PacBio sequencing of human fecal samples uncovers the DNA methylation landscape of 22 673 gut phages.

Gut phages have an important impact on human health. Methylation plays key roles in DNA recognition, gene expression regulation and replication for phages. However, the DNA methylation landscape of gut phages is largely unknown. Here, with PacBio sequencing (2120×, 4785 Gb), we detected gut phage methylation landscape based on 22 673 gut phage genomes, and presented diverse methylation motifs and methylation differences in genomic elements. Moreover, the methylation rate of phages was associated with taxonomy and host, and N6-methyladenine methylation rate was higher in temperate phages than in virulent phages, suggesting an important role for methylation in phage-host interaction. In particular, 3543 (15.63%) phage genomes contained restriction-modification system, which could aid in evading clearance by the host. This study revealed the DNA methylation landscape of gut phage and its potential roles, which will advance the understanding of gut phage survival and human health.

Systematic review and meta-analysis of the effects of exercise on cognitive impairment and neuroprotective mechanisms in diabetes mellitus animal models.

This study aims to assess the effects of exercise on cognitive impairment behavioral performance and neuroprotective mechanisms in diabetes mellitus (DM) animal models. PubMed, Embase, Web of Science, China National Knowledge Infrastructure (CNKI), Wanfang Database, VIP Database (VIP), and China Biomedical Literature Database (CBM) were systematically searched for studies investigating the impact of exercise on cognitive impairment in animal models of diabetes mellitus (DM) from the inception of these databases through July 2023. Rigorous quality assessments were conducted on the included literature. Primary outcome measures comprised fasting blood glucose (FBG) levels and performance in the Morris water maze test, while secondary outcomes focused on mechanisms related to neuroprotection. Statistical analysis of outcome data was conducted using RevMan 5.3 and R software. A total of 17 studies were included, encompassing 399 animals. The results of the meta-analysis of primary outcome measures revealed that, compared to the control group, exercise effectively reduced fasting blood glucose (FBG) levels in diabetic animal models. In the Morris water maze experiment, exercise also significantly decreased the escape latency of diabetic animal models, increased the number of platform crossings, improved the percentage of time spent in the target quadrant, extended the time spent in the target quadrant, and enhanced swimming speed. Meta-analysis of secondary outcome measures indicated that exercise effectively reduced Aß deposition, attenuated oxidative stress, enhanced synaptic function, suppressed cellular apoptosis and neuroinflammation, and promoted neurogenesis. Exercise represents a promising non-pharmacological therapy with a positive impact on diabetes-related cognitive function and neuroprotection. Moreover, this study provides a theoretical foundation for further preclinical and clinical trials.

Uncovering 1058 Novel Human Enteric DNA Viruses Through Deep Long-Read Third-Generation Sequencing and Their Clinical Impact.

BACKGROUND & AIMS: Lack of viral reference genomes poses a challenge to virome study. We investigated human gut virome and its clinical implication by ultra-deep metagenomic sequencing. METHODS: We extracted sufficient viral DNA from human feces for ultra-deep PacBio sequencing (>10 µg) and Illumina sequencing (>1 µg). Upon de novo assembly and 6 stages of strict filtering, viral genomes were generated and validated in 3 cohorts of 2819 published fecal metagenomes. Diagnostic performance of assembled viruses for colorectal cancer were tested in a training cohort and 2 independent validation cohorts. Virus mapping ratio, evolutionary history, and virus status (lytic or temperate) were also examined. RESULTS: The mean amount of extracted viral DNA increased by 14-fold compared with previous protocols. We obtained PacBio long reads and Illumina short reads with 290-fold higher depth than previous studies. We assembled and validated 1178 contigs as complete viral genomes, of which 1058 were newly identified. Thirteen viral genomes (398-839 kb) that are longer than the largest bacteriophage found in humans (393 kb) were discovered. Phylogenetic tree was constructed based on Hidden Markov Models alignment scores of 4 conserved viral proteins. Incorporating our assembled genomes into the National Center for Biotechnology Information database improved the mapping ratio of published metagenomes ≤18 times. Lytic viruses (75.9% ± 12.2% of total) were predominantly present in our sample. A biomarker panel of 14 novel viruses could discriminate patients with colorectal cancer from controls with an area under the receiver operating characteristics curve of 0.87 in the training cohort, which was validated with areas under the receiver operating characteristics curve of 0.85 and 0.73 in 2 independent cohorts. CONCLUSIONS: We uncovered 1058 novel human gut viruses. These findings can contribute to clinical diagnosis, current viral reference genome, and future virome investigation.

