Ketone body ß-hydroxybutyrate ameliorates colitis by promoting M2 macrophage polarization through the STAT6-dependent signaling pathway.

BACKGROUND: Ketone body ß-hydroxybutyrate (BHB) has received more and more attentions, because it possesses a lot of beneficial, life-preserving effects in the fields of clinical science and medicine. However, the role of BHB in intestinal inflammation has not yet been investigated. METHODS: Colonic mucosa of inflammatory bowel disease (IBD) patients and healthy controls were collected for evaluation of BHB level. Besides, the therapeutic effect of exogenous BHB in a murine model of acute dextran sulfate sodium (DSS)-induced colitis were assessed by body weight change, colon length, disease activity index, and histopathological sections. The regulatory effectors of BHB were analyzed by RT-qPCR, immunofluorescence, and microbe analysis in vivo. Moreover, the molecular mechanism of BHB was further verified in bone marrow-derived macrophages (BMDMs). RESULTS: In this study, significantly reduced BHB levels were found in the colonic mucosa from IBD patients and correlated with IBD activity index. In addition, we demonstrated that the administration of exogenous BHB alleviated the severity of acute experimental colitis, which was characterized by less weight loss, disease activity index, colon shortening, and histology scores, as well as decreased crypt loss and epithelium damage. Furthermore, BHB resulted in significantly increased colonic expression of M2 macrophage-associated genes, including IL-4Ra, IL-10, arginase 1 (Arg-1), and chitinase-like protein 3, following DSS exposure, suggesting an increased M2 macrophage skewing in vivo. Moreover, an in vitro experiment revealed that the addition of BHB directly promoted STAT6 phosphorylation and M2 macrophage-specific gene expression in IL-4-stimulated macrophages. Besides, we found that BHB obviously increased M2 macrophage-induced mucosal repair through promoting intestinal epithelial proliferation. However, the enhancement effect of BHB on M2 macrophage-induced mucosal repair and anti-inflammation was completely inhibited by the STAT6 inhibitor AS1517499. CONCLUSIONS: In summary, we show that BHB promotes M2 macrophage polarization through the STAT6-dependent signaling pathway, which contributes to the resolution of intestinal inflammation and the repair of damaged intestinal tissues. Our finding suggests that exogenous BHB supplement may be a useful therapeutic approach for IBD treatment.

Activating cryptic biosynthetic gene cluster through a CRISPR-Cas12a-mediated direct cloning approach.

Direct cloning of biosynthetic gene clusters (BGCs) from microbial genomes facilitates natural product-based drug discovery. Here, by combining Cas12a and the advanced features of bacterial artificial chromosome library construction, we developed a fast yet efficient in vitro platform for directly capturing large BGCs, named CAT-FISHING (CRISPR/Cas12a-mediated fast direct biosynthetic gene cluster cloning). As demonstrations, several large BGCs from different actinomycetal genomic DNA samples were efficiently captured by CAT-FISHING, the largest of which was 145 kb with 75% GC content. Furthermore, the directly cloned, 110 kb long, cryptic polyketide encoding BGC from Micromonospora sp. 181 was then heterologously expressed in a Streptomyces chassis. It turned out to be a new macrolactam compound, marinolactam A, which showed promising anticancer activity. Our results indicate that CAT-FISHING is a powerful method for complicated BGC cloning, and we believe that it would be an important asset to the entire community of natural product-based drug discovery.

Radiosensitivity of colorectal cancer and radiation-induced gut damages are regulated by gasdermin E.

Although radiotherapy is an important clinical option available for colorectal cancer (CRC), its use is restricted due to low radiosensitivity of CRC and high toxicity to surrounding normal tissues. The purpose of this study is to investigate the molecular mechanism by which CRC is not sensitive to radiation and radiation causes toxicity to surrounding normal tissues. Here we found that GSDME was silenced in CRC but markedly expressed in their surrounding normal tissues. GSDME determines radiation-induced pyroptosis in CRC cells and normal epithelial cells through the caspase-3-dependent pathway. GSDME expression sensitizes radioresistant CRC cells to radiation. In the homograft model, after radiation treatment, the tumor volume and weight were significantly decreased in GSDME-expressed homograft tumors compared to GSDME-knockout homograft tumors. On the mechanism, radiation induced GSDME-mediated pyroptosis in CRC cells, which recruited and activated NK cells to enhance antitumor immunity. In addition, GSDME-knockout mice were protected from radiation-induced weight loss and tissue damages in the intestine, stomach, liver and pancreas compared to wild-type control littermates. In summary, we show that GSDME determines CRC radiosensitivity and radiation-related toxicity to surrounding normal tissues through caspase-3-dependent pyroptosis. Our finding reveals a previously unrecognized link between radiation and pyroptosis.

