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Rotor syndrome is a rare, benign, inherited disorder that is commonly associated with mild hyperbilirubinemia. It is caused by bi-allelic pathological variants in both SLCO1B1 and SLCO1B3 genes, causing defective OATP1B1 and OATP1B3 in the sinusoidal membrane and interrupted bilirubin uptake of the hepatocytes. We report five Taiwanese pediatric and adult patients aged 5–32 years presenting with conjugated hyperbilirubinemia, and were found to have genetic variants of SLCO1B1 and SLCO1B3. Two also had history of prolonged neonatal jaundice. Genetic analysis using panel-based next generation sequencing revealed three patients with homozygous mutations c.1738C>T (p.R580∗) in SLCO1B1 and a transposon LINE-1 insertion in SLCO1B3, one patient with homozygous mutations for another haplotype, c.757C>T (p.R253∗) in SLCO1B1 and c.1747+1G>A in SLCO1B3. Another patient had heterozygous c.1738C>T (p.R580∗) in SLCO1B1 linked with a LINE-1 insertion in SLCO1B3, and heterozygous c.757C>T (p.R253∗) in SLCO1B1 linked with c.1747+1G>A in SLCO1B3. In conclusion, we present the first time of genetic diagnosis of Rotor syndrome in Taiwan. Advanced genetic testing has enhanced the diagnosis of rare diseases with mild symptoms.

ABSTRACT: Objectives: This study aimed to explore associations between the molecular characterization of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and disease severity in ambulatory and hospitalized patients in two main Colombian epicentres during the first year of the coronavirus disease 2019 pandemic. Methods: In total, 1000 patients with SARS-CoV-2 infection were included in this study. Clinical data were collected from 997 patients, and 678 whole-genome sequences were obtained by massively parallel sequencing. Bivariate, multi-variate, and classification and regression tree analyses were run between clinical and genomic variables. Results: Age >88 years, and infection with lineages B.1.1, B.1.1.388, B.1.523 or B.1.621 for patients aged 71–88 years were associated with death [odds ratio (OR) 6.048036, 95% confidence interval (CI) 1.346567–32.92521; P=0.01718674]. The need for hospitalization was associated with higher age and comorbidities. The hospitalization rate increased significantly for patients aged 38–51 years infected with lineages A, B, B.1.1.388, B.1.1.434, B.1.153, B.1.36.10, B.1.411, B.1.471, B.1.558 or B.1.621 (OR 8.368427, 95% CI 2.573145–39.10672, P=0.00012). Associations between clades and clinical outcomes diverged from previously reported data. Conclusions: Infection with lineage B.1.621 increased the hospitalization and mortality rates. These findings, plus the rapidly increasing prevalence in Colombia and other countries, suggest that lineage B.1.621 should be considered as a ‘variant of interest’. If associated disease severity is confirmed, possible designation as a ‘variant of concern’ should be considered.

The rodent gut microbiota is a known reservoir of antimicrobial resistance, yet the distribution of antibiotic resistance genes (ARGs) within rodent cecal microbial communities and the specific bacterial species harboring these ARGs remain largely underexplored. This study employed high-throughput sequencing of 122 samples from five distinct rodent species to comprehensively profile the diversity and distribution of ARGs and to identify the bacterial hosts of these genes. A gene catalog of the rodent cecal microbiome was constructed, comprising 22,757,369 non-redundant genes. Analysis of the microbial composition and diversity revealed that Bacillota and Bacteroidota were the dominant bacterial phyla across different rodent species, with significant variations in species composition among the rodents. In total, 3703 putative antimicrobial resistance protein-coding genes were identified, corresponding to 392 unique ARG types classified into 32 resistance classes. The most enriched ARGs in the rodent cecal microbiome were associated with multidrug resistance, followed by glycopeptide and elfamycin antibiotics. Procrustes analysis demonstrated a correlation between the structure of the microbial community and the resistome. Metagenomic assembly-based host tracking indicated that most ARG-carrying contigs originated from the bacterial family Oscillospiraceae. Additionally, 130 ARGs showed significant correlations with mobile genetic elements. These findings provide new insights into the cecal microbiota and the prevalence of ARGs across five rodent species. Future research on a wider range of wild rodent species carrying ARGs will further elucidate the mechanisms underlying the transmission of antimicrobial resistance., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Background: The prognostic role of circulating tumor DNA (ctDNA)-based molecular residual disease (MRD) detection and its utility for postsurgical risk stratification has been reported in colorectal cancer. In this study, we explored the use of ctDNA-based MRD detection in patients with colorectal liver metastases (CLM), for whom the survival benefit of adjuvant chemotherapy (ACT) after surgical resection remains unclear., Methods: Patients with CLM without extrahepatic disease from the GALAXY study (UMIN000039205) were included. The disease-free survival (DFS) benefit of ACT was evaluated in MRD-positive and -negative groups after adjusting for age, gender, number, and size of liver metastases, RAS status, and previous history of oxaliplatin for primary cancer. ctDNA was detected using a personalized, tumor-informed 16-plex polymerase chain reaction-next-generation sequencing (mPCR-NGS) assay. ctDNA-based MRD status was evaluated 2-10 weeks after curative surgery, before the start of ACT., Results: Among 6061 patients registered in GALAXY, 190 surgically resected CLM patients without any preoperative chemotherapy were included with a median follow-up of 24 months (1-48 months). ctDNA positivity in the MRD window was 32.1% (61/190). ACT was administered to 25.1% (48/190) of patients. In the MRD-positive group, 24-month DFS was higher for patients treated with ACT [33.3% versus not reached, adjusted hazard ratio (HR): 0.07, P < 0.0001]; whereas no benefit of ACT was seen in the MRD-negative group (24-month DFS: 72.3% versus 62.2%, adjusted HR: 0.68, P = 0.371). Multivariate analysis showed that the size of liver metastases (HR: 3.94, P = 0.031) was prognostic of DFS in the MRD-positive group. In the MRD-negative group, however, none of the clinicopathological factors were prognostic of DFS., Conclusions: Our data suggest that ACT may offer notable clinical benefits in MRD-positive patients with CLM. MRD status-based risk stratification could be potentially incorporated in future clinical trials for CLM., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Indolent natural killer cell lymphoproliferative disorder of the gastrointestinal tract (iNKLPD-GI) is an uncommon, recently recognized lymphoid proliferation of mature NK cells primarily manifesting in the GI tract. Unlike NK/T lymphoma, iNKLPD-GI exhibits a rather indolent clinical course, underscoring the need for cautious management to prevent unnecessary interventions. However, clinical and molecular features of this entity have not been thoroughly understood. This study aimed to add more information to the current knowledge of this disease. Seven patients with iNKLPD-GI were included in our study. Clinical data included initial symptoms, endoscopic manifestations, pathological features, and therapies. Besides, next-generation sequencing was arranged to explore the underlying genetic mechanism of this disease. In our study, iNKLPD-GI in the urinary bladder was first identified. Edema of extremities (3, 42.8 %) was the most prevalent onset symptom which was reported for the first time. Pathological and immunohistological features were found to display the phenotype of NK cells. Unlike extranodal NK/T cell lymphoma, Epstein-Barr virus-encoded small RNA (EBER) were negative in all patients. Moreover, we found that two patients harbored JAK3 mutation. Apart from JAK3 K563&#95;C565del previously reported in the literature, we discovered new JAK3 mutation sites. Other mutations including BRAF, KRAS, and SH2B3 were also identified. In conclusion, iNKLPD-GI was an indolent atypical NK-cell proliferation with diverse clinical characteristics. "Watch and wait" therapy was preferable to intense chemotherapy. Recurrent JAK3 mutation may be the underlying mechanism responsible for the neoplastic nature of the disease and may serve as a potential target for patients with severe symptoms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Introduction: In the dynamic landscape of metastatic NSCLC (mNSCLC) management, marked by several frontline options and the integration of next generation sequencing (NGS) for informed decision-making, barriers persist despite advancements. This includes challenges in clinical trial recruitment. To gain global insights into clinicians' practices, we conducted a survey on their testing and management approaches for patients with mNSCLC., Methods: The survey, conducted from July 12 to August 20, 2023, utilized multiple-choice questions and qualitative research questions, employing the Likert Scale for comprehensive insights., Results: A total of 127 individuals responded, with 72% affiliated with academic health systems, and 55% practicing in the USA. Regarding testing practices, 93% consistently ordered NGS for non-squamous histology, while 54% did so for squamous cell histology. Concurrent tissue and liquid biopsies were routinely ordered by 28%, while 39% reported ordering both testing platforms concurrently for select cases only. Respondents cited logistical barriers, such as insufficient tissue and lack of infrastructure, as the most common hindrance to molecular testing (76%), followed by reimbursement challenges (56%) and concerns about delayed turnaround time (50%). While most respondents were confident in interpreting NGS results, 22% lacked confidence. Concerning treatment decisions, 72% preferred awaiting molecular testing results before initiating systemic therapy. Less than 50% routinely referred patients for clinical trials in the frontline setting for mNSCLC. For patients with disease expressing high PD-L1 levels, most oncologists preferred pembrolizumab monotherapy. For disease with low PD-L1 expression, a platinum doublet chemotherapy regimen combined with pembrolizumab was favored. In disease cases with negative PD-L1 expression, a platinum doublet chemotherapy regimen with pembrolizumab was preferred. Key factors influencing oncologists' preferred immune checkpoint inhibitor (ICI) included experience with one ICI over another, preferred status per national guidelines, availability of trial data with a significant follow-up period, and consideration of drug cost., Conclusion: Although this study demonstrates an improved awareness and adoption of ordering NGS for the management of mNSCLC, it underscores the persistence of various barriers that must be addressed to improve upon the quality of care for patients diagnosed with mNSCLC., Competing Interests: Disclosure AS: None. HJW: Howard Jack West reports a relationship with Summit Therapeutics plc that includes: employment. Howard Jack West reports a relationship with AbbVie Inc that includes: consulting or advisory. Howard Jack West reports a relationship with Amgen Inc that includes: consulting or advisory. Howard Jack West reports a relationship with Genetech Biotech Co Ltd that includes: consulting or advisory. Howard Jack West reports a relationship with Gilead Sciences Inc that includes: consulting or advisory. Howard Jack West reports a relationship with Merck & Co Inc that includes: consulting or advisory. Howard Jack West reports a relationship with Mirati Therapeutics Inc that includes: consulting or advisory. Howard Jack West reports a relationship with Regeneron Pharmaceuticals Inc that includes: consulting or advisory. Howard Jack West reports a relationship with AstraZeneca Pharmaceuticals LP that includes: speaking and lecture fees. Howard Jack West reports a relationship with Merck & Co Inc that includes: speaking and lecture fees. EKS: Eric K. Singhi reports a relationship with AstraZeneca Pharmaceuticals LP that includes: consulting or advisory. Eric K. Singhi reports a relationship with Novocure that includes: consulting or advisory. Eric K. Singhi reports a relationship with Amgen Inc that includes: consulting or advisory. Eric K. Singhi reports a relationship with Janssen Pharmaceuticals Inc that includes: consulting or advisory. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Forensic Biology is contingent upon matching DNA profiles between a crime sample and a reference sample. There are several capillary electrophoresis kits available to generate a short tandem repeat (STR) profile from DNA samples, while newer methods using massively parallel sequencing are slowly being implemented in forensic laboratories worldwide. During evaluation of a newer capillary electrophoresis kit, Applied Biosystems™ VeriFiler™ Plus, a discordance was observed in the Penta D locus. The previous kit, Promega PowerPlex 21® System produced a 13.4,14 genotype, whilst VeriFiler™ Plus produced a 14,14 genotype. An expanded investigation into Penta D microvariant alleles revealed that multiple discordances were observed for DNA profiles containing larger x.4 variants. There was full concordance between PowerPlex® 21 and QIAGEN Investigator® 26plex, however discordances were observed between VeriFiler™ Plus and the other three kits tested, including the massively parallel sequencing kit, Verogen ForenSeq® MainstAY. Notably, four of these discordances resulted in null alleles with the VeriFiler™ Plus kit. A review of the Penta D DNA sequences in MainstAY revealed fully concordant microvariant alleles involved deletions within the repeat region, whilst variability in the discordances observed were dependent on the location of the variation outside the repeat region and the analysis method used. Variations observed within the 5' flanking region produced the same allele designation across all capillary electrophoresis kits. However, deletions within the 3' region either produced a null allele for VeriFiler™ Plus where the deletion is thought to overlap the primer binding site, or microvariant alleles for the PowerPlex® 21 and Investigator 26plex kits, which produced longer Penta D amplicons. The discovery of these variations in the Penta D flanking sequences is informative as it increases the awareness of Penta D discordances between different kit chemistries in nominated reference DNA profile comparisons and DNA database searching and matching alike, and provides support for this phenomenon when providing evidence as to the admissibility of such results in trial proceedings., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier B.V. All rights reserved.)

