Outcomes of three-piece rigid scleral fixated intraocular lens implantation in subjects with deficient posterior capsule following complications in manual small incision cataract surgery.

Objective: To evaluate the surgical visual outcomes of three-piece rigid scleral fixated intraocular lens (SFIOL) implantation in subjects with deficient posterior capsule following complications of cataract extraction. Design: Retrospective 4-year cohort study. Participants: Data from 174 eyes that underwent SFIOL combined with pars plana vitrectomy (PPV) between January 2018 and March 2022 and follow-up exams were included. Methods: Demographic characteristics including primary indications for surgery, history of trauma, laterality, baseline and best-corrected visual acuity (BCVA), refraction as spherical equivalent (SE), intraocular pressure (IOP), duration of follow-up, and complications were analyzed. Results: The mean preoperative BCVA was 1.38 ± 0.46 logarithm of the minimum angle of resolution (logMAR), which improved significantly to 0.37 ± 0.22 logMAR. The baseline refractive status measured in spherical equivalent (SE) was 4.1 ± 6.2 Diopters (D), and the postoperative status was -0.4 ± 0.97 D. Early postoperative complications included hypotony (n = 1; 0.57%, vitreous hemorrhage (n = 3; 1.72%), elevated IOP (n = 8; 4.59%), mild dilated pupil (n = 1; 0.57%) and corneal edema (n = 16; 9.19%). Late complications included in this study were retinal detachment (n = 1; 0.57%), cystoid macular edema (CME) (n = 1; 0.57%), primary glaucoma (n = 1; 0.57%), secondary glaucoma (n = 13; 7.47%), zonular dehiscence (n = 3; 1.72%), retinal pigment epithelium (RPE) changes (n = 3; 1.72%), choroidal coloboma (n = 2; 1.14%), posterior dislocation of posterior chamber IOL (PCIOL) (n = 1; 0.57%), corneal decompensation (n = 1; 0.57%), retinal hemorrhage (n = 1; 0.57%), macular hole (n = 1; 0.57%), chronic uveitis (n = 1; 0.57%), mild non-proliferative diabetic retinopathy (NPDR) (n = 3; 1.72%), and mild NPDR with diabetic macular edema (DME) (n = 1; 0.57%). Conclusion: Integrating IOL implantation with vitrectomy various posterior segment complications were resolved in the same setting without attempting a second surgery.

Effect of Au@SiO2 core shell nanoparticles on HG-induced oxidative stress triggered apoptosis in keratinocytes.

Growing evidences suggest that excess generation of highly reactive free oxygen/nitrogen radicals (ROS/RNS) are largely due to hyperglycemia causes oxidative stress. Further, excess accumulation of ROS/RNS in cellular compartments aggravates the development and progression of diabetes and its associated complications. Impaired wound healing in diabetic condition is a known vital complication all around the world. Thus, an antioxidant agent having the potential for hindering the oxidative/nitrosative stress triggered diabetic skin complication is required. The present investigation was carried out to understand the impact of silica coated gold nanoparticle (Au@SiO2 NPs) on high glucose (HG)-induced keratinocyte complications. We demonstrated that HG environment enhanced the ROS and RNS accumulations and reduced in cellular antioxidant capacities in keratinocte cells, however, Au@SiO2 NPs treatment restored the HG effect. Furthermore, excess production of ROS/RNS was associated with mitochondrial dysfunction, characterized by loss of mitochondrial membrane potential (ΔΨm), and increased in mitochondrial mass, which was restored by Au@SiO2 NPs treatment in keratinocyte cells. In addition, HG-induced excess production of ROS/RNA caused an increased in the biomolecules damage including lipid peroxidation (LPO), and protein carbonylation (PC), 8-oxoguanine DNA glycosylase-1 (OGG1) expression and increased 8-hydroxydeoxyguanosine (8-OHdG) accumulations in DNA, leading to activation of ERK1/2MAPK, AKT and tuberin pathway, inflammatory reaction, and finally apoptotic cell death. In conclusion, our findings showed that Au@SiO2 NPs treatment improved the HG-induced keratinocytes injury by suppressing the oxidative/nitrosative stress, elevating the antioxidant defence system, thereby inhibiting the inflammatory mediators and apoptosis, which may be a therapeutic cure for the diabetic keratinocyte problems.

