An enzymatically activated AND-gate DNA logic circuit for tumor cells recognition via multi-microRNAs detection.

The DNA-based logic circuit, constructed to mimic biochemical reaction networks, is highly significant in detecting biomarkers at the molecular level. The differences in the expression levels of microRNAs (miRNAs) within different types of cells provide hope for distinguishing cell subtypes. However, reliance on a single miRNA often leads to unreliable results. Herein, we constructed an enzyme-triggered cascade logic circuit based on the AND gate, which is capable of generating corresponding fluorescence signals in the presence of target miRNAs. The introduction of apurinic/apyrimidinic (AP) sites effectively reduces the likelihood of false signal generation. Amplification of the fluorescence signal relies on the catalytic hairpin assembly and the repetitive reuse of the multicomponent nucleic acid enzyme (MNAzyme). We demonstrated that the logic circuit can not only distinguish cancer cells from normal cells but also identify different types of cancer cells. The programmability of the logic circuits and the simplicity of the assay system allow us to modify the functional sequences to recognize different types of biomarkers, thus providing a reference for the identification of various cell subtypes.

A Simple Technique of Tunnel Constructing for Occluding Splenic Vessels During Laparoscopic Splenectomy in Patients with Cirrhosis.

Purpose: Dissecting and ligating the splenic artery is crucial for bleeding control during laparoscopic splenectomy (LS). However, for patients with portal hypertension from liver cirrhosis, it is difficult for identification and ligation because the splenic vessel is circuitous and dilated. The aim of this study was to describe a simple technique of constructing a tunnel behind the tail of the pancreas for occluding the splenic vessels during LS in patients with portal hypertension. Materials and Methods: The clinical data of 61 patients who underwent LS from April 2016 to January 2017 were retrospectively analyzed. In 27 patients, the tunnel construction (TC) behind the tail of the pancreas approach was performed owning to difficulty in dissecting and ligating the splenic artery (TC group), including 17 patients who received the TC method directly and 10 patients who received the TC method after trying to dissect the splenic artery. The remaining 34 patients underwent traditional ligating of the splenic artery (LA group). The peri- and postoperative outcomes of operative time, blood loss, conversion rate, postoperative oral diet intake, postoperative hospital stay, and postoperative complication rate of the two groups were analyzed. All the operations were completed by the same group of surgeons. Results: All 61 operations were successfully completed. Compared with patients in the LA group, patients in the TC group had less blood loss (120.37 ± 40.74 mL versus 162.65 ± 87.47 mL; t = -2.317, P = .024). There was no statistical difference of operative time, conversion rate, complication rate, postoperative hospital stays, and follow-up between the two groups. Conclusions: The technique of constructing a tunnel behind the tail of the pancreas for occluding the splenic vessels was effective and safe in those patients whose splenic artery was difficult to dissect and ligate.

Direct interaction between Rab5a and Rab4a enhanced epidermal growth factor-stimulated proliferation of gastric cancer cells.

BACKGROUND: Gastric cancer (GC) is one of the leading causes of cancer-related death worldwide. Although targeted therapies such as antibodies against human epidermal growth factor receptor 2 or vascular endothelial growth factor receptor 2 have been widely used in the treatment of metastatic cancer, the overall outcomes are poor. Therefore, elucidation of the mechanism underlying cancer progression is important to improve prognosis. Overexpression of the Rab5a gene has been confirmed to correlate with tumorigenesis of many cancers, but the mechanism underling, especially of GC, is still unclear. AIM: To investigate the effects of Rab5a overexpression on the tumorigenesis of GC. METHODS: First, the expression levels of Rab5a and Rab4a in primary tumorous tissues of GC patients diagnosed between 2015 and 2018 were analyzed. Then we constructed HGC-27 cell lines overexpressing green fluorescent protein-Rab5a or red fluorescent protein-Rab4a and investigated the interaction between Rab5a or Rab4a using Western blotting, co-immunoprecipitation, confocal microscopy, and colocalization analysis. Finally, epidermal growth factor-stimulated proliferation of these cell lines was analyzed using cell counting kit-8 cell viability assay. RESULTS: Compared with normal gastric tissues, the expression levels of Rab5a and Rab4a increased progressively both in paracancerous tissues and in advanced cancerous tissues. Epidermal growth factor could promote the proliferation of HGC-27 cells, especially Rab5a-overexpressing HGC-27 cells. Notably, Rab5a and Rab4a co-overexpression promoted the proliferation of HGC-27 cells to the greatest extent. Further analysis identified a direct interaction between Rab5a and Rab4a in HGC-27 cells. CONCLUSION: Co-overexpression of Rab5a and Rab4a in GC may promote the endosomal recycling of epidermal growth factor receptor, which in turn contributes to poor prognosis and tumor progression in GC patients. Inhibition of Rab5a or Rab4a expression might be a promising therapy for refractory GC.

