and malignancies. A wide spectrum of mutations has been identified in the WAS gene responsible for a broad variety of clinical phenotypes. By using targeted next-generation sequencing (t-NGS), we identified in a 2-month-old boy with thrombocytopenia and immunological alterations a 4-nucleotide deletion from position +3 to +6 of intron 8 (c.777 + 3_777 + 6delGAGT) of WAS , currently classified on ClinVar as a variant of uncertain significance. The in-vitro characterization of the variant revealed the complete retention of intron 8 in the mature transcript, suggesting a splicing defect due to the loss of a splice donor site at the 5′-end of intron 8. By sequencing the polymerase chain reaction product, we identified a premature stop at codon 269; thus, consequently, no Wiskott-Aldrich syndrome protein (WASp) was detectable in peripheral blood mononuclear cells from the patient. Due to the total absence of a full-length WASp, it is expected that the patient will develop the severe form of the disease, although further monitoring is needed to better define his phenotype. [ABSTRACT FROM AUTHOR]

Background: Early diagnosis of pulmonary tuberculosis can greatly reduce the harm caused by the disease. However, traditional diagnostic methods have various shortcomings in diagnosing pulmonary tuberculosis. Currently, with the increasing popularity, iteration, and decreasing costs of Next-generation sequencing (NGS) testing technology, NGS is being more widely applied in the diagnosis of pulmonary tuberculosis. Case presentation: A 29-year-old male presented with "fever accompanied by cough for more than 20 days." Multiple chest CT scans revealed progressive enlargement of the right hilar lymph nodes and thickening of the interlobular septa in the right upper lobe. Routine testing of bronchoalveolar lavage fluid, search for tuberculosis bacilli, bacterial and fungal cultures, X-pert MTB/RIF, and multiplex PCR-based targeted Next-generation sequencing (mp-tNGS) results were all inconclusive. Finally, bronchoalveolar lavage fluid was sent for hybrid capture-based targeted Next-generation sequencing (hc-tNGS) testing, and special staining of the enlarged lymph nodes confirmed the diagnosis of pulmonary tuberculosis. Conclusion: The hc-tNGS has significant value in diagnosing pulmonary tuberculosis, especially in cases that are difficult to detect with other methods. In the future, this could gradually become a routine diagnostic method for pulmonary tuberculosis, enhancing the accuracy of early diagnosis. [ABSTRACT FROM AUTHOR]

Advances in high-throughput sequencing (HTS) have made it a powerful resource for the conservation of threatened species, providing information at both population and individual levels to inform management decisions. In South America, however, the application of HTS in conservation has been limited, primarily due to challenges in funding and access to advanced genomic equipment and analytical expertise. Darwin's fox (Lycalopex fulvipes), endemic to Chile's Valdivian Temperate Rainforest, is the most endangered canid in South America with a small and declining population estimated at less than 1000 mature individuals. Despite its endangered status, significant knowledge gaps remain. Here we highlight the potential of HTS to address these challenges, such as clarifying its taxonomy, demographic history, geographic distribution, population structure, genetic diversity, and pathogen exposure. Integrating molecular data into conservation planning will be pivotal in ensuring the long-term survival of Darwin's fox by identifying priorities for targeted management interventions, highlighting areas of critical habitat for conservation, and guiding genetic rescue efforts to enhance genetic diversity and resilience. [ABSTRACT FROM AUTHOR]

Background: The capability of mNGS in diagnosing suspected LRTIs and characterizing the respiratory microbiome in lung cancer patients requires further evaluation. Methods: This study evaluated mNGS diagnostic performance and utilized background microbial sequences to characterize LRT microbiome in these patients. GSVA was used to analyze the potential functions of identified genera. Results: Bacteria were the most common pathogens (n=74) in LRTIs of lung cancer patients, and polymicrobial infections predominated compared to monomicrobial infections (p<0.001). In diagnosing LRTIs in lung cancer patients, the pathogen detection rate of mNGS (83.3%, 70/84) was significantly higher than that of sputum culture (34.5%, 29/84) (p<0.001). This result was consistent with that of non-lung cancer patients (p<0.001). Furthermore, in the specific detection of bacteria (95.7% vs. 22.6%) and fungi (96.0% vs. 22.2%), the detection rate of mNGS was also significantly higher than that of CMTs mainly based on culture (p<0.001, p<0.001). However, in the detection of CMV/EBV viruses, there was no significant difference between the detection rate of mNGS and that of viral DNA quantification (p = 1.000 and 0.152). mNGS analysis revealed Prevotella , Streptococcus , Veillonella , Rothia , and Capnocytophaga as the most prevalent genera in the LRT of lung cancer patients. GSVA revealed significant correlations between these genera and tumor metabolic pathways as well as various signaling pathways including PI3K, Hippo, and p53. Conclusion: mNGS showed a higher pathogen detection rate than culture-based CMTs in lung cancer patients with LRTIs, and also characterizing LRT microbiome composition and revealing potential microbial functions linked to lung carcinogenesis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Cassava (Manihot esculenta, Crantz), is a staple food and the main source of calories for many populations in Africa, but the plant is beset by several damaging viruses. So far, eight families of virus infecting cassava have been identified; the Geminiviridae (ssDNA viruses responsible for cassava mosaic disease, CMD) and Potyviridae (ssRNA + viruses responsible for cassava brown streak disease, CBSD) families being the most damaging to cassava in Africa. In several cassava-growing regions, the co-existence of species and strains from these two families results in a complex epidemiological situation making it difficult to correctly identify the viruses in circulation and delaying the implementation of disease management schemes. Nevertheless, the development of next generation sequencing (NGS) methods has revolutionized plant virus detection and identification. One NGS method that has been successfully used in virus detection and identification is ribodepleted RNA sequencing. Unfortunately, the relatively high cost makes it difficult to upscale this method to large epidemiological surveys and limits its adoption as a diagnostic tool. Results: Here, we develop a high-throughput sequencing protocol, named Ribo-M-Seq, that combines plant rRNA ribodepletion, cDNA synthesis, tagging with a 96 multiplexing scheme and Illumina sequencing. We evaluated the protocol on a series of cassava samples with a known assemblage of viruses. After confirming that the protocol was suitable for ribodepletion, we demonstrated it was possible to detect RNA and DNA viruses via identification of near full-size genomes. Additional phylogenetic analyses confirmed the presence of begomoviruses and ipomoviruses responsible for CMD and CBSD, respectively. We also detected a recently described ampelovirus (Manihot esculenta-associated virus) that was not detected in previous analyses. Conclusions: The use of the Ribo-M-Seq protocol will pave the way for large-scale sample analyses of collections with potentially complex viromes, such as those collected in the West African cassava integrated pest management program. [ABSTRACT FROM AUTHOR]