High glucose-induced increasing reactive nitrogen species accumulation triggered mitochondrial dysfunction, inflammation, and apoptosis in keratinocytes.

Growing evidence indicates that skin injuries are a common complication of diabetes. However, the cellular and molecular mechanisms of high glucose (HG) environment trigger nitrosative stress-mediated inflammation and apoptosis in keratinocytes remains unknown. Here we investigated whether reactive nitrogen species (RNS) induced by HG environment restrain antioxidant activity, and mitochondrial dysfunction leading to inflammation, and apoptosis via stress signaling pathways in keratinocytes. Our results established that the HG environment enhanced the production of nitric oxide (NO) and peroxynitrite anion (ONOO-) by inducible NO synthase (iNOS) in keratinocytes. Overproduction of RNS in HG environment suppress the antioxidants activity leading to mitochondrial dysfunction, characterized by loss of mitochondrial membrane potential (ΔΨm), increase in mitochondrial mass, decrease in mitochondrial transcription factor A(TFAM), increase in mitochondrial DNA (mtDNA) displacement loop (D-loop) and decrease in glycolytic flux concentration, which was attenuated by pharmacological inhibitors of NO/ONOO-, Nω-Nitro-l-argininemethyl ester hydrochloride (NAME)/hydralazine hydrochloride (Hyd.HCl). Excess production of RNS in HG environment restrained 8-oxoguanine DNA glycosylase-1 (OGG1) expression and increased 8-hydroxydeoxyguanosine (8-OHdG) accumulations in DNA were regulated by NO or ONOO-. Further, HG-induced RNA production caused an increase in the production of inflammatory mediators accompanied by activation of ERK1/2MAPK/Akt/tuberin-mTOR/IRF3 signaling cascade, lipid peroxidation (LPO), and protein carbonylation (PC) reactions followed by breakdown the cell-cell communication and apoptosis. Pre-treatment of cell with NAME/Hyd.HCl, diminished the expression of ERK1/2MAPK/Akt/tuberin-mTOR/IRF3, inflammatory mediators, and attenuated apoptosis in keratinocytes. Together, our results indicated that excess production of RNS in HG environment triggered inflammation and apoptosis, mediated by activation of ERK1/2MAPK/Akt/tuberin-mTOR/IRF3 signaling cascades in keratinocytes.

Inflammation and apoptosis, two key events induced by hyperglycemia mediated reactive nitrogen species in RGC-5 cells.

Nitrosative stress plays a critical role in retinal injury in high glucose (HG) environment of eye, but the mechanisms remain poorly understood. Here we tested the hypothesis that HG induced reactive nitrogen species (RNS) production acts as a key functional mediator of antioxidant depletion, mitochondrial dysfunction, biomolecule damage, inflammation and apoptosis. Our findings illustrated that exposure of cultured RGC-5 cells to HG significantly disrupts the antioxidant defense mechanism and mitochondrial machineries by increasing the loss of mitochondrial membrane potential (ΔÑ°M) and elevating mitochondrial mass. Furthermore, we used biochemical tools to analyze the changes in metabolites, sulfur amino acids (SAAs) such as L-glutathione (GSH) and L-cysteine (Cys), in the presence of HG environment. These metabolic changes were followed by an increase in glycolytic flux that is phosphofructokinase-2 (PFK-2) activity. Moreover, HG exposure results in a significant disruption of protein carbonylation (PC) and lipid peroxidation (LPO), downregulation of OGG1 and increase in 8-OHdG accumulations in RGC-5 cells. In addition, our results demonstrated that HG environment coinciding with increased expression of inflammatory mediators, cell cycle deregulation, decreased in cell viability and expression of FoxOs, increased lysosomal content leading to apoptosis. Pre-treatment of selective inhibitors of RNS significantly reduced the HG-induced cell cycle deregulation and apoptosis in RGC-5 cells. Collectively, these results illustrated that accumulated RNS exacerbates the antioxidant depletion, mitochondrial dysfunction, biomolecule damage, inflammation and apoptosis induced by HG exposure in RGC-5 cells. Treatment of pharmacological inhibitors attenuated the HG induced in retinal cells.

