Generation Sequencing Analysis Research Articles

Diversity and Potential Cross-Species Transmission of Rotavirus A in Wild Animals in Yunnan, China

Rotavirus A (RVA) is the primary enteric pathogen of humans and many other species. However, RVA interspecies transmission remains poorly understood. In this study, we conducted a comprehensive screening and genotyping analysis of RVA in 1706 wild animal samples collected from various regions within Yunnan Province, China. A total of 24 samples, originating from wild boars, rodents, bats, and birds tested positive for RVA. Next generation sequencing and phylogenetic analyses revealed a high degree of genetic diversity and reassortment, particularly for VP4 and VP7. Strains isolated from wild boars and rodents exhibited gene segments with high similarity to those found in humans and other mammalian RVA strains, indicating that RVA may undergo interspecies transmission and reassortment, resulting in novel strains with potential risks for human infection. This study provides critical data for understanding the transmission mechanisms and the RVA host range, and highlights the pivotal role of wildlife in viral evolution and dissemination. These findings have significant implications for public health policies and emphasize the need for enhanced surveillance to prevent interspecies RVA transmission.

Kojic Acid Derivative as an Antimitotic Agent That Selectively Kills Tumour Cells

Background/Objectives: The primary method used to pharmacologically arrest cancer development and its metastasis is to disrupt the cell division process. There are a few approaches that may be used to meet this objective, mainly through inhibiting DNA replication or mitosis. Despite intensive studies on new chemotherapeutics, the biggest problem remains the side effects associated with the inhibition of cell division in non-tumoural host cells. Methods: The efficacy and selectivity of the kojic acid derivative (L1) was studied in vitro with the use of tumoural (Caco2, SW480, HT29, T98G) and non- tumoural (HEK293T, RAW) cell lines. Light and electron microscopy observations were supported by the next generation sequencing (NGS), cytoflow, and spectroscopy analysis of mRNA and biomolecules, respectively. Results: The light and electron microscopy observations showed that L1 treatment leads to significant morphological changes in Caco2 cells, which are characteristic of mitosis arrest. Moreover, the fluorescent tubulin staining revealed the formation of tubulin ring structure associated with the apoptotic stage. Mitotic exit into apoptosis was further conformed by the cytoflow of early/late apoptosis stages and caspase-3 analysis. NGS investigation showed differentiated expressions of genes involved in mitosis and apoptosis processes. The observed IC50 in tumoural cell lines were as follows: Caco2 (IC50 = 68.2 mM), SW480 (IC50 = 15.5 mM), and HT29 (IC50 = 4.7 mM). Conclusions: The findings presented here suggest that L1 could be a valid candidate for oral prevention and/or chemotherapy in colorectal cancer. Considering high selectivity of L1 versus tumoural cell lines, more in-depth mechanistic studies could reveal unknown stages in carcinogenesis.

Prolonged clinical response is possible with regorafenib in refractory osteosarcoma: A case report

<b>Background:</b> Treatment options for metastatic unresectable osteosarcoma are limited, and there is no standard approach after the failure of first-line chemotherapy. Conventional chemotherapy in the second and subsequent lines typically yield a median progression-free survival (PFS) of less than 4 months, with objective response rates ranging from 3 to 29%. The activity of anti-vascular endothelial growth factor receptor multi-tyrosine kinase inhibitors (TKIs) in bone sarcomas has been demonstrated. We present a case of metastatic osteosarcoma exhibiting a durable response to regorafenib treatment.<br /> <b>Case: </b>We discuss a 30-year-old male with metastatic osteosarcoma who had extensive bone and lung metastases and was ineligible for metastasectomy. The patient had received all active chemotherapy regimens in the early and metastatic stages except ifosfamide. Next generation sequencing analysis of resected tumor tissue revealed a possibly pathogenic missense mutation (P.G472E) in NOTCH1 gene. Due to the presence of heart failure with a reduced ejection fraction, chemotherapy was not considered, and regorafenib was initiated. After four cycles of regorafenib, a partial metabolic response was achieved. The patient was followed up without progression under regorafenib for 15 months, without requiring dose reduction. Unfortunately, the treatment had to be interrupted for an extended period due to challenges with recurrent tumor infection and the necessity for repeated surgeries. Following successful wound site control, we restarted full-dose regorafenib and achieved a metabolic partial response again.<br /> <b>Conclusion: </b>To our knowledge, this case represents the longest achieved PFS with regorafenib in metastatic osteosarcoma. TKIs can provide a durable clinical response in metastatic osteosarcoma patients. Clinical and molecular biomarkers are required to determine which patients are most likely to benefit from these treatments.