The evolution of primary melanoma to lymph node and distant metastasis is incompletely understood. We examined the genomic diversity in melanoma progression in matched primary melanomas and lymph node and distant metastases from 17 patients. FISH analysis revealed cancer cell fractions with monotonic copy number alterations, including PHIP gain and PTEN loss, in the metastatic cascade. By contrast, the cancer cell fraction with copy number alterations for BPTF and MITF was reduced in lymph node metastases but increased in distant metastases. Separately, the cancer cell fraction with NCOA3 copy number alteration was comparable between primary tumors and lymph node metastases yet increased in distant metastases. These results suggest enrichment of the phosphoinositide 3-kinase and MITF pathways in the transition through the metastatic cascade. By contrast, next-generation sequencing analysis did not identify a consistent pattern of changes in variant allele frequency while revealing several intriguing findings, including decreased variant allele frequency in distant metastases and distinct drivers in lymph node versus distant metastases. These results provide evidence that distant melanoma metastasis does not always emanate from lymph node metastasis. These results enhance our understanding of clonal patterns of melanoma metastasis, with possible implications for targeted therapy and metastasis competency., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Numerous studies have demonstrated a correlation between p53 overexpression and diminished survival in gastric cancer patients. However, conflicting findings exist, and we hypothesize that these discrepancies arise from the cancer's complexity and heterogeneity, coupled with a lack of consensus on aberrant p53 expression., Methods: We enrolled a cohort of 187 patients with surgically resected gastric cancer. Patient categorization was based on Epstein-Barr virus (EBV), microsatellite instability (MSI), and Lauren classification (intestinal, diffuse and mixed). Utilizing an incremental algorithm, we evaluated p53 immunohistochemical (IHC) patterns in all 187 cases, while next-generation sequencing was successfully performed on 152 cases to identify TP53 mutations (mutTP53)., Results: MutTP53 was identified in 32 % of the 152 cases, comprising 36 missense, 5 nonsense, and 7 frameshift alterations. Missense mutations predominantly correlated with p53 overexpression, while nonsense and frameshifting alterations related to null expression. Trial calculations indicated that null expression and a p53 IHC cutoff at >40 % offered the best prediction of mutTP53 (kappa coefficient, 0.427), with the highest agreement (0.524) observed in diffuse type and the lowest (0.269) in intestinal type. Null expression and a p53 IHC cutoff at >10 %, but not mutTP53 per se, provided the optimal prediction of survival outcome (p = 0.043), particularly in diffuse type (p = 0.044). Multivariate analysis showed that aberrant p53 IHC expression was not an independent prognostic factor., Conclusions: P53 IHC patterns are predictive biomarkers for mutTP53 and gastric cancer outcomes, where a prerequisite involves a nuanced approach considering cutoff values and molecular-histologic subtyping., Competing Interests: Declaration of competing interest All coauthors do not have conflict of interest., (Copyright © 2024 Asian Surgical Association and Taiwan Society of Coloproctology. Published by Elsevier B.V. All rights reserved.)

This study evaluates the performance of analysing surface DNA samples using massively parallel sequencing (MPS) compared to traditional capillary electrophoresis (CE). A total of 30 samples were collected from various surfaces in an office environment and were analysed with CE and MPS. These were compared against 60 reference samples (office inhabitants). To identify contributors, likelihood ratios (LRs) were calculated for MPS and CE data using the probabilistic genotyping software MPSproto and EuroForMix respectively. Although a higher number of sequences/peaks were observed per DNA profile in MPS compared to CE, LR values were found to be lower for MPS data formats. This might be the result of the increased complexity of MPS data, along with a possible elevation of unknown alleles and/or artefacts. The study highlights avenues for improving MPS data quality and analysis to facilitate more robust interpretation of challenging casework-like samples., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Forensic genetic analyses aim to retrieve as much information as possible from biological trace material recovered from crime scenes. While standard short tandem repeat (STR) profiling is essential to individualize biological traces, its significance is diminished in crime scenarios where the presence of a suspect's DNA is acknowledged by all parties. In such cases, forensic (m)RNA analysis can provide crucial contextualizing information on the source level about a trace's composition, i.e., body fluids/tissues, and has therefore emerged as a powerful tool for modern forensic investigations. However, the question which of several suspects contributed a specific component (body fluid) to a mixed trace cannot be answered by RNA analysis using conventional methods. This individualizing information is stored within the sequence of the mRNA transcripts. Massively parallel sequencing (MPS) represents a promising alternative, offering not only higher multiplex capacity, but also the typing of individual coding region SNPs (cSNPs) to enable the assignment of contributors to mixture components, thereby reducing the risk of association fallacies. Herein, we describe the development of an extensive mRNA/cSNP panel for targeted sequencing on the IonTorrent S5 platform. Our panel comprises 30 markers for the detection of six body fluids/tissues (blood, saliva, semen, skin, vaginal and menstrual secretion), along with 70 linkage-controlled cSNPs for contributor assignment. It exhibited high reliable detection sensitivity with RNA inputs down to 0.75 ng and a conservatively calculated probability of identity of 0.03 - 6 % for individual body fluid-specific cSNP profiles. Limitations and areas for future work include RNA-related allele imbalances, inclusion of markers to correctly identify rectal mucosa and the optimization of specific markers. In summary, our new panel is intended to be a major step forward to interpret biological evidence at sub-source and source level based on cSNP attribution of a body fluid component to a suspect and victim, respectively., Competing Interests: Declaration of Competing Interest All authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Abstract: We previously reported a better outcome in adult and pediatric T-cell acute lymphoblastic leukemia (T-ALL) harboring NOTCH1 and/or FBXW7 mutations without alterations of K-N-RAS and PTEN genes. Availability of high-throughput next-generation sequencing (NGS) strategies led us to refine the outcome prediction in T-ALL. Targeted whole-exome sequencing of 72 T-ALL-related oncogenes was performed in 198 adults with T-ALLs in first remission from the GRAALL-2003/2005 protocols and 242 pediatric patients with T-ALLs from the FRALLE2000T. This approach enabled the identification of, to our knowledge, the first NGS-based classifier in T-ALL, categorizing low-risk patients as those with N/F, PHF6, or EP300 mutations, excluding N-K-RAS, PI3K pathway (PTEN, PIK3CA, and PIK3R1), TP53, DNMT3A, IDH1/2, and IKZF1 alterations, with a 5-year cumulative incidence of relapse (CIR) estimated at 21%. Conversely, the remaining patients were classified as high risk, exhibiting a 5-year CIR estimated at 47%. We externally validated this stratification in the pediatric cohort. NGS-based classifier was highly prognostic independently of minimal residual disease (MRD) and white blood cell (WBC) counts, in both adult and pediatric cohorts. Integration of the NGS-based classifier into a comprehensive risk-stratification model, including WBC count at diagnosis and MRD at the end of induction, enabled the identification of an adverse-risk subgroup (25%) with a 5-year CIR estimated at 51%, and a favorable-risk group (32%) with a 5-year CIR estimated at 12%. NGS-based stratification combined with WBC and MRD sharpens the prognostic classification in T-ALL and identifies a new subgroup of patients who may benefit from innovative therapeutic approaches. The GRAALL-2003/2005 studies were registered at www.ClinicalTrials.gov as #NCT00222027 and #NCT00327678., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Advances in Thyroid Pathology: WHO CLASSIFICATION 2022, BETHESDA SYSTEM 2023, MOLECULAR BIOLOGY AND MOLECULAR TESTING: Thyroid pathology has experienced significant advances with the publication of the 5th edition of the World Health Organization classification of endocrine tumors in 2022 and the third edition of the Bethesda system for thyroid cytopathology in 2023. At the same time, the availability of next-generation sequencing data coupled with numerous translational research projects have considerably increased our knowledge of the genomics and mechanics of thyroid cancers, enabling us to refine prognosis and propose new targeted therapies. In this review, we will take up the main new features of the WHO 2022 and Bethesda 2023 classifications, as well as molecular biology findings, with an emphasis on the practical implications for clinicians., Competing Interests: Liens d’intérêts S. Moog, M. Amine Bani, A. Al Ghuzlan et V. Suciu déclarent ne pas avoir de liens d’intérêts. L. Lamartina déclare des liens d’intérêts pour des financements pour participation à un congrès de la part des laboratoires AAA Novartis et Ipsen; pour participation à des tumor boards de la part des laboratoires Eisai et Ipsen; pour des honoraires perçus de la part des laboratoires Eisai, Lilly et Roche. Cet article fait partie du supplément Prise en charge des cancers thyroïdiens en 2024 : avancées diagnostiques et thérapeutiques réalisé avec le soutien institutionnel de Lilly., (Copyright © 2024 Elsevier Masson SAS. Tous droits réservés. Published by Elsevier Masson SAS. All rights reserved.)

