Protective autophagy enhances antistress ability through AMPK/ULK1 signaling pathway in human immortalized keratinocytes.

Keratinocytes, located in the outermost layer of human skin, are pivotal cells to resist environmental damage. Cellular autophagy plays a critical role in eliminating damaged organelles and maintaining skin cell homeostasis. Low-dose 5-Aminolevulinic acid photodynamic therapy (ALA-PDT) has been demonstrated to enhance skin's antistress ability; however, the regulatory mechanisms of autophagy in keratinocytes remain unclear. In this study, we treated immortalized human keratinocytes (HaCaT cells) with low-dose ALA-PDT (0.5 mmol/L, 3 J/cm2). Through RNA-sequencing analysis, we identified that low-dose ALA-PDT modulated autophagy-related pathways in keratinocytes and pinpointed Unc-51-like kinase 1 (ULK1) as a key gene involved. Western blot results revealed that low-dose ALA-PDT treatment upregulated the expression of autophagy-related proteins Beclin-1 and LC3-II/LC3-I ratio. Notably, low-dose ALA-PDT regulated autophagy by inducing an appropriate level of reactive oxygen species (ROS), transiently reducing mitochondrial membrane potential, and decreasing adenosine triphosphate production; all these processes functioned on the AMP-activated protein kinase (AMPK)/ULK1 pathway to activate autophagy. Finally, we simulated external environmental damage using ultraviolet B (UVB) at a dose of 60 mJ/cm2 and observed that low-dose ALA-PDT mitigated UVB-induced cell apoptosis; however, this protective effect was reversed when using the autophagy inhibitor 3-methyladenine. Overall, these findings highlight how low-dose ALA-PDT enhances antistress ability in HaCaT cells through controlling ROS generation and activating the AMPK/ULK1 pathway to arouse cellular autophagy.

Absence of Tissue-Sparing Effects in Partial Proton FLASH Irradiation in Murine Intestine.

Ultra-high dose rate irradiation has been reported to protect normal tissues more than conventional dose rate irradiation. This tissue sparing has been termed the FLASH effect. We investigated the FLASH effect of proton irradiation on the intestine as well as the hypothesis that lymphocyte depletion is a cause of the FLASH effect. A 16 × 12 mm2 elliptical field with a dose rate of ~120 Gy/s was provided by a 228 MeV proton pencil beam. Partial abdominal irradiation was delivered to C57BL/6j and immunodeficient Rag1-/-/C57 mice. Proliferating crypt cells were counted at 2 days post exposure, and the thickness of the muscularis externa was measured at 280 days following irradiation. FLASH irradiation did not reduce the morbidity or mortality of conventional irradiation in either strain of mice; in fact, a tendency for worse survival in FLASH-irradiated mice was observed. There were no significant differences in lymphocyte numbers between FLASH and conventional-dose-rate mice. A similar number of proliferating crypt cells and a similar thickness of the muscularis externa following FLASH and conventional dose rate irradiation were observed. Partial abdominal FLASH proton irradiation at 120 Gy/s did not spare normal intestinal tissue, and no difference in lymphocyte depletion was observed. This study suggests that the effect of FLASH irradiation may depend on multiple factors, and in some cases dose rates of over 100 Gy/s do not induce a FLASH effect and can even result in worse outcomes.

Addition of Losartan to FOLFIRINOX and Chemoradiation Reduces Immunosuppression-Associated Genes, Tregs, and FOXP3+ Cancer Cells in Locally Advanced Pancreatic Cancer.

