Next-generation Sequencing Analysis Research Articles

B-191 Genomic Profile of Circulating Tumor DNA in Advanced Lung Cancer: Comparison with Tissue DNA

Abstract Background The most common cancer in Korea is thyroid cancer, followed by lung cancer, and the most common cancer in men is lung cancer. The cancer with the highest mortality rate is lung cancer. Recently, the precision medical approach is becoming a major treatment strategy for lung cancer. For precision medical approach, the usefulness of next-generation sequencing (NGS) analysis targeting circulating tumor DNA (ctDNA) is being emphasized. Therefore, in this study, NGS analysis was performed on blood and tissues of advanced lung cancer patients to compare and analyze genetic mutation patterns to confirm the usefulness of ctDNA NGS assay. Methods A total of 101 patients with advanced lung cancer (stage 3 or 4) were recruited, and ctDNA NGS assays were performed on their plasma using a Pan100 panel (Dxome). Tissues from primary lung cancer, metastatic lymph nodes, or metastatic organs of 22 patients were analyzed using ONCOaccuPanel (NGeneBio). All variants were classified into four tiers based on the level of clinical significance, according to the standards and guidelines established by the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists. Results In the ctDNA NGS assay of 101 patients, 70 (69.3%) harbored 154 tier 1 or 2 variants. Mutations in TP53 (45, 29.2%) and EGFR (23, 14.9%) were the most frequently detected, followed by mutations in RB1, CHEK2, ATM, JAK2, ATR, DNMT3A, and KRAS. Among the 22 patients whose tissue was examined, tier 1 or 2 variants were detected in both the plasma and tissue of 14 patients and only in the tissue of six patients. All genes for these variants were targeted using both methods. Identical variants were detected in the plasma and tissue of six of the 14 patients, and discordant results were observed in the remaining eight patients. Twenty copy number variations were detected in 13 of the 101 patients, with copy number gain of EGFR being the most common (6, 30%). Fusion genes were detected in five of the 101 patients, two of which were not detected in the tissue. Overall, 74 (73.3%) of the 101 patients had positive ctDNA NGS assay results. Conclusions Although the NGS assay with ctDNA is less sensitive than that with tissue DNA, some genetic alterations are detected only in ctDNA NGS assays; therefore, it is useful to apply the ctDNA NGS assay as a complement.

Identification of 10 differentially expressed genes involved in the tumorigenesis of cervical cancer via next-generation sequencing.

The incidence and mortality of cervical cancer remain high in female malignant tumors worldwide. There is still a lack of diagnostic and prognostic markers for cervical carcinoma. This study aimed to screen differentially expressed genes (DEGs) between normal and cervical cancer tissues to identify candidate genes for further research. Uterine cervical specimens were resected from our clinical patients after radical hysterectomy. Three patients' transcriptomic datasets were built by the next generation sequencing (NGS) results. DEGs were selected through the edgeR and DESeq2 packages in the R environment. Functional enrichment analysis, including GO/DisGeNET/KEGG/Reactome enrichment analysis, was performed. Normal and cervical cancer tissue data from the public databases TCGA and GTEx were collected to compare the expression levels of 10 selected DEGs in tumor and normal tissues. ROC curve and survival analysis were performed to compare the diagnostic and prognostic values of each gene. The expression levels of candidate genes were verified in 15 paired clinical specimens via quantitative real-time polymerase chain reaction. There were 875 up-regulated and 1,482 down-regulated genes in cervical cancer samples compared with the paired adjacent normal cervical tissues according to the NGS analysis. The top 10 DEGs included APOD, MASP1, ACKR1, C1QTNF7, SFRP4, HSPB6, GSTM5, IGFBP6, F10 and DCN. GO, DisGeNET and Reactome analyses revealed that the DEGs were related to extracellular matrix and angiogenesis which might influence tumorigenesis. KEGG enrichment showed that PI3K-Akt signaling pathway might be involved in cervical cancer tumorigenesis and progression. The expression levels of selected genes were decreased in tumors in both the public database and our experimental clinical specimens. All the candidate genes showed excellent diagnostic value, and the AUC values exceeded 0.90. Additionally, APOD, ACKR1 and SFRP4 expression levels could help predict the prognosis of patients with cervical cancer. In this study, we selected the top 10 DEGs which were down-regulated in cervical cancer tissues. All of them had dramatically diagnostic value. APOD, ACKR1 and SFRP4 were associated with the survivals of cervical cancer. C1QTNF7, HSPB6, GSTM5, IGFBP6 and F10 were first reported to be candidate genes of cervical carcinoma.

