Congenital thrombocytopenia/platelet disorders are heterogeneous disorders of platelet number and/or function. Pathogenic variants in the genes implicated in megakaryocyte differentiation and platelet formation cause thrombocytopenia in these patients. Recent advances have elucidated several causative genes for these disorders, but identifying the underlying causative genes remains challenging. Patients with these disorders often receive inappropriate treatments, including glucocorticoids and splenectomy, for chronic immune thrombocytopenia (ITP). In Japan, we have developed a diagnostic system using high-throughput DNA sequencing with a multigene panel and established a registry. Between 2018 and 2023, 245 patients were enrolled and analyzed. Pathogenic variants in 17 genes (42 MYH9, 19 ANKRD26, 17 ITGA2B/ITGB3, 8 ACTN1, 8 WAS, 6 ETV6, 6 VWF, 5 CYCS, and 14 others) were identified in 125 patients (51.0%). An additional 29 patients (11.8%) had suspected pathogenic variants under investigation. We also found that immature platelet fraction (IPF%) is useful in the differential diagnosis because the median IPF% in MYH9 disorders, 48.7%, was significantly higher than in all other groups (chronic ITP, 13.4%; controls, 2.6%). The results of this study provide new insight into congenital thrombocytopenia/platelet disorders.

Tumor heterogeneity refers to the diversity observed among tumor cells: both between different tumors (inter-tumor heterogeneity) and within a single tumor (intra-tumor heterogeneity). These cells can display distinct morphological and phenotypic characteristics, including variations in cellular morphology, metastatic potential and variability treatment responses among patients. Therefore, a comprehensive understanding of such heterogeneity is necessary for deciphering tumor-specific mechanisms that may be diagnostically and therapeutically valuable. Innovative and multidisciplinary approaches are needed to understand this complex feature. In this context, proteogenomics has been emerging as a significant resource for integrating omics fields such as genomics and proteomics. By combining data obtained from both Next-Generation Sequencing (NGS) technologies and mass spectrometry (MS) analyses, proteogenomics aims to provide a comprehensive view of tumor heterogeneity. This approach reveals molecular alterations and phenotypic features related to tumor subtypes, potentially identifying therapeutic biomarkers. Many achievements have been made; however, despite continuous advances in proteogenomics-based methodologies, several challenges remain: in particular the limitations in sensitivity and specificity and the lack of optimal study models. This review highlights the impact of proteogenomics on characterizing tumor phenotypes, focusing on the critical challenges and current limitations of its use in different clinical and preclinical models for tumor phenotypic characterization.

Oxygen conditions in the lung determine downstream organ functionality by setting the partial pressure of oxygen, regulating the redox homeostasis and by activating mediators in the lung that can be propagated in the blood stream. Examples for such mediators are secreted soluble or vesicle-bound molecules (proteins and nucleic acids) that can be taken up by remote target cells impacting their metabolism and signaling pathways. MicroRNAs (miRNAs) have gained significant interest as intercellular communicators, biomarkers and therapeutic targets in this context. Due to their high stability in the blood stream, they have also been attributed a role as \"memory molecules\" that are able to modulate gene expression upon repeated (stress) exposures. In this study, we aimed to identify and quantify released miRNAs from lung microvascular endothelial cells in response to different oxygen conditions. We combined next-generation sequencing (NGS) of secreted miRNAs and cellular mRNA sequencing with bioinformatic analyses in order to delineate molecular events on the cellular and extracellular level and their putative interdependence. We show that the identified miRNA networks have the potential to co-mediate some of the molecular events, that have been observed in the context of hypoxia, hyperoxia, intermittent hypoxia and intermittent hypoxia/hyperoxia.

BACKGROUND: A significant portion of South Korea\'s population, approximately a quarter, owns pets, with dogs being the most popular choice among them. However, studies analyzing the fecal organism communities of dogs in South Korea are lacking, and limited efforts have been exerted to identify pathogens with potential zoonotic implications. Therefore, this study aimed to investigate potential pathogens using metabarcoding analysis and evaluate the risk of zoonotic diseases in dog feces in Seoul, South Korea.
METHODS: Fecal samples were collected from both pet and stray dogs in the Mapo district of Seoul. Next-generation sequencing (NGS) was utilized, employing 16S rRNA amplicon sequencing to identify prokaryotic pathogens, and 18S rRNA amplicon sequencing for eukaryotic pathogens. The data obtained from the QIIME2 pipeline were subjected to various statistical analyses to identify different putative pathogens and their compositions.
RESULTS: Significant variations in microbiota composition were found between stray and pet dogs, and putative prokaryotic and eukaryotic pathogens were identified. The most prevalent putative bacterial pathogens were Fusobacterium, Helicobacter, and Campylobacter. The most prevalent putative eukaryotic pathogens were Giardia, Pentatrichomonas, and Cystoisospora. Interestingly, Campylobacter, Giardia, and Pentatrichomonas were found to be significantly more prevalent in stray dogs than in pet dogs. The variation in the prevalence of potential pathogens in dog feces could be attributed to environmental factors, including dietary variances and interactions with wildlife, particularly in stray dogs. These factors likely contributed to the observed differences in pathogen occurrence between stray and pet dogs.
CONCLUSIONS: This study offers valuable insights into the zoonotic risks associated with dog populations residing in diverse environments. By identifying and characterizing putative pathogens in dog feces, this research provides essential information on the impact of habitat on dog-associated pathogens, highlighting the importance of public health planning and zoonotic risk management.