Benchmarking for genotyping and imputation using degraded DNA for forensic applications across diverse populations

Advancements in sequencing and laboratory technologies have enabled forensic genetic analysis on increasingly low quality and degraded DNA samples. However, existing computational methods applied to genotyping and imputation for generating DNA profiles from degraded DNA have not been tested for forensic applications. Here we simulated sequencing data of varying qualities–coverage, fragment lengths, and deamination patterns–from forty individuals of diverse genetic ancestries. We used this dataset to test the performance of commonly used genotype and imputation methods (SAMtools, GATK, ATLAS, Beagle, and GLIMPSE) on five different SNP panels (MPS-plex, FORCE, two extended kinship panels, and the Human Origins array) that are used for forensic and population genetics applications. For genome mapping and variant calling with degraded DNA, we find use of parameters and methods (such as ATLAS) developed for ancient DNA analysis provides a marked improvement over conventional standards used for next generation sequencing analysis. We find that ATLAS outperforms GATK and SAMtools, achieving over 90 % genotyping accuracy for the four largest SNP panels with coverages greater than 10X. For lower coverages, decreased concordance rates are correlated with increased rates of heterozygosity. Genotype refinement and imputation improve the accuracy at lower coverages by leveraging population reference data. For all five SNP panels, we find that using a population reference panel representative of worldwide populations (e.g., the 1000 Genomes Project) results in increased genotype accuracies across genetic ancestries, compared to ancestry-matched population reference panels. Importantly, we find that the low SNP density of commonly used forensics SNP panels can impact the reliability and performance of genotype refinement and imputation. This highlights a critical trade-off between enhancing privacy by using panels with fewer SNPs and maintaining the effectiveness of genomic tools. We provide benchmarks and recommendations for analyzing degraded DNA from diverse populations with widely used genomic methods in forensic casework.

NCMP-10. EFFECT OF EXTENT OF RESECTION ON GLIOMA SEIZURE OUTCOMES BASED ON MOLECULAR GROUPS

Abstract INTRODUCTION Increased extent of resection (EOR) is a known predictor of seizure freedom in glioma patients. However, it is unknown how tumor genomic alterations affect EOR-induced seizure control. OBJECTIVE To evaluate the genotype-specific impact of EOR on post-operative seizure control METHODS Records of 553 glioma patients who underwent tumor resection from 2012-2023 were reviewed. All patient tumors were analyzed with next generation sequencing and FISH analysis. The unsupervised machine learning algorithm non-negative matrix factorization (NMF) was used to cluster the genomic data. Clinical variables including extent of resection of contrast enhancing tumor and non-enhancing tumor, temozolomide therapy, and radiation therapy were analyzed using Cox Proportional Hazards models. RESULTS NMF clustering of tumor sequencing data revealed four molecular groups: Group 1 (n=56): CDKN2a loss, EGFR copy number gain or mutation, TERT promoter (TERTp) mutation, and MGMT methylated; Group 2 (n=27): IDH mutation, TP53 mutation, and MGMT methylated; Group 3 (n=60): TP53 mutation, TERTp mutation and MGMT methylated; Group 4 (n=244): MGMT unmethylated. Thresholds in the post-operative residual non-enhancing tumor volume (NETV) were identified which marked the most difference in seizure control. For patients in Group 1, the NETV threshold that led to improved epilepsy outcomes was <94.7 cc (p=0.047), whereas the threshold for Group 4 was <89.1 cc (p=0.016). When analysis was conducted for Group 2, the threshold of 10.2 cc failed to reach significance (p=0.1). Interestingly, the threshold analysis for Group 3 found that having a NETV of > 10.1 cc showed improved seizure outcomes (p=0.016). A multivariable cox proportional hazard analysis found that twelve-month seizure freedom was predictive of better OS in group 1 (HR 0.02, p=0.05) and group 4 (HR 0.21, p=1.63e-9). CONCLUSION Our study reveals clinically distinct molecular groups of glioma that display different seizure responses. This data suggests a tumor-specific approach to aggressiveness of extent of resection in order to achieve optimal seizure control.

Third generation sequencing analysis detects significant differences in duodenal microbiome composition between functional dyspepsia patients and control subjects.