Introduction: Cunninghamella elegans infections cause rare and severe mucormycosis. Bordetella bronchiseptica and Pneumocystis jirovecii relate to pneumonia. They are all clinically uncommon pathogens and no reports of co-infections have been reported. Case presentation: Here we present a case of a 67-year-old male patient who initially presented with fever, chills, and mild cough. B. bronchiseptica, P. jirovecii, Aspergillus fumigatus, and human alphaherpesvirus 1 (HSV1) were detected by clinical metagenomic next-generation sequencing (mNGS) of his bronchoalveolar lavage fluid (BLAF). Despite receiving anti-infective treatment, the patient rapidly developed respiratory failure and was transferred to the intensive care unit. Subsequent mNGS testing further revealed the presence of C. elegans, indicating that different pathogens played dominant roles at various stages of the disease progression. The routine culture also identified several of the above pathogens, but the results were reported much later than those of mNGS. Eventually, imaging findings and symptoms of the patient improved with comprehensive antibiotic coverage, and he was transferred to a lower-level hospital for rehabilitation treatment. Conclusions: This is the first detailed report of the combined infection of B. bronchiseptica, P. jirovecii, and C. elegans. During the treatment process, we also observed rare and unusual neurological side effects: visual and auditory hallucinations, restlessness, and aphasia. Also, the case indicates that traditional methods are insufficient for the etiological diagnosis needs of critical and severe patient populations, and timely use of mNGS should be recommended. [ABSTRACT FROM AUTHOR]

Background: Seedless grapes are in high demand for fresh and dry fruit consumption. Seedlessness in grapes (Vitis vinifera L.) is triggered by two different mechanisms: stenospermocarpy and parthenocarpy. However, the key regulators of seed development and their targets in grapes are not well characterized. The present study used the seeded grape hybrid ARI 516 and its seedless mutant to understand the molecular mechanisms controlling the seedless phenotype in grapes. Results: Gametogenesis studies demonstrated that the seedless mutant exhibits pollen sterility due to abnormal pollen morphology, significantly low viability, and a complete lack of germination ability. The macrogametophyte in the seedless mutant was significantly smaller than in ARI 516. Transcriptomic comparisons were performed during three developmental stages, including pre-flowering stage E-L 15, anthesis stage E-L 23, and berry formation E-L 31, to study altered developmental processes in the seedless mutant and ARI 516. Genes downregulated in the seedless mutant were enriched in the male gametophyte development-related pathways, which may cause pollen sterility. The RNAseq results were validated by qRT-PCR. Genome sequence data was also used to identify induced mutations in the seedless mutant, which revealed three homozygous and 25 heterozygous InDels in the genes related to male gametophyte development. RNAseq and genome sequencing data collectively indicate parthenocarpic seedless phenotype due to aberrant developmental and physiological processes involved in pollen formation, maturation and germination in the seedless mutant of ARI 516. Conclusion: The study showed the downregulation of transcription factors and their target genes involved in cell division, gibberellin biosynthesis and signalling, cell wall development, organization, and pollen germination. This study represents a comprehensive attempt to identify putative candidate genes associated with parthenocarpic pollen sterility in grapes using genomic approaches. [ABSTRACT FROM AUTHOR]

Background: This study was designed to characterize gut microbiota changes of the patients with gastric cancer before and after the gastrectomy during their hospital staying periods. Methods: 16S ribosomal RNA (rRNA) gene sequencing was used to evaluate differences in gut microbiota among patients with gastric cancer before and after the gastrectomy by comparing gut microbiota α diversity, β diversity, and structure composition at different taxonomic levels. Results: A total of 120 fecal specimens were collected from 60 patients. There was no significant difference in Chao1 index, Shannon index, and Simpson index before and after gastrectomy (all P > 0.05). At the phylum level, the gut microbiota in the gastrectomy group showed less abundance of Bacteroidota , Synergistota , and Verrucomicrobiota but with higher abundance of Campylobacter , Actinobacteria , and Bacillota. At the genus level, the gut microbiota in the gastrectomy group showed less abundance of flora Bacteroides , Faecalibacterium , Blautia , and Lachnospiraceae nk4a136 group but with higher abundance of Campylobacter , Porphyromona , Finegordia , Dialist , Anaerococcus , and Corynebacterium. Conclusions: There was no significant change in the diversity of intestinal flora before and after surgery. However, significant changes in the structure of intestinal flora before and after surgery were occurred. [ABSTRACT FROM AUTHOR]

Flavors and fragrances are essential for product quality, yet they are highly susceptible to contamination due to high moisture content and rich nutrients. This study investigates microbial growth, pH changes, volatile compound dynamics, and microbial community changes during the storage of flavors and fragrances. Results indicate that total viable counts (TVC) remained stable for the first three days but increased rapidly afterward, exceeding the acceptable limit of 5 log CFU/mL by day 7. The pH levels initially rose slightly, followed by a steady decline, which indicates spoilage progression. Gas chromatography–mass spectrometry (GC–MS) analysis revealed significant degradation of key aromatic compounds, such as 5-hydroxymethylfurfural (5-HMF), vanillin, and its derivative ethyl vanillin. Whole genome shotgun (WGS) sequencing demonstrated a marked increase in microbial community richness and diversity as storage progressed, with a notable shift in composition. Early storage stages were dominated by fungal species from the Ascomycota phylum, while later stages saw a rise in spoilage-associated bacteria, particularly from the Firmicutes and Proteobacteria phyla. Throughout the storage process, Zygosaccharomyces and its dominant species, Zygosaccharomyces bailii , remained prevalent, though their average relative abundance decreased from 81.26 to 32.29%. In addition, the bacterial species Oceanobacillus sojae and Niallia nealsonii showed significant increases in relative abundance, suggesting that bacteria were one of the key contributors to the spoilage of flavors and fragrances. Functional analysis based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database indicated a shift in metabolic pathways within the microbial community, with heightened metabolic activity correlating with spoilage. These findings provide valuable insights for improving storage methods and quality control of flavors and fragrances. [ABSTRACT FROM AUTHOR]

Myxobacteria are a special kind of Gram-negative bacteria that can slide and produce a variety of bioactive substances against bacteria, fungi, and viruses. It has great development and research value in medicine and agriculture. Although myxobacteria have become a research hotspot at home and abroad, there are few systematic studies on the relationship between its diversity, geographical location, and environment factors. In order to solve these problems, 133 soil samples were collected from the east to the west of Inner Mongolia Autonomous Region and divided into five groups. The water content, pH, organic matter, available phosphorus, hydrolytic nitrogen, and available potassium content of soil samples were determined by national standards and other methods. The quantitative assessment of the abundance of myxobacteria in the soil sample was performed by quantitative real-time PCR. The composition of myxobacteria in the soil was determined by 16S rRNA high-throughput sequencing technology to analyze the differences in the community structure of myxobacteria among different groups, explore the relationship between the diversity of myxobacteria resources and the distribution and physical and chemical properties of the soil, and predict and analyze its community function. The results showed that there were abundant myxobacteria resources in the soils of Inner Mongolia, and the average relative abundance of myxobacteria in the soil samples from the central part of Inner Mongolia was higher than that in the eastern and western parts, but the richness and diversity of samples from the central to eastern regions were significantly higher than those from the western regions. The myxobacteria resources in the whole region included 10 families and 22 genera, among which the dominant genera were Labilitrix , Sandaracinus , Archangium , and Haliangium. The analysis of the species composition of myxobacteria among different groups found that the distribution of soil and soil type had an impact on the species composition of the samples. The species with significant differences in relative abundance among the five groups of samples were Labilitrix , Archangium , Sandaracinus , Minicystis , Polyangium , and Myxococcus. In addition, the latitude and longitude of the sampling point and the soil pH, water content, available phosphorus content and organic matter content had a greater impact on the myxobacteria community structure of samples, while the altitude of the soil sample and the contents of available potassium and alkaline nitrogen had a relatively small impact. Our data suggest that the distribution, type and nutrient composition of soil samples have an impact on the relative abundance and species composition of myxobacteria community. The completion of this work can provide basic data for the in-depth study of myxobacteria in the soil. [ABSTRACT FROM AUTHOR]

