Diagnostic efficacy of elevated serum angiotensin-converting enzyme and lymphopenia between presumed sarcoid uveitis and presumed tubercular uveitis.

PURPOSE: To evaluate the diagnostic efficacy of elevated serum angiotensin-converting enzyme (sACE) and lymphopenia in presumed sarcoid and tubercular uveitis. METHODS: A single-centre retrospective study was conducted on a cohort of 755 adult patients with uveitis between January 2019 and June 2020. Demographic, clinical and laboratory data were retrieved from our hospital database. Measurements of serum angiotensin-converting enzyme (sACE) and lymphocyte counts were analysed. RESULTS: The mean age of the patients was 41 ± 13 years. Presumed sarcoid uveitis was diagnosed in 50 (7%) patients, presumed tubercular uveitis in 222 (29.4%) and other uveitic entities noted in 483 (64%). Intermediate and posterior uveitis were the most common anatomical diagnosis in presumed sarcoid uveitis (59% and 20%, respectively) and in presumed tubercular uveitis (46% and 38%, respectively). Elevated sACE was noted in 76% of presumed sarcoid uveitis and 46% in presumed tubercular uveitis. The combination of high serum angiotensin-converting enzyme along with lymphopenia was only in 17% in presumed sarcoid uveitis and 9.7% in presumed tubercular uveitis. sACE was found to be a significant risk factor for presumed sarcoid uveitis with an odds ratio of 3.603 (p < 0.002), and in presumed tubercular uveitis odds ratio was not significant with odds ratio of 1.19. Lymphopenia was not found to be a significant factor in both groups. CONCLUSION: Elevated sACE activity was an independent risk factor for presumed sarcoid uveitis over lymphopenia alone or in combination with lymphopenia.

RAGE silencing deters CML-AGE induced inflammation and TLR4 expression in endothelial cells.

The Nε-(carboxymethyl)lysine (CML), the predominant advanced glycation end products (AGEs) in diabetes and its RAGE induced cytokine release has been well explored. But the CML mediated multiple AGEs receptor expression is still not understood and the role played by RAGE silencing in modulating CML generated pro-inflammatory cytokines in micro and macrovascular endothelial cells is yet to be studied. HUVEC and HREC cells were exposed with CML for 24 h. RAGE, AGER1, AGER2, Gal-3, TLR4, TLR2, CD36, FEEL-1, FEEL-2, and chemokine HMGB1 were quantified by either qPCR/western blotting. The receptor's expression was also determined in control vs diabetic retina. Expression of pro-inflammatory genes, ROS, and mitochondrial membrane potential change were assessed using ELISA, DCFDA, and JC-1 method respectively. RAGE expression was silenced either by Si-RAGE or neutralising antibody with anti-RAGE and expression of other AGE receptors, adaptors, and signalling pathway were studied compared with Si-Control. CML activated RAGE, TLR4, HMGB1(p < 0.001) and Gal-3 (p < 0.05) expression in both micro and macro vascular cells. Cadaveric diabetic retinal tissues also showed increased expression of RAGE, TLR4 and HMGB1 (p < 0.05). RAGE silencing significantly reduced TLR4, HMGB1 (p < 0.05) expression and inhibited the phosphorylation of NFκB and ERK1/2 in both these cells. The TLR4 adaptors MyD88 and TIRAP (p < 0.05) showed down regulation on RAGE silencing. This study shows CML induces AGE receptors expression as observed in diabetic retina and RAGE silencing down regulated TLR4 signalling and cytokine release by partly modulating TLR4 adaptors which needs further validation. From this study we speculate targeting the TLR4 adaptors like MyD88 and TIRAP can be a potential therapeutic target for reducing diabetic induced vascular complications.

Exogenous administration of hydrogen sulfide alleviates homocysteine induced inflammation in ARPE-19 cells.

