Long-term effects of fecal microbiota transplantation on gut microbiota after Helicobacter pylori eradication with bismuth quadruple therapy: A randomized controlled trial.

BACKGROUND: Eradicating Helicobacter pylori infection by bismuth quadruple therapy (BQT) is effective. However, the effect of BQT and subsequent fecal microbiota transplant (FMT) on the gut microbiota is less known. MATERIALS AND METHODS: This prospective randomized controlled trial was conducted at a tertiary hospital in China from January 2019 to October 2020, with the primary endpoints the effect of BQT on the gut microbiota and the effect of FMT on the gut microbiota after bismuth quadruple therapy eradication therapy. A 14-day BQT with amoxicillin and clarithromycin was administered to H. pylori-positive subjects, and after eradication therapy, patients received a one-time FMT or placebo treatment. We then collected stool samples to assess the effects of 14-day BQT and FMT on the gut microbiota. 16 s rDNA and metagenomic sequencing were used to analyze the structure and function of intestinal flora. We also used Gastrointestinal Symptom Rating Scale (GSRS) to evaluate gastrointestinal symptom during treatment. RESULTS: A total of 30 patients were recruited and 15 were assigned to either FMT or placebo groups. After eradication therapy, alpha-diversity was decreased in both groups. At the phylum level, the abundance of Bacteroidetes and Firmicutes decreased, while Proteobacteria increased. At the genus level, the abundance of beneficial bacteria decreased, while pathogenic bacteria increased. Eradication therapy reduced some resistance genes abundance while increased the resistance genes abundance linked to Escherichia coli. While they all returned to baseline by Week 10. Besides, the difference was observed in Week 10 by the diarrhea score between two groups. Compared to Week 2, the GSRS total score and diarrhea score decreased in Week 3 only in FMT group. CONCLUSIONS: The balance of intestinal flora in patients can be considerably impacted by BQT in the short term, but it has reverted back to baseline by Week 10. FMT can alleviate gastrointestinal symptoms even if there was no evidence it promoted restoration of intestinal flora.

ZO-1 and IL-1RAP Phosphorylation: Potential Role in Mediated Brain-Gut Axis Dysregulation in Irritable Bowel Syndrome-like Stressed Mice.

Background and Objectives: Irritable Bowel Syndrome (IBS) is a common gastrointestinal disorder often exacerbated by stress, influencing the brain-gut axis (BGA). BGA dysregulation, disrupted intestinal barrier function, altered visceral sensitivity and immune imbalance defects underlying IBS pathogenesis have been emphasized in recent investigations. Phosphoproteomics reveals unique phosphorylation details resulting from environmental stress. Here, we employ phosphoproteomics to explore the molecular mechanisms underlying IBS-like symptoms, mainly focusing on the role of ZO-1 and IL-1RAP phosphorylation. Materials and Methods: Morris water maze (MWM) was used to evaluate memory function for single prolonged stress (SPS). To assess visceral hypersensitivity of IBS-like symptoms, use the Abdominal withdrawal reflex (AWR). Colonic bead expulsion and defecation were used to determine fecal characteristics of the IBS-like symptoms. Then, we applied a phosphoproteomic approach to BGA research to discover the molecular mechanisms underlying the process of visceral hypersensitivity in IBS-like mice following SPS. ZO-1, p-S179-ZO1, IL-1RAP, p-S566-IL-1RAP and GFAP levels in BGA were measured by western blotting, immunofluorescence staining, and enzyme-linked immunosorbent assay to validate phosphorylation quantification. Fluorescein isothiocyanate-dextran 4000 and electron-microscopy were performed to observe the structure and function of the intestinal epithelial barrier. Results: The SPS group showed changes in learning and memory ability. SPS exposure affects visceral hypersensitivity, increased fecal water content, and significant diarrheal symptoms. Phosphoproteomic analysis displayed that p-S179-ZO1 and p-S566-IL-1RAP were significantly differentially expressed following SPS. In addition, p-S179-ZO1 was reduced in mice's DRG, colon, small intestine, spinal and hippocampus and intestinal epithelial permeability was increased. GFAP, IL-1ß and p-S566-IL-1RAP were also increased at the same levels in the BGA. And IL-1ß showed no significant difference was observed in serum. Our findings reveal substantial alterations in ZO-1 and IL-1RAP phosphorylation, correlating with increased epithelial permeability and immune imbalance. Conclusions: Overall, decreased p-S179-ZO1 and increased p-S566-IL-1RAP on the BGA result in changes to tight junction structure, compromising the structure and function of the intestinal epithelial barrier and exacerbating immune imbalance in IBS-like stressed mice.

