Direct Analysis from Phase-Separated Liquid Samples using ADE-OPI-MS: Applicability to High-Throughput Screening for Inhibitors of Diacylglycerol Acyltransferase 2.

The primary goal of high-throughput screening (HTS) is to rapidly survey a broad collection of compounds, numbering from tens of thousands to millions of members, and identify those that modulate the activity of a therapeutic target of interest. For nearly two decades, mass spectrometry has been used as a label-free, direct-detection method for HTS and is widely acknowledged as being less susceptible to interferences than traditional optical techniques. Despite these advantages, the throughput of conventional MS-based platforms like RapidFire or parallel LC-MS, which typically acquire data at speeds of 6-30 s/sample, can still be limiting for large HTS campaigns. To overcome this bottleneck, the field has recently turned to chromatography-free approaches including MALDI-TOF-MS and acoustic droplet ejection-MS, both of which are capable of throughputs of 1 sample/second or faster. In keeping with these advances, we report here on our own characterization of an acoustic droplet ejection, open port interface (ADE-OPI)-MS system as a platform for HTS using the membrane-associated, lipid metabolizing enzyme diacylglycerol acyltransferase 2 (DGAT2) as a model system. We demonstrate for the first time that the platform is capable of ejecting droplets from phase-separated samples, allowing direct coupling of liquid-liquid extraction with OPI-MS analysis. By applying the platform to screen a 6400-member library, we further demonstrate that the ADE-OPI-MS assay is suitable for HTS and also performs comparably to LC-MS, but with an efficiency gain of >20-fold.

High-Throughput Bioassays using "Dip-and-Go" Multiplexed Electrospray Mass Spectrometry.

A multiplexed system based on inductive nanoelectrospray mass spectrometry (nESI-MS) has been developed for high-throughput screening (HTS) bioassays. This system combines inductive nESI and field amplification micro-electrophoresis to achieve a "dip-and-go" sample loading and purification strategy that enables nESI-MS based HTS assays in 96-well microtiter plates. The combination of inductive nESI and micro-electrophoresis makes it possible to perform efficient in situ separations and clean-up of biological samples. The sensitivity of the system is such that quantitative analysis of peptides from 1-10 000 nm can be performed in a biological matrix. A prototype of the automation system has been developed to handle 12 samples (one row of a microtiter plate) at a time. The sample loading and electrophoretic clean-up of biosamples can be done in parallel within 20 s followed by MS analysis at a rate of 1.3 to 3.5 s per sample. The system was used successfully for the quantitative analysis of BACE1-catalyzed peptide hydrolysis, a prototypical HTS assay of relevance to drug discovery. IC50 values for this system were in agreement with LC-MS but recorded in times more than an order of magnitude shorter.

GPR120 suppresses adipose tissue lipolysis and synergizes with GPR40 in antidiabetic efficacy.

GPR40 and GPR120 are fatty acid sensors that play important roles in glucose and energy homeostasis. GPR40 potentiates glucose-dependent insulin secretion and demonstrated in clinical studies robust glucose lowering in type 2 diabetes. GPR120 improves insulin sensitivity in rodents, albeit its mechanism of action is not fully understood. Here, we postulated that the antidiabetic efficacy of GPR40 could be enhanced by coactivating GPR120. A combination of GPR40 and GPR120 agonists in db/db mice, as well as a single molecule with dual agonist activities, achieved superior glycemic control compared with either monotherapy. Compared with a GPR40 selective agonist, the dual agonist improved insulin sensitivity in ob/ob mice measured by hyperinsulinemic-euglycemic clamp, preserved islet morphology, and increased expression of several key lipolytic genes in adipose tissue of Zucker diabetic fatty rats. Novel insights into the mechanism of action for GPR120 were obtained. Selective GPR120 activation suppressed lipolysis in primary white adipocytes, although this effect was attenuated in adipocytes from obese rats and obese rhesus, and sensitized the antilipolytic effect of insulin in rat and rhesus primary adipocytes. In conclusion, GPR120 agonism enhances insulin action in adipose tissue and yields a synergistic efficacy when combined with GPR40 agonism.