Plasma homocysteine (Hcy) is an independent risk factor for Age related macular degeneration (AMD) and an inducer of inflammation. Homocysteine catabolism releases hydrogen sulfide (H2S). H2S has controversial effects on inflammation. In this study we have analysed the endogenous and exogenous H2S in modulating inflammation using adult retinal pigment epithelial (ARPE-19) cells as an in vitro model for AMD. ARPE-19 cells were treated with various concentrations of Hcy (15, 30 and 50 µM) for 3 h. Expression of Hcy transulfuration genes (CBS, CSE) by qPCR and western blot. H2S levels were measured using Free Radical Analyzer System (WPI, USA). The inflammatory markers (IL-6 and IL-8) were evaluated using real-time PCR and ELISA. Hcy exposure increased CBS protein expression, hydrogen sulfide levels and pro-inflammatory cytokines, modulating CBS by silencing did not alter H2S levels, but inhibition of CSE with PAG inhibited H2S production and decreased cytokine (IL-6 and IL-8) levels. On the contrary exogenous supply of hydrogen sulfide with NaHS and by compound 1c showed anti-inflammatory effects even in the presence of Hcy. This study shows that exogenous delivery of H2S decreases inflammation in retinal pigment epithelial cells on exposure to Hcy in ARPE-19 cells.

Polarity of Hydrated Phosphatidylcholine Headgroups.

The headgroup (H) stratum (sometimes called the polar region) of membrane bilayers is a relevant yet poorly understood solvation phase for small molecules and macromolecules interacting with the membranes. Solvation of compounds in bilayer strata is characterized experimentally by wide- and small-angle X-ray scattering, neutron diffraction, and various NMR techniques. The quantification is tedious and only available for a limited set of small molecules. Our recently published model of liposome partitioning of small molecules shows that solvation of compounds in the H-stratum of fluid phosphatidylcholine (PC) bilayers correlates well with their solvation in hydrated diacetyl phosphatidylcholine (DAcPC), and solvation in the core (C) depends in a similar way on that in n-hexadecane. These two correlations became a basis for a model describing the location of compounds in the H- and C-strata and at the connecting interface as a nonlinear function of the fragment solvation characteristics of the compounds. In this study, refractivity of hydrated DAcPC phases with varying water contents was measured and polarity was determined using the steady-state fluorescence of indole and Nile Red. The results were compared with the published data obtained by other techniques for PC bilayers in liposomes or on solid supports. The demonstrated qualitative agreement, as well as the polarity and refractivity dependencies on the DAcPC concentration, supports the suitability of hydrated DAcPC as the H-stratum surrogate. Interestingly, depending on hydrations typical for the H-strata of fluid PC bilayers, the dielectric constant could decrease significantly from 31.0 to 7.3 for 16 and 8 water molecules per headgroup, respectively. Although additional experiments are needed for confirmation, this observation could help set proper dielectric constant magnitudes in continuum-based computational models of accumulation and crossing of the PC bilayers with varying hydration levels thanks to the temperature or the structure of fatty acid chains.

Ocular distribution of antioxidant enzyme paraoxonase & its alteration in cataractous lens & diabetic retina.

BACKGROUND & OBJECTIVES: The enzyme paraoxonase (PON), an antioxidant enzyme that has both arylesterase and thiolactonase activity, is well studied in cardiovascular diseases. Although a few studies have shown altered PON activity in ocular diseases such as age-related macular degeneration and diabetic retinopathy, but the tissue-wise expression of PON in its three gene forms has not been studied. This study was conducted to see the ocular distribution of PON for any altered expression in ocular pathologies such as in cataract and diabetes mellitus. METHODS: Immunohistochemistry (IHC) of the ocular tissues was done for localizing all three forms of the PON in the human donor eyeballs. The PON arylesterase (PON-AREase) and thiolactonase (PON-HCTLase) activities were determined by spectrophotometry in kinetic mode, and the mRNA expression of the PON genes (PON1-3) was determined by reverse transcription-polymerase chain reaction. RESULTS: IHC showed the presence of both PON1 and 2 in all the ocular tissues and PON3 was seen only in retina. The mRNA expression analysis showed that PON2 and PON3 were present in all the tissues, whereas PON1 was seen only in ciliary and retina. Both the PON-AREase and PON-HCTLase activities were detected in all ocular tissues and was in the order of lens>retina>choroid>ciliary body>iris. The expression and activity were studied in cataractous lens and in diabetic retina of the donor eyes. A significant decrease in PON-AREase activity was seen in cataractous lens (P<0.05) but not in diabetic retina, and there was an increase in PON- HCTLase activity (P<0.05) only in diabetic retina. Bioinformatic studies and in vitro experiments indicated that advanced glycation end products (AGE) such as carboxymethyl -lysine might decrease the PON- AREase activity of the PON. INTERPRETATION & CONCLUSIONS: Distribution of PON enzyme and its activity in ocular tissues is reported here. The study revealed maximal PON activity in lens and retina, which are prone to higher oxidative stress. Differential activities of PON were observed in the lens and retinal tissues from cataractous and diabetic patients, respectively.