Life satisfaction and its influencing factors for bedridden patients with stroke.

OBJECTIVES: This study aimed to evaluate the life satisfaction of bedridden patients with stroke and explore its relationship with demographic, social, and medical factors. MATERIAL AND METHODS: This multicenter cross-sectional study was conducted in two steps. The Longshi scale was used to select the study population and assess patients' ability to perform activities of daily living. Subsequently, a multidimensional questionnaire was used to obtain the participants' information and evaluate their level of life satisfaction. The chi-squared test and binary logistic regression methods were employed to analyze the factors influencing the life satisfaction of bedridden patients with stroke. RESULTS: A total of 3,639 bedridden patients with stroke were included in this study, of them, only 27.2% reported satisfaction with their current lives. Factors associated with higher life satisfaction include female sex, older age, and primary school education or lower (P<0.05). Patients who had experienced a single stroke episode had chronic diseases, and rated their health as good were more satisfied with their lives than those who did not. The results of the binary logistic regression confirmed that age, education, religion, household income, cohabitation, social participation, number of chronic diseases, self-rated health status, and disability level significantly influenced the life satisfaction of bedridden patients with stroke (P<0.05). CONCLUSION: Our study showed that the overall life satisfaction of bedridden patients with stroke was low, with several factors influencing their life satisfaction. Therefore, effective measures should be implemented to improve life satisfaction and quality of life.

Cigarette smoke promotes colorectal cancer through modulation of gut microbiota and related metabolites.

OBJECTIVE: Cigarette smoking is a major risk factor for colorectal cancer (CRC). We aimed to investigate whether cigarette smoke promotes CRC by altering the gut microbiota and related metabolites. DESIGN: Azoxymethane-treated C57BL/6 mice were exposed to cigarette smoke or clean air 2 hours per day for 28 weeks. Shotgun metagenomic sequencing and liquid chromatography mass spectrometry were parallelly performed on mice stools to investigate alterations in microbiota and metabolites. Germ-free mice were transplanted with stools from smoke-exposed and smoke-free control mice. RESULTS: Mice exposed to cigarette smoke had significantly increased tumour incidence and cellular proliferation compared with smoke-free control mice. Gut microbial dysbiosis was observed in smoke-exposed mice with significant differential abundance of bacterial species including the enrichment of Eggerthella lenta and depletion of Parabacteroides distasonis and Lactobacillus spp. Metabolomic analysis showed increased bile acid metabolites, especially taurodeoxycholic acid (TDCA) in the colon of smoke-exposed mice. We found that E. lenta had the most positive correlation with TDCA in smoke-exposed mice. Moreover, smoke-exposed mice manifested enhanced oncogenic MAPK/ERK (mitogen-activated protein kinase/extracellular signal­regulated protein kinase 1/2) signalling (a downstream target of TDCA) and impaired gut barrier function. Furthermore, germ-free mice transplanted with stools from smoke-exposed mice (GF-AOMS) had increased colonocyte proliferation. Similarly, GF-AOMS showed increased abundances of gut E. lenta and TDCA, activated MAPK/ERK pathway and impaired gut barrier in colonic epithelium. CONCLUSION: The gut microbiota dysbiosis induced by cigarette smoke plays a protumourigenic role in CRC. The smoke-induced gut microbiota dysbiosis altered gut metabolites and impaired gut barrier function, which could activate oncogenic MAPK/ERK signalling in colonic epithelium.

Microbial Community Heterogeneity Within Colorectal Neoplasia and its Correlation With Colorectal Carcinogenesis.

