Generation Sequencing Research Articles

The correlation between blastocyst morphological parameters and chromosomal euploidy, aneuploidy and other chromosomal abnormalities following pre-implantation genetic testing-a single center retrospective study.

To examine the association between blastocyst morphology and chromosomal status utilizing pre-implantation genetic testing for aneuploidy (PGT-A). A single-center retrospective cohort study including 169 in-vitro fertilization cycles that underwent PGT-A using Next Generation Sequencing (2017-2022). Blastocysts were morphologically scored based on Gardner and Schoolcraft's criteria. Chromosomal analysis results included: euploid; aneuploid (single or double); segmental; mosaic; and complex (≥ 3 chromosome abnormalities). We examined associations between morphological parameters and chromosomal statuses of biopsied embryos utilizing multivariate logistic regression. Overall, 855 blastocysts underwent PGT-A (PGT-A alone: N = 804; unaffected PGT for monogenic disease (PGT-M) embryos along with PGT-A: N = 51). Of these, 826 were successfully analyzed, with 321 euploid embryos (38.86%). Various morphological parameters (embryo quality, inner cell mass (ICM), trophectoderm (TE), and expansion stage) were more frequent within the double (n = 72, 8.72%), complex (n = 97, 11.74%), mosaic (n = 139, 16.83%), and segmental aneuploidy (n = 28, 3.39%) groups, with similar associations between different morphological parameters and single aneuploidy (n = 169, 20.46%). Utilizing multivariate logistic regression, higher expansion, embryo quality, and TE and ICM grades, were associated with increased odds of euploidy (versus non-euploidy). Higher expansion was a positive predictor of single versus double aneuploidy (aOR 2.94, 95% CI 1.52-5.56, p = 0.001); and higher ICM grade was a positive predictor of single versus complex aneuploidy (aOR 2.86, 95% CI 1.15-7.12, p = 0.024). No morphological parameter was found to be associated with single versus mosaic aneuploidy. Various morphological parameters are associated with euploidy and different aneuploidy statuses of pre-implantation blastocysts. These findings may aid in the selection of the assumed best chromosomally structured blastocyst for transfer when PGT-A is not performed.

Neuropilin1-dependent paracrine signaling of cancer cells mediated by miRNA exosomal cargo

BackgroundNeuropilin-1 (NRP1) is a transmembrane protein involved in surface receptor complexes for a variety of extracellular signals. NRP1 expression in human cancers is associated with prominent angiogenesis and advanced progression stage. However, the molecular mechanisms underlying NRP1 activity in the tumor microenvironment remain unclear. Notably, diffusible forms of NRP1 in the extracellular space have been reported, but their functional role is poorly understood.MethodsExtracellular vesicles (EV) were isolated from conditioned media of diverse cancer cells. The quality of exosome-enriched preparations was validated by the presence of specific markers in western blotting, as well as by light scattering and nanoparticle tracking analysis. Wound healing, transwell, and digital real-time migration assays were carried out to assess the activity of cancer cell-derived exosomes in the regulation of endothelial cells. RNA interference was applied to obtain NRP1 knock-down, and cDNA transfer to achieve its overexpression, in exosome-releasing cells. The micro-RNA profile carried by exosomes was investigated by Next Generation Sequencing. miRNA-Scope in situ hybridization was used to assess the transfer of miRNA exosome cargo to target cells, and immunofluorescence analysis revealed expression regulation of targeted proteins. miRNA activity was blocked by the use of specific antago-miRs.ResultsIn this study, we show that diverse human cancer cells release NRP1 embedded in exosome-like small extracellular vesicles, which mediate a previously unknown NRP1-dependent paracrine signaling mechanism regulating endothelial cell migration. By transcriptomic analysis of the cargo of NRP1-loaded exosomes, we found a significant enrichment of miR-210-3p, known to promote tumor angiogenesis. Gene knock-down and overexpression experiments demonstrated that the loading of miR-210-3p into exosomes is dependent on NRP1. Data furthermore indicate that the exosomes released through this NRP1-driven mechanism effectively transfer miR-210-3p to human endothelial cells, causing paracrine downregulation of the regulatory cue ephrin-A3 and promotion of cell migration. The mechanistic involvement of miR-210-3p in this pathway was confirmed by applying a specific antago-miR.ConclusionsIn sum, we unveiled a previously unknown NRP1-dependent paracrine signaling mechanism, mediated by the loading of pro-angiogenic miR-210-3p in exosomes released by cancer cells, which underscores the relevance of NRP1 in controlling the tumor microenvironment.