PURPOSE: Adding losartan (LOS) to FOLFIRINOX (FFX) chemotherapy followed by chemoradiation (CRT) resulted in 61% R0 surgical resection in our phase II trial in patients with locally advanced pancreatic cancer (LAPC). Here we identify potential mechanisms of benefit by assessing the effects of neoadjuvant LOS on the tumor microenvironment. EXPERIMENTAL DESIGN: We performed a gene expression and immunofluorescence (IF) analysis using archived surgical samples from patients treated with LOS+FFX+CRT (NCT01821729), FFX+CRT (NCT01591733), or surgery upfront, without any neoadjuvant therapy. We also conducted a longitudinal analysis of multiple biomarkers in the plasma of treated patients. RESULTS: In comparison with FFX+CRT, LOS+FFX+CRT downregulated immunosuppression and pro-invasion genes. Overall survival (OS) was associated with dendritic cell (DC) and antigen presentation genes for patients treated with FFX+CRT, and with immunosuppression and invasion genes or DC- and blood vessel-related genes for those treated with LOS+FFX+CRT. Furthermore, LOS induced specific changes in circulating levels of IL-8, sTie2, and TGF-ß. IF revealed significantly less residual disease in lesions treated with LOS+FFX+CRT. Finally, patients with a complete/near complete pathologic response in the LOS+FFX+CRT-treated group had reduced CD4+FOXP3+ regulatory T cells (Tregs), fewer immunosuppressive FOXP3+ cancer cells (C-FOXP3), and increased CD8+ T cells in pancreatic ductal adenocarcinoma lesions. CONCLUSIONS: Adding LOS to FFX+CRT reduced pro-invasion and immunosuppression-related genes, which were associated with improved OS in patients with LAPC. Lesions from responders in the LOS+FFX+CRT-treated group had reduced Tregs, decreased C-FOXP3 and increased CD8+ T cells. These findings suggest that LOS may potentiate the benefit of FFX+CRT by reducing immunosuppression.

Peripheral blood CD4+ T cell count predicts recurrence of condyloma acuminatum after photodynamic therapy in HIV-positive patients.

BACKGROUND: Few studies have reported postoperative relapse of condyloma acuminatum (CA) after 5-aminolevulinic acid photodynamic therapy (ALA-PDT) in human immunodeficiency virus (HIV) positive patients. METHODS: The clinical data of HIV-positive CA patients treated with ALA-PDT from October 2018 to December 2019 were analyzed retrospectively. Univariate and multivariate Cox regression was used to analyze the variables related to postoperative recurrence. Pearson correlation test was employed to analyze the correlation between CD4+ T cell count and postoperative recurrence rate. Kaplan-Meier method was used to compare the CA recurrence after ALA-PDT in low CD4 group and high CD4 group. RESULTS: A total of 38 HIV-positive patients with CA were included in the study. Among them, 26 patients experienced CA recurrence within 6 months, and the recurrence rate was 68.4%. CD4+ T cell count was 187.0 (79.0-596.0) cells/µl in relapsed patients and 406.0 (89.0-612.0) cells/µl in non-relapsed patients, showing a statistically significant difference (p = .005). Pearson correlation coefficient analysis revealed a negative correlation between CD4+ T cell count and postoperative recurrence rate (p = .005, r = -.443). Univariate regression analysis showed that CD4+ T cell count was correlated with postoperative recurrence, hazard ratio (HR) was 0.99 [95% Confidence interval (CI) = 0.99-1.0, p = .012]. Multivariate Cox regression analysis showed that with the low CD4+ T cell count as the reference, the high CD4+ T cell count was negatively correlated with postoperative recurrence (HR = 0.09, 95% CI 0.01-0.87, p = .038). CONCLUSIONS: Peripheral blood CD4+ T cell count can predict the CA recurrence rate after ALA-PDT in HIV-positive patients.

Vonoprazan non-inferior to lansoprazole in treating duodenal ulcer and eradicating Helicobacter pylori in Asian patients.

