Machine learning identifies cell-free DNA 5-hydroxymethylation biomarkers that detect occult colorectal cancer in PLCO Screening Trial subjects.

Background: Colorectal cancer (CRC) is a leading cause of cancer-related mortality, and CRC detection through screening improves survival rates. A promising avenue to improve patient screening compliance is the development of minimally-invasive liquid biopsy assays that target CRC biomarkers on circulating cell-free DNA (cfDNA) in peripheral plasma. In this report, we identify cfDNA biomarker candidate genes bearing the epigenetic mark 5-hydroxymethylcytosine (5hmC) that diagnose occult CRC up to 36 months prior to clinical diagnosis using the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial samples. Methods: Archived PLCO Trial plasma samples containing cfDNA were obtained from the National Cancer Institute (NCI) biorepositories. Study subjects included those who were diagnosed with CRC within 36 months of blood collection (i.e., case, n = 201) and those who were not diagnosed with any cancer during an average of 16.3 years of follow-up (i.e., controls, n = 402). Following the extraction of 3 - 8 ng cfDNA from less than 300 microliters plasma, we employed the sensitive 5hmC-Seal chemical labeling approach, followed by next-generation sequencing (NGS). We then conducted association studies and machine-learning modeling to analyze the genome-wide 5hmC profiles within training and validation groups that were randomly selected at a 2:1 ratio. Results: Despite the technical challenges associated with the PLCO samples (e.g., limited plasma volumes, low cfDNA amounts, and long archival times), robust genome-wide 5hmC profiles were successfully obtained from these samples. Association analyses using the Cox proportional hazards models suggested several epigenetic pathways relevant to CRC development distinguishing cases from controls. A weighted Cox model, comprised of 32-associated gene bodies, showed predictive detection value for CRC as early as 24-36 months prior to overt tumor presentation, and a trend for increased predictive power was observed for blood samples collected closer to CRC diagnosis. Notably, the 5hmC-based predictive model showed comparable performance regardless of sex and self-reported race/ethnicity, and significantly outperformed risk factors such as age and obesity according to BMI (body mass index). Additionally, further improvement of predictive performance was achieved by combining the 5hmC-based model and risk factors for CRC. Conclusions: An assay of 5hmC epigenetic signals on cfDNA revealed candidate biomarkers with the potential to predict CRC occurrence despite the absence of clinical symptoms or the availability of effective predictors. Developing a minimally-invasive clinical assay that detects 5hmC-modified biomarkers holds promise for improving early CRC detection and ultimately patient survival through higher compliance screening and earlier intervention. Future investigation to expand this strategy to prospectively collected samples is warranted.

5-Hydroxymethylcytosine signals in serum are a predictor of chemoresistance in high-grade serous ovarian cancer.

OBJECTIVE: The genome-wide profiling of 5-hydroxymethylcytosines (5hmC) on circulating cell-free DNA (cfDNA) has revealed promising biomarkers for various diseases. The purpose of this study was to investigate 5hmC signals in serum cfDNA and identify novel predictive biomarkers for the development of chemoresistance in high-grade serous ovarian cancer (HGSOC). We hypothesized that 5hmC profiles in cfDNA reflect the development of chemoresistance and elucidate pathways that may drive chemoresistance in HGSOC. Moreover, we sought to identify predictors that would better stratify outcomes for women with intermediate-sensitive HGSOC. METHODS: Women diagnosed with HGSOC and known platinum sensitivity status were selected for this study. Nano-hmC-Seal was performed on cfDNA isolated from archived serum samples, and differential 5hmC features were identified using DESeq2 to establish a model predictive of chemoresistance. RESULTS: A multivariate model consisting of three features (preoperative CA-125, largest residual implant after surgery, 5hmC level of OSGEPL), stratified samples from intermediate sensitive, chemo-naive women diagnosed with HGSOC into chemotherapy-resistant- and sensitive-like strata with a significant difference in overall survival (OS). Independent analysis of The Cancer Genome Atlas data further confirmed that high OSGEPL1 expression is a favorable prognostic factor for HGSOC. CONCLUSIONS: We have developed a novel multivariate model based on clinico-pathologic data and a cfDNA-derived 5hmC modified gene, OSGEPL1, that predicted response to platinum-based chemotherapy in intermediate-sensitive HGSOC. Our multivariate model applies to chemo-naïve samples regardless if the patint was treated with adjuvant or neoadjuvant chemotherapy. These results merit further investigation of the predictive capability of our model in larger cohorts.