BACKGROUND & AIMS: Gut microbial dysbiosis has pivotal involvement in colorectal cancer (CRC). However, the intratumoral microbiota and its association with CRC progression remain elusive. We aimed to determine the microbial community architecture within a neoplasia (CRC or adenoma) and its contribution to colorectal carcinogenesis. METHODS: We collected 436 tissue biopsies from patients with CRC (n = 36) or adenoma (n = 32) (2-6 biopsies from a neoplasia plus 2-5 biopsies from adjacent normal tissues per individual). Microbial profiling was performed using 16S ribosomal RNA gene sequencing with subsequent investigation of microbiota diversities and heterogeneity. The correlation between microbial dysbiosis and host genetic alterations (KRAS mutation and microsatellite instability) in all neoplasia biopsies was also analyzed. RESULTS: We discovered that intra-neoplasia microbial communities are heterogeneous. Abundances of some CRC-associated pathobionts (eg, Fusobacterium, Bacteroides, Parvimonas, and Prevotella) were found to be highly varied within a single neoplasia. Correlation of such heterogeneity with CRC development revealed alterations in microbial communities involving microbes with high intra-neoplasia variation in abundance. Moreover, we found that the intra-neoplasia variation in abundance of individual microbes changed along the adenoma-carcinoma sequence. We further determined that there was a significant difference in intra-neoplasia microbiota between biopsies with and without KRAS mutation (P < .001) or microsatellite instability (P < .001), and illustrated the association of intratumoral microbial heterogeneity with genetic alteration. CONCLUSIONS: We demonstrated that intra-neoplasia microbiota is heterogeneous and correlated with colorectal carcinogenesis. Our findings provide new insights on the contribution of gut microbiota heterogeneity to CRC progression.

Streptococcus thermophilus Inhibits Colorectal Tumorigenesis Through Secreting ß-Galactosidase.

BACKGROUND & AIMS: Streptococcus thermophilus was identified to be depleted in patients with colorectal cancer (CRC) by shotgun metagenomic sequencing of 526 multicohort fecal samples. Here, we aim to investigate whether this bacterium could act as a prophylactic for CRC prevention. METHODS: The antitumor effects of S thermophilus were assessed in cultured colonic epithelial cells and in 2 murine models of intestinal tumorigenesis. The tumor-suppressive protein produced by S thermophilus was identified by mass spectrometry and followed by ß-galactosidase activity assay. The mutant strain of S thermophilus was constructed by homologous recombination. The effect of S thermophilus on the gut microbiota composition was assessed by shotgun metagenomic sequencing. RESULTS: Oral gavage of S thermophilus significantly reduced tumor formation in both Apcmin/+ and azoxymethane-injected mice. Coincubation with S thermophilus or its conditioned medium decreased the proliferation of cultured CRC cells. ß-Galactosidase was identified as the critical protein produced by S thermophilus by mass spectrometry screening and ß-galactosidase activity assay. ß-Galactosidase secreted by S thermophilus inhibited cell proliferation, lowered colony formation, induced cell cycle arrest, and promoted apoptosis of cultured CRC cells and retarded the growth of CRC xenograft. The mutant S thermophilus without functional ß-galactosidase lost its tumor-suppressive effect. Also, S thermophilus increased the gut abundance of known probiotics, including Bifidobacterium and Lactobacillus via ß-galactosidase. ß-Galactosidase-dependent production of galactose interfered with energy homeostasis to activate oxidative phosphorylation and downregulate the Hippo pathway kinases, which partially mediated the anticancer effects of S thermophilus. CONCLUSION: S thermophilus is a novel prophylactic for CRC prevention in mice. The tumor-suppressive effect of S thermophilus is mediated at least by the secretion of ß-galactosidase.

Thermo-Induced Coalescence of Dual Cores in Double Emulsions for Single-Cell RT-PCR.

Single-cell reverse-transcription polymerase chain reaction (RT-PCR) has shown significant promise for transcriptional profiling of heterogeneous cells. However, currently developed microfluidic droplet-based methodologies for single-cell RT-PCR often require complex chip design to accommodate the associated multistep processes as well as customized detection platforms for high-throughput analysis. Herein, we proposed a dual-core double emulsion (DE)-based method to streamline the single-cell RT-PCR through thermo-induced coalescence of the dual cores. The dual-core DEs were produced by pairing two water-in-oil single emulsions containing a single-cell/lysis buffer and RT-PCR mix, respectively. After complete lysis of single cells in one of the cores, the dual-core DEs were merged by gentle heating, made possible by the optimized glycerol concentration present in the cores. Upon the coalescence of dual cores, the alkaline lysis buffer present in the core of the cell lysate was neutralized by the reaction buffer presented in the RT-PCR core, allowing TaqMan assay-based RT-PCR to occur effectively within the DEs. To demonstrate the potential of this streamlined dual-core platform, AKR1B10-positive A549 cells and AKR1B10-negative HEK293 cells were investigated via the TaqMan assay. Subsequently, specific transcript of AKR1B10 was readily available for quantitative profiling at the single-cell level using a commercially available flow cytometer in a high-throughput manner.

