Using CO2 and oxidants for in situ regeneration of permeable reactive barriers for leachate-contaminated groundwater.

Groundwater contamination near landfills is commonly caused by leachate leakage, and permeable reactive barriers (PRBs) are widely used for groundwater remediation. However, the deactivation and blockage of the reactive medium in PRBs limit their long-term effectiveness. In the current study, a new methodology was proposed for the in situ regeneration of PRB to remediate leachate-contaminated groundwater. CO2 coupled with oxidants was applied for the dispersion and regeneration of the fillers; by injecting CO2 to disperse the fillers, the permeability of the PRB was increased and the oxidants could flow evenly into the PRB. The results indicate that the optimum filler proportion was zero-valent iron (ZVI)/zeolites/activated carbon (AC) = 3:8:10 and the optimum oxidant proportion was COD/Na2S2O8/H2O2/Fe2+ = 1:5:6:5; the oxidation system of Fe2+/H2O2/S2O82- has a high oxidation efficiency and persistence. The average regeneration rate of zeolites was 72.71%, and the average regeneration rate of AC was 68.40%; the permeability of PRB also increased. This technology is effective for the remediation of landfills in China that have large contaminated areas, an uneven pollutant concentration distribution, and a long pollution duration. The purification mode of long-term adsorption and short-time in situ oxidation can be applied to the remediation of long-term high-concentration organically polluted groundwater, where pollution sources are difficult to cut off.

Olink proteomics analysis uncovers inflammatory proteins in patients with different states of bipolar disorder.

STUDY DESIGN: A prospective study. BACKGROUND: This study aims to investigate the relationship between different states of bipolar disorder (BD) and plasma inflammatory proteins, which may be used as their biomarkers. MATERIALS AND METHODS: We totally collected admission plasma from 16 healthy subjects and 32 BD patients, including 16 patients with BD manic episodes (BD-M) and 16 patients with BD depressive episodes (BD-D). Ten samples in each group were analyzed by proximity extension assays of 92 inflammation-related proteins, and all samples were verified by ELISA. Receiver-operating characteristic (ROC) curve analysis was performed to identify the diagnostic ability and cut-off values of potential biomarkers. RESULTS: Our findings showed that BD patients had significantly higher levels of IL6, MCP-1, TGF-α, IL8, and IL10-RB in comparison with healthy subjects, and their cut-off values were 0.531 pg/ml, 0.531 pg/ml, 0.469 pg/ml, 0.406 pg/ml, and 0.406 pg/ml, respectively. The levels of IL6, MCP-1, TGF-α, and IL8 in BD-M patients were significantly greater than in healthy individuals, and their cut-off values were 0.813 pg/ml, 0.688 pg/ml, 0.438 pg/ml, and 0.625 pg/ml, respectively. Moreover, we found cut-off values of 0.500 pg/mL and 0.688 ng/mL for TGF-α and ß-NGF, respectively, even though the levels in the BD-D group were much higher than in the control group. Furthermore, BD-M patients had significantly higher levels of IL6, FGF-19, IFN-γ, and IL-17C in comparison with BD-D patients. Likewise, 0.687 pg/ml, 0.500 pg/ml, 0.438 pg/ml, and 0.375 pg/ml were their cut-off values, respectively. Our findings also showed that the combination of these proteins had the highest diagnostic accuracy. CONCLUSIONS: Our findings showed that plasma inflammatory proteins were related to BD and its subtypes, which may be utilized as potential biomarkers of different stages of BD. Furthermore, we also found their cut-off values and their combinations to have the highest diagnostic accuracy, providing clinicians with a new method to rapidly differentiate BD and its subtypes and manage early targeted interventions.

Analysis of complete blood count and derived inflammatory indicators for bipolar disorder patients with different states.