Background: Fulminant myocarditis (FM), a critical cardiac disease, is characterised by atypical initial symptoms and rapid progression and tends to lead to cardiomyocyte degeneration or necrosis. Reliable biological markers for FM screening remain lacking. Circular RNAs (circRNAs) are highly stable in peripheral blood due to their special closed-loop structure, and reports have described their involvement in regulating inflammatory responses and cell injury in cardiomyocytes. This study attempted to identify the abnormal expression of circRNAs in the peripheral blood of patients with FM and to evaluate the potential diagnostic value. Methods: Peripheral blood was collected from 5 children with FM and 5 sex- and age-matched healthy controls; total RNA was extracted from each sample, and the extracted RNA from each group was pooled. After RNase R treatment, high-throughput sequencing was performed to screen for differentially expressed circRNAs in the peripheral blood of patients. Biological function classification and enrichment analysis were used to explore the main action pathways involving differentially expressed circRNAs. A lipopolysaccharide (LPS)-induced cardiomyocyte inflammation model was used to explore the effect of inflammation on the expression of these dysregulated circRNAs. Receiver operating characteristic (ROC) curves were used to evaluate the potential clinical value of FM-related circRNAs as biomarkers in a large sample of patients. Results: CircRNA expression profiling of peripheral blood samples from patients revealed 6,435 and 3,678 circRNAs with upregulated and downregulated expression, respectively, compared with healthy controls. The expression of 1,749 circRNAs did not significantly differ between the groups. GO and KEGG analysis revealed that the genes encoding the dysregulated circRNAs were associated with various biological functions related to the risk of FM development, including infectious diseases, the immune system, and signal transduction. The high expression of hsa_circ_0064338 (circ_PPARG) was confirmed in both the myocardial cell inflammation model and peripheral blood from a large sample of FM patients. ROC curve analysis showed that the level of circ_PPARG in peripheral blood had a good ability to distinguish patients with FM from healthy controls. Conclusions: Large numbers of abnormally regulated circRNAs were identified in peripheral blood from patients with FM; among these, the highly expressed circ_PPARG could distinguish patients from healthy controls to a certain extent. It is expected to become a potential clinical biomarker of FM in the future. [ABSTRACT FROM AUTHOR]

Introduction: Immunoglobulin (Ig) refers to the nomenclature for all antibody proteins produced by B lymphocytes. The genetic locus encoding Ig is critical for vertebrate humoral immune responses and diverse antibody repertoires. Despite the critical role of buffaloes as livestock and their significance in disease transmission, the Ig loci of this species have not been thoroughly annotated. This study aimed to systematically characterize the Ig loci in buffaloes and their unique features, providing a foundation for understanding buffalo immune function. Methods: The genomic assembly of Murrah buffalo (NDDB_SH_1) was analyzed to annotate Ig loci. Annotation criteria included functional motifs, RSS sequences, and structural features of V, D, J, and C genes. The CDR3 repertoires were constructed using genomic DNA extracted from spleen samples of five healthy buffaloes. High-throughput sequencing of multiplex PCR products enabled repertoire analysis, and MiXCR software was used for alignment and CDR3 extraction. Repertoire diversity, gene usage, and clonal frequencies were analyzed using the Immunarch R package. Results: The IgH locus spans approximately 667 kb on chromosome 20, containing two D-J-C clusters, 54 VH genes, 10 DH genes, 8 JH genes, and 9 CH genes. The Igκ locus, located on chromosome 12, encompasses 24 Vκ genes, 5 Jκ genes, and 1 Cκ gene, while the Igλ locus on chromosome 17 includes 71 Vλ genes, 3 Jλ genes, and 3 Cλ genes. We also conducted a detailed examination of the buffalo IgH CDR3 repertoire, revealing the presence of ultra-long CDR3 sequences, a biased usage of certain V genes, and a high-frequency usage of IgHJ1-4 genes. Furthermore, we identified a set of shared clonotypes across the samples, highlighting commonalities in the buffalo antibody repertoire. Conclusion: These findings contribute to the understanding of buffalo immune function and provide insights into the evolution and diversity of ruminant immunoglobulin genes. [ABSTRACT FROM AUTHOR]

This case report presents a patient with metastatic adenocarcinoma of unclear primary focus at initial presentation and revealed lung adenocarcinoma in subsequent follow-up. The patient has been surviving for more than 10 years after pemetrexed-based treatment and local radiotherapy. Sequential gene tests showed kirsten rat sarcoma viral oncogene homolog (KRAS) G13D mutation and epidermal growth factor receptor (EGFR) 19ins. To further investigate the correlation between pemetrexed efficacy and genetic polymorphisms, genotyping tests on folate-metabolism-related genes [methylenetetrahydrofolate reductase (MTHFR) (C677T) and MTHFR (A1298C)] were performed, revealing that the patient exhibited the T/T genotype for MTHFR (C677T) and the A/A genotype for MTHFR (A1298C). The clinical data and gene analysis were discussed with literature review to explain the underlying explanation for the long survival. [ABSTRACT FROM AUTHOR]

Introduction: Little was known about the characteristics of low respiratory tract (LRT) microbiota of refractory M. pneumoniae pneumonia (RMPP) in children before and after the COVID-19 pandemic. Methods: Forty-two children diagnosed with RMPP in 2019 (Y2019 group) and 33 children diagnosed with RMPP in 2023 (Y2023 group), entered into the study. The characteristics of the clinical findings were examined, and the LRT microbiota was analyzed by metagenomic next generation sequencing. Results: The ratio of consolidate, atelectasis, lung necrosis, and erythema multiforme in Y2023 group was significantly higher than that in Y2019 (P <0.05). Mycoplasmoides pneumoniae was the top species of the LRT microbiota in both groups. The rate of macrolide resistance MP in Y2023 was significantly higher than that in Y2019 (P <0.05), and the mutant site was all 23S rRNA A2063G. There were no significant differences in α-diversity and β-diversity of LRT microbiota between Y2019 and Y2023 group. Trichoderma citrinoviride, Canine mastadenovirus A, Ralstonia pickettii, Lactococcus lactis, Pseudomonas aeruginosa were the biomarkers of LRT microbiota in children with RMPP of Y2023. The abundance of Mycoplasmoides pneumoniae positively correlated with the levels of D-dimer and LDH, negatively correlated with the counts of CD3+ T cells, CD8+ T cells, CD19+ B cells and CD16+CD56+ NK cells. Discussion: Our study showed that high abundance of MP was correlated with the severity of RMPP and decrease of immune cells. Trichoderma citrinoviride, Canine mastadenovirus A, Ralstonia pickettii, Lactococcus lactis, Pseudomonas aeruginosa were the biomarkers in microbiota of LRT in children with RMPP post COVID-19 era. [ABSTRACT FROM AUTHOR]

