Clinical Application of Single-Surgeon, Three-Port, Laparoscopic Resection for Colorectal Cancer with Natural Orifice Specimen Extraction.

Reduced-port laparoscopic surgery (RPLS) has been widely used for the radical resection of gastrointestinal tumors. Single-surgeon, three-port, laparoscopic radical resection for sigmoid colon or high rectal cancer with natural orifice specimen extraction surgery (NOSES) has the advantage of a small incision, quick postoperative recovery, and short hospital stay. Yet, there are still only a few reports on NOSES. This paper describes the indications, preoperative preparations, surgical steps, and precautions for single-surgeon, three-port, laparoscopic radical resection of the sigmoid colon and high rectal cancer, and intraoperative specimen collection through the natural orifice. The protocol focuses on the steps of radical dissection and the main technical points of resection and reconstruction. At the same time, a procedure for fixing an anvil seat by self-traction of extracorporeal silk thread, used for purse-string suture fixation after the proximal anvil was placed in the abdominal cavity, was creatively improved. This operation could effectively avoid problems such as an insufficient proximal intestinal tube, shaking off the anvil seat, and weak purse-string suture during a single operation. The surgical care had less variability and was easy to perform, effectively avoiding postoperative anastomotic leakage and bleeding due to excessive intraoperative anastomotic tissue. This surgery could be widely promoted in primary hospitals.

The efficacy of irinotecan supplementation for colorectal cancer: A meta-analysis of randomized controlled studies.

BACKGROUND: The efficacy of irinotecan as the adjunctive therapy to fluorouracil and leucovorin remains controversial in patients with colorectal cancer. We conduct this meta-analysis to explore the efficacy of irinotecan supplementation for colorectal cancer. METHODS: We have searched PubMed, EMBASE, Web of science, EBSCO, and Cochrane library databases through March 19, 2020, and included randomized controlled trials assessing the efficacy of irinotecan plus fluorouracil and leucovorin for colorectal cancer. RESULTS: Five randomized controlled trials were included in the meta-analysis. Compared with fluorouracil and leucovorin for colorectal cancer, irinotecan supplementation could significantly improve progression-free survival rate (hazard ratio = 0.72; 95% confidence interval [CI] = 0.58-0.90; P = .003), median progression-free survival (standard mean difference = -0.30; 95% CI = -0.44 to -0.15; P < .0001), overall survival rate (hazard ratio = 0.77; 95% CI = 0.66-0.90; P = .001), and objective response (risk ratio [RR] = 0.57; 95% CI = 0.49-0.66; P < .00001) and decrease progressive disease (RR = 2.10; 95% CI = 1.40-3.14; P = .0003), but revealed no obvious effect on complete response (RR = 0.88; 95% CI = 0.33-2.29; P = .79). The incidence of grade ≥3 adverse events in irinotecan supplementation group was increased compared to control group (RR = 0.67; 95% CI = 0.57-0.79; P < .00001). CONCLUSIONS: Irinotecan as the adjunctive therapy to fluorouracil and leucovorin can increase the survival and objective response of patients with colorectal cancer, but the incidence of grade ≥3 adverse events is found to be increased after irinotecan supplementation.

Effectiveness of aquatic exercise in lower limb osteoarthritis: a meta-analysis of randomized controlled trials.

The objective of this study was to evaluate the short-term and follow-up effectiveness of aquatic training on the health status of lower limb osteoarthritis. Randomized controlled trials (RCTs) on related topics were systematically searched in PubMed, Embase, Web of Science, the Cochrane Library, Physiotherapy Evidence Database (PEDro), the China National Knowledge Infrastructure and Wanfang databases from inception to January 2021. RevMan 5.3 was used for statistical analysis, and the standardized mean difference (SMD) was used to present pooled effect sizes. As a result, 19 RCTs (1592 patients) were included. Compared with unsupervised home exercise or usual care (land-based training excluded), aquatic training showed short-term pain relief (SMD, -0.54; 95% CI, -0.81 to -0.28), physical function improvement (SMD, -0.64; 95% CI, -1.00 to -0.28), stiffness reduction (SMD, -0.40; 95% CI, -0.79 to -0.01) and improved function in sport and recreation (SMD, -0.30; 95% CI, -0.59 to -0.02). Analyses restricted to patients with knee osteoarthritis only also confirmed the positive effects of aquatic training on most dimensions excluding physical function. At medium-term follow-ups, improvements in physical function and function in sport and recreation were observed. No significant difference was observed between arms in the above four outcomes at long-term follow-ups. All studies reported no major adverse event with relation to aquatic training, and the minor adverse events were not common. It is concluded that aquatic training likely has short-term benefits on pain, physical function, stiffness and sport ability in lower limb osteoarthritis patients, but these positive effects may not last long.