Purposes: This study aimed to analyze the predictive ability of the complete blood count and derived inflammatory indicators for BD patients with different states to identify potential biomarkers. Methods: We collected the data of BD in-patients from January 2021 to March 2023. The complete blood count and derived inflammatory indicators were computed by univariate analysis, logistic regression analysis, and receiver operating characteristic (ROC) curve analysis. Results: In terms of BD patients, the levels of MON (p<0.0001), hs-CRP (p = 0.018), and NLR (p = 0.002) were independent risk factors in logistic regression analysis, as well as the cut-off values were 0.405 109/L, 2.600 mg/L, and 2.321, respectively. Regarding BD-M patients, the levels of MON (p<0.0001), hs-CRP (p = 0.012), and NLR (p = 0.002) were predictors in logistic regression analysis, and the cut-off values were, respectively, 0.505 109/L, 2.600 mg/L, and 2.620. Additionally, the levels of NLR (p = 0.006) and MHR (p<0.0001) were important indicators for BD-D and the cut-off values were 1.735 and 0.487, respectively. Furthermore, our findings showed that the level of MON (p = 0.001) was related to BD-mixed and the cut-off value was 0.340 109/L. Notably, MON+hs-CRP + NLR, MON+hs-CRP + NLR, and NLR + MHR had the highest diagnostic accuracy to predict BD, BD-M, and BD-D patients, respectively. Conclusion: Our findings showed that distinct inflammatory indicators were closely associated with BD and its different states. Additionally, we also identified their cut-off values and optimal combined predictive indicators in different states of BD, helping us improve diagnostic accuracy and better assess them to manage early targeted interventions.

Beneficial use of dredged sediments as a resource for mine reclamation: A case study of Lake Dianchi's management in China.

Dredging is one of the most effective methods for inhibiting the endogenous contamination of natural lakes. However, both the amount and the scope of dredging will be restricted if the disposal of the dredged sediment incurs considerable environmental and economic costs. The use of dredged sediments as a post-mining soil amendment for mine reclamation benefits both sustainable dredging and ecological restoration. This study incorporates a field planting experiment with a life cycle assessment to confirm the practical effectiveness of sediment disposal via mine reclamation, as well as its environmental and economic superiority over other alternative scenarios. The results show that the sediment offered plentiful organic matter and nitrogen for mine substrate, stimulating plant growth and increasing photosynthetic carbon fixation density, followed by enhanced plant root absorption and an improved soil immobilization effect on heavy metals. A 2:1 ratio of mine substrate to sediment is recommended to significantly promote the yield of ryegrass while reducing levels of groundwater pollution and soil contaminant accumulation. Due to the significant reduction in electricity and fuel, mine reclamation had minimal environmental impacts on global warming (2.63 × 10-2 kg CO2 eq./kg DS), fossil depletion (6.81 × 10-3 kg oil eq./DS), human toxicity (2.29 × 10-5 kg 1,4-DB eq/kg DS), photochemical oxidant formation (7.62 × 10-5 kg NOx eq./kg DS), and terrestrial acidification (6.69 × 10-5 kg SO2 eq./kg DS). Mine reclamation also had a lower cost (CNY 0.260/ kg DS) than cement production (CNY 0.965/kg DS) and unfired brick production (CNY 0.268/kg DS). The use of freshwater for irrigation and electricity for dehydration were the key factors in mine reclamation. Through this comprehensive evaluation, the disposal of dredged sediment for mine reclamation was verified to be both environmentally and economically feasible.

Chemotherapy reinforces anti-tumor immune response and enhances clinical efficacy of immune checkpoint inhibitors.

New evidence suggests that the clinical success of chemotherapy is not merely due to tumor cell toxicity but also arises from the restoration of immunosurveillance, which has been immensely neglected in previous preclinical and clinical researches. There is an urgent need for novel insights into molecular mechanisms and regimens that uplift the efficacy of immunotherapy since only a minority of cancer patients are responsive to immune checkpoint inhibitors (ICIs). Recent findings on combination therapy of chemotherapy and ICIs have shown promising results. This strategy increases tumor recognition and elimination by the host immune system while reducing immunosuppression by the tumor microenvironment. Currently, several preclinical studies are investigating molecular mechanisms that give rise to the immunomodulation by chemotherapeutic agents and exploit them in combination therapy with ICIs in order to achieve a synergistic clinical activity. In this review, we summarize studies that exhibit the capacity of conventional chemotherapeutics to elicit anti-tumor immune responses, thereby facilitating anti-tumor activities of the ICIs. In conclusion, combining chemotherapeutics with ICIs appears to be a promising approach for improving cancer treatment outcomes.