BACKGROUND AND AIM: Duodenal ulcers, especially caused by increasingly drug-resistant Helicobacter pylori, are a concern in Asia. We compared oral vonoprazan versus lansoprazole for efficacy (healing duodenal ulcers) and safety in non-Japanese Asian patients. METHODS: In this phase 3, randomized (1:1), double-blind, double-dummy, parallel-group, non-inferiority study (April 5, 2017, to July 19, 2019), patients with ≥ 1 endoscopically confirmed duodenal ulcer, at 52 hospitals (China, South Korea, and Taiwan), received vonoprazan 20 mg once daily (QD) or lansoprazole 30 mg QD for 6 weeks maximum. Patients with H. pylori received bismuth-containing quadruple therapy including vonoprazan 20 mg twice daily (BID) or lansoprazole 30 mg BID, for 2 weeks, followed by vonoprazan or lansoprazole monotherapy QD (4 weeks maximum). Endpoints were endoscopically confirmed duodenal ulcer healing (Week 4/6; primary) and H. pylori eradication (4 weeks post-treatment; secondary); non-inferiority margins were -6% and -10%, using a two-sided 95% confidence interval (CI). RESULTS: Of 533 enrolled patients, one was lost to follow-up and one withdrew (full analysis set: 531 patients [vonoprazan, n = 263; lansoprazole, n = 268]; 85.4% = H. pylori positive). Vonoprazan was non-inferior to lansoprazole for duodenal ulcer healing (96.9% vs 96.5%; difference 0.4% [95% CI -3.00, 3.79]). H. pylori eradication rates were 91.5% (vonoprazan) and 86.8% (lansoprazole; difference 4.7% [95% CI -1.28, 10.69]). Vonoprazan and lansoprazole were well tolerated, with similar safety profiles, no new safety signals; no deaths occurred. CONCLUSIONS: Vonoprazan was well tolerated and non-inferior to lansoprazole for duodenal ulcer healing and achieved H. pylori eradication above the clinically meaningful threshold (90%), in non-Japanese Asian patients.

A novel nomogram based on a retrospective study of 346 patients to predict the recurrence risk of condyloma acuminatum after 5-aminolevulinic acid photodynamic therapy.

Condyloma acuminatum (CA) is a sexually transmitted disease caused by human papillomavirus (HPV) often with high recurrence rate after treatment. This study aimed to construct and evaluate a nomogram model containing three clinical parameters to predict the recurrence risk of CA after 5-aminolevulinic acid photodynamic therapy (ALA-PDT). A predictive model was established based on a training cohort of 346 CA patients treated with ALA-PDT between January 2013 and July 2018. A validation cohort of 123 CA patients was recruited from August 2018 to December 2019. The least absolute shrinkage and selection operator (LASSO) regression model was used to optimize the clinical feature selection. A nomogram predicting the recurrence of CA after ALA-PDT was constructed based on the predictors identified by LASSO regression. C-index and area under the curve (AUC) values were used to evaluate the discrimination. Calibration was evaluated with a calibration curve. The net benefit was performed via decision curve analysis (DCA). In the training cohort, 55 (15.89%) patients experienced recurrences after ALA-PDT. Predictors selected by LASSO regression were concomitant human immunodeficiency virus (HIV) infection [Hazard ratio (HR) = 4.4; 95% confidence interval (CI), 2.5-7.9; p < 0.001], skin and mucosa as affected area (HR = 1.7; 95% CI, 0.9-3.1; p = 0.109), and more than one time of CO2 laser therapy (HR = 6.3; 95% CI, 2.8-13.9; p < 0.001). The nomogram showed a good performance in predicting recurrence as the C-indexes were 0.843 (95% CI, 0.799-0.887) in the training cohort, and 0.831 (95% CI, 0.727-0.934) in the validation cohort. The AUCs of the nomogram were 0.85 in training and 0.8 in validation. DCA confirmed the nomogram was clinically useful when the intervention was determined at the non-adherence possibility threshold of 5%. This nomogram can provide individualized prediction for the recurrence risk of CA in patients treated by ALA-PDT.

Multiple-type HPV infection predicting condyloma acuminatum recurrence after aminolevulinic acid photodynamic therapy.

