A toolbox for genetic manipulation in intestinal Clostridium symbiosum.

Gut microbes are closely related with human health, but remain much to learn. Clostridium symbiosum is a conditionally pathogenic human gut bacterium and regarded as a potential biomarker for early diagnosis of intestinal tumors. However, the absence of an efficient toolbox that allows diverse genetic manipulations of this bacterium limits its in-depth studies. Here, we obtained the complete genome sequence of C. symbiosum ATCC 14940, a representative strain of C. symbiosum. On this basis, we further developed a series of genetic manipulation methods for this bacterium. Firstly, following the identification of a functional replicon pBP1 in C. symbiosum ATCC 14940, a highly efficient conjugative DNA transfer method was established, enabling the rapid introduction of exogenous plasmids into cells. Next, we constructed a dual-plasmid CRISPR/Cas12a system for genome editing in this bacterium, reaching over 60 % repression for most of the chosen genes as well as efficient deletion (>90 %) of three target genes. Finally, this toolbox was used for the identification of crucial functional genes, involving growth, synthesis of important metabolites, and virulence of C. symbiosum ATCC 14940. Our work has effectively established and optimized genome editing methods in intestinal C. symbiosum, thereby providing strong support for further basic and application research in this bacterium.

Bilayer Silk Fibroin/Sodium Alginate Scaffold Delivered hUC-MSCs to Enhance Skin Scarless Healing and Hair Follicle Regeneration with the IRE1/XBP1 Pathway Inhibition.

Efficient local delivery of mesenchymal stem cells (MSCs) is a decisive factor for their application in regeneration processes. Here, we prepared a biomimetic bilayer silk fibroin/sodium alginate (SF/SA) scaffold to deliver human umbilical mesenchymal stem cells (hUC-MSCs) for wound healing. An SA membrane was prepared by the casting method on the upper layer of the scaffold to simulate the dense epidermal structure. On the lower layer, porous materials simulating the loose structure of the dermis were formed by the freeze-drying method. In vitro, the scaffold was proven to have a high-density pore structure, good swelling property, and suitable degradation rate. The hUC-MSCs could survive on the scaffold for up to 14 days and maintain cell stemness for at least 7 days. In vivo, SF/SA scaffolds loaded with hUC-MSCs (M-SF/SA) were applied to full-thickness defect wounds and compared with the local injection of hUC-MSCs. The M-SF/SA group showed excellent therapeutic efficacy, characterized by induction of macrophage polarization, regulation of TGF-ß expression and collagen components, and enhancement of vascular regeneration, thereby preventing scar formation and promoting hair follicle regeneration. Furthermore, the expression of endoplasmic reticulum stress markers IRE1, XBP1, and CHOP was inhibited significantly in M-SF/SA treatment. In conclusion, the bilayer SF/SA scaffold is an ideal delivery platform for hUC-MSCs, and the M-SF/SA system could locally promote scarless skin healing and hair follicle regeneration by alleviating the IRE1/XBP1 signal pathway.

METTL14 modulates glycolysis to inhibit colorectal tumorigenesis in p53-wild-type cells.

The frequency of p53 mutations in colorectal cancer (CRC) is approximately 40-50%. A variety of therapies are being developed to target tumors expressing mutant p53. However, potential therapeutic targets for CRC expressing wild-type p53 are rare. In this study, we show that METTL14 is transcriptionally activated by wild-type p53 and suppresses tumor growth only in p53-wild-type (p53-WT) CRC cells. METTL14 deletion promotes both AOM/DSS and AOM-induced CRC growth in mouse models with the intestinal epithelial cell-specific knockout of METTL14. Additionally, METTL14 restrains aerobic glycolysis in p53-WT CRC, by repressing SLC2A3 and PGAM1 expression via selectively promoting m6 A-YTHDF2-dependent pri-miR-6769b/pri-miR-499a processing. Biosynthetic mature miR-6769b-3p and miR-499a-3p decrease SLC2A3 and PGAM1 levels, respectively, and suppress malignant phenotypes. Clinically, METTL14 only acts as a beneficial prognosis factor for the overall survival of p53-WT CRC patients. These results uncover a new mechanism for METTL14 inactivation in tumors and, most importantly, reveal that the activation of METTL14 is a critical mechanism for p53-dependent cancer growth inhibition, which could be targeted for therapy in p53-WT CRC.