An IRF1-dependent Pathway of TNFα-induced Shedding in Intestinal Epithelial Cells.

BACKGROUND: Shedding of intestinal epithelial cells [IECs] is a potent cause of barrier loss which plays an important role in the pathogenesis of inflammatory bowel disease [IBD]. TNFα can induce IEC shedding, but little is known about this process. METHODS: To investigate the molecular mechanism regulating IEC shedding, mice lacking interferon regulatory factor1 [IRF1], caspase-3, or gasdermin E [GSDME] and their control wild-type [WT] littermates were intravenously injected with tumour necrosis factor alpha [TNFα] to establish an IEC shedding model. A dual-luciferase reporter assay and a chromatin immunoprecipitation assay were used to determine the role of IRF1 in regulating caspase-3 expression. RESULTS: TNFα administration induced obvious IEC shedding in WT mice, but IRF1-/- and caspase-3-/-mice were completely protected from TNFα-induced IEC shedding. As a critical transcription factor, IRF1 was found to be required for caspase-3 expression in IECs by binding to IRF1-binding sites in the caspase-3 promoter. In WT mice, plasma membrane integrity was disrupted in shed IECs; these cells were swollen and contained GSDME-N terminal [NT] fragments which are responsible for the induction of pyroptosis. However, in GSDME-/- mice, plasma membrane integrity was not disrupted in shed IECs, which were not swollen and did not contain GSDME-NT, indicating that GSDME converted TNFα-induced IEC shedding into a pyroptotic cell death process. In addition, IRF1 deficiency resulted in decreases in mucosal inflammation and mucosal bacteria levels in TNFα-challenged colons. CONCLUSIONS: IRF1 deficiency maintains intestinal barrier integrity by restricting TNFα-induced IEC shedding.

Gasdermin-E-mediated pyroptosis participates in the pathogenesis of Crohn's disease by promoting intestinal inflammation.

Crohn's disease (CD) is a kind of refractory intestinal inflammatory diseases. Pyroptosis was recently identified as a gasdermin-mediated proinflammatory cell death. However, it is unclear whether gasdermin-mediated pyroptosis participates in the pathogenesis of CD. Here, we show that the pyroptosis-inducing fragment GSDME N-terminal is obviously detected in the inflamed colonic mucosa but not in the uninflamed mucosa of patients with CD, suggesting that GSDME-mediated pyroptosis may be correlated with intestinal mucosal inflammation in CD. To investigate the role of GSDME in colitis development, Gsdme-/- mice and wild-type (WT) littermate controls were treated with 2,4,6-trinitrobenzenesulfonic acid (TNBS) to induce colitis. We found that Gsdme-/- mice exhibit less-severe intestinal inflammation than WT controls do. Furthermore, our results indicate that GSDME-mediated epithelial-cell pyroptosis induces intestinal inflammation through the release of proinflammatory intracellular contents. In summary, we show that GSDME participates in the pathogenesis of CD through GSDME-mediated pyroptosis to release proinflammatory cytokines.

Polyketide pesticides from actinomycetes.

Natural product derived pesticides have increased in popularity worldwide because of their high efficacy, eco-friendly nature and favorable safety profile. The development of polyketide pesticides from actinomycetes reflects this increase in popularity in the past decades. These pesticides, which include avermectins, spinosyns, polynactins, tetramycin and their analogues, have been successfully applied in crop protection. Moreover, the advance of biotechnology has led to continuous improvement in the discovery and production processes. In this review, we summarize these polyketide pesticides, their activities and provide insight into their development. We also discuss engineering strategies and the current status of industrial production for these pesticides. Given that actinomycetes are known to produce a wide range of bioactive secondary metabolites, the description of pesticide development and high yield strain improvement presented herein will facilitate further development of these valuable polyketide pesticides from actinomycetes.