BACKGROUND: Aminolevulinic acid photodynamic therapy (ALA-PDT) has been widely used in the treatment of condyloma acuminatum (CA), but there is a lack of reports on clinical factors related to CA relapse after photodynamic therapy. METHODS: The clinical data of patients with CA treated with ALA-PDT from April 2018 to December 2019 were retrospectively analyzed, including HPV type, follow-up time and recurrence within 6 months after treatment. The patients were classified into single-type HPV infection and multiple-type HPV infection groups. Besides, the patients were also classified into high-risk HPV infection, low-risk HPV infection, and low + high-risk HPV infection groups. Univariate and multivariate COX regression was performed to analyze whether HPV type was related with CA relapse after photodynamic therapy. RESULTS: A total of 161 CA patients who underwent ALA-PDT were analyzed in this study. CA recurred in 20 patients within 6 months after treatment, with a recurrence rate of 12.4%. Of them, the patients with multiple-type HPV infection made up 85%. Multivariate COX regression analysis revealed that multiple-type HPV infection was associated with CA recurrence (HR:5.0; 95% CI: 1.1-21.4; P = 0.032). Of the patients with CA recurrence, 70% developed low + high-risk infections, a proportion significantly higher than that in patients without CA relapse (16.3%, P < 0.001). Using low-risk infection as a reference, low + high-risk infection was positively correlated with postoperative recurrence (HR: 6.7; 95% CI: 1.6-26.2; P = 0.006). CONCLUSION: Multiple-type HPV infection were closely associated with CA recurrence after photodynamic therapy.

Correlation between vascular endothelial growth factor pathway and immune microenvironment in head and neck squamous cell carcinoma.

BACKGROUND: Immunotherapy is a crucial therapeutic approach in oncology. However, most patients with head and neck squamous cell carcinoma (HNSCC) do not derive benefit from immunotherapy. Vascular endothelial growth factor (VEGF)/VEGF Receptor 2 (VEGFR2) signaling pathway is one of the most important pathways regulating angiogenesis in tumor. The combination of immunotherapy and anti-angiogenic therapy is considered to improve efficacy of immunotherapy. The correlation between VEGF signaling pathway and tumor immune microenvironment in HNSCC patients is unclear. METHODS: We utilized RNA sequencing and clinical data of HNSCC patients from the TCGA database to study the correlation between VEGF signaling pathway and tumor immune microenvironment, on aspect of immune cell infiltration, immune-related gene expression profiling and immune-related biological pathways. RESULTS: We observed that VEGF signaling pathway is positively correlated with immune cell infiltration, immune-related gene expression profiles, and the prognosis of HNSCC patients. The functional enrichment analysis of differentially expressed genes between different VEGF score subtypes detected multiple immune-related biological processes. CONCLUSION: Our findings suggested that combining anti-VEGF signaling pathway agents with immunotherapy, such as immune checkpoint inhibitors (ICI) therapy, may exhibit encouraging benefits in HNSCC.

Prognostic Value of an Immune-Related Gene Signature in Oral Squamous Cell Carcinoma.

The prognosis and immunotherapy response rates are unfavorable in patients with oral squamous cell carcinoma (OSCC). The tumor microenvironment is associated with tumor prognosis and progression, and the underlying mechanisms remain unclear. We obtained differentially expressed immune-related genes from OSCC mRNA data in The Cancer Genome Atlas (TCGA) database. Overall survival-related risk signature was constructed by univariate Cox regression analysis and LASSO Cox regression analysis. The prognostic performance was validated with receiver operating characteristic (ROC) analysis and Kaplan-Meier survival curves in the TCGA and Gene Expression Omnibus (GEO) datasets. The risk score was confirmed to be an independent prognostic factor and a nomogram was built to quantify the risk of outcome for each patient. Furthermore, a negative correlation was observed between the risk score and the infiltration rate of immune cells, as well as the expression of immunostimulatory and immunosuppressive molecules. Functional enrichment analysis between different risk score subtypes detected multiple immune-related biological processes, metabolic pathways, and cancer-related pathways. Thus, the immune-related gene signature can predict overall survival and contribute to the personalized management of OSCC patients.

Arecoline suppresses epithelial cell viability by upregulating tropomyosin-1 through the transforming growth factor-ß/Smad pathway.