MODY, or maturity-onset diabetes of the young, is a group of monogenic diseases characterized by autosomal dominant inheritance of a non-insulin-dependent form of diabetes that classically manifests in adolescence or in young adults under 25 years of age. MODY is a rare cause of diabetes, accounting for 1% of all cases, and is often misdiagnosed as type 1 or type 2 diabetes. It is of great importance to accurately diagnose MODY, as this allows for the most appropriate treatment of patients and facilitates early diagnosis for them and their families. This disease has a high degree of phenotypic and genetic polymorphism. The most prevalent forms of the disease are attributed to mutations in three genes: GCK (MODY 2) and (HNF)1A/4A (MODY 3 and MODY 1). The remaining MODY subtypes, which are less prevalent, have been identified by next generation sequencing (NGS) in the last decade. Mutations in the GCK gene result in asymptomatic, stable fasting hyperglycemia, which does not require specific treatment. Mutations in the HNF1A and HNF4A genes result in pancreatic β-cell dysfunction, which in turn causes hyperglycemia. This often leads to diabetic angiopathy. The most commonly prescribed drugs for the treatment of hyperglycemia are sulfonylurea derivatives. Nevertheless, with advancing age, some patients may require insulin therapy due to the development of resistance to sulfonylurea drugs. The strategy of gene therapy for monogenic forms of MODY is still an experimental approach, and it is unlikely to be widely used in the clinic due to the peculiarities of MODY structure and the high genetic polymorphism and clinical variability even within the same form of the disease. Furthermore, there is a lack of clear gene-phenotypic correlations, and there is quite satisfactory curability in the majority of patients. This review presents the main clinical and genetic characteristics and mutation spectrum of common and rarer forms of MODY, with a detailed analysis of the field of application of AVV vectors in the correction of hyperglycemia and insulin resistance. [ABSTRACT FROM AUTHOR]

Objectives Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is compromising gonorrhoea treatment, and enhanced N. gonorrhoeae AMR and genome-based epidemiological surveillance is imperative. Molecular tests are replacing N. gonorrhoeae culture internationally, excluding possibilities to perform WGS. We describe and evaluate a novel approach using a custom SureSelectXTHS Target-Enrichment probe panel automated on the Magnis NGS Prep System and Illumina sequencing to generate accurate N. gonorrhoeae genomes directly from clinical urogenital and extragenital specimens. Methods One hundred thirteen clinical N. gonorrhoeae -positive APTIMA Combo 2 (AC2) specimens (with 89 linked N. gonorrhoeae isolates) were included. DNA was extracted using QIAsymphony DSP Virus/Pathogen kit. Amplisens multiplex RT–PCR assay (AM-PCR) identified 105 (92.9%) of the AC2 specimens as N. gonorrhoeae positive, which were further examined. Sequence libraries for AC2 specimens were prepared on the Magnis NGS Prep System using the Magnis SureSelectXTHS Reagent kit for Illumina paired-end platforms. Paired-end sequencing was performed on Illumina platforms. Results Seventy-four of the 105 (70.5%) AC2 samples remained N. gonorrhoeae positive with a cycle threshold <20 in the AM-PCR and subjected to SureSelectXTHS target enrichment and subsequently Illumina WGS. Seventy-two (97.3%) of all target-enriched specimens were successfully genome-sequenced. All linked AC2 specimens and N. gonorrhoeae isolates from the same anatomical site had identical AMR determinants and molecular epidemiological sequence types. Conclusions We show that custom SureSelectXTHS target enrichment automated on the Magnis NGS Prep System, followed by Illumina sequencing, enables culture-independent genome-based surveillance of N. gonorrhoeae AMR and molecular epidemiology. This novel methodological advancement provides an efficient and accurate WGS of N. gonorrhoeae directly from clinical urogenital and extragenital NAAT specimens. [ABSTRACT FROM AUTHOR]

The Tibetan Plateau has a large number of hot springs with varying temperatures and hydrochemistry, high elevation, and limited nitrogenous nutrition. Nitrogen-fixing bacteria (NFB) can fix N2 to form ammonia and thus provide bioavailable nitrogen. However, there is limited knowledge about the distribution of NFB and its influencing factors in Tibetan hot springs. Here, we measured hydrochemical variables of the hot springs with a wide temperature range (32–77 °C) in the Qucai and Daggyai geothermal zones on the Tibetan Plateau and investigated the composition of NFB using high-throughput sequencing of 16S rRNA and nifH genes. The Cl−/SO42− ratio in Qucai hot springs was higher than that in Daggyai, indicating that Qucai and Daggyai hot springs were more affected by the supply of liquid and gaseous phases, respectively. The NFB communities consisted predominantly of Nirtospirae, Chloroflexi, Deltaproteobacteria and an unidentified clade, with the last three acting as the main NFB with over 42% of the communities (the proportions are significantly larger than those found in hot springs of other geothermal regions). This demonstrates the uniqueness of NFB communities in Tibetan hot springs. NFB richness was limited by temperature in the studied Tibetan hot springs and was significantly lower than in low-elevation geothermal regions. The NFB community was predominantly affected by hydrochemistry, in contrast to the entire prokaryotic community, which was primarily influenced by temperature. This study expands our current understanding of NFB distribution and diversity as well as biogeochemical process in geothermal spring environments. [ABSTRACT FROM AUTHOR]

5-Methylcytosine (m5C) plays a pivotal role in various RNA metabolic processes, including RNA localization, stability, and translation. Current high-throughput sequencing technologies for m5C site identification are resource-intensive in terms of cost, labor, and time. As such, there is a pressing need for efficient computational approaches. Many existing computational methods rely on intricate hand-crafted features, requiring unavailable features, often leading to suboptimal prediction accuracy. Addressing these challenges, we introduce a novel deep-learning method, Trans-m5C. We first categorize m5C sites into NSUN2-dependent and NSUN6-dependent types for independent feature extraction. Subsequently, meticulously crafted transformer neural networks are employed to distill global features. The prediction of m5C sites is then accomplished using a discriminator built from a multi-layer perceptron. A rigorous evaluation for the performance of Trans-m5C on experimentally validated m5C data from human and mouse species reveals that our method offers a competitive edge over both baseline and existing methodologies. • Integrated Transformer neural network into the Trans-m5C model. • First to predict NSUN2- and NSUN6-dependent m5C sites with high precision. • Trans-m5C outperforms existing models for human and mouse species. • Provides step-by-step downloadable codes for researchers. [ABSTRACT FROM AUTHOR]

• CHiTA is a scarless high-throughput ribozyme characterization assay. • We provide a guide for implementing CHiTA to study large pools of ribozymes. • We discuss potential applications and adaptations of CHiTA for various uses. Small self-cleaving ribozymes are catalytic RNAs that cleave their phosphodiester backbone rapidly and site-specifically, without the assistance of proteins. Their catalytic properties make them ideal targets for applications in RNA pharmaceuticals and bioengineering. Consequently, computational pipelines that predict or design thousands of self-cleaving ribozyme candidates have been developed. Traditional experimental techniques for verifying the activity of these putative ribozymes, however, are low-throughput and time intensive. High-throughput (HT) pipelines that employ next-generation sequencing (NGS) analyze the activity of these thousands of ribozymes simultaneously. Until recently, the application of these HT pipelines has been limited to studying all single and double mutants of a select representative ribozyme. Unfortunately, this prevents the exploration of candidates having different lengths, circular permutations, and auxiliary stem-loops. Moreover, pipelines that analyze ribozymes en masse often include transcription of non-native flanking sequences that preclude accurate assessment of the intrinsic rate of ribozyme self-cleavage. To overcome these limitations, we developed a HT pipeline, " C leavage Hi gh- T hroughput A ssay (CHiTA)", which employs NGS and massively parallel oligonucleotide synthesis (MPOS) to characterize ribozyme activity for thousands of candidates in a scarless fashion. Herein, we describe detailed strategies and protocols to implement CHiTA to measure the activity of putative ribozymes from a wide range of ribozyme classes. [ABSTRACT FROM AUTHOR]