Aspirin Reduces Colorectal Tumor Development in Mice and Gut Microbes Reduce its Bioavailability and Chemopreventive Effects.

BACKGROUND & AIMS: Alterations in the intestinal microbiota affect development of colorectal cancer and drug metabolism. We studied whether the intestinal microbiota affect the ability of aspirin to reduce colon tumor development in mice. METHODS: We performed studies with APCmin/+ mice and mice given azoxymethane and dextran sulfate sodium to induce colorectal carcinogenesis. Some mice were given antibiotics to deplete intestinal microbes, with or without aspirin, throughout the entire experiment. Germ-free mice were studied in validation experiments. Colon tissues were collected and analyzed by histopathology, quantitative reverse-transcription polymerase chain reaction, and immunoblots. Blood samples and gut luminal contents were analyzed by liquid chromatography/mass spectrometry and an arylesterase activity assay. Fecal samples were analyzed by 16S ribosomal RNA gene and shotgun metagenome sequencing. RESULTS: Administration of aspirin to mice reduced colorectal tumor number and load in APCmin/+ mice and mice given azoxymethane and dextran sulfate sodium that had been given antibiotics (depleted gut microbiota), but not in mice with intact microbiota. Germ-free mice given aspirin developed fewer colorectal tumors than conventionalized germ-free mice given aspirin. Plasma levels of aspirin were higher in mice given antibiotics than in mice with intact gut microbiota. Analyses of luminal contents revealed that aerobic gut microbes, including Lysinibacillus sphaericus, degrade aspirin. Germ-free mice fed L sphaericus had lower plasma levels of aspirin than germ-free mice that were not fed this bacterium. There was an inverse correlation between aspirin dose and colorectal tumor development in conventional mice, but this correlation was lost with increased abundance of L sphaericus. Fecal samples from mice fed aspirin were enriched in Bifidobacterium and Lactobacillus genera, which are considered beneficial, and had reductions in Alistipes finegoldii and Bacteroides fragili, which are considered pathogenic. CONCLUSIONS: Aspirin reduces development of colorectal tumors in APCmin/+ mice and mice given azoxymethane and dextran sulfate sodium, depending on the presence of intestinal microbes. L sphaericus in the gut degrades aspirin and reduced its chemopreventive effects in mice. Fecal samples from mice fed aspirin were enriched in beneficial bacteria, with reductions in pathogenic bacteria.

Gavage of Fecal Samples From Patients With Colorectal Cancer Promotes Intestinal Carcinogenesis in Germ-Free and Conventional Mice.