Regulate the hydrophobic motif to enhance the non-classical secretory expression of Pullulanase PulA in Bacillus subtilis.

Bacillus subtilis has been widely used as a prokaryotic host for the secretory expression of heterologous proteins. In this study, a pullulanase (PulA) from Anoxybacillus sp. LM18-11 was firstly identified to be expressed in Bacillus subtilis 1A751 through non-classical secretion pathway. Results showed that both the N- and C-terminal regions of PulA were essential for its soluble expression. To explore its specific structural basis of secretion in B. subtilis, we revealed a hydrophobic motif A501-H507 which is vital for the secretion of the whole protein of PulA. Through a series of site-specific mutagenesis, the triple-sites mutants R503E/I506E/H507E and R503E/I506Y/H507E showed the highest extracellular activity (160.07 U/mL) and total activity (243.37 U/mL) which was 1.71 times and 1.55 times higher than those of PulA. The highest secretion rate of mutant I506E/H507E was more than 50% which was 34.72% higher comparing with that of PulA. The glutamic acid substitution on these three key surface sites which decreased the surface hydrophobicity of that region was confirmed to be beneficial to improve the secretory expression of PulA. This novel discovery for the secretory expression of PulA in B. subtilis would make a new perspective on regulating a kind of non-classical secretion in B. subtilis.

LncRNA CRNDE attenuates chemoresistance in gastric cancer via SRSF6-regulated alternative splicing of PICALM.

De novo and acquired resistance, which are mainly mediated by genetic alterations, are barriers to effective routine chemotherapy. However, the mechanisms underlying gastric cancer (GC) resistance to chemotherapy are still unclear. We showed that the long noncoding RNA CRNDE was related to the chemosensitivity of GC in clinical samples and a PDX model. CRNDE was decreased and inhibited autophagy flux in chemoresistant GC cells. CRNDE directly bound to splicing protein SRSF6 to reduce its protein stability and thus regulate alternative splicing (AS) events. We determined that SRSF6 regulated the PICALM exon 14 skip splice variant and triggered a significant S-to-L isoform switch, which contributed to the expression of the long isoform of PICALM (encoding PICALML). Collectively, our findings reveal the key role of CRNDE in autophagy regulation, highlighting the significance of CRNDE as a potential prognostic marker and therapeutic target against chemoresistance in GC.

The effect of VNS on the rehabilitation of stroke: A meta-analysis of randomized controlled studies.

INTRODUCTION: The efficacy of vagus nerve stimulation (VNS) for the rehabilitation of stroke remains controversial. We conduct a systematic review and meta-analysis to explore the influence of VNS on the rehabilitation of stroke. METHODS: We search PubMed, EMbase, Web of science, EBSCO, and Cochrane library databases through March 2020 for randomized controlled trials (RCTs) assessing the effect of VNS on the rehabilitation of stroke. This meta-analysis is performed using the random-effect model. RESULTS: Three RCTs are included in the meta-analysis. Overall, compared with control group in stroke, VNS is associated with significantly improved FMA-UE (SMD = 3.86; 95% CI = 1.19 to 6.52; P = 0.005) and Motor Function Test (SMD = 0.33; 95% CI = 0.04 to 0.62; P = 0.03), but has no obvious impact on Box and Block Test (SMD = -0.31; 95% CI = -3.48 to 2.86; P = 0.85), Nine-Hole Peg Test (SMD = 8.35; 95% CI = -40.59 to 57.28; P = 0.74), atrial fibrillation (RR = 3.46; 95% CI = 0.39 to 30.57; P = 0.26) or adverse events (RR = 0.59; 95% CI = 0.21 to 1.61; P = 0.30). CONCLUSIONS: VNS may be beneficial to the rehabilitation of stroke.