Achieving efficient digestion faster with Flash Digest: potential alternative to multi-step detergent assisted in-solution digestion in quantitative proteomics experiments.

RATIONALE: In quantitative analysis of protein biomarkers and therapeutic proteins by liquid chromatography/mass spectrometry (LC/MS), it is a preferred and well-established approach to digest with proteolytic enzymes to produce smaller peptide fragments which are more suitable for LC/MS analysis than the intact protein. In-solution digestion is one widely used method for protein digestion. Proteolytically resistant proteins often require digestion times that extend beyond normal working hours and prohibit same day analysis. We evaluated the performance of an immobilized enzyme reactor (IMER) to determine if this technology could reduce method development time, digestion time and increase throughput. METHODS: We digested human plasma samples using a commercially available IMER, Flash Digest, and compared it to an in-solution digestion method for analysis of three different apolipoprotein biomarkers APOE, APOC2, and APOC3. The plasma digests were analyzed via LC/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM). Value assigned calibrators were selected over a relevant physiological concentration range for each protein of interest. Quality control samples (QCs) and 'unknown' human plasma samples were analyzed with both methods. RESULTS: Flash Digest significantly reduced digestion time for APOC3, the most proteolytically resistant of the three proteins, to 30 min compared with overnight used with in-solution digestion. The Flash Digest achieved comparable digestion efficiency with minimal method development and reduced sample preparation time. Both methods showed linearity over a physiologically relevant concentration range. Precision was evaluated and a percentage coefficient of variance (% CV) less than 8% was obtained during intra-day reproducibility evaluation for all three apolipoproteins with Flash Digest. Concentrations observed for QCs and unknown samples using Flash Digest were comparable to the in-solution method. CONCLUSIONS: An IMER such as Flash Digest may be a potential alternative to in-solution digestion to accelerate digestion of proteolytically resistant proteins in a quantitative proteomics experiments, reduce method development time and increase throughput. Copyright © 2016 John Wiley & Sons, Ltd.

Invasive fungal rhinosinusitis with palatal erosion in an elderly edentulous patient with uncontrolled diabetes: report of a rare case.

OBJECTIVES: To report a rare case of chronic invasive fungal rhinosinusitis with palatal erosion. BACKGROUND: Restoring and maintaining oral health of diabetic elderly patients with increased risk of infections is a challenge to the dentist. Patients suffering from uncontrolled diabetes are susceptible to fungal infections. Palatal erosion due to fungal rhinosinusitis is rare. MATERIALS AND METHODS: Case report of a 65 years old illiterate female patient from low socio-economic strata, suffering from uncontrolled diabetes and poor systemic health presenting with chronic invasive fungal rhinosinusitis leading to palatal erosion. CONCLUSION: Such a case is a diagnostic challenge to a dentist. Therefore understanding the disease process and its possible outcomes is desirable. The treatment warrants a multidisciplinary approach.

Glucagon receptor antagonism induces increased cholesterol absorption.

Glucagon and insulin have opposing action in governing glucose homeostasis. In type 2 diabetes mellitus (T2DM), plasma glucagon is characteristically elevated, contributing to increased gluconeogenesis and hyperglycemia. Therefore, glucagon receptor (GCGR) antagonism has been proposed as a pharmacologic approach to treat T2DM. In support of this concept, a potent small-molecule GCGR antagonist (GRA), MK-0893, demonstrated dose-dependent efficacy to reduce hyperglycemia, with an HbA1c reduction of 1.5% at the 80 mg dose for 12 weeks in T2DM. However, GRA treatment was associated with dose-dependent elevation of plasma LDL-cholesterol (LDL-c). The current studies investigated the cause for increased LDL-c. We report findings that link MK-0893 with increased glucagon-like peptide 2 and cholesterol absorption. There was not, however, a GRA-related modulation of cholesterol synthesis. These findings were replicated using structurally diverse GRAs. To examine potential pharmacologic mitigation, coadministration of ezetimibe (a potent inhibitor of cholesterol absorption) in mice abrogated the GRA-associated increase of LDL-c. Although the molecular mechanism is unknown, our results provide a novel finding by which glucagon and, hence, GCGR antagonism govern cholesterol metabolism.