Sequencing of N6-methyl-deoxyadenosine at single-base resolution across the mammalian genome.

Although DNA N6-methyl-deoxyadenosine (6mA) is abundant in bacteria and protists, its presence and function in mammalian genomes have been less clear. We present Direct-Read 6mA sequencing (DR-6mA-seq), an antibody-independent method, to measure 6mA at base resolution. DR-6mA-seq employs a unique mutation-based strategy to reveal 6mA sites as misincorporation signatures without any chemical or enzymatic modulation of 6mA. We validated DR-6mA-seq through the successful mapping of the well-characterized G(6mA)TC motif in the E. coli DNA. As expected, when applying DR-6mA-seq to mammalian systems, we found that genomic DNA (gDNA) 6mA abundance is generally low in most mammalian tissues and cells; however, we did observe distinct gDNA 6mA sites in mouse testis and glioblastoma cells. DR-6mA-seq provides an enabling tool to detect 6mA at single-base resolution for a comprehensive understanding of DNA 6mA in eukaryotes.

Analysis of genome-wide 5-hydroxymethylation of blood samples stored in different anticoagulants: opportunities for the expansion of clinical resources for epigenetic research.

BACKGROUND: Elucidating epigenetic mechanisms could provide new biomarkers for disease diagnosis and prognosis. Technological advances allow genome-wide profiling of 5-hydroxymethylcytosines (5hmC) in liquid biopsies. 5hmC-Seal followed by NGS is a highly sensitive technique for 5hmC biomarker discovery in cfDNA. Currently, 5hmC Seal is optimized for EDTA blood collection. We asked whether heparin was compatible with 5hmC Seal as many clinical and biobanked samples are stored in heparin. METHODS: We obtained 60 samples in EDTA matched to 60 samples in heparin from the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial. Samples were comprised of 30 controls and 30 individuals who were later diagnosed with colon cancer. We profiled genome-wide 5hmC in cfDNA using 5hmC-Seal assay followed by NGS. The 5hmC profiling data from samples collected in EDTA were systematically compared to those in heparin across various genomic features. RESULTS: cfDNA isolation and library construction appeared comparable in heparin vs. EDTA. Typical genomic distribution patterns of 5hmC, including gene bodies and enhancer markers, were comparable in heparin vs. EDTA. 5hmC analysis of cases and controls yielded highly correlated differential features suggesting that both anticoagulants were compatible with 5hmC Seal assay. CONCLUSIONS: While not currently recommended for the 5hmC-Seal protocol, blood samples stored in heparin were successfully used to generate analysable and biologically relevant genome-wide 5hmC profiling. Our findings are the first to support opportunities to expand the biospecimen resource to heparin samples for 5hmC Seal and perhaps other PCR-based technologies in epigenetic research.

A lncRNA from the FTO locus acts as a suppressor of the m6A writer complex and p53 tumor suppression signaling.

N6-methyladenosine (m6A) of mRNAs modulated by the METTL3-METTL14-WTAP-RBM15 methyltransferase complex and m6A demethylases such as FTO play important roles in regulating mRNA stability, splicing, and translation. Here, we demonstrate that FTO-IT1 long noncoding RNA (lncRNA) was upregulated and positively correlated with poor survival of patients with wild-type p53-expressing prostate cancer (PCa). m6A RIP-seq analysis revealed that FTO-IT1 knockout increased mRNA m6A methylation of a subset of p53 transcriptional target genes (e.g., FAS, TP53INP1, and SESN2) and induced PCa cell cycle arrest and apoptosis. We further showed that FTO-IT1 directly binds RBM15 and inhibits RBM15 binding, m6A methylation, and stability of p53 target mRNAs. Therapeutic depletion of FTO-IT1 restored mRNA m6A level and expression of p53 target genes and inhibited PCa growth in mice. Our study identifies FTO-IT1 lncRNA as a bona fide suppressor of the m6A methyltransferase complex and p53 tumor suppression signaling and nominates FTO-IT1 as a potential therapeutic target of cancer.

PETCH-DB: a Portal for Exploring Tissue-specific and Complex disease-associated 5-Hydroxymethylcytosines.