Multiplex genetic engineering improves endogenous expression of mesophilic α-amylase gene in a wild strain Bacillus amyloliquefaciens 205.

A wild strain Bacillus amyloliquefaciens 205 was screened for its high activity of α-amylase. A mesophilic α-amylase encoding gene amyE-205 was revealed and analyzed by genome sequencing. In order to facilitate plasmid transformation to strain 205, an interspecific plasmid transformation method was improved with 5-13 times higher in transformants than that of electronic transformation. A series of CRISPR genome editing tools have been successfully constructed for gene knockout, transcript repression and activation in 205 genome. At this basis, sporulation related genes spo0A and spoIIAC were knockout and suppressed with CRISPR/Cas9 and CRISPR/dCas9 respectively. The double knockout strain 205spo- was eliminated sporulation with 22.8% increasing of α-amylase activity. The optimal binding site G8 for dCas9-ω has been confirmed in the transcript activation. When amyE-205 was over-expressed with high copy plasmid pUC980-2, its whole upstream sequences containing G8 were also cloned. Whereafter, dCas9-ω was used to activate amyE-205 expression both at genome and plasmid. The final engineered strain 205PG8spo- achieved 784.3% promotion on α-amylase activity than the starting strain 205. The novel genetic tool box containing an efficient interspecific transformation method and functional CRISPR systems, superadded the multiplex regulation strategies used in strain modification would be also applicative in many Bacillus species.

Enhanced Heterologous Production of Glycosyltransferase UGT76G1 by Co-Expression of Endogenous prpD and malK in Escherichia coli and Its Transglycosylation Application in Production of Rebaudioside.

Steviol glycosides (SGs) with zero calories and high-intensity sweetness are the best substitutes of sugar for the human diet. Uridine diphosphate dependent glycosyltransferase (UGT) UGT76G1, as a key enzyme for the biosynthesis of SGs with a low heterologous expression level, hinders its application. In this study, a suitable fusion partner, Smt3, was found to enhance the soluble expression of UGT76G1 by 60%. Additionally, a novel strategy to improve the expression of Smt3-UGT76G1 was performed, which co-expressed endogenous genes prpD and malK in Escherichia coli. Notably, this is the first report of constructing an efficient E. coli expression system by regulating prpD and malK expression, which remarkably improved the expression of Smt3-UGT76G1 by 200% as a consequence. Using the high-expression strain E. coli BL21 (DE3) M/P-3-S32U produced 1.97 g/L of Smt3-UGT76G1 with a yield rate of 61.6 mg/L/h by fed-batch fermentation in a 10 L fermenter. The final yield of rebadioside A (Reb A) and rebadioside M (Reb M) reached 4.8 g/L and 1.8 g/L, respectively, when catalyzed by Smt3-UGT76G1 in the practical UDP-glucose regeneration transformation system in vitro. This study not only carried out low-cost biotransformation of SGs but also provided a novel strategy for improving expression of heterologous proteins in E. coli.

LASP1 interacts with N-WASP to activate the Arp2/3 complex and facilitate colorectal cancer metastasis by increasing tumour budding and worsening the pattern of invasion.

LIM and SH3 protein 1 (LASP1) is a metastasis-related protein reported to enhance tumour progression in colorectal cancer (CRC). However, the underlying mechanism is still elusive. As the major biological and pathological functions of LASP1 are accomplished by its LIM and SH3 domains via protein-protein interactions, a yeast two-hybrid system was employed to screen novel LASP1-interacting proteins. N-WASP, a member of the Wiskott-Aldrich syndrome protein (WASP) family, was screened and identified as a LASP1-interacting protein overexpressed in CRC tissues. N-WASP could stimulate the migration and invasion of CRC cells in vitro and increase the formation of subcutaneous tumours, mesenteric implanted tumours and hepatic metastatic tumours. N-WASP could interact with and activate the Arp2/3 complex to stimulate actin polymerization, thus changing the migratory and invasive capabilities of CRC cells. The interaction of LASP1 with N-WASP did not influence the expression of N-WASP but recovered the reduced actin polymerization induced by N-WASP silencing. High N-WASP expression was detected in most clinical colorectal samples, and it was positively correlated with the expression of LASP1 and ARP3, as well as the tumour budding and pattern of invasion, but negatively correlated with host lymphocytic response. Our study suggests a new mechanism for LASP1-mediated CRC metastasis determined by exploring LASP1-interacting proteins and identifies N-WASP as a potential therapeutic target for CRC.