Objectives: This study aims to discover the role of lncRNA MIR17HG, referred to as MIR17HG, in cisplatin resistance for cholangiocarcinoma (CCA). Methods: QRT-PCR was conducted to measure the expression of MIR17HG in cisplatin-resistant/sensitive CCA cells and clinical CCA specimens. Log-rank test was used to analyze the survival curve. Cck8-assay and flow cytometry were employed to detect the sensitivity of CCA cells to cisplatin and the apoptosis rate following different treatments, respectively. The next-generation sequencing was carried out to get gene transcripts after silencing MIR17HG in HCCC-9810 cells. The LncBase database was used to predict the target miRNA of MIR17HG, and MS2 RIP assay and dual luciferase assay were conducted to confirm their binding. MiRwalk database and the RNA sequencing data were utilized to screen the key genes regulated by MIR17HG/miR-138-5p axis and a dual luciferase assay was performed to confirm the binding site of miR-138-5p with AKAP9. Immunoblotting was further employed to give assistant evidence. Rescue experiments were performed to observe the function of miR-138-5p and AKAP9 in MIR17HG-induced cisplatin resistance. Results: MIR17HG overexpression predicts cisplatin resistance and poor prognosis in CCA. MIR17HG could bind with miR-138-5p to release AKAP9, thereby inhibiting cisplatin-induced apoptosis and promoting cisplatin resistance in CCA. MIR17HG silencing in CCA cells leads to expression alteration of genes, which are enriched in platinum resistance-related pathways. Conclusions: LncRNA MIR17HG regulates platinum resistance-associated genes and promotes cisplatin resistance partially via the miR-138-5p/AKAP9 axis by inhibiting cisplatin-induced apoptosis in CCA. [ABSTRACT FROM AUTHOR]

Liquid biopsies are a minimally invasive approach to obtain biomarkers including cell-free DNA (cfDNA) from peripheral blood. To date, there are limited and conflicting studies evaluating their role in thyroid cancer. Our study evaluated the utility of cfDNA in advanced thyroid cancers. Patients aged >18 years with metastatic medullary thyroid cancer (MTC), poorly differentiated thyroid cancer (PDTC) or anaplastic thyroid cancer (ATC) were enrolled in this prospective study between 2020 and 2024. As part of standard care, sequencing of germline and tumoral DNA was conducted, and patients with germline mutations were excluded from the study. Whole blood samples were collected in Streck cell-free DNA BCT tubes, and cfDNA was extracted from plasma using an EZ1&2 ccfDNA kit and EZ2 Connect. The extracted cfDNA was then sequenced across 50 key cancer-related genes using an Oncomine Precision Assay panel on an Ion Torrent Genexus integrated sequencer. Forty patients were included: 27 MTC, 2 ATC and 11 PDTC. Tumoral mutations were detected in 36 of the included patients (90%): cfDNA detected mutations in 18/36 patients (13 MTC, 1 ATC and 4 PDTC (50%)). The sensitivity of cfDNA was 86% (6/7) pre-tyrosine kinase inhibitor therapy and reduced to 54% on therapy (13/24), suggestive of the lack of tumor-derived DNA shedding with a strong on-target treatment efficacy. The median cfDNA concentration was higher in samples with a detected mutation (n = 43) than those without (n = 26), 11.91 ng/mL vs 5.81 ng/mL, respectively, while an increasing cfDNA was associated with worse progression-free survival (P < 0.01). cfDNA is a novel biomarker with potential to monitor disease progression in patients with advanced thyroid cancers. [ABSTRACT FROM AUTHOR]

River ecosystems are intricately connected to the biogeochemical processes mediated by sediment bacterial communities. The Three Gorges Reservoir, a critical freshwater resource, has seen limited research on bacterial community composition, particularly in comparative studies between its mainstream and tributaries. High-throughput sequencing was utilized to examine bacterial diversity and community structure in the sediments of the Wanzhou section. The analysis uncovered significant spatiotemporal heterogeneity and revealed similar community structures between the mainstream and tributaries, with Proteobacteria, Actinobacteria, and Bacteroidetes as the predominant phyla. Although no significant phylum-level differences were observed among the top five dominant species, their relative abundances showed notable variations. These differences are attributed to the reservoir's cyclical water storage and release, which alter nutrient concentrations and induce seasonal variations in bacterial communities. The intrinsic growth characteristics of microorganisms and their adaptive responses to environmental changes are identified as key factors influencing bacterial community differentiation. These insights enhance the understanding of bacterial dynamics in freshwater sediments and contribute to ecological assessments and environmental management in the Three Gorges Reservoir area. [ABSTRACT FROM AUTHOR]

Background: To develop efficient selection strategies and improve the discovery of promising ligands, it is highly desirable to analyze the sequence composition of naïve phage display libraries and monitor the evolution of their peptide content during successive rounds of amplification. In the current study, we performed a comparative analysis of the compositional features in different lots of the same naïve phage display library and monitored alterations in their peptide compositions during three rounds of amplification. Methods: We conducted three rounds of duplicate serial amplification of two different lots of the Ph.D.™-12 phage display library. DNA from the samples was subjected to Next-Generation Sequencing (NGS) using an Illumina platform. The NGS datasets underwent a variety of bioinformatic analyses using Python and MATLAB scripts. Results: We observed substantial heterogeneity in the sequence composition of the two lots indicated by differences in the enhanced percentage of wildtype clones, reduced diversity (number of unique sequences), and increased enrichment factors (EFs) during amplification as well as by observing no common sequence between lots and decreased number of common sequences between the naïve library and the consecutive rounds of amplification for each lot. We also found potential propagation-related target-unrelated peptides (TUPs) with the highest EFs in the two lots, which were displayed by the fastest-propagating phage clones. Furthermore, motif analysis of the most enriched subpopulation of amplified libraries led to the identification of some motifs hypothesized to contribute to the increased amplification rates of the respective phage clones. Conclusion: Our results highlight tremendous heterogeneity in the peptide composition of different lots of the same type of naïve phage display library, and the divergent evolution of their compositional features during amplification rounds at the amino acid, peptide, and motif levels. Our findings can be instrumental for phage display researchers by bringing fundamental insights into the vast extent of non-uniformity between phage display libraries and by providing a clear picture of how these discrepancies can lead to different evolutionary fates for the peptide composition of phage pools, which can have profound impacts on the outcome of phage display selections through biopanning. [ABSTRACT FROM AUTHOR]