New scientific techniques and methods are always needed to link the perpetrators to the incident or the crime scene. Recent microbiota studies based on NGS (Next-generation sequencing) show that various biological samples from crime scenes have the potential to be used in forensic investigations. Especially when DNA traces belonging to more than one person are insufficient to fully determine the genetic profile, a secret sample, such as a microbiota sample created by the suspect's touch, can be used. In this preliminary study, a fictionalized experimental model was designed to investigate the transfer and persistence of the hand microbiome on the knife handle, which has a high potential to be used in criminal incidents, by metagenomic analysis methods. In addition, it was aimed to determine the transfer of specific bacterial species identified only to the person among the five participants onto the knife handle and their persistence over time. In the first stage of the research, samples were collected from the hands of 5 volunteer participants using the swap method, including their palms. Then, after each participant held a different knife, samples were collected from the knife handles via swabs from different angles of the knives at 4 and 24 h and analyzed by metagenomic methods. The findings of this preliminary study showed that the heatmap graphs generated after UniFrac distance analysis were not successful in establishing any similarity between the hand samples and the post-transfer knife handle samples. Nonetheless, it was observed that the transfer of bacterial species detected in the hand samples to knives differed according to the individuals and some bacterial species were transferred to the knife samples held by the participants. The number of bacterial species detected that are specific to each participant's hand sample was 302 in total, and it was determined that a total of 8.28 % of these bacterial species were transferred to the knife handle samples of the 4th hour and 6.95 % to the knife samples of the 24th hour. In the presented study, considering the transfer of some bacterial species in the hand microbiome, which are effective in the variation between individuals, onto the knife; It has been evaluated that some rare bacterial species can be important potential markers to associate the object with the perpetrator., Competing Interests: Declaration of competing interest All authors have contributed significantly to manuscript and all authors agree that they agree with the content of the article. The authors declare that there is no financial support or relationship that may create a conflict of interest., (Copyright © 2024 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.)

Giardia duodenalis is a globally distributed intestinal parasite that commonly infects both humans and animals. G. duodenalis is a species complex, which includes eight assemblages that vary both in genetic structure and host specificity. The prevalence of mixed-assemblage G. duodenalis cysts on food, an understudied infection route for G. duodenalis, remains unknown. In the present study, a method able to detect G. duodenalis mixed-assemblage infections using next-generation amplicon sequencing (NGS) of the beta-giardin gene was applied in combination with the US-FDA's BAM Chapter 19b protocol for the detection of G. duodenalis from fresh produce to ascertain the limit of detection of G. duodenalis on leafy greens. Ready-to-eat baby Romaine lettuce was inoculated with 5 (n = 5), 20 (n = 10), 100 (n = 10), 200 (n = 10), or 1,000 (n = 10) G. duodenalis cysts of the assemblage B strain H3. Detection of G. duodenalis was successful in 100% of the samples seeded with 1,000, 200, and 100 cysts, in 50% of the samples seeded with 20 cysts, and in none of the samples seeded with 5 cysts. We thus demonstrate robust detection of G. duodenalis on packaged leafy greens using the BAM Chapter 19B method coupled with assemblage-sensitive NGS. This protocol provides a new diagnostic tool useful for both prevalence studies and outbreak investigations involving fresh produce that may assist in better describing the role of G. duodenalis in foodborne illness and in protecting consumers from contaminated fresh produce., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Inc.)

Ice, water, and sediment represent three interconnected habitats in lake ecosystems, and bacteria are crucial for maintaining ecosystem equilibrium and elemental cycling across these habitats. However, the differential characteristics and driving mechanisms of bacterial community structures in the ice, water, and sediments of seasonally frozen lakes remain unclear. In this study, high-throughput sequencing technology was used to analyze and compare the structure, function, network characteristics, and assembly mechanisms of bacterial communities in the ice, water, and sediment of Wuliangsuhai, a typical cold region in Inner Mongolia. The results showed that the bacterial communities in the ice and water phases had similar diversity and composition, with Proteobacteria, Bacteroidota, Actinobacteria, Campilobacterota, and Cyanobacteria as dominant phyla. The bacterial communities in sediments displayed significant differences from ice and water, with Chloroflexi, Proteobacteria, Firmicutes, Desulfobacterota, and Acidobacteriota being the dominant phyla. Notably, the bacterial communities in water exhibited higher spatial variability in their distribution than those in ice and sediment. This study also revealed that during the frozen period, the bacterial community species in the ice, water, and sediment media were dominated by cooperative relationships. Community assembly was primarily influenced by stochastic processes, with dispersal limitation and drift identified as the two most significant factors within this process. However, heterogeneous selection also played a significant role in the community composition. Furthermore, functions related to nitrogen, phosphorus, sulfur, carbon, and hydrogen cycling vary among bacterial communities in ice, water, and sediment. These findings elucidate the intrinsic mechanisms driving variability in bacterial community structure and changes in water quality across different media phases (ice, water, and sediment) in cold-zone lakes during the freezing period, offering new insights for water environmental protection and ecological restoration efforts in such environments., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: This study characterized the bacteriome in primary endodontic infection (PEI) with apical periodontitis (AP), identified core and rare bacteriome species and community diversity metrics, and analyzed the relationship between the bacteriome composition, diversity and features, and patient variables., Methods: Twenty-seven patients with PEI and AP were sampled. The DNA was extracted and quantified using quantitative polymerase chain reaction. Raw V3-V4 amplicon sequencing data were processed with the DADA2 pipeline to generate amplicon sequence variants, and taxonomic assignment of the amplicon sequence variants up to the species level was done against the Human Oral Microbiome Database. Core bacteriome and differential abundance analyses were performed using ANCOM. Alpha diversity was determined using Chao1, Shannon, and Simpson indexes. LeFse analysis was used to identify abundant taxa. Sparse Estimation of Correlations among Microbiomes analysis estimated linear and nonlinear relationships among bacteria., Results: Of 27, 24 root canal samples were analyzed, and 3 root canal sampling were filtered out with a low read count. The bacterial phyla with top mean relative abundance were Bacteroidetes, Firmicutes, Synergistetes, Fusobacteria, and Actinobacteria. A total of 113 genera and 215 species were identified. The samples were gathered into 3 clusters. LefSe analysis identified differences in abundant taxa between distinct age, gender, symptomatology, and lesion size groups. Sparse Estimation of Correlations among Microbiomes distance analysis indicated Slackia exigua as the node with the highest degree., Conclusions: The bacteriome in PEI with AP among the patients in this study was complex and displayed high microbial heterogeneity. Moreover, age, gender, symptomatology, and lesion size were associated with differences in bacteriome features in PEI with AP., (Copyright © 2024 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Since the advent of Hi-C in 2009, a plethora of high-throughput sequencing methods have emerged to profile the three-dimensional (3D) organization of eukaryotic genomes, igniting the era of 3D genomics. In recent years, the genomic resolution achievable by these approaches has dramatically increased and several single-cell versions of Hi-C have been developed. Moreover, a new repertoire of tools not based on proximity ligation of digested chromatin has emerged, enabling the investigation of the higher-order organization of chromatin in the nucleus. In this review, we summarize the expanding portfolio of 3D genomic technologies, highlighting recent developments and applications from the past three years. Lastly, we present an outlook of where this technology-driven field might be headed., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objectives: Hepatitis C virus (HCV) disproportionately affects people who inject drugs (PWID) worldwide. Despite carrying a high HCV burden, little is known about transmission dynamics in low- and middle-income countries., Methods: We recruited PWID from Nairobi and coastal cities and towns of Mombasa, Kilifi, and Malindi in Kenya at needle and syringe programs. Next-generation sequencing data from HCV hypervariable region 1 were analyzed using Global Hepatitis Outbreak and Surveillance Technology to identify transmission clusters., Results: HCV strains belonged to genotype 1a (n = 64, 46.0%), 4a (n = 72, 51.8%) and mixed HCV/1a/4a (n = 3, 2.2%). HCV/1a was dominant (61.2%) in Nairobi, whereas HCV/4a was dominant in Malindi (85.7%) and Kilifi (60.9%), and both genotypes were evenly identified in Mombasa (45.3% for HCV/1a and 50.9% for HCV/4a). Global Hepatitis Outbreak and Surveillance Technology identified 11 transmission clusters involving 90 cases. Strains in the two largest clusters (n = 38 predominantly HCV/4a and n = 32 HCV/1a) were sampled from all four sites., Conclusions: Transmission clusters involving 64.7% of cases indicate an effective sampling of major HCV strains circulating among PWID. Large clusters involving 77.8% of clustered strains from Nairobi and Coast suggest successful introduction of two ancestral HCV/1a and HCV/4a strains to PWID and the existence of a widespread transmission network in the country. The disruption of this network is essential for HCV elimination., Competing Interests: Declarations of competing interest The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Introduction: Autosomal Recessive Polycystic Kidney Disease (ARPKD) is a rare paediatric disease primarily caused by sequence variants in PKHD1. ARPKD presents with considerable clinical variability relating to the type of PKHD1 sequence variant, but not its position. Animal models of Polycystic Kidney Disease (PKD) suggest a complex genetic landscape, with genetic modifiers as a potential cause of disease variability., Methods: To investigate in an unbiased manner the molecular mechanisms of ARPKD and identify potential indicators of disease severity, Whole Exome Sequencing (WES) and RNA-Sequencing (RNA-Seq) were employed on human ARPKD kidneys and age-matched healthy controls., Results: WES confirmed the clinical diagnosis of ARPKD in our patient cohort consisting of ten ARPKD kidneys. Sequence variant type, nor position of PKHD1 sequence variants, was linked to disease severity. Sequence variants in genes associated with other ciliopathies were detected in the ARPKD cohort, but only PKD1 could be linked to disease severity. Transcriptomic analysis on a subset of four ARPKD kidneys representing severe and moderate ARPKD, identified a significant number of genes relating to WNT signalling, cellular metabolism and development. Increased expression of WNT signalling-related genes was validated by RT-qPCR in severe and moderate ARPKD kidneys. Two individuals in our cohort with the same PKHD1 sequence variants but different rates of kidney disease progression, with displayed transcriptomic differences in the expression of WNT signalling genes., Conclusion: ARPKD kidney transcriptomics highlights changes in WNT signalling as potentially significant in ARPKD manifestation and severity, providing indicators for slowing down the progression of ARPKD., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Taylor Richards reports financial support was provided by PKD Charity UK. Paraskevi Goggolidou reports equipment, drugs, or supplies were provided by PKD Charity UK. Patricia Wilson reports a relationship with PKD Charity UK that includes: board membership. Patricia Wilson is an Editorial Board member for BBA Molecular Basis of Disease. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier B.V. All rights reserved.)