High-level extracellular production of an alkaline pectate lyase in E. coli BL21 (DE3) and its application in bioscouring of cotton fabric.

A high heterologous expression of an alkaline pectate lyase (APL) pelNK93I in E. coli was obtained through optimizing the lactose feeding and fed-batch fermentation. The highest soluble APL activity produced by E. coli BL21 (pET22b-pelNK93I) was 10,181 U/mL which is the highest level so far. On this basis, to improve the extracellular yield of APL, optimized glycine feeding was used to achieve elevated extracellular production of pelNK93I. The highest extracellular APL activity produced by E. coli BL21 (pET22b-pelNK93I) was 6357 U/mL which was also relatively higher than that in previous reports. The final productivity of APL was 282.8 U/mL/h in the fermentation of E. coli BL21 (pET22b-pelNK93I) in a 10 L fermenter. Thus the current study has provided a cost-effective method for the over-expression and preparation of alkaline pectate lyase pelNK93I for its industrial applications. Moreover, pelNK93I (4 U/mL) used for bioscouring increased cottonseed husk removal and radial capillary effect of cotton fabric by 37.63% and 47.06%, respectively, making it a promising enzyme in green textile technology.

High copy number and highly stable Escherichia coli-Bacillus subtilis shuttle plasmids based on pWB980.

BACKGROUND: pWB980 derived from pUB110 is a promising expression vector in Bacillus for its high copy number and high stability. However, the low transformation rate of recombinant plasmids to the wild cells limited the application of it. On the basis of pWB980, constructing an E. coli-B. subtilis shuttle plasmid could facilitate the transformation rate to Bacillus cells. Because the insertion site for E. coli replication origin sequence (ori) is not unique in pWB980, in order to investigate the best insertion site, eight shuttle plasmids (pUC980-1 ~ pUC980-8) containing all possible insertion sites and directions were constructed. RESULTS: The results showed that all the selected insertion sites could be used to construct shuttle plasmid but some sites required a specific direction. And different insertion sites led to different properties of the shuttle plasmids. The best shuttle plasmids pUC980-1 and pUC980-2, which showed copies more than 450 per cell and segregational stabilities up to 98%, were selected for heterologous expressions of an alkaline pectate lyase gene pelN, an alkaline protease spro1 and a pullulanase gene pulA11, respectively. The highest extracellular activities of PelN, Spro1 and PulA11 were up to 5200 U/mL, 21,537 U/mL and 504 U/mL correspondingly after 54 h, 60 h and 48 h fermentation in a 10 L fermentor. Notably, PelN and Spro1 showed remarkably higher yields in Bacillus than previous reports. CONCLUSION: The optimum ori insertion site was the upstream region of BA3-1 in pWB980 which resulted in shuttle plasmids with higher copy numbers and higher stabilities. The novel shuttle plasmids pUC980-1 and pUC980-2 will be promising expression vectors in B. subtilis. Moreover, the ori insertion mechanism revealed in this work could provide theoretical guidance for further studies of pWB980 and constructions of other shuttle plasmids.

Transanal versus transabdominal specimen extraction in laparoscopic rectal cancer surgery: a retrospective analysis from China.

