No abstract

Allostery is a common mechanism of controlling many biological processes such as enzyme catalysis, signal transduction, and metabolic regulation. The use of allostery to regulate protein activity is an important and promising strategy in drug discovery and biological network regulation. In order to modulate protein activity by allostery, predictive methods need to be developed to discover allosteric binding sites. In the present study, we developed a new approach to identify allosteric sites in proteins based on the coarse-grained two-state Go̅ model. Starting from the concept that allostery is a conformation population shift process, we first constructed an ensemble of two functional states of a protein and tuned the energy landscape to bias one state. We then added perturbations to a binding site and monitored the population distribution of the new ensemble. If population redistribution occurred, then the binding perturbed site was predicted as a potential allosteric site. Our approach successfully identified all the known allosteric sites in a set of test proteins. Several new allosteric sites in the test proteins were also predicted. By use of one of the new allosteric sites predicted from Escherichia coli phosphoglycerate dehydrogenase (PGDH), novel allosteric regulating molecules were screened by molecular docking and enzymatic assay. Three novel allosteric inhibitors were discovered and their binding modes were confirmed by mutation experiments and competitive assay. The IC50 of the strongest inhibitor discovered was 21 μM, which is comparable to that of the native allosteric inhibitor l-serine. The novel allosteric site discovered in PGDH is l-serine-independent, and inhibitors targeting this site can be used as novel regulators of the E. coli serine synthesis pathway. Our approach for allosteric site prediction is generally applicable and the predicted sites can be used in discovering novel allosteric regulating molecules.

No abstract

The weakened tumour colonization of attenuated Salmonella has severely hampered its clinical development. In this study, we investigated whether an anti-inflammation and antiangiogenesis compound triptolide could improve the efficacy of VNP20009, a highly attenuated Salmonella strain, against mice melanoma. By comparing the effects of conventional VNP20009 monotherapy and a combination therapy that uses both triptolide and VNP20009, we found that triptolide significantly improved the tumour colonization of VNP20009 by reducing the number of infiltrated neutrophils in the melanoma, which led to a larger necrotic area in the melanoma. Moreover, the combination therapy suppressed tumour angiogenesis by reducing the expression of VEGF in a synergistic manner, retarding the growth of the melanoma. Our study revealed that triptolide could significantly enhance the antitumour effect of VNP20009 by modulating tumour angiogenesis and the host immune response, providing a new understanding of the strategy to improve Salmonella-mediated tumour therapy.

Ischemic stroke is one of the leading causes of brain disease, with high morbidity, disability, and mortality. MicroRNAs (miRNAs) have been identified as vital gene regulators in various types of human diseases. Accumulating evidence has suggested that aberrant expression of miRNAs play critical roles in the pathologies of ischemic stroke. Yet, the precise mechanism by which miRNAs control cerebral ischemic stroke remains unclear. In the present study, we explored whether miR-455 suppresses neuronal death by targeting TRAF3 in cerebral ischemic stroke. The expression levels of miR-455 and TRAF3 were detected by quantitative real-time polymerase chain reaction and Western blot. The role of miR-455 in cell death caused by oxygen–glucose deprivation (OGD) was assessed using Cell Counting Kit-8 (CCK-8) assay. The influence of miR-455 on infarct volume was evaluated in mouse brain after middle cerebral artery occlusion (MCAO). Bioinformatics softwares and luciferase analysis were used to find and confirm the targets of miR-455. The results showed that the expression levels of miR-455 significantly decreased in primary neuronal cells subjected to OGD and mouse brain subjected to MCAO. In addition, forced expression of miR-455 inhibited neuronal death and weakened ischemic brain infarction in focal ischemia-stroked mice. Furthermore, TRAF3 was proved to be a direct target of miR-455, and miR-455 could negatively suppress TRAF3 expression. Biological function analysis showed that TRAF3 silencing displayed the neuroprotective effect in ischemic stroke and could enhance miR-455-induced positive impact on ischemic injury both in vitro and in vivo. Taken together, miR-455 played a vital role in protecting neuronal cells from death by downregulating TRAF3 protein expression. These findings may represent a novel latent therapeutic target for cerebral ischemic stroke.