Epigenetic modifications play critical roles in gene regulation and disease pathobiology. Highly sensitive enabling technologies, including microarray- and sequencing-based approaches have allowed genome-wide profiling of cytosine modifications in DNAs in clinical samples to facilitate discovery of epigenetic biomarkers for disease diagnosis and prognosis. Historically, many previous studies, however, did not distinguish the most investigated 5-methylcytosines (5mC) from other modified cytosines, especially the biochemically stable 5-hydroxymethylcytosines (5hmC), which have been shown to have a distinct genomic distribution and regulatory role from 5mC. Notably, during the past several years, the 5hmC-Seal, a highly sensitive chemical labeling technique, has been demonstrated to be a powerful tool for genome-wide profiling of 5hmC in clinically feasible biospecimens (e.g. a few milliliter of plasma or serum). The 5hmC-Seal technique has been utilized by our team in biomarker discovery for human cancers and other complex diseases using circulating cell-free DNA (cfDNA), as well as the characterization of the first 5hmC Human Tissue Map. Convenient access to the accumulating 5hmC-Seal data will allow the research community to validate and re-use these results, potentially providing novel insights into epigenetic contribution to a range of human diseases. Here we introduce the PETCH-DB, an integrated database that was implemented to provide 5hmC-related results generated using the 5hmC-Seal technique. We aim the PETCH-DB to be a central portal, which will be available to the scientific community with regularly updated 5hmC data in clinical samples to reflect current advances in this field. Database URL http://petch-db.org/.

PETCH-DB: a Portal for Exploring Tissue-specific and Complex disease-associated 5-Hydroxymethylcytosines.

Epigenetic modifications play critical roles in gene regulation and disease pathobiology. Highly sensitive enabling technologies, including microarray- and sequencing-based approaches have allowed genome-wide profiling of cytosine modifications in DNAs in clinical samples to facilitate discovery of epigenetic biomarkers for disease diagnosis and prognosis. Historically, many previous studies, however, did not distinguish the most investigated 5-methylcytosines (5mC) from other modified cytosines, especially the biochemically stable 5-hydroxymethylcytosines (5hmC), which have been shown to have a distinct genomic distribution and regulatory role from 5mC. Notably, during the past several years, the 5hmC-Seal, a highly sensitive chemical labeling technique, has been demonstrated to be a powerful tool for genome-wide profiling of 5hmC in clinically feasible biospecimens (e.g. a few milliliter of plasma or serum). The 5hmC-Seal technique has been utilized by our team in biomarker discovery for human cancers and other complex diseases using circulating cell-free DNA (cfDNA), as well as the characterization of the first 5hmC Human Tissue Map. Convenient access to the accumulating 5hmC-Seal data will allow the research community to validate and re-use these results, potentially providing novel insights into epigenetic contribution to a range of human diseases. Here we introduce the PETCH-DB, an integrated database that was implemented to provide 5hmC-related results generated using the 5hmC-Seal technique. We aim the PETCH-DB to be a central portal, which will be available to the scientific community with regularly updated 5hmC data in clinical samples to reflect current advances in this field. Database URL http://petch-db.org/.

Azospirillum Aestuarii sp. nov., a Novel Nitrogen-Fixting and Aerobic Denitrifying Bacteria Isolated from an Estuary of a Freshwater River.

A novel Gram-staining negative, aerobic, rod-shaped bacterium, designated strain YIM DDC1T, was isolated from an estuary sediment sample of Dongda River flowing into Dianchi lake in Yunnan, southwest China. The strain displayed growth at 10-40 °C (optimum of 28 °C), pH 5.0-9.0 (optimum of 7.0-8.0) and in presence of 0-3% (w/v) NaCl (optimum of 0-1%). Strain YIM DDC1T comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and two unidentified aminolipids as the predominant polar lipids; the ubiquinone 10 as the major respiratory quinone; and summed feature 8 (C18:1ω6c and/or C18:1ω7c), summed feature 3 (C16:1ω7c and/or C16:1ω6c) and C18:1 2-OH as the major cellular fatty acids. Analysis of 16S rRNA showed that YIM DDC1T represents a member of the genus Azospirillum, and was closely related to A. brasilense ATCC 29145 T (98.9%), A. baldaniorum Sp245T (98.2%), A. argentinense Az39T (98.2%) and A. formosense CC-Nfb-7 T (98.2%). The draft genome size was 7.15 Mbp with a 68.4% G + C content. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain YIM DDC1T and the aforementioned closely related strains exhibited similarity in the range of 93.8-93.5% and 53.7-52.7%, respectively. nif gene cluster (nifHDK) and denitrification genes ((napA, nirS, nirK, norBC and nosZ) detected in the genome indicated its potential nitrogen fixation and full-fledged denitrifying function. Based on combined genotypic and phenotypic data, strain YIM DDC1T represents a novel species of the genus Azospirillum, for which the name Azospirillum aestuarii sp. nov. is proposed. The type strain is YIM DDC1T (= KCTC 42887 T = CGMCC 1.17325 T).