Functional dyspepsia (FD) is a multifactorial disorder as its development may be based on several different pathophysiological mechanisms. Interaction of gut microbiome with the host has been proposed as a potential mechanism involved in the disease's pathogenesis. We aimed to characterize microbiome profiling on duodenal luminal content (DLC) of FD patients and compare it to that of controls (CG) and patients with irritable bowel syndrome (IBS). Outpatients fulfilling Rome IV criteria for FD, IBS, and control group (CG) underwent upper gastrointestinal endoscopy and 2 cc of duodenal aspirate (3rd - 4th part) was aspirated in sterile traps. Duodenal microbiome was assessed after DNA extraction and 16S gene-based sequencing on Oxford Nanopore MinION followed by EPI2ME analysis (ONT/Metrich-ore Ltd). Bioanalysis of the microbiome (alpha-, beta-diversity, comparisons of relative abundances for all taxonomic ranks) was implemented in Python. Multiple group means comparisons were performed with one-way Analysis of Variance (ANOVA) and Kruskal-Wallis test with Tuckey's and Dunn's post hoc tests respectively, in case of significance (P-value <0.05). 20 subjects with FD (8 females; age 49.9 ± 13.5 yrs.), 20 with IBS (14 females; age 57.6 ± 14.8 yrs.) and 10 CG (6 females; age 49.2 ± 13.8 yrs.) had their DLC analyzed. The α-diversity index of subjects with FD was significantly lower compared to controls (Shannon's index, p = 0.0218) and similar to that of patients with IBS. Principal Coordinate Analysis (PCoA) generated from species relative abundances (beta-diversity) showed no difference in the DLC profile of subjects with FD and IBS when compared to controls (p = 0.513). Compared to controls, the relative abundance (RA) of Chloroflexota phylum was lower in subjects with FD (p = 0.017) and IBS (p = 0.026), respectively. Additionally, the RA of the Rhodothermota and Thermotogota phyla was lower in FD (p = 0.017 and p = 0.018, respectively) but not in IBS patients (p = 0.15 and p = 0.06, respectively) compared to controls. Interestingly, the RA of specific taxa from Chloroflexota, Rhodothermota and Thermotogota phyla were consistently lower in subjects with FD when compared to CG but similar to IBS, during analysis of all the subsequent major ranks of taxonomy. At the class level, there were significant differences in Syntrophobacteria, Acidithiobacillia, Cytophagia and Flavobacteriia between the FD and CG groups (p < 0.05), but no such difference between FD and IBS was found. Finally, multiple significant differences at the order, family, genus and species level between the FD and CG groups were also detected. A positive relationship between the RA of Streptococcus and those from genus Granulicatella was observed both in FD (p = 0.014) and IBS (p = 0.014) patients. The microbiome profiling from duodenal luminal content of FD patients is significantly different to that of controls, including lower microflora diversity, different microflora structure/composition and specific taxa. Similar differences in the DLC between FD and IBS patients were not evident.

First time evaluation of myocardium microRNA profile in Anderson-Fabry disease: implications in the pathological processes

Abstract Background Anderson Fabry disease (AFD) is an X-linked lysosomal disorder, characterized by progressive sphingolipid storage and organ dysfunction. Cardiac involvement is characterized by left ventricular hypertrophy, inflammation and fibrosis and is leading cause of mortality. MicroRNAs (miRNAs) are emerging as promising biomarkers in medicine, as they are dysregulated in many human diseases, offering opportunities to improve our understanding of disorders and discover new therapeutic targets. However, so far, only a few studies have evaluated the role of miRNAs in Fabry disease, and cardiac tissue miRNAs have never been assessed. Purpose To determine the specific tissue miRNA profile analyzing myocardial tissue of AFD patients and to evaluate their relationship with the severity of cardiac involvement. Methods 19 AFD patients (10 females [53%]; median age 53 years [IQR 43-63]) undergoing right ventricular endomyocardial biopsy (n=15) or surgical septal myectomy (n=4), standard cardiac magnetic resonance (1.5T, cines for maximum wall thickness (MWT) and indexed LV mass (LVMi) calculation) and high-sensitivity Troponin I (TnI) dosage were included and compared to 7 endomyocardial biopsies of controls. Myocardial samples were fixed in formalin and embedded in paraffin. RNA was extracted from 3-5 slides of tissue and used to generate small RNA libraries (Qiagen) for Next Generation Sequencing analysis on Illumina sequencer. Raw data were normalized and analyzed using DEseq2 pipeline. Correlations were performed using Pearson r; a p &amp;lt; 0.05 was considered statistically significant. Results In AFD group median MWT was 13 mm [IQR 8-17], LVMi was 72 g/m2 [IQR 46-97]. TnI was increased in 9 patients (47%) and NT-proBNP in 8 (42%). 6 patients were on specific AFD treatment. We identified 9 miRNAs differentially expressed in AFD patients compared to controls; miRPath analysis revealed that these miRNAs are associated to pathways involved in AFD pathogenesis (autophagy and mTOR signalling, HIF-1, apoptosis, TGF-beta signalling and inflammation) (figure 1). Moreover, we found 10 miRNAs that were differentially expressed in maximal wall thickness (&amp;lt;10 mm, 10-15 mm, &amp;gt;15 mm) groups (figure 2), in particular 3 miRNAs that progressively increased at the increasing of thickness. Among these miRNAs, miR-21-5p showed the strongest correlation with myocardial hypertrophy (r=0.869, p value&amp;lt;0.001 for MWT; r=0.905, p&amp;lt;0.001 for LVMi) and with cardiac damage (r=0.831, p&amp;lt;0.001 for TnI). Additionally, miR-21-5p had a modest correlation with age (r=0.518, p=0.05) and did not differ between patients on specific AFD treatment and patients not treated (p=0.660). Conclusions This study for the first time evaluated the myocardial miRNA profile in AFD patients and identified miR-21-5p as a cardiac biomarker correlating with clinical heart involvement, both in terms of left ventricular hypertrophy (MWT, LVMi) and myocardium damage (troponin).Figure 1Figure 2