CONTEXT: Oral submucous fibrosis (OSF) is a chronic and progressive disease. Arecoline, present in betel nuts, has been proposed as a vital aetiological factor. However, the underlying mechanism remains unclear. OBJECTIVES: This research elucidates the expression of tropomyosin-1 (TPM1) and its regulation mechanism in HaCaT cells treated with arecoline. MATERIALS AND METHODS: HaCaT cells were assigned into three groups: (1) Control; (2) Treated with arecoline (0.16 mM) for 48 h (3) Treated with arecoline (0.16 mM) and transfected with small interfering RNA (siRNA) for TPM1 (50 nM) for 48 h. CCK8, cell cycle, and apoptosis phenotypic analyses were performed. PCR and western blot analyses were performed to detect the expression level of TPM1 and examine the related signalling pathway. RESULTS: The IC50 of arecoline was approximately 50 µg/mL (0.21 mM). The arecoline dose (0.16 mM) and time (48 h) markedly increased TPM1 expression at the mRNA and protein levels in HaCaT cells. Arecoline suppressed the cell growth, caused cell cycle arrest at the G1 phase, and induced cell apoptosis in HaCaT cells. siRNA-mediated knockdown of TPM1 attenuated the effect of arecoline on cell proliferation, apoptosis, and cell cycle arrest at the G1 phase. Furthermore, blocking of the transforming growth factor (TGF)-ß receptor using SB431542 significantly suppressed TPM1 expression in the cells treated with arecoline. DISCUSSION AND CONCLUSIONS: Arecoline suppresses HaCaT cell viability by upregulating TPM1 through the TGF-ß/Smad signalling pathway. This research provides a scientific basis for further study of arecoline and TPM1 in OSF and can be generalised to broader pharmacological studies. TPM1 may be a promising molecular target for treating OSF.

Cytokines secreted by arecoline activate fibroblasts that affect the balance of TH17 and Treg.

BACKGROUND: Oral submucous fibrosis (OSF) is a chronic progressive oral disease with cancerous tendency. Arecoline plays an important role in the pathogenesis of OSF. Fibroblasts (FBs) are the primary cells involved in the pathogenesis of OSF. There is a change in CD4+ IL-17+ helper T cells (Th17) and CD4+ CD25+ Foxp3+ regulatory T cells (Treg) in OSF patients, but the molecular mechanisms of are not clearly understood. In this work, we studied the molecular mechanisms. METHODS: Enzyme digestion was used to extract primary FBs, and immunofluorescence was used to identify FBs. Cytotoxic experiment was then performed to determine the effect of arecoline on FB activity. Enzyme-linked immunosorbent assay (ELISA) was used to detect changes in the amount of cytokines. In addition, we treated human peripheral blood mononuclear cells (PBMC) with the above cytokines and detected their changes. Flow cytometry was used to detect the changes of Th17 and Treg, and quantitative-polymerase chain reaction (Q-PCR) was used to detect the changes of RORγt and Foxp3. RESULTS: We have found that the stimulation of arecoline on FBs increased interleukin-2, interleukin-6, and interleukin-21 (IL-2, IL-6, and IL-21) while decreased transforming growth cytokine-ß (TGF-ß). After the cytokine-containing supernatant was co-cultured with PBMC, the cytometry results showed that Th17 was increased, while Treg was significantly decreased and Q-PCR results showed that RORγt expression was increased and Foxp3 expression was decreased. CONCLUSION: The arecoline can affect inflammatory cytokines produced by FBs, which then act on immune cells Th17 and Treg, and make them change.

Achieving the composite end-point of glycated hemoglobin <7.0% without weight gain or hypoglycemia with once-weekly dulaglutide in Chinese patients with type 2 diabetes: A post-hoc analysis.