Gut single-cell eukaryotes (GSCEs) are residents of billions of people, yet their roles in gut ecology remain largely unknown. Understanding GSCEs is crucial to avoiding misguided eradication efforts that could disrupt gut microbiome homeostasis. Retrospective analyses suggest links between GSCEs, gut bacteria, and host health, but expose methodological pitfalls. Current methods for studying GSCEs are underdeveloped, accentuating the need for more reference genomes and improved bioinformatic pipelines. Blastocystis is often studied in isolation, which comes with a risk of misattributing effects that could be due to other GSCEs. Gut single-celled eukaryotes (GSCEs) are found in billions of people worldwide, but we still know little about their functions and relationships in human gut ecology. Lately, retrospective analysis of bacterial data obtained by next-generation sequencing (NGS) methods has been used to identify links between GSCEs, gut bacteria, host metabolism, and host phenotypical traits, suggesting possible direct or indirect associations to favorable gut microbiome features and other health parameters. Here, we highlight some of the pitfalls related to the research strategy typically used so far and propose action points that could pave the way for a more accurate understanding of GSCEs in human health and disease. [ABSTRACT FROM AUTHOR]

Despite being present in many North American forest understories, the ectomycorrhizal (ECM) fungal communities associated with Corylus shrubs have received no prior study. To address this knowledge gap, we characterized the ECM fungal communities on roots of Corylus shrubs as well as co-occurring Quercus and Pinus trees in Minnesota, USA. ECM-colonized root tips from pairs of Corylus shrubs and four ECM tree species, Quercus macrocarpa, Quercus ellipsoidalis, Pinus strobus, and Pinus resinosa, growing in close proximity (<1 m), were sampled at the Cedar Creek Ecosystem Science Reserve. ECM fungal communities were assessed using high-throughput sequencing of the ITS2 region. ECM fungal operational taxonomic unit (OTU) richness was equivalent among the two Quercus species and their associated Corylus shrubs, but significantly higher on P. strobus–associated Corylus shrubs compared with P. strobus, P. resinosa, and P. resinosa–associated Corylus shrubs. ECM fungal community composition on Corylus shrubs largely mirrored that on each of the Quercus and Pinus species, although the two Pinus communities were significantly different from each other. Further, the same ECM fungal OTUs were commonly encountered on paired Corylus–tree host samples, suggesting a high potential for co-colonization by the same fungal individuals. Collectively, these results support the growing consensus that woody understory plants often associate with similar ECM fungal communities as co-occurring tree hosts regardless of phylogenetic relatedness. [ABSTRACT FROM AUTHOR]

The boundary between polygenic and monogenic diabetes is less distinct than previously believed, with oligogenic forms providing a bridge between these forms. The advent of large-scale next-generation sequencing has enabled the identification of rare variants in several genes causing oligogenic type 2 diabetes. Identification of these rare oligogenic variants has been achieved through two primary approaches: targeted studies of candidate genes enhanced by functional analyses, and large-scale exome- or genome-wide sequencing studies. The demarcation between monogenic and polygenic type 2 diabetes (T2D) is less distinct than previously believed. Notably, recent research has highlighted a new entity, that we suggest calling oligogenic forms of T2D, serving as a genetic link between these two forms. In this opinion article, we have reviewed scientific advances that suggest categorizing genes involved in oligogenic T2D. Research focused on polygenic T2D has faced challenges in deepening our comprehension of the pathophysiology of T2D due to the inability to directly establish causal links between a signal and the molecular mechanisms underlying the disease. However, the study of oligogenic forms of T2D has illuminated distinct causal connections between genes and disease risk, thereby indicating potential new drug targets. [ABSTRACT FROM AUTHOR]

Cryptic MHC-associated peptides (MAPs) originate from various genomic elements that generate short, rapidly degraded polypeptides that are very efficient substrates for MHC I processing. The systematic exploration of the cryptic immunopeptidome is best achieved by integrating RNA-Seq, Ribo-Seq, and mass spectrometry analyses. In several cancer types, most tumor-specific antigens are cryptic MAPs resulting from epigenetic changes and splicing aberrations. Transposable elements are genetic parasites that contribute to the cryptic immunopeptidome and regulate thymic development and function. They are highly expressed in thymic medullary cells, crucial for establishing self-tolerance. Viruses generate cryptic MAPs that can be highly immunogenic and should be considered when designing RNA vaccines. Peptides presented by MHC proteins regulate all aspects of T cell biology. These MHC-associated peptides (MAPs) form what is known as the immunopeptidome and their comprehensive analysis has catalyzed the burgeoning field of immunopeptidomics. Advances in mass spectrometry (MS) and next-generation sequencing have facilitated significant breakthroughs in this area, some of which are highlighted in this article on the cryptic immunopeptidome. Here, 'cryptic' refers to peptides and proteins encoded by noncanonical open reading frames (ORFs). Cryptic MAPs derive mainly from short unstable proteins found in normal, infected, and neoplastic cells. Cryptic MAPs show minimal overlap with cryptic proteins found in whole-cell extracts. In many cancer types, most cancer-specific MAPs are cryptic. [ABSTRACT FROM AUTHOR]

Purpose: Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, but the therapies are not satisfactory. This study aimed to find AMD specific features through the analysis of high-throughput sequencing. Methods: In this study, we integrated six projects containing single-cell RNA sequencing (scRNA-seq) data to perform a comprehensive analysis for AMD samples in the tissues of retina and retinal pigment epithelium/choroid, and in the positions of macula and periphery. Differentially expressed genes (DEGs) were analyzed and crucial signaling pathways were identified across cell types and between the macula and periphery. The intercellular signaling transduction among cell types were inferred by "CellChat" to build cell–cell communication network under normal and AMD conditions, and verified at the transcriptional level. The CD31+ endothelial cells were obtained to evaluate the enrichment of KEGG pathways in atrophic and neovascular AMD, and GSVA was adopted to discover differential metabolic signals in each AMD type. Results: Thirteen major cell types were identified in the integrated scRNA-seq data. Although no disease-specific cell type or differential cell proportion was found, DEGs and enriched pathways were shown in cell-type- and position-dependent manners. Severe impairment of endothelial cell-mediated cell interactions was found in the signaling transduction network of the macula, and compromised cell interactions were observed in the periphery. Furthermore, distinct signaling pathways and metabolic states were uncovered in atrophic and neovascular AMD. Striking reduction in energy metabolism, lipid metabolism, and oxidative stress was indicated in the atrophic AMD. Conclusion: Conclusively, we discover aberrant signals and metabolic pathways in AMD samples, providing insight into mechanisms and potential therapeutic targets for the AMD treatment. [ABSTRACT FROM AUTHOR]

Expanded carrier screening (ECS) is a preventive genetic test that enables couples to know their risk of having a child affected by certain monogenetic diseases. This study aimed to evaluate the carrier frequency for rare monogenic diseases in the general Chinese population and the impacts of ECS on their reproductive decisions and pregnancy outcomes. This single-center study was conducted between September 2022 and April 2023. An ECS panel containing 224 recessive genes was offered to 1,499 Chinese couples from the general population who were at early gestational ages or planned to conceive. Overall, 55.0 % of the individuals carried for at least one recessive condition. There were 16 autosomal recessive (AR) genes with a carrier frequency of ≥1/100 and 22 AR genes with a carrier frequency of <1/100 to ≥1/200. The most common AR and X-linked diseases were GJB2-related non-syndromic hearing loss, and hemolytic anemia, respectively. Fifty-five couples (3.67 %; 1 in 27.3) were at increased risk of having an affected child with 19 pregnant at the time of testing. Of these, 10 opted for amniocentesis, and four affected pregnancies were identified, with three of them being terminated. This study not only provides valuable information about the recessive genetic landscape, but also establishes a solid foundation for couple-based ECS in a real clinical setting. [ABSTRACT FROM AUTHOR]