Structure-based prediction of drug distribution across the headgroup and core strata of a phospholipid bilayer using surrogate phases.

Solvation of drugs in the core (C) and headgroup (H) strata of phospholipid bilayers affects their physiological transport rates and accumulation. These characteristics, especially a complete drug distribution profile across the bilayer strata, are tedious to obtain experimentally, to the point that even simplified preferred locations are only available for a few dozen compounds. Recently, we showed that the partition coefficient (P) values in the system of hydrated diacetyl phosphatidylcholine (DAcPC) and n-hexadecane (C16), as surrogates of the H- and C-strata of the bilayer composed of the most abundant mammalian phospholipid, PC, agree well with the preferred bilayer location of compounds. High P values are typical for lipophiles accumulating in the core, and low P values are characteristic of cephalophiles preferring the headgroups. This simple pattern does not hold for most compounds, which usually have more even distribution and may also accumulate at the H/C interface. To model complete distribution, the correlates of solvation energies are needed for each drug state in the bilayer: (1) for the H-stratum it is the DAcPC/W P value, calculated as the ratio of the C16/W and C16/DAcPC (W for water) P values; (2) for the C-stratum, the C16/W P value; (3) for the H/C interface, the P values for all plausible molecular poses are characterized using the fragment DAcPC/W and C16/W solvation parameters for the parts of the molecule embedded in the H- and C-strata, respectively. The correlates, each scaled by two Collander coefficients, were used in a nonlinear, mass-balance based model of intrabilayer distribution, which was applied to the easily measurable overall P values of compounds in the DMPC (M = myristoyl) bilayers and monolayers as the dependent variables. The calibrated model for 107 neutral compounds explains 94% of experimental variance, achieves similar cross-validation levels, and agrees well with the nontrivial, experimentally determined bilayer locations for 27 compounds. The resulting structure-based prediction system for intrabilayer distribution will facilitate more realistic modeling of passive transport and drug interactions with those integral membrane proteins, which have the binding sites located in the bilayer, such as some enzymes, influx and efflux transporters, and receptors. If only overall bilayer accumulation is of interest, the 1-octanol/W P values suffice to model the studied set.

Unveiling the therapeutic potential of a mutated paraoxonase 2 in diabetic retinopathy: Defying glycation, mitigating oxidative stress, ER stress and inflammation.

Paraoxonase 2 (PON2) is an intracellular anti-oxidant protein ubiquitously expressed in all cells and reduces reactive oxygen species, endoplasmic reticulum (ER) stress, further improves mitochondrial function and thereby shows anti-apoptotic function. In diabetes and its complications this PON gets glycated and becomes in effective. The PON activity is reported to be reduced in diabetic retinopathy and we have earlier showed Carboxy methyl lysine (AGE) decreased PON2 expression and activity in Human retinal endothelial cells (HREC) . In this study, we have designed and developed a mutated PON2 by in silico and in vitro approach which can resist glycation. Where in glycation-prone residues in PON2 was predicted using in silico analyses and a mutated PON2 was developed using in vitro site directed mutagenesis (SDM) assay mPON2 (mutant PON2-PON2-K70A) and its efficacy was compared with wPON2 (wild type PON2). CML glycated wPON2 and reduced its activity when compared with mPON2 in HREC confirmed by immunoprecipitation and in vitro experiments. Additionally, mPON2 interaction efficiency with its substrates was higher than wPON2 by insilico assay and demonstrated enhanced inhibition against CML-induced oxidative stress, ER stress, pro-inflammation, and mitochondrial fission than wPON2 by invitro assay. Further mPON2 showed increased inhibition of phosphorylation of NFĸB induced by CML. Our investigation establishes that the over expression of mPON2 in HREC can defy glycation and therefore mitigate ER stress and inflammation against CML than endogenous wPON2. These findings imply that mPON2 can be a beneficial therapeutic target against diabetic retinopathy.

Copper Dyshomeostasis And Diabetic Complications: Chelation Strategies For Management.