High glucose-induced ROS accumulation is a critical regulator of ERK1/2-Akt-tuberin-mTOR signalling in RGC-5 cells.

Hyperglycemia and oxidative stress are the primary stressors that elicit mitochondria specific cell stress in diabetes. Here we hypothesized that elevated level of ROS in high glucose (HG) environment, trigger mitochondrial stress by damaging mitochondrial DNA (mtDNA), altering inflammatory mediators, and neurodegenerative markers via stress signalling pathway in retinal ganglion cells (RGC-5). Mechanistically, our findings illustrated that the HG environment increases the ROS production in retinal cells leading to the disruption of antioxidant defence mechanism, and altering mitochondrial machinery such as an increase in loss of mitochondrial membrane potential (ΔΨm), increase in mitochondrial mass, and increase in mtDNA fragmentation. Furthermore, fragmented mtDNA escape from mitochondria into the cytosol, where it engaged with cyclic GMP-AMP synthase (cGAS) and stimulator of IFN gene (STING) phosphorylation and activate interferon regulatory factor 3 (IRF3) via ERK1/2-Akt-tuberin-mTOR dependent pathways. Our results further indicate that siRNA-mediated gene silencing of tuberin suppresses the strong downregulation of tuberin-mTOR-IRF3 activation. HG environment resulted in activation of IRF3, coinciding with the increased expression of inflammatory mediators and neurodegenerative markers. Pre-treatment of N-acetyl-l-cysteine (NAC) or ERK1/2 or phosphoinositide3-kinase (PI3-K)/Akt inhibitors in RGC-5 cells significantly reduced the HG-induced IRF3 expression and declined the expression of neurodegenerative markers. Collectively, our results demonstrates that HG-induced over production of ROS, disrupts the antioxidant defence mechanism and mitochondrial dysfunction, leading to alterations of inflammatory mediators and neurodegenerative markers through the ERK1/2-Akt-tuberin-mTOR dependent signalling pathway in RGC-5 cells.

CEES-induced ROS accumulation regulates mitochondrial complications and inflammatory response in keratinocytes.

Reactive oxygen species (ROS) is mainly produced as a by-product from electron transport chain (ETC) of mitochondria and effectively eliminated by cellular antioxidants. However, 2-chloroethyl ethyl sulfide (CEES) exposure to keratinocytes declined antioxidant capacity and increased accumulation of ROS triggered alteration of mitochondrial activity and apoptosis is lacking. Our findings demonstrated that the electron leakage from the impaired ETC, leading to the accumulation of ROS was gradually elevating with increasing concentration of CEES exposure, which decline the activity of superoxide dismutase (SOD), manganese SOD (MnSOD) and copper-zinc SOD (Cu-ZnSOD) in keratinocytes. Further, excess accumulation of ROS, decreased the mitochondrial membrane potential (ΔΨm) and increased the mitochondrial mass with increasing dose of CEES. CEES exposure provoked the decrease in expression of transcription factor A mitochondrial (TFAM), augmented mitochondrial DNA (mtDNA) damage and altered the mtDNA-encoded oxidative phosphorylation (OXPHOS) subunits. Moreover, fragmented mtDNA translocated into cytosol, where it activated cGAS-STING and interferon regulatory factor3 (IRF3), coinciding with the increased expression of inflammatory mediators and alteration of cell-to-cell communication markers. Pre-treatment of N-acetyl-l-cysteine (NAC) or L-Nω-nitroarginine methyl ester (NAME), hydralazine hydrochloride (Hyd·HCl) or ERK1/2 or phosphoinositide3-kinase (PI3-K)/Akt inhibitors in keratinocyte cells significantly restored the CEES effect. Our findings suggest that CEES-induced mitochondrial ROS production and accumulation leads to mitochondrial dysfunction and inflammatory response in keratinocytes. However, treatment of antioxidants or ERK1/2 or PI3-K/Akt inhibitors is a novel therapeutic option for the keratinocytes complication.

High glucose augments ROS generation regulates mitochondrial dysfunction and apoptosis via stress signalling cascades in keratinocytes.

