Design and Development of Acetylthiocholine Electrochemical Biosensor Based on Zinc Oxide–Cerium Oxide Nanohybrid Modified Platinum Electrode

Acetylcholinesterase (AChE) enzyme has been predominantly used for the detection of pesticides and metal ions. But, these sensors respond to pesticides as well as metal ions at certain concentration, which results in poor selectivity. Hence in this work, the amount of thiocholine produced during AChE inhibition has been estimated to detect the residual activity of AChE enzyme in-turn to enhance the efficiency of the biosensor. In this context, Pt/ZnO–CeO₂/AChE/Chitosan based biosensor has been developed for sensitive voltammetric quantification of thiocholine in AChE. The sensor exhibited enhanced electron transfer rate, good conductivity and biocompatibility. Both the intrinsic and extrinsic parameters were simultaneously optimized using second order polynomial regression to get the best conditions for ATCh determination. Under optimized experimental conditions, the redox peak current was linear over the concentration range of 0.1–1.5 mM with detection and quantification limit of 0.05 and 0.15 μM respectively and the sensitivity of 1.47 μA mM^?1.     

Accuracy of four dental age estimation methods in determining the legal age threshold of 18 years among South Indian adolescents and young

The aim of this study was to compare the accuracy, specificity and sensitivity of four commonly used methods of dental age estimation in a sample of south Indian adolescents and young adults aged between 14 and 30 years, with an age threshold of 18 years, using receiver operating characteristic curves (ROC) and the area under the curve (AUC). A total of 1070 orthopantomograms (535 males and 535 females) of adolescents and young adults of south Indian origin were collected retrospectively and interpreted. The effectiveness of each method was evaluated by using sensitivity (Se), specificity (Sp), likelihood ratios (LR+ and LR-) and AUC. Among all methods, I3M< 0.08 resulted in better values of AUC, Se and Sp which were 0.950, 91.5%, 97.8% and 0.950, 88.5% and 98.6% in males and females, respectively. For “stage H” of Demirjian’s system, the AUC, Se and Sp were 0.940, 84.9%, 97.7% and 0.930, 79.9% and 98.5% in males and females, respectively. The use of the Olze et al “stage 1 (or higher)” root pulp visibility and “stage D” of third molar eruption were not recommended in the studied population due to the greater percentage of third molars with incomplete mineralization in younger age groups and impaction. Taking into account the values of Se, Sp, both positive and negative LRs, we recommend the use of the cut-off value of I3M< 0.08 to discriminate adults and minors in south Indian adolescents and young adults.     

Living donor liver transplantation: selection, perioperative care, and outcome

A perception that living donor liver transplantation can be accomplished with an acceptable donor complication rate and recipient survival rate has led to the acceptance of living donor liver transplantation as a viable alternative to decreased deceased donor transplantation. Careful candidate evaluation and selection has been crucial to the success of this procedure. Advancements in the understanding of the lobar nature of the liver and of liver regeneration have advanced the surgical technique. Initial attempts at adult-to-adult donation utilized the left hepatic lobe, but now have evolved into use of the right hepatic lobe. Size matching is very important to successful graft function in the recipient. There is great concern regarding morbidity and mortality in donors. Biliary complications and infections continue to be among the most highly reported complications, although rates vary among centers and countries. Reports of single center complications have ranged from 9% to 67%. A survey of centers in the United States in 2003 reported complications of 10%. A series from our institution reported complications arising in 13 (33%) of 39 patients. A review focused on documenting donor deaths found 33 living liver donor deaths worldwide. The much publicized immediate postoperative mishap of 2002 that resulted in a donor's death resulted in a drop in the utilization of living donor liver transplantation in the United States, from which this procedure has never fully recovered. The future development and expansion of living donor liver transplantation depends on open communication regarding donor complications and deaths. Close immediate postoperative monitoring and meticulous management will remain an essential aspect in limiting donor complications and deaths.     

Components of the phosphatidylserine endoplasmic reticulum to plasma membrane transport mechanism as targets for KRAS inhibition in pancreatic cancer

KRAS is mutated in 90% of human pancreatic ductal adenocarcinomas (PDACs). To function, KRAS must localize to the plasma membrane (PM) via a C-terminal membrane anchor that specifically engages phosphatidylserine (PtdSer). This anchor-binding specificity renders KRAS–PM localization and signaling capacity critically dependent on PM PtdSer content. We now show that the PtdSer lipid transport proteins, ORP5 and ORP8, which are essential for maintaining PM PtdSer levels and hence KRAS PM localization, are required for KRAS oncogenesis. Knockdown of either protein, separately or simultaneously, abrogated growth of KRAS-mutant but not KRAS–wild-type pancreatic cancer cell xenografts. ORP5 or ORP8 knockout also abrogated tumor growth in an immune-competent orthotopic pancreatic cancer mouse model. Analysis of human datasets revealed that all components of this PtdSer transport mechanism, including the PM-localized EFR3A-PI4KIIIα complex that generates phosphatidylinositol-4-phosphate (PI4P), and endoplasmic reticulum (ER)–localized SAC1 phosphatase that hydrolyzes counter transported PI4P, are significantly up-regulated in pancreatic tumors compared to normal tissue. Taken together, these results support targeting PI4KIIIα in KRAS-mutant cancers to deplete the PM-to-ER PI4P gradient, reducing PM PtdSer content. We therefore repurposed the US Food and Drug Administration–approved hepatitis C antiviral agent, simeprevir, as a PI4KIIIα inhibitor In a PDAC setting. Simeprevir potently mislocalized KRAS from the PM, reduced the clonogenic potential of pancreatic cancer cell lines in vitro, and abrogated the growth of KRAS-dependent tumors in vivo with enhanced efficacy when combined with MAPK and PI3K inhibitors. We conclude that the cellular ER-to-PM PtdSer transport mechanism is essential for KRAS PM localization and oncogenesis and is accessible to therapeutic intervention.