BACKGROUND & AIMS: Altered gut microbiota is implicated in development of colorectal cancer (CRC). Some intestinal bacteria have been reported to potentiate intestinal carcinogenesis by producing genotoxins, altering the immune response and intestinal microenvironment, and activating oncogenic signaling pathways. We investigated whether stool from patients with CRC could directly induce colorectal carcinogenesis in mice. METHODS: We obtained stored stool samples from participants in a metagenome study performed in Hong Kong. Conventional (male C57BL/6) mice were given azoxymethane to induce colon neoplasia after receiving a course of antibiotics in drinking water. Mice were gavaged twice weekly with stool from 5 patients with CRC or 5 healthy individuals (controls) for 5 weeks. Germ-free C57BL/6 mice were gavaged once with stool from 5 patients with CRC or 5 controls. We collected intestinal tissues from mice and performed histology, immunohistochemistry, expression microarray, quantitative polymerase chain reaction, immunoblot, and flow cytometry analyses. We performed 16S ribosomal RNA gene sequencing analysis of feces from mice. RESULTS: Significantly higher proportions of conventional mice fed with stool from individuals with CRC than control stool developed high-grade dysplasia (P < .05) and macroscopic polyps (P < .01). We observed a higher proportion of proliferating (Ki-67-positive) cells in colons of germ-free mice fed with stool from patients with CRC vs those fed with stool from controls (P < .05). Feces from germ-free and conventional mice fed with stool from patients with CRC vs controls contained different microbial compositions, with lower richness in mice fed with stool from patients with CRC. Intestines collected from conventional and germ-free mice fed with stool from patients with CRC had increased expression of cytokines that modulate inflammation, including C-X-C motif chemokine receptor 1, C-X-C motif chemokine receptor 2, interleukin 17A (IL17A), IL22, and IL23A. Intestines from conventional and germ-free mice fed with stool from patients with CRC contained higher proportions of T-helper 1 (Th1) cells (2.25% vs 0.44%) and Th17 cells (2.08% vs 0.31%) (P < .05 for each) than mice fed with stool from controls. Real-time polymerase chain reaction arrays revealed up-regulation of genes involved in cell proliferation, stemness, apoptosis, angiogenesis, invasiveness, and metastasis in mice fed with stool from patients with CRC. CONCLUSIONS: We fed stool samples from patients with CRC and heathy individuals to germ-free mice and conventional mice with azoxymethane. We found stool from patients with CRC to increase the numbers of polyps, levels of intestinal dysplasia and proliferation, markers of inflammation, and proportions of Th1 and Th17 cells in colon, compared with stool from individuals without CRC. This study provides evidence that the fecal microbiota from patients with CRC can promote tumorigenesis in germ-free mice and mice given a carcinogen.

[Application of PeriCam PSI blood flow imaging perfusion system in rat brain ischemia reperfusion injury models].

OBJECTIVE: : To study the feasibility and effect of PeriCam PSI system guiding the establishment of ischemia/reperfusion injury model in rats. METHODS: : A total of 70 adult male Sprague-Dawley rats were divided into the control group(n=6), PSI monitoring group(n=34) and traditional operation group(n=30). Ischemia reperfusion model was established with reference to improve Zea-Longa line plug method. After the model established, the blood flow to the brain of control group, PSI monitoring group (ischemic 2 h, 24 h reperfusion) were observed and recorded respectively with PSI. The rats were then executed after 24 h, and the 2,3,5-triphenyltetrazolium chloride (TTC) staining and HE staining were used to observe the brain tissue. RESULTS: : The survival rate and modeling success rate of PSI monitoring group were higher than those of the traditional operation group(all P<0.05). The blood perfusion in the brain and the distribution of blood vessels were clearly observed in the control group, and the data were normal. In 2 h ischemic group, the arterial flow was interrupted in the right cerebral artery, and the blood flow in the middle arterial blood supply was significantly decreased than that in the control group(P<0.05). After the recovery of 24 h, the artery in the right side of the brain was restored to blood flow, but the blood flow in the partial supply area decreased, unable to recover to normal level. The TTC staining results indicated that there were obvious infarcts in the right brain tissue of PSI monitoring group,and the infarct area was more stable than that of the traditional operation group. The results of HE staining showed that the structure of brain tissue in the control group was normal, and the morphological rules of nerve cells were not change. While in brain tissue from PSI monitoring group, cortex and ischemia half dark stripe, nerve cell degeneration, necrosis and glial fiber disintegration, liquefaction, and light color, screen mesh in ischemic central area were observed. CONCLUSIONS: : PSI system can guide ischemia reperfusion model building and improve the success rate of the model.

SeqKit2: A Swiss army knife for sequence and alignment processing.

In the era of ubiquitous high-throughput sequencing studies, there is a growing need for analysis tools that are not just performant but also comprehensive and user-friendly enough to cater to both novice and advanced users. This article introduces SeqKit2, the next iteration of the widely used sequence analysis tool SeqKit, featuring expanded functionality, performance optimizations, and support for additional compression methods. Retaining a pragmatic subcommand architecture, SeqKit2 represents substantial enhancement through the inclusion of 19 additional subcommands, expanding its overall repertoire to a total of 38 in eight categories. The new subcommands add functionality such as amplicon processing and robust, error-tolerant parsing of sequence records. In addition, three subcommands designed for real-time analysis are added for periodic monitoring of properties of FASTQ and Binary Alignment/Map alignment records and real-time streaming from multiple sequence files. The performance of SeqKit2 is benchmarked against the old version of SeqKit, Bioawk, Seqtk, and SeqFu tools. SeqKit2 consistently outperforms its predecessor, albeit with marginally higher memory usage, while maintaining competitive runtimes against other tools. With its broad functionality, proven usability, and ongoing development driven by user feedback, we hope that bioinformaticians will find SeqKit2 useful as a "Swiss army knife" of sequence and alignment processing-equally adept at facilitating ad hoc analyses and seamlessly integrating into larger pipelines.

