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.

Inhibition of HSP20 ameliorates steatotic liver disease by stimulating ERK2-dependent autophagy.

Heat shock protein 20 (HSP20) emerges as a novel regulator of autophagy in the heart. Nonetheless, the detailed function of HSP20 in liver and its effect on autophagy remain unknown. Here, we observed that HSP20 expression is increased in liver tissues from mice and patients with metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as nonalcoholic fatty liver disease (NAFLD). Liver-specific downregulation of HSP20 mitigates hepatic steatosis and insulin resistance in obese mice, while upregulating HSP20 promotes lipid deposition and hepatocyte cell death. Mechanistically, Liquid chromatography tandem mass spectrometry (LC-MS/MS) revealed that HSP20 interacts with phosphorylated extracellular regulated protein kinases 2 (ERK2) and prevents its dephosphorylation by dual specificity phosphatase 6 (DUSP6), leading to ERK2-mediated repression of autophagy, resulting in aggravated saturated fatty acid (SFA)-triggered hepatocyte death. Importantly, such adverse effects could be ameliorated by ERK inhibitor. Our data reveals a framework of how HSP20 increases susceptibility of SFA-induced liver injury through enhancing ERK2 phosphorylation, which represents a plausible therapeutic intervention to combat MASLD.

Synergistic action of cavity and catalytic sites in etched Pd-Cu2O octahedra to augment the peroxidase-like activity of Cu2O nanoparticles for the colorimetric detection of isoniazid and ascorbic acid.

Despite the widespread use of nanozyme-based colorimetric assays in biosensing, challenges such as limited catalytic efficiency, inadequate sensitivity to analytes, and insufficient understanding of the structure-activity relationship still persist. Overcoming these hurdles by enhancing the inherent enzyme-like performance of nanozymes using the unique attributes of nanomaterials is still a significant obstacle. Here, we designed and constructed Pd-Cu2O nanocages (Pd-Cu2O NCs) by selectively etching the vertices of the copper octahedra to enhance the peroxidase-like (POD-like) activity of Cu2O nanoparticles. The improved catalytic activity of Pd-Cu2O NCs was attributed to their high specific surface area and abundant catalytic sites. Mechanistic studies revealed that reactive oxygen species (ROS) intermediates (•OH) were generated through the decomposition of H2O2, resulting in POD-like activity of the Pd-Cu2O NCs. The designed Pd-Cu2O NCs can oxidize 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2, producing a blue oxidation product (oxTMB). The oxidation reaction was inhibited and led to a significant bleaching of the blue color in the presence of reducing substances isoniazid (INH) and ascorbic acid (AA). Based on these principles, we developed a colorimetric sensing platform for the detection of INH and AA, exhibiting good sensitivity and stability. This work provided a straightforward approach to the structural engineering of nanomaterials and the enhancement of enzyme-mimicking properties.

Facile synthesis of core-shell Co-MOF with hierarchical porosity for enhanced electrochemical detection of furaltadone in aquaculture water.

Metal-organic frameworks (MOFs) exhibited huge application potential in electrochemical analysis field, how to facilely and effectively boost the electrochemical sensing activity of MOFs materials still face enormous challenges. In this work, core-shell Co-MOF (Co-TCA@ZIF-67) polyhedrons with hierarchical porosity was easily synthesized via simple chemical etching reaction by selecting thiocyanuric acid as the etching reagent. Benefiting from the introduction of mesopores and thiocyanuric acid/Co2+ complex on the surface of ZIF-67 frameworks, the property and functions of the pristine ZIF-67 was seriously tailored. Compared with the pristine ZIF-67, the as-resulted Co-TCA@ZIF-67 nanoparticles displayed greatly enhanced physical adsorption capacity and electrochemical reduction activity toward the antibiotic drug furaltadone. As a result, a novel furaltadone electrochemical sensor with high sensitivity was fabricated. The linear detection range was from 50 nM to 5 µM with sensitivity of 110.40 µA-1 µM-1 cm-2 and detection limit of 12 nM. This work demonstrated chemical etching strategy is truly a facile and effective way to modify the electrochemical sensing performance of MOFs-based materials, and we believed the chemically etched MOFs materials will play a stronger role in terms of food safety and environmental conservation.