Mitochondria are fascinating structures of the cellular compartments that generate energy to run the cells. However, inherent disorders of mitochondria due to diabetes can cause major disruption of metabolism that produces huge amount of reactive oxygen species (ROS). Here we study the elevated level of ROS provoked by high glucose (HG) environment triggered mitochondrial dysfunction, inflammatory response and apoptosis via stress signalling pathway in keratinocytes. Our results demonstrated that elevated glucose level in keratinoctes, increase the accumulations of ROS and decrease in cellular antioxidant capacities. Moreover, excess production of ROS was associated with mitochondrial dysfunction, characterized by loss of mitochondrial membrane potential (ΔΨm), increase in mitochondrial mass, alteration of mitochondrial respiratory complexes, cytochrome c (Cyt c) release, decrease in mitochondrial transcription factor A (TFAM) and increase in mitochondrial DNA (mtDNA) fragmentation. Damaged mtDNA escaped into the cytosol, where it engaged the activation of ERK1/2, PI3K/Akt, tuberin and mTOR via cGAS-STING leading to IRF3 activation. Pre-treatment of pharmacological inhibitors, ERK1/2 or PI3K/Akt suppressed the IRF3 activation. Furthermore, our results demonstrated that activation of IRF3 in HG environment coinciding with increased expression of inflammatory mediators. Excess production of ROS interfered with decreased in cell viability, increased lysosomal content and expression of FoxOs, leading to cell cycle deregulation and apoptosis. Pre-treatment of N-acetyl-l-cysteine (NAC) significantly reduced the HG-induced cell cycle deregulation and apoptosis in keratinocytes. In conclusion, increased oxidative stress underlies the decrease in antioxidant capacities and mitochondrial dysfunction in HG environment correlate with inflammation response and apoptosis via ERK1/2-PI3K/Akt-IRF3 pathway in keratinoctes.

Molecular characterization, constitutive expression and GTP binding mechanism of Cirrhinus mrigala (Hamilton, 1822) Myxovirus resistance (Mx) protein.

Myxovirus resistance (Mx) proteins represents the subclass of the dynamin superfamily of large Guanosine triphosphates (GTPases), play esential role in intracellular vesicle trafficking, endocytosis, organelle homeostasis and mitochondria distribution. These proteins are key players of the vertebrate immune system, induced by type-I and type-III interferons (IFN) of infected host and inhibit viral replication by sequestering its nucleoprotein. In the present study, we report the sequencing and characterization of Cirrhinus mrigala Mx protein (CmMx) for the first time and observed its constitutive expression in different tissues for a period of fourteen days. The synthetic peptide, LSGVALPRGTGI, was dissolved in PBS and injected into a rabbit and the antibody raised against CmMx was used to study the level of its expression. The full length of the CmMx cDNA is 2244 bp with a molecular mass of 70.9 kDa and a predicted isoelectric point of 8.25. The 627 amino acids polypeptide formed of three main functional domains: N-terminal GTPase domain (GD), a middle domain (MD) and GTPase effector domain (GED) with carboxy terminal leucine zipper motif. The 3D models of CmMx protein was modeled based on available close structural homologs and further validated through molecular dynamics (MD) simulations. MD study revealed the importance of G-domain responsible for recognition of GTP, which perfectly corroborate with earlier studies. MM/PBSA binding free energy analysis displayed that van der Waals and electrostatic energy were the key driving force behind molecular recognition of GTP by CmMx protein. The results from this study will illuminate more lights into the ongoing research on myxovirus resistance protein and its role in inhibition of viral replication in other eukaryotic system as well.

Relevance of Erk1/2-PI3K/Akt signaling pathway in CEES-induced oxidative stress regulates inflammation and apoptosis in keratinocytes.