Deep learning-based analysis of EGFR mutation prevalence in lung adenocarcinoma H&E whole slide images.

EGFR mutations are a major prognostic factor in lung adenocarcinoma. However, current detection methods require sufficient samples and are costly. Deep learning is promising for mutation prediction in histopathological image analysis but has limitations in that it does not sufficiently reflect tumor heterogeneity and lacks interpretability. In this study, we developed a deep learning model to predict the presence of EGFR mutations by analyzing histopathological patterns in whole slide images (WSIs). We also introduced the EGFR mutation prevalence (EMP) score, which quantifies EGFR prevalence in WSIs based on patch-level predictions, and evaluated its interpretability and utility. Our model estimates the probability of EGFR prevalence in each patch by partitioning the WSI based on multiple-instance learning and predicts the presence of EGFR mutations at the slide level. We utilized a patch-masking scheduler training strategy to enable the model to learn various histopathological patterns of EGFR. This study included 868 WSI samples from lung adenocarcinoma patients collected from three medical institutions: Hallym University Medical Center, Inha University Hospital, and Chungnam National University Hospital. For the test dataset, 197 WSIs were collected from Ajou University Medical Center to evaluate the presence of EGFR mutations. Our model demonstrated prediction performance with an area under the receiver operating characteristic curve of 0.7680 (0.7607-0.7720) and an area under the precision-recall curve of 0.8391 (0.8326-0.8430). The EMP score showed Spearman correlation coefficients of 0.4705 (p = 0.0087) for p.L858R and 0.5918 (p = 0.0037) for exon 19 deletions in 64 samples subjected to next-generation sequencing analysis. Additionally, high EMP scores were associated with papillary and acinar patterns (p = 0.0038 and p = 0.0255, respectively), whereas low EMP scores were associated with solid patterns (p = 0.0001). These results validate the reliability of our model and suggest that it can provide crucial information for rapid screening and treatment plans.

Molecular Profiling of Low-Grade Appendiceal Mucinous Neoplasms (LAMN).

Low-grade appendiceal mucinous neoplasia (LAMN) represents a relatively rare tumor of the appendix typically diagnosed incidentally through appendectomy for acute appendicitis. In cases where perforation occurs, mucinous content may disseminate into the abdominal cavity, leading to the development of pseudomyxoma peritonei (PMP). The primary objective of this study was to elucidate the molecular characteristics associated with various stages of LAMN and PMP. DNA was extracted from LAMN, primary PMPs, recurrent PMPs, and adenocarcinomas originating from LAMN. The subsequent analysis involved the examination of mutational hotspot regions within 50 cancer-related genes, covering over 2800 COSMIC mutations, utilizing amplicon-based next-generation sequencing (NGS). Our findings revealed activating somatic mutations within the MAPK-signaling pathway across all tumors examined. Specifically, 98.1% of cases showed mutations in KRAS, while one tumor harbored a BRAF mutation. Additionally, GNAS mutations were identified in 55.8% of tumors, with no significant difference observed between LAMN and PMP. While LAMN rarely displayed additional mutations, 42% of primary PMPs and 60% of recurrent PMPs showed additional mutations. Notably, both adenocarcinomas originating from LAMN showed mutations within TP53. Furthermore, 7.7% (4/52) of cases exhibited a potentially targetable KRAS G12C mutation. In four patients, NGS analysis was performed on both primary PMP and recurrent PMP/adenocarcinoma samples. While mutations in KRAS and GNAS were detected in almost all samples, 50% of recurrent cases displayed an additional SMAD4 mutation, suggesting a notable alteration during disease progression. Our findings indicate two key points: First, mutations within the MAPK pathway, particularly in KRAS, are evident across all tumors, along with a high frequency of GNAS mutations. Second, progression toward PMP or adenocarcinoma is associated with an accumulation of additional mutations within common oncogenic pathways.