UNASSIGNED: To explore the clinical value of metagenomic next-generation sequencing (mNGS) in diagnosis and treatment of periprosthetic joint infection (PJI) after total knee arthroplasty (TKA).
UNASSIGNED: Between April 2020 and March 2023, 10 patients with PJI after TKA were admitted. There were 3 males and 7 females with an average age of 69.9 years (range, 44-83 years). Infection occurred after 8-35 months of TKA (mean, 19.5 months). The duration of infection ranged from 16 to 128 days (mean, 37 days). The preoperative erythrocyte sedimentation rate (ESR) was 15-85 mm/1 h (mean, 50.2 mm/1 h). The C reactive protein (CRP) was 4.4-410.0 mg/L (mean, 192.8 mg/L). The white blood cell counting was (3.4-23.8)×10 9/L (mean, 12.3×10 9/L). The absolute value of neutrophils was (1.1-22.5)×10 9/L (mean, 9.2×10 9/L). After admission, the joint fluid was extracted for bacterial culture method and mNGS test, and sensitive antibiotics were chosen according to the results of the test, and the infection was controlled in combination with surgery.
UNASSIGNED: Seven cases (70%) were detected as positive by bacterial culture method, and 7 types of pathogenic bacteria were detected; the most common pathogenic bacterium was Streptococcus lactis arrestans. Ten cases (100%) were detected as positive by mNGS test, and 11 types of pathogenic bacteria were detected; the most common pathogenic bacterium was Propionibacterium acnes. The difference in the positive rate between the two methods was significant ( P=0.211). Three of the 7 patients who were positive for both the bacterial culture method and the mNGS test had the same results for the type of pathogenic bacteria, with a compliance rate of 42.86% (3/7). The testing time (from sample delivery to results) was (4.95±2.14) days for bacterial culture method and (1.60±0.52) days for mNGS test, and the difference was significant ( t=4.810, P<0.001). The corresponding sensitive antibiotic treatment was chosen according to the results of bacterial culture method and mNGS test. At 3 days after the one-stage operation, the CRP was 6.8-48.2 mg/L (mean, 23.6 mg/L); the ESR was 17-53 mm/1 h (mean, 35.5 mm/1 h); the white blood cell counting was (4.5-8.1)×10 9/L (mean, 6.1×10 9/L); the absolute value of neutrophils was (2.3-5.7)×10 9/L (mean, 4.1×10 9/L). All patients were followed up 12-39 months (mean, 23.5 months). One case had recurrence of infection at 6 months after operation, and the remaining 9 cases showed no signs of infection, with an infection control rate of 90%.
UNASSIGNED: Compared with bacterial culture method, mNGS test can more rapidly and accurately detect pathogenic bacteria for PJI after TKA, which is important for guiding antibiotics combined with surgical treatment of PJI.
UNASSIGNED: 探讨宏基因组二代测序（metagenomic next-generation sequencing，mNGS）在人工全膝关节置换术（total knee arthroplasty，TKA）后假体周围感染（periprosthetic joint infection，PJI）诊治中的临床价值。.
UNASSIGNED: 2020年4月—2023年3月，收治10例TKA术后PJI患者。男3例，女7例；年龄44～83岁，平均69.9岁。置换术后8～35个月发生感染，平均19.5个月；感染病程16～128 d，平均37 d。术前红细胞沉降率（erythrocyte sedimentation rate，ESR）15～85 mm/1 h，平均50.2 mm/1 h；C 反应蛋白（C reactive protein，CRP）4.4～410.0 mg/L，平均192.8 mg/L；白细胞计数（3.4～23.8）×10 9/L，平均12.3×10 9/L；中性粒细胞绝对值（1.1～22.5）×10 9/L，平均9.2×10 9/L。入院后抽取关节液行细菌培养及mNGS检测，根据检测结果调整敏感抗生素，并结合手术控制感染。.
UNASSIGNED: 细菌培养检测阳性7例（70%），共检出7种病原菌，最常见病原菌为停乳链球菌。mNGS检测阳性10例（100%），共检出11种病原菌，最常见病原菌为痤疮丙酸杆菌。两种方法检测阳性率差异有统计学意义（ P=0.211）。7例细菌培养法和mNGS检测均为阳性患者中，3例病原菌类型结果完全一致，符合率42.86%（3/7）。细菌培养检测时间（送样本至出结果）为（4.95±2.14） d，mNGS检测为（1.60±0.52）d，差异有统计学意义（ t=4.810， P<0.001）。根据细菌培养及mNGS检测培养结果采取敏感抗生素治疗。一期术后3 d CRP为6.8～48.2 mg/L，平均23.6 mg/L；ESR 17～53 mm/1 h，平均35.5 mm/1 h；白细胞计数（4.5～8.1）×10 9/L，平均6.1×10 9/L；中性粒细胞绝对值（2.3～5.7）×10 9/L，平均4.1×10 9/L。患者均获随访，随访时间12～39个月，平均23.5个月。1例术后6个月感染复发，其余9例均未出现感染征象，感染控制率为90%。.
UNASSIGNED: 与细菌培养相比，mNGS能更快速准确地检测TKA术后PJI病原菌，对指导抗生素联合手术治疗PJI具有重要意义。.