Erythropoiesis and Gene Expression Analysis in Erythroid Progenitor Cells Derived from Patients with Hemoglobin H/Constant Spring Disease.

Hemoglobin H/Constant Spring (Hb H/CS) disease represents a form of non-deletional Hb H disease characterized by chronic hemolytic anemia that ranges from moderate to severe and may lead to transfusion-dependent thalassemia. To study the underlying mechanisms of this disease, we conducted an analysis of erythropoiesis and gene expression in erythroid progenitor cells derived from CD34+ hematopoietic stem/progenitor cells from patients with Hb H/CS disease and normal controls. Twelve patients with Hb H/CS disease and five normal controls were enrolled. Peripheral blood samples were collected to isolate CD34+ hematopoietic stem/progenitor cells for the analysis of cell proliferation and differentiation. Six samples from patients with Hb H/CS disease and three controls were subsequently studied for gene expression by next generation sequencing analysis. Erythroid progenitor cells derived from patients with Hb H/CS disease exhibited a trend towards increased rates of erythroid proliferation and decreased cell viability compared to those from controls. Moreover, erythroid progenitor cells derived from patients with Hb H/CS disease demonstrated delayed terminal differentiation. Gene expression profiling revealed elevated levels of genes encoding molecular chaperones, including the heat shock protein genes (HSPs) and the chaperonin containing TCP-1 subunit genes (CCTs) in the Hb H/CS disease group. In summary, erythroid progenitor cells derived from patients with Hb H/CS disease exhibit a trend towards heightened erythroid proliferation, diminished cell viability, and delayed terminal differentiation. Additionally, the increased expression of genes encoding molecular chaperones was observed, providing information on potential underlying pathophysiological mechanisms.

46P Congenital myopathies and muscular dystrophies in the UK: a comprehensive next generation sequencing analysis of frequencies over an 8-year period (2016-2023)

46P Congenital myopathies and muscular dystrophies in the UK: a comprehensive next generation sequencing analysis of frequencies over an 8-year period (2016-2023)

β-galactosidase producing Lactiplantibacillus spp in intestinal microbiome mouse diarrhea model and metagenomic analyses

Probiotics have attained significant interest in recent years as a result of their gut microbiome modulation and gastrointestinal health benefits. Numerous fermented foods contain LAB with GRAS grade and probiotic bacteria. The most promising isolates with high potentiality were selected for 16S rRNA gene sequence analysis and identified as Lactiplantibacillus plantarum, L. fermentum, L. pentosus and Lactiplantibacillus sp. The present study was conducted to determine the gastrointestinal symptoms after the consumption of milk and dairy products, lactose intolerance status using mice model. Post-weaning Balb/c mice were orally administered with probiotic bacteria to assess their efficiency in reducing lactose intolerance symptoms. They exhibited significantly reduced total feces weight and slower intestinal motility and they efficiently alleviated diarrhea symptoms within 6 h of lactose challenge in lactose intolerance-induced mice. Next generation sequence analysis was performed using intestinal content of model treated with L. plantarum GV54 and GV64, the intestinal bacterial diversity was studied using Illumina NGS analysis (16S rRNA hypervariable regions [V3 and V4]). NGS report confirmed the existence of the dominant phyla of the gut microbiome in mice are Bacteroidetes, Firmicutes and Proteobacteria. Due to the influence of the probiotic bacteria in the model group (GV54 and GV64), maximum abundance of phylum Firmicutes were observed when compared to the control group, whereas control group showed more abundance of phylum Bacteriodetes. However, the relative abundance of Bacteroidetes in the model group was decreased and Firmicutes abundance was increased. This indicates the genus abundance of Lactobacillus (Lactiplantibacillus) species or Lactiplantibacillus plantarum GV54 and GV64 bacteria. After treatment with probiotic bacteria Lactobacillus (Lactiplantibacillus) species, the probiotic bacterial abundance was increased consequently genera Helicobacter, Clostridiales unclassified and Desulfovibrio were decreased in the model groups. Probiotic bacteria has a positive impact on the regulation of intestinal microbial diversity in the tested mice system and it reflects that the intestinal microbiota of mice is dynamic and has positive impact on gut health. Considering this point, it is possible to promote Lactiplantibacillus spp. as probiotics and therapeutic food development at industrial scale.