Preparation and Anti-tumor Study of Dextran 70,000-Selenium Nanoparticles and Poloxamer 188-Selenium Nanoparticles.

The anti-tumor effect of selenium nanoparticles (SeNPs) has received more and more attention. However, the clinical application of SeNPs is not optimistic due to the poor stability. To improve the stability of SeNPs, many polymers are used to modify the SeNPs. However, most of the polymers are not approved by FDA. It is significant to develop a SeNPs product with good stability for clinic application. Dextran 70,000 (T70) and poloxamer 188 (P188) are FDA-approved pharmaceutical injection excipients. In this study, we decorate SeNPs with T70 and P188 and assess the physicochemical characterization, storage stability, and anti-tumor activities of T70-SeNPs and P188-SeNPs. Transmission electron microscopy (TEM) shows that T70-SeNPs and P188-SeNPs are spherical particles with particle sizes of 110 nm and 60 nm respectively. Fourier-Transform Infrared Spectra (FT-IR) show that T70 or P188 can interact with SeNPs through hydrogen bonding. Stability study shows that P188-SeNPs freeze-dried powder and T70-SeNPs freeze-dried powder remain stable at 4℃ for 6 months. T70-SeNPs and P188-SeNPs can aggregate in cell matrix and play an anti-tumor role to HepG2 by promoting apoptosis, increasing reactive oxygen species (ROS) content and reducing mitochondrial membrane potential (MMP). This study can provide reference for industrial production of SeNPs products.

TRAPPC4 regulates the intracellular trafficking of PD-L1 and antitumor immunity.

Tumor cells evade T cell-mediated immunosurveillance via the interaction between programmed death-1 (PD-1) ligand 1 (PD-L1) on tumor cells and PD-1 on T cells. Strategies disrupting PD-1/PD-L1 have shown clinical benefits in various cancers. However, the limited response rate prompts us to investigate the molecular regulation of PD-L1. Here, we identify trafficking protein particle complex subunit 4 (TRAPPC4), a major player in vesicular trafficking, as a crucial PD-L1 regulator. TRAPPC4 interacts with PD-L1 in recycling endosomes, acting as a scaffold between PD-L1 and RAB11, and promoting RAB11-mediated recycling of PD-L1, thus replenishing its distribution on the tumor cell surface. TRAPPC4 depletion leads to a significant reduction of PD-L1 expression in vivo and in vitro. This reduction in PD-L1 facilitates T cell-mediated cytotoxicity. Overexpression of Trappc4 sensitizes tumor cells to checkpoint therapy in murine tumor models, suggesting TRAPPC4 as a therapeutic target to enhance anti-tumor immunity.

Synbindin restrains proinflammatory macrophage activation against microbiota and mucosal inflammation during colitis.

OBJECTIVE: As a canonical membrane tethering factor, the function of synbindin has been expanding and indicated in immune response. Here, we investigated the role of synbindin in the regulation of toll-like receptor 4 (TLR4) signalling and macrophage response to microbiota during colitis. DESIGN: Three distinct mouse models allowing global, myeloid-specific or intestinal epithelial cell-specific synbindin heterozygous deletion were constructed and applied to reveal the function of synbindin during dextran sodium sulfate (DSS) colitis. Effects of synbindin on TLR4 signalling and macrophage activation in response to bacterial lipopolysaccharide (LPS) or Fusobacterium nucleatum were evaluated. The colocalisation and interaction between synbindin and Rab7b were determined by immunofluorescence and coimmunoprecipitation. Synbindin expression in circulating monocytes and intestinal mucosal macrophages of patients with active IBD was detected. RESULTS: Global synbindin haploinsufficiency greatly exacerbated DSS-induced intestinal inflammation. The increased susceptibility to DSS was abolished by gut microbiota depletion, while phenocopied by specific synbindin heterozygous deletion in myeloid cells rather than intestinal epithelial cells. Profoundly aberrant proinflammatory gene signatures and excessive TLR4 signalling were observed in macrophages with synbindin interference in response to bacterial LPS or Fusobacterium nucleatum. Synbindin was significantly increased in intestinal mucosal macrophages and circulating monocytes from both mice with DSS colitis and patients with active IBD. Interleukin 23 and granulocyte-macrophage colony-stimulating factor were identified to induce synbindin expression. Mechanistic characterisation indicated that synbindin colocalised and directly interacted with Rab7b, which coordinated the endosomal degradation pathway of TLR4 for signalling termination. CONCLUSION: Synbindin was a key regulator of TLR4 signalling and restrained the proinflammatory macrophage activation against microbiota during colitis.