Effect of percutaneous nephrolithotripsy under guidance of B-ultrasound for the treatment of complex renal calculi.

The purpose of this study was to investigate the effect of percutaneous nephrolithotripsy guided by B-ultrasound in the treatment of complex renal calculi. A total of 82 patients with complex renal calculi were selected, who underwent percutaneous nephrolithotripsy guided by B-ultrasound. The average operation time was 101.52±8.35 minutes, the average intraoperative bleeding volume was 132.86±7.22 ml, the average hospital stay was 10.47 ±1.68 days, the primary calculi clearance rate was 85.37% (70 cases), and the secondary calculi clearance rate was 95.12% (78 cases). Postoperative complications occurred in 18 (21.95%) cases, including fever in 13 (15.85%), residual stone in 4 (4.88%), and delayed bleeding in 1 (1.22%) case. The findings show that percutaneous nephrolithotripsy guided by B-ultrasound is safe and effective in the treatment of complex renal calculi and is suitable for popularisation and application in primary hospitals.

State of the art of bone biomaterials and their interactions with stem cells: Current state and future directions.

BACKGROUND: There has been a rapid increase in bone tissue regeneration since the concept of "tissue engineering." Stem cell-based biomaterials have revolutionized the field of tissue regeneration. Biomaterials play an essential part in bone regeneration through their crucial substratum for cell differentiation, cohesion, and proliferation by manipulating cells. Numerous studies have been carried out in order to create a biomaterial with diverse biological and physical characteristics. Furthermore, they developed a cell microenvironment with the desired pore magnitude to stimulate stem cells to transform them from artificial to biological microenvironments. PURPOSE AND SCOPE: The current review aims to give a comprehensive overview of stem cells and biomaterials in bone tissue regeneration. SUMMARY: Initially, bone biology and its interaction with stem cells and biomaterials are briefly explained. Following that, the behavior and mechanism of biomaterials influencing the stem cells during bone tissue regeneration are emphasized. Lastly, the future outlook for tackling the current challenges for designing biomaterials/stem cell materials for bone tissue engineering (TE) is discussed. CONCLUSION: Compatible biomaterial for bone regeneration requires evaluating the structure, matrix, composition, flexibility, and nature of native bone tissue defects. The concept of TE offers a platform for designing biologically, physically, and chemically biocompatible biomaterials for stem cells to proliferate and differentiate. Currently, stem cells are increasingly used for TE with a promising outcome due to their self-renewal and differentiation potential. Furthermore, they can secrete biological-active compounds and modulate the fate and behavior of other cells in native tissues. Bone TE may flourish more rapidly and efficiently using stem cells.

The role of long non-coding RNAs and circular RNAs in bone regeneration: Modulating miRNAs function.

Although bone is a self-healing organ and is able to repair and restore most fractures, large bone fractures, about 10%, are not repairable. Bone grafting, as a gold standard, and bone tissue engineering using biomaterials, growth factors, and stem cells have been developed to restore large bone defects. Since bone regeneration is a complex and multiple-step process and the majority of the human genome, about 98%, is composed of the non-protein-coding regions, non-coding RNAs (ncRNAs) play essential roles in bone regeneration. Recent studies demonstrated that long ncRNAs (lncRNAs) and circular RNAs (circRNAs), as members of ncRNAs, are widely involved in bone regeneration by interaction with microRNAs (miRNAs) and constructing a lncRNA or circRNA/miRNA/mRNA regulatory network. The constructed network regulates the differentiation of stem cells into osteoblasts and their commitment to osteogenesis. This review will present the structure and biogenesis of lncRNAs and circRNAs, the mechanism of bone repair, and the bone tissue engineering in bone defects. Finally, we will discuss the role of lncRNAs and circRNAs in osteogenesis and bone fracture healing through constructing various lncRNA or circRNA/miRNA/mRNA networks and the involved pathways.