Diagnostic and Prognostic Value of Serum miR-296-5p and miR-28-3p in Human Gastric Cancer.

Background: Previous studies reported the use of microRNAs (miRNAs) as diagnostic and/or prognostic biomarkers for various cancers, including gastric cancer (GC). This study evaluated the diagnostic and prognostic significance of serum miR-296-5p and miR-28-3p in GC. Materials and Methods: Serum samples of 90 patients with GC and 90 healthy individuals, and 20 pairs of tissue specimens from patients with GC were collected. The expression of miR-296-5p and miR-28-3p in both the serum and tissue samples were detected using quantitative real-time polymerase chain reaction analysis. The diagnostic and prognostic values of miR-296-5p and miR-28-3p were evaluated by using receiver operating characteristic curve and Kaplan-Meier analyses, respectively. Results: Compared with the healthy controls, the expression of miR-296-5p in the serum and tissues of patients with GC was significantly upregulated, whereas that of miR-28-3p was significantly downregulated. High miR-296-5p and low miR-28-3p levels in the serum significantly correlated with larger tumor size (>5 cm), lymph node metastasis, and TNM stage III+IV. The area under the curve values of miR-296-5p and miR-28-3p were 0.919 and 0.911, respectively, with high sensitivity and specificity. Kaplan-Meier survival curves showed that patients with GC with high level of miR-296-5p or low level of miR-1236-3p in the serum had the poorest overall survival. COX analysis showed that lymphatic metastasis, high miR-296-5p expression, and low miR-28-3p expression are independent parameters indicating poor prognosis in GC. Conclusion: Our findings indicate that serum miR-296-5p and miR-28-3p levels are potential biomarkers in the diagnosis and prognosis of GC.

Characterization and genomic analysis of a novel halovirus infecting Chromohalobacter beijerinckii.

Bacteriophages function as a regulator of host communities and metabolism. Many phages have been isolated and sequenced in environments such as the ocean, but very little is known about hypersaline environments. Phages infecting members of the genus Chromohalobacter remain poorly understood, and no Chromohalobacter phage genome has been reported. In this study, a halovirus infecting Chromohalobacter sp. F3, YPCBV-1, was isolated from Yipinglang salt mine. YPCBV-1 could only infect host strain F3 with burst size of 6.3 PFU/cell. It could produce progeny in 5%-20% (w/v) NaCl with an optimal concentration of 10% (w/v), but the optimal adsorption NaCl concentration was 5%-8% (w/v). YPCBV-1 is sensitive to pure water and depends on NaCl or KCl solutions to survive. YPCBV-1 stability increased with increasing salinity but decreased in NaCl saturated solutions, and it has a broader salinity adaptation than the host. YPCBV-1 has a double-stranded DNA of 36,002 bp with a G + C content of 67.09% and contains a total of 55 predicted ORFs and no tRNA genes. Phylogenetic analysis and genomic network analysis suggested that YPCBV-1 is a novel Mu-like phage under the class Caudoviricetes. Auxiliary metabolic gene, SUMF1/EgtB/PvdO family non-heme iron enzyme, with possible roles in antioxidant was found in YPCBV-1. Moreover, DGR-associated genes were predicted in YPCBV-1 genome, which potentially produce hypervariable phage tail fiber. These findings shed light on the halovirus-host interaction in hypersaline environments.

METTL4-mediated nuclear N6-deoxyadenosine methylation promotes metastasis through activating multiple metastasis-inducing targets.