A versatile biosensing platform coupling CRISPR-Cas12a and aptamers for detection of diverse analytes.

Rapid and sensitive detection of various analytes is in high demand. Apart from its application in genome editing, CRISPR-Cas also shows promises in nucleic acid detection applications. To further exploit the potential of CRISPR-Cas for detection of diverse analytes, we present a versatile biosensing platform that couples the excellent affinity of aptamers for broad-range analytes with the collateral single-strand DNA cleavage activity of CRISPR-Cas12a. We demonstrated that the biosensors developed by this platform can be used to detect protein and small molecule in human serum with a complicated background, i.e., the tumor marker alpha fetoprotein and cocaine with the detection limits of 0.07 fmol/L and 0.34 µmol/L, respectively, highlighting the advantages of simplicity, sensitivity, short detection time, and low cost compared with the state-of-the-art biosensing approaches. Altogether, this biosensing platform with plug-and-play design show great potential in the detection of diverse analytes.

HMGB1 released from GSDME-mediated pyroptotic epithelial cells participates in the tumorigenesis of colitis-associated colorectal cancer through the ERK1/2 pathway.

BACKGROUND: Pyroptosis is a form of proinflammatory gasdermin-mediated programmed cell death. Abnormal mucosal inflammation in the intestine is a critical risk factor for colitis-associated colorectal cancer (CAC). However, it is unknown whether pyroptosis participates in the development of CAC. METHODS: To investigate the role of gasdermin E (GSDME)-mediated pyroptosis in the development of CAC, Gsdme-/- mice and their wild-type (WT) littermate controls were challenged with azoxymethane (AOM) and dextran sodium sulfate (DSS) to induce a CAC model. Neutralizing antibodies against high-mobility group box protein 1 (HMGB1) were used to determine the role of HMGB1 in CAC. To identify the role of ERK1/2 in HMGB1-induced colon cancer cell proliferation, we performed western blotting and CCK8 assays using the ERK1/2-specific inhibitor U0126 in CT26 colon cancer cells. RESULTS: In the CAC model, Gsdme-/- mice exhibited reduced weight loss and colon shortening, attenuated rectal prolapse, and reduced tumor numbers and sizes compared to WT littermates. Furthermore, treatment with neutralizing anti-HMGB1 antibodies decreased the numbers and sizes of tumors, ERK1/2 activation and proliferating cell nuclear antigen (PCNA) expression in AOM/DSS-challenged WT mice. In addition, our in vitro experiments demonstrated that HMGB1 induced proliferation and PCNA expression in CT26 colon cancer cells through the ERK1/2 pathway. CONCLUSION: GSDME-mediated pyroptosis promotes the development of CAC by releasing HMGB1, which induces tumor cell proliferation and PCNA expression through the ERK1/2 pathway. This finding reveals a previously unrecognized link between pyroptosis and CAC tumorigenesis and offers new insight into CAC pathogenesis.

Genetic Variations at rs3129891 and rs77005575 are Associated With Reduced Expression of Enteric α-defensins in IBD Patients.