New methodologies for studying lipid synthesis and turnover: looking backwards to enable moving forwards.

Our ability to understand the pathogenesis of problems surrounding lipid accretion requires attention towards quantifying lipid kinetics. In addition, studies of metabolic flux should also help unravel mechanisms that lead to imbalances in inter-organ lipid trafficking which contribute to dyslipidemia and/or peripheral lipid accumulation (e.g. hepatic fat deposits). This review aims to outline the development and use of novel methods for studying lipid kinetics in vivo. Although our focus is directed towards some of the approaches that are currently reported in the literature, we include a discussion of the older literature in order to put "new" methods in better perspective and inform readers of valuable historical research. Presumably, future advances in understanding lipid dynamics will benefit from a careful consideration of the past efforts, where possible we have tried to identify seminal papers or those that provide clear data to emphasize essential points. This article is part of a Special Issue entitled: Modulation of Adipose Tissue in Health and Disease.

Statins inhibit insulin-like growth factor action in first trimester placenta by altering insulin-like growth factor 1 receptor glycosylation.

The rapid rise in obesity, metabolic syndrome and type 2 diabetes is one of the major healthcare problems of the Western world. Affected individuals are often treated with statins (3-hydroxy-3-methylglutaryl co-enzyme A [HMG CoA] reductase inhibitors) to reduce circulating cholesterol levels and the risk of developing cardiovascular disease; given the evolving demographic profile of these conditions, such drugs are increasingly prescribed to women of reproductive age. We have previously shown that exposure of placental tissue to statins inhibits the action of insulin-like growth factors (IGF)-I and -II which are key regulators of trophoblast proliferation and placental development. N-linked glycans in the IGF receptor, IGF1R, influence its presentation at the cell surface. This study aimed to determine whether statins, which are known to affect N-glycosylation, modulate IGF1R function in placenta. Treatment of first trimester villous tissue explants with statins (pravastatin or cerivastatin) or inhibitors of N-glycosylation (tunicamycin, deoxymannojirimycin or castanospermine) altered receptor distribution in trophoblast and attenuated proliferation induced by IGF-I or IGF-II (Ki67; P < 0.05, n = 5). Decreased binding of Phaseolus vulgaris lectin and phytohaemagglutinin to IGF1R immunoprecipitated from treated explants demonstrated reduced levels of complex N-linked glycans. Co-incubation of tissue explants with statins and farnesyl pyrophosphate (which increases the supply of dolichol intermediates), prevented statin-mediated disruption of IGF1R localization and reversed the negative effect on IGF-mediated trophoblast proliferation. These data suggest that statins attenuate IGF actions in the placenta by inhibiting N-linked glycosylation and subsequent expression of mature IGF1R at the placental cell surface.

In vivo isotopically labeled atherosclerotic aorta plaques in ApoE KO mice and molecular profiling by matrix-assisted laser desorption/ionization mass spectrometric imaging.

RATIONALE: The ability to quantify rates of formation, regression and/or remodeling of atherosclerotic plaque should facilitate a better understanding of the pathogenesis and management of cardiovascular disease. In the current study, we coupled a stable isotope labeled tracer protocol with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to examine spatial and temporal lipid dynamics in atherosclerotic plaque. METHODS: To promote plaque formation in the aorta region, ApoE KO mice were fed a high cholesterol diet (0.15% cholesterol) and orally dosed with (2,2,3,4,4,6-d(6))-cholesterol over several weeks. Tissue sections of ~10 µm thickness were analyzed by MALDI-MSI using matrix deposition by either chemical sublimation or acoustic droplet ejection. RESULTS: MALDI-MSI yielded distinct spatial distribution information for a variety of lipid classes including specific lysophosphatidylcholines typically associated with atherosclerosis-related tissue damage such as phospholipase 2 (Lp-PLA(2)) that mediate chemotactic responses to inflammation (e.g. LPC 16:0, LPC 18:0 and LPC 18:1) as well as free cholesterol and cholesteryl esters that contribute to atheroma formation. MALDI mass spectra acquired from aorta tissue sections clearly distinguished non-esterified and esterified versions of (2,2,3,4,4,6-d(6))-cholesterol within aortic plaque regions and showed distinct spatial accumulation of the cholesterol tracer. CONCLUSIONS: The ability to couple stable isotope based protocols with MALDI-MSI enables a novel strategy to characterize the effects of therapeutic treatments on atherosclerotic plaque formation, regression and potential remodeling of the complex lipid components with high chemical specificity and spatiotemporal information.