Cholangiocarcinoma (CCA) is a life-threatening disease that impacts patients worldwide. In Southeast Asian countries, the liver fluke Opisthorchis viverrini plays a major role in inducing carcinogenesis of the bile ducts. Due to its asymptomatic nature, O. viverrini infections are rarely treated, consequently leading to the development of advanced stages of CCA before diagnosis. Despite the current use of exosomal microRNAs (miRNA) as diagnostic biomarkers for the early detection of many types of cancer, the applications for miRNA remain limited with CCA. Circulating exosomes, membranous vesicles essential for intercellular communication, were found to contain unique miRNA. In this study, we conducted next-generation sequencing (Ion Torrent PGM) and bioinformatics to characterize and compare the contents of exosomal miRNA derived from the plasma of CCA patients, O. viverrini-infected patients, and healthy individuals, as well as to identify and validate key molecules as markers for screening the diagnosis of CCA and O. viverrini infection. The obtained results showed the success of using NGS technology in discovering exosomal miRNAs, specifically miR-194-5p and miR-192-5p, both of which were upregulated in the O. viverrini-infected group. Interestingly, miR-192-5p was upregulated while miR-194-5p was downregulated in CCA, suggesting their potential use as biomarkers for screening CCA and O. viverrini infection, especially in O. viverrini-endemic areas., Competing Interests: Declaration of competing interest The authors declared no competing interests for this study, (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objectives: The aim of this study was to quantitatively and comprehensively investigate the combined effects of arginine and fluoride on the suppression of pathogenicity using an in situ biofilm model and next-generation sequencing (NGS)., Methods: Using the in situ model, dental biofilms were formed and the viable bacterial counts and arginine activity in the arginine- and fluoride-containing dentifrice and control groups were measured. We also compared their effects on the bacterial microbiota and predictive functional factors in the control, arginine (arg), and arginine + fluoride (argF) groups using NGS analysis., Results: Compared to the control treatment, the use of 8 % arginine and 1450 ppm fluoride toothpaste resulted in significantly high oral NH 4 + concentrations without affecting the number of viable bacteria (P < 0.05). NGS analysis revealed that the oral microbiota of the control, arg, and argF groups were significantly different. Heat map analysis of the predicted functional factors revealed that the arg group had different properties from the other groups and activated specific substrate metabolic pathways; contrastingly, argF treatment inhibited the activity of these pathways and prevented an increase in the abundance of bacterial genera that utilize substrates such as sucrose, suggesting the synergistic effect of arginine and fluoride., Conclusions: This study indicates that the combination of arginine and fluoride has a synergistic effect on the bacterial microbiota and pathogenicity of dental biofilms compared with arginine alone., Clinical Significance: Our findings suggest that the combination of arginine and fluoride could be used as an effective prebiotic and may inhibit the growth of bacteria associated with dental diseases., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier Ltd.)

Genome-wide platelet transcriptomics is increasingly used to uncover new aspects of platelet biology and as a diagnostic and prognostic tool. Nevertheless, platelet isolation methods for transcriptomic studies are not standardized, introducing challenges for cross-study comparisons, data integration, and replication. In this prospective multicenter study, called "Standardizing Platelet Transcriptomics for Discovery, Diagnostics, and Therapeutics in the Thrombosis and Hemostasis Community (STRIDE)" by the International Society on Thrombosis and Haemostasis Scientific and Standardization Committees, we assessed how 3 of the most commonly used platelet isolation protocols influence metrics from next-generation bulk RNA sequencing and functional assays. Compared with washing alone, more stringent removal of leukocytes by anti-CD45 beads or PALL filters resulted in a sufficient quantity of RNA for next-generation sequencing and similar quality of RNA sequencing metrics. Importantly, stringent removal of leukocytes resulted in the lower relative expression of known leukocyte-specific genes and the higher relative expression of known platelet-specific genes. The results were consistent across enrolling sites, suggesting that the techniques are transferrable and reproducible. Moreover, all 3 isolation techniques did not influence basal platelet reactivity, but agonist-induced integrin αIIbβ 3 activation is reduced by anti-CD45 bead isolation compared with washing alone. In conclusion, the isolation technique chosen influences genome-wide transcriptional and functional assays in platelets. These results should help the research community make informed choices about platelet isolation techniques in their own platelet studies., Competing Interests: Declaration of competing interests M.T.R. reports patents pending or issued on using platelet transcriptomics. All other authors have no conflicts to declare., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This study conducted a thorough analysis of the myofiber type composition in the extensor digitorum longus muscle (EDL) and soleus muscle (SOL) of Kazakh horses, across different genders (male and female). The results showed significant differences in myofiber type composition between EDL and SOL, with a higher proportion of Type I fibers in SOL muscles and a greater prevalence of Type II fibers in EDL muscles. Additionally, the myofiber diameter in Kazakh horses was relatively small, potentially related to the tenderness and edible quality of their muscles. Using high-throughput sequencing technology, we constructed 32 cDNA sequencing libraries and obtained high-quality read data. Gene expression analysis revealed 278 and 372 differentially expressed genes (DEGs) in EDL and SOL muscles, respectively, including genes related to muscle contraction, metabolism, and development. Intersection analysis of DEGs between genders showed that 60 DEGs were significantly different in both male and female horses. GO annotation and KEGG analysis further elucidated the roles of these DEGs in muscle structure, function, and cellular signaling. Protein-protein interaction (PPI) network analysis and identification of hub genes provided new insights into the molecular mechanisms underlying muscle growth and development. Finally, the reliability of the DEGs data was validated through quantitative real-time PCR (qRT-PCR). This study not only enhances our understanding of the biological characteristics of horse muscles but also provides potential molecular targets for improving horse muscle performance and health., Competing Interests: Declaration of competing interest All authors declare that they have no conflict of interest., (Copyright © 2023. Published by Elsevier Ltd.)