BACKGROUND AND AIMS: Human enteric antimicrobial peptides composed predominantly of human enteric α-defensins (HD5 and HD6) are important in the mucosal antimicrobial barrier. Previous studies have identified that genetic variations at rs2066844, rs2066845, rs2066847 are associated with diminished enteric α-defensins in ileal Crohn's disease (CD). However, genetic variations associated with enteric antimicrobial peptides in colonic inflammatory bowel disease (IBD) remain unclear. To investigate it, we compared the colonic expression of antimicrobial peptides with respect to genotypes at 22 IBD-associated single-nucleotide polymorphisms (SNPs). MATERIALS AND METHODS: In total, 16 controls and 102 colonic IBD patients including 42 ulcerative colitis (UC) and 60 CD were studies. Mutation assay was performed to determine their genotypes at 22 IBD-associated SNPs. Real-time PCR was performed to determine the colonic mRNA expression of HD5, HD6, lysozyme, and secretory phospholipase A2. RESULTS: Mutant genotypes at rs2066844, rs2066845, rs2066847 were not found, and only SNPs rs3129891 and rs77005575 were associated with enteric α-defensin expression in colonic IBD. In both inflamed and noninflamed tissues, colonic expression of HD5 and HD6 was significantly decreased in UC and CD patients carrying rs3129891 homozygous mutant genotype. And their colonic expression was significantly decreased in inflamed but not noninflamed tissues from UC patients carrying rs77005575 homozygous mutant genotype. However, both lysozyme and secretory phospholipase A2 in UC and CD were unaffected by rs3129891 and rs77005575 genotypes. CONCLUSIONS: As enteric α-defensins play critical roles in the mucosal antimicrobial barrier, their reduced expression may partly explain the microbial-induced mucosal inflammation in colonic IBD patients, especially in patients carrying rs3129891 and rs77005575 mutant genotypes.

YAP triggers the Wnt/ß-catenin signalling pathway and promotes enterocyte self-renewal, regeneration and tumorigenesis after DSS-induced injury.

Impaired epithelial regeneration is a crucial pathophysiological feature of ulcerative colitis (UC). Yes-associated protein (YAP1) appears to control cell proliferation and differentiation. In this study, we sought to identify the roles of YAP in intestinal epithelial cell (IEC) self-renewal, regeneration and tumorigenesis. We first observed that YAP was significantly reduced in 62.5% (45/72) of human UC tissues and it was dramatically enhanced during epithelial regeneration in a murine colitis model. Using lentiviral infection, we established a YAP-overexpression (YAPWT) mouse model. We then found that after tissue injury, YAPWT mice had increased epithelial cell self-renewal capacity and drastically restored intestinal crypt structure. Strikingly, these mice were more susceptible to colitis-associated cancer (CAC) in chemically induced carcinoma. Mechanistically, YAP and ß-catenin showed increased nuclear co-localization during regeneration after inflammation. Overexpressing YAP significantly improved IEC 'wound-healing' ability and increased the expression of both ß-catenin and the transcriptional targets of Wnt signalling Lgr5 and cyclin D1, whereas silencing ß-catenin in YAPWT cells attenuated this effect. Remarkably, we observed that YAP could directly interact with ß-catenin in the nucleus and formed a transcriptional YAP/ß-catenin/TCF4 complex; Lgr5 and cyclin D1 were confirmed to be the target genes of this complex. In contrast, cancer cell proliferation and tumour development were suppressed by the phospho-mimetic YAP mutant. In summary, nuclear YAP-driven IEC proliferation could control epithelial regeneration after inflammation and may serve as a potential therapeutic target in UC. However, excessive YAP activation promoted CAC development.

MiR-199a/b-3p inhibits gastric cancer cell proliferation via down-regulating PAK4/MEK/ERK signaling pathway.

BACKGROUND: Gastric cancer (GC) is one of the most frequent malignant tumors and the molecular mechanism underlying its proliferation remains far from completely understood. Although accumulating evidence shows that abnormal expression of microRNA (miRNA) is involved in tumorigenesis, the role of specific miRNAs involved in GC remains elusive. MiR-199a/b-3p functions as a tumor suppressor in diverse cancers, but its expression, function, and mechanism in GC remain unclear. Our aim is to explore miR-199a/b-3p expression and its role in regulating GC cell proliferation. METHODS: Real-time PCR was performed to determine miR-199a/b-3p expression in GC tissues and normal adjacent tissues as well as normal gastric mucosal cell line GES-1 and GC cell lines MGC-803 and SGC-7901. MTT assay and Western blot were performed to determine cell proliferation and expression of PAK4, p-MEK and p-ERK, respectively. MiR-199a/b-3p mimics-transfected assay and PAK-specific siRNA assay were performed to determine their function in cell proliferation, respectively. GC xenograft nude mice were used to determine miR-199a/b-3p function in cell proliferation. RESULTS: MiR-199a/b-3p expression was significantly decreased in GC tissues and GC cell lines MGC-803 and SGC-7901. MiR-199a/b-3p over-expression and PAK4 silencing inhibited cell proliferation and diminished the activation of p-MEK and p-ERK in MGC-803 and SGC-7901 cells, and miR-199a/b-3p over-expression reduced PAK4 expression. MiR-199a/b-3p over-expression suppressed MGC-803 cell growth and PAK4 expression in nude mice. CONCLUSIONS: miR-199a/b-3p inhibits GC cell proliferation via down-regulating PAK4/MEK/ERK signaling pathway and may be a novel prognostic biomarker and a potential therapeutic target for GC patients.