Mechanical stimuli-mediated modulation of bone cell function-implications for bone remodeling and angiogenesis.

Bone remodeling, expressed as bone formation and turnover, is a complex and dynamic process closely related to its form and function. Different events, such as development, aging, and function, play a critical role in bone remodeling and metabolism. The ability of the bone to adapt to new loads and forces has been well known and has proven useful in orthopedics and insightful for research in bone and cell biology. Mechanical stimulation is one of the most important drivers of bone metabolism. Interestingly, different types of forces will have specific consequences in bone remodeling, and their beneficial effects can be traced using different biomarkers. In this narrative review, we summarize the major mediators and events in bone remodeling, focusing on the effects of mechanical stimulation on bone metabolism, cell populations, and ultimately, bone health.

Modulating immune microenvironment during bone repair using biomaterials: Focusing on the role of macrophages.

Bone is a self-regenerative tissue that can repair small defects and fractures. In large defects, bone tissue is unable to provide nutrients and oxygen for repair, and autologous grafting is used as the gold standard. As an alternative method, the bone tissue regeneration approach uses osteoconductive biomaterials to overcome bone graft disadvantages. However, biomaterials are considered as foreign components that can stimulate host immune responses. Although traditional principles have been aimed to minimize immune reactions, the design of biomaterials has steadily shifted toward creating an immunomodulatory microenvironment to harness immune cells and responses to repair damaged tissue. Among immune cells, macrophages secrete various immunomodulatory mediators and crosstalk with bone-forming cells and play key roles in bone tissue engineering. Macrophage polarization toward M1 and M2 subtypes mediate pro-inflammatory and anti-inflammatory responses, respectively, which are crucial for bone repairing at different stages. This review provides an overview of the crosstalk between various immune cells and biomaterials, macrophage polarization, and the effect of physicochemical properties of biomaterials on the immune responses, especially macrophages, in bone tissue engineering.

Fuxin Granules ameliorate diabetic nephropathy in db/db mice through TGF-ß1/Smad and VEGF/VEGFR2 signaling pathways.

Diabetic nephropathy (DN) is a common disease, and patients often do not have satisfactory treatments. We investigated therapeutic effects of Fuxin Granules(FX) on DN and potential molecular mechanisms. We orally administered doses of FX to db/db mice for 10 weeks and measured total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol. H&E, PAS, Masson, and Oil Red O staining were used to observe the structure of kidneys and calculate indices of kidney function. We used pharmacological analysis to investigate potential mechanisms of FX. Relative mRNA and protein levels in the TGF-ß1/Smad, TGF-ß1/Smad, and VEGF/VEGFR2 pathways were examined. TC, TG, and LDL-C were markedly reduced, lipid accumulation was low, fibrosis reduced, kidney atrophy improved, kidney lipid droplet number significantly reduced, and glomerular filtration function improved by FX treatment. Multi-channel therapeutic effects in DN through the TGF-ß1/Smad and VEGF/VEGFR2 signaling pathways occurred, and FX substantially reduced expression of TGF-ß1 in the glomeruli. FX significantly inhibited TGF-ß1, Smad2/3 total protein levels, Smad2/3 phosphorylation mRNA levels of TGF-ß1, Smad2, and Smad3. eNOS, VEGFA, and VEGFR2 expression was regulated, levels of VEGFA and VEGFR2 were decreased, and FX increased eNOS. FX ameliorated symptoms of DN, resulting in marked improvement in hyperglycemia and hyperlipidemia and optimized structure and function of kidneys in db/db mice. FX efficacy was associated with the TGF-ß1/Smad and VEGF/VEGFR2 signaling pathways. We verified this potential mechanism and hope that this study will provide benefits for the clinical treatment of DN.

Cryptotanshinone Inhibits ERα-Dependent and -Independent BCRP Oligomer Formation to Reverse Multidrug Resistance in Breast Cancer.