Copy Number Amplification of DNA Damage Repair Pathways Potentiates Therapeutic Resistance in Cancer.

Rationale: Loss of DNA damage repair (DDR) in the tumor is an established hallmark of sensitivity to DNA damaging agents such as chemotherapy. However, there has been scant investigation into gain-of-function alterations of DDR genes in cancer. This study aims to investigate to what extent copy number amplification of DDR genes occurs in cancer, and what are their impacts on tumor genome instability, patient prognosis and therapy outcome. Methods: Retrospective analysis was performed on the clinical, genomics, and pharmacogenomics data from 10,489 tumors, matched peripheral blood samples, and 1,005 cancer cell lines. The key discoveries were verified by an independent patient cohort and experimental validations. Results: This study revealed that 13 of the 80 core DDR genes were significantly amplified and overexpressed across the pan-cancer scale. Tumors harboring DDR gene amplification exhibited decreased global mutation load and mechanism-specific mutation signature scores, suggesting an increased DDR proficiency in the DDR amplified tumors. Clinically, patients with DDR gene amplification showed poor prognosis in multiple cancer types. The most frequent Nibrin (NBN) gene amplification in ovarian cancer tumors was observed in 15 out of 31 independent ovarian cancer patients. NBN overexpression in breast and ovarian cancer cells leads to BRCA1-dependent olaparib resistance by promoting the phosphorylation of ATM-S1981 and homology-dependent recombination efficiency. Finally, integration of the cancer pharmacogenomics database of 37 genome-instability targeting drugs across 505 cancer cell lines revealed significant correlations between DDR gene copy number amplification and DDR drug resistance, suggesting candidate targets for increasing patient treatment response. Principal Conclusions: DDR gene amplification can lead to chemotherapy resistance and poor overall survival by augmenting DDR. These amplified DDR genes may serve as actionable clinical biomarkers for cancer management.

LncRNA differentiation antagonizing non-protein coding RNA promotes proliferation and invasion through regulating miR-135a/NLRP37 axis in pancreatic cancer.

Background It is well-known that long-chain non-coding RNA (LncRNA) plays an important role in the development of tumor. DANCR, which is one crucial part of the lncRNA family, has been shown to be involved in the invasion of various tumors. However, its molecular mechanism in pancreatic cancer remains unknown. Methods qRT-PCR was used to detect the expression of DANCR, miR-135a mRNA in pancreatic cancer tissues or cells. E-cadherin and NLRP3 protein levels were measured by Western Blot. CCK-8, cell scratch, and transwell assays were applied to detect the proliferation and invasion of pancreatic cancer cells. Bioinformatical analysis and luciferase assay were performed to explore the relationship among DANCR, miR-135a and NLRP3. Results In pancreatic cancer, DANCR was up-regulated while miR-135a was down-regulated. The over-expression of DANCR promoted the proliferation and invasion of pancreatic cancer cells. A negative relationship was found between DANCR and miR-135a expression. Moreover, we found that miR-135a reversed the effects of DANCR in the promoting of pancreatic cancer cells, which was achieved by regulating the downstream protein of NLRP3. The correlations among DANCR, miR-135a and NLRP3 were confirmed in animal experiments. Conclusion DANCR promoted proliferation and invasion through the regulating of miR-135a / NLRP3 axis in pancreatic cancer cell. Our results suggest that DANCR may be a potential target for the therapy of pancreatic cancer.

Detection of BRAF V600E mutation in fine-needle aspiration fluid of papillary thyroid carcinoma by droplet digital PCR.