Push-down automata as sequential generators of highly entangled states

Abstract We exploit the duality between quantum channels and sequentially generated states to construct families of highly entangled states that undergo phase transitions. These highly entangled states can be sequentially generated by collecting the emitted radiation from an open quantum system. In the dual perspective, the open system is regarded as a quantum-state-generating machine. A nontrivial class of such machines are quantum push-down automata, which can be used to create highly entangled states including the Motzkin state parametrically faster than adiabatic evolution. We construct generalizations of the Motzkin state that can be efficiently sequentially generated.

Detection of human noroviruses in sewage by next generation sequencing in Shandong Province, 2019–2021

BackgroundHuman noroviruses are the major cause of acute gastroenteritis and exhibit considerable genetic diversity. Next generation sequencing (NGS) analysis based on environmental surveillance has been proved to be an effective method in norovirus surveillance.MethodsBetween January 2019 and December 2021, 36 sewage samples were collected and analyzed using real-time quantitative PCR to detect noroviruses. Partial VP1 region was amplified and subjected to NGS analysis to assess the abundance and genetic characterization of various norovirus genotypes across different samples.ResultsA total of 23 norovirus genotypes were identified, including 9 genotypes of GI, 13 genotypes of GII and 1 genotype of GIX. The most frequently detected genotypes were GI.5 (86.11%), GII.2 (86.11%), GII.4 (63.89%), GII.17 (58.33%), and GII.13 (55.56%). Additionally, some rare genotypes, such as GI.7, GII.5, GII.9, and GII.16, which had not been previously reported in Shandong, were identified. No significant differences were observed in genotypic diversity or viral copy numbers in sewage samples when comparing pre- and post-COVID-19 periods. A total of 379 partial VP1 sequences were obtained, with the means sequence identity within a genotype of Shandong sequences ranging from 92.69 to 98.37% and a coefficient of variation ranging from 1.46 to 6.73%. Phylogenetic analysis indicated that local noroviruses within each genotype comprised multiple co-circulating lineages.ConclusionsOur data demonstrate that sewage contains noroviruses with considerable high diversities. NGS based environmental surveillance greatly improves the understanding of norovirus circulation and should be encouraged.

Testosterone Modulation of Muscle Transcriptomic Profile During Lifestyle Therapy in Older Men with Obesity and Hypogonadism

ABSTRACTBackgroundTestosterone replacement therapy (TRT) added to lifestyle therapy can mitigate weight‐loss–induced reduction of muscle mass and bone mineral density (BMD) in older men with obesity and hypogonadism.ObjectiveTo investigate the molecular mechanisms underlying the attenuation of muscle and BMD loss in response to TRT during intensive lifestyle intervention in this high‐risk older population.MethodsAmong 83 older (≥ 65 years) men with obesity (BMI ≥ 30 kg/m2) and hypogonadism (early AM testosterone persistently < 300 ng/dL) associated with frailty (Modified Physical Performance Test score ≤ 31) randomized into 26‐week lifestyle therapy plus testosterone (LT+TRT) or placebo (LT+Pbo) in the LITROS trial, 38 underwent serial muscle biopsies for the muscle transcriptomics substudy.ResultsDespite similar ~10% weight loss, lean body mass and thigh muscle volume decreased less in LT+TRT than LT+Pbo (−2% vs. −4%, respectively; p = 0.04). Hip BMD was preserved in LT+TRT compared with LT+Pbo (0.4% vs. −1.3%; p = 0.03). Muscle strength increased similarly in LT+TRT and LT+Pbo (23% vs. 24%; p = 0.95). Total testosterone increased more in LT+TRT than LT+Pbo (133% vs. 32%; p = 0.005). Based on Next Generation Sequencing, of the 39 160 and 39 115 genes detected in LT+TRT and LT+Pbo, respectively, 195 were differentially expressed in LT+TRT and 158 in LT+Pbo. Gene Ontology enrichment analyses revealed that in LT+TRT, just four muscle‐related pathways (muscle organ development, muscle organ morphogenesis, regulation of skeletal muscle contraction, muscle atrophy) were downregulated and one pathway (muscle system process) was upregulated. In contrast, in LT+Pbo, nine muscle‐related pathways (muscle system process, muscle tissue development, muscle organ development, skeletal muscle tissue development, skeletal muscle organ development, skeletal muscle cell differentiation, muscle organ morphogenesis, response to stimuli involved in regulation of muscle adaptation, muscle atrophy) and one pathway related to bone (bone mineralization involved in bone maturation) were downregulated. Muscle system process was upregulated in LT+TRT but downregulated in LT+Pbo. RT‐PCR analyses showed that LT+TRT resulted in a higher expression of MYOD1 (p = 0.02) and WNT4 (p = 0.02), key genes involved in muscle and bone metabolism, respectively, compared with LT+Pbo. We also observed significantly higher mRNA expression of MYBPH (p = 0.006), SCN3B (p = 0.02) and DSC2 (p = 0.01), genes involved in the muscle system process, in response to LT+TRT compared with LT+Pbo.ConclusionThe addition of TRT to lifestyle therapy mitigates the weight‐loss–induced reduction of muscle mass and BMD via countering the weight‐loss–induced downregulation of genes involved in muscle and bone anabolism.