Both long-term anti-estrogen therapy and estrogen receptor-negative breast cancer contribute to drug resistance, causing poor prognosis in breast cancer patients. Breast cancer resistance protein (BCRP) plays an important role in multidrug resistance. Here, we show that cryptotanshinone (CPT), an anti-estrogen compound, inhibited the oligomer formation of BCRP on the cell membrane, thus blocking its efflux function. The inhibitory effect of CPT on BCRP was dependent on the expression level of estrogen receptor α (ERα) in ERα-positive breast cancer cells. Furthermore, ERα-negative breast cancer cells with high expression of BCRP were also sensitive to CPT because CPT was able to bind to BCRP and inhibit its oligomer formation on the cell membrane, suggesting that the high level of BCRP expression is crucial for CPT to reverse drug resistance. The combination of CPT and chemotherapeutic agents displayed enhanced anticancer effects. The results suggest that CPT is a novel BCRP inhibitor via blocking the oligomer formation of BCRP on the cell membrane. CPT is able to inhibit the activity of BCRP in an ERα-dependent and -independent manner, sensitizing breast cancer cells to chemotherapy.

Role of regulatory B cells in gastric cancer: Latest evidence and therapeutics strategies.

Gastric cancer (GC) is the second most common cancer globally and kills about 700,000 people annually. Today's knowledge clearly shows a close and complicated relationship between the tumor microenvironment (TME) and the immune system. The immune system components can both stimulate tumor growth and inhibit tumor cells. However, numerous of these mechanisms are not yet fully understood. As an essential immune cell in humoral immunity, B lymphocytes can play a dual role during various pathologic states, including infections, autoimmune diseases, and cancer, depending on their phenotype and environmental signals. Inherently, B cells can inhibit tumor growth by producing antibodies as well as the presentation of tumor antigens. However, evidence suggests that a subset of these cells termed regulatory B cells (Bregs) with an inhibitory phenotype can suppress anti-tumor responses and support the tumor growth by producing anti-inflammatory cytokines and the expression of inhibitory molecules. Therefore, in this review, the role of Bregs in the microenvironment of GC and treatment strategies based on targeting this subset of B cells have been investigated.

In vivo behavior of bioactive glass-based composites in animal models for bone regeneration.

This review presents the recent advances and the current state-of-the-art of bioactive glass-based composite biomaterials intended for bone regeneration. Composite materials comprise two (or more) constituents at the nanometre scale, in which typically, one constituent is organic and functions as the matrix phase and the other constituent is inorganic and behaves as the reinforcing phase. Such materials, thereby, more closely resemble natural bio-nanocomposites such as bone. Various glass compositions in combination with a wide range of natural and synthetic polymers have been evaluated in vivo under experimental conditions ranging from unloaded critical-sized defects to mechanically-loaded, weight-bearing sites with highly favourable outcomes. Additional possibilities include controlled release of anti-osteoporotic drugs, ions, antibiotics, pro-angiogenic substances and pro-osteogenic substances. Histological and morphological evaluations suggest the formation of new, highly vascularised bone that displays signs of remodelling over time. With the possibility to tailor the mechanical and chemical properties through careful selection of individual components, as well as the overall geometry (from mesoporous particles and micro-/nanospheres to 3D scaffolds and coatings) through innovative manufacturing processes, such biomaterials present exciting new avenues for bone repair and regeneration.

Nanotechnology shaping stem cell therapy: Recent advances, application, challenges, and future outlook.

Currently, stem cell nanotechnology is one of the novel and exciting fields. Certain experimental studies conducted on the interaction of stem cells with nanostructures or nanomaterials have made significant progress. The significance of nanostructures, nanotechnology, and nanomaterials in the development of stem cell-based therapies for degenerative diseases and injuries has been well established. Specifically, the structure and properties of nanomaterials affecting the propagation and differentiation of stem cells have become a new interdisciplinary frontier in material science and regeneration medicines. In the current review, we highlight the recent major progress in this field, explore the application prospects, and discuss the issues, approaches, and challenges, to improve the applications of nanotechnology in the research and development of stem cells.

Microbiota-Derived Short-Chain Fatty Acids Promote LAMTOR2-Mediated Immune Responses in Macrophages.