OBJECTIVES: Papillary thyroid carcinoma (PTC) accounts for 85% of thyroid carcinoma, which is the most common endocrine tumor. For the diagnosis of PTC, ultrasound-guided fine needle aspiration (FNA) with pathological evaluation is the standard test and BRAF V600E mutation is the most common molecular marker associated with the occurrence, progression and poor clinicopathological characteristics of PTC. However, because of the small amount of the tumor cells obtained by FNA for pathological evaluation or BRAF V600E mutation detection, more sensitive and accurate methods are required. Our study aimed to investigate the performance of droplet digital PCR (ddPCR) in detecting BRAF V600E mutation in FNA samples from PTC patients. METHODS: One hundred and sixty suspected thyroid cancer patients were enrolled, including 146 PTC patients, 2 follicular thyroid carcinoma (FTC) and 12 benign patients, identified by FNA biopsy according to the NCCN clinical practice guidelines of Thyroid Carcinoma. ddPCR and amplification-refractory mutation system (ARMS, AmoyDx) were used to detect BRAFV600E mutation and the results were compared. RESULTS: ddPCR had high reproducibility (CV0.1% = 22.82% and CV10% = 4.85%) and the detection sensitivity can reach 1­2 copies/µl (0.01%). Among the 160 patients, 128 BRAF V600E mutations were detected, including 4 ARMS negative patients and 3 benign cases [corrected]. CONCLUSIONS: Our results demonstrated that ddPCR could be used in detecting BRAF V600E mutation from FNA fluid samples with higher sensitivity and accuracy than ARMS.

Digital droplet polymerase chain reaction analysis of common viruses in the aqueous humour of patients with Posner-Schlossman syndrome in Chinese population.

BACKGROUND: To compare the detection results consistency of quantitative polymerase chain reaction (qPCR) and digital droplet polymerase chain reaction (ddPCR), and determine the value of ddPCR for viral detection in the aqueous humour. METHODS: A total of 130 aqueous humour samples were collected, including 60 patients with Posner-Schlossman syndrome (PSS) in case group and 70 elderly patients with senile cataract in control group. The target nucleic acid fragments of human cytomegalovirus (HCMV), herpes simplex virus, Epstein-Barr virus and varicella zoster virus in aqueous humour were analysed by qPCR and ddPCR, respectively, for the diagnosis and curative effect monitoring of pathogen-induced PSS. Samples with inconsistent results were verified by next-generation sequencing. RESULTS: There were 27 and 20 HCMV-positive cases detected in the case group by ddPCR and qPCR, respectively. ddPCR increased the sensitivity for the HCMV virus detection from 400 to 100 copies/mL. No other pathogens were found in this study. The results of ddPCR were consistent with that of next generation sequencing. The mean (SD) of Lg (HCMV copies/mL) detected by ddPCR and qPCR were 1.66 (1.92) and 1.10 (1.61), respectively (P < 0.001). Compared with qPCR, results of ddPCR showed better consistency with validity of clinical treatment. All patients with ddPCR-positive results had good validity on antiviral therapy, exhibiting anterior chamber inflammation remission, resolution of corneal oedema and good IOP control within 1 month. CONCLUSIONS: HCMV was the leading cause of pathogen-induced PSS in the Chinese population. ddPCR was a promising tool for early detection, accurate diagnosis and therapeutic validity monitoring of pathogen-induced PSS. The high sensitivity of ddPCR could avoid repeated anterior chamber tap.

Cathepsin B inhibition ameliorates the non-alcoholic steatohepatitis through suppressing caspase-1 activation.