2-Chloroethyl ethyl sulfide (CEES) is a well-known chemical warfare agent that induces cellular stress in exposed individuals. However, molecular mechanisms of CEES-induced oxidative stress-mediated metabolic deregulation are not clearly elucidated. Here we investigated CEES-induced free radical production act as key functional mediators of metabolic stress via Erk1/2 mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3-kinase (PI3K/Akt) signaling cascades in keratinocytes. We observed that CEES exposure disrupts the cellular antioxidant defense capacities leading to increase in free oxygen and nitrogen radical accumulation in keratinocytes. These unusual cellular abnormalities initiate cellular stress via Erk1/2-PI3K/Akt signaling pathways. Biochemical tools were used to analyze the changes in metabolites including sulfur amino acids (SAAs), namely, L-glutathione (GSH) and L-cysteine (Cys), in the presence of selective inhibitors of reactive oxygen/nitrogen species (ROS/RNS), Erk1/2, or PI3K/Akt after CEES exposure. Importantly, these metabolite changes were accompanied by a decrease in the glycolytic flux, consistent with the observed decrease in 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) concentration and these CEES-induced phenomena were attenuated by pretreatment of Erk1/2 or PI3-K/Akt inhibitors. On the other hand, CEES exposure disrupts the protein carbonylation (PC) and lipid peroxidation (LPO) in keratinocytes leading to inflammation, crash of the cell-cell communication, cell cycle deregulation, and apoptosis via Erk1/2-PI3K/Akt pathways. However, pretreatment of Erk1/2 or PI3K/Akt inhibitors attenuated the CEES action. Collectively, these results illustrated that accumulated free radicals act as key functional mediators for inflammation, and apoptosis via Erk1/2-PI3K/Akt regulatory signaling cascades induced by CEES exposure. Treatment of pharmacological Erk1/2-PI3K/Akt inhibitors attenuated the CEES-induced keratinocyte injury that may provide the basis for the development of therapeutic strategy to work against CEES exposure.

Oxidative stress, antioxidant capacity, biomolecule damage, and inflammation symptoms of sickle cell disease in children.

BACKGROUND: The phenotypic expression of sickle cell disease (SCD) is a complex pathophysiologic condition. However, sickle erythrocytes might be the cause for multiple sources of pro-oxidant processes with consequent linked to chronic and systemic oxidative stress. Herein, we explored the SCD phenomena could be the result in formation of oxidative stress as well as inflammation in children. MATERIAL AND METHODS: Blood samples of 147 SCD subjects were evaluated. A control group was formed of 156 individuals without SCD. Different oxidative stress markers and inflammatory mediators were measured by using various biochemical techniques. Plasma samples were collected from blood for the measurement of antioxidants and reactive oxygen species (ROS). RESULTS: The levels of plasma hydroxyl radical (HO•), and nitric oxide (NO) production were higher in SCD children in compared to control groups. The plasma antioxidants capacities such as superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione peroxidase (GPx) and protein thiol levels were significantly reduced in SCD children. The plasma lipid peroxidation, protein carbonylation, DNA damage markers were significantly altered in different age groups of SCD children. Further, our results showed that SCD children have chronic inflammatory disease due to persistent alteration of haemoglobin content, reticulocyte, total bilirubin, platelet, creatinine, leukocytes, and altered expression of inflammatory mediators in compared to control groups. CONCLUSION: SCD children have high oxidative stress, and conversely, decreased antioxidant activity. Decrease in antioxidant activity might explained the reduction in lipid peroxidation, protein carbonylation and increased inflammation, which in turn intensify the symptoms of SCD in children.

Prevalence of ocular morbidity among children aged 17 years or younger in the eastern India.

PURPOSE: Childhood ocular morbidity involves a spectrum of eye diseases that critically impact the mental development, future education and quality of life. However, there is limited evidence about the early detection and appropriate treatment of ocular morbidity in children <20 years. This study was aimed to assess the prevalence and make a comparison between the different types of ocular morbidity in children of both sexes in the age group of 6-17 years in the eastern India. METHODS: A cross-sectional survey of ocular morbidity among children <17 years of age who presented at the Department of Ophthalmology, Kalinga Institute of Medical Sciences, Bhubaneswar, and Vision Care Center for Retina, Bhubaneswar, in the eastern India between January 2015 and March 2018 was accomplished. Demographic information, visual acuity, type of eye injury, refractive errors and other detailed ophthalmic examination were screened. RESULTS: A total of 633 children (age 6-17 years) were examined in this study. The majority of cases were observed in children of age 12-17 years, accounting for almost close to half of all the cases. The prevalence of ocular morbidity was 45.92% in males and 53.97% in females. The most common ocular morbidity in children encountered was refractive error (54.62%), followed by congenital abnormalities (9%), allergic conjunctivitis (8.52%) and traumatic eye injury (7.1%). There was an increase in ocular morbidity with age, especially the refractive error and congenital abnormalities. CONCLUSION: A large number of ocular morbidity was observed in children of age <17 years. Since most of this morbidity was preventable or treatable, reasonable service for ocular morbidity and early age screening are effective methods to reduce this load. Moreover, health education for the prevention of childhood ocular morbidity and, at the same time, early presentation of children to ophthalmic hospitals for the treatment of eye disorders are essential.