Next Generation Sequencing and Genetic Analyses Reveal Factors Driving Evolution of Sweetpotato Viruses in Uganda.

Sweetpotato (Ipomoea batatas L.) is an essential food crop globally, especially for farmers facing resource limitations. Like other crops, sweetpotato cultivation faces significant production challenges due to viral infections. This study aimed to identify and characterize viruses affecting sweetpotato crops in Uganda, mostly those associated with sweetpotato virus disease (SPVD). Infected leaf samples were collected from farmers' fields in multiple districts spanning three regions in Uganda. MiSeq, a next-generation sequencing platform, was used to generate reads from the viral nucleic acid. The results revealed nine viruses infecting sweetpotato crops in Uganda, with most plants infected by multiple viral species. Sweet potato pakakuy and sweet potato symptomless virus_1 are reported in Uganda for the first time. Phylogenetic analyses demonstrated that some viruses have evolved to form new phylogroups, likely due to high mutations and recombination, particularly in the coat protein, P1 protein, cylindrical inclusion, and helper component proteinase regions of the potyvirus. The sweet potato virus C carried more codons under positive diversifying selection than the closely related sweet potato feathery mottle virus, particularly in the P1 gene. This study provides valuable insights into the viral species infecting sweetpotato crops, infection severity, and the evolution of sweet potato viruses in Uganda.

Exogenous Janus Kinase 617 Codon Influences Small Noncoding RNAs and Gene Expression in Ba/F3 Cells.

Myeloproliferative neoplasms (MPNs) are blood cancers caused by mutations that originate from hematopoietic stem cells. More than 50%-90% of MPN patients had a dominant negative valine (V) to phenylalanine (F) mutation at the Janus kinase 617 codon (JAK2V617F) within the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway; however, this mutation was also found in a high percentage of the general population, its penetrance varied, and its onset was shown to be polygenic. Consequently, it is still unknown what molecular mechanism underlies the MPN transformation produced by JAK2V617F. Patients with MPN have been shown to have dysregulation of noncoding RNAs, such as microRNA (miRNA) and PIWI-interacting RNA (piRNA), although there is not any concrete proof that JAK2V617F alone is responsible for the aberrant regulation of miRNA and piRNA. Human wild type versus V617F-mutated JAK2 are expressed in mouse Ba/F3 cells, and the expressed small and total RNAs were subjected to next generation sequencing analysis to determine the direct induction. Differentially expressed miRNAs, gene expression, and transcript and gene variations were found between exogenously expressed JAK2 and JAK2V617F in Ba/F3 cells. The differently expressed variations contained enriched transposable elements and piRNAs, indicating a rearranged epigenome. The results of the pathway analysis show that the transformation that self-validated the chosen sequencing target genes is impacted by the JAK-STAT pathway. The induction route is functionally conserved, according to exogenously produced miRNA and gene expression. These results may clarify how the JAK2V617F induces transformation.

Identification of biomarker associated with Trop2 in breast cancer: implication for targeted therapy

PurposeTrop2, a cell membrane glycoprotein, is overexpressed in almost all epithelial cancers. This study aimed to explore the mutational characteristics and significance of Trop2 in breast cancer (BC).MethodsPatients diagnosed with BC (n = 77) were enrolled to investigate expression level and clinical characteristics of Trop2. Database of cBioPortal and Kaplan–Meier Plotter were used to evaluate the effects of Trop2 (TACSTD2) genomic ateration and mRNA expression levels on disease-free survival (DFS) and relapse-free survival (RFS), respectively. Based on next generation sequencing analysis, the Trop2 mutation characteristics of BC patients were deeply depicted. In addition, Trop2 expression, mutation and methylation signature associated with Trop2 mutations were analyzed.ResultsTrop2 mutation and high expression of Trop2 were predictive biomarker for shorter DFS and RFS in BC. The positive rate of Trop2 expression in these 77 BC patients was 96.1% (74/77). Based on the Trop2 expression level, the patients were classified into Trop2 negative group, medium expression group and high expression group. The mutation frequencies of MAP3K1, NOTCH2, PTEN and MAGI2 were significantly higher in Trop2 medium expression group than high expression group. Moreover, we investigated the effect of the Trop2 mutations on other genes, including co-expressed genes, differentially mutated genes, differentially expressed genes, gene methylation and phosphorylation. We found that MED8, DPH2, KDM4A, EBNA1BP2, USP1, IPO13, CGAS, PRKAA2, NCOA7, ASCC3 and ABRACL were differentially expressed, mutated and methylated between Trop2 mutation group and wild group.ConclusionMAP3K1, NOTCH2, PTEN and MAGI2 mutations were significantly different between Trop2 medium expression and Trop2 high expression BC patients. The effects of Trop2 mutation on the expression, variation, methylation, and phosphorylation of other genes were comprehensively revealed. High expression level of Trop2 and Trop2 mutation were predictive biomarker for poor prognosis and targeted therapy in BC.