Non-alcoholic fatty liver disease (NAFLD) has emerged as the most common chronic liver disease. NLRP3 inflammasome activation has been widely studied in the pathogenesis of NAFLD. Cathepsin B (CTSB) is a ubiquitous cysteine cathepsin, and the role of CTSB in the progression and development of NAFLD has received extensive concern. However, the exact roles of CTSB in the NAFLD development and NLRP3 inflammasome activation are yet to be evaluated. In the present study, we used methionine choline-deficient (MCD) diet to establish mice NASH model. CTSB inhibitor (CA-074) was used to suppress the expression of CSTB. Expressions of CTSB and caspase-1 were evaluated by immunohistochemical staining. Serum IL-1ß and IL-18 levels were also determined. Palmitic acid was used to stimulate Kupffer cells (KCs), and protein expressions of CTSB, NLRP3, ASC (apoptosis-associated speck-like protein containing CARD), and caspase-1 in KCs were detected. The levels of IL-1ß and IL-18 in the supernatant of KCs were evaluated by enzyme-linked immunosorbent assay (ELISA). Our results showed that CTSB inhibition improved the liver function and reduced hepatic inflammation and ballooning, and the levels of pro-inflammatory cytokines IL-1ß and IL-18 were decreased. The expressions of CTSB and caspase-1 in liver tissues were increased in the NASH group. In in vitro experiments, PA stimulation could increase the expressions of CTSB and NLRP3 inflammasome in KCs, and CTSB inhibition downregulated the expression of NLRP3 inflammasome in KCs, when challenged by PA. Moreover, CTSB inhibition effectively suppressed the expression and activity of caspase-1 and subsequently secretions of IL-1ß and IL-18. Collectively, these results suggest that CTSB inhibition limits NLRP3 inflammasome-dependent NASH formation through regulating the expression and activity of caspase-1, thus providing a novel anti-inflammatory signal pathway for the therapy of NAFLD.

Rapid detection of CALR type 1 and type 2 mutations using PNA-LNA clamping loop-mediated isothermal amplification on a CD-like microfluidic chip.

Bleeding and thrombosis represent common complications in myeloproliferative neoplasms (MPN) and significantly contribute to morbidity and mortality. Molecular markers, including CALR mutations, were considered not only as diagnostic markers, but also as risk factors for bleeding and thrombosis associated with MPN, especially for patients in remote primary hospitals. We sought to develop an easy-to-use assay for the rapid detection of CALR type 1 (CALR-1) and type 2 (CALR-2) mutations in Philadelphia chromosome-negative MPN patients. Peptide nucleic acid-locked nucleic acid (PNA-LNA) clamping loop-mediated isothermal amplification (LAMP) assays were established, which were integrated into a centrifugal compact disc (CD) microfluidic platform. A total of 158 clinical blood samples were tested simultaneously by this microfluidic platform and an in-house real time PCR assay. The detection performance of the LAMP arrays was validated and conflicting results were identified by Sanger sequencing. The results suggested that the LAMP methods we developed exhibited good sensitivity, specificity, and precision. By real time fluorescence assay the detection limit for CALR-1 and CALR-2 mutations could reach as low as 1% and 0.5% respectively, and 10% and 5% respectively by visual method. There were no nonspecific background amplifications among different detection systems. For the CALR-1 and CALR-2 LAMP detection systems, intra-batch CV values of 1% mutated plasmid were 10.56% and 10.51% respectively, and the inter-batch CV values were 19.55% and 18.39%, respectively. The products were all analyzed by melting curve analysis and electrophoresis followed by Sanger sequencing analysis, which were consistent with the database sequences. The microfluidic platform could complete rapid detection of CALR-1/2 mutations within 60 min. The results of clinical samples detected by our CD-like microfluidic chipLAMP assay and rtPCR assay suggested that 133 samples were CALR wild type, 15 were CALR-1 mutation type, and 9 were CALR-2 mutation type. The correlation coefficient value (Kendall's tau_b) of the two assays was 0.99. Interestingly, by the newly established detection platform, we were surprised to find that one patient of Chinese origin harbored both CALR-1 and CALR-2 mutations. This result was verified by Sanger sequencing analysis. The LAMP detection systems developed herein displayed good sensitivity, specificity, and stability. Additionally, the detection results could be directly judged by color changes of the reaction systems without any auxiliary equipment. Thus, the platform we developed has the potential of being widely used in remote and economically undeveloped areas in the future.

S-Adenosylmethionine attenuates bile duct early warm ischemia reperfusion injury after rat liver transplantation.