Influences of Ipomoea batatas Anti-Cancer Peptide on Tomato Defense Genes.

This study investigates the impact of IbACP (Ipomoea batatas anti-cancer peptide) on defense-related gene expression in tomato leaves, focusing on its role in plant defense mechanisms. Previously, IbACP was isolated from sweet potato leaves, and it was identified as a peptide capable of inducing an alkalinization response in tomato suspension culture media. Additionally, IbACP was found to regulate the proliferation of human pancreatic adenocarcinoma cells. Elucidate IbACP's molecular influence on defense-related gene expression in tomato leaves using next-generation sequencing analysis. To assess the impact of IbACP on defense-related gene expression, transcriptome data were analyzed, encompassing various functional categories such as photosynthesis, metabolic processes, and plant defense. Semi-quantitative reverse-transcription polymerase chain reaction analysis was employed to verify transcription levels of defense-related genes in tomato leaves treated with IbACP for durations ranging from 0 h (control) to 24 h. IbACP induced jasmonic acid-related genes (LoxD and AOS) at 2 h, with a significant up-regulation of salicylic acid-dependent gene NPR1 at 24 h. This suggested a temporal antagonistic effect between jasmonic acid and salicylic acid during the early hours of IbACP treatment. Downstream ethylene-responsive regulator genes (ACO1, ETR4, and ERF1) were consistently down-regulated by IbACP at all times. Additionally, IbACP significantly up-regulated the gene expressions of suberization-associated anionic peroxidases (TMP1 and TAP2) at all time points, indicating enhanced suberization of the plant cell wall to prevent pathogen invasion. IbACP enhances the synthesis of defense hormones and up-regulates downstream defense genes, improving the plant's resistance to biotic stresses.

HEARRING group genetic marker study: genetic background of CI patients

Background While cochlear implantation (CI) and electric acoustic stimulation (EAS) have a positive outcome in most cases, their effectiveness varies depending on the etiology of the hearing loss. Among the various etiologies, genetic factors are the leading cause of hearing loss and may impact CI and EAS outcomes. Aims/Objectives To reveal the genetic background of the hearing loss in CI/EAS patients in each ethnic population, we undertook a multi-center study involving the genetic testing of hearing loss in CI/EAS patients from 10 centers. Material and Methods Saliva samples and clinical information for the patients and their family members were obtained and next-generation sequencing analysis using a panel carrying 63 deafness genes was then performed. Results Genetic testing successfully identified the causative gene variants in 54.5% (48/88) of patients with pre-lingual onset hearing loss (onset under 6 years) and in 12% (12/95) of those with late-onset hearing loss (onset at 6 years or more). Conclusions and Significance We clearly indicated that genetic factors are the most common cause of hearing loss regardless of ethnic background. Saliva-based genetic testing is a useful tool for multi-center studies seeking to clarify the genetic causes of hearing loss in CI or EAS patients between countries separated by distance.

Fish diversity assessment through conventional morphological identification and recent advances in Saudi Arabia: A review

Fish identification in the Red Sea, particularly in Saudi Arabia, has a long history. Because of the vast fish diversity in Saudi Arabia, proper species identification is required. Indeed, identifying fish species is critical for biodiversity conservation, food and drug safety, and sustainable fishery management. Numerous approaches have been used to identify fish species, including conventional morphological identification, next-generation sequencing (NGS), nanopore sequencing, DNA barcoding, and environmental DNA analysis. In this review, we collected as much scientific information as possible on species identification in Saudi Arabia. Our findings suggest that the identification process has advanced and spread rapidly and broadly, as evidenced by the discovery of new fish species in Saudi Arabia. The advantages and disadvantages of each method were discussed as part of a comprehensive comparison. This study aimed to provide further scientific knowledge to promote the growth of fish diversity worldwide. Keywords: fish diversity, identification process, Saudi Arabia.