BACKGROUND: DNA N6-methyldeoxyadenosine (6mA) is rarely present in mammalian cells and its nuclear role remains elusive. RESULTS: Here we show that hypoxia induces nuclear 6mA modification through a DNA methyltransferase, METTL4, in hypoxia-induced epithelial-mesenchymal transition (EMT) and tumor metastasis. Co-expression of METTL4 and 6mA represents a prognosis marker for upper tract urothelial cancer patients. By RNA sequencing and 6mA chromatin immunoprecipitation-exonuclease digestion followed by sequencing, we identify lncRNA RP11-390F4.3 and one novel HIF-1α co-activator, ZMIZ1, that are co-regulated by hypoxia and METTL4. Other genes involved in hypoxia-mediated phenotypes are also regulated by 6mA modification. Quantitative chromatin isolation by RNA purification assay shows the occupancy of lncRNA RP11-390F4.3 on the promoters of multiple EMT regulators, indicating lncRNA-chromatin interaction. Knockdown of lncRNA RP11-390F4.3 abolishes METTL4-mediated tumor metastasis. We demonstrate that ZMIZ1 is an essential co-activator of HIF-1α. CONCLUSIONS: We show that hypoxia results in enriched 6mA levels in mammalian tumor cells through METTL4. This METTL4-mediated nuclear 6mA deposition induces tumor metastasis through activating multiple metastasis-inducing genes. METTL4 is characterized as a potential therapeutic target in hypoxic tumors.

First Report of Anthracnose Crown Rot on Strawberry (Fragaria × ananassa Duch.) Caused by Colletotrichum pandanicola in Yunnan Provence, China.

Strawberry (Fragaria × ananassa Duch.), a widely grown octoploid species, is one of the most important economic fruit crops and has been widely cultivated in the world, including China. In December 2021, a serious crown rot disease (approximately 50% incidence) was observed in strawberry (cultivar Miaoxiang) plantations in Qujing City, Yunnan Province, China. Symptoms observed on aboveground part withered rapidly, reddish-brown marbled necrosis on crown. The roots were healthy and strong, but the plants finally died. To isolate the causal agent of this disease, crown tissues from five strawberry plants showing typical symptoms were cut into pieces of 5×5 mm, and the pieces were surface-sterilized with 75% ethanol for 45 s followed by 2.5% NaClO for 3 min and rinsed thrice with sterile water, and then placed onto potato dextrose agar (PDA) for 7 days at 25 ºC. After 3 to 4 days, extended single hyphal tips from the tissues were transferred to PDA and incubated for 7 days at 25 ºC. The colonies were initially white, later became somewhat zonate, velvety, cyan gray on the upper side and cyan ink pigment ring on the reverse side of plates, with concentric rings of salmon sporodochia. Many yellowish or orange creamy conidial droplets formed on PDA after 14 days at 25 ºC. Fifty-nine isolates were obtained, and three isolates QLYRR1, QLMCR9, and QLMCR39 were selected for further experiments. Conidia were hyaline, cylindrical with rounded ends, 12.17-19.35×3.71-6.30 µm (average±SD, 15.24±1.37×5.09±0.45 µm, n=150), L/W ratio = 2.99. The three isolates were molecularly identified using the genomic regions of internal transcribed spacer (ITS), actin (ACT), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin (TUB2) genes, and the sequences were deposited in GenBank (accession nos. QLYRR1, QLMCR9, QLMCR39: ON668272, ON668256, ON668257[ITS], ON684302, ON684300, ON684301[ACT], ON684316, ON684314, ON684315[CHS-1], ON684292, ON684290, ON684291[GAPDH], ON684286, ON684284, ON684285[TUB2]). The phylogenetic analysis of experimental strains was performed by Maximum-likelihood (ML) tree and Bayesian inference (BI) method. Nucleotide sequences exhibited three isolates were clustered with the ex-type strain C. pandanicola strain MFLUCC 170571T found in Thailand, C. pandanicola strains (SAUCC201152, SAUCC200204) found in Shandong Province, and the holotype stain C. parvisporum YMF 1.06942T found in Guangxi Province, China. Morphologically, isolates were easily distinguished from C. parvisporum by the colony on PDA and the size of conidia (Yu et al. 2022). Morphological characteristics and phylogenetic analyses revealed that QLYRR1, QLMCR9, and QLMCR39 belong to C. pandanicola, the members of the C. gloeosporioides species complex (Tibpromma et al. 2018; Mu et al. 2021). Koch's postulates were tested by strawberry plants (two cultivars, Akihime and Miaoxiang) in vivo, strawberry plants were tested for the three isolates by spraying 1×106 conidia/mL suspension on three seedlings. Three seedlings sprayed with sterile distilled water were served as control. All of the plants were transferred to a glasshouse with a 28/20 °C day/night temperature range and natural sunlight. After 6 weeks, QLYRR1-, QLMCR9-, and QLMCR39-sprayed seedlings were stunted and developed typical wilt symptoms similar to those observed in the field with the incidence for 3, 3, and 3 seedlings, respectively. The negative control remained asymptomatic. The fungi were reisolated again from lesions of diseased plants and leaves with 100% frequency, and morphological characteristics and tested gene sequences were identical to the original isolates in this note, thus fulfilling Koch's postulates. C. pandanicola was described from the healthy leaves of Pandanus sp. and the lesion fruits of Juglans regia. To our knowledge, this is the first report confirming C. pandanicola causes anthracnose crown rot on strawberries in China. C. pandanicola has the potential for causing serious losses to the strawberry industry, and research is needed on management strategies to minimize losses.