Muscone alleviates neuronal injury via increasing stress granules formation and reducing apoptosis in acute ischemic stroke.

As the main bioactive component of musk, muscone has been reported to have marked protective effects in treating acute ischemic stroke (AIS). However, the specific anti-stroke mechanism of muscone still needs further research. In the current investigation, the PC12 cells OGD/R and the rat transient MCAO/R models were utilized as the AIS models. Serum hepatic and renal functional indexes (ALT, AST, BUN, and Cr) and cell viability were determined to select the appropriate muscone concentrations for in vitro and in vivo experiments. TTC, Hematoxylin and eosin (H&E), and Live/Dead staining were utilized to evaluate the protective effects of muscone in injured tissues and cells. Western blotting analysis, TUNEL staining, propidium iodide, and annexin V staining were applied to detect the anti-apoptotic effect of muscone. Double-label immunofluorescence staining of T-cell intracellular antigen-1 (TIA1) and Ras-GAP SH3 domain-binding protein 1 (G3BP1) was performed to observe whether muscone regulated the SG formation level. Molecular docking, TIA1 silencing and TIA1 overexpression experiments were employed to investigate the molecular mechanism underlying the regulation of SG formation by muscone. The 2, 3, 5-Triphenyl-tetrazolium chloride (TTC) staining and live/dead staining showed the AIS injury level of MCAO/R rat and the OGD/R PC12 cells were attenuated by muscone administration. The muscone significantly minimized the apoptosis rate in MCAO/R rats and OGD/R PC12 cells following flow cytometry analysis, western blotting analysis, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The double-label immunofluorescence staining data revealed that muscone promoted the SG formation level in OGD/R PC12 cells and the cortex MCAO/R rats. The results of molecular docking, TIA1 silencing and TIA1 overexpression experiments revealed that muscone could bind to TIA1 protein and regulate its expression level, thereby promoting the formation of stress granules and exerting a protective effect against AIS injury. This study indicated that the significant protective effect of muscone in reducing apoptosis levels might be via promoting SG formation under AIS conditions. This study further explores the therapeutic effect and anti-apoptosis mechanism of muscone in AIS, which may provide a potential candidate drug for the clinical treatment of AIS injury.

Mechanistic Insights into How the Single Point Mutation Change the Autoantibody Repertoire.

A recent study showed that just one point mutation F33 to Y in the complementarity-determining region 1 of heavy chain (H-CDR1) could lead to the auto-antibody losing its DNA binding ability. However, the potential molecular mechanisms have not been well elucidated. In this study, we investigated how the antibody lost the DNA binding ability caused by mutation F33 to Y in the H-CDR1. We found that the electrostatic force was not the primary driving force for the interaction between anti-DNA antibodies and the antigen single strand DNA (ssDNA), and that the H-CDR2 largely contributed to the binding of antigen ssDNA, even larger than H-CDR1. The H-F33Y mutation could increase the hydrogen-bond interaction but impair the pi-pi stacking interaction between the antibody and ssDNA. We further found that F33H, W98H and Y95L in the wiletype antibody could form the stable pi-pi stacking interaction with the nucleotide bases of ssDNA. However, the Y33 in mutant could not form the parallel sandwich pi-pi stacking interaction with the ssDNA. To further confirm the importance of pi-pi stacking, the wildtype antibody and the mutants (F33YH, F33AH, W98AH and Y95AL) were experimentally expressed in CHO cells and purified, and the results from ELISA clearly showed that all the mutants lost the ssDNA binding ability. Taken together, our findings may not only deepen the understanding of the underlying interaction mechanism between autoantibody and antigen, but also broad implications in the field of antibody engineer.

[A prospective comparative study on effectiveness of single versus continuous adductor canal block combined with local infiltration anesthesia in unicompartmental knee arthroplasty].