Cuproptosis, An Emerging Concept In The Field Of Diabetes Research, Presents A Novel And Promising Perspective For The Effective Management Of Diabetes Mellitus And Its Associated Complications. Diabetes, Characterized By Chronic Hyperglycemia, Poses A Substantial Global Health Burden, With An Increasing Prevalence Worldwide. Despite Significant Progress In Our Understanding Of This Complex Metabolic Disorder, Optimal Therapeutic Strategies Still Remain Elusive. The Advent Of Cuproptosis, A Term Coined To Describe Copper-Induced Cellular Cell Death And Its Pivotal Role In Diabetes Pathogenesis, Opens New Avenues For Innovative Interventions. Copper, An Indispensable Trace Element, Plays A Pivotal Role In A Myriad Of Vital Biological Processes, Encompassing Energy Production, Bolstering Antioxidant Defenses, And Altered Cellular Signaling. However, In The Context Of Diabetes, This Copper Homeostasis Is Perturbed, Driven By A Combination Of Genetic Predisposition, Dietary Patterns, And Environmental Factors. Excessive Copper Levels Act As Catalysts For Oxidative Stress, Sparking Intricate Intracellular Signaling Cascades That Further Exacerbate Metabolic Dysfunction. In This Review, We Aim To Explore The Interrelationship Between Copper And Diabetes Comprehensively, Shedding Light On The Intricate Mechanisms Underpinning Cuproptosis. By Unraveling The Roles Of Copper Transporters, Copper-Dependent Enzymes, And Cuproptotic Signaling Pathways, We Seek To Elucidate Potential Therapeutic Strategies That Harness The Power Of Copper Modulation In Diabetes Management. This Insight Sets The Stage For A Targeted Approach To Challenge The Complex Hurdles Posed By Diabetes, Potentially Transforming Our Therapeutic Strategies In The Ongoing Fight Against This Pervasive Global Health Concern.

Structural determinants of drug partitioning in surrogates of phosphatidylcholine bilayer strata.

The knowledge of drug concentrations in bilayer headgroups, core, and at the interface between them is a prerequisite for quantitative modeling of drug interactions with many membrane-bound transporters, metabolizing enzymes and receptors, which have the binding sites located in the bilayer. This knowledge also helps understand the rates of trans-bilayer transport because balanced interactions of drugs with the bilayer strata lead to high rates, while excessive affinities for any stratum cause a slowdown. Experimental determination of bilayer location is so tedious and costly that the data are only available for some fifty compounds. To extrapolate these valuable results to more compounds at a higher throughput, surrogate phases have been used to obtain correlates of the drug affinities for individual strata. We introduced a novel system, consisting of a diacetyl phosphatidylcholine (DAcPC) solution with the water content of the fluid bilayer as the headgroup surrogate and n-hexadecane (C16) representing the core. The C16/DAcPC partition coefficients were measured for 113 selected compounds, containing structural fragments that are frequently occurring in approved drugs. The data were deconvoluted into the ClogP-based fragment solvation characteristics and processed using a solvatochromic correlation. Increased H-bond donor ability and excess molar refractivity of compounds promote solvation in the DAcPC phase as compared to bulk water, contrary to H-bond acceptor ability, dipolarity/polarizability, and volume. The results show that aromates have more balanced distribution in bilayer strata, and thus faster trans-bilayer transport, than similar alkanes. This observation is in accordance with the frequent occurrence of aromatic rings in approved drugs and with the role of rigidity of drug molecules in promoting intestinal absorption. Bilayer locations, predicted using the C16/DAcPC system, are in excellent agreement with available experimental data, in contrast to other surrogate systems.

Structural determinants of drug partitioning in n-hexadecane/water system.