Background: Young women with breast cancer (YWBC; ≤40 years) often have a poorer prognosis than older women with breast cancer (OWBC; ≥65 years). We explored molecular features of tumors from YWBC and OWBC to identify a biologic connection for these patterns., Materials and Methods: We retrospectively analyzed the molecular profiles of 1879 breast tumors. Testing included immunohistochemistry (IHC), in situ hybridization (ISH), and next-generation sequencing. Statistical analyses included Pearson's chi 2 test for comparisons, with significance defined as FDR (false discovery rate)-P < .05., Results: TP53 and BRCA1 somatic mutations were more common in YWBC tumors than in OWBC tumors (53%, 42%; P = .0001, FDR-P = .0025 and 7%, 2%; P = .0001, FDR-P = .0025; respectively). Conversely, OWBC tumors had higher androgen receptor expression (55%, 45%; P = .0002, FDR-P = .0025) higher PD-L1 expression detected by IHC (8%, 5%; P = .0476, FDR-P = .2754), and more frequent PIK3CA mutations (33%, 17%; P = < .0001, FDR-P = < .0001). Among HR+/HER2- samples, YWBC had more gene amplifications in FGF3 (27%, 10%; P = .0353, FDR-P = .2462), FGF4 (27%, 9%; P = .0218, FDR-P = .1668), FGF19 (30%, 12%; P = .034, FDR-P = .2462) and CCND1 (37%, 18%; P = .0344, FDR-P = .2462) than OWBC., Conclusions: Our data suggest distinct molecular aberrations exist between YWBC and OWBC. Exploiting these molecular changes could refine our treatment strategies in YWBC and OWBC., Competing Interests: Disclosure Arielle L. Heeke: consulting (Caris Life Sciences). Wei Sha: no relevant conflicts to disclose. Rebecca Feldman: employee (Caris Life Sciences) Julie Fisher: no relevant conflicts to disclose. Lejla Hadzikadic-Gusic: no relevant conflicts to disclose. James T. Symanowski: no relevant conflicts to disclose. Richard L. White, Jr: no relevant conflicts to disclose. Antoinette R. Tan: meeting travel and accommodations (Caris Life Sciences)., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The rot caused by pathogens during the storage of table grapes is an important factor that affects the development of the grape industry and food safety, and it cannot be ignored. The development of innovative methods for pathogen control should be based on a comprehensive understanding of the overall microbial community changes that occur during grape storage. The study aims to investigate the relationship between the native microbiota (including beneficial, pathogenic and spoilage microorganisms) on grape surfaces and the development of disease during grape storage. In this study, the bacteria and fungi present on grape surfaces were analyzed during storage under room temperature conditions using high-throughput sequencing. During the storage of grapes at room temperature, observable diseases and a noticeable decrease in quality were observed at 8 days. Microbial community analysis showed that 4996 bacterial amplicon sequence variants (ASVs) and 488 fungal ASVs were determined. The bacterial richness exhibited an initial increase followed by a subsequent decrease. However, the diversity exhibited a distinct pattern of gradual decrease. The fungal richness and community diversity both exhibit a gradual decrease during the storage of grapes. Fungal β-diversity analysis showed that despite the absence of rot and the healthy state of grapes on the first and fourth days, the fungal β-diversity exhibited a significant difference. The analysis of changes in genera abundances suggested that Candidatus Profftella and Aspergillus exhibited dominance in the rotting grape at 16 days, which are the main pathogens that caused disease in the present study. The co-occurrence networks among the microbial showed that the Candidatus proftella genera has a positive correlation with Aspergillus niger, indicating that they work together to cause disease and promote growth in grapes. Predicting the function of bacterial communities found that the microorganisms associated with lipid metabolism at 4 days play an important role in the process of postharvest decay of grapes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The rhizosphere environment of plants, which harbors halophilic bacterial communities, faces significant challenges in coping with environmental stressors, particularly saline soil properties. This study utilizes a high-throughput 16S rRNA gene-based amplicon sequencing to investigate the variations in bacterial community dynamics in rhizosphere soil (RH), root surface soil (RS), root endophytic bacteria (PE) compartments of Suaeda salsa roots, and adjoining soils (CK) across six locations along the eastern coast of China: Nantong (NT), Yancheng (YC), Dalian (DL), Tianjin (TJ), Dongying (DY), and Qingdao (QD), all characterized by chloride-type saline soil. Variations in the physicochemical properties of the RH compartment were also evaluated. The results revealed significant changes in pH, electrical conductivity, total salt content, and ion concentrations in RH samples from different locations. Notably, the NT location exhibited the highest alkalinity and nitrogen availability. The pH variations were linked to HCO 3 - accumulation in S. salsa roots, while salinity stress influenced soil pH through H + discharge. Despite salinity stress, enzymatic activities such as catalase and urease were higher in soils from various locations. The diversity and richness of bacterial communities were higher in specific locations, with Proteobacteria dominating PE samples from the DL location. Additionally, Vibrio and Marinobacter were prevalent in RH samples. Significant correlations were found between soil pH, salinity, nutrient content, and the abundance and diversity of bacterial taxa in RH samples. Bioinformatics analysis revealed the prevalence of halophilic bacteria, such as Bacillus, Halomonas, and Streptomyces, with diverse metabolic functions, including amino acid and carbohydrate metabolisms. Essential genes, such as auxin response factor (ARF) and GTPase-encoding genes, were abundant in RH samples, suggesting adaptive strategies for harsh environments. Likewise, proline/betaine transport protein genes were enriched, indicating potential bioremediation mechanisms against high salt stress. These findings provide insight into the metabolic adaptations facilitating resilience in saline ecosystems and contribute to understanding the complex interplay between soil conditions, bacterial communities, and plant adaptation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

DNA mixtures are a common sample type in forensic genetics, and we typically assume that contributors to the mixture are unrelated when calculating the likelihood ratio (LR). However, scenarios involving mixtures with related contributors, such as in family murder or incest cases, can also be encountered. Compared to the mixtures with unrelated contributors, the kinship within the mixture would bring additional challenges for the inference of the number of contributors (NOC) and the construction of probabilistic genotyping models. To evaluate the influence of potential kinship on the individual identification of the person of interest (POI), we conducted simulations of two-person (2 P) and three-person (3 P) DNA mixtures containing unrelated or related contributors (parent-child, full-sibling, and uncle-nephew) at different mixing ratios (for 2 P: 1:1, 4:1, 9:1, and 19:1; for 3 P: 1:1:1, 2:1:1, 5:4:1, and 10:5:1), and performed massively parallel sequencing (MPS) using MGIEasy Signature Identification Library Prep Kit on MGI platform. In addition, in silico simulations of mixtures with unrelated and related contributors were also performed. In this study, we evaluated 1): the MPS performance; 2) the influence of multiple genetic markers on determining the presence of related contributors and inferring the NOC within the mixture; 3) the probability distribution of MAC (maximum allele count) and TAC (total allele count) based on in silico mixture profiles; 4) trends in LR values with and without considering kinship in mixtures with related and unrelated contributors; 5) trends in LR values with length- and sequence-based STR genotypes. Results indicated that multiple numbers and types of genetic markers positively influenced kinship and NOC inference in a mixture. The LR values of POI were strongly dependent on the mixing ratio. Non- and correct-kinship hypotheses essentially did not affect the individual identification of the major POI; the correct kinship hypothesis yielded more conservative LR values; the incorrect kinship hypothesis did not necessarily lead to the failure of POI individual identification. However, it is noteworthy that these considerations could lead to uncertain outcomes in the identification of minor contributors. Compared to length-based STR genotyping, using sequence-based STR genotype increases the individual identification power of the POI, concurrently improving the accuracy of mixing ratio inference using EuroForMix. In conclusion, the MGIEasy Signature Identification Library Prep kit demonstrated robust individual identification power, which is a viable MPS panel for forensic DNA mixture interpretations, whether involving unrelated or related contributors., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