Gold nanoparticles reduce high glucose-induced oxidative-nitrosative stress regulated inflammation and apoptosis via tuberin-mTOR/NF-κB pathways in macrophages.

Hyperglycemia is a risk factor for cardiovascular mortality and morbidity, and directly responsible for exacerbating macrophage activation and atherosclerosis. We showed that gold nanoparticles (AuNPs) reduce the high glucose (HG)-induced atherosclerosis-related complications in macrophages via oxidative-nitrosative stress-regulated inflammation and apoptosis. The effects of AuNPs on oxidative-nitrosative stress markers such as cellular antioxidants were attenuated by HG exposure, leading to reduction in the accumulation of reactive oxygen/nitrogen species in cellular compartments. Further, these abnormalities of antioxidants level and reactive oxygen/nitrogen species accumulations initiate cellular stress, resulting in the activation of nuclear factor κB (NF-κB) via ERK1/2mitogen-activated protein kinase (MAPK)/Akt/tuberin-mammalian target of rapamycin (mTOR) pathways. The activated NF-κB stimulates inflammatory mediators, which subsequently subdue biomolecules damage, leading to aggravation of the inflammatory infiltration and immune responses. Treatment of AuNPs inhibits the intracellular redox-sensitive signaling pathways, inflammation, and apoptosis in macrophages. Together, our results indicate that AuNPs may modulate HG-induced oxidative-nitrosative stress. These effects may be sealed tight due to the fact that AuNPs treatment reduces the activation of NF-κB by ERK1/2MAPK/Akt/tuberin-mTOR pathways-mediated inflammatory genes expression and cellular stress responses, which may be beneficial for minimizing the atherosclerosis.

Hyperglycemia-Induced Oxidative-Nitrosative Stress Induces Inflammation and Neurodegeneration via Augmented Tuberous Sclerosis Complex-2 (TSC-2) Activation in Neuronal Cells.

Diabetes is a systemic disease mainly characterized by chronic hyperglycemia and with extensive and long-lasting spiteful complications in central nervous systems (CNS). Astrocytes play an important role in the defense mechanism of CNS, with great ability of withstanding accumulation of toxic substances. Apart from functional disorders, hyperglycemia leads to slow progressive structural abnormalities in the CNS through oxidative stress pathways. However, the molecular mechanism by which neurons die under oxidative stress induced by high glucose (HG) remains largely unclear. Here, we report that HG-induced inflammation and neurodegeneration in brain tissues, brain astrocytes (C6), and pheochromocytoma (PC-12) cells are cultured in HG conditions. Our results show that the increases in phosphorylation of Akt and ERK1/2MAPK are associated with increased accumulations of reactive oxygen species (ROS) in neuronal cells, which simultaneously enhanced phosphorylations of tuberous sclerosis complex-2 (TSC-2) and mammalian target of rapamycin (mTOR) in the diabetic brain and in HG-exposed neuronal cells. Pharmacologic inhibition of Akt or ERK1/2 or siRNA-mediated gene silencing of TSC-2 suppressed the strong downregulation of TSC-2-mTOR activation. Findings of this study also demonstrate that HG resulted in phosphorylation of NF-κB, coinciding with the increased production of inflammatory mediators and activation of neurodegenerative markers. Pretreatment of cells with antioxidants, phosphoinositide3-kinase (PI3-K)/Akt, and ERK1/2 inhibitors significantly reduced HG-induced TSC-2 phosphorylation and restored NF-κB protein expression leading to decreased production of inflammatory mediators and neurodegenerative markers. These results illustrate that ROS functions as a key signaling component in the regulatory pathway induced by elevated glucose in neuronal cell activation leading to inflammation and neurodegeneration.