ALK rearranged Spitz melanocytoma: a clinicopathological and molecular genetic analysis of two cases

Objective: To investigate the clinicopathological, immunohistochemical and molecular characteristics of cutaneous ALK-rearranged Spitz melanocytoma. Methods: Two cases of cutaneous ALK-rearranged Spitz melanocytoma from outside hospital consultations in Department of Pathology, Affiliated Cancer Hospital of Fudan University in August 2020 and in Shanghai Ackermann Medical Laboratory in June 2022 were collected. The clinicopathological features, immunophenotypes and molecular profiles of two patients with cutaneous Spitzoid melanocytic tumor harboring ALK-rearrangement were analyzed. The literatures were reviewed. Results: The study included an 8-year-old boy and an 11-year-old girl, who presented with a polypoid lesion in the skin of right thigh and left auricle measuring 1.0 cm and 1.2 cm, respectively. Histologically, they were composed of medium to large-sized epithelioid to plump spindle cells, arranged in nested, plexiform or fascicular patterns in the superficial dermis. The neoplastic cells had abundant eosinophilic cytoplasm with round to ovoid vesicular nuclei containing prominent eosinophilic nucleoli. One case showed mild to moderate nuclear pleomorphism and mitotic activity (average, 2/mm2). Immunohistochemically, the epithelioid and plump spindle cells showed diffuse and strong staining of S-100 protein, SOX10, and ALK (D5F3 and 1A4), but did not express HMB45, PNL2 and MiTF. ALK-rearrangement was detected by fuorescence in situ hybridization in both cases. Subsequent next generation sequence (NGS) analysis identified KANK1::ALK and TPM3:ALK fusions. At 34 and 14 months after surgical resection, both patients remained well with no signs of recurrence or metastasis. Conclusions: ALK-rearranged Spitz melanocytoma represents a morphologically and genetically distinct subset of Spitz melanocytoma, characterized clinically by predilection in children and adolescents, with Spitzoid morphology in plexiform pattern, positive immunohistochemical stains, and rearrangement of ALK. As some cases show atypical features and high mitotic activity, a distinction from Spitz melanoma is warranted.

AB003. Clinical analysis of central nervous system (CNS) World Health Organization (WHO) grade 4 gliomas with IDH-mutant versus IDH-wildtype focused on CDKN2A homozygous deletion.

We are primarily investigating the prognostic role of cell-cycle-dependent kinase inhibitor (CDKN)-2A homozygous deletion in central nervous system (CNS) World Health Organization (WHO) grade 4 gliomas. Additionally, traditional prognostic factors for grade 4 gliomas will be examined, and our results will be validated. We conducted a retrospective analysis of glioma cohorts in our institute. Medical records were reviewed for 142 glioblastoma patients for 15 years, and pathological slides were examined again for the updated diagnosis according to the 2021 WHO classification of CNS tumors. The isocitrate dehydrase (IDH) mutation and CDKN2A deletion were examined by next generation sequencing (NGS) analysis using ONCO accuPanel®. Traditional prognostic factors including age, WHO performance status, extent of resection, and O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation were examined. After the exclusion of 6 patients with poor status of pathologic samples, 136 glioblastoma that were diagnosed by previous WHO criteria were changed into 29 (21.3%) astrocytoma, IDH-mutant, CNS WHO grade 4 and 107 (78.7%) glioblastoma, IDH-wildtype, CNS WHO grade 4. Among them, 61 patients (56.0%) had CDKN2A deletion. Group A with IDH-wildtype and CDKN2A deletion had a mean overall survival (OS) of 15.70 months [95% confident interval (CI): 13.86-17.54], group B with IDH-mutant and CDKN2A deletion had a mean OS of 19.37 months (95% CI: 13.43-25.30), group C with IDH-wildtype and intact CDKN2A had a mean OS of 22.63 months (95% CI: 20.10-25.17), and group D with IDH-mutant and intact CDKN2A had a mean OS of 33.38 months (95% CI: 29.35-37.40). Multifactor analysis showed following factors were independently associated with OS: age [≥50 vs. <50 years; hazard ratio (HR) 4.642], extent of resection (gross total resection vs. others; HR 5.523), WHO performance (0, 1 vs. 2; HR 5.007), MGMT promoter methylation, (methylated vs. unmethylated; HR 5.075), IDH mutation (mutant vs. wildtype; HR 6.358), and CDKN2A deletion (absence vs. presence; HR 13.452). The presenting study suggests that CDKN2A deletion should play a powerful prognostic role in CNS WHO grade 4 gliomas as well as low-grade glioma. Even if CNS WHO grade 4 gliomas had mutant IDH, they can have poor clinical outcomes due to CDKN2A deletion.

