Profiling of mismatch discrimination in RNAi enabled rational design of allele-specific siRNAs

Silencing specificity is a critical issue in the therapeutic applications of siRNA, particularly in the treatment of single nucleotide polymorphism (SNP) diseases where discrimination against single nucleotide variation is demanded. However, no generally applicable guidelines are available for the design of such allele-specific siRNAs. In this paper, the issue was approached by using a reporter-based assay. With a panel of 20 siRNAs and 240 variously mismatched target reporters, we first demonstrated that the mismatches were discriminated in a position-dependent order, which was however independent of their sequence contexts using position 4th, 12th and 17th as examples. A general model was further built for mismatch discrimination at all positions using 230 additional reporter constructs specifically designed to contain mismatches distributed evenly along the target regions of different siRNAs. This model was successfully employed to design allele-specific siRNAs targeting disease-causing mutations of PIK3CA gene at two SNP sites. Furthermore, conformational distortion of siRNA-target duplex was observed to correlate with the compromise of gene silencing. In summary, these findings could dramatically simplify the design of allele-specific siRNAs and might also provide guide to increase the specificity of therapeutic siRNAs

A flow-through electroporation chip integrated with viable cell sorting based on dielectrophoresis

Flow-through electroporation on a chip is a promising technique to introduce molecules into cells in applications of biological research, drug delivery and gene therapy. During the electroporation process, the electrical field also causes large amount of non-viable cells, which significantly influence the following biological procedure. Off-chip separation of the variable cells increases experimental complexity dramatically and results in further cell death. In this paper, we have developed a flow-through electroporation chip, in which dielectrophoresis (DEP) is employed to sort the viable cells and non-viable cells on chip. For the standard expression cell line HEK-293a (Human embryonic kidney cells), the ratio of the viable cells in the sorted sample was increased to 90% from 20% in the as-electroporated sample.http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000312912800250&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=8e1609b174ce4e31116a60747a720701Engineering, Electrical & ElectronicNanoscience & NanotechnologyEICPCI-S(ISTP)

A Prognostic Framework for Wheel Treads Integrating Parameter Correlation and Multiple Uncertainties

As crucial rotary components of high-speed trains, wheel treads in realistic operation environment usually suffer severe cyclic shocks, which damage the health status and ultimately cause safety risks. Timely and precise health prognosis based on vibration signals is an effective technology to mitigate such risks. In this work, a new parameter-related Wiener process model is proposed to capture multiple uncertainties existed in on-site prognosis of wheel treads. The proposed model establishes a quantitative relationship between degradation rate and variations, and integrates uncertainties via heterogeneity analysis of both criterions. A maximum-likelihood-based method is presented to initialize the unknown model parameters, followed by a recursive update algorithm with fully utilization of historical lifetime information. An investigation of real-world wheel tread signals demonstrates the superiority of the proposed model in accuracy improvement

Mesenchymal Stem Cells Protect Nucleus Pulposus Cells from Compression-Induced Apoptosis by Inhibiting the Mitochondrial Pathway

Objective. Excessive apoptosis of nucleus pulposus cells (NPCs) induced by various stresses, including compression, contributes to the development of intervertebral disc degeneration (IVDD). Mesenchymal stem cells (MSCs) can benefit the regeneration of NPCs and delay IVDD, but the underlying molecular mechanism is poorly understood. This study aimed to evaluate the antiapoptosis effects of bone marrow-derived MSC (BMSC) on rat NPCs exposed to compression and investigate whether the mitochondrial pathway was involved. Methods. BMSCs and NPCs were cocultured in the compression apparatus at 1.0 MPa for 36 h. Cell viability, apoptosis, mitochondrial function, and the expression of apoptosis-related proteins were evaluated. Results. The results showed that coculturing with BMSCs increased the cell viability and reduced apoptosis of NPCs exposed to compression. Meanwhile, BMSCs could relieve the compression-induced mitochondrial damage of NPCs by decreasing reactive oxygen species level and maintaining mitochondrial membrane potential as well as mitochondrial integrity. Furthermore, coculturing with BMSCs suppressed the activated caspase-3 and activated caspase-9, decreased the expressions of cytosolic cytochrome c and Bax, and increased the expression of Bcl-2. Conclusions. Our results suggest that BMSCs can protect against compression-induced apoptosis of NPCs by inhibiting the mitochondrial pathway and thus enhance our understanding on the MSC-based therapy for IVDD

Temperature Distribution in Ridge Structure InGaN Laser Diodes and Its Influence on Device Characteristics

Time-dependent thermal simulation of ridge-geometry InGaN laser diodes is carried out with a two-dimensional model. A high temperature in the waveguide layer and a large temperature step between the regions under and outside the ridge are generated due to the poor thermal conductivity of the sapphire substrate and the large threshold current and voltage. The temperature step is thought to have a strong influence on the characteristics of the laser diodes. Time-resolved measurements of light-current curves,spectra, and the far-field pattern of the InGaN laser diodes under pulsed operation are performed. The results show that the thermal lensing effect improves the confinement of the higher order modes and leads to a lower threshold current and a higher slope efficiency of the device while the high temperature in the active layer results in a drastic decrease in the slope efficiency

RNA binding protein TIAR modulates HBV replication by tipping the balance of pgRNA translation