Suitability of different cytological preparations for molecular analysis of advanced NSCLC.

Around 85% of non-small cell lung cancers (NSCLCs) are diagnosed at an advanced stage (IIIB to IV), where therapeutic options depend on molecular analysis. However, diagnostic material for molecular testing is often represented by cytological samples which are generally scarce and span a wide range of preparation types. Thus, the primary objective is to efficiently manage materials for molecular profiling. This study aims to evaluate the suitability of different cytological samples to assess morphological and molecular characteristics of advanced NSCLC. Sixty-seven cytological samples obtained from patients with advanced NSCLC were utilized. The series encompassed different procedure types (fine-needle aspiration cytology, transbronchial needle aspiration, effusions) processed by cell blocks in 54% (n=36), direct smears in 33% (n=22), and Liquid Based Cytology (LBC) in 13% (n=9). Cytological diagnoses were routinely performed and molecular analysis were conducted using NGS and RT-PCR methods. Adequate quantity and quality of nucleic acids were obtained from all the samples, allowing molecular profiling. Combined Next Generation Sequencing (NGS) and Real Time-Polymerase Chain Reaction (RT-PCR) analysis showed wild-type profiles in 62.7% (n=42) and mutated profiles in 37.3% (n=25) of the samples. Kirsten Rat Sarcoma Virus (KRAS) mutations were identified in 19.5% (n=13) of samples, Epidermal Growth Factor Receptor (EGFR) mutations in 10.4% (n=7) and v-raf murine sarcoma viral oncogene homolog B (BRAF) mutations in 2.9% (n=2). Identified chromosomal alterations were v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (ERBB2) duplication in 2,9% (n=2). The cytological sample types examined in this study proved to be suitable for molecular testing, in addition to conventional morphologic diagnosis, showing versatility and adaptability to different clinical contexts. Molecular testing on cytological samples is accurate and fast, representing a valid tool for molecular profiling of advanced NSCLC.

Next generation sequencing of multiple SARS-CoV-2 infections in the Omicron Era

Since the emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the need for an effective vaccine has appeared crucial for stimulating immune system responses to produce humoral/cellular immunity and activate immunological memory. It has been demonstrated that SARS-CoV-2 variants escape neutralizing immunity elicited by previous infection and/or vaccination, leading to new infection waves and cases of reinfection. The study aims to gain into cases of reinfections, particularly infections and/or vaccination-induced protection. We conducted a retrospective descriptive study using data collected during the SARS-CoV-2 pandemic. This analysis involved Reverse Transcriptase Quantitative Polymerase Chain Reaction (RT-qPCR) and Next Generation Sequencing (NGS). RT-qPCR was performed on 416,466 naso-oropharyngeal swabs, with 10,380 samples further analyzed using NGS technology. RT-qPCR identified 350 cases of reinfection, of which 228 were subjected to detailed analysis via NGS. Our findings revealed two interesting cases involving pediatric patients who were not vaccinated. Positive results were observed in these cases within a short interval (< 60 days) and the “nature” of the infection, whether attributable to Reinfection or Viral Persistence, was investigated. Specifically, we discuss a case involving an unvaccinated 18-month-old child, which may represent one of the earliest instances of BA.5/BA.5 reinfection identified worldwide.

Child with hypocalcaemia: a rare case of HDR syndrome

Barakat syndrome (also known as HDR syndrome) is an autosomal dominant disorder characterized by the triad of Hypoparathyroidism (H), nerve Deafness (D) and/or renal disease caused by mutation of the GATA3 gene located at chromosome 10p15. This syndrome is very rare with exact prevalence not known and only few cases are reported in the literature. 4-month-old baby girl, diagnosed as hypoparathyroidism on supplements, presented with seizures due to hypocalcemia. On detailed evaluation, child was found to have bilateral hearing loss. Facial dysmorphisms were noted in the form of bulbous nose with everted nares, long philtrum, thin upper lip, low set ears and bilateral non paralytic convergent squint. External genitalia showed perianal groove with an anteriorly placed anus. Startle response was blunted. Other systemic examination was within normal limits. Routine blood investigations revealed low serum calcium (calcium (total) 4.8 mg/dl) and high serum phosphorous level (11.3 mg/dl). Serum parathormone level was 4.4pg/ml and vitamin D levels were insufficient. Child was managed with IV calcium correction and 1,25 (OH)2 VIT D3. The brainstem evoked response audiometry (BERA) showed severe hearing loss. Next - generation sequencing showed a heterozygous missense variant in exon 4 of the GATA 3 gene which was suggestive of Hypoparathyroidism, sensorineural deafness, and renal dysplasia syndrome. This case report aims to provide awareness of rare inherited conditions in a patient with abnormal physical and laboratory findings even though their initial presentation was seizure and hypocalcemia.