Low expression of lncRNA APTR promotes gastric cancer progression.

BACKGROUND: Gastric cancer (GC) is one of the most common cancers worldwide and the majority of GC patients are diagnosed at advanced stages due to the lack of early detection biomarkers. LncRNAs have been shown to play important roles in various diseases and could be predictive biomarkers and therapeutic targets. Our study demonstrated that low expression of lncRNA APTR could promote gastric cancer progression. METHODS: Differentiated expressed lncRNAs were identified through analyzing TCGA paired GC RNA sequencing data. LncRNA APTR's clinical relevance was analyzed using the TCGA dataset and GEO datasets. APTR expression in patient samples was detected through qPCR. The proliferation, colony formation, and migration of GC cells were tested. Bioinformatic analyses were performed to explore APTR-affected signaling pathways in GC. RESULTS: LncRNA APTR is lower expressed in gastric tumor samples and low expression of APTR predicts a poor diagnosis and outcome in GC patients. Silencing APTR promotes gastric cancer proliferation and invasiveness. APTR expression is negatively correlated with inflammatory signaling in the gastric tumor microenvironment. CONCLUSION: Our study showed that low expression of lncRNA APTR in gastric cancer is correlated with tumorigenesis and poor diagnosis and prognosis, which is a potential biomarker for gastric cancer patients' diagnosis and treatment.

Gene Mutational Clusters in the Tumors of Colorectal Cancer Patients With a Family History of Cancer.

Introduction: Family history is a high-risk factor for colorectal cancer (CRC). The risk comes not only from known germline mutations but also from the other family-related mechanisms. Uncovering them would be an important step to improve the diagnosis and treatment of these patients. Method: Samples from 168 patients with advanced CRC were collected and applied to next-generation sequencing of 624 pan-cancer genes. Genomic mutations and significantly mutated genes were identified. Significantly mutated genes and co-mutated genes were used to cluster patients. For each cluster of patients, mutational signatures were extracted. The identified mutational signatures were further validated in the other independent cohort. Result: Significantly mutated genes including TP53, APC, KRAS, and SMAD4 were found associated with tumor mutational burden and microsatellite instability. LRP1, ACVR2A, and SETBP1 were found co-mutated. Patients with mutations in LRP1, ACVR2A, and SETBP1 tend to have a family history of cancer. Those patients tended to have right-sided tumors with high tumor mutational burden and microsatellite instability. Among them, signature analysis identified two possible etiologies, SBS10a (defective polymerase epsilon exonuclease domain) and SBS6 (defective DNA mismatch repair and microsatellite unstable tumors). These signatures were also found in another independent cohort. Conclusion: The gene cluster (LRP1, ACVR2A, and SETBP1) could be a good biomarker of these patients with a family risk, which was characterized by right-sidedness, high tumor mutational burden, and high microsatellite instability.

Comparative Analysis of the Microbial Community Structures Between Healthy and Anthracnose-Infected Strawberry Rhizosphere Soils Using Illumina Sequencing Technology in Yunnan Province, Southwest of China.