AIMS/INTRODUCTION: To assess the effect of dulaglutide (DU) 1.5/0.75 mg in comparison with glimepiride (GLIM) or insulin glargine (GLAR) on the composite end-point in Chinese type 2 diabetes patients. MATERIALS AND METHODS: Post-hoc analyses of two randomized phase III trials (NCT01644500 and NCT01648582) were carried out using Fisher's exact test. The primary composite end-point was the number of patients reaching glycated hemoglobin (HbA1c) <7.0%, without weight gain and hypoglycemia. Secondary composite end-points included the number of patients reaching HbA1c <7.0% without weight gain and HbA1c <7.0% without hypoglycemia. RESULTS: Data of 1,147 Chinese type 2 diabetes patients were analyzed (NCT01644500 = 556; NCT01648582 = 591). In each analyzed trial, 40-48% of patients received DU (1.5 mg), 30-39% of patients received DU (0.75 mg) and 15-20% of patients on active comparators (GLIM/GLAR) reached the primary composite end-point at week 26 (P < 0.001 for DU vs GLIM/GLAR). At 52 weeks, 26% of patients that received DU (1.5 mg), 23% of patients that received DU (0.75 mg) and 7% of patients that received GLAR attained the primary composite end-point (P < 0.001 for DU vs GLAR). A similar trend of results was found for secondary composite end-points. CONCLUSIONS: Dulaglutide is found to be an effective therapeutic alternative for Chinese type 2 diabetes patients. Compared with GLIM/GLAR, significantly greater proportions of patients on DU attained the HbA1c target of <7.0% without weight gain or hypoglycemia.

Arecoline suppresses epithelial cell viability through the Akt/mTOR signaling pathway via upregulation of PHLPP2.

Arecoline, the major active ingredient of the betel nut, is involved in the pathogenesis of oral submucous fibrosis. However, the underlying mechanism of this pathogenesis remains unclear. In this study, we found that arecoline suppresses the cell proliferation of the HaCaT epithelial cell and induces cell cycle arrest at the G1/S phase with an IC50 of 50 µg/mL. Furthermore, we found that arecoline reduces the protein level of cyclin D1, but it has no effect on its mRNA level and protein stability, implying that arecoline may modulate the translation of cyclin D1. We also observed the downregulation of the Akt/mTOR signaling pathway after treatment with arecoline, which may be related to the translation of cyclin D1. RNA-seq analysis identified that PHLPP2, the direct upstream target of Akt, is significantly upregulated after arecoline treatment. siRNA-mediated knockdown of PHLPP2 recovered the phosphorylation state of Akt, as well as attenuated the effect of arecoline on cell viability. Thus, our study revealed the crucial role of PHLPP2 in arecoline-induced cell viability suppression.

Efficacy and safety of once-weekly dulaglutide versus insulin glargine in mainly Asian patients with type 2 diabetes mellitus on metformin and/or a sulphonylurea: A 52-week open-label, randomized phase III trial.

AIM: To compare the efficacy and safety of once-weekly dulaglutide with that of insulin glargine in combination with metformin and/or a sulphonylurea in mainly Asian patients with type 2 diabetes mellitus (T2DM). MATERIALS AND METHODS: In this 52-week, randomized, parallel-arm open-label study, we enrolled patients aged ≥18 years with T2DM for at least 6 months and a glycated haemoglobin (HbA1c) concentration ≥53.0 mmol/mol (7.0%) and ≤96.7 mmol/mol (11.0%). The primary outcome was change in HbA1c from baseline to week 26 to determine non-inferiority of dulaglutide 1.5 mg versus glargine. RESULTS: A total of 774 patients from China, South Korea, Mexico and Russia were randomly assigned (1:1:1) to dulaglutide 1.5 mg, dulaglutide 0.75 mg or glargine treatment groups. The patients' mean age was 55 years and the average T2DM duration was ~8 years. The least squares mean (SE) changes from baseline in HbA1c at 26 weeks were - 18.9 (0.73) mmol/mol (-1.73 [0.067]%) for dulaglutide 1.5 mg and -14.5 (0.73) mmol/mol (-1.33 [0.067]%) for dulaglutide 0.75 mg, compared with -12.7 (0.73) mmol/mol (-1.16 [0.067]%) for glargine. Statistical criteria for superiority were met with both dulaglutide 1.5 mg and dulaglutide 0.75 mg. More patients in the dulaglutide 1.5 and 0.75 mg groups achieved HbA1c target <53.0 mmol/mol (<7.0%) than in the glargine group at week 26 (P < 0.001 and P = 0.004, respectively). Body weight decreased with dulaglutide and increased with glargine. The incidence and rate of total hypoglycaemia were lower with dulaglutide versus glargine. Gastrointestinal adverse events, including diarrhoea and nausea, were the most frequently reported for patients taking dulaglutide. CONCLUSIONS: Once-weekly dulaglutide provides greater improvement in HbA1c, with weight loss and less hypoglycaemia, than once-daily insulin glargine in a population of mainly Asian patients with T2DM who had failed to achieve optimal glycaemic control on metformin and/or a sulphonylurea.