X-linked microhaplotypes (X-MHs) have the potential to be a valuable supplementary tool in complex kinship identification or the resolution of DNA mixtures, because they bring together the distinctive genetic pattern of X chromosomal markers and the benefits of microhaplotypes (MHs). In this study, we used the 1000 Genome database to screen and select 63 X-MHs; 18 MHs were filtered out though a batch sequencing assessment of the DNA samples collected from 112 unrelated Chinese Han individuals. The resulting 45-plex panel performed well in comprehensive assessments including repeatability, sensitivity, species specificity, resistance to PCR inhibitors or degradation, mutation rate, and accuracy in detecting DNA mixture samples. The minimum amount of DNA template that can be tested with this panel is 0.5 ng. Additionally, the alleles of the minor contributor can be accurately detected when the mixture rate is larger than 1:9 in female-male mixture or 1:19 in male-male mixture. Then, we calculated population parameters on each MH based on the allele frequency data obtained from the sequence results of the aforementioned 112 unrelated samples. Combining these parameters on each MH, it can be calculated that TDP m , TDP f , CPET, CPED FM , CPED FF and CNCEP 3 of the 45-plex system were 1-8.99×10 -13 , 1-1.62×10 -19 , 0.9999999995, 0.9999981, 0.9955, 0.9999971 and 0.99940, respectively, indicating that the panel is capable in personal identification and parentage testing. To reveal the unique advantage of X-MHs in the analyses of complex kinship and male DNA mixture, further assessments were made. For complex kinship identification, 22 types of individual pairs with different second-degree kinship were simulated and different types of likelihood ratios (LR) were calculated for each. The results revealed that the panel can achieve accuracy of approximately 70 %∼80 % when dividing each of the three types of second-degree kinships into three or four groups. Theoretically, such sub-division cannot be done by using independent autosomal markers. For male DNA mixture analysis without suspects, the maximum likelihood ratio strategy was derived and employed in the estimation of the number of male contributors (NOMC). Simulations were conducted to verify the efficacy of the 45-plex panel in the field and to compare it with autosomal markers by assuming the 45 MHs as autosomal ones. The results showed that X-MHs can achieve higher accuracy in the estimation of NOMC than autosomal ones when the mixed males were unrelated. The results highlighted the unique value of X-linked MHs in complex kinship and male mixture analyses., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: the human leukocyte antigen (HLA) loci have been widely characterized to be associated with viral infectious diseases. Several studies including various ethnic groups and populations suggested associations between certain HLA alleles and SARS-CoV-2 infection. Despite the numerous associations identified, the role of HLA polymorphisms in determining the individual response to SARS-CoV-2 infection is controversial among different Saudi populations., Method: Here, we performed HLA typing by next-generation sequencing to investigate if variations in polymorphic HLA genes are linked to COVID-19 severity in the Saudi population. Namely, we analyzed HLA loci at allele level in 575 Saudi patients with SARS-CoV-2 infection. HLA class I and class II frequencies in patients were compared with allele frequency data from healthy Saudi population., Results: in our cohort HLA-A* 02:01:01 G was associated with mild disease but was not associated with moderate and severe disease. HLA-B* 51:01:01 G was protective from severe disease while HLA-B* 50:01:01 G, HLA-C* 06:02:01 G and HLA-DRB1 * 07:01:01 G were associated with risk to severe disease as well as the total COVID-19 cohort. HLA-DRB1 * 15:01:01 G was associated with risk to all severity groups., Conclusion: in conclusion, we found significant associations between HLA alleles and COVID-19 disease severity in Saudis. Further studies are warranted to include HLA typing in the workup for any new COVID-19 patients., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Common capillary malformations are red vascular skin lesions, most commonly associated with somatic activating GNAQ or GNA11 mutations. We focused on capillary malformations lacking such a mutation to identify previously unreported genetic causes. We used targeted next-generation sequencing on 82 lesions. Bioinformatic analysis allowed the identification of 9 somatic pathogenic variants in PIK3R1 and PIK3CA, encoding for the regulatory and catalytic subunits of phosphoinositide 3-kinase, respectively. Recharacterization of these lesions unraveled a common phenotype: a pale capillary malformation associated with visible dilated veins. Primary endothelial cells from 2 PIK3R1-mutated lesions were isolated, and PI3k-Akt-mTOR and RAS-RAF-MAPK signaling were assessed by western blot. This unveiled an abnormal increase in Akt phosphorylation, effectively reduced by PI3K pathway inhibitors, such as mTOR, Akt, and PIK3CA inhibitors. The effects of mutant PIK3R1 were further studied using zebrafish embryos. Endothelium-specific expression of PIK3R1 mutants resulted in abnormal development of the posterior capillary-venous plexus. In summary, capillary malformation associated with visible dilated veins emerges as a clinical entity associated with somatic pathogenic variants in PIK3R1 or PIK3CA (nonhotspot). Our findings suggest that the activated Akt signaling can be effectively reversed by PI3K pathway inhibitors. In addition, the proposed zebrafish model holds promise as a valuable tool for future drug screening aimed at developing patient-tailored treatments., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Tuberculosis (TB) remains a global public health threat, and the development of rapid and precise diagnostic tools is the key to enabling the early start of treatment, monitoring response to treatment, and preventing the spread of the disease., Objectives: An overview of recent progress in host- and pathogen-based TB diagnostics., Sources: We conducted a PubMed search of recent relevant articles and guidelines on TB screening and diagnosis., Content: An overview of currently used methods and perspectives in the following areas of TB diagnostics is provided: immune-based diagnostics, X-ray, clinical symptoms and scores, cough detection, culture of Mycobacterium tuberculosis and identifying its resistance profile using phenotypic and genotypic methods, including next-generation sequencing, sputum- and non-sputum-based molecular diagnosis of TB and monitoring of response to treatment., Implications: A brief overview of the most relevant advances and changes in international guidelines regarding screening and diagnosing TB is provided in this review. It aims at reviewing all relevant areas of diagnostics, including both pathogen- and host-based methods., (Copyright © 2023 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Introduction: To evaluate the antimicrobial activity of Triton irrigation versus 4% sodium hypochlorite (NaOCl) utilizing a direct contact test and an extracted tooth model., Methods: In the first experiment, a direct contact test was conducted to compare bacterial DNA removal and microbial diversity changes following irrigation with 4% NaOCl or Triton. Hydroxyapatite and dentin discs were inoculated with subgingival human-derived dental plaque for 2 weeks utilizing the Center for Disease Control biofilm reactor and subsequently challenged with the root canal irrigants for 5 minutes. In the second experiment, teeth contaminated with a multispecies biofilm (n = 24) were assigned into two treatment groups, NaOCl or Triton irrigation. Samples were obtained for quantitative real-time polymerase chain reaction and next-generation sequencing analysis before and after instrumentation. The Shannon and Chao1 indices were used to measure alpha diversity. The Bray-Curtis dissimilarity and ANOSIM was used to measure beta diversity. Differences in abundances of genera were evaluated using Kruskal-Wallis test with Bonferroni corrections., Results: The direct contact test revealed no significant differences in the bacterial load based on 16S rRNA gene molecules/μL, reads, or differences in the Shannon index among groups. In the extracted tooth model, a bacterial load reduction of log 10 3.08 ± 0.69 and 2.76 ± 0.91 were found for NaOCl and Triton, respectively (P = .348). Next-generation sequencing showed fewer reads, lower Chao1, and beta diversity values when pretreatment and post-treatment samples were assessed in both experimental groups (P < .0001). The Kruskal-Wallis analysis found that 17 genera of bacteria were over-represented in minimal values in the Triton post-treatment group, 14 of these genera represented less than 1% of the microbial community., Conclusions: Both irrigants had limited antimicrobial activity in the direct contact test. When used in conjunction with mechanical instrumentation both irrigants were able to reduce the bacterial DNA load and diversity in comparison with pretreatment communities. The NaOCl irrigation, followed by ethylenediaminetetraacetic acid flush, was more effective in decreasing DNA counts from low-abundance organisms., (Copyright © 2024 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Tripos is a large dinoflagellate genus widely distributed in the world's oceans. Morphology-based species identification is inconclusive due to high morphological intraspecific variability. Metabarcoding analysis has been demonstrated to be effective for species identification and tracking their spatiotemporal dynamics. However, accumulating evidence suggests high levels of intragenomic variations (IGVs) are common in many algae, leading to concerns about overinterpretation of molecular diversity in metabarcoding studies. In this project, we evaluated and compared IGVs in Tripos species by conducting the first high-throughput sequencing (HTS) of 18S rDNA V4 of Tripos single cells. High numbers of haplotypes (19-172) were identified in each of the 30 Tripos cells. Each cell contained one dominant haplotype with high relative abundance and many haplotypes with lower abundances. Thus, the presence of multiple minor haplotypes substantially overestimate the molecular diversity identified in metabarcoding analysis, which encompass not only interspecific and intraspecific diversities, but high levels of IGVs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Human anaplasmosis cases, caused by Anaplasma phagocytophilum, are increasing in the United States. This trend is explained, in part, by expansion in the geographic range of the primary vector, Ixodes scapularis. Multiple variants of A. phagocytophilum have been identified in field collected ticks, but only a single variant (human active, or "Ap-ha," variant) has been shown to be pathogenic in humans. Until recently, laboratory methods used to differentiate variants were cumbersome and seldomly used in large scale assessments of the pathogen's geographic distribution. As a result, many surveys reported A. phagocytophilum without segregating variants. Lack of discrimination among A. phagocytophilum variants could lead to overestimation of anaplasmosis risk to humans. Next Generation Sequencing (NGS) assays were recently developed to efficiently detect multiple Ixodes scapularis-borne human pathogens including Ap-ha. In this study, we utilized NGS to detect and differentiate A. phagocytophilum variants (Ap-ha vs. non ha) in host-seeking I. scapularis nymphs and adults collected across 23 states in the eastern United States from 2012 to 2023 as part of national tick surveillance efforts and research studies. Many of the included ticks were tested previously using a TaqMan PCR assay that could detect A. phagocytophilum but could not differentiate variants. We retested A. phagocytophilum infected ticks with NGS to differentiate variants. Anaplasma phagocytophilum (any variant) was identified in 165 (35 %) of 471 counties from which ticks were tested, whereas Ap-ha was detected in 70 (15 %) of 469 counties where variants were differentiated. Both variants were identified in 32 % (n = 40) of 126 counties with either variant detected. Among states where A. phagocytophilum (any variant) was detected, prevalence ranged from 2 % to 19 % in unfed adults and from 0.2 % to 7.8 % in unfed nymphs; prevalence of Ap-ha variant ranged from 0.0 % to 16 % in adults, and 0.0 % to 4.6 % in nymphs., (Copyright © 2024. Published by Elsevier GmbH.)