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.

Time series analysis revealed prognostic value of continuous nasopharyngeal SARS-CoV-2 nucleic acid quantification for COVID-19: A retrospective study of >3000 COVID-19 patients from 2 centers.

BACKGROUND: Early stratification of disease progression remains one of the major challenges towards the post-coronavirus disease 2019 (COVID-19) era. The clinical relevance of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid load is debated due to the heterogeneity in patients' underlying health conditions. We determined the prognostic value of nasopharyngeal viral load dynamic conversion for COVID-19. METHODS: The cycling threshold (Ct) values of 28,937 nasopharyngeal SARS-CoV-2 RT-PCRs were retrospectively collected from 3,364 COVID-19 patients during hospitalization and coordinated to the onset of disease progression. The ROC curve was utilized to determine the predictive performance of the rate of Ct value alteration between two consecutive RT-PCR runs within 48 h (ΔCt%) for disease transformation across patients with different COVID-19 severity and immune backgrounds, and further validated with 1,860 SARS-CoV-2 RT-PCR results from an independent validation cohort of 262 patients. For the 67 patients with severe COVID-19, Kaplan-Meier analysis was performed to evaluate the difference in survival between patients stratified by the magnitude of Ct value alteration between the late and early stages of hospitalization. RESULTS: The kinetics of viral nucleic acid conversion diversified across COVID-19 patients with different clinical characteristics and disease severities. The ΔCt% is a clinical characteristic- and host immune status-independent indicator for COVID-19 progression prediction (AUC = 0.79, 95 % CI = 0.76 to 0.81), which outperformed the canonical blood test markers, including c-reactive protein (AUC = 0.57, 95 % CI = 0.53 to 0.61), serum amyloid A (AUC = 0.61, 95 % CI = 0.54 to 0.68), lactate dehydrogenase (AUC = 0.61, 95 % CI = 0.56 to 0.67), d-dimer (AUC = 0.56, 95 % CI = 0.46 to 0.66), and lymphocyte count (AUC = 0.62, 95 % CI = 0.58 to 0.66). Patients with persistent high SARS-CoV-2 viral load (an increase of mean Ct value < 50 %) during the first 3 days of hospitalization demonstrated a significantly unfavorable survival (HR = 0.16, 95 % CI = 0.04 to 0.65, P = 2.41 × 10-3). CONCLUSIONS: Viral nucleic acid dynamics of SARS-CoV-2 eliminates the inter-patient variance of basic health conditions and therefore, can serve as a prognostic marker for COVID-19.

A diagnostic accuracy study comparing RNA LAMP, direct LAMP, and rapid antigen testing from nasopharyngeal swabs.

Introduction: During the coronavirus disease 2019 (COVID-19) pandemic, the early detection and isolation of individuals infected with severe acute respiratory syndrome coronavirus disease 2 (SARS-CoV-2) through mass testing can effectively prevent disease transmission. SARS-CoV-2 nucleic acid rapid detection based on loop-mediated isothermal amplification (LAMP) may be appropriate to include in testing procedures. Methods: We used 860 nasopharyngeal specimens from healthcare workers of Huashan Hospital and COVID-19 patients collected from April 7th to 21st, 2022, to assess the clinical diagnostic performance of the LAMP assay marketed by Shanghai GeneSc Biotech and compared it to the result of a rapid antigen test (RAT) head-to-head. Results: Overall, the diagnostic performance of LAMP assay and RAT were as follows. The LAMP assay represented higher sensitivity and specificity than RAT, especially in the extracted RNA samples. The sensitivity was 70.92% and 92.91% for direct LAMP and RNA-LAMP assay, respectively, while the specificity was 99.86% and 98.33%. The LAMP assay had overall better diagnostic performance on the specimens with relatively lower C t values or collected in the early phase (≤7 days) of COVID-19. The combination of LAMP assay and RAT improved diagnostic efficiency, providing new strategies for rapidly detecting SARS-CoV-2. Conclusion: The LAMP assay are suitable for mass screenings of SARS-CoV-2 infections in the general population.