Increasing NO level regulates apoptosis and inflammation in macrophages after 2-chloroethyl ethyl sulphide challenge.

Generation of nitric oxide (NO) in cellular compartments acts in a redox-dependent manner to counteract oxidative stress either by directly acting as an antioxidant through scavenging superoxide anions (O2-), to form peroxynitrite (ONOO-) or acting as a signaling molecule, altering gene expression that triggers various physiological processes. However, the molecular mechanisms of macrophage activation and NO production leads to apoptosis and inflammation after 2-chloroethyl ethyl sulphide (CEES) exposure remains unclear. We showed that CEES exposure in macrophages increased the O2- production. Also CEES exposure transiently increases the NO production and ONOO- accumulation via expression of inducible NO synthase (iNOS). Simultaneously, CEES exposure caused a significant reduction in cellular antioxidants and modulate lipid peroxidation (LPO), and protein carbonylation (PC) reactions, which was correlated with the increased level of NO and ONOO- accumulation. Mechanistic studies showed the DNA damage, 8-oxoGDNA glycosylase (OGG1) down regulation and 8-hydroxydeoxyguanosine (8-OHdG) accumulations in DNA, which was also confirmed by phosphorylation of ATM, ATR and H2A.X. Elevated levels of NO/ONOO- plays an important role in apoptosis, and alteration of cell cycle regulatory proteins in macrophages after CEES exposure. Moreover, CEES exposure to macrophage cells enhanced the transcriptional activities of inflammatory mediators such as TNFα, IL-1α, ICAM, CX3CL1, CCL8, and CXCL10, which were linked with NO/ONOO- accumulation. These results showed a mechanistic explanation of how NO/ONOO- cooperate to conduct apoptosis and inflammatory signals in macrophages after CEES challenged. Further, the protective effects of NO/ONOO- inhibitors may provide the basis for the development of a therapeutic strategy to counteract exposure to CEES.

Lactobacillus acidophilus attenuates Aeromonas hydrophila induced cytotoxicity in catla thymus macrophages by modulating oxidative stress and inflammation.

The pathogenesis of Aeromonas hydrophila, a potent fish pathogen, is attributed to its ability to cause motile aeromonad septicaemia leading to apoptosis in a myriad of fish species, including freshwater carp Catla catla. However, the underlying mechanism of antagonistic activity of probiotics against A. hydrophila induced apoptosis is not elucidated due to lack of appropriate in-vitro models. This study reported that the exposure of catla thymus macrophages (CTM) to A. hydrophila markedly induced cellular injuries as evidenced by elevated levels of reactive oxygen species (ROS), reactive nitrogen species (RNS), increased apoptosis, DNA damage and decreased cellular viability. Flow cytometry analysis and Annexin-V/propidium iodide assay further confirmed increased ROS positive cells leading to cell death after infection. The quantitative real-time PCR analysis, also revealed upregulation of inducible nitric-oxide synthase (iNOS), pro-inflammatory cytokine (TNFα), cyclooxygenase2 (COX-2) and downregulation of anti-inflammatory cytokine (IL-10). Pretreatment of cells with probiotic, Lactobacillus acidophilus attenuated A. hydrophila induced apoptosis as evident from the decrease in the levels of ROS, RNS and DNA damage. Significant increase (P≤0.05) in expression of TNFα and IL-10 and decrease in iNOS and COX-2 was observed on probiotic stimulation. In-vivo study using catla fingerlings confirmed similar pattern of ROS, iNOS, NO production and cytokine expression in thymus. This study provides a comprehensive insight into the mechanistic basis of L. acidophilus induced macrophage mediated inflammatory response against A. hydrophila in CTM cells. Further, it speculates the possibility of using cost-effective in-vitro models for screening probiotic candidates of therapeutic potential in aquaculture industry.

Hyperglycemia-induced inflammation caused down-regulation of 8-oxoG-DNA glycosylase levels in murine macrophages is mediated by oxidative-nitrosative stress-dependent pathways.