Surrogate phases have been widely used as correlates for modeling transport and partitioning of drugs in biological systems, taking advantage of chemical similarity between the surrogate and the phospholipid bilayer as the elementary unit of biological phases, which is responsible for most of the transport and partitioning. Solvation in strata of the phospholipid bilayer is an important drug characteristic because it affects the rates of absorption and distribution, as well as the interactions with the membrane proteins having the binding sites located inside the bilayer. The bilayer core can be emulated by n-hexadecane (C16), and the headgroup stratum is often considered a hydrophilic phase because of the high water content. Therefore, we tested the hypothesis that the C16/water partition coefficients (P) can predict the bilayer locations of drugs and other small molecules better than other surrogate systems. Altogether 514 PC16/W values for nonionizable (458) and completely ionized (56) compounds were collected from the literature or measured, when necessary. With the intent to create a fragment-based prediction system, the PC16/W values were factorized into the fragment solvation parameters (f) and correction factors based on the ClogP fragmentation scheme. A script for the PC16/W prediction using the ClogP output is provided. To further expand the prediction system and reveal solvation differences, the fC16/W values were correlated with their more widely available counterparts for the 1-octanol/water system (O/W) using solvatochromic parameters. The analysis for 50 compounds with known bilayer location shows that the available and predicted PC16/W and PO/W values alone or the PC16/O values representing their ratio do not satisfactorily predict the preference for drug accumulation in bilayer strata. These observations indicate that the headgroups stratum, albeit well hydrated, does not have solvation characteristics similar to water and is also poorly described by the O/W partition characteristics.

Paraoxonase 2 protects against the CML mediated mitochondrial dysfunction through modulating JNK pathway in human retinal cells.

BACKGROUND: Paraoxonase 2 (PON2) a known anti-apoptotic protein, has not been explored against Nε-(carboxymethyl)lysine (CML), induced mitochondrial dysfunction and apoptosis in human retinal cells. Hence this present study aims to investigate the potential role of PON2 in mitigating CML-induced mitochondrial dysfunction in these cells. METHODS: PON2 protein was quantified in HRECs (Human retinal endothelial cells), ARPE-19 (Retinal pigment epithelial cells) cells upon CML treatment and also in cadaveric diabetic retina vs respective controls. ROS production, mitochondrial membrane potential (MMP), mitochondrial permeability transition pore (mPTP) opening, the release of Cyt-c, Bax, Caspase-3, Fis1, Mfn1, Mfn2, mitochondrial morphology, and the signaling pathway was assessed using DCFDA, JC-1, CoCl2, immunofluorescence or western blotting analysis in both loss-of-function or gain-of-function experiments. RESULTS: PON2 protein was downregulated in HREC and ARPE-19 cells upon CML treatment as well as in the diabetic retina (p = 0.035). Decrease in PON2 augments Fis1 expression resulting in fragmentation of mitochondria and enhances the ROS production, decreases MMP, facilitates mPTP opening, and induces the release of Cyt-c, which activates the pro-apoptotic pathway. Whereas PON2 overexpression similar to SP600125 (a specific JNK inhibitor) was able to decrease Fis1 (p = 0.036) and reverse the Bcl-2 and Bax ratio, and inhibit the JNK1/2 signaling pathway. CONCLUSION: Our results confirm that PON2 has an anti-apoptotic role against the CML mediated mitochondrial dysfunction and inhibits apoptosis through the JNK-Fis1 axis. GENERAL SIGNIFICANCE: We hypothesis that enhancing PON2 may provide a better therapeutic potential against diabetic vascular disease.

Effect of advanced glycation end product on paraoxonase 2 expression: Its impact on endoplasmic reticulum stress and inflammation in HUVECs.

BACKGROUND: Paraoxonase (PON), an antioxidant enzyme, comprises of three members (PON1, 2 and 3). Hyperglycemia accelerates formation of AGE in diabetes which mediates endothelial dysfunction. PON1 is studied in diabetes due to its association with HDL, lipid peroxidation and vascular complications but PON2 is not explored much. Recently decreased PON2 activity is reported in monocytes of Type 2 diabetes mellitus (T2DM) patients but its regulation by factors like high glucose and AGE has not been studied. AIM: The aim of the current study is to identify the effect of AGEs on PON2 expression and activity and its implications on endothelial dysfunction in HUVECs. MAIN METHODS: HUVECs were exposed to Glycated albumin (GA)/Carboxymethyl lysine (CML) for 24 h to check for PON2 expression. The ER stress markers GRP78 and IRE1α, pro-inflammatory genes MCP-1, IL-6, IL-8, ICAM1, VCAM1 were assessed by qPCR, western blotting/ELISA. Endothelial-leukocyte adhesion and ROS were assessed using Calcein AM and DCFDA method. One-way ANOVA and student's t-test was done using Graphpad Prism. KEY FINDINGS: AGE exposure significantly decreased PON2 expression and activity with increased oxidative stress, ER stress and inflammation. PON2 overexpression significantly reduced ROS, ER stress and inflammation as well as inhibited NFκB, and ERK1/2, phosphorylation induced by GA/CML treatment. Concomitantly, silencing of PON2 accelerated AGEs induced effects. SIGNIFICANCE: This is the first study to show that both GA and CML down regulates the PON2 expression and activity in HUVECs and over expression of PON2 alleviates AGEs induced endothelial dysfunction.