Horse Meat Microbiota: Determination of Biofilm Formation and Antibiotic Resistance of Isolated Staphylococcus Spp.

Domestic horses could be bred for leisure activities and meat production, as is already the case in many countries. Horse meat is consumed in various countries, including Kazakhstan and Kyrgyzstan, and with the increase in this consumption, horses are registered as livestock by the Food and Agricultural Organization. In this study, horse meat microbiota of horse samples (n = 56; 32 samples from Kazakhstan and 24 samples from Kyrgyzstan) from two countries, Kazakhstan (n = 3) and Kyrgyzstan (n = 1), were investigated for the first time by next-generation sequencing and metabarcoding analysis. The results demonstrated that Firmicutes, Proteobacteria, and Actinobacteria were the dominant bacterial phyla in all samples. In addition, three (5.4%) Staphylococcus strains were isolated from the Uzynagash region, Kazakhstan. Staphylococcus strains were identified as Staphylococcus warneri, S. epidermidis, and S. pasteuri by partial 16S rRNA DNA gene Sanger sequencing. All three Staphylococcus isolates were nonbiofilm formers; only the S. pasteuri was detected as multidrug-resistant (resistant to penicillin, cefoxitin, and oxacillin). In addition, S. pasteuri was found to carry mecA, mecC, and tetK genes. This is the first study to detect potentially pathogenic Staphylococcus spp. in horse meat samples originating from Kazakhstan. In conclusion, it should be carefully considered that undercooked horse meat may pose a risk to consumers in terms of pathogens such as antibiotic-resistant Staphylococcus isolates.

Triple-targeted therapy of dabrafenib, trametinib, and osimertinib for the treatment of the acquired BRAF V600E mutation after progression on EGFR-tyrosine kinase inhibitors in advanced EGFR-mutated non-small cell lung cancer patients.

The B-Raf proto-oncogene, serine/threonine kinase (BRAF) V600E mutation is responsible for approximately 3% of acquired resistance mechanisms to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) in advanced EGFR-mutant non-small cell lung cancer (NSCLC). This study investigated the efficacy and safety of a triple-targeted therapy combining EGFR/BRAF/mitogen-activated protein kinase kinase (MEK) inhibitors of dabrafenib, trametinib, and osimertinib in NSCLC patients with acquired BRAF V600E mutation after EGFR-TKI treatment. A multi-center retrospective review of medical records was performed to analyze EGFR-mutated advanced Chinese NSCLC patients who acquired the BRAF V600E mutation following EGFR-TKI treatment. All patients subsequently received dabrafenib, trametinib, and osimertinib. The clinical characteristics, progression-free survival (PFS), and adverse events (AEs) were documented. The in-vivo drug response of patient-derived organoids (PDOs) was observed. Next-generation sequencing (NGS) was performed upon progression to triple-targeted therapy. Thirteen patients with BRAF V600E mutations were included. Following triple-targeted therapy, the corresponding objective response rate and disease control rate were 61.5% and 92.3%, respectively. The median PFS was 13.5 months (95% confidence interval: 6.6-20.4). PDOs derived from one patient's tumor sample were established, revealing that the triple-targeted therapy had a significantly lower half-maximal inhibitory concentration (IC50) value compared to other regimens. The tumor growth inhibitory rate was 99.36% for dabrafenib, trametinib, and osimertinib; 99.25% for osimertinib plus vemurafenib; 98.92% for osimertinib, encorafenib, and cetuximab; and 62.83% for pemetrexed plus carboplatin. NGS analysis identified major resistance mechanisms following the triple-targeted therapy, including the EGFR-dependent pathway, EGFR and BRAF V600E-dependent pathway, and an off-target mechanism. EGFR/BRAF/MEK triple-targeted therapy is an effective and safe approach for treating EGFR-mutated NSCLC patients resistant to EGFR-TKIs with acquired BRAF V600E mutations.

Clinical and Genomic Characterization of ERBB2-Altered Gallbladder Cancer: Exploring Differences Between an American and a Chilean Cohort.