INTRODUCTION: Comparison of transanal specimen extraction (TSE) and transabdominal specimen extraction (TASE) in laparoscopic rectal surgery is still sparsely reported. Trauma, pain, scarring, and bad psychological suggestion have long been considered an inevitable outcome of surgery. For laparoscopic rectal cancer surgery, whether TSE or TASE is beneficial in terms of technical platforms, indications, contraindications, technical requirements for aseptic operation, tumor-free operation, prevention and treatment of complications still has not reached a unified consensus and standards. Recently, comparison of TSE and TASE in laparoscopic rectal surgery has still been sparsely reported. AIM: In this study, we retrospectively analyzed the short-term outcomes of TSE and TASE in laparoscopic rectal surgery in a single institution in southern China. MATERIAL AND METHODS: Patients who underwent laparoscopic radical rectal cancer surgery using either TSE or TASE were recruited. Data, including patient demographics, perioperative and postoperative variables, were analyzed retrospectively. RESULTS: Sixty-seven patients were included in this study. Thirty patients underwent TSE and 37 patients underwent TASE. The two groups were similar in demographics and tumor characteristics. Postoperative complications were similar in both groups, except that wound infection was lower for the TSE group (p = 0.122). The TSE group had a better cosmetic result with no abdominal incision and no differences in circumferential margins, distal resection margins or completeness of total mesorectal excision. CONCLUSIONS: Laparoscopic TSE is recommended in the treatment of rectal cancer with similar oncologic outcomes compared with conventional TASE. It is mini-invasive surgery and has the advantage of better cosmetic results. There is a need for further randomized studies to refine the applicability of laparoscopic TSE in rectal cancer.

Gut microbiota-stimulated cathepsin K secretion mediates TLR4-dependent M2 macrophage polarization and promotes tumor metastasis in colorectal cancer.

Metastasis is a complex process that requires the interaction between tumor cells and their microenvironment. As an important regulator of intestinal microenvironment, gut microbiota plays a significant role in the initiation and progression of colorectal cancer (CRC), but the underlying mechanisms remain elusive. In this study, a metastasis-related secretory protein cathepsin K (CTSK) was identified as a vital mediator between the imbalance of intestinal microbiota and CRC metastasis. We implanted MC38 cells into the cecal mesentry of antibiotic-treated mice with intragastrically administration of E. coli to mimic gut microbiota imbalance. The bigger primary tumors and more liver metastatic foci were detected in the E. coli group accompanied with high LPS secretion and CTSK overexpression compared with that in the control group. CTSK contributes to the aggressive phenotype of CRC cells both in vitro and in vivo. Silencing CTSK or administration of Odanacatib, a CTSK-specific inhibitor, totally abolished the CTSK-enhanced migration and motility of CRC cells. Interestingly, the tumor-secreted CTSK could bind to toll-like receptor 4 (TLR4) to stimulate the M2 polarization of tumor-associated macrophages (TAMs) via an mTOR-dependent pathway. Recombinant CTSK could neither stimulate CRC growth and metastasis, nor induce M2 macrophage polarization in TRL4-/- mice. Meanwhile, CTSK could stimulate the secretion of cytokines by M2 TAMs including IL10 and IL17, which, in turn, promote the invasion and metastasis of CRC cells through NFκB pathway. Clinically, overexpression of CTSK in human CRC tissues is always accompanied with high M2 TAMs in the stroma, and correlated with CRC metastasis and poor prognosis. Our current research identified CTSK as a mediator between the imbalance of gut microbiota and CRC metastasis. More importantly, we illustrated a CTSK-mediated-positive feedback loop between CRC cells and TAMs during metastasis, prompting CTSK as a novel predictive biomarker and feasible therapeutic target for CRC.

Tumor-secreted dickkopf2 accelerates aerobic glycolysis and promotes angiogenesis in colorectal cancer.