Significant progress has been made in recent years in the development of techniques for Next Generation Sequencing (NGS), or Massively Parallel Sequencing (MPS), of forensically relevant short tandem repeat (STR) loci. However, as these technologies are investigated and adopted by forensic laboratories, new challenges unfold that require further scrutiny. In the analysis of DNA profiles generated using the MiSeq FGx sequencing system, we have observed noise sequences with relatively high readcounts that are challenging to distinguish from genuine alleles. These high read count noise sequences appear as allele sequences with one or a few substituted bases compared to a known allele sequence within the profile. An examination of ForenSeq DNA Signature Prep Kit STR noise sequences revealed that the substituted base of a parent allele can align to the same position on the sequence across noise sequences. This suggests that these substitution events occur at specific positions within the amplicon, resulting in multiple noise reads with substitutions at the same position. Mapping of the noise events onto the original raw read positions revealed a high number of events, or "noise spikes", occurring at specific positions within a given sequencing run. These noise spikes affected reads across the entire run, agnostic of locus or sample, while the position, occurrence, and amplitude of the spikes differed across runs. The majority of noise sequences with high read counts in a DNA profile were generated from base changes at these spike positions, and could be classified as "noise spike artefacts". In this paper we present evidence of the noise spike artefacts and their genesis during the sequencing process in the sequencing-by-synthesis (SBS) cycles, as well as the methods developed to detect them. The information and methods will assist laboratories with detecting noise spikes in MiSeq FGx sequencing runs, differentiating authentic allele sequences from noise spike artefacts, and developing protocols for analyst review and handling of MiSeq FGx data., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to disclose., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Microhaplotypes (MHs), comprising two or more single-nucleotide polymorphisms in a short fragment, are promising forensic markers owing to their remarkable polymorphic nature. Several studies have demonstrated the utility of MHs through massively parallel sequencing (MPS). Nevertheless, the background noise level associated with MHs in MPS, which imposes a practical detection limit for the system, remains uninvestigated. Currently, unique molecular identifier (UMI) systems are known to effectively mitigate background noise by tracking original DNA molecules and facilitating PCR and MPS error corrections. Hence, this study aimed to design a UMI-based amplicon sequencing system, designated MH-UMIseq, which can amplify 46 MHs simultaneously and generate MPS libraries in four steps: barcoding PCR, nuclease reaction, boosting PCR, and indexing PCR. The performance of the MH-UMIseq system was evaluated using the Illumina NextSeq 550 and MiniSeq systems with 31 sets for 5 ng, 1 ng, and 200 pg of input DNA. The fgbio toolkit was used in conjunction with STRait Razor 3.0 and Visual Microhap to analyze the UMI data on MHs. The corresponding average not suppressed noise proportion of MH-UMIseq were 0.1 %, 0.3 %, and 0.7 % for 5 ng, 1 ng, and 200 pg of DNA, respectively, which substantially suppressed the background noise for more than 1 ng of DNA. Interestingly, the proportion of not suppressed noise in MH-UMIseq notably decreased as the amount of input DNA increased. The number of UMI families was proportional to the copy number of the template DNA and closely correlated with the system resolution. Therefore, the resolution of MH-UMIseq system is expected to be higher than that of conventional MPS for the deconvolution of mixtures containing more than 1 ng of DNA., Competing Interests: Declaration of Competing Interest A part of the results was included in the doctoral dissertation of Ye-Lim Kwon, the first author., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objectives: Non-tuberculous mycobacteria (NTM) frequently colonize the airways of patients with bronchiectasis; however, there has been limited research into airway microbiota composition and predisposing factors for NTM detection during acute bronchiectasis exacerbations., Methods: This study enrolled 34 patients with bronchiectasis experiencing acute exacerbations. Metagenomic next-generation sequencing was used to detect microbiota in bronchoalveolar lavage fluid (BALF), and bioinformatics methods were used for the comparative analysis of meaningful microbiota in the BALF of patients with acute exacerbations of bronchiectasis. A correlation analysis was conducted to identify susceptibility factors for NTM in patients with bronchiectasis., Results: Compared with patients with community-acquired pneumonia, patients with bronchiectasis had higher detection rates of NTM (38.2%), Pseudomonas aeruginosa, and Haemophilus influenzae. Patients with NTM-positive bronchiectasis had lower body mass index and lipid profiles than patients who were NTM-negative. Metagenomic next-generation sequencing of BALF revealed patients who were NTM-positive had increased relative abundance of Rothia and other anaerobic genera compared with patients who were NTM-negative. Patients who were NTM-positive also showed higher levels of Streptococcus parasanguinis at the species level. Elevated Rothia mucilaginosa and S. parasanguinis correlated with decreased percentages of clusters of differentiation 3+ T lymphocytes and clusters of differentiation 3+ T-cell subgroups in peripheral blood., Conclusions: NTM colonization increases the risk of acute bronchiectasis exacerbations. Low body mass index, lipid levels, and isolation of R. mucilaginosa and S. parasanguinis in BALF are susceptibility factors for NTM colonization in patients with bronchiectasis., Competing Interests: Declarations of competing interest The authors have no competing interest to declare. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Baloxavir acid (BXA) is a pan-influenza antiviral that targets the cap-dependent endonuclease of the polymerase acidic (PA) protein required for viral mRNA synthesis. To gain a comprehensive understanding on the molecular changes associated with reduced susceptibility to BXA and their fitness profile, we performed a deep mutational scanning at the PA endonuclease domain of an A (H1N1)pdm09 virus. The recombinant virus libraries were serially passaged in vitro under increasing concentrations of BXA followed by next-generation sequencing to monitor PA amino acid substitutions with increased detection frequencies. Enriched PA amino acid changes were each introduced into a recombinant A (H1N1)pdm09 virus to validate their effect on BXA susceptibility and viral replication fitness in vitro. The I38 T/M substitutions known to confer reduced susceptibility to BXA were invariably detected from recombinant virus libraries within 5 serial passages. In addition, we identified a novel L106R substitution that emerged in the third passage and conferred greater than 10-fold reduced susceptibility to BXA. PA-L106 is highly conserved among seasonal influenza A and B viruses. Compared to the wild-type virus, the L106R substitution resulted in reduced polymerase activity and a minor reduction of the peak viral load, suggesting the amino acid change may result in moderate fitness loss. Our results support the use of deep mutational scanning as a practical tool to elucidate genotype-phenotype relationships, including mapping amino acid substitutions with reduced susceptibility to antivirals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Abstract Background Colorectal cancer patients have an increased risk of developing venous thromboembolism (VTE), resulting in increased morbidity and mortality. Because the exact mechanism is yet unknown, risk prediction is still challenging; therefore, new biomarkers are needed. MicroRNAs (miRNAs) are small, relatively stable RNAs, that regulate a variety of cellular processes, and are easily measured in body fluids. Objective The aim of this study was to identify novel tumor‐expressed miRNAs associated with VTE. Methods In a cohort of 418 colorectal cancer patients diagnosed between 2001 and 2015 at the Leiden University Medical Center, 23 patients (5.5%) developed VTE 1 year before or after cancer diagnosis. Based on availability of frozen tumor material, tumor cells of 17 patients with VTE and 18 patients without VTE were isolated using laser capture microdissection and subsequently analyzed on the Illumina sequencing platform NovaSeq600 using 150‐bp paired‐end sequencing. Cases and controls were matched on age, sex, tumor stage, and grade. Differential miRNA expression was analyzed using edgeR. Results A total of 547 miRNAs were detected. Applying a 1.5‐fold difference and false discovery rate of

Objective: The aim of this work was to characterize the genetic abnormalities and prenatal diagnosis indications in one fetus with Cri-du-Chat syndrome with codependent 10q24.2-q26.3 duplication in prenatal screening. Materials and methods: A 31-year-old woman had a second trimester serum screening that indicated the fetus was at low risk. During this pregnancy, the woman underwent amniocentesis at 18+4 weeks' gestation because of adverse fertility history and nuchal fold thickening. Cytogenetic analysis and next-generation sequencing analysis were simultaneously performed to provide genetic analysis of fetal amniotic fluid. According to abnormal results, parental chromosome karyotype of peripheral blood was performed to analysis. Results: CNV-seq detected a 14.00 Mb deletion at 5p15.33-p15.2 and a 34.06 Mb duplication at 10q24.2-q26.3 in the fetus. Cytogenetic analysis of the fetus revealed a karyotype of 46, XY, der(5) t(5;10) (p15.2;q26.3). The karyotype of pregnant women was 46,XX,t(5;10) (p15.2;q24.2). The pregnancy was subsequently terminated after sufficient informed consent. Conclusion: This is the first study that reports prenatal diagnosis of a Cri-du-Chat syndrome with concomitant 10 q24.2-q26.3 duplication. Adverse pregnancy history has to be as an important indicator for prenatal diagnosis, and the genetic factors of abnormal pregnancy should be identified before next pregnancy. Nuchal fold thickening is closely related to fetal abnormalities. Combined with ultrasonography, the use of CNV-seq will improve the diagnosis of submicroscopic chromosomal aberrations in fetuses with congenital anomalies.

Most existing wastewater surveillance studies that focus on viruses have identified a large fraction of bacteriophages. Identifying bacteria by considering bacteriophage-host interactions is a novel method for detecting bacterial pathogens circulating in a community, using wastewater surveillance. This study aims to identify human-related bacterial pathogens in municipal wastewater collected in metro Detroit, using high-throughput sequencing and bioinformatics. Untreated municipal wastewater samples were collected on August 11, 2020, and bacteriophages were concentrated using the VIRus ADsorption-ELution (VIRADEL) method. Bacteriophage-related contigs in samples ranged from 15.53 % to 18.91 %, with 2477 classified and 8853 unclassified contigs. Most identified bacteriophages were from Caudoviricetes and Malgrandaviricetes classes belonging to 19 families. Hosts of bacteriophages were predicted with the PhaBOX (CHERRY) tool. The results indicated that out of the 2477 classified phages, 2373 were associated with known bacterial hosts. Also, out of 8853 unclassified bacteriophages, 8421 were associated with known bacterial hosts, and the remaining 432 were with unknown bacterial hosts. Among all bacteriophage-associated hosts, 399 were identified as pathogenic bacteria at the species level. Approximately, 85 % of the identified pathogenic bacteria are reported to be associated with human diseases. Genome quality assessments showed that 15 bacteriophages had nearly complete genomes, which were further analyzed to understand bacteriophage-bacteria interactions in wastewater. Identified hosts of these complete-genome phages included human pathogens such as Salmonella enterica, Bacillus cereus, Achromobacter xylosoxidans, and Escherichia coli. The S. enterica bacteriophage (k141&#95;1005294) genomic map was annotated, and responsible open reading frames (ORFs) were characterized to illustrate bacteriophage behavior during infection of pathogenic bacteria in untreated wastewater. To the best of our knowledge, this is the first attempt to characterize human bacterial pathogens in wastewater through bacteriophage-pathogen interactions. Novel bioinformatic approaches enhance pathogen detection and improve the understanding of community wastewater microbiomes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this study., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Abstract Background The occurrence of mosaicism in hemophilia A (HA) has been investigated in several studies using different detection methods. Objectives To characterize and compare the ability of AmpliSeq/Ion Torrent sequencing and droplet digital polymerase chain reaction (ddPCR) for mosaic detection in HA. Methods Ion Torrent sequencing and ddPCR were used to analyze 20 healthy males and 16 mothers of sporadic HA patients. Results An error‐rate map over all coding positions and all positions reported as mutated in the F8‐specific mutation database was produced. The sequencing produced a mean read depth of >1500X where >97% of positions were covered by >100 reads. Higher error frequencies were observed in positions with A or T as reference allele and in positions surrounded on both sides with C or G. Seventeen of 9319 positions had a mean substitution error frequency >1%. The ability to identify low‐level mosaicism was determined primarily by read depth and error rate of each specific position. Limit of detection (LOD) was 1% require repeated testing and mononucleotide repeats with more than four repeat units need an alternative analysis strategy. Mosaicism was detected in 1 of 16 mothers and confirmed using ddPCR. Conclusions Deep sequencing using an AmpliSeq/Ion Torrent strategy allows for simultaneous identification of disease‐causing mutations in patients and mosaicism in mothers. ddPCR has high sensitivity but is hampered by the need for mutation‐specific design.