Reports in the field of robotic surgery for gastric cancer are increasing. However, studies only on patients with advanced gastric cancer (AGC) are lacking. This retrospective study was to compare the short-term outcomes of robotic-assisted distal gastrectomy (RADG) and laparoscopic-assisted distal gastrectomy (LADG) with D2 lymphadenectomy for AGC. From December 2014 to November 2019, 683 consecutive patients with AGC underwent mini-invasive assisted distal gastrectomy. Propensityscore matching (PSM) analysis was conducted to reduce patient selection bias. Short-term outcomes were compared between the two groups. The clinical features were well matched in the PSM cohort. Compared with the LADG group, the RADG group was associated with less operative blood loss, a lower rate of postoperative blood transfusion, less volume of abdominal drainage, less time to remove abdominal drainage tube, retrieved more lymph node, and lower rates of surgical complications and pancreatic fistula (P <0.05). However, the time to recovery bowel function, the length of postoperative stay, the rates of other subgroups of complications and unplanned readmission were similar between the two groups (P > 0.05). This study suggests that RADG is a safe and feasible technique with better short-term outcomes than LADG for AGC.Gastric cancer continues to be a major public health problem worldwide, especially in developing countries, such as China 1,2 . Every year, approximately 680,000 new patients are diagnosed with gastric cancer in China; among them, more than 80% of these patients have advanced-stage disease 1,3 . For patients with advanced gastric cancer (AGC), multidisciplinary comprehensive treatment is usually required, and gastrectomy with lymph node dissection is currently considered to be the only curable treatment 4 .In the past few decades, with the development of science and technology, much advanced surgical equipment has been invented, particularly in the field of mini-invasive surgery (MIS). Since laparoscopy-assisted Billroth I gastrectomy was first reported in 1994, several randomized controlled trials (RCTs) have demonstrated that laparoscopic gastrectomy (LG) for AGC is a safe and feasible technique with better short-term outcomes than and similar oncological outcomes to open gastrectomy [5][6][7][8][9] . Although LG has obtained greater acceptance among abdominal surgeons, because of the limitations of laparoscopic instruments, it is difficult to perform precisely, as is the case with D2 lymphadenectomy 10,11 .Robotic surgery systems, as another MIS method, were invented to overcome the drawbacks of laparoscopy and are becoming increasingly accepted by abdominal surgeons. Since Hashizume et al. first reported the use of robotic surgery for gastric cancer in 2002, a number of studies have shown the safety and advantages of robotic

Attenuated Salmonella typhimurium (S. typhimurium) strains can selectively grow and express exogenous genes in tumors for targeted therapy. We engineered S. typhimurium strain VNP20009 to secrete tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) under the control of a hypoxia-induced nirB promoter and examined the efficacy of Salmonella-mediated targeted expression of TRAIL in mice bearing melanoma tumor and in TRAIL-resistant RM-1 tumor. We found that VNP preferentially accumulated in tumor tissues and the nirB promoter effectively drove targeted expression of TRAIL. Compared with recombinant TRAIL protein and VNP20009 combination therapy, VNP20009 expressing TRAIL significantly suppressed melanoma growth but failed to suppress RM-1 tumor growth. Furthermore, we confirmed that VNP20009 expressing TRAIL yielded its antitumor effect by inducing melanoma apoptosis. Our findings indicate that Salmonella-mediated tumor-targeted therapy with TRAIL could reduce tumor growth and extend host survival. (Cancer Sci 2012; 103: 325-333) T umor tissues consume a greater amount of oxygen than that supplied by the tumor-feeding blood vessels.(1) Normal tissues typically have median oxygen tensions of 40-70 mmHg, whereas that of most solid tumors is <10 mmHg. Clinically, poor oxygenation is a major indicator of adverse prognosis for solid tumors, which frequently contain large regions with low oxygen concentrations.(2-6) One contributing factor to the adverse outcome is the inadequate distribution of antitumor agents to the hypoxic areas of a tumor where blood flow is sluggish or highly irregular.(2) Strategies for improving tumor oxygenation have met with rather limited success.(7) Specific killing of hypoxic tumor cells by hypoxia-selective tumoricidal agents or modalities offers a separate strategy that views the presence of hypoxia in tumors not only as a determinant for poor prognosis but also as an opportunity for tumor-specific treatment.Selective targeting of tumors where hypoxia exists enhances targeted concentration of therapeutic agents of interest in cancer cells while minimizing damage to the normal tissues, regardless of whether these therapeutic agents are chemotherapeutic drugs or recombinant proteins or ectopic genes. Attenuated Salmonella typhimurium (S. typhimurium) strains have been shown to selectively grow and express exogenous genes in tumors for targeted therapy. (8)(9)(10)(11)(12) We have previously shown that the facultative anaerobe S. typhimurium strain VNP20009 can replicate in hypoxic tumors (13)(14)(15)(16) and is synergistic with cyclophosphamide against melanoma in a mouse tumor model. (17) Because the bacteria could preferentially accumulate in the hypoxic tumor microenvironment, there have been attempts to engineer controllable expression of therapeutic molecules such as prodrugconverting enzymes and antigens. (18)(19)(20)(21) Tumor necrosis factor-related apoptosis-inducing ligand can selectively induce apoptosis in cancer cells and is effective against many cancers, includin...

No abstract