Molecular HLA typing techniques are currently undergoing a rapid evolution. While real-time PCR is established as the standard method in tissue typing laboratories regarding allocation of solid organs, next generation sequencing (NGS) for high-resolution HLA typing is becoming indispensable but is not yet suitable for deceased donors. By contrast, high-resolution typing is essential for stem cell transplantation and is increasingly required for questions relating to various disease associations. In this multicentre clinical study, the TGS technique using nanopore sequencing is investigated applying NanoTYPE™ kit and NanoTYPER™ software (Omixon Biocomputing Ltd., Budapest, Hungary) regarding the concordance of the results with NGS and its practicability in diagnostic laboratories. The results of 381 samples show a concordance of 99.58% for 11 HLA loci, HLA-A, -B, -C, -DRB1, -DRB3, -DRB4, -DRB5, -DQA1, -DQB1, -DPA1 and -DPB1. The quality control (QC) data shows a very high quality of the sequencing performed in each laboratory, 34,926 (97.15%) QC values were returned as \'passed\', 862 (2.4%) as \'inspect\' and 162 (0.45%) as \'failed\'. We show that an \'inspect\' or \'failed\' QC warning does not automatically lead to incorrect HLA typing. The advantages of nanopore sequencing are speed, flexibility, reusability of the flow cells and easy implementation in the laboratory. There are challenges, such as exon coverage and the handling of large amounts of data. Finally, nanopore sequencing presents potential for applications in basic research within the field of epigenetics and genomics and holds significance for clinical concerns.

The gut microbiota significantly contributes to human health and well-being. The aim of this study was to evaluate the stability and resilience of a consortium composed of three next-generation probiotics (NGPs) candidates originally found in the human gut. The growth patterns of Akkermansia muciniphila, Bacteroides thetaiotaomicron, and Faecalibacterium prausnitzii were studied both individually and consortium. The growth kinetics of Akkermansia muciniphila (A. muciniphila), Bacteroides thetaiotaomicron (B. thetaiotaomicron), and Faecalibacterium prausnitzii (F. prausnitzii) were characterized both individually and in consortium using isothermal microcalorimetry and 16S ribosomal RNA next-generation sequencing. The consortium reached stability after three passages and demonstrated resilience to changes in its initial composition. The concentration of butyrate produced was nearly twice as high in the consortium compared to the monoculture of F. prausnitzii. The experimental conditions and methodologies used in this article are a solid foundation for developing further complex consortia.

Various antibiotic-resistant bacteria (ARB) are known to induce repeated pulmonary infections and increase morbidity and mortality. A thorough knowledge of antibiotic resistance is imperative for clinical practice to treat resistant pulmonary infections. In this study, we used a reads-based method and an assembly-based method according to the metagenomic next-generation sequencing (mNGS) data to reveal the spectra of ARB and corresponding antibiotic resistance genes (ARGs) in samples from patients with pulmonary infections. A total of 151 clinical samples from 144 patients with pulmonary infections were collected for retrospective analysis. The ARB and ARGs detection performance was compared by the reads-based method and assembly-based method with the culture method and antibiotic susceptibility testing (AST), respectively. In addition, ARGs and the attribution relationship of common ARB were analyzed by the two methods. The comparison results showed that the assembly-based method could assist in determining pathogens detected by the reads-based method as true ARB and improve the predictive capabilities (46% > 13%). ARG-ARB network analysis revealed that assembly-based method could promote determining clear ARG-bacteria attribution and 101 ARGs were detected both in two methods. 25 ARB were obtained by both methods, of which the most predominant ARB and its ARGs in the samples of pulmonary infections were Acinetobacter baumannii (ade), Pseudomonas aeruginosa (mex), Klebsiella pneumoniae (emr), and Stenotrophomonas maltophilia (sme). Collectively, our findings demonstrated that the assembly-based method could be a supplement to the reads-based method and uncovered pulmonary infection-associated ARB and ARGs as potential antibiotic treatment targets.