[Effect of carcinoma-associated fibroblasts on cancer occurrence and development].

Tumor microenvironment has been confirmed to play an important role in the occurrence, invasion and metastasis of many kinds of tumors. Carcinoma-associated fibroblasts (CAFs) are the primary type of host cells in the tumor microenvironment. CAFs have an assignable role in tumor development. CAFs create a suitable "soil" for tumor origination, secrete a large amount of growth factors promoting tumor growth and angiogenic factors promoting tumor angiogenesis. In addition, CAFs attract a large number of inflammatory cytokines, and secrete a great quantity of soluble products promoting tumor cell invasion and metastasis. Therefore, CAFs may become new targets for targeted cancer therapy, and provide new ideas for the clinical cancer comprehensive treatment.

Boosting the electrochemical performance of Li-rich Mn-based cathode materials via oxygen vacancy and spinel phase integration.

Li-rich Mn-based oxide cathodes (LMOs) are regarded as one of the most prospective high energy density cathodes due to the reversible anion redox reaction, which gives them a very high capacity. However, LMOs materials usually have problems like low initial coulombic efficiency (ICE) and poor cycling performance during cycling, which are associated with irreversible surface O2 release and unfavourable electrode/electrolyte interface side reactions. Herein, an innovative and scalable NH4Cl-assisted gas-solid interfacial reaction treatment technique is employed to construct oxygen vacancies and spinel/layered heterostructures simultaneously on the surface of LMOs. The synergistic effect of the oxygen vacancy and the surface spinel phase can not only effectively enhance the redox properties of the oxygen anion and inhibit irreversible oxygen release, but also effectively mitigate the side reactions at the electrode/electrolyte interface, inhibit the formation of CEI films and stabilize the layered structure. The electrochemical performance of the treated NC-10 sample improved significantly, showing an increase in ICE from 77.4 % to 94.3 % and excellent rate capability and cycling stability, with a capacity retention of 77.9 % after 400 cycles at 1 C. This oxygen vacancy and spinel phase integration strategy offers an exciting prospect and avenue for improving the integrated electrochemical performance of LMOs.

Parabacteroides distasonis uses dietary inulin to suppress NASH via its metabolite pentadecanoic acid.

Non-alcoholic steatohepatitis (NASH) is the severe form of non-alcoholic fatty liver disease, and is characterized by liver inflammation and fat accumulation. Dietary interventions, such as fibre, have been shown to alleviate this metabolic disorder in mice via the gut microbiota. Here, we investigated the mechanistic role of the gut microbiota in ameliorating NASH via dietary fibre in mice. Soluble fibre inulin was found to be more effective than insoluble fibre cellulose to suppress NASH progression in mice, as shown by reduced hepatic steatosis, necro-inflammation, ballooning and fibrosis. We employed stable isotope probing to trace the incorporation of 13C-inulin into gut bacterial genomes and metabolites during NASH progression. Shotgun metagenome sequencing revealed that the commensal Parabacteroides distasonis was enriched by 13C-inulin. Integration of 13C-inulin metagenomes and metabolomes suggested that P. distasonis used inulin to produce pentadecanoic acid, an odd-chain fatty acid, which was confirmed in vitro and in germ-free mice. P. distasonis or pentadecanoic acid was protective against NASH in mice. Mechanistically, inulin, P. distasonis or pentadecanoic acid restored gut barrier function in NASH models, which reduced serum lipopolysaccharide and liver pro-inflammatory cytokine expression. Overall this shows that gut microbiota members can use dietary fibre to generate beneficial metabolites to suppress metabolic disease.