Gallbladder cancer (GBC) is a biliary tract malignancy characterized by its high lethality. Although the incidence of GBC is low in most countries, specific areas such as Chile display a high incidence. Our collaborative study sought to compare clinical and molecular features of GBC cohorts from Chile and the United States with a focus on ERBB2 alterations. Patients were accrued at Memorial Sloan Kettering Cancer Center (MSK) or the Pontificia Universidad Católica de Chile (PUC). Clinical information was retrieved from medical records. Genomic analysis was performed by the next-generation sequencing platform MSK-Integrated Mutation Profiling of Actionable Cancer Targets. A total of 260 patients with GBC were included, 237 from MSK and 23 from PUC. There were no significant differences in the clinical characteristics between the patients identified at MSK and at PUC except in terms of lithiasis prevalence which was significantly higher in the PUC cohort (85% v 44%; P = .0003). The prevalence of ERBB2 alterations was comparable between the two cohorts (15% v 9%; P = .42). Overall, ERBB2 alterations were present in 14% of patients (8% with ERBB2 amplification, 4% ERBB2 mutation, 1.5% concurrent amplification and mutation, and 0.4% ERBB2 fusion). Notably, patients with GBC that harbored ERBB2 alterations had better overall survival (OS) versus their ERBB2-wild type counterparts (22.3 months v 11.8 months; P = .024). The prevalence of lithiasis seems to be higher in Chilean versus US patients with GBC. A similar prevalence of ERBB2 alterations of overall 14% and better OS suggests that a proportion of them could benefit from human epidermal growth factor receptor type 2-targeted therapies. The smaller cohort of Chile, where the disease prevalence is higher, is a reminder and invitation for the need of more robust next-generation sequencing analyses globally.

Detecting Alu Element Insertion Variant in RP1 Gene Using Whole Genome Sequencing in Patients with Retinitis Pigmentosa.

Background/Objectives:Alu element insertion in the exon 4 of the RP1 gene was newly identified through whole genome sequencing (WGS). This was not detected in previous next-generation sequencing (NGS) analysis. We report three cases of Korean retinitis pigmentosa (RP) patients with compound heterozygous variants including Alu element insertion in the RP1 gene, indicating that Alu element insertion could be a cause of RP; Methods: Among patients diagnosed with RP having variants in the RP1 gene in the Asan Medical Center, WGS was additionally performed for genetically unsolved cases in previous NGS analysis to detect any presence of Alu element insertion. For cases detected to have Alu element insertion in the exon 4 of the RP1 gene, genetic and clinical characteristics were analyzed; Results: Among 16 patients with RP, 3 patients were detected to have Alu element insertion in the RP1 gene. Alu element insertion in the RP1 gene was also detected using WGS. It was revealed to be a pathogenic variant. Therefore, RP1 gene mutation was the confirmed genetic cause of RP for these three cases and genetic counseling was enabled for them; Conclusions: Alu element insertion in the RP1 gene could be a genetic cause of autosomal recessive RP patients with compound heterozygous variants. Through WGS, the identification of this pathogenic variant was possible. Confirmation is needed to check the presence of Alu element insertion in patients with compound heterozygous variants in the RP1 gene.

Higher Microbial Abundance and Diversity in Bronchus-Associated Lymphoid Tissue (BALT) Lymphomas than in Non-Cancerous Lung Tissues.