P-05 17Β-HYDROXYSTEROID DEHYDROGENASE 3 DEFICIENCY: TWO CASE REPORTS

Abstract Introduction 17-β-hydroxysteroid dehydrogenase type3 (17β-HSD3) is an enzyme that produces testosterone from androstenedione, encoded by the HSD17B3 gene. Its deficiency causes 46 XY disorder of sex development (DSD) with autosomal recessive inheritance. 46 XY individuals with this enzyme deficiency can exhibit a wide range of phenotypes, ranging from normal female external genitalia to ambiguous genitalia. In early childhood, the diagnosis might be clinically indistinguishable from other causes of 46 XY DSD such as partial androgen insensitivity syndrome (AIS) and 5α-reductase type 2 deficiency. Laboratory diagnosis is based on a low testosterone/androstenedione ratio. The diagnosis is confirmed by molecular genetic testing. If the diagnosis is missed in early infancy, patients present with severe virilization symptoms and amenorrhea during puberty. We present two siblings followed and managed with a presumed diagnosis of AIS until the genetic testing was performed. Clinical Case 1 A 4-month-old patient with female external genitalia was operated on with suspicion of bilateral inguinal hernia, and pathologic investigation revealed immature testicular tissue. No uterus and ovaries could be detected by pelvic ultrasound. The karyotype analysis was consistent with 46 XY, and the patient was followed up with the diagnosis of AIS. The patient’s parents were first cousins. The patient was started on estradiol replacement when she was 13 years old. At the age of 21, she underwent vaginoplasty due to a 2 cm long vaginal canal. A genetic analysis was available when the patient was 21 years old. Next generation sequencing revealed a previously reported homozygous c.277+5g&amp;gt;A mutation in exon 3 of HSD17B3 gene. The diagnosis was established as 17β-HSD3 deficiency. Clinical Case 2 After the index case was diagnosed with AIS karyotype analyses were performed on three other siblings at the time. The patient’s 7 years old sibling, who had a complete female-appearing genitalia, had a karyotype of 46XY. As the presumed diagnosis was AIS, bilateral orchiectomy was performed after counselling. Estradiol replacement was started at the age of 14 and complete breast development was achieved. Vaginal length was reported as 8 cm. Further genetic analysis was performed at the age of 27 and it revealed that the two siblings shared the same genetic mutation. Both preferred to retain the female gender identity during adulthood. Two other female siblings with a karyotype of 46XX and both parents had a heterozygous c.277+5g&amp;gt;A mutation in the HSD17B3 gene. Conclusion Although clinical evaluation and laboratory test results might point out to 17-β-HSD3 deficiency, the definite diagnosis is established with genetic testing. As these patients might develop male gender identity, awareness of the disease and early genetic testing is of importance for the prevention of misdiagnoses, counselling for gender identity, deciding on surgical interventions, and management of these patients.

Clinical and imaging characteristics of 135 cases of infectious sacroiliitis: a retrospective cohort study in China.