Selective covalent capture of a DNA sequence corresponding to a cancer-driving C>G mutation in the KRAS gene by a chemically reactive probe: optimizing a cross-linking reaction with non-canonical duplex structures.

Covalent reactions are used in the detection of various biological analytes ranging from low molecular weight metabolites to protein-protein complexes. The detection of specific nucleic acid sequences is important in molecular biology and medicine but covalent approaches are less common in this field, in part, due to a deficit of simple and reliable reactions for the covalent capture of target sequences. Covalent anchoring can prevent the denaturation (melting) of probe-target complexes and causes signal degradation in typical hybridization-based assays. Here, we used chemically reactive nucleic acid probes that hybridize with, and covalently capture, a target sequence corresponding to a cancer-driving variant of the human KRAS gene. Our approach exploits a reductive amination reaction to generate a stable covalent attachment between an abasic site in the probe strand and a guanine mutation at position 35 in the KRAS gene sequence. Importantly, systematic variation of the probe sequence in a manner that formally introduces non-canonical structures such as bulges and mispairs into the probe-target duplex led to probes with dramatically improved cross-linking properties. An optimized abasic site-containing probe enabled simultaneous quantitative detection of both mutant and wild-type KRAS sequences in mixtures.

Single Locked Nucleic Acid-enhanced nanopore genetic discrimination of pathogenic serotypes and cancer driver mutations.

Rapid and accurate detection of single-nucleotide polymorphism (SNP) in pathogenic mutants is crucial for broad fields from food safety monitoring to disease diagnostics and prognosis. Here, we developed a nanopore single-molecule sensor, coupled with the locked nucleic acid (LNA) technique, to accurately discriminate SNPs for detection of Shiga toxin producing Escherichia coli (STEC) O157:H7 pathogen serotype, and cancer-derived driver mutations EGFR L858R and KRAS G12D. This sensitive method, with a simplified, low cost, easy-to-operate LNA design, can be applied in food science and medical detection that need rapid and accurate determination of genetic variations.

Generation and Single-Molecule Characterization of a Sequence-Selective Covalent Cross-Link Mediated by Mechlorethamine at a C-C Mismatch in Duplex DNA for Discrimination of a Disease-Relevant Single Nucleotide Polymorphism.

Many strategies for the detection of nucleic acid sequence rely upon Watson-Crick hybridization of a probe strand to the target strand, but the reversible nature of nucleic acid hybridization presents an inherent challenge: short probes that provide high target specificity have relatively low target affinity resulting in signal losses. Sequence-specific covalent cross-linking reactions have the potential to provide both selective target capture and durable signal. We explore a novel approach involving sequence-specific covalent cross-linking of a probe to target DNA combined with single-molecule nanopore detection of the cross-linked DNA. Here, we exploited the selective reaction of mechlorethamine at a C-C mismatch for covalent capture of a target DNA sequence corresponding to a cancer-driving mutation at position 1799 of the human BRAF kinase gene. We then demonstrated that the α-hemolysin protein nanopore can be employed for the unambiguous, single-molecule detection of the cross-linked probe-target complex. Cross-linked DNA generates an unmistakable deep and persistent current block (≥5 s) that is easily distinguished from the microsecond and millisecond blocks generated by translocation of single-stranded DNA and uncross-linked duplexes through the nanopore.

Efficacy and safety of dulaglutide monotherapy compared with glimepiride in East-Asian patients with type 2 diabetes in a multicentre, double-blind, randomized, parallel-arm, active comparator, phase III trial.