Carnobacterium maltaromaticum boosts intestinal vitamin D production to suppress colorectal cancer in female mice.

Carnobacterium maltaromaticum was found to be specifically depleted in female patients with colorectal cancer (CRC). Administration of C. maltaromaticum reduces intestinal tumor formation in two murine CRC models in a female-specific manner. Estrogen increases the attachment and colonization of C. maltaromaticum via increasing the colonic expression of SLC3A2 that binds to DD-CPase of this bacterium. Metabolomic and transcriptomic profiling unveils the increased gut abundance of vitamin D-related metabolites and the mucosal activation of vitamin D receptor (VDR) signaling in C. maltaromaticum-gavaged mice in a gut microbiome- and VDR-dependent manner. In vitro fermentation system confirms the metabolic cross-feeding of C. maltaromaticum with Faecalibacterium prausnitzii to convert C. maltaromaticum-produced 7-dehydrocholesterol into vitamin D for activating the host VDR signaling. Overall, C. maltaromaticum colonizes the gut in an estrogen-dependent manner and acts along with other microbes to augment the intestinal vitamin D production to activate the host VDR for suppressing CRC.

Parvimonas micra promotes colorectal tumorigenesis and is associated with prognosis of colorectal cancer patients.

Large-scale fecal shotgun metagenomic sequencing revealed the high abundance of Parvimonas micra in colorectal cancer (CRC) patients. We investigated the role and clinical significance of P. micra in colorectal tumorigenesis. The abundance of P. micra was examined in 309 fecal samples and 165 colon biopsy tissues of CRC patients and healthy subjects. P. micra was significantly enriched in fecal samples from 128 CRC patients compared to 181 healthy subjects (P < 0.0001); and in colon tissue biopsies from 52 CRC patients compared to 61 healthy subjects (P < 0.0001). Multivariate analysis showed that P. micra is an independent risk factor of poor survival in CRC patients (Hazard Ratio: 1.93). P. micra strain was isolated from feces of a CRC patient. Apcmin/+ mice gavaged with P. micra showed significantly higher tumor burden and tumor load (both P < 0.01). Consistently, gavage of P. micra significantly promoted colonocyte proliferation in conventional mice, which was further confirmed by germ-free mice. P. micra colonization up-regulated genes involved in cell proliferation, stemness, angiogenesis and invasiveness/metastasis; and enhanced Th17 cells infiltration and expression of Th17 cells-secreted cytokines (Il-17, Il-22, and Il-23) in the colon of Apcmin/+, conventional and germ-free mice. P. micra-conditioned medium significantly promoted the differentiation of CD4+ T cells to Th17 cells (IL-17+CD4+ phenotype) and enhanced the oncogenic Wnt signaling pathway. In conclusion, P. micra promoted colorectal tumorigenesis in mice by inducing colonocyte proliferation and altering Th17 immune response. P. micra may act as a prognostic biomarker for poor survival of CRC patients.

Monoclonal antibodies against nucleophosmin mutants: potentials for the detection of acute myeloid leukemia.

Nucleophosmin (NPM1) gene mutations resulting in cytoplasmic delocalization of Nucleophosmin (NPMc+) are the most common genetic alteration in acute myeloid leukemia (AML). Here, we attempted to prepare monoclonal antibodies (mAbs) against NPM1 mutation A (NPM-mA) and investigated the mAbs' clinical utility in immunohistochemical detection of NPMc+AML. The pET-32a-NPM-mA vector with the whole open reading frame of the NPM-mA gene was constructed. E.coli BL21 transformed with the vector were induced to express the NPM-mA recombinant protein. BALB/c mice were immunized with the recombinant NPM-mA. Positive clones were selected by indirect ELISA and the mAbs were obtained. Immunohistochemistry was performed to detect the NPMc+ in bone marrow smears from 10 AML patients with NPM-mA. The results showed that the pET-32a-NPM-mA vector was successfully constructed and the NPM-mA recombinant protein was used to immunize the mice. Two positive clones (2G3 and 3F9) were selected. The mAbs against NPM-mA were raised, but did cross-react with wild type NPM1. The mAbs can be used to detect the cytoplasmic dislocation of NPM1 in all AMLs carrying NPM-mA. Our results show that anti-NPM-mA mAbs were produced. Though they would cross-react with wild type NPM1, the mAbs may still have potential in the detection of NPMc+AMLs.