To study the clinical, imaging, and computed tomography (CT)-guided biopsy pathology of patients with infectious sacroiliitis (ISI). We retrospectively analysed 135 patients diagnosed with ISI between 2008 and 2020, comprehensively evaluating clinical characteristics, laboratory test outcomes, pathological examination results, and magnetic resonance images (MRI). Among the 135 patients with ISI, 90 (66.7%) were diagnosed with pyogenic sacroiliitis (PSI), 28 (20.7%) with tuberculous sacroiliitis (TSI), and 17 (12.6%) with brucella infectious sacroiliitis (BSI); 69 (51.1%) met the 2009 ASAS criteria for axial spondyloarthritis. The rate of back pain is lower in the PSI group (18.9%) than in the TSI (39.2%) and BSI (64.7%) groups. The Berlin MRI scoring in the sacroiliac joint showed erosion and bone marrow oedema in the PSI (3.11 ± 2.33, 8.55 ± 2.66) and TSI (3.14 ± 2.08, 7.88 ± 3.90) groups, with higher values than in the BSI group (1.62 ± 1.54, 5.23 ± 3.05). The erythrocyte sedimentation rate (ESR) was higher in the PSI (52.71 ± 29.63mm/h) and TSI (56.22 ± 19.39mm/h) groups than in the BSI group (33.29 ± 25.12mm/h). Our study is crucial because all patients underwent CT-guided sacroiliac joint biopsy; 130 patients (86.7%) had positive results through tissue culture and pathological examination, and one (0.8%) had a confirmed Malassezia fungal infection by tissue next generation sequencing. Two patients (1.5%) were diagnosed through blood culture, and another (0.8%) was diagnosed through the brucellosis agglutination test. ISI is diagnostically challenging as it can mimic spondyloarthritis, particularly when relying exclusively on imaging and clinical parameters. CT-guided sacroiliac joint biopsy is indispensable as a diagnostic intervention for precisely differentiating infectious sacroiliitis and identifying the specific pathogens involved, especially in female patients who exhibit negative HLA-B27 status and manifest systemic symptoms such as fever, elevated ESR, and unilateral lesions on imaging. Key Points • To the best of our knowledge, this is the largest single-centre cohort study on infectious sacroiliitis in China. • CT-guided biopsy of the sacroiliac joint is necessary for identifying infectious sacroiliitis and pathogens. • This study provides insights into the clinical and imaging features of infectious sacroiliitis based on a large number of cases.

Clinical and molecular characteristics and long-term outcomes of pediatric intracranial meningiomas: a comprehensive analysis from a single neurosurgical center

BackgroundMeningioma represents the most common intracranial tumor in adults. However, it is rare in pediatric patients. We aimed to demonstrate the clinicopathological characteristics and long-term outcome of pediatric meningiomas (PMs).MethodWe enrolled 74 patients with intracranial PMs and analyzed their clinicopathological characteristics. Targeted next generation sequencing was used to detect alterations in meningioma relevant genes. Progression-free survival (PFS) was compared between PMs and adult meningiomas (AMs). Univariate and multivariate Cox analyses were employed to evaluate the predictive values of clinicopathological characteristics. A nomogram was constructed and its predictive accuracy evaluated.Result40 females (54.1%) and 34 males (45.9%) patients, with the gender ratio of 1.18:1, were identified. 9 (12.2%) cases were clinically diagnosed as NF2-related Schwannomatosis (NF2-SWN), while 65 (87.8%) were sporadic. Ventricular location was found in 16 patients (21.6%). 19 patients (25.7%) experienced recurrence during a median follow-up period of 33 months (range 2 -145.25 months). The 3-, 5-, and 8-year PFS rates was 74.74%, 74.74%, and 59.38%, respectively. The PFS of the PM and AM cohorts were not significantly different, with or without propensity score matching. NF2 mutation was observed in 33 sporadic PMs (52.4%), whereas alterations in other genes (AKT1, TRAF7, SMO, PIK3CA, KLF4) frequently mutated in AMs, were not identified. The proportion of NF2 mutation in PMs was significantly lower in the skull base than other locations (p = 0.02). One anaplastic PM harbored TERT promoter mutation. Of note, in sporadic PMs, NF2 mutations were not significantly associated with PFS (p = 0.434) or overall survival (OS) (p = 0.60). The multivariate Cox analysis showed NF2-SWN (p < 0.001) and extent of resection (p = 0.013) to be independently associated with the PFS of PMs. Our prognostic model showed predictive accuracy for long-term PFS in PMs as the 3-, 5- and 8-year Area Under the Curve (AUC) was 0.927, 0.930, and 0.870, respectively.ConclusionPM was characterized by its relative male predominance, ventricular location, NF2-SWN, and NF2 mutation. Of note, PMs had similar prognosis to AMs and NF2 alteration was not significantly associated with PFS in PMs.

Positive sputum fungal culture, fungal sensitisation and airway microbial diversity in asthmatic children.

Sensitisation to thermotolerant fungi such as Aspergillus fumigatus and Candida albicans which can colonise the airways is associated with poor lung function in children with asthma. Dysbiosis of bacteria and fungi in the airway microbiome has been reported between health and asthma but has yet to be characterised for fungal sensitised asthmatic children. We investigated if microbial diversity of the airways is altered in fungal sensitised school-age asthmatic children. Sputum samples from children with asthma who were fungal sensitised (n=22) and non-fungal sensitised (n=17) along with children without asthma (n=15), aged 5-16 years were profiled by traditional microbiological culture, modified fungal culture, bacterial 16S and fungal ITS2 next generation sequencing. Microbiota were compared between groups using alpha/beta diversity and differential abundance analysis. Bacterial alpha diversity was significantly lower in asthma compared to disease controls and in stable compared to acute asthma. Fungal alpha and beta diversity did not change between asthma states and disease controls, but alpha diversity was significantly lower in asthma samples from patients with positive A. fumigatus culture. Children sensitised to fungi had similar microbial diversity compared to non-sensitised children. However, in children not sensitised to fungi, those with a positive airway fungal culture had significantly lower fungal alpha diversity and bacterial beta differences compared to children with negative fungal culture. Fungal sensitisation did not alter bacterial or fungal microbiota in the airways of asthmatic children. However, positive airway fungal culture was associated with significant changes in microbial diversity, particularly in non-fungal sensitised children with asthma.