Anthracnose caused by Colletotrichum spp. was widespread in recent years and resulted in great damage to strawberry production. Soil microbial communities were key contributors to host nutrition, development, and immunity; however, the difference between the microbial communities of healthy and anthracnose-infected strawberry rhizosphere soils remains unclear. In this study, the Illumina sequencing technique was used to comparatively study the prokaryotic and fungal community compositions and structures between healthy and anthracnose-infected strawberry rhizosphere soils in Yuxi, Yunnan Province. Both microbial community diversities and richness of anthracnose-infected strawberry rhizosphere soils were higher than those of healthy strawberry rhizosphere soils. A total of 2,518 prokaryotic and 556 fungal operational taxonomic units (OTUs) were obtained at the 97% similarity threshold. Proteobacteria, Thaumarchaeota, and Acidobacteria were the dominant prokaryotic phyla; Ascomycota, unclassified_k__Fungi, and Mortierellomycota were the dominant fungal phyla. The relative abundances of beneficial bacterial phyla Actinobacteria and Firmicutes, genera Streptomyces, Azospirillum, and Bacillus were significantly reduced in anthracnose-infected strawberry rhizosphere soils; the relative abundance of beneficial fungal species Trichoderma asperellum shows a similar tendency with bacterial abundance. Besides Colletotrichum, 15 other potential fungal pathogen genera and seven fungal pathogen species were identified; among the potential pathogen genera and species, eight pathogen genera and Fusarium oxysporum showed significant differences between healthy and anthracnose-infected strawberry rhizosphere soils. The results suggested that strawberry planted in this area may be infected by other fungal pathogens except for Colletotrichum spp. Our present research will provide theoretical basis and data reference for the isolation and identification of strawberry pathogens and potential probiotics in future works.

FTO mediates LINE1 m6A demethylation and chromatin regulation in mESCs and mouse development.

N6-methyladenosine (m6A) is the most abundant internal modification on mammalian messenger RNA. It is installed by a writer complex and can be reversed by erasers such as the fat mass and obesity-associated protein FTO. Despite extensive research, the primary physiological substrates of FTO in mammalian tissues and development remain elusive. Here, we show that FTO mediates m6A demethylation of long-interspersed element-1 (LINE1) RNA in mouse embryonic stem cells (mESCs), regulating LINE1 RNA abundance and the local chromatin state, which in turn modulates the transcription of LINE1-containing genes. FTO-mediated LINE1 RNA m6A demethylation also plays regulatory roles in shaping chromatin state and gene expression during mouse oocyte and embryonic development. Our results suggest broad effects of LINE1 RNA m6A demethylation by FTO in mammals.

Nutrient levels and prokaryotes affect viral communities in plateau lakes.

Viruses are the most abundant organisms in aquatic environments. Recent advances of viral metagenomic have greatly expanded our understanding of aquatic viral communities. However, little is known about the difference of viral communities and driving factors in freshwater lake. This study seeks to understand the spatio-temporal variation, differences, and driving factors of viral communities in two plateau lakes (Dianchi and Fuxian Lakes) with significant nutritional differences. The viral communities exhibited apparent seasonal variation in Dianchi Lake, while seasonal influences on the viral communities were greater than location-based influences. Two-thirds of all detected viral taxa were shared in two lakes, but there was variation in the composition of viral communities. Correlations between prokaryotic communities, environmental factors and viral communities were analyzed. The nutrients, chlorophyll a were primarily environmental parameters affecting viral communities, and the prokaryotic community was significantly correlated with the viral community. In addition, several viruses infecting humans were identified in two lakes, with the most abundant being Herpesviridae and Poxviridae. Overall, these findings provide information on the dynamics, composition, and differences of viral and prokaryotic communities in plateau lakes with different nutrient levels. These results suggest that nutritional levels and prokaryotic communities could play an important role in shaping viral communities in freshwater lakes.

The METTL5-TRMT112 N6-methyladenosine methyltransferase complex regulates mRNA translation via 18S rRNA methylation.