SARS-CoV-2 activates lung epithelial cell proinflammatory signaling and leads to immune dysregulation in COVID-19 patients.

BACKGROUND: The outbreak of Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection has become a global health emergency. We aim to decipher SARS-CoV-2 infected cell types, the consequent host immune response and their interplay in lung of COVID-19 patients. METHODS: We analyzed single-cell RNA sequencing (scRNA-seq) data of bronchoalveolar lavage fluid (BALF) samples from 10 healthy donors, 6 severe COVID-19 patients and 3 mild recovered patients. The expressions of SARS-CoV-2 receptors (ACE2 and TMPRSS2) were examined among different cell types. The immune cells infiltration patterns, their expression profiles, and interplays between immune cells and SARS-CoV-2 target cells were further investigated. FINDINGS: Compared to healthy controls, ACE2 and TMPRSS2 expressions were significantly higher in lung epithelial cells of COVID-19 patients, in particular club and ciliated cells. SARS-CoV-2 activated pro-inflammatory genes and interferon/cytokine signaling in these cells. In severe COVID-19 patients, significantly higher neutrophil, but lower macrophage in lung was observed along with markedly increased cytokines expression compared with healthy controls and mild patients. By contrast, neutrophil and macrophage returned to normal level whilst more T and NK cells accumulation were observed in mild patients. Moreover, SARS-CoV-2 infection altered the community interplays of lung epithelial and immune cells: interactions between the club and immune cells were higher in COVID-19 patients compared to healthy donors; on the other hand, immune-immune cells interactions appeared the strongest in mild patients. INTERPRETATION: SARS-CoV-2 could infect lung epithelium, alter communication patterns between lung epithelial cells and immune system, and drive dysregulated host immune response in COVID-19 patients. FUNDING: This project was supported by National Key R&D Program of China (No. 2018YFC1315000/2018YFC1315004), Science and Technology Program Grant Shenzhen (JCYJ20170413161534162), HMRF Hong Kong (17160862), RGC-CRF Hong Kong (C4039-19G), RGC-GRF Hong Kong (14163817), Vice-Chancellor's Discretionary Fund CUHK and CUHK direct grant, Shenzhen Virtual University Park Support Scheme to CUHK Shenzhen Research Institute.

Microtubule associated protein 9 inhibits liver tumorigenesis by suppressing ERCC3.

BACKGROUND: Chromosomal instability plays an important part in cancer, but its genetic basis in liver tumorigenesis remains largely unclear. We aimed to characterize the mechanistic significance and clinical implication of mitotic regulator microtubule-associated protein 9 (MAP9) in hepatocellular carcinoma (HCC). METHODS: The biological functions of MAP9 were determined by in vitro tumorigenicity assays. Systematic MAP9 knockout mouse (MAP9∆/∆) and hepatocyte-specific MAP9 knockout mouse (MAP9∆/∆hep) were generated to confirm the role of MAP9 in HCC. The clinical impact of MAP9 was assessed in primary HCC tissue samples. FINDINGS: We found that MAP9 was frequently silenced in HCC tissue samples. The transcriptional silence of MAP9 in liver cancer cell lines and tissue samples was mediated by its promoter hypermethylation. MAP9 promoter hypermethylation or downregulation was associated with poor survival and recurrence in patients with HCC. Mechanistically, ectopic expression of MAP9 in LO2 and HepG2 cell lines impaired cell proliferation, colony formation, migration and invasion, and induced cell apoptosis and cycle arrest, whereas knockdown of MAP9 in Miha cell line showed the opposite effects. We found that MAP9∆/∆ mice spontaneously developed a liver hyperplastic nodule and MAP9∆/∆hep accelerated diethylnitrosamine-induced HCC formation. The tumour suppressive effect of MAP9 in HCC was mediated by downregulating excision repair cross-complementation group 3 (ERCC3), a nucleotide excision repair gene. Restoration of ERCC3 expression possessed an oncogenic potency and abrogated the tumour suppressive effects of MAP9. INTERPRETATION: MAP9 is a novel tumour suppressor in HCC by inhibiting ERCC3 expression, and serves as a prognostic factor in HCC patients.