Formalin-fixed paraffin-embedded (FFPE) tissue represents a valuable source for translational cancer research. However, the widespread application of various downstream methods remains challenging. Here, we aimed to assess the feasibility of a genomic and gene expression analysis workflow using FFPE breast cancer (BC) tissue. We conducted a systematic literature review for the assessment of concordance between FFPE and fresh-frozen matched tissue samples derived from patients with BC for DNA and RNA downstream applications. The analytical performance of three different nucleic acid extraction kits on FFPE BC clinical samples was compared. We also applied a newly developed targeted DNA Next-Generation Sequencing (NGS) 370-gene panel and the nCounter BC360® platform on simultaneously extracted DNA and RNA, respectively, using FFPE tissue from a phase II clinical trial. Of the 3701 initial search results, 40 articles were included in the systematic review. High degree of concordance was observed in various downstream application platforms. Moreover, the performance of simultaneous DNA/RNA extraction kit was demonstrated with targeted DNA NGS and gene expression profiling. Exclusion of variants below 5% variant allele frequency was essential to overcome FFPE-induced artefacts. Targeted genomic analyses were feasible in simultaneously extracted DNA/RNA from FFPE material, providing insights for their implementation in clinical trials/cohorts.

Objective: To explore the feasibility of constructing an objective tinnitus subtype model based on peripheral blood differentially expressed genes (DEGs) using a combination of Weighted Gene Co-expression Network Analysis (WGCNA) and Random Forest algorithm (RF). Methods: From October 2019 to June 2020, peripheral blood DEGs were obtained from 37 patients (from the Third Affiliated Hospital of Sun Yat-sen University)with chronic subjective high-frequency tinnitus (21 unbothersome type, 16 bothersome type) and 20 healthy volunteers through high-throughput sequencing. WGCNA was used to construct gene modules with different expression patterns and analyze their relationships with tinnitus characteristics. Subsequently, RF was employed to build subtype models, which were evaluated by the area under the receiver operating characteristic curve (AUC), accuracy, and F1-score. Results: A total of 12 351 intergroup DEGs were divided into 9 gene modules. Among them, MEblue, MEgreen, and MEbrown showed significant negative correlations with the healthy volunteer group, while MEpink showed a significant positive correlation with the tinnitus distress group. The \"Tinnitus vs. Normal\" and \"Compensatory vs. Decompensatory\" subtype models, based on MEblue and MEpink respectively, both had AUCs greater than 0.80, accuracies above 90%, and F1-scores above 0.90, indicating good performance. Conclusions: Peripheral blood DEGs are potential biological indicators for objective classification of subjective tinnitus. The combined application of WGCNA and the Random Forest algorithm should be a viable approach to constructing an objective tinnitus subtype model. However, further exploration and refinement are needed to validate the model\'s generalizability, cross-dataset performance, and algorithm optimization.
目的： 探讨联合加权基因共表达网络分析（Weighted Gene Co-expression Network Analysis，WGCNA）及随机森林算法构建基于外周血差异表达基因（differentially expressed genes，DEGs）的主观性耳鸣客观分型模型的可行性。 方法： 2019年10月至2020年6月期间，对中山大学附属第三医院37例慢性主观性高频耳鸣患者（代偿型21例，失代偿型16例）及20名健康志愿者通过高通量测序获得外周血DEGs。采用WGCNA构建不同表达模式的基因模块，并分析各自与耳鸣特征之间的关系。随后采用随机森林算法构建分型模型，并通过受试者工作特征曲线下面积（area under the curve，AUC）、准确度和F1-score对分型性能进行评价。 结果： 12 351个组间DEGs被分成9个基因模块，其中MEblue、MEgreen和MEbrown与健康志愿者组呈负相关，MEpink与耳鸣困扰组呈正相关。基于MEblue及MEpink分别构建“耳鸣-正常”及“代偿-失代偿”分型模型，AUC均>0.80，准确度均>90%，F1-score均>0.90，分型性能良好。 结论： 外周血DEGs是慢性主观性耳鸣客观分型的潜在生物学指标，而WGCNA和随机森林算法的联合应用是构建慢性主观性耳鸣客观分型模型的可行方案。但模型的外延、跨数据集性能的验证，以及模型算法的优化仍需进一步探索并完善。.