Aim: Congenital hypothyroidism (CH) is the most common endocrine disorder of the newborn; it is seen in every 3000-4000 births. Genetic features can guide treatment for patients with in situ glands. The present study aimed to contribute to the literature on CH variants and to show the benefit that genetic analysis can provide to patients in follow-up., Method: A total of 52 patients (47 families) diagnosed with CH were included in the study. Overall, 32 target genes involved in thyroid physiology were investigated by next-generation sequencing (NGS)., Results: In total, 29 (55 %) of the patients were male, and the rate of dysgenesis was 19.2 %. In this study, 29 of 52 patients had at least one variant in one gene involved in CH (n = 29, 33 different variants) (Including likely benign variants and variants of unknown significance). There were 21 patients (40.3 %) with gland in situ. The most common variant was DUOX2 (20 %). The second most common variants were those in the TPO and TG genes (15 % and 15 %, respectively); 41.1 % of these were variants of uncertain significance (VUS), 26.4 % were pathogenic, 23.5 % were likely benign, and 11.7 % were likely pathogenic. On the basis of their zygosity, we identified 73.5 % heterozygous, 17.6 % homozygous, and 8.9 % combined heterozygous variants. There were mutant variants in two genes in six patients and three in one patient., Conclusion: This study found a variant in 55 % of the patients and shed light on the etiology of some cases of CH. The frequency of VUS was high. Although variants were identified in this study, their implication in the etiology of CH is not certain and, for most of the patients, it is also not sufficient for explaining the pathology with the current state of knowledge., Competing Interests: Declaration of competing interest Authors state no conflict of interest., (Copyright © 2024 French Society of Pediatrics. Published by Elsevier Masson SAS. All rights reserved.)

Objectives: The prevalence of respiratory infectious diseases has changed in the post-COVID-19 epidemic era, and mycoplasma pneumoniae (MP) infection in children has attracted wide attention., Methods: Children hospitalized for pneumonia in Wuhan, China, in 2023 were enrolled. Respiratory secretions were obtained for the targeted next-generation sequencing (tNGS) including mutation of MP. Pulmonary inflammation was divided into bronchopneumonia and pulmonary consolidation/atelectasis according to lung computed tomography imaging., Results: Of the 667 pediatric pneumonia, 478 were MP positive (72%). The positive rate of MP detected by tNGS increased from April, and MP had become the primary pathogen of pneumonia in children in 2023. The 23S rRNA mutations were all A2063G, accounting for 85% of detected MP. The clinical symptoms of the mutant and wild-type strains were similar, with half of them experiencing atelectasis and lung consolidation. Early bronchoscopic lavage combined with azithromycin in pediatric pulmonary consolidation was an effective therapy strategy, which could be an alternative selection to MP pneumonia treatment., Conclusions: A2063G mutant strain MP was the primary pathogen of mycoplasma pneumoniae in children recently, which was often complicated by extra-pulmonary symptoms and complications., Competing Interests: Declaration of competing interest The authors have no competing interest to declare., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Microbiological safety and quality of beef is crucial as beef can serve as a reservoir for a variety of bacteria, including spoilage-related and foodborne pathogens. Controlling microbial contamination is a critical aspect of food quality and safety, but it is difficult to prevent as there are several potential sources of contamination from production to distribution. In this study, the microbiological ecology of cattle/beef and associated environmental samples (n = 69) were trace-investigated to reveal microbiome shifts in cattle/beef and possible cross-contaminants throughout the entire supply chain using 16S rRNA gene sequencing. Pseudomonas, Psychrobacter, and Acinetobacter, known as spoilage bacteria, opportunistic pathogens, or antibiotic-resistant bacteria, were the main microorganisms present in cattle/beef, and Staphylococcus became abundant in the final products. The dominance of Acinetobacter and Pseudomonas was noticeable in the slaughtered carcasses and slaughterhouse environment, indicating that the slaughterhouse is a critical site where hygienic practices are required to prevent further contamination. Taxonomic similarities between cattle/beef and several environmental samples, as well as diversity analysis, presented a high potential for microbial transmission. Source tracking identified environmental samples that primarily contributed to the microbiota of cattle/beef. Farm floor (48%), workers' gloves (73%), and carcass splitters (20%) in the slaughterhouse were found to be major sources influencing the microbiome of cattle/beef at the farm, slaughterhouse, and processing plant, respectively. These findings demonstrated the dynamics of bacterial communities in cattle/beef according to stage and detected potential contamination sources, which may aid in a better understanding and control of microbial transmission in beef production., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Objectives: The International Consensus Classification (ICC) and 5th Edition of the World Health Organization Classification (WHO-5) made substantive updates to the classification of myeloid neoplasms. This study compares the systems in a series of myeloid neoplasms with increased blasts, analyzing implications for diagnostic workflow and reporting., Methods: Bone marrow biopsies categorized as myelodysplastic syndrome with excess blasts (MDS-EB) or acute myeloid leukemia (AML) by WHO-R4 were identified. Results of morphology review, karyotype, fluorescence in situ hybridization, and next-generation sequencing were compiled. Cases were retrospectively re-classified by WHO-5 and ICC., Results: 46 cases were reviewed. 28 cases (61 %) had ≥20 % blasts, with the remaining cases having 5-19.5 % blasts. The most common differences in classification were 1) the designation of MDS versus MDS/AML (10/46, 22 %) for cases with 10-19 % blasts and 2) the ICC's designation of TP53 variants as a separate classifier for AML (8/46, 17 %). Bi-allelic/multi-hit TP53 alterations were identified in 15 cases (33 %). Variants of potential germline significance were identified in 29 (63 %) cases., Conclusions: While terminology differences between WHO-5 and ICC exist, both systems invoke similar opportunities for improved reporting: standardized classification of pathogenic variants (notably TP53), streamlined systems to evaluate for potential germline variants, and integrated reporting of morphologic and genetic data., Competing Interests: Declaration of competing interest The authors have no financial relationships or conflicts of interest to disclose., (Copyright © 2024. Published by Elsevier Inc.)

Background: We assessed the potential role of serial circulating tumor DNA (ctDNA) as a biomarker to monitor treatment response to primary systemic therapy (PST) in breast cancer and evaluated the predictive value of ctDNA to further identify patients with residual disease., Methods: We prospectively enrolled 208 plasma samples collected at three time points (before PST, after 2 cycles of treatment, before surgery) of 72 patients with stage Ⅱ-III breast cancer. Somatic mutations in plasma samples were identified using a customized 128-gene capture panel with next-generation sequencing. The correlation between early change in ctDNA levels and treatment response or long-term clinical outcomes was assessed., Results: 37 of 72 (51.4%) patients harbored detectable ctDNA alterations at baseline. Patients with complete response showed a larger decrease in ctDNA levels during PST. The median relative change of variant allele fraction (VAF) was -97.4%, -46.7%, and +21.1% for patients who subsequently had a complete response (n = 11), partial response (n = 11), and no response (n = 15) (p = 0.0012), respectively. In addition, the relative change of VAF between the pretreatment and first on-treatment blood draw exhibited the optimal predictive value to tumor response after PST (area under the curve, AUC = 0.7448, p = 0.02). More importantly, early change of ctDNA levels during treatment have significant prognostic value for patients with BC, there was a significant correlation between early decrease of VAF and longer recurrence-free survival compared to those with an VAF increase (HR = 12.54; 95% CI, 2.084 to 75.42, p = 0.0063)., Conclusion: Early changes of ctDNA are strongly correlated with therapeutic efficacy to PST and clinical outcomes in BC patients. The integration of preoperative ctDNA evaluation could help improving the perioperative management for BC patients receiving PST., Competing Interests: Declaration of Competing interest JH declares grants from Hanhui Pharmaceutical Co., LTD. All other authors report that they have no conflicts to declare., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

DNA methylation is an important epigenetic modification involved in gene regulation. Advances in the next generation sequencing technology have enabled the retrieval of DNA methylation information at single-base-resolution. However, due to the sequencing process and the limited amount of isolated DNA, DNA-methylation-data are often noisy and sparse, which complicates the identification of differentially methylated regions (DMRs), especially when few replicates are available. We present a varying-coefficient model for detecting DMRs by using single-base-resolved methylation information. The model simultaneously smooths the methylation profiles and allows detection of DMRs, while accounting for additional covariates. The proposed model takes into account possible overdispersion by using a beta-binomial distribution. The overdispersion itself can be modeled as a function of the genomic region and explanatory variables. We illustrate the properties of the proposed model by applying it to two real-life case studies., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The epitranscriptome encompasses a diverse array of dynamic and reversible RNA modifications, affecting both coding and noncoding RNAs. Over 170 types of RNA chemical modifications have been identified, underscoring the need for innovative detection methods to deepen our understanding of RNA modification roles and mechanisms. In particular, the base resolution and quantitative information on RNA modifications are critical for understanding the regulation and functions of RNA modifications. Based on detection throughput and principles, existing quantitative RNA modification detection methods can be categorized into two groups, including next-generation sequencing and nanopore direct RNA sequencing. In this review, we focus on methodologies for elucidating the base resolution and stoichiometric information of RNA modifications. In addition, we further discuss the challenges and the potential prospects of the quantitative RNA modification detection methods., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest in this work., (Copyright © 2024 Elsevier Ltd. All rights reserved.)