Metagenomic next-generation sequencing (mNGS) was previously established as a method that can increase the pathogen identification rate in patients with severe community-acquired pneumonia (SCAP). What is the impact on clinical outcomes of mNGS of BAL fluid (BALF) in patients with SCAP in the ICU? A multicenter randomized controlled open-label clinical trial was conducted in 10 ICUs. Patients were randomized in a 1:1 ratio to undergo BALF assessment with conventional microbiological tests (CMTs) only (ie, the CMT group) or BALF assessment with both mNGS and CMTs (ie, the mNGS group). The primary outcome was the time to clinical improvement, defined as the time from randomization to either an improvement of two points on a six-category ordinal scale or discharge from the ICU, whichever occurred first. A total of 349 patients were randomized to treatment between January 1, 2021, and November 18, 2022; 170 were assigned to the CMT group and 179 to the mNGS group. In the intention-to-treat analysis, the time to clinical improvement was better in the mNGS group than in the CMT group (10 days vs 13 days; difference, –2.0 days; 95% CI, –3.0 to 0.0 days). Similar results were obtained in the per-protocol analysis. The proportion of patients with clinical improvement within 14 days was significantly higher in the mNGS group (62.0%) than in the CMT group (46.5%). There was no significant difference in other secondary outcomes. We found that compared with the use of CMTs alone, mNGS combined with CMTs reduced the time to clinical improvement for patients with SCAP. Chinese Clinical Trial Registry, ChiCTR; www.chictr.org.cn/index.html ; ChiCTR2000037894. [ABSTRACT FROM AUTHOR]

The clinicopathological spectrum of undifferentiated round cell sarcomas of bone and soft tissues is expanding after the 5th edition of the WHO classification. A 23-year-old male patient presented with a lump in his left thigh of 3 months' duration. Radiological examination revealed a well-defined, solid-cystic lobulated, soft tissue lesion in the proximal medial region of his left thigh, measuring 7.7 cm in the largest dimension. The referring diagnosis was an epithelioid sarcoma. Histopathological review of the tumor sections revealed a cellular tumor composed of malignant epithelioid to focally "rhabdoid-like" cells in a variable hyalinized and myxoid stroma with geographic areas of necrosis. In addition, there were areas reminiscent of hemangiopericytomatous vasculature. By immunohistochemistry, the tumor cells were diffusely positive for CD34, focally and distinctly for pan keratin (AE1/AE3). INI1/SMARCB1 and SMARCA4 (BRG1) were diffusely positive (normal). Next-generation sequencing with a wide sarcoma panel revealed EWSR1exon8::ZBT44exon4 fusion. The present example constitutes the first malignant epithelioid tumor with a hemangiopericytomatous growth pattern, exhibiting this rare fusion. The differential diagnoses of this tumor and their corresponding immunohistochemical profile are discussed. This example highlights the value of NGS in unraveling rare fusions and in differentiating these tumors from their several mimics. [ABSTRACT FROM AUTHOR]

To investigate the bacterial community structure and physicochemical characteristics of different types of Daqu in the Binzhou region, this study employed traditional pure culture methods, high-throughput sequencing technology, and conventional physicochemical assays for analysis. The research results indicate that Enterococcus faecium and Bacillus licheniformis emerged as the main LAB and Bacillus species in Daqu from Binzhou region, respectively. In addition, high-throughput sequencing revealed significant differences in bacterial community structure between the two types of Daqu (P < 0.01). Compostibacillus and Sebaldella were identified as the biomarkers and potential key strains of high- and medium-temperature Daqu, respectively, and high-temperature Daqu demonstrated higher microbial complexity and stability than medium-temperature Daqu. Physicochemical assays demonstrated that the a* value, Daqu skin hardness, Daqu core hardness, density, starch content, and aminophenol content being significantly higher in high-temperature Daqu (P < 0.05), meanwhile, the L* value, water activity, water content, protein content, liquefaction power, and saccharification power were found to be significantly lower in high-temperature Daqu (P < 0.05). And there was significant association between dominant genera and the physicochemical indexes of Daqu (P = 0.001). It can thus be seen that there were significant differences between the microbial communities and physicochemical indicators of different types of Daqu in the Binzhou region. The results of this study are of great significance for further analyzing the differences between different types of Daqu and improving their quality. [ABSTRACT FROM AUTHOR]

Objectives: Depression is a widely prevalent mental disorder, and nutritional interventions play an increasingly important role in its treatment. In this paper, effects of linoleic acid (LA) on depressive behavior in mice induced by gut microbiome disorders were investigated. Methods: Fifty C57BL/6J male mice were randomly separated into five groups, control group (CK), ceftriaxone sodium group (CRO), low-dose linoleic acid group (LLA, 1 g/kg), medium-dose linoleic acid group (MLA, 2 g/kg), and high-dose linoleic acid group (HLA, 5 g/kg). In the LLA, MLA, and HLA groups, mice were treated with ceftriaxone sodium (CRO) to induce depressive behaviors, followed by LA administration. Behavioral tests were used to evaluate depressive behavior. High-throughput sequencing and Hematoxylin–eosin (H&E) staining in gut microenvironment were carried out. ELISA kits were used to measure brain inflammatory factors, and 5-hydroxy-tryptamine (5-HT). Gas chromatography and western blot were used to determine fatty acids compositions and the enzymes expression involved in lipid metabolism in brain respectively. Results: The results showed that 10 weeks CRO treatment contribute to depressive behavior, gut microbiome disturbance, and serotonin system disturbance. LLA and MLA improved the depressive-like behavior, and significantly increased the levels of 5-HT1A, 5-HTT and 5-HT in the hippocampus. LLA was found to improve the diversity of gut microbiome and alleviate colon tissue damage. Meantime, LLA increased the content of linoleic acid, improved the expression of FADS2 and COX-2, increased IL-10 levels, and decreased IL-6 levels in the brain. Discussion: LA alleviated depressive behavior in mice by improving the gut microenvironment, regulate fatty acid metabolism, and modulate inflammation. [ABSTRACT FROM AUTHOR]

Brucellosis with neurological symptoms at onset is rare in children and is frequently misdiagnosed or overlooked due to nonspecific clinical presentations, particularly in non-endemic areas. We report a case of neurobrucellosis in a child from a non-pastoral area, diagnosed via metagenomic next-generation sequencing (mNGS). The patient presented with headache and altered consciousness, accompanied by fever, projectile vomiting, seizures, and urinary incontinence. Physical examination indicated possible nuchal rigidity. Cerebrospinal fluid (CSF) analysis showed colorless and clear fluid, with a white blood cell count of 259 × 10⁶/L, 4.6% polymorphonuclear cells, positive protein qualitative test, protein level of 0.78 g/L, and glucose level of 1.66 mmol/L. Initial diagnosis suggested central nervous system infection, and empirical treatment led to improvement in consciousness. However, after a few days of stable body temperature, the patient experienced recurrent fever. Ultimately, mNGS of CSF identified Brucella melitensis, confirming neurobrucellosis. Following treatment with ceftriaxone, doxycycline, and rifampin, the patient's clinical symptoms improved significantly, and follow-up CSF analysis showed normalization of cell counts. This case highlights the early diagnostic utility of mNGS in CSF for neurobrucellosis and its role in differential diagnosis. [ABSTRACT FROM AUTHOR]