Determination of low levels of 2H-labeling using high-resolution mass spectrometry: application in studies of lipid flux and beyond.

RATIONALE: The ability to measure low levels of (2)H-labeling is important in studies of metabolic flux, e.g. one can estimate lipid synthesis by administering (2)H2O and then measuring the incorporation of (2)H into fatty acids. Unfortunately, the analyses are complicated by the presence of more abundant naturally occurring stable isotopes, e.g. (13)C. Conventional approaches rely on coupling gas chromatographic separation of lipids with either quadrupole-mass spectrometry (q-MS) and/or pyrolysis-isotope ratio mass spectrometry (IRMS). The former is limited by high background labeling (primarily from (13)C) whereas the latter is not suitable for routine high-throughput analyses. METHODS: We have contrasted the use of continuous flow-pyrolysis-IRMS against high-resolution mass spectrometry (i.e. Qq-FT-ICR MS) for measuring the (2)H-enrichment of fatty acids and peptides. RESULTS: In contrast to IRMS, which requires ~30 min per analysis, it is possible to measure the (2)H-enrichment of palmitate via direct infusion high-resolution mass spectrometry (HRMS) in ~3 min per sample. In addition, Qq-FT-ICR MS enabled measurements of the (2)H-enrichment of peptides (which is not possible using IRMS). CONCLUSIONS: High-resolution mass spectrometry can be used to measure low levels of (2)H-labeling so we expect that this approach will enhance studies of metabolic flux that rely on (2)H-labeled tracers, e.g. (2)H2O. However, since the high-resolution analyses require greater amounts of a given analyte one potential limitation centers on the overall sensitivity. Presumably, future advances can overcome this barrier.

Lipidome of atherosclerotic plaques from hypercholesterolemic rabbits.

The cellular, macromolecular and neutral lipid composition of the atherosclerotic plaque has been extensively characterized. However, a comprehensive lipidomic analysis of the major lipid classes within atherosclerotic lesions has not been reported. The objective of this study was to produce a detailed framework of the lipids that comprise the atherosclerotic lesion of a widely used pre-clinical model of plaque progression. Male New Zealand White rabbits were administered regular chow supplemented with 0.5% cholesterol (HC) for 12 weeks to induce hypercholesterolemia and atherosclerosis. Our lipidomic analyses of plaques isolated from rabbits fed the HC diet, using ultra-performance liquid chromatography (UPLC) and high-resolution mass spectrometry, detected most of the major lipid classes including: Cholesteryl esters, triacylglycerols, phosphatidylcholines, sphingomyelins, diacylglycerols, fatty acids, phosphatidylserines, lysophosphatidylcholines, ceramides, phosphatidylglycerols, phosphatidylinositols and phosphatidylethanolamines. Given that cholesteryl esters, triacylglycerols and phosphatidylcholines comprise greater than 75% of total plasma lipids, we directed particular attention towards the qualitative and quantitative assessment of the fatty acid composition of these lipids. We additionally found that sphingomyelins were relatively abundant lipid class within lesions, and compared the abundance of sphingomyelins to their precursor phosphatidylcholines. The studies presented here are the first approach to a comprehensive characterization of the atherosclerotic plaque lipidome.

Enhanced data-independent analysis of lipids using ion mobility-TOFMSE to unravel quantitative and qualitative information in human plasma.