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.

Can drip irrigation under mulch be replaced with shallow-buried drip irrigation in spring maize production systems in semiarid areas of northern China?

BACKGROUND: In recent years, shallow-buried drip irrigation in spring maize production has gradually replaced drip irrigation under plastic films and has become a common irrigation method in semiarid areas of northern China. RESULT: Two years of field experiments were carried out in the semiarid area of western Jilin province to compare the two drip irrigation methods in the spring maize production system. The treatments included MW1 (drip irrigation under mulch + moderate irrigation amount), MW2 (drip irrigation under mulch + high irrigation amount), SM1 (shallow-buried drip irrigation + moderate irrigation amount), and SM2 (shallow-buried drip irrigation + high irrigation amount). The maize yields were significantly higher under mulch than under shallow-buried drip irrigation, but there was no interaction between mulch and the irrigation amount. Drip irrigation under mulch greatly improved spring maize N, P and K uptake compared with that under shallow-buried drip irrigation. The agronomic-use efficiency, recovery-use efficiency, and partial factor productivity were ranked in the order of MW2 > MW1 > SW2 > SW1. The water-use efficiency of drip irrigation under mulch was 7.44% and 6.82% higher than that of shallow-buried drip irrigation under the moderate and high irrigation levels, respectively. However, considering the costs of the plastic mulch, there was no significant (P < 0.05) difference in economic benefits between the SW and MW treatments. CONCLUSION: Drip irrigation under plastic film provides greater advantages for production, but shallow-buried drip irrigation may be a suitable method for farmers until fertigation technology is further optimized and the problem of plastic film pollution is solved. © 2020 Society of Chemical Industry.

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.

Magnesium accumulation, partitioning and remobilization in spring maize (Zea mays L.) under magnesium supply with straw return in northeast China.

BACKGROUND: Magnesium (Mg) has important effects on maize growth, and the application of Mg fertilizer with straw return inevitably has an impact on Mg absorption in maize. RESULTS: A two-year field trial was conducted to investigate the effects of Mg fertilizers with straw return on Mg accumulations, partitioning and remobilization in maize (Zea mays L.) in northeast China. The treatments included: (i) JM3 (straw + Mg fertilizer), (ii) JM0 (straw + no Mg fertilizer), (iii) WM3 (no straw + Mg fertilizer), and (iv) WM0 (no Mg fertilizer + no straw). The results showed that the highest Mg accumulation stage in maize was prominent between the tasseling stage (VT) and blister stage (R2), and JM3 treatment accumulated 13.3% and 26.6% more Mg on average than those of the WM3 and WM0, respectively. Magnesium remobilization in distinct organs was highest in JM3 and there were significant differences between treatments. The total contribution to the grain for the JM3 treatment was higher by 6.0% and 17.9% on average than those for the WM3 and WM0, respectively. The grain yield of JM3 treatment was 0.5% and 5.3% higher than that of WM3 and WM0, respectively. CONCLUSION: Generally, these outcomes indicated that there was an interaction between Mg fertilizer and maize straw. The application of Mg fertilizer significantly promoted the accumulation, distribution to the maize organs, and the remobilization of Mg. The combination of straw return and Mg application further increased the accumulation of Mg in the grain. And all these lead to an increase in yield. © 2020 Society of Chemical Industry.

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.

Effects of equal chemical fertilizer substitutions with organic manure on yield, dry matter, and nitrogen uptake of spring maize and soil nitrogen distribution.