Objective: To compare the early analgesic effects and the impact on knee joint function recovery after unicompartmental knee arthroplasty (UKA) between single adductor canal block (SACB) and continuous adductor canal block (CACB) combined with local infiltration anesthesia (LIA) using a prospective study. Methods: The patients with knee osteoarthritis admitted between April 2022 and December 2023 were enrolled as a subject. Among them, 60 patients met the selection criteria and were enrolled in the study. They were randomly assigned to the SACB group or CACB group in a ratio of 1:1 using a random number table method. There was no significant difference between the two groups ( P>0.05) in terms of age, gender, height, body mass, body mass index, affected side, and preoperative resting visual analogue scale (VAS) score and active VAS score, Oxford knee score (OKS), and American Hospital of Special Surgery (HSS) score. All patients received multimodal analgesia management using LIA combined with SACB or CACB. The operation time, pain related indicators (resting and activity VAS scores, number and timing of breakthrough pain, opioid consumption), joint function related indicators (quadriceps muscle strength, knee range of motion, OKS score, and HSS score), as well as postoperative block complications and adverse events were recorded and compared between the two groups. Results: There was no significant difference in the operation time between the two groups ( P<0.05). All patients in the two groups were followed up with a follow-up time of (9.70±4.93) months in the SACB group and (12.23±5.05) months in the CACB group, and the difference was not significant ( P>0.05). The CACB group had a significant lower resting VAS score at 24 hours after operation compared to the SACB group ( P<0.05). There was no significant difference in resting and active VAS scores between the two groups at other time points ( P>0.05). The CACB group had a significantly lower incidence of breakthrough pain compared to the SACB group [9 cases (30.00%) vs. 17 cases (56.67%); P<0.05). However, there was no significant difference in the timing of breakthrough pain occurrence and opioid consumption between the two groups ( P>0.05). Four cases in the SACB group and 7 cases in the CACB group experienced adverse events, with no significant difference in the incidence of adverse events between the two groups ( P>0.05). The CACB group had significantly better knee joint mobility than the SACB group at 1 and 2 days after operation ( P<0.05). There was no significant difference between the two groups in knee joint mobility on 0 day after operation and quadriceps muscle strength and OKS and HSS scores at different time points ( P>0.05). Conclusion: In UKA, the analgesic effects and knee joint function recovery are similar when compared between LIA combined with SACB and LIA combined with CACB. However, SACB is simpler to perform and can avoid adverse events such as catheter displacement and dislocation. Therefore, SACB may be a better choice.

Immunosuppressive regulatory cells in cancer immunotherapy: restrain or modulate?

Immunosuppressive regulatory cells (IRCs) play important roles in negatively regulating immune response, and are mainly divided into myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). Large numbers of preclinical and clinical studies have shown that inhibition or reduction of IRCs could effectively elevate antitumor immune responses. However, several studies also reported that excessive inhibition of IRCs function is one of the main reasons causing the side effects of cancer immunotherapy. Therefore, the reasonable regulation of IRCs is crucial for improving the safety and efficiency of cancer immunotherapy. In this review, we summarised the recent research advances in the cancer immunotherapy by regulating the proportion of IRCs, and discussed the roles of IRCs in regulating tumour immune evasion and drug resistance to immunotherapies. Furthermore, we also discussed how to balance the potential opportunities and challenges of using IRCs to improve the safety of cancer immunotherapies.

Fat mass and obesity-associated protein regulates RNA methylation associated with spatial cognitive dysfunction after chronic cerebral hypoperfusion.

RNA methylation can epigenetically regulate learning and memory. However, it is unclear whether RNA methylation plays a critical role in the pathophysiology of Vascular dementia (VD). Here, we report that expression of the fat mass and obesity associated gene (FTO), an RNA demethylase, is downregulated in the hippocampus in models of VD. Through prediction and dual-luciferase reporters validation studies, we observed that miRNA-711 was upregulated after VD and could bind to the 3'-untranslated region of FTO mRNA and regulate its expression in vitro. Methylated RNA immunoprecipitation (MeRIP)-qPCR assay and functional study confirmed that Syn1 was an important target gene of FTO. This suggests that FTO is an important regulator of Syn1. FTO upregulation by inhibition of miR-711 in the hippocampus relieves synaptic association protein and synapse deterioration in vivo, whereas FTO downregulation by miR-711 agomir in the hippocampus leads to aggravate the synapse deterioration. FTO upregulation by inhibition of miR-711 relieves cognitive impairment of rats VD model, whereas FTO downregulation by miR-711 deteriorate cognitive impairment. Our findings suggest that FTO is a regulator of a mechanism underlying RNA methylation associated with spatial cognitive dysfunction after chronic cerebral hypoperfusion.