ABSRTACTKlebsiella pneumoniae is a common cause of human-pneumonia-derived sepsis with high morbidity and mortality. The microbiota promotes and maintains host immune homeostasis. The mechanisms by which the gut microbiota affects the host defenses in the respiratory system systematically, however, remain poorly understood. Here, we show that gut microbiota depletion increases susceptibility to extracellular K. pneumoniae infections in terms of increased bacterial burdens in lung and decreased survival rates. Oral supplementation with gut microbiota-derived short-chain fatty acids (SCFAs), subsequently activating G protein-coupled receptor 43 (GPCR43), enhances a macrophage's capacity to phagocytose invading K. pneumoniae Furthermore, SCFAs and GPR43 increase macrophage bacterial clearance by upregulating LAMTOR2, which is further identified as an antibacterial effector and elucidated to facilitate phagosome-lysosome fusion and extracellular signal-regulated kinase (ERK) phosphorylation. Lastly, conditional ablation of Lamtor2 in macrophages decreases their antimicrobial activity, even though mice were pretreated with exogenous SCFA supplementation.IMPORTANCE These observations highlight that SCFAs promote macrophage elimination of K. pneumoniae via a LAMTOR2-dependent signal pathway and suggest that it is possible to intervene in K. pneumoniae pneumonia by targeting the gut microbiota.

Rhein modulates host purine metabolism in intestine through gut microbiota and ameliorates experimental colitis.

Background: Gut microbiota, which plays a crucial role in inflammatory bowel diseases (IBD), might have therapeutic benefits for ulcerative colitis or Crohn's disease. Targeting gut microbiota represents a new treatment strategy for IBD patients. Rhein is one of the main components of rhubarb and exhibits poor oral bioavailability but still exerts anti-inflammatory effects in some diseases. Therefore, we investigated the effect of rhein on colitis and studied its possible mechanisms. Methods: The chronic mouse colitis model was induced by four rounds of 2% dextran sulfate sodium (DSS) treatment. The mice were treated with 50 mg/kg and 100 mg/kg rhein daily, body weight, colon length, histological score, inflammatory cytokines in serum or intestine, and fecal lipocalin 2 concentration were determined. Th17 cell, Th1 cell and Th2 cell infiltration in the mesenteric lymph node were analyzed by flow cytometry. Metabolic profiles were collected by non-targeted metabolomics and key metabolic pathways were identified using MetaboAnalyst 4.0. We also assessed intestinal barrier permeability and performed 16s rDNA sequencing. Lactobacillus sp. was cultured, and fecal microbiota transplantation (FMT) was employed to evaluate the contribution of gut microbiota. Results: Rhein could significantly alleviate DSS-induced chronic colitis. Uric acid was identified as a crucial modulator of colitis and rhein treatment led to decreased uric acid levels. We determined that rhein changed purine metabolism indirectly, while the probiotic Lactobacillus was involved in the regulation of host metabolism. Uric acid resulted in a worsened intestinal barrier, which could be rescued by rhein. We further confirmed that rhein-treated gut microbiota was sufficient to relieve DSS-induced colitis by FMT. Conclusion: We showed that rhein could modulate gut microbiota, which indirectly changed purine metabolism in the intestine and subsequently alleviated colitis. Our study has identified a new approach to the clinical treatment of colitis.

Alterations in the Gut Microbiome and Cecal Metabolome During Klebsiella pneumoniae-Induced Pneumosepsis.

Klebsiella (K.) pneumoniae is a common cause of pneumonia-derived sepsis in human and is associated with high morbidity and mortality. The microbiota promotes and maintains host immune homeostasis during bacterial infections. However, the mechanisms by which the gut microbiota affects immune responses in the lung still remain poorly understood. Here, we performed cecal metabolomics sequencing and fecal 16s rRNA sequencing in K. pneumoniae-infected mice and uninfected controls and showed that K. pneumoniae infection led to profound alterations in the gut microbiome and thus the cecal metabolome. We observed that the levels of Lactobacillus reuteri and Bifidobacterium pseudolongum were significantly decreased in K. pneumoniae-infected mice. Spearman correlation analysis showed that alterations in the richness and composition of the gut microbiota were associated with profound changes in host metabolite concentrations. Further, short-chain fatty acids (SCFAs), including acetate, propionate, and butyrate, were detected in cecal contents and serum by gas chromatography-mass spectrometry (GC-MS). We observed that the concentrations of these three SCFAs were all lower in the infected groups than in the untreated controls. Lastly, oral supplementation with these three SCFAs reduced susceptibility to K. pneumoniae infections, as indicated by lower bacterial burdens in the lung and higher survival rates. Our data highlight the protective roles of gut microbiota and certain metabolites in K. pneumoniae-pneumonia and suggests that it is possible to intervene in this bacterial pneumonia by targeting the gut microbiota.