AIMS: To compare the efficacy and safety of once-weekly glucagon-like peptide-1 receptor agonist dulaglutide 1.5 and 0.75 mg with glimepiride in East-Asian patients with type 2 diabetes (T2D). MATERIALS AND METHODS: In this phase III, multinational, multicentre, double-blind, randomized, parallel-arm, 26-week study, patients with inadequate glycaemic control were randomized 1:1:1 to once-weekly dulaglutide 1.5 or 0.75 mg or daily glimepiride (1-3 mg/d). The primary endpoint was assessment of the non-inferiority of dulaglutide (1.5 mg), as measured by change in glycated haemoglobin (HbA1c), compared with glimepiride using a 0.4% non-inferiority margin. RESULTS: A total of 737 patients were randomized (dulaglutide 1.5 mg, n = 244; dulaglutide 0.75 mg, n = 248; glimepiride, n = 245). At week 26, both doses of dulaglutide were non-inferior and also superior to glimepiride for HbA1c reduction from baseline with a least squares mean difference of -6.34 mmol/mol (95% confidence interval [CI] -8.31, -4.26) or -0.58% (95% CI -0.76, -0.39) for dulaglutide 1.5 mg and -3.50 mmol/mol (95% CI -5.47, -1.42) or -0.32% (95% CI -0.50, -0.13) for dulaglutide 0.75 mg (P < .001). A greater proportion of patients in the dulaglutide 1.5 mg group achieved the HbA1c target of <53 mmol/mol (<7.0%) compared with the glimepiride group (74.1% vs 57.4%; P < .001). The mean body weight decreased (P < .005) and total hypoglycaemia rates were lower (P < .001) in the dulaglutide groups compared with the glimepiride group. The most common drug-related adverse events in both dulaglutide groups (≥5% of patients) included diarrhoea, nausea, increased lipase, decreased appetite, abdominal distension and vomiting. CONCLUSIONS: Dulaglutide (both doses) demonstrated superior glycaemic control vs glimepiride, with a favourable tolerability and safety profile in East-Asian patients with T2D.

Single Locked Nucleic Acid-Enhanced Nanopore Genetic Discrimination of Pathogenic Serotypes and Cancer Driver Mutations.

Accurate and rapid detection of single-nucleotide polymorphism (SNP) in pathogenic mutants is crucial for many fields such as food safety regulation and disease diagnostics. Current detection methods involve laborious sample preparations and expensive characterizations. Here, we investigated a single locked nucleic acid (LNA) approach, facilitated by a nanopore single-molecule sensor, to accurately determine SNPs for detection of Shiga toxin producing Escherichia coli (STEC) serotype O157:H7, and cancer-derived EGFR L858R and KRAS G12D driver mutations. Current LNA applications that require incorporation and optimization of multiple LNA nucleotides. But we found that in the nanopore system, a single LNA introduced in the probe is sufficient to enhance the SNP discrimination capability by over 10-fold, allowing accurate detection of the pathogenic mutant DNA mixed in a large amount of the wild-type DNA. Importantly, the molecular mechanistic study suggests that such a significant improvement is due to the effect of the single-LNA that both stabilizes the fully matched base-pair and destabilizes the mismatched base-pair. This sensitive method, with a simplified, low cost, easy-to-operate LNA design, could be generalized for various applications that need rapid and accurate identification of single-nucleotide variations.

Mitogen-activated protein kinase signaling pathway in oral cancer.

The mitogen-activated protein kinase (MAPK) signaling pathway is associated with tumor cell proliferation, differentiation, apoptosis, angiogenesis, invasion and metastasis. The present review assesses the involvement of the MAPK signaling pathway in oral cancer progression and invasion based on analysis of individual sub-pathways and their mechanisms of action. The regulation of this pathway for targeted oral cancer therapy is explored and the challenges confronting this, as well as corresponding potential solutions, are discussed. Exploring this pathway with an emphasis on its components, subfamilies, sub-pathways, interactions with other pathways and clinical practice modes may improve oral cancer treatment.