A case of de novo neuroendocrine prostate cancer presented with elevated level of serum CEA carrying BRCA2 mutation: case report and literature review

BackgroundDe novo neuroendocrine prostate cancer (NEPC) is a rare subtype of prostate cancer (PCa) and few markers are available for screening and monitoring. Potential circulating or fluid markers might facilitate early diagnosis thus improving prognosis of NEPC, especially for de novo NEPC.Case presentationA man of 71-year was presented with elevated level of serum carcinoembryonic antigen (CEA) (1296.5 ng/ml) and normal PSA (0.47ng/ml). Gastrointestinal endoscopy showed no signs of gastric or colorectal cancer. Fluorodeoxyglucose positron emission tomography-computed tomography (FDG PET-CT) and prostate-specific membrane antigen PET-CT (PSMA PET-CT) indicated prostate cancer with metastases including pelvic lymph nodes, bone as well as lung metastases. Biopsy of prostate revealed mixed carcinoma including small cell neuroendocrine carcinoma (SCNEC) and adenocarcinoma (Gleason score of 4 + 5). Immunohistochemistry (IHC) staining and next generation sequencing demonstrated a strong expression of chromogranin A (CgA), synaptophysin (SYN) and CEA, and a germline mutation in BRCA2, respectively. After a prostatic massage, an increased level of CEA (137 ng/ml vs 5 ng/ml) was detected in urine. Olaparib, a Poly ADP-ribose polymerase inhibitor (PARPi), combined with androgen deprivation therapy (ADT) were administrated. FDG PET-CT indicated tumor regression in both quantity and size three months later, and CEA levels of serum and urine decreased to 23 ng/ml and 2.4 ng/ml 4 months later, respectively.ConclusionThis is the first report of a de novo NEPC presented with an elevated level of CEA, in which CEA was also detected in urine specimen post a prostatic massage. After a combination treatment of ADT for 3 months, levels of CEA in both serum and urine decreased sharply when tumor regressed radiologically. CEA might be a marker of screening and monitoring of NEPC.

Detection and genomic characterisation of foot-and-mouth disease virus serotypes circulating in Cameroon using environmental sampling

Foot-and-mouth disease virus (FMDV) is a highly contagious, economically important disease of livestock and wildlife species. Active monitoring and understanding the epidemiology of FMDV underpin the foundations of control programmes. In many endemic areas, however, veterinary resources are limited, resulting in a requirement for simple sampling techniques to increase and supplement surveillance efforts. In this study, environmental sampling was used for the first time at livestock markets and abattoirs across Cameroon to assess the opportunities for broad scale, non-invasive disease surveillance at such sites. Environmental samples (n = 1994) were collected from six locations across Cameroon between May and July 2019. Concurrent with environmental sampling, a questionnaire was used to gather descriptive information on the use and practices of market and abattoir sites. Samples were screened for the presence of FMDV RNA using a pan-serotype FMDV specific real-time RT-PCR assay. Positive samples were characterised at the genomic level using next generation sequencing in combination with a novel probe-based enrichment strategy. A total of 173/1994 (8.68%) environmental samples were found to be positive for FMDV RNA. Genome length sequences were obtained from environmental samples, with phylogenetically relevant capsid sequences obtained from 14 samples, with representatives of serotypes O (n = 6), A (n = 7) and SAT 2 (n = 3). The questionnaire results revealed that animals in Cameroon can be transported long distances to markets and abattoirs, with varying levels of control and biosecurity practices in place. The approaches used in this study have highlighted that environmental sampling is an effective and non-invasive approach to assessing FMDV presence. Furthermore, the study has demonstrated that livestock markets, abattoirs and trucks could be targeted for the introduction of biosecurity interventions as well as providing opportunities for carrying out disease surveillance. Information resulting from such surveillance could provide valuable knowledge of circulating viruses within a region of interest, aiding strategic approaches for surveillance and control of FMDV.