Angiogenesis is a fundamental process that involves in tumor progression and metastasis. Vascular endothelial growth factor (VEGF) family and their receptors are identified as the most prominent regulators of angiogenesis. However, the clinical efficacy of anti-VEGF/VEGFR therapy is not ideal, prompting the needs to further understand mechanisms behind tumor angiogenesis. Here, we found that Dickkopf associated protein 2 (DKK2), a secretory protein highly expressed in metastatic colorectal cancer tissues, could stimulate angiogenesis via a classic VEGF/VEGFR independent pathway. Methods: DKK2 was screened out from microarray data analyzing gene expression profiles of eight pairs of non-metastatic and metastatic human colorectal cancer (CRC) tissues. Immunofluorescence histochemical staining (IHC) was used to detect the expression of DKK2 and angiogenesis in CRC tissues. Chicken chorioallantoic membrane (CAM) assay and Human umbilical vein endothelial cells (HUVEC) tubule formation assay was used for in vitro and in vivo angiogenesis study, respectively. Lactate and glucose concentration in the culture medium was measured by enzyme-linked immunosorbent assay (ELISA). Luciferase reporter assay was used to verify the interaction between miR-493-5p and the 3'UTR of DKK2. Results: DKK2 could stimulate angiogenesis via accelerating the aerobic glycolysis of CRC cells, through which lactate is produced from glucose and accumulated in tumor microenvironment. Lactate functions as the final executor of DDK2 to stimulate tube formation of endothelial cells, and blockage of lactate secretion by lactate transporter (MCT) inhibitors dramatically neutralize the progression and metastasis of CRC both in vitro and in vivo. DKK2 could cooperate with lipoprotein receptor-related protein 6, which is required for glucose uptake, and activated the downstream mTOR signal pathway to accelerate lactate secretion. In addition, the expression of DKK2 is switched on via the demethylation of miR-493-5p, which allows the dissociated of miR-493-5p from the 3'-UTRs of DKK2 and initiates its stimulatory role on CRC progression in an autocrine or paracrine manner. Conclusion: DKK2 promotes tumor metastasis and angiogenesis through a novel VEGF-independent, but energy metabolism related pathway. DKK2 might be a potential anti-angiogenic target in clinical treatment for the advanced CRC patients.

Ethanol effects on the overexpression of heterologous catalase in Escherichia coli BL21 (DE3).

A novel method involving ethanol-induced increase in the heterologous recombinant protein expression in E. coli cells was commonly used in recent studies. However, the detailed mechanism of this method is still to be revealed. This work used comparative transcriptomic analysis and numerous experiments to uncover the mechanism of ethanol effects on the expression of heterologous catalase in the recombinant strain E. coli BL21 (pET26b-katA). The key regulatory genes malK and prpD were found to have the most significant effects on the expression of heterologous catalase. Thus, the maltose ABC transporter and carbon metabolism from propanoate metabolism to citrate cycle were found to be the main regulatory pathways activated by ethanol to enhance the synthesis of heterologous proteins. Based on these mechanisms, a universally applicable E. coli expression host strain for improving the expression of heterologous proteins might be constructed.

High expression of uPA related to p38MAPK in esophageal cancer indicates poor prognosis.

BACKGROUND: The aim of the study was to investigate the relationship between urokinase-type plasminogen activator (uPA) and mitogen-activated protein kinase 38 (p38MAPK), and preliminarily analyze their relationship with clinical characteristics of esophageal cancer. MATERIALS AND METHODS: Immunohistochemistry and Western blot were used to detect the expressions of uPA and p38MAPK in patients with esophageal cancer. The relationship between them and clinicopathological features was analyzed by chi-squared test and Spearman correlation. Prognosis was performed using Kaplan-Meier and Cox proportional hazard models analysis. RESULTS: The expressions of uPA and p38MAPK proteins were significantly higher in esophageal squamous cell carcinoma or adenocarcinoma than in normal esophageal mucosa tissue (both P<0.0001). The expression of uPA was significantly correlated with the depth of invasion of esophageal cancer (P=0.0067), tumor size (P=0.0364), and pathological stage (P<0.0001); p38MAPK expression vs esophageal cancer tissue type (P=0.0043), esophageal cancer infiltration depth (P=0.0097), tumor size (P=0.0015), and pathological stage (P<0.0001). Both were not significantly associated with lymph node staging, gender, age, and esophageal cancer histological type. There was a positive correlation between uPA and p38MAPK expressions (r=0.7301, P=0.0104). Kaplan-Meier analysis showed that the overall survival time of patients with positive expression of uPA or p38MAPK protein was significantly shorter, and the time of recurrence or metastasis of esophageal cancer was significantly earlier in patients with uPA-positive expression. Multivariate analysis of Cox model showed that uPA, p38MAPK, and pathological staging were independent factors influencing survival. CONCLUSION: The expressions of uPA and p38MAPK may play an important role in the progression of esophageal cancer, and there is a close relationship between the two proteins, which may be one of the prognostic indicators.