Nasal microbiome and the effect of nasal decolonization with a novel povidone-iodine antiseptic solution: a prospective and randomized clinical trial

The aim of this study was to assess the profile of nasal microbiome and evaluate the effect of a specific nasal decolonization solution on the microbiome. We conducted a randomized, placebo-controlled, and parallel-group clinical study of 50 volunteers aged 18 years and older. The subjects were randomly assigned to receive a nasal antiseptic solution, containing povidone-iodine as the main ingredient, (n = 25) or a control solution (n = 25). Nasal swabs were obtained before application (baseline) and at 3 timepoints after application (5 min, 2 h, 24 h). Nasal swabs were subjected to next generation sequencing analysis and cultured in agar plates. At baseline, there were substantial associations between anaerobic species, Corynebacterium spp., Staphylococcus spp., and Dolosigranulum spp. Then, a high bioburden reduction was observed after the application of povidone-iodine (log10 3.68 ± 0.69 at 5 min; log10 3.57 ± 0.94 at 2 h; log10 1.17 ± 1.40 at 24 h), compared to the control. The top species affected by the treatment were Cutibacterium acnes, Staphylococcus, and Corynebacterium species. None of the subjects experienced any adverse effects, nor increases in mucociliary clearance time. Antiseptic solutions applied to the anterior nares can transiently and markedly reduce the bioburden of the nose. The registration number for this clinical trial is NCT05617729.

Effects of oxidized LDL versus IL-1ß/TNF-ɑ/INFɣ on human gingival mesenchymal stem cells properties.

Oxidized low-density lipoprotein (oxLDL) is an important player in the course of metabolic inflammatory diseases. oxLDL was identified in the gingival crevicular fluid, denoting possible associations between oxLDL-induced inflammation and periodontal disease. The current investigation compared for the first-time direct effects of oxLDL to a cytokine cocktail of IL-1ß/TNF-ɑ/INF-γ on gingival mesenchymal stem cells' (G-MSCs) attributes. Human third passage G-MSCs, isolated from connective tissue biopsies (n = 5) and characterized, were stimulated in three groups over 7 days: control group, cytokine group (IL-1β[1 ng/mL], TNF-α[10 ng/mL], IFN-γ[100 ng/mL]), or oxLDL group (oxLDL [50 μg/mL]). Next Generation Sequencing and KEGG pathway enrichment analysis, stemness gene expression (NANOG/SOX2/OCT4A), cellular proliferation, colony-formation, multilinear potential, and altered intracellular pathways were investigated via histochemistry, next-generation sequencing, and RT-qPCR. G-MSCs exhibited all mesenchymal stem cells' characteristics. oxLDL group and cytokine group displayed no disparities in their stemness markers (p > .05). Next-generation-sequencing revealed altered expression of the TXNIP gene in response to oxLDL treatment compared with controls (p = .04). Following an initial boosting for up to 5 days by inflammatory stimuli, over 14 day, cellular counts [median count ×10-5 (Q25/Q75)] were utmost in control - [2.6607 (2.0804/4.5357)], followed by cytokine - [0.0433 (0.0026/1.4215)] and significantly lowered in the oxLDL group [0.0274 (0.0023/0.7290); p = .0047]. Osteogenic differentiation [median relative Ca2+ content(Q25/Q75)] was significantly lower in cytokine - [0.0066 (0.0052/0.0105)] compared to oxLDL - [0.0144 (0.0108/0.0216)] (p = .0133), with no differences notable for chondrogenic and adipogenic differentiation (p > .05). Within the current investigation's limitations, in contrast to cytokine-mediated inflammation, G-MSCs appear to be minimally responsive to oxLDL-mediated metabolic inflammation, with little negative effect on their differentiation attributes and significantly reduced cellular proliferation.

An overview of next generation sequencing strategies and genomics tools used for tuberculosis research.