Improved ChIP Sequencing for H3K27ac Profiling and Super-Enhancer Analysis Assisted by Fluorescence-Activated Sorting of Formalin-Fixed Paraffin-Embedded Tissues

Archived clinical formalin-fixed paraffin-embedded tissue (FFPE) is valuable for the study of tumor epigenetics. Although protocol of chromatin immunoprecipitation coupled with next generation sequencing (NGS) (ChIP-seq) using FFPE samples has been established, removal of interference signals from non-target cell components in the samples is still needed. In this study, the protocol of ChIP-seq with purified cells from FFPE lymphoid tissue of nodal T follicular helper cell lymphoma, angioimmunoblastic type (nTFHL-AI) after fluorescence-activated cell sorting (FACS) was established and optimized. Essential steps included single cell preparation, heat treatment enhancing antigen retrieval and labeling, cell sorting, chromatin shearing, ChIP and NGS. Through assistance of FACS, we successfully isolated tumor cells from FFPE lymph node samples of nTFHL-AI and profiled super-enhancers (SEs) mapping by enrichment of H3K27ac signals. The data indicated that the SEs mapping of the sorted cells was different from that of the entire unsorted tissue sample. The H3K27ac signals with cell lineage specificity from background cell components were successfully removed, and the remaining SEs mapping was more similar to T follicular helper cell in an unsupervised clustering analysis, rather than the primary tissue. In addition, we also evaluated the protocol using cultured pure cell lines, and the results indicated that the sequencing data obtained through this protocol had high fidelity and reproducibility. These results show that ChIP-seq for H3K27ac profiling and SEs mapping assisted by FACS with pathological FFPE tissue is available for research of histone modification. Precise epigenetic characteristics of the tumor cell can be described with this protocol.

Human embryo implantation: The complex interplay between endometrial receptivity and the microbiome.

The endometrial and vaginal microbiota have co-evolved with the reproductive tract and play a key role in both health and disease. However, the difference between endometrial and vaginal microbiota, as well as their association with reproductive outcomes in women undergoing frozen embryo transfer, remains unclear. 120 women who underwent in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) and whole embryo freezing were enrolled. The vaginal and uterine microbiome were sequenced during the first frozen thawed embryo transfer. Based on whether or not they were pregnant after embryo transfer, women were assigned into two groups, and the microbiome of their reproductive tract was compared. The V3-V4 regions of the 16S rRNA gene were examined in the samples using the Next Generation Sequencing method. In the vagina, the non-pregnant group had higher bacterial species richness and diversity, with significantly lower Lactobacillus levels (91.66 % & 74.50 %) and higher Gardnerella levels (3.92 % & 12.12 %) than pregnant group (P < 0.05). In the uterine cavity, the diversity of uterine microbiome between pregnant group and non-pregnant group showed no significant differences. However, dramatic decrease in Lactobacillus (37.27 % & 33.45 %) and Pseudomonas (9.80 % & 4.08 %) were observed in the non-pregnant group (P < 0.05). There may be a correlation between the composition of female reproductive tract microbiome and the reproductive outcomes of patients with frozen-thawed embryo transfer. Lactobacillus-dominated microbiome in reproductive tract is more likely to be associated with higher clinical and ongoing pregnancy rate.

A single RBM20 missense variant is a potential contributor to dilated cardiomyopathy and/or isolated left ventricular dilatation in the Emilia Romagna region of Italy.

non-syndromic dilated cardiomyopathy (DCM) is found to correlate with a genetic cause in 30-40 % of cases. The identification of a causative gene variant can guide treatment options and cascade testing of at-risk family members. Cardiomyopathy multigene panels are routinely used to identify the genetic cause, but often detect variants of uncertain significance (VUS). Pathogenic variants in RBM20 have been reported to account for 2-6 % of familial DCM, but geographic differences can be relevant. we collected clinical information, cardiac imaging and family history of 101 individuals with DCM, non-dilated left ventricular cardiomyopathy (NDLVC) and isolated left ventricular dilatation from the Emilia-Romagna Region of Italy. Every subject underwent genetic testing through a next generation sequencing panel of genes related to cardiomyopathies. Analysis of single nucleotide polymorphisms (SNP) was used for haplotype analyses. in our cohort, seven individuals (7 %) carried the same heterozygous variant in RBM20 (chr10-110,821,350-G-A; c.2731G > A; p.Val911Met). The referring laboratories reported four further subjects, for a total of 11 unrelated individuals with DCM or isolated left ventricular dilatation from the same geographical area carrying the same variant. These individuals showed high arrhythmic burden and a possible unfavorable evolution towards advanced heart failure. According to guidelines, this variant is classified as VUS; however, its absence in a large local control database and its clinical consistency among affected subjects supports its contributing role. SNP analysis unveiled a common haplotype in the carriers of this variant, suggesting a founder effect. We emphasize the importance of this finding in terms of diagnosis, management and cascade testing of family members.