NOD2 up-regulates TLR2-mediated IL-23p19 expression via NF-κB subunit c-Rel in Paneth cell-like cells.

IL-23p19 plays important roles in intestinal antimicrobial immunity, while its over-expression can lead to intestinal inflammation. However, the bacterial compounds and the type of pattern recognition receptor involved in the inducible expression of IL-23p19 in Paneth cells remain unclear. Here we show that the mRNA expression of IL-23p19 was increased in Paneth cell (PC)-like cells stimulated by Toll-like receptor 2 (TLR2) ligands, peptidoglycan (PGN) and Pam3CSK4, and was further increased in the presence of nucleotide-binding oligomerization domain 2 (NOD2)-ligand muramyl dipeptide (MDP). However, its mRNA expression was decreased in NOD2-knockdown PC-like cells. Additionally, the c-Rel activation was increased in Pam3CSK4- or PGN-stimulated PC-like cells, but the PGN-induced c-Rel activation was decreased in NOD2-knockdown PC-like cells and had no significant difference compared with Pam3CSK4-induced c-Rel activation. Our results suggest that NOD2 up-regulates TLR2-mediated IL-23p19 expression via increasing c-Rel activation in PC-like cells. This finding might provide us with a novel therapeutic target for inflammatory bowel disease to inhibit IL-23p19 over-expression via the NOD2-c-Rel pathway.

RASSF1A inhibits gastric cancer cell proliferation by miR-711- mediated downregulation of CDK4 expression.

Although interaction with DNA repair proteins has demonstrated that RASSF1A is a tumour suppressor gene, much attention has been directed in recent years towards its roles in regulating the cell cycle. However, the precise mechanism remains unclear. Uncovering how RASSF1A participates in regulating the cell cycle is critical to exploring effective therapeutic targets for gastric cancer. Here we show that RASSF1A could regulate 14 miRNAs' expression in the typical human gastric cancer line SGC-7901, of which miR-711 was upregulated the most. Moreover, for SGC-7901 cells, miR-711 was found to downregulate CDK4 expression, and to arrest the cell cycle in the G1 phase. Our results suggest that RASSF1A inhibits the proliferation of gastric cancer cells by upregulating the expression of miR-711, which arrested gastric cancer cells in the G1 phase by downregulating expression of CDK4. This finding might provide us with a novel therapeutic target for gastric cancer by increasing RASSF1A expression via miR-711 regulation.

Analytic Study on the Characteristics of Infrared Spectrum of Oil Pool Flame.

In virtue of the severity and scale of the pollution caused by oil pool flame, space remote sensing can provide us a new way of monitoring in real time the oil pool flame pollution. Space remote sensing monitoring is based on the analysis of target spectrum characteristics. Due to lack of adequate researches on the characteristics of infrared spectrum of oil pool flame, this paper carries out the analytical study on flame spectrums of several types of oil, mixed oil and other combustible objects in outdoor space by establishing all-flame infrared testing system with the spectrum range of 1～14 µm. The results show that the spectrum curves of oil pool flame of 92# gasoline, 95# gasoline, 0# diesel, aviation kerosene and lube have similar features, that there exist characteristics emission peaks at the area of certain wave lengths­H2O characteristics emission peak for 1.1, 2.4, 2.8 and 6.3 µm, CO2 characteristics emission peak for 4.2 and 4.5 µm, C­H stretching vibration emission peak for 3.4 µm, and no obvious characteristics peak for spectrum curves of 6.3 µm and above; that there is no obvious difference in the spectrum of oil pool flame among the mixtures of 92# gasoline and 0# diesel at different proportions, that the comparison of the flame spectrum of 92# gasoline with that of wood and paper shows that there appears a characteristics emission peak at 3.4 µm; that though the flame spectrum of alcohol has similar radiated emission near 3.4 µm, the proportion of its radiation intensity to that of CO2 at 4.5 µm is far less than that for the flame spectrum of 92# gasoline; that the flame spectrum of honeycomb briquette is similar to that of gray body radiation. The differences in flame spectrum among all kinds of combustible materials are closely linked to their chemical compositions and burning reaction mechanisms. Comparative analysis on the spectrum characteristics at continuous area, intermission area and flue gas area shows that C­H stretching vibration peak only exists in continuous area, which proves that the emission peak is caused by the combustible reaction of oil and gas. This result is in line with the mechanism of oil pool combustion reaction. The experimental conclusion is of great significance in the remote-sensing recognition of oil pool flame based on the analysis of spectrum characteristics.