It is well known that the majority of the extranodal marginal zone lymphomas of mucosa-associated lymphoid tissues (MALT lymphomas) are associated with microbiota, e.g., gastric MALT lymphoma with Helicobacter pylori. In general, they are very sensitive to low-dose radiotherapy and chemotherapeutic agents. The microbiota profile is not clearly elucidated in bronchus-associated lymphoid tissue (BALT) lymphoma, a rare type of MALT lymphoma in the lung. Thus, this study aimed to clarify the intratumor microbiome in BALT lymphoma using the third-generation NGS method. DNAs were extracted from 12 formalin-fixed paraffin-embedded (FFPE) tumor tissues obtained from BALT lymphoma patients diagnosed between 1990 and 2016. 16S rRNA gene was amplified by polymerase chain reaction. Amplicons were sequenced using a Nanopore platform. Next-generation sequencing analysis was performed to assess microbial profiles. For comparison, FFPE specimens from nine non-cancerous lung tissues were also analyzed. Specific bacterial families including Burkholderiaceae, Bacillaceae, and Microbacteriaceae were associated with BALT lymphoma by a linear discriminant analysis effect size approach. Although the number of specimens was limited, BALT lymphomas exhibited significantly higher microbial abundance and diversity with distinct microbial composition patterns and correlation networks than non-cancerous lung tissues. This study provides the first insight into intratumor microbiome in BALT lymphoma using the third-generation NGS method. A distinct microbial composition suggests the presence of a unique tumor microenvironment of BALT lymphoma.

Identification of Microorganisms (Fungi) in Fermented Ganjang from Korea, China, and Japan Using Next-Generation Sequencing Analysis

Identification of Microorganisms (Fungi) in Fermented Ganjang from Korea, China, and Japan Using Next-Generation Sequencing Analysis

Cancer associated variant enrichment CAVE, a gene agnostic approach to identify low burden variants in chronic lymphocytic leukemia

Intratumoral heterogeneity is an important clinical challenge because low burden clones expressing specific genetic alterations drive therapeutic resistance mechanisms. We have developed CAVE (cancer-associated variant enrichment), a gene-agnostic computational tool to identify specific enrichment of low-burden cancer driver variants in next-generation sequencing (NGS) data. For this study, CAVE was applied to TP53 in chronic lymphocytic leukemia (CLL) as a cancer model. Indeed, as TP53 mutations are part of treatment decision-making algorithms and low-burden variants are frequent, there is a need to distinguish true variants from background noise. Recommendations have been published for reliable calling of low-VAF variants of TP53 in CLL and the assessment of the background noise for each platform is essential for the quality of the testing. CAVE is able to detect specific enrichment of low-burden variants starting at variant allele frequencies (VAFs) as low as 0.3%. In silico TP53 dependent and independent analyses confirmed the true driver nature of all these variants. Orthogonal validation using either ddPCR or NGS analyses of follow-up samples confirmed variant identification. CAVE can be easily deployed in any cancer-related NGS workflow to detect the enrichment of low-burden variants of clinical interest.

Endophytic Bacteria and Fungi Associated with Polygonum cuspidatum in the Russian Far East.

Polygonum cuspidatum, alternatively known as Fallopia japonica or Reynoutria japonica, is a perennial herb belonging to the Polygonaceae family. Commonly called Japanese knotweed or Asian knotweed, this plant is native to East Asia, particularly in regions such as Korea, China, and Japan. It has successfully adapted to a wide range of habitats, resulting in it being listed as a pest and invasive species in several countries in North America and Europe. This study focuses on analysing the composition of the bacterial and fungal endophytic communities associated with Japanese knotweed growing in the Russian Far East, employing next-generation sequencing (NGS) and a cultivation-based method (microbiological sowing). The NGS analysis showed that the dominant classes of endophytic bacteria were Alphaproteobacteria (28%) and Gammaproteobacteria (28%), Actinobacteria (20%), Bacteroidia (15%), and Bacilli (4%), and fungal classes were Agaricomycetes (40%), Dothideomycetes (24%), Leotiomycetes (10%), Tremellomycetes (9%), Pezizomycetes (5%), Sordariomycetes (3%), and Exobasidiomycetes (3%). The most common genera of endophytic bacteria were Burkholderia-Caballeronia-Parabukholderia, Sphingomonas, Hydrotalea, Methylobacterium-Metylorubrum, Cutibacterium, and Comamonadaceae, and genera of fungal endophytes were Marasmius, Tuber, Microcyclosporella, Schizothyrium, Alternaria, Parastagonospora, Vishniacozyma, and Cladosporium. The present data showed that the roots, leaves, and stems of P. cuspidatum have a greater number and diversity of endophytic bacteria and fungi compared to the flowers and seeds. Thus, the biodiversity of endophytic bacteria and fungi of P. cuspidatum was described and analysed for the first time in this study.