RATIONALE: Lipids are involved in various biochemical and signaling pathways, cell structure and function, and the pathophysiology of many diseases. We took advantage of ion mobility spectrometry (IMS) in conjunction with ultra-performance liquid chromatography (UPLC) and high-resolution mass spectrometry to gain quantitative and deeper qualitative structural insight within a single experiment. METHODS: Human plasma lipid extracts were analyzed using an Acquity UPLC system coupled to a Synapt G2-HDMS mass spectrometer system. The ion mobility gas employed was helium for the helium cell (150 mL/min) and nitrogen (80 mL/min) for the T-wave drift tube. The wave height for the T-wave cell was ramped in a linear fashion between 5-40 V. The mass spectra were acquired in an electrospray positive ionization mode. RESULTS: We resolved chromatographically co-eluting lipids further by ion mobility tube drift time and then subjected them to low- and high-energy fragmentation without pre-selecting respective precursor species. The fragment ions produced in a high-energy mode were aligned with their precursor ions in a low-energy mode. By aligning intact molecular spectra and fragment spectra for these lipids at a given ion mobility drift time and chromatographic retention time, we were able to obtain much cleaner fragment ion spectra for structural elucidation. For quantitative analysis we obtained a dynamic linear range from 0.002 to 2 µg/mL with and without an additional dimension of IMS. CONCLUSIONS: The additional dimension of IMS allowed us to perform quantitative and qualitative analysis within a single experiment in a relatively high-throughput manner thus providing deeper structural insights into lipids of biological interest and resulting in an information-rich dataset.

Posterior Microphthalmos Pigmentary Retinopathy Syndrome.

Purpose: To report a case of a rare disease entity Posterior Microphthalmos Pigmentary Retinopathy Syndrome (PMPRS) in a 47-year-old female with a brief review of literature. Case Report: A 47-year-old woman presented with a history of defective vision with an associated difficulty in night vision. Clinical workup was done, which included a thorough ocular examination showing diffuse pigmentary mottling of fundus, ocular biometry showing short axial length with normal anterior segment dimensions, electroretinography showing extinguished response, optical coherence tomography showing foveoschisis, and ultrasonography showing thickened sclera-choroidal complex. Findings were consistent with those reported by other authors with PMPRS. Conclusion: Posterior microphthalmia with or without other ocular and systemic associations should be suspected in cases with high hyperopia. It is mandatory to carefully examine the patient at presentation and close follow-ups are needed to maintain visual function.

In vivo D2O labeling to quantify static and dynamic changes in cholesterol and cholesterol esters by high resolution LC/MS.

High resolution LC/MS-MS and LC/APPI-MS methods have been established for the quantitation of flux in the turnover of cholesterol and cholesterol ester. Attention was directed toward quantifying the monoisotopic mass (M0) and that of the singly deuterated labeled (M+1) isotope. A good degree of isotopic dynamic range has been achieved by LC/MS-MS ranging from 3-4 orders of magnitude. Correlation between the linearity of GC/MS and LC atmospheric pressure photoionization (APPI)-MS are complimentary (r² = 0.9409). To prove the viability of this particular approach, male C57Bl/6 mice on either a high carbohydrate (HC) or a high fat (HF) diet were treated with ²H2O for 96 h. Gene expression analysis showed an increase in the activity of stearoyl-CoA desaturase (Scd1) in the HC diet up to 69-fold (P < 0.0008) compared with the HF diet. This result was supported by the quantitative flux measurement of the isotopic incorporation of ²H into the respective cholesterol and cholesterol ester (CE) pools. We concluded that it is possible to readily obtain static and dynamic measurement of cholesterol and CEs in vivo by coupling novel LC/MS methods with stable isotope-based protocols.

Anacetrapib promotes reverse cholesterol transport and bulk cholesterol excretion in Syrian golden hamsters.

Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester (CE) and triglyceride between HDL and apoB-containing lipoproteins. Anacetrapib (ANA), a reversible inhibitor of CETP, raises HDL cholesterol (HDL-C) and lowers LDL cholesterol in dyslipidemic patients; however, the effects of ANA on cholesterol/lipoprotein metabolism in a dyslipidemic hamster model have not been demonstrated. To test whether ANA (60 mg/kg/day, 2 weeks) promoted reverse cholesterol transport (RCT), ³H-cholesterol-loaded macrophages were injected and (3)H-tracer levels were measured in HDL, liver, and feces. Compared to controls, ANA inhibited CETP (94%) and increased HDL-C (47%). ³H-tracer in HDL increased by 69% in hamsters treated with ANA, suggesting increased cholesterol efflux from macrophages to HDL. ³H-tracer in fecal cholesterol and bile acids increased by 90% and 57%, respectively, indicating increased macrophage-to-feces RCT. Mass spectrometry analysis of HDL from ANA-treated hamsters revealed an increase in free unlabeled cholesterol and CE. Furthermore, bulk cholesterol and cholic acid were increased in feces from ANA-treated hamsters. Using two independent approaches to assess cholesterol metabolism, the current study demonstrates that CETP inhibition with ANA promotes macrophage-to-feces RCT and results in increased fecal cholesterol/bile acid excretion, further supporting its development as a novel lipid therapy for the treatment of dyslipidemia and atherosclerotic vascular disease.

Identifying static and kinetic lipid phenotypes by high resolution UPLC-MS: unraveling diet-induced changes in lipid homeostasis by coupling metabolomics and fluxomics.

A novel method to differentiate diet-induced alterations in plasma lipid phenotypes "static (concentration of lipids) and kinetic (endogenous production, e.g., denovo lipogenesis)" was employed. C57Bl6 mice were randomized into 2 groups and fed either a high-carbohydrate, low-fat (HC) or a carbohydrate-free, high-fat diet (HF) diet for 13 days; D(2)O was administered via intraperitoneal injection and then adding D(2)O to the drinking water for 96 h. Principal component analysis (PCA) revealed differences in the plasma lipid content, for example, triglycerides (TG) 50:2, 50:3, and 52:2 were up-regulated in mice fed the HC diet, whereas TG 52:4, 52:1, 54:5, 54:3, 54:4, and 54:2 were higher in animals fed the HF diet. However, although the fractional contribution of synthesis was ~10-fold lower in HF vs HC fed mice, changes in TG concentration were not entirely mediated by altered de novo lipogenesis. In addition, the ability to couple isotope labeling measurements with PCA analyses revealed cases where there were no differences in the concentration of a compound but its source was substantially altered. In summary, this strategy determined (i) the presence/absence of differences in concentration and (ii) the contribution of different pathways and synthesis that could affect lipid biology in a mouse model respectively.

Attenuation of Slc27a5 gene expression followed by LC-MS measurement of bile acid reconjugation using metabolomics and a stable isotope tracer strategy.

The purpose of this study was to evaluate the use of high resolution LC-MS together with metabolomics and D(4)-cholic acid (D(4)-CA) as a metabolic tracer to measure the metabolism and reconjugation of bile acids (BAs) in vitro and in vivo. Metabolic tracers are very important because they allow for the direct detection (substrate-to-product) of small and significant biological perturbations that may not be apparent when monitoring "static" endogenous levels of particular metabolites. Slc27a5, also known as fatty acid transport protein 5 (FATP5), is the hepatic BA-CoA ligase involved in reconjugating BAs during enterohepatic BA recycling. Using Slc27a5-cKD mice, silencing of ∼90% gene expression was achieved followed by reduction in the reconjugation of D(4)-CA to D(4)-taurocholic acid (D(4)-TCA), as well as other conjugated BA metabolites in plasma (p = 0.0031). The method described allowed a rapid measure of many D(4) and endogenous BA. Analysis of bile resulted in the detection of 39 BA metabolites from a 13 min analytical run. Finally, the utilization of a novel high resolution mass spectrometry method in combination with metabolomics and a stable isotope metabolic tracer allowed for the detection of targeted and untargeted BAs following silencing of the Slc27a5 gene in primary hepatocytes and in mice.