In order to maintain high yields and protect the environment, the replacement of chemical fertilizers with organic ones has received increasing attention in recent years. A 2-year field experiment (2015-2016) was carried out to assess the effects of substituting equal amounts of mineral fertilizer with organic manure on the yield, dry matter (DM), and nitrogen (N) uptake of spring maize (Zea mays L.) and on the mineral N (Nmin) distribution in the soil profile. The treatments included chemical fertilizer; different amounts of maize straw, cow manure, and chicken manure; and an unfertilized control (CK). Compared with the chemical fertilizer treatments, equal amounts of substitutions with cow manure or chicken manure increased production, and a 25% nutrient substitution resulted in the best yield increase. Straw return had no effect on maize production, and 100% straw return resulted in reduced production. The N accumulation and DM content both exhibited a slow-fast-slow growth trend throughout the various growth stages, and the average N uptake and DM accumulation in response to the treatments followed the order of chicken manure > cow manure > chemical fertilizer > straw return > CK. The Nmin content in the profile not only increased as the Nmin application rate increased but also showed greater increases at certain depths than at the surface, indicating that excessive N led to leaching. These results suggest that an appropriate proportion of organic substitution not only provides enough nutrients but also improves the soil environment and leads to increased yields. This technique represents a practical method of continuously increasing production and reducing the risk of N leaching.

Ultrasmall HKUST-1 nanoparticles decorated graphite nanosheets for highly sensitive electrochemical sensing of DNA damage biomarker 8-hydroxy-2'-deoxyguanosine.

Herein, graphite nanosheets (GN) were first prepared through simple liquid-phase exfoliation of graphite powder in N, N-dimethylacetamide (DMAC). After then, ultrasmall Cu-based metal organic frame (HKUST-1) nanoparticles (less than 5 nm) were in-situ anchored on the surface of graphite nanosheets with high degree of dispersion. Due to the intrinsic structural advantages of the as-synthesized HKUST-1 nanoparticles decorated graphite nanosheets (HKUST-1/GN) hybrids, including superior electron transfer ability and the greatly enhanced adsorption property, HKUST-1/GN shows excellent electrochemical sensing performance toward DNA damage biomarker 8-hydroxy-2'-deoxyguanosine with fast detection speed (∼240 s), wide linear window (10 nM-1 µM), high sensitivity (346857 µA mM-1 cm-2), low detection limit (∼2.5 nM), and good reproducibility. As a result, a highly sensitive electrochemical sensing platform for the detection of DNA damage biomarker 8-hydroxy-2'-deoxyguanosine was fabricated basing the as-prepared HKUST-1/GN hybrids. What is more, the developed electrochemical method was successfully used for the detection of real samples and exhibited satisfied result.

In Situ Synthesis of a Sandwich-like Graphene@ZIF-67 Heterostructure for Highly Sensitive Nonenzymatic Glucose Sensing in Human Serums.

Metal-organic frameworks (MOFs) have been extensively studied in recent years due to their tunable porosity, huge specific area, and controllable structure. The rich metal centers and large specific area have endowed MOFs with excellent electrochemical activity due to the multiple valence states, but the poor electronic conductivity of MOFs seriously impedes their electrocatalytic performance. Here, a polyhedral Co-based zeolite imidazole frame [Co(mim)2] n (denoted as ZIF-67, mim = 2-methylimidazole) is in situ loaded on the two sides of physically exfoliated graphene nanosheets (GSs) at room temperature, and sandwich-like GS@ZIF-67 hybrids with an ordered nanostructure are easily obtained. Compared with each individual component, the as-synthesized GS@ZIF-67 hybrids exhibit higher electrochemical activity toward glucose oxidation. Besides, the hierarchical nanocomposites also show better electrocatalytic performance compared with the same ratio of a physical mixture of GSs and ZIF-67, further demonstrating the synergistic effect between ZIF-67 and GSs. Thus, a highly sensitive nonenzymatic glucose electrochemical sensor is proposed with a linear range of 1-805.5 µM, sensitivity of 1521.1 µA Mm-1 cm-2, detection limit of 0.36 µM (S/N = 3), and excellent stability and selectivity. More importantly, the newly fabricated sensor is also successfully applied for glucose determination in human serums with satisfactory results, suggesting its promising potential toward glucose detection in real samples.

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-1Beta 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-1Beta 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-1Beta 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-1Beta 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

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.