Betulinic acid attenuated bleomycin-induced pulmonary fibrosis by effectively intervening Wnt/ß-catenin signaling.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a fatal and progressive fibrotic lung disease lacking a validated and effective therapy. Aberrant activation of the Wnt/ß-catenin signaling cascade plays the key role in the pathogenesis of IPF. Betulinic acid is a natural pentacyclic triterpenoid molecule that has excellent antitumor and antiviral activities. HYPOTHESIS: We hypothesized that BA has an anti-pulmonary fibrosis effect mediated by the suppression of the Wnt/ß-catenin pathway. Study design Pulmonary fibrosis markers were detected in vitro and in vivo to confirm the antifibrotic effect of BA. The Wnt/ß-catenin pathway-related proteins were overexpressed to determine the effect of BA on Wnt signaling. METHODS AND RESULTS: BA dose-dependently inhibited Wnt3a-induced fibroblast activation in vitro. Moreover, BA decreased Wnt3a- and LiCl-induced transcriptional activity, as assessed by the TOPFlash assay in fibroblasts, and repressed the expression of the Wnt target genes cyclin D1, axin 2, and S100A4. Further investigation indicated that BA restrained the nuclear accumulation of ß-catenin, mainly by increasing the phospho-ß-catenin ratio (S33/S37/T41 and S45), inhibited the phosphorylation of DVL2 and LRP, and decreased the levels of Wnt3a and LRP6. In agreement with the results of the in vitro assays, the in vivo experiments indicated that BA significantly decreased bleomycin-induced pulmonary fibrosis in mice and suppressed myofibroblast activation by inhibiting Wnt/ß-catenin signaling. CONCLUSION: BA may directly interfere with the Wnt/ß-catenin pathway to subsequently repress myofibroblast activation and pulmonary fibrosis.

Chiral Separation by NACE Using Polyol Derivative-Boric Acid Complexes.

Nonaqueous capillary electrophoresis (NACE) is an effective method for chiral separation. Many polyol derivatives (e.g., D-(+)-xylose, lactobionic acid, diacetone-D-mannitol, L-sorbose, and D-gluconic acid δ-lactone) can react with boric acid in methanol to produce polyol derivative-boric acid complexes which can be utilized as chiral selectors of enantioseparations. The enantiomers of more than a dozen basic analytes can be resolved under the optimized NACE using these chiral selectors.

Revealing the interaction between intrauterine adhesion and vaginal microbiota using high­throughput sequencing.

Intrauterine adhesion (IUA) is one of the most common diseases of the reproductive system. Due to the high postoperative recurrence rate of IUA, it is crucial to identify the possible causes of pathogenesis and recurrence of this disease. In the present study, a high­throughput sequencing approach was applied to compare the vaginal microbiota between healthy women [healthy vaginal secretion (HVS) group] and patients with IUA [intrauterine adhesion patients' vaginal secretion (IAVS) group]. The results indicated that IUA had little effect on the number of vaginal bacterial species. However, at the phylum level, patients with IUA had a significantly lower percentage of Firmicutes and a higher percentage of Actinobacteria than the HVS group (P<0.05). At the genus level, ~50% of patients with IUA were found to have a marked reduction in probiotic Lactobacillus accompanied by an overgrowth of pathogenic Gardnerella and Prevotella (P<0.05), and the Principal Coordinates Analysis confirmed that 10/20 samples in the IAVS group were scattered far away from the HVS group. Therefore, it was concluded that the interaction between IUA and vaginal microbiota greatly influenced the vaginal diversity of patients with IUA. In order to increase the recovery rate and lower the recurrence rate of IUA, increasing the vaginal Lactobacillus population should be considered.

Evaluation of the Treatment Effect of Aloe vera Fermentation in Burn Injury Healing Using a Rat Model.