Cannabinol (CBN) Influences the Ion Channels and Synaptic-Related Genes in NSC-34 Cell Line: A Transcriptomic Study.

Neurological disorders such as Alzheimer's, Parkinson's, amyotrophic lateral sclerosis, and schizophrenia are associated with altered neuronal excitability, resulting from dysfunctions in the molecular architecture and physiological regulation of ion channels and synaptic transmission. Ion channels and synapses are regarded as suitable therapeutic targets in modern pharmacology. Cannabinoids have received great attention as an original therapeutic approach for their effects on human health due to their ability to modulate the neurotransmitter release through interaction with the endocannabinoid system. In our study, we explored the effect of cannabinol (CBN) through next-generation sequencing analysis of NSC-34 cell physiology. Our findings revealed that CBN strongly influences the ontologies related to ion channels and synapse activity at all doses tested. Specifically, the genes coding for calcium and potassium voltage-gated channel subunits, and the glutamatergic and GABAergic receptors (Cacna1b, Cacna1h, Cacng8, Kcnc3, Kcnd1, Kcnd2, Kcnj4, Grik5, Grik1, Slc17a7, Gabra5), were up-regulated. Conversely, the genes involved into serotoninergic and cholinergic pathways (Htr3a, Htr3b, Htr1b, Chrna3, Chrnb2, Chrnb4), were down-regulated. These findings highlight the influence of CBN in the expression of genes involved into ion influx and synaptic transmission.

Differential genotoxicity of Polygoni Multiflori in rat and human: insights from Ames test and S9 metabolic activation system

The Ames test is used worldwide to initially screen the mutagenic potential of new chemicals. In the standard Ames test, S. typhimurium strains (TA100, TA98, TA1535, and TA1537) and Escherichia coli (WP2uvrA) are treated with substances with/without cytochrome P450s (CYPs)-induced rat S9 fractions for identifying mutagens and pro-mutagens. However, many substances show completely different toxicity patterns depending on whether the liver S9 fraction belongs to rats or humans. The natural product Polygoni Multiflori Radix (PMR) can also show bacterial reverse mutation, followed by the rat or human liver S9 fraction. While PMR elicits reverse mutations in the TA1537 strain in rat liver S9 but not in human liver S9, this mechanism has not been verified yet. To explain this, the differences in metabolic enzymes compositions commonly observed between rats and humans have been implicated. This study aimed to explore the key factors that cause differences in the genotoxicity of PMR between rat and human liver S9 metabolic enzymes. The results of next-generation sequencing (NGS) analysis showed that both rat and human metabolic enzymes caused similar mutations in TA1537. However, when the metabolic enzymes in each S9 fraction were analyzed using ion mobility tandem mass spectrometry (IM-MS), rat- and human-specific enzymes were identified among the cytochrome (CYP) family, especially aryl hydrocarbon receptor (AHR)-related CYPs. These findings suggest that CYP1A1 isoforms contribute to the mechanism of PMR in the Ames test. Therefore, an in vitro Ames test might be more reliable in predicting genotoxicity for both rodents and humans. This will also help overcome the limitations of laboratory animal-based toxicity evaluations, which provide unreliable results due to interspecies differences between humans and rodents.

Retrospective clinical and microbiologic analysis of metagenomic next-generation sequencing in the microbiological diagnosis of cutaneous infectious granulomas