Case report on two diabetic donor eyes with no retinopathy: Clinicopathological and molecular studies.

We were intrigued to analyze donor eyes of two individuals without retinopathy even after 40 years of type 2 diabetes mellitus. Targeted molecular factors associated with angiogenesis and the key antioxidant enzymes in retinal tissue were analyzed. Accordingly PEDF, Adiponectin and Paraoxonase 2 showed augmented mRNA expression in both the retina with no significant change in VEGF expression. Vitreous showed increased PEDF protein in donor 1 and Adiponectin in donor 2 with no change in VEGF protein. This study highlights the profile of specific molecular factors that contribute to the non-development of diabetic retinopathy changes in these individuals.

Novel bis-(arylsulfonamide) hydroxamate-based selective MMP inhibitors.

A series of bis-(arylsulfonamide) hydroxamate inhibitors were synthesized. These compounds exhibit good potency against MMP-7 and MMP-9 depending on the nature, steric bulk, and substitution pattern of the substituents in the benzene ring. In general, the preliminary structure-activity relationships (SAR) suggest that among the DAPA hydroxamates (i) electron-rich benzene rings of the sulfonamides may produce better inhibitors than electron-poor analogs. However, potential H-bond acceptors can reverse the trend depending on the isozyme; (ii) isozyme selectivity between MMP-7 and -9 can be conferred through steric bulk and substitution pattern of the substituents in the benzene ring, and (iii) the MMP-10 inhibition pattern of the compounds paralleled that for MMP-9.

Scalable Synthesis and Purification of Acetylated Phosphatidyl Choline Headgroup.

The acetylated headgroup of the most abundant mammalian phospholipid, 1,2-diacetyl-3-sn-phosphatidyl choline (DAcPC), has several important applications in research. For instance, it can be dissolved in the same amount of water as in the fluid PC bilayer, to create a surrogate of a PC headgroup stratum, for studying solvation of small molecules and the influence of their structure on the process. In contrast to PC derivatives with longer acyl chains, DAcPC does not self-aggregate, rendering the aqueous solution homogeneous and suitable for simplified analyses of interactions of molecules with the headgroups. Several studies have been published where DAcPC was used in a crudely purified form. Here we describe a one-step preparation of DAcPC from commercially available bulk chemicals and purification of the product by crystallization and washing. The process gives a good yield and is easily scalable. The availability of enantiopure, crystalline DAcPC could open the door to more extensive biochemical, pharmacological, and nutritional studies of this interesting chemical.

Enantioselective conjugate radical addition to alpha'-hydroxy enones.

Enantioselective conjugate radical addition to alpha'-hydroxy alpha,beta-unsaturated ketones, compounds containing bidentate donors, has been investigated. It has been found that radical additions to alpha'-hydroxy alpha,beta-unsaturated ketones in the presence of Mg(NTf2)2 and bisoxazoline ligand 5a proceeded cleanly, yielding the addition products in high chemical yields and good enantiomeric excesses.

Paraoxonase enzyme protects retinal pigment epithelium from chlorpyrifos insult.

Retinal pigment epithelium (RPE) provides nourishment and protection to the eye. RPE dysfunction due to oxidative stress and inflammation is one of the major reason for many of the retinal disorders. Organophosphorus pesticides are widely used in the agricultural, industrial and household activities in India. However, their effects on the eye in the context of RPE has not been studied. In this study the defense of the ARPE19 cells exposed to Chlorpyrifos (1 nM to 100 µM) in terms of the enzyme paraoxonase (PON) was studied at 24 hr and 9 days of treatment. Chlorpyrifos was found to induce oxidative stress in the ARPE19 cells as seen by significant increase in ROS and decrease in glutathione (GSH) levels without causing cell death. Tissue resident Paraoxonase 2 (PON2) mRNA expression was elevated with chlorpyrifos exposure. The three enzymatic activities of PON namely, paraoxonase (PONase), arylesterase (PON AREase) and thiolactonase (PON HCTLase) were also found to be significantly altered to detoxify and as an antioxidant defense. Among the transcription factors regulating PON2 expression, SP1 was significantly increased with chlorpyrifos exposure. PON2 expression was found to be crucial as ARPE19 cells showed a significant loss in their ability to withstand oxidative stress when the cells were subjected to chlorpyrifos after silencing PON2 expression. Treatment with N-acetyl cysteine positively regulated the PON 2 expression, thus promoting the antioxidant defense put up by the cells in response to chlorpyrifos.