Warm ischemia reperfusion injury (IRI) plays a key role in biliary complication, which is a substantial vulnerability of liver transplantation. The early pathophysiological changes of IRI are characterized by an excessive inflammatory response. S-Adenosylmethionine (SAM) is an important metabolic intermediate that modulates inflammatory reactions; however, its role in bile duct warm IRI is not known. In this study, male rats were treated with or without SAM (170 µmol/kg body weight) after orthotopic autologous liver transplantation. The histopathological observations showed that bile duct injury in the IRI group was more serious than in the SAM group. The alanine aminotransferase (ALT), alkaline phosphatase (ALP) and direct bilirubin (DBIL) levels in the serum of the IRI group were significantly increased compared to the SAM group (P < .05). Simultaneously, SAM effectively improved the survival of the transplant recipients. Furthermore, the H2O2 and malondialdehyde (MDA) of the IRI group were much higher compared to the SAM group (P < .05). The GSH/GSSG ratio in the SAM group was significantly increased by SAM treatment compared to the IRI group (P < .05). SAM administration significantly inhibited macrophage infiltration in liver and bile duct tissues, down-regulated TNF-α levels and up-regulated IL-10 expression in bile duct tissues compared to the IRI group (P < .05). The number of apoptotic biliary epithelial cells and caspase-3-positive cells in IRI rat livers were much higher compared to those in SAM-treated rats at 24 h after liver transplantation (P < .05). These data suggested that SAM protected bile ducts against warm IRI by suppressing oxidative stress, inflammatory reactions and apoptosis of biliary epithelial cells after liver transplantation.α.

Laparoscopic liver hanging maneuver through the retrohepatic tunnel on the right side of the inferior vena cava combined with a simple vascular occlusion technique for laparoscopic right hemihepatectomy.

BACKGROUND: Laparoscopic hepatectomy has been performed in many hospitals, with the development of the laparoscopic operation technique. However, performing complex laparoscopic hepatectomy, such as right hemihepatectomy, is still a challenge. The aim of this study was to describe the application of a simple vascular occlusion technique and new liver hanging maneuver (LHM) in complex laparoscopic hepatectomy, which are both advocated by Chen Xiaoping for open hepatectomy. METHODS: The clinical data of 29 consecutive patients who underwent laparoscopic right hemihepatectomy (LRH) from October 2014 to October 2016 were retrospectively analyzed. During operation, the vascular occlusion technique without hilus dissection and LHM through the retrohepatic avascular tunnel on the right side of the inferior vena cava were used. RESULT: All 29 operations were successfully performed laparoscopically, while adopting Chen's methods. The study consisted of 23 patients with hepatocellular carcinoma, four patients with intrahepatic cholangiocarcinoma, and two patients with hepatic metastasis of colonic carcinoma. The tumor size was 12.4 ± 1.9 cm. The operation time of LRH was 190.3 ± 49.9 min. The intraoperative blood loss of LRH was 281.7 ± 117.8 mL; five patients required blood transfusion, and the amount of blood transfusion was 300.0 ± 89.4 mL. No case was converted to open surgery, and no death occurred. All resulted in R0 resections. The median free margin was 20.1 ± 10.8 mm. The time of postoperative oral diet intake was 2.10 ± 0.96 days. The complication rate was 17.2%. The average hospital stay after operation was 10.0 ± 2.9 days. CONCLUSION: Complex hepatectomy is a bloodless procedure that can be performed under a laparoscope safely using Chen's methods of vascular occlusion technique and LHM.

Intronic miR-301 feedback regulates its host gene, ska2, in A549 cells by targeting MEOX2 to affect ERK/CREB pathways.

miR-301 is localized in the first intron of ska2, whose function has not been clarified. Here, a new circuit model in which intronic miR-301 regulates the transcription and function of its host gene through a feedback mechanism has been described. Our results showed that blocking of miR-301 in A549 cells leads to a decrease in the expression of the host gene, ska2. Further analysis showed that miR-301 targets MEOX2 to affect the ERK/CREB pathway. CREB directly regulates the expression of the host gene, ska2. In addition, the inhibition of miR-301 or ska2 resulted in an increase of the mitotic index and a decrease in colony formation in soft agar, which may be related to lung tumorigenesis.

[Transfer RNAs inhibit the growth of L929 cells in vitro].