The close association between IL­12Rß2 and p38MAPK, and higher expression in the early stages of NSCLC, indicates a good prognosis for survival.

Interleukin­12 receptor (IL­12R) and p38 mitogen­activated protein kinase (p38MAPK) serve an important role in non­small cell lung cancer (NSCLC). It has previously been suggested that IL­12Rß2 may be involved in key regulatory pathways and interacts with the p38MAPK signaling pathway. The present study aimed to elucidate the possible association and roles of IL­12Rß2 and p38MAPK in NSCLC. The protein expression levels of IL­12Rß2 and p38MAPK were measured in 230 NSCLC tissue samples by immunohistochemistry (IHC) and western blot analyses. In addition, an immunofluorescence assay was used to observe the expression levels of these proteins in A549 and H358 cells. The associations between IL­12Rß2, p38MAPK and clinical characteristics, were evaluated by Pearson χ2 and Spearman correlation tests. Kaplan­Meier plots (log­rank test) and Cox proportional hazard models were used to analyze overall survival (OS). Compared with in benign pulmonary tissues, the expression levels of IL­12Rß2 and p38MAPK were not demonstrated to be significantly different in I+II pathological tumor­node­metastasis (pTNM) stage NSCLC tissues; however, reduced expression was detected in III+IV pTNM stage NSCLC tissues. Analysis of the association between advanced stage pTNM and the expression of both proteins demonstrated a significantly decreased Allred score (both P<0.0001), which was confirmed by IHC and western blot analyses. The IHC results demonstrated a significant correlation between IL­12Rß2 and p38MAPK expression (r=0.415, P=0.0143). By analyzing IL­12Rß2, p38MAPK expression and clinical characteristics, it was identified that IL­12Rß2 was significantly associated with gender (P=0.0168), age (P=0.0341), histological type (P<0.0001) and pTNM stage (P<0.0001). p38MAPK demonstrated a strong association with gender (P=0.0082) and pTNM stage (P<0.0001). The results of a Kaplan­Meier analysis indicated that positive IL­12Rß2 and p38MAPK expression was associated with increased OS compared with negative protein expression. The Cox proportional hazard models revealed that IL­12Rß2 and p38MAPK predicted a long OS. To the best of our knowledge, the present study is the first to reveal a close association between IL­12Rß2 and p38MAPK, and their possible function in NSCLC progression. It further demonstrated that expression of both proteins was lower with advanced pTNM staging, whereas a high expression of both proteins was associated with improved prognosis in NSCLC.

Construction of a plasmid interspecific transfer system in Bacillus species with the counter-selectable marker mazF.

Bacillus sp. strains as attractive hosts for the production of heterologous secretory proteins usually play important roles in bio-industry. However, low transformation efficiency of exogenous plasmids limited the application of Bacillus species. Here, a novel plasmid interspecific transfer system, with high transformation efficiency, high positive rate, and convenient manipulation, has been successfully constructed. A high electrotransformation efficiency strain Bacillus subtilis F-168 containing the counter-selectable marker mazF was used as the plasmid donor strain in this transfer method. A shuttled plasmid pBE980 and its recombinant plasmids pBE980::pulA and pBE980::HSPA were successfully transferred into the recipient Bacillus strains (Bacillus amyloliquefaciens 66, Bacillus licheniformis 124 and Bacillus megaterium 258) by this method. After co-culturing the donor cells (OD600nm = 1.3-1.7) and the recipient cells (OD600nm = 0.5-0.9) for 24 h in 22 °C, more than 1.0 × 105 positive transformants were obtained and a interspecific transformation efficiency of 1.0 × 10-3. It would provide a new approach for genetic manipulation in Bacillus strains and accelerate the research progress of the wild Bacillus strains in bio-industry.

Over expression of GPR30, indicating poor prognosis and promoting proliferation, upregulates Beclin-1 expression via p38MAPK signaling in esophageal squamous cell carcinoma progression.