Binding affinity prediction for ligands and receptors forming tautomers and ionization species: inhibition of mitogen-activated protein kinase-activated protein kinase 2 (MK2).

Treatment of ionization and tautomerism of ligands and receptors is one of the unresolved issues in structure-based prediction of binding affinities. Our solution utilizes the thermodynamic master equation, expressing the experimentally observed association constant as the sum of products, each valid for a specific ligand-receptor species pair, consisting of the association microconstant and the fractions of the involved ligand and receptor species. The microconstants are characterized by structure-based simulations, which are run for individual species pairs. Here we incorporated the multispecies approach into the QM/MM linear response method and used it for structural correlation of published inhibition data on mitogen-activated protein kinase (MAPK)-activated protein kinase (MK2) by 66 benzothiophene and pyrrolopyridine analogues, forming up to five tautomers and seven ionization species under experimental conditions. Extensive cross-validation showed that the resulting models were stable and predictive. Inclusion of all tautomers and ionization ligand species was essential: the explained variance increased to 90% from 66% for the single-species model.

Cellular quantitative structure-activity relationship (Cell-QSAR): conceptual dissection of receptor binding and intracellular disposition in antifilarial activities of Selwood antimycins.

We present the cellular quantitative structure-activity relationship (cell-QSAR) concept that adapts ligand-based and receptor-based 3D-QSAR methods for use with cell-level activities. The unknown intracellular drug disposition is accounted for by the disposition function (DF), a model-based, nonlinear function of a drug's lipophilicity, acidity, and other properties. We conceptually combined the DF with our multispecies, multimode version of the frequently used ligand-based comparative molecular field analysis (CoMFA) method, forming a single correlation function for fitting the cell-level activities. The resulting cell-QSAR model was applied to the Selwood data on filaricidal activities of antimycin analogues. Their molecules are flexible, ionize under physiologic conditions, form different intramolecular H-bonds for neutral and ionized species, and cross several membranes to reach unknown receptors. The calibrated cell-QSAR model is significantly more predictive than other models lacking the disposition part and provides valuable structure optimization clues by factorizing the cell-level activity of each compound into the contributions of the receptor binding and disposition.

Homocysteine induces oxidative stress in young adult central retinal vein occlusion.

OBJECTIVES: High levels of plasma homocysteine have been reported to be toxic to the vascular endothelium, thereby creating an environment of hypercoagulability and occlusion. Elevated homocysteine has been reported as a risk factor for young adult central retinal vein occlusion (CRVO) cases. This study aimed to see if oxidative stress is an independent risk factor or is homocysteine dependent. METHODS: 23 young adult CRVO patients and 54 age and sex-matched controls were included in the study. Oxidative stress markers thiobarbituric acid-reacting substance (TBARS), superoxide dismutase (SOD), total thiols, glutathione peroxidase and total antioxidant capacity (TAC) were estimated. The effect of homocysteine (25-200 µM) on cultured bovine retinal endothelial cells (BREC) on oxidative stress parameter TBARS was measured. RESULTS: There was a significant increase in the plasma TBARS in CRVO cases compared with controls (p=0.000). SOD and TAC were significantly lower in CRVO cases than controls (p=0.000, p=0.022). There was a significant negative correlation between TAC and TBARS (p=0.00) and a significant positive correlation between homocysteine and TBARS (p=0.029). Nominal regression analysis showed that TAC and homocysteine influence TBARS significantly. The in-vitro study in BREC cells revealed that homocysteine increased the TBARS dose and time dependently. CONCLUSION: TBARS and homocysteine are known to be independent risk factors for CRVO. TBARS can be influenced by both homocysteine and TAC, thereby contributing to the aetiopathology of CRVO by increasing oxidative stress.