Tuberculosis (TB) is a grave public health concern and is considered the foremost contributor to human mortality resulting from infectious disease. Due to the stringent clonality and extremely restricted genomic diversity, conventional methods prove inefficient for in-depth exploration of minor genomic variations and the evolutionary dynamics operating in Mycobacterium tuberculosis (M.tb) populations. Until now, the majority of reviews have primarily focused on delineating the application of whole-genome sequencing (WGS) in predicting antibiotic resistant genes, surveillance of drug resistance strains, and M.tb lineage classifications. Despite the growing use of next generation sequencing (NGS) and WGS analysis in TB research, there are limited studies that provide a comprehensive summary of there role in studying macroevolution, minor genetic variations, assessing mixed TB infections, and tracking transmission networks at an individual level. This highlights the need for systematic effort to fully explore the potential of WGS and its associated tools in advancing our understanding of TB epidemiology and disease transmission. We delve into the recent bioinformatics pipelines and NGS strategies that leverage various genetic features and simultaneous exploration of host-pathogen protein expression profile to decipher the genetic heterogeneity and host-pathogen interaction dynamics of the M.tb infections. This review highlights the potential benefits and limitations of NGS and bioinformatics tools and discusses their role in TB detection and epidemiology. Overall, this review could be a valuable resource for researchers and clinicians interested in NGS-based approaches in TB research.

Aquatic conditions & bacterial communities as drivers of the decomposition of submerged remains

Aquatic decomposition, as a forensic discipline, has been largely under-investigated as a consequence of the highly complex and influential variability of the water environment. The limitation to the adaptability of scenario specific results justifies the necessity for experimental research to increase our understanding of the aquatic environment and the development of post-mortem submersion interval (PMSI) methods of estimation. This preliminary research aims to address this contextual gap by assessing the variation in the bacterial composition of aquatic biofilms as explained by water parameter measurements over time, associated with clothed and bare decomposing remains.As part of three field investigations, a total of 9 still-born piglets (n = 3, per trial) were used as human analogues and were submerged bare or clothed in either natural cotton or synthetic nylon. Changes in the bacterial community composition of the water surrounding the submerged remains were assessed at 4 discrete time points post submersion (7, 14, 21 and 28 days) by 16 S rRNA gene Next Generation Sequencing analysis and compared to coinciding water parameter measurements (i.e. conductivity, total dissolved solids (TDS), salinity, pH, and dissolved oxygen (DO)).Bacterial diversity was found to change over time and relative to clothing type, where significant variation was observed between synthetic nylon samples and bare/cotton samples. Seasonality was a major driver of bacterial diversity, where substantial variation was found between samples collected in early winter to those collected in mid - late winter. Water parameter measures of pH, salinity and DO were identified to best explain the global bacterial community composition and their corresponding dynamic trajectory patterns overtime.Further investigation into bacterial community dynamics in accordance with varying environmental conditions could potentially lead to the determination of influential extrinsic factors that may drive bacterial activity in aquatic decomposition. Together with the identification of potential bacterial markers that complement the different stages of decomposition, this may provide a future approach to PMSI estimations.

Transcriptomic analysis of the effect of remote ischaemic conditioning in an animal model of necrotising enterocolitis

Necrotising enterocolitis (NEC) has a complex pathophysiology but the common end-point is ischaemia reperfusion injury (IRI) and intestinal necrosis. We have previously reported that RIC significantly reduces the intestinal injury in a rat model of NEC. Here we describe the changes in intestinal mRNA occurring in the intestine of animals exposed to IRI, both with and without RIC. Related rat-pups were randomly assigned to four groups: SHAM, IRI only, RIC only and RIC + IRI. IRI animals, underwent 40 min of intestinal ischaemia, and 90 min of reperfusion. Animals that underwent RIC had three cycles of 5 min of alternating ischaemia/reperfusion by means of a ligature applied to the hind limb. Samples from the terminal ileum were immediately stored in RNA-preserving media for later next generation sequencing and transciptome analysis using R v 3.6.1. Differential expression testing showed that 868 genes differentially expressed in animals exposed to RIC alone compared to SHAM and 135 in the IRI and RIC group compared to IRI alone. Comparison between these two sets showed that 25 genes were differentially expressed in both groups. Pro-inflammatory molecules: NF-ĸβ2, Cxcl1, SOD2 and Map3k8 all show reduced expression in response to RIC. Targeted gene analysis revealed increased expression in PI3K which is part of the so-called RISK-pathway which is a key part of the protective mechanisms of RIC in the heart. Overall, this transcriptomic analysis shows that RIC provides a protective effect to the intestine via anti-inflammatory pathways. This could be particularly relevant to treating and preventing NEC.