Integrating functional proteomics and next generation sequencing reveals potential therapeutic targets for Taiwanese breast cancer

Integrating functional proteomics and next-generation sequencing (NGS) offers a comprehensive approach to unraveling the molecular intricacies of breast cancer. This study investigates the functional interplay between genomic alterations and protein expression in Taiwanese breast cancer patients. By analyzing 61 breast cancer samples using tandem mass tag (TMT) labeling and mass spectrometry, coupled with whole-exome sequencing (WES) or targeted sequencing, we identified key genetic mutations and their impact on protein expression. Notably, pathogenic variants in BRCA1, BRCA2, PTEN, and PIK3CA were found to be clinically relevant, potentially guiding targeted therapy decisions. Additionally, we discovered trans correlations between specific gene alterations (FANCA, HRAS, PIK3CA, MAP2K1, JAK2) and the expression of 22 proteins, suggesting potential molecular mechanisms underlying breast cancer development and progression. These findings highlight the power of integrating proteomics and NGS to identify potential therapeutic targets and enhance personalized medicine strategies for Taiwanese breast cancer patients.

Gut Microbiota Profile in Endometriosis Patients at Dr. M. Djamil Padang, West Sumatra, Indonesia

Objective: Endometriosis (EMS) is a chronic gynecological condition affecting up to 10% of women worldwide. The pathogenesis associated with hormonal imbalance, immune dysregulation, and changes in the gut microbiota. Gut microbiota dysbiosis may drive chronic inflammation that contributes to the progression of EMS. This study hypothesizes that certain gut microbiota profiles are associated with these factors in EMS patients, potentially developing non-invasive diagnostics and more targeted therapies. Methods: This is an observational analytical study with a case-control design conducted at Dr. M. Djamil Padang General Hospital in May 2024 with a sample size of 44 people. Case samples were women aged 18–40 years who were diagnosed with endometriosis based on the inclusion criteria, while the control group were women of reproductive age without a diagnosis of endometriosis. Data testing used the Next Generation Sequencing method, microbiome analysis test, and SPSS test. Results: The majority of EMS patients were aged 30-37 years, married, and had Nullipara obstetric status. In addition, the gut microbiota was dominated by the phyla Bacteroidetes and Firmicutes. Conclusion: There is a relationship between gut microbiota dysbiosis and the incidence of endometriosis, namely an increase in Bacteroidetes and a decrease in Firmicutes Keywords: endometriosis, dysbiosis, bacteroides, firmicutes, gut microbiota

Next generation sequencing (NGS)-based molecular panel analysis for metastatic prostate cancer: how often can we detect druggable mutations? : NGS for metastatic adenocarcinoma of the prostate

Prostate cancer guidelines recommend molecular analysis of biomaterial following resistance to first-line systemic therapy in order to identify druggable mutations. We report on our results of molecular analysis of tissue specimens via next generation sequencing (NGS) in men with metastatic castration resistant prostate cancer (mCRPC). In all, 311 mCRPC patients underwent NGS analysis from biopsy samples of progressive metastatic lesions or archival radical prostatectomy specimens. NGS analysis was either performed using a panel of 18 prostate cancer-specific amplicons or via the TS0500 panel. Of the 311 biopsies, 299 (96%) revealed sufficient DNA content for NGS analysis independent on the specimen origin. Biopsies were taken from prostate (31%), lymph nodes (26%), visceral (17%) or osseous (18%) metastases. In 223 (75%) and 76 (25%) patients activating/inhibiting and no mutations were identified, respectively. Most frequently, mutations of HRD genes including a positive HRD score and p53 were identified in 22% of patients each. About 50% of HRD gene mutations were pathogenic and treatment with PARP inhibitors was initiated. Although the majority of p53 alterations were inactivating mutations, 3 patients demonstrated gain-of-function mutations resulting in an inactivation of ATM. Activating androgen receptor mutations and inactivating PTEN mutations were identified in 42 (14%) and 24 (8%) patients, respectively. Specific AR mutations resulted in a switch of hormonal therapy. Mutations of mismatch repair deficiency genes/MSI high were identified in 5 patients resulting in the administration of pembrolizumab. Addition of the TSO 500 panel identified additional mutations in 4.5% of patients and only 2% of the total cohort would have benefitted from this large panel with the identification of druggable mutations. Druggable mutations were identified in one third of mCRPC patients using an 18 amplicon-panel analyzed via NGS. Based on our data, molecular analysis of AR mutations or mutations of the HRD genes should be performed following progression after first-line hormonal therapy. A more extensive molecular analysis seems to be useful following progression to the standard sequential hormonal, cytotoxicand radioligand therapies. Use of the expensive TSO 500 panel seems to be of additional therapeutic value in only a minority of patients.