AIM: To explore the effects of tRNA on the growth of mammalian cells. METHODS: L929, NIH3T3, MCF-7 and PC12 cells were seeded in 96 well culture plate individually, and incubated at 37 degrees C in 5% CO2 for 4 h, the tRNAs from different species were added to the culture media individually. After certain time of incubation, the viability of the cells was evaluated by the MTT methods. Sub-confluent L929 cells were incubated with 200 microg/ml ytRNA for different times, then the cells were pooled and analyzed with flow cytometry assay. RESULTS: tRNA specifically inhibited the growth of L929 cells in a dose-dependent manner. The sizes of tRNA-treated cells showed larger sizes and longer processes than those of untreated cells. Flow cytometric analysis further showed that most of tRNA-treated cells were arrested in S phase of the cell cycle. CONCLUSION: The cell growth inhibitory effects of tRNAs were caused mainly by their degraded fragments. The results suggested that tRNA or its degraded fragments might play important roles in regulation of cell proliferation.

3'-Poly(A) tail enhances siRNA activity against exogenous reporter genes in MCF-7 cells.

RNA interference (RNAi) is a powerful tool in the study of gene function. We added poly(A) tails to the 3' ends of siRNA antisense strands by in vitro transcription, and investigated the silencing effects of poly (A)-tailed siRNAs on enhanced green fluorescence protein (EGFP) and red fluorescence protein (RFP) genes. The results of this study showed that siRNAs with single-stranded 3'-poly(A) tails at antisense strands had noticeably stronger silencing effect on exogenous reporter genes than their corresponding parental forms. The enhanced silencing effect appears to be related to the length of poly(A) but was non siRNA sequence-specific. Furthermore, our results demonstrate that weakly-activated PKR and reduced stability of mRNAs of exogenous reporter genes in vivo may be the possible mechanisms of this non-specific enhanced silencing effect. Our findings are likely to be of value in designing siRNAs with enhanced activity.

Identification of the RNA chaperone activity of recombinant human tumor necrosis factor alpha in vitro.

RNA chaperones are defined as proteins that aid in the process of RNA folding by processing misfolding or by resolving misfolded structures. Although RNA chaperones are ubiquitous and abundant in all living organisms and viruses, there are no any reports that a cytokine has such RNA chaperone activity. Here, we demonstrate for the first time that recombinant human tumor necrosis factor alpha (rhTNF-alpha), a well-known cytokine, has RNA chaperone activity in vitro. rhTNF-alpha binds random 68 nt RNAs strongly at the minimal concentration of 10 microM with a broad sequence specificity. Our results also show that rhTNF-alpha facilitates annealing and strand exchange, and promotes the cleavage of a 17-nucleotide substrate S by hammerhead ribozyme HH16. The role of TNF-alpha as an RNA chaperone in vivo is not clear, but we propose that TNF-alpha may play an important role as an RNA chaperone during the process of some infectious and inflammatory diseases.

RNA aptamers specific for bovine thrombin.

Bovine thrombin is widely used in clinical wound healing after surgery. There is 85% homology between bovine thrombin and human thrombin, so most antibodies against bovine thrombin cross-react with human thrombin. Rare antibodies against bovine thrombin but not cross-reacting with human thrombin have been reported. RNA ligands (aptamers) have been used to bind to target molecules with sometimes higher specificity than antibodies. Here we report the isolation of aptamers specific for bovine thrombin by systematic evolution of ligands by exponential enrichment (SELEX) from an RNA pool containing a 25-nucleotide randomized region. After seven rounds of selection, two aptamers specific for bovine thrombin were identified with a K(d) of 164 and 240 nM, respectively. Significantly, these aptamers do not bind to human thrombin. Secondary structure prediction revealed potential stem-loop structures for these RNAs. Both RNA aptamers inhibit only bovine thrombin-catalyzed fibrin clot formation in vitro. Competition assay results suggested that the RNA aptamers might bind to the electropositive domain of bovine thrombin, that is, heparin-binding site, instead of fibrinogen-recognition exosite. The resulting bovine-specific thrombin inhibitor might be used in some clinical applications when bovine thrombin activity needs to be contained or in research where human and bovine thrombin need to be distinguished.