Ribosomal RNAs (rRNAs) have long been known to carry chemical modifications, including 2'O-methylation, pseudouridylation, N6-methyladenosine (m6A), and N6,6-dimethyladenosine. While the functions of many of these modifications are unclear, some are highly conserved and occur in regions of the ribosome critical for mRNA decoding. Both 28S rRNA and 18S rRNA carry single m6A sites, and while the methyltransferase ZCCHC4 has been identified as the enzyme responsible for the 28S rRNA m6A modification, the methyltransferase responsible for the 18S rRNA m6A modification has remained unclear. Here, we show that the METTL5-TRMT112 methyltransferase complex installs the m6A modification at position 1832 of human 18S rRNA. Our work supports findings that TRMT112 is required for METTL5 stability and reveals that human METTL5 mutations associated with microcephaly and intellectual disability disrupt this interaction. We show that loss of METTL5 in human cancer cell lines and in mice regulates gene expression at the translational level; additionally, Mettl5 knockout mice display reduced body size and evidence of metabolic defects. While recent work has focused heavily on m6A modifications in mRNA and their roles in mRNA processing and translation, we demonstrate here that deorphanizing putative methyltransferase enzymes can reveal previously unappreciated regulatory roles for m6A in noncoding RNAs.

Pleckstrin 2 is a potential drug target for colorectal carcinoma with activation of APC/ß­catenin.

The tumor suppressor gene adenomatous polyposis coli (APC) is frequently inactivated or absent in colorectal carcinoma (CRC). Loss­of­function of APC promotes the expression of ß­catenin, which is critical for CRC development. Since ß­catenin acts as an important transcription factor, blockage of ß­catenin may have side effects, including impairment of tissue homeostasis and regeneration, thus limiting the application of ß­catenin inhibitors for the treatment of patients with CRC. Therefore, identifying a novel substrate of APC/ß­catenin may provide essential clues to develop effective drugs. Small interfering RNA technology and lentivirus­mediated overexpression were performed for knockdown and overexpression of pleckstrin 2 (PLEK2) in CRC cells. Cell Counting Kit­8 and colony formation assays, and cell cycle analysis and cell apoptosis detection were used to detect the capacity of cell proliferation, cell cycle distribution and apoptosis. The present study demonstrated that the APC/ß­catenin signaling cascade transcriptionally activated PLEK2 in CRC cells. PLEK2 expression was markedly increased in CRC tissues. There was an inverse correlation between APC and PLEK2 expression in patients with CRC. In vitro, overexpression of PLEK2 increased the proliferation of CRC cells. Opposite results were observed in the cells with knockdown of PLEK2. Furthermore, PLEK2 promoted cell cycle progression and suppressed apoptosis. In summary, upregulation of PLEK2 contributed to CRC proliferation and colony formation activated by the APC/ß­catenin signal pathway. Targeting PLEK2 may be important for the treatment of patients with CRC with activation of the APC/ß­catenin signaling pathway.

Carcinoma-associated fibroblasts promote the proliferation and metastasis of osteosarcoma by transferring exosomal LncRNA SNHG17.

Cancer-associated fibroblasts (CAFs) serve as a predominant regulator in the tumor microenvironment. However, the crosstalk between CAFs and OS cells remains mostly unclear. Recent studies explored that long non-coding RNA (LncRNAs) involved in regulating osteosarcoma (OS) formation and development, but their functions in CAFs are unknown. Here, we first investigated the SNHG17 was upregulated in OS tissues and correlated with the poor prognosis through the integrating clinical data. We then evaluated the function of SNHG17 in vitro using the stable SNHG17-depleted OS cells. HOS cells with SNHG17 knocked down were performed to generate the OS xenograft model. Through immunohistochemistry assay and TUNEL apoptosis assay, the role of SNHG17 on OS development was assessed in vivo. We then examined the SNHG17 expression in exosomes derived from CAFs, normal fibroblasts (NFs), and tumor tissues from the OS clinical samples. The interaction among SNHG17, miR-2861, and MMP2 was predicted by bioinformatics analysis and identified by RIP and luciferase assays. The cell proliferation, migration, and apoptosis of SJSA-1 and HOS cells co-cultured with CAFs-derived exosomes were assessed by CCK-8 and colony formation assays. We found that SNHG17 was upregulated in the tumor tissues and presented a pro-tumorigenic effect on OS both in vitro and in vivo. It also was an essential exosomal cargo of CAFs and could affect OS cell proliferation and migration in vitro. CAFs-released exosomal SNHG17 acted as an essential molecular sponge for miR-2861 in OS cells. Moreover, MMP2 was a direct target of miR-2861 and was regulated by SNHG17. Overall, our findings identified that SNHG17 was an essential exosomal cargo of OS-related CAFs that contributes to proliferation and metastasis of OS, supporting the therapeutic potency of targeting the crosstalk between cancer cells and CAFs.