Volatile oil of Angelica sinensis Radix improves cognitive function by inhibiting miR-301a-3p targeting Ppp2ca in cerebral ischemia mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Angelica Sinensis Radix (ASR) is a commonly used Chinese medicine known for its effects on tonifying blood, promoting blood circulation, and alleviating pain associated with menstrual regulation. Additionally, it has been used in the treatment of vascular cognitive impairment (VCI). The primary pharmacodynamic agent within ASR is volatile oil of Angelica Sinensis Radix (VOASR), which has demonstrated efficacy in combating cognitive impairment, although its mechanism remains unclear. OBJECTIVE: This study aimed to elucidate the potential molecular mechanisms underlying VOASR's improvement of cognitive function in cerebral ischemic mice. METHODS: A model of cerebral ischemic mice was established through unilateral common carotid artery occlusion (UCCAO) surgery, followed by intervention with VOASR. Cognitive function was assessed using the Morris water maze (MWM) test, while RT-qPCR was utilized to measure the differential expression of miR-301a-3p in the hippocampus. To evaluate cognitive function and hippocampal protein differences, wild-type mice and miR-301a-3p knockout mice were subjected to the MWM test and iTRAQ protein profiling. The relationship between miR-301a-3p and potential target genes was validated through a Dual-Luciferase Reporter experiment. RT-qPCR and Western blot were employed to determine the differential expression of Ppp2ca and synaptic plasticity-related proteins in the mouse hippocampus. RESULTS: Intervention with VOASR significantly improved cognitive impairment in cerebral ischemic mice and reduced the expression of miR-301a-3p in the hippocampus. Our findings suggest that miR-301a-3p may regulate cognitive function by targeting Ppp2ca. Furthermore, VOASR intervention led to an increase in the expression of Ppp2ca and synaptic plasticity-related proteins. CONCLUSION: Our study indicates that VOASR may be involved in regulating cognitive function by inhibiting miR-301a-3p, consequently increasing the expression of Ppp2ca and synaptic plasticity proteins. These results provide a new target and direction for the treatment of cognitive dysfunction.

Geraniin restricts inflammasome activation and macrophage pyroptosis by preventing the interaction between ASC and NLRP3 to exert anti-inflammatory effects.

Geraniin, a chemical component of the traditional Chinese medicine geranii herba, possesses anti-inflammatory and anti-oxidative activities. However, its anti-inflammatory role in managing NLRP3 inflammasome and pyroptosis remains to be elucidated. To investigate the anti-inflammation mechanism of geraniin, LPS-primed macrophages were incubated with classical activators of NLRP3 inflammasome (such as ATP, Nigericin, or MSU crystals), and MSU crystals were injected into the ankle joints of mice to establish an acute gouty arthritis model. The propidium iodide (PI) staining results showed that geraniin could restrain cell death in the ATP- or nigericin-stimulated bone marrow-derived macrophages (BMDMs). Geraniin decreased the release of lactate dehydrogenase (LDH) and interleukin (IL)-1ß from cytoplasm to cell supernatant. Geraniin also inhibited the expression of caspase-1 p20, IL-1ß in cell supernatant and N-terminal of gasdermin D (GSDMD-NT) while blocking the oligomerization of ASC to form speck. The inhibitory effects of geraniin on caspase-1 p20, IL-1ß, GSDMD-NT, and ASC speck were not observed in NLRP3 knockout (NLRP3-/-) BMDMs. Hence, the resistance of geraniin to inflammasome and pyroptosis was contingent upon NLRP3 presence. Geraniin reduced reactive oxygen species (ROS) production and maintained mitochondrial membrane potential while preventing interaction between ASC and NLRP3 protein. Additionally, geraniin diminished MSU crystal-induced mouse ankle joint swelling and IL-1ß expression. Geraniin blocked the recruitment of neutrophils and macrophages to the synovium of joints. Our results demonstrate that geraniin prevents the assembly of ASC and NLRP3 through its antioxidant effect, thereby inhibiting inflammasome activation, pyroptosis, and IL-1ß release to provide potential insights for gouty arthritis targeted therapy.