Down-regulation of human enteric antimicrobial peptides by NOD2 during differentiation of the paneth cell lineage.

Ileal Crohn's disease (CD) arising from the alteration of intestinal homeostasis is characterized by two features, namely a decrease in Paneth cell-produced antimicrobial peptides that play a key role in maintaining this balance and an increase in NOD2, an intracellular sensor. Although mutations in NOD2 are highly correlated with the incidence of CD, the physiological role of NOD2 in intestinal immunity remains elusive. Here, we show that NOD2 can down-regulate the expression of human enteric antimicrobial peptides during differentiation of the Paneth cell lineage. This finding, which links the decrease of human enteric antimicrobial peptides to increased NOD2 in ileal CD patients, provides a new view into the pathogenesis of ileal CD.

Regulation of human enteric α-defensins by NOD2 in the Paneth cell lineage.

Human enteric α-defensins (HD5 and HD6), major antimicrobial peptides produced by Paneth cells in the intestine, play important roles in intestinal innate immunity. Since their expression is decreased in Crohn's disease (CD), with decreased expression being more pronounced in the presence of NOD2 mutations, it would be extremely interesting to investigate the mechanism by which NOD2 may regulate expression of human enteric α-defensins. Here we show that although NOD2 by itself can slightly up-regulate expression of enteric α-defensins mainly through activation of the NF-κB pathway, it can strongly down-regulates their expression during differentiation of the Paneth cell lineage mainly by inhibiting activation of the MAPK pathway. Since NOD2 is over-expressed in CD and mutant NOD2 cannot result in NF-κB activity, our finding can provide an explanation of the previous observation showing decreased expression of human enteric α-defensin in CD and even more so in the presence of NOD2 mutations. In addition, this finding provides a new view on the function of NOD2 in regulating intestinal innate immunity.

Increased expression and possible role of chitinase 3-like-1 in a colitis-associated carcinoma model.

AIM: To investigate the possible role of chitinase 3-like-1 (CHI3L1) in the progression of colitis-associated carcinoma (CAC). METHODS: Thirty-four Balb/c mice were randomly assigned to five groups, including the control, CAC control, CAC + caffeine, colitis control and colitis + caffeine. Three animals were sacrificed every two weeks for blinded macroscopic inspection, histological analysis, and total RNA extraction. An immunofluorescent assay was performed using specimens from the colitis control and colitis + caffeine groups to investigate whether the protective effect of caffeine was associated with less oxidative DNA damage. In vitro, HT29 cells pre-stimulated with different concentrations of recombinant CHI3L1 protein and H2O2 were loaded with the DCFH-DA fluorescent probe to determine the effect of CHI3L1 on intracellular reactive oxygen species production. RESULTS: CHI3L1 mRNA was increased during the progression of colon carcinogenesis. Tumors were mostly located in the distal end of the colon where the expression of CHI3L1 was higher than in the proximal colon. Caffeine-treated mice developed fewer tumors and milder inflammation than untreated mice. CHI3L1 protein increased reactive oxygen species in HT29 cells when exposed to H2O2. CONCLUSION: Caffeine reduces tumor incidence by decreasing oxidative DNA damage. CHI3L1 may contribute to CAC by increasing reactive oxygen species production.