BackgroundCutaneous infectious granulomas (CIG) are localized and chronic skin infection caused by a variety of pathogens such as protozoans, bacteria, worms, viruses and fungi. The diagnosis of CIG is difficult because microbiological examination shows low sensitivity and the histomorphological findings of CIG caused by different pathogens are commonly difficult to be distinguished.ObjectiveThe objective of this study is to explore the application of mNGS in tissue sample testing for CIG cases, and to compare mNGS with traditional microbiological methods by evaluating sensitivity and specificity.MethodsWe conducted a retrospective study at the Department of Dermatology of Sun Yat-sen Memorial Hospital, Sun Yat-sen University from January 1st, 2020, to May 31st, 2024. Specimens from CIG patients with a clinical presentation of cutaneous infection that was supported by histological examination were retrospectively enrolled. Specimens were delivered to be tested for microbiological examinations and mNGS.ResultsOur data show that mNGS detected Non-tuberculosis mycobacteria, Mycobacterium tuberculosis, fungi and bacteria in CIG. Compared to culture, mNGS showed a higher positive rate (80.77% vs. 57.7%) with high sensitivity rate (100%) and negative predictive value (100%). In addition, mNGS can detect more pathogens in one sample and can be used to detect variable samples including the samples of paraffin-embedded tissue with shorter detective time. Of the 21 patients who showed clinical improvement within a 30-day follow-up, eighteen had their treatments adjusted, including fifteen who continued treatment based on the results of mNGS.ConclusionsmNGS could provide a potentially rapid and effective alternative detection method for diagnosis of cutaneous infectious granulomas and mNGS results may affect the clinical prognosis resulting from enabling the patients to initiate timely treatment.

A novel GFP-based strategy to quantitate cellular spatial associations in HSV-1 viral pathogenesis.

Periodic reactivation of herpes simplex virus type 1 (HSV-1) triggers immune responses that result in corneal scarring (CS), known as herpes stromal keratitis (HSK). Despite considerable research, fully understanding HSK and eliminating it remains challenging due to a lack of comprehensive analysis of HSV-1-infected immune cells in both corneas and trigeminal ganglia (TG). We engineered a recombinant HSV-1 expressing green fluorescent protein (GFP) in the virulent McKrae virus strain that does not require corneal scarification for efficient virus replication (GFP-McKrae). Next-generation sequencing (NGS) analysis, along with in vitro and in vivo assays, showed that GFP-McKrae virus was similar to WT-McKrae virus. Furthermore, corneal cells infected with GFP-McKrae were quantitatively analyzed using image mass cytometry (IMC). The single-cell reconstruction data generated cellular maps of corneas based on the expression of 25 immune cell markers in GFP-McKrae-infected mice. Corneas from mock control mice showed the presence of T cells and macrophages, whereas corneas from GFP-McKrae-infected mice on days 3 and 5 post-infection (PI) exhibited increased immune cells. Notably, on day 3 PI, increased GFP expression was observed in closely situated clusters of DCs, macrophages, and epithelial cells. By day 5 PI, macrophages and T cells became prominent. Finally, immunostaining methods detected HSV-1 or GFP and gD proteins in latently infected TG. This study presents a valuable strategy for identifying cellular spatial associations in viral pathogenesis and holds promise for future therapeutic applications.IMPORTANCEThe goal of this study was to establish quantitative approaches to analyze immune cell markers in HSV-1-infected intact corneas and trigeminal ganglia from primary and latently infected mice. This allowed us to define spatial and temporal interactions between specific immune cells and their potential roles in virus replication and latency. To accomplish this important goal, we took advantage of the utility of GFP-McKrae virus as a valuable research tool while also highlighting its potential to uncover previously unrecognized cell types that play pivotal roles in HSV-1 replication and latency. Such insights will pave the way for developing targeted therapeutic approaches to tackle HSV-1 infections more effectively.

In Vitro Selection and Characterization of a Light-up DNA Aptamer for Thiazole Orange.

A new DNA aptamer that binds to the target Thiazole Orange-biotin (TO1-biotin) was isolated after nine rounds of in vitro selection. The selection was performed on streptavidin-coated beads with the target bound to the surface and with free dye in solution in higher selection rounds to select for slower off-rate binding. Using next-generation sequencing (NGS), the libraries after the 4th and 9th rounds of selection were sequenced to identify enriched sequences. Several sequence families emerged, showing superior fluorescence enhancement and high affinity for the target compared to the other families obtained by NGS analysis. These sequence families were further studied to understand the binding interactions better. Primary sequence and secondary structure analysis tools were used to identify a hypothetical three-tiered G-quadruplex motif for these families. This indicates that the TO1-biotin DNA aptamer identified here uses a similar ligand-binding topology to the original Mango RNA aptamer.