Predominant P3-Type Solid-Solution Phase Transition Enables High-Stability O3-Type Na-Ion Cathodes.

Layered O3-type oxides are one of the most promising cathode materials for Na-ion batteries owing to their high capacity and straightforward synthesis. However, these materials often experience irreversible structure transitions at elevated cutoff voltages, resulting in compromised cycling stability and rate performance. To address such issues, understanding the interplay of the composition, structure, and properties is crucial. Here, we successfully introduced a P-type characteristic into the O3-type layered structure, achieving a P3-dominated solid-solution phase transition upon cycling. This modification facilitated a reversible transformation of the O3-P3-P3' structure with minimal and gradual volume changes. Consequently, the Na0.75Ni0.25Cu0.10Fe0.05Mn0.15Ti0.45O2 cathode exhibited a specific capacity of approximately 113 mAh/g, coupled with exceptional cycling performance (maintaining over 70% capacity retention after 900 cycles). These findings shed light on the composition-structure-property relationships of Na-ion layered oxides, offering valuable insights for the advancement of Na-ion batteries.

Allosensitisation in NHP results in cross-reactive anti-SLA antibodies not detected by a lymphocyte-based flow cytometry crossmatch.

Xenotransplantation is a potential option for individuals for whom an acceptable human allograft is unavailable. Individuals with broadly reactive HLA antibodies due to prior exposure to foreign HLA are potential candidates for a clinical xenotransplant trial. It remains controversial if allosensitisation results in the development of cross-reactive antibodies against SLA. This may require increased histocompatibility scrutiny for highly sensitised individuals prior to enrollment in a clinical trial. Serum samples were obtained from non-human primates sensitised via serial skin transplantation from maximally MHC-mismatched donor, as reported. Sera from pre- and post-allosensitisation timepoints were assessed in a flow crossmatch (FXM) for IgM and IgG binding to pig splenocytes with or without red blood cell adsorption. Xenoreactive antibodies were eluted from pig splenocytes and screened on a single antigen HLA bead assay. A MHC Matchmaker algorithm was developed to predict potential conserved amino acid motifs among the pig, NHP, and human. Our sensitised NHP model was used to demonstrate that allosensitisation does not result in an appreciable difference in xenoreactive antibody binding in a cell-based FXM. However, antibody elution and screening on single antigen HLA beads suggest the existence of potential cross-reactive antibodies against SLA. The cross-reactive IgG after allosensitisation were predicted by comparing the recipient Mamu alleles against its previous allograft donor Mamu alleles and the donor pig SLA alleles. Our study suggests that allosensitisation could elevate cross-reactive antibodies, but a more sensitive assay than a cell-based FXM is required to detect them. The MHC Matchmaker algorithm was developed as a potential tool to help determine amino acid motif conservation and reactivity pattern.

Stabilization of TGF-ß Receptor 1 by a Receptor-Associated Adaptor Dictates Feedback Activation of the TGF-ß Signaling Pathway to Maintain Liver Cancer Stemness and Drug Resistance.

Dysregulation of the transforming growth factor-ß (TGF-ß) signaling pathway regulates cancer stem cells (CSCs) and drug sensitivity, whereas it remains largely unknown how feedback regulatory mechanisms are hijacked to fuel drug-resistant CSCs. Through a genome-wide CRISPR activation screen utilizing stem-like drug-resistant properties as a readout, the TGF-ß receptor-associated binding protein 1 (TGFBRAP1) is identified as a TGF-ß-inducible positive feedback regulator that governs sensitivity to tyrosine kinase inhibitors (TKIs) and promotes liver cancer stemness. By interacting with and stabilizing the TGF-ß receptor type 1 (TGFBR1), TGFBRAP1 plays an important role in potentiating TGF-ß signaling. Mechanistically, TGFBRAP1 competes with E3 ubiquitin ligases Smurf1/2 for binding to TGFΒR1, leading to impaired receptor poly-ubiquitination and proteasomal degradation. Moreover, hyperactive TGF-ß signaling in turn up-regulates TGFBRAP1 expression in drug-resistant CSC-like cells, thereby constituting a previously uncharacterized feedback mechanism to amplify TGF-ß signaling. As such, TGFBRAP1 expression is correlated with TGFΒR1 levels and TGF-ß signaling activity in hepatocellular carcinoma (HCC) tissues, as well as overall survival and disease recurrence in multiple HCC cohorts. Therapeutically, blocking TGFBRAP1-mediated stabilization of TGFBR1 by selective inhibitors alleviates Regorafenib resistance via reducing CSCs. Collectively, targeting feedback machinery of TGF-ß signaling pathway may be an actionable approach to mitigate drug resistance and liver cancer stemness.