BACKGROUND: Beclin-1 and GPR30, both very important proteins, have been associated with tumor development. In our pre-experiment, the co-expression of GPR30 and Beclin-1 was observed in esophageal squamous cell carcinoma (ESCC), an observation not reported in other studies. The aim of our research was to investigate the relationship of these two proteins in the further progression of ESCC. METHODS: The over expression of GPR30 and Beclin-1 proteins was observed and confirmed by immunohistochemistry and immunofluorescence arrays. By interfering with GPR30 and p38 MAPK expression in EC-109, KYSE510, and KYSE3 cell lines, MTT and a scratch wound healing assay were used to investigate the impact of the GPR30 protein on the proliferative and migrative abilities of ESCC cells. A co-immunoprecipitation assay was used to observe the interaction between the p38 MAPK and Beclin-1 proteins; meanwhile, at a different time, in each group, the GPR30, MAPK, p ERK1/2, p38 MAPK, and Beclin-1 proteins were analyzed. The correlation between GPR30, Beclin-1 expression levels, and the clinical characteristics were evaluated by Mann-Whitney and chi-square tests. Using Kaplan-Meier plots and the Cox proportional hazard model analysis, we determined overall survival (OS) and progression free survival (PFS). RESULTS: GPR30 and Beclin-1 were over expressed significantly in ESCC (both p=0.0000) and were distributed into cytoplasms the most (the former p=0.0223, latter p=0.0018). In contrast to the non-agonist group, the abilities of GPR30 in promoting cell proliferation and metastasis were observed in the agonist group, and the effects could be blocked by p38MAPK inhibitors. In further assays, GPR30 agonists, via binding to GPR30, up-regulated Beclin-1, MAPK, and p38 MAPK expression, and Beclin-1 expression was reversed in the p38MAPK inhibitor group. In the GPR30 agonist group, an interaction between p38MARK and Beclin-1 was observed, but no similar results were observed in the non-agonist group. The high expression of both GPR30 and Beclin-1 was observed with p-stage and pT advancing (both p<0.0001). In a Kaplan-Meier analysis, compared to GPR30's negative expression, high expression identified a group of patients with the shortest overall survival (OS, p=0.0072) and progression free survival (PFS, p=0.0074). The Cox proportional hazard models revealed that they predicted a short time to OS (p=0.0125). CONCLUSION: Over expression of GPR30 up-regulated Beclin-1 expression and indicated a poor prognosis and may promote ESCC development via p38 MAPK in ESCC progression.

Irisin protects against neuronal injury induced by oxygen-glucose deprivation in part depends on the inhibition of ROS-NLRP3 inflammatory signaling pathway.

Recent studies found that irisin, a newly discovered skeletal muscle-derived myokine during exercise, is also synthesized in various tissues of different species and protects against neuronal injury in cerebral ischemia. The NOD-like receptor pyrin 3 (NLRP3) inflammasome play an important role in detecting cellular damage and mediating inflammatory responses to aseptic tissue injury during ischemic stroke. However, it is unclear whether irisin is involved in the regulation of NLRP3 inflammasome activation during ischemic stroke. In the present study, PC12 neuronal cells were exposed to oxygen-glucose deprivation (OGD), exogenous irisin (12.5, 25, 50nmol/L) or NLRP3 inhibitor glyburide (50, 100, 200µmol/L) were used as an intervention reagent, NLRP3 was over-expressed or suppressed by transfection with a NLRP3 expressing vector or NLRP3-specifc siRNA, respectively. Our data showed that both irisin and its precursor protein fibronectin type III domain containing 5 (FNDC5) expression were significantly down-regulated (p<0.05); but oxidative stress and ROS-NLRP3 inflammasome signaling were activated by OGD (p<0.05); treatment with irisin or inhibition of NLRP3 reversed OGD-induced oxidative stress and inflammation (p<0.05). However, these irisin-mediated effects were blunted by over-expression NLRP3 (p<0.05). Taken together, our results firstly revealed that irisin mitigated OGD-induced neuronal injury in part via inhibiting ROS-NLRP3 inflammatory signaling pathway, suggesting a likely mechanism for irisin-induced therapeutic effect in ischemic stroke.