[CRISPR/Cas9-based knockout of GPR43 gene in RAW264.7 cells inhibits their phagocytosis to Klebsiella pneumoniae].

Objective To construct cell line RAW264.7 with stable knockout of GPR43 gene using CRISPR/Cas9 system, and explore the role and mechanism of GPR43 gene in Klebsiella pneumoniae infection. Methods Three pairs of small-guide RNA (sgRNA) targeting the GPR43 gene were designed and inserted into plasmid pLenticrisprV2. The recombinant plasmid pLenticrisprV2 containing sgRNA was packaged using a lentivirus packaging system. RAW264.7 cells were transfected with viruses, and monoclonal cells were screened using puromycin. The genomic DNA was extracted from the amplified monoclonal cells. The GPR43 gene-related sequences were sequenced and compared with the wild-type GPR43 gene to confirm the cell line with successful knockout (GPR43-/- RAW264.7 cells). The expression of GPR43 protein was detected by Western blotting. After GPR43-/- RAW264.7 cells were transfected with Klebsiella pneumoniae, the changes in the expression of interleukin-1ß (IL-1ß), IL-6 and tumor necrosis factor-α (TNF-α) in the cells were detected using real-time quantitative PCR. Additionally, the phagocytic capacity of RAW264.7 cells after GPR43 knockout was observed. Results Western blotting confirmed that GPR43 protein was not expressed in the selected monoclonal cells, and DNA sequencing showed that 34 bases were missing at the insertion site of sgRNA, which proved that GPR43 gene was successfully knocked out. After GPR43-/- RAW264.7 cells were transfected with Klebsiella pneumoniae, the levels of IL-1ß, IL-6 and TNF-α expression in the cells were all lower than those in the control group, and the phagocytic capacity of GPR43-/- RAW264.7 cells to Klebsiella pneumoniae decreased. Conclusion CRISPR/Cas9-based knockout of GPR43 gene in RAW264.7 cells can inhibit their phagocytosis for Klebsiella pneumoniae and production of inflammatory cytokines.

Effect of the transdifferentiation of BECs into myofibroblasts on the pathogenesis of secondary cholestatic hepatic fibrosis.

The present study investigated the effect of the transdifferentiation of bile duct epithelial cells (BECs) into myofibroblasts on the pathogenesis of secondary cholestatic hepatic fibrosis and examined the underlying mechanisms. A total of 60 male rats with hepatic fibrosis were randomly divided into two groups: A secondary cholestatic hepatic fibrosis model group induced by ligation of the bile duct (BDL) and a sham group, which only underwent segregation of the choledochus. Rats in the BDL group were dynamically observed after week 1, 2, 3 and 4 post-BDL, and the remaining rats were sacrificed after week 5 to determine histological changes and hydroxyproline content. The cellular co-localization of cytokeratin (CK)7/α-smooth muscle actin (SMA) or α-SMA/desmin was detected by immunofluorescence staining and laser confocal microscopy, while the protein expression levels of CK7, α-SMA and desmin were determined by western blot analysis. Sirius red staining was also performed and quantified. The results revealed a significant correlation between the protein expression of CK7 and α-SMA (r=0.9692, P<0.01). Furthermore, a predominant correlation between the number of cells stained for CK7/α-SMA and collagen deposition in liver tissues was identified, while the correlation of cells with co-localized α-SMA and desmin was less pronounced. The transdifferentiation of BECs into myofibroblasts may be a key pathological factor in secondary cholestatic hepatic fibrosis formation.