MicroRNA-301a knockout attenuates peripheral nerve regeneration by delaying Wallerian degeneration.

Our recent study demonstrated that knockout of microRNA-301a attenuates migration and phagocytosis in macrophages. Considering that macrophages and Schwann cells synergistically clear the debris of degraded axons and myelin during Wallerian degeneration, which is a prerequisite for nerve regeneration, we hypothesized that microRNA-301a regulates Wallerian degeneration and nerve regeneration via impacts on Schwann cell migration and phagocytosis. Herein, we found low expression of microRNA-301a in intact sciatic nerves, with no impact of the microRNA-301a knockout on nerve structure and function. By contrast, we found significant upregulation of microRNA- 301a in injured sciatic nerves. We established a sciatic nerve crush model in microRNA-301a knockout mice, which exhibited attenuated morphological and functional regeneration following sciatic nerve crush injury. The microRNA-301a knockout also led to significantly inhibited Wallerian degeneration in an in vivo sciatic nerve-transection model and in an in vitro nerve explant block model. Schwann cells with the microRNA-301a knockout showed inhibition of phagocytosis and migration, which was reversible under transfection with microRNA-301a mimics. Rescue experiments involving transfection of microRNA-301a-knockout Schwann cells with microRNA-301a mimics or treatment with the C-X-C motif receptor 4 inhibitor WZ811 indicated the mechanistic involvement of the Yin Yang 1/C-X-C motif receptor 4 pathway in the role of microRNA-301a. Combined with our previous findings in macrophages, we conclude that microRNA-301a plays a key role in peripheral nerve injury and repair by regulating the migratory and phagocytic capabilities of Schwann cells and macrophages via the Yin Yang 1/C-X-C motif receptor 4 pathway.

Non-alcoholic steatohepatitis-related liver cirrhosis is increasing in China: A ten-year retrospective study

OBJECTIVE: Little is known about metabolic factors in cirrhotic patients in China. Therefore, we aimed to quantify the prevalence of both metabolic factors and non-alcoholic steatohepatitis-related liver cirrhosis in China. METHODS: The medical records of 1,582 patients diagnosed with liver cirrhosis from June 2003 to July 2013 at Daping Hospital (Chongqing, China) were retrospectively reviewed through a computer-generated search. RESULTS: Serum hepatitis B virus surface antigen was present in 1,083 (68.5%) patients, and hepatitis B was found to be the only etiological factor in 938 (59.3%) of all patients. Obesity, diabetes mellitus, and arterial hypertension were observed in 229 (14.5%), 159 (10.1%), and 129 (8.2%) patients, respectively. From 2012-2013, the proportion of non-alcoholic steatohepatitis-related liver cirrhosis increased to 3.2%, whereas the average proportion of non-alcoholic steatohepatitis-related liver cirrhosis in the previous ten years was 1.9%. The incidence of hepatocellular carcinoma was much higher in males than in females (6.3% vs. 3.7%, respectively, p=0.036). Obesity and diabetes mellitus did not significantly increase the incidence of hepatocellular carcinoma in the whole cirrhotic group. The presence of hepatitis B virus was the only risk factor for hepatocellular carcinoma in cirrhotic patients (p<0.001). CONCLUSIONS: Although hepatitis B virus remains the main etiology of liver cirrhosis in China, steatohepatitis-related liver cirrhosis is increasing in frequency. Hepatitis B virus was the sole significant risk factor for hepatocellular carcinoma in the whole cirrhotic group in the present study, in contrast to obesity and diabetes mellitus, for which only a trend of increased hepatocellular carcinoma was found. .