RAS proteins are small GTPases that switch between active GTP-bound and inactive GDP-bound states, regulating cell proliferation, differentiation, and apoptosis. RAS is regulated by guanine nucleotide exchange factors that promote GDP–GTP exchange, thereby activating RAS, and GTPase-activating proteins (GAPs), which stimulate intrinsic RAS GTPase activity to return it to its inactive state. Approximately 20% of human cancers express oncogenic RAS with mutations at residues 12, 13, or 61 (1), which prevent RASGAPs from stimulating GTP hydrolysis, rendering RAS constitutively active. The RAS isoforms, HRAS, NRAS, KRAS4A, and KRAS4B (hereafter referred to as KRAS), have near-identical G-domains, which are implicated in guanine nucleotide binding and effector interaction. However, they have different C termini and membrane anchors, which contribute to their differential signaling outputs (2). KRAS is the most-frequently mutated isoform in cancer and hence represents the major clinical concern, especially in pancreatic, colon, and non–small cell lung cancers (NSCLCs) in which mutant KRAS is expressed in ∼90%, ∼50%, and ∼25% of cases, respectively (3).RAS proteins must localize to the plasma membrane (PM) and organize into nanoclusters for biological activity (4–8), whereby RAS.GTP recruits its effectors to PM nanoclusters, leading to downstream pathway activation. KRAS interacts with the PM via its C-terminal membrane anchor that comprises a farnesyl-cysteine-methyl-ester and a polybasic domain (PBD) of six contiguous lysines (9–11). Together, the KRAS PBD sequence and prenyl group define a combinatorial code for lipid binding, resulting in a membrane anchor that specifically interacts with asymmetric species of phosphatidylserine (PtdSer) that contain one saturated and one desaturated acyl chain (8, 12–14). Since PtdSer binding specificity is hardwired into its anchor structure, KRAS–PM interactions are PtdSer dependent. KRAS that partitions into the cytosol following endocytosis is captured by PDEδ, which, upon interacting with ARL2, is released to the recycling endosome (RE) for forward transport back to the PM (15). Capture of KRAS by the RE is again PtdSer dependent; therefore, abrogating PtdSer delivery to the PM will reduce PM and RE PtdSer content, abrogating both KRAS PM binding and KRAS recycling back to the PM. In sum, KRAS–PM localization, nanoclustering, and signaling capacity are all exquisitely dependent on PM PtdSer levels.Previous attempts at preventing KRAS–PM localization to inhibit its function include the development of farnesyltransferase inhibitors (FTIs), which inhibit the first posttranslational processing step that generates the KRAS membrane anchor. FTIs were clinically unsuccessful since KRAS can alternatively be geranylgeranylated by geranylgeranyl transferase1 when cells are treated with FTIs, allowing for continued PM localization (2, 16, 17). We recently leveraged the dependence of KRAS on PM PtdSer to inhibit KRAS signaling by targeting the cellular machinery that actively maintains PM PtdSer levels (18). Genetic knockdown (KD) of ORP5 or ORP8, two lipid transporters that function at endoplasmic reticulum (ER)–PM membrane contact sites to transport PtdSer to the PM (Fig. 1), mislocalized KRAS from the PM and reduced nanoclustering of any remaining KRAS. Consequently, ORP5/8 KD decreased proliferation and anchorage-independent growth of multiple KRAS-dependent pancreatic cancer cell lines. In this study, we examine the effects of ORP5/8 genetic KD and knockout (KO) on tumor growth in vivo and provide compelling evidence that these proteins are essential for tumor maintenance in KRAS-dependent pancreatic cancer. ORP5/8 function by exchanging phosphoinositide-4-phosphate (PI4P) synthesized on the PM by PI4KIIIα for PtdSer synthesized in the ER (19, 20). We demonstrate both in vitro and in vivo that PI4KIIIα inhibitors can potently inhibit oncogenic KRAS function. One such inhibitor is simeprevir, a US Food and Drug Administration (FDA)–approved antiviral agent used for the treatment of hepatitis C, that may have potential for repurposing as a therapeutic for mutant KRAS-driven cancers.Open in a separate windowFig. 1.ORP5 and ORP8 transport PtdSer to the PM. ORP5 and ORP8 exchange ER PtdSer with PM PI4P. This is driven by a PI4P concentration gradient whereby PM PI4P levels are kept high by PI4KIIIα and low at the ER by SAC1P, which hydrolyzes PI4P. ORP, oxysterol-binding protein-related protein; PI4KIIIα, class III PI4 kinase alpha; and SAC1P, SAC1-like phosphatidylinositide phosphatase.