Burn injury is a growing medical problem associated with public health, and few effective agents are available for treatment of this disease. In the present study, a burn injury rat model was developed and the accelerated effect of Aloe vera fermentation on burn injury healing was evaluated. Our results indicated that Aloe vera fermentation could markedly reduce the DPPH (56.12%), O2·- (93.5%), ·OH (76.12%), Fe2+ chelation (82%), and oxygen-reduction activity (0.28 µg/ml) and significantly inhibited the growth of pathogens S. typhimurium ATCC 13311 (inhibition zone diameter: 14 mm), S. enteritidis ATCC13076 (IZD: 13 mm), S. flexneri ATCC 12022 (IZD: 18 mm), E. coli 44102 (IZD: 10 mm), L. monocytogenes ATCC 19111 (IZD: 18 mm), S. dysenteriae 301 (IZD: 20 mm), S. aureus COWAN1 (IZD: 19 mm), and P. acnes ATCC 11827 (IZD: 25 mm) in vitro. The in vivo results indicated that Aloe vera fermentation produced more eosinophils and fibroblasts and less vessel proliferation compared with the model group on the 14th day, which had greatly accelerated burn injury healing via shedding of the scab and promoting hair growth. ELISA results indicated that Aloe vera fermentation had significantly reduced the production of proinflammatory factors TNF-α and IL-1ß (p < 0.05) and greatly enhanced the yield of anti-inflammatory factor IL-4 in animal serum (p < 0.05). In addition, the high-throughput sequencing results indicated that Aloe vera fermentation obviously increased the percentage of Firmicutes (65.86% vs. 49.76%), while reducing the number of Bacteroidetes (27.60% vs. 45.15%) compared with the M group at the phylum level. At the genus level, Aloe vera fermentation increased the probiotic bacteria Lactobacillus (3.13% vs. 2.09%) and reduced the pathogens Prevotella (10.60% vs.18.24%) and Blautia (2.91% vs. 16.41%) compared with the M group. Therefore, we concluded that the use of Aloe vera fermentation significantly accelerates burn injury healing via reduction of the severity of inflammation and through modification of gut microbiota.

Triptolide modulates tumour-colonisation and anti-tumour effect of attenuated Salmonella encoding DNase I.

The strong human immunity and the associated toxicities of attenuated Salmonella severely limit the clinical use of Salmonella in tumour suppression. In the present study, we constructed an engineered VNP20009-DNase I strain and evaluated the synergistic effects of triptolide (TPL) and VNP20009-DNase I against melanoma in mice. Our results indicated that TPL could significantly inhibit the cell growth and cell migration and significantly enhanced the apoptosis rate of B16F10 cells in vitro. The in vivo results indicated that TPL markedly improved tumour colonisation of VNP20009-DNase I and led to a larger necrotic area in the melanoma. Moreover, the combination therapy significantly suppressed tumour volume and prolonged the life span of mice (P < 0.05) by upregulating the expression of Bcl-2/Bax and Caspase-3 and by downregulating the TLR4/NF-κB signalling, the expression of p-AKT/AKT and the production of proinflammatory factors. Therefore, the sound synergistic anti-tumour effects of TPL and VNP20009-DNase I indicate that the unconventional application of TPL and biological agents, approved by the China Food and Drug Administration (CFDA), can result in improved anti-cancer therapeutic outcomes.

Synbindin deficiency inhibits colon carcinogenesis by attenuating Wnt cascade and balancing gut microbiome.

The molecular mechanisms that control the development of colorectal cancer (CRC) remain poorly defined. Here we show Synbindin promoted CRC oncogenesis by activating Wnt signaling and altering gut microbiome. Synbindin upregulation in human CRCs was associated with poor patient prognosis. Intestine-specific disruption of Synbindin balanced the disturbed gut microbiota and protected mice against tumor formation in the colitis-associated cancer (CAC) model. The protective role was compromised after gut microbiota depletion. In host, increased goblet cells and mucin2 expression, together with increased intestinal epithelial cells (IECs) apoptosis and decreased epithelial proliferation were observed. Further transcriptomic sequencing identified Wnt signaling a major regulatory node downstream of Synbindin. Combined molecular and cellular characterizations revealed that Synbindin confers Disheveled-3 (DVL3)-based signalosome assembly and acts as a modular scaffold for DVL3 and Axin2 complex, orchestrating the intensity of Wnt signaling. These findings identify a critical role of Synbindin in gut microbiome composition and Wnt signaling activation in colorectal carcinogenesis, and highlight Synbindin as an adaptor protein with multifaceted roles.