A recent trend in some wineries is the return to using spontaneous fermentation, but it is not clear whether winery flora or vineyard microorganisms drive fermentation. We compared fungal communities during the spontaneous fermentation of wine produced in a winery and in a laboratory with sterilized equipment using three grape cultivars (Chardonnay, Merlot, and Muscat Bailey A) obtained from the same harvest. High-throughput sequencing analysis based on the ITS1 region showed that Saccharomyces cerevisiae was the dominant species in winery batches at the end of fermentation, but it was not always dominant in laboratory batches. The number of laboratory batches where S. cerevisiae reached more than 50% at the end of fermentation was only 10 of 26. Consistent with this, in the grape juice/must before fermentation, S. cerevisiae accounted for 1.71% of fungal species identified in winery batches and 0.04% in laboratory batches. In addition, in laboratory-based winemaking, juice clarification of Chardonnay and cold maceration of Merlot influenced the microbial communities observed during fermentation. Our findings suggest that S. cerevisiae present in the winery environment participates at an early stage of fermentation, leading to its dominance at the end in wine produced by spontaneous fermentation in a winery. • Fungal community was compared between winery and laboratory spontaneous fermentation. • In winery fermentations, Saccharomyces cerevisiae ultimately became dominant. • In laboratory fermentations, S. cerevisiae was not always the dominant species. • S. cerevisiae present in the winery environment seems to participate in fermentation. • Conditions such as juice clarification affect microbial communities in fermentation. [ABSTRACT FROM AUTHOR]

Objectives: The ability to differentiate epileptic- and non-epileptic events is challenging due to a lack of reliable molecular seizure biomarker that provide a retrospective diagnosis. Here, we use next generation sequencing methods on whole blood samples to identify changes in RNA expression following seizures. Methods: Blood samples were obtained from 32 patients undergoing video electroencephalogram (vEEG) monitoring. Blood samples were collected in PaxGene tubes at baseline (admission) and following a seizure event (4–6 h and 24 h later or discharge). EEG and video of clinical events were reviewed by the clinical team and study epileptologist and were classified as epileptic seizure, psychogenic nonepileptic spell (PNES), or other. RNA was extracted from blood and RNA expression was determined using RNA-sequencing. Results: We show significant differences in RNA profiles between patients that did or did not experience an epileptic seizure. Compared to baseline patients with PNES show large increases in RNA expression 4–6 h and 24 h post seizure. Conversely, genes that changed following epileptic seizure showed more modest changes associated with a decrease in immune system function. Transcript usage was changed between patients with PNES and epileptic seizure at all three time points examined. Lists of genes differentially expressed following PNES or epileptic seizure vs. all baseline samples were used as classifiers for prediction. Models generated using random forest and radial support vector machine algorithms were 100% accurate at predicting both PNES and epileptic seizures. Significance: These data suggest that blood gene expression changes may have utility to retrospectively identify patients who have suffered a seizure or seizure-like event as a cause of transient loss of consciousness. [ABSTRACT FROM AUTHOR]

Cell-free DNA to determine the fetal RHD genotype from the maternal circulation was first described in 1993. High throughput assays using polymerase chain reaction technology were introduced in Europe and gained widespread acceptance in the management of the Rhesus alloimmunized pregnancy. The specificity and sensitivity of these assays approached 99%. As confidence was gained with these results, Scandinavian countries began to employ cell-free DNA for fetal RHD typing as an integral component of their introduction of antenatal Rhesus immune globulin in non-alloimmunized pregnancies. Since 40% of RhD-negative pregnant women will carry an RhD-negative fetus, doses of Rhesus immune globulin were conserved. Recently 2 U.S. companies have introduced cell-free DNA assays for RHD as part of their noninvasive prenatal testing assays. Both utilize next generation sequencing and have developed methodologies to detect the aberrant RHD pseudogene and the hybrid RHD-CE-D s genotype. In addition, excellent correlation studies with either neonatal genotyping or serology have been reported. The manufacturer of RhoGAM has recently announced a national shortage. Given the current availability of reliable cell-free DNA assays for determining the RHD status of the fetus, the time has come to implement this strategy to triage the antenatal use of Rhesus immune globulin in the U.S. [ABSTRACT FROM AUTHOR]

Hypophosphatemic rickets is a rare bone disease characterized by short stature, bone deformities, impaired bone mineralization, and dental problems. Most commonly, hypophosphatemic rickets is caused by pathogenic variants in the X-chromosomal PHEX gene, but autosomal dominant and recessive forms also exist. We investigated a Finnish family in which the son (index, 29 yr) and mother (56 yr) had hypophosphatemia since childhood. Both patients had typical clinical, radiographic, and biochemical features of hypophosphatemic rickets, including a pathological fracture in the son. Gene panels and whole-exome sequencing did not reveal any pathogenic variants in the known hypophosphatemia genes. Therefore, we performed whole genome sequencing and identified a deep intronic variant (c.2147 + 1197A > G) in PHEX. Both the affected individuals, but none of the unaffected family members, had the same variant, as confirmed by Sanger sequencing. According to RT-PCR, whole transcriptomic data, and in silico analyses, the variant led to a new splice donor site in intron 21 and an 84 basepair pseudoexon between exons 21 and 22, likely leading to the synthesis of abnormal PHEX protein. Our study underscores the importance of intronic PHEX variants in X-linked hypophosphatemia (XLH). In patients with features of XLH but negative gene panel or whole-exome sequencing results, the combination of whole-genome sequencing and whole transcriptomics should be considered to detect possible deep intronic variants. The methodologies presented have the potential to be used more widely in other rare diseases. [ABSTRACT FROM AUTHOR]

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The role of circular RNAs (circRNAs) in sepsis-induced lung injury is not clear. This study investigated the role and molecular mechanism of a novel circRNA in sepsis-induced lung injury and explored its prognostic value in patients with sepsis. In this study, aberrant circRNA expression profiling in lung tissues from mice with sepsis-induced lung injury was analyzed using high-throughput sequencing. circRNA-Cacna1d was verified by qRT-PCR, and its biological function in sepsis-induced lung injury was validated in vitro and in vivo. The interactions among circRNA-Cacna1d, microRNAs (miRNAs), and their downstream genes were verified. Furthermore, the clinical value of circRNA-Cacna1d in peripheral blood from patients with sepsis was also evaluated. We found that circRNA-Cacna1d expression was significantly increased in lung tissues of mice with sepsis and in microvascular endothelial cells after LPS challenge. circRNA-Cacna1d knockdown alleviated inflammatory response and ameliorated the permeability of vascular endothelium, thereby mitigating sepsis-induced lung injury and significantly improving the survival rate of mice with sepsis. Mechanistically, circRNA-Cacna1d directly interacted with miRNA-185-5p and functioned as a miRNA sponge to regulate the RhoA/ROCK1 signaling pathway. The expression level of circRNA-Cacna1d in patients with early sepsis was significantly higher than that in the healthy control subjects. Higher levels of circRNA-Cacna1d in patients with sepsis were associated with increased disease severity and poorer outcomes. In conclusions, circRNA-Cacna1d may play a role in sepsis-induced lung injury by regulating the RhoA/ROCK1 axis by acting as a miRNA-185-5p sponge. circRNA-Cacna1d is a potential therapeutic target for sepsis-induced lung injury and a prognostic biomarker in sepsis. [ABSTRACT FROM AUTHOR]