Abstract The pregenomic RNA (pgRNA) of hepatitis B virus (HBV) serves not only as a bicistronic message RNA to translate core protein (Cp) and DNA polymerase (Pol), but also as the template for reverse transcriptional replication of viral DNA upon packaging into nucleocapsid. Although it is well known that pgRNA translates much more Cp than Pol, the molecular mechanism underlying the regulation of Cp and Pol translation efficiency from pgRNA remains elusive. In this study, we systematically profiled HBV nucleocapsid- and pgRNA-associated cellular proteins by proteomic analysis and identified TIA-1-related protein (TIAR) as a novel cellular protein that binds pgRNA and promotes HBV DNA replication. Interestingly, loss- and gain-of-function genetic analyses showed that manipulation of TIAR expression did not alter the levels of HBV transcripts nor the secretion of HBsAg and HBeAg in human hepatoma cells supporting HBV replication. However, Ribo-seq and PRM-based mass spectrometry analyses demonstrated that TIAR increased the translation of Pol but decreased the translation of Cp from pgRNA. RNA immunoprecipitation (RIP) and pulldown assays further revealed that TIAR directly binds pgRNA at the 5’ stem-loop (ε). Moreover, HBV replication or Cp expression induced the increased expression and redistribution of TIAR from the nucleus to the cytoplasm of hepatocytes. Our results thus imply that TIAR is a novel cellular factor that regulates HBV replication by binding to the 5’ ε structure of pgRNA to tip the balance of Cp and Pol translation. Through induction of TIAR translocation from the nucleus to the cytoplasm, Cp indirectly regulates the Pol translation and balances Cp and Pol expression levels in infected hepatocytes to ensure efficient viral replication

Liver Tumor Markers, HALP Score, and NLR: Simple, Cost-Effective, Easily Accessible Indexes for Predicting Prognosis in ICC Patients after Surgery

Introduction: To investigate the prognostic significance of liver tumor markers, the hemoglobin, albumin, lymphocyte, and platelet (HALP) score; neutrophil-to-lymphocyte ratio (NLR); and platelet-to-lymphocyte ratio (PLR), for predicting the specific site of recurrence or metastasis after surgery in patients with intrahepatic cholangiocarcinoma (ICC). Methods: In total, 162 patients with pathologically proven ICC who underwent curative surgery at Sun Yat-sen University Cancer Center between April 2016 and April 2020 were analyzed. Clinicopathological characteristics were collected retrospectively. The Kaplan–Meier method was used to analyze the overall survival (OS) and recurrence-free survival (RFS). Significant clinical factors were examined by univariate analysis and multivariate analysis and analyzed by receiver operating characteristic (ROC) curve analysis. Results: The cutoff values for the HALP score, NLR, and PLR were determined to be 43.63, 3.73, and 76.51, respectively, using the surv_cutpoint function of survminer using RFS as the target variable. In multivariate analysis, vascular invasion, pathology nerve tract invasion, and carbohydrate antigen 19-9 (CA19-9) levels were independent prognostic factors of OS, whereas the tumor number, pathology microvascular invasion, pathology differentiation, CA19-9 levels, and NLR were independent prognostic factors of RFS. For the whole recurrence analysis, the carcinoembryonic antigen (CEA) index exhibited the largest ROC curve area of all (AUC = 0.590), and the alpha-fetoprotein (AFP) index exhibited the smallest ROC curve area (AUC = 0.530). The HALP score exhibited the largest ROC curve area of all in predicting intrahepatic recurrence (AUC = 0.588), the NLR showed the best predictive value in predicting lymph node metastasis (AUC = 0.703), and the AUC of the CA19-9 index was the largest of all variables in predicting distant metastasis (AUC = 0.619). Conclusions: Our study showed that CA19-9, CEA, HALP score, and NLR are easily accessible, reliable, cost-effective indexes for predicting the specific site of recurrence or metastasis after surgery in ICC patients. Patients with high HALP scores and NLR have a higher risk of intrahepatic and lymph node metastasis recurrence

Molecular Mechanism Behind the Resistance of the G1202R-Mutated Anaplastic Lymphoma Kinase to the Approved Drug Ceritinib

Anaplastic lymphoma kinase (ALK) has been regarded as an essential target for the treatment of nonsmall cell lung cancer (NSCLC). However, the emergence of the G1202R solvent front mutation that confers resistance to the drugs was reported for the first as well as the second generation ALK inhibitors. It was thought that the G1202R solvent front mutation might hinder the drug binding. In this study, a different fact could be clarified by multiple molecular modeling methodologies through a structural analogue of ceritinib (compound 10, Cpd-10) that is reported to be a potent inhibitor against the G1202R mutation. Herein, molecular docking, accelerated molecular dynamics (aMD) simulations in conjunction with principal component analysis (PCA), and free energy map calculations were used to produce reasonable and representative initial conformations for the conventional MD simulations. Compared with Cpd-10, the binding specificity of ceritinib between ALK wild-type (ALK^WT) and ALK G1202R (ALK^G1202R) are primarily controlled by the conformational change of the P-loop- and A-loop-induced energetic redistributions, and the variation is nonpolar interactions, as indicated by conventional MD simulations, PCA, dynamic cross-correlation map (DCCM) analysis, and free energy calculations. Furthermore, the umbrella sampling (US) simulations were carried out to make clear the principle of the dissociation processes of ceritinib and Cpd-10 toward ALK^WT and ALK^G1202R. The calculation results suggest that Cpd-10 has similar dissociation processes from both ALK^WT and ALK^G1202R, but ceritinib is more easily dissociated from ALK^G1202R than from ALK^WT, thus less residence time is responsible for the ceritinib resistance. Our results suggest that both the binding specificity and the drug residence time should be emphasized in rational drug design to overcome the G1202R solvent front mutation of ALK resistance