Comparative evaluation of anatomical and functional outcomes using brilliant blue G versus triamcinolone assisted ILM peeling in macular hole surgery in Indian population.

PURPOSE: To compare anatomical and functional outcomes using brilliant blue G (BBG) vs triamcinolone acetonide (TA)-assisted ILM peeling in macular hole surgery (MHS). STUDY DESIGN: Simple, comparative, retrospective, non-randomised, interventional single-centre study. METHODS: Ninety-four eyes of 94 patients with idiopathic macular holes (≥ stage 2) who underwent MHS at our centre were included. Patients with failed macular holes, post-traumatic macular holes, history of previous vitreoretinal surgery, high myopia (6 dioptres or more) or any other macular pathology potentially limiting visual acuity, such as diabetic retinopathy or age-related macular degeneration, were excluded. An OCT evaluation of hole status was followed by pars plana vitrectomy for each of these eyes. Those who underwent TA-assisted ILM peeling were considered as group 1 and those with BBG-assisted ILM peeling were considered as group 2. Primary outcome measures included anatomical hole closure and functional success in terms of change in visual acuity of ≥2 LogMAR lines. Various preoperative factors were also evaluated. RESULTS: Anatomical hole closure was achieved in 85 eyes (90.43%) and visual gain in 78 eyes (82.9%). Mean postoperative follow-up duration was 16.14 ± 1.95 months. No significant difference was found in anatomical and functional success between the two groups. Group 1 had a significantly higher incidence of postoperative glaucoma. Duration of symptoms of <12 months (p = 0.004) and preoperative visual acuity ≤1.0 LogMAR were related to anatomical success. However, greater visual gain was found in patients with chronic holes (≥12 months) (p = 0.046) and poor preoperative visual acuity (>1.0 LogMAR) (p = 0.001). CONCLUSION: BBG-assisted ILM peeling offers an effective alternative to triamcinolone, with the added advantage of marked enhancement of vitreoretinal interface contrast with comparable hole closure rates and visual outcomes.

High-Throughput Mass Spectrometry for Hit Identification: Current Landscape and Future Perspectives.

For nearly two decades mass spectrometry has been used as a label-free, direct-detection method for both functional and affinity-based screening of a wide range of therapeutically relevant target classes. Here, we present an overview of several established and emerging mass spectrometry platforms and summarize the unique strengths and performance characteristics of each as they apply to high-throughput screening. Multiple examples from the recent literature are highlighted in order to illustrate the power of each individual technique, with special emphasis given to cases where the use of mass spectrometry was found to be differentiating when compared with other detection formats. Indeed, as many of these examples will demonstrate, the inherent strengths of mass spectrometry-sensitivity, specificity, wide dynamic range, and amenability to complex matrices-can be leveraged to enhance the discriminating power and physiological relevance of assays included in screening cascades. It is our hope that this review will serve as a useful guide to readers of all backgrounds and experience levels on the applicability and benefits of mass spectrometry in the search for hits, leads, and, ultimately, drugs.

Aurolab aqueous drainage implant in the vitreous cavity: Our modifications over the conventional technique of glaucoma implant surgery.

Glaucoma drainage devices (GDDs) are used for managing refractory glaucoma due to failed trabeculectomy, neovascular glaucoma, traumatic glaucoma, and secondary glaucoma post keratoplasty. Aurolab aqueous drainage implant (AADI) is a nonvalved drainage implant conventionally implanted with the tube placed in the anterior chamber. Studies about the outcome of the various aqueous drainage devices implanted in the anterior chamber have reported complications such as tube extrusion, migration, blockage, erosion, and corneal decompensation. We propose modifying the conventional GDD implantation technique by placing the tube in the vitreous cavity, thereby negating the risk of anterior segment complications in patients with refractory glaucoma whose anterior segment is already compromised. Another novel approach implemented in this technique was making a scleral tunnel instead of using a scleral or corneal patch graft to cover the tube to prevent its migration. This article describes the surgical steps of this technique and its advantages, along with a surgical video.