High glucose-induced increase in production of reactive oxygen/nitrogen species (ROS/RNS) is recognized as a major cause of the clinical complications associated with diabetes. ROS/RNS apart from being redox agents, cause an unwanted severe physiological load to cells, also act as cellular messengers, and play a key role in activation of circulating macrophages. However, the molecular mechanisms of activation of macrophages by hyperglycemic conditions are currently unclear. In the present study, we report that high glucose (HG) causes a dramatic increase in the production of inflammatory cytokines and chemokines, at least in part through enhanced mRNA transcription. The increase in levels of inflammatory cytokines/chemokines corresponds to increased levels of ROS/RNS, which is accompanied by increased activities of Akt, ERK1/2, tuberin, down regulation of 8-oxoG-DNA glycosylase (OGG1), and increase in 8-hydroxydeoxyguanosine (8-OHdG) accumulation in DNA. Elevated levels of ROS/RNS are triggering alteration in antioxidants level, biomolecules damage, cell cycle dysregulation, and apoptosis in macrophage cells. Pretreatment of antioxidants caused decrease in the levels of ROS/RNS leads to an increase in the levels of antioxidants, decrease in biomolecules damage, alterations in Akt, ERK1/2, tuberin, upregulation of OGG1, and decrease in 8-OHdG accumulations in DNA. Further, antioxidants treatments inhibit the effects of HG on the transcriptional activity of cytokines and chemokines. Our results demonstrate that intracellular signaling pathways mediated by ROS/RNS are linked to each other by elevated glucose in macrophages activation leading to inflammation. These findings provide a mechanistic explanation of how ROS/RNS cooperate to conduct inflammatory intracellular signals in macrophages related complications in hyperglycemic conditions.

Expression of Mx Gene in Cirrhinus mrigala (Hamilton, 1822) to OmpC Protein of Aeromonas hydrophila and Bacterial Infection.

The aims of this study were to identify alternative myxovirus (Mx) stimulatory compounds in Cirrhinus mrigala and to characterize the kinetics and intensity of their stimulated responses by semi-quantitative RT-PCR. Mx transcripts were measured in C. mrigala injected with Aeromonas OmpC (outer membrane protein) at a dose 0.4 mg/fish. At day 1, day 2, day 3, day 5, day 10, day 20 and day 30, samples were collected from kidney, spleen, liver, heart brain, gill, intestine and muscle for the study of Mx transcript and housekeeping gene ß-actin. Similarly, Mx gene expression was also studied in Aeromonas hydrophila-infected fish for a period of 10 days. Mx/ß-actin ratio was constitutively expressed from all the organs of OmpC-vaccinated fish. The expression was significantly highest (P ≤ 0.05) in spleen, followed by liver, kidney, intestine, gill, heart, muscle and brain. The expression was highest in day 2 except spleen (on day 3) and subsequently reduced up to day 30. Control fish also showed Mx expression. Similarly, A. hydrophila-infected fish showed Mx/ß-actin ratio upregulated significantly in the spleen and kidney on day 5, liver on day 2 and intestine on day 3. This study revealed that OmpC of A. hydrophila and its infection could stimulate the antiviral Mx gene of C. mrigala.

Aldose reductase expression as a risk factor for cataract.

Aldose reductase (AR) is thought to play a role in the pathogenesis of diabetic eye diseases, including cataract and retinopathy. However, not all diabetics develop ocular complications. Paradoxically, some diabetics with poor metabolic control appear to be protected against retinopathy, while others with a history of excellent metabolic control develop severe complications. These observations indicate that one or more risk factors may influence the likelihood that an individual with diabetes will develop cataracts and/or retinopathy. We hypothesize that an elevated level of AR gene expression could confer higher risk for development of diabetic eye disease. To investigate this hypothesis, we examined the onset and severity of diabetes-induced cataract in transgenic mice, designated AR-TG, that were either heterozygous or homozygous for the human AR (AKR1B1) transgene construct. AR-TG mice homozygous for the transgene demonstrated a conditional cataract phenotype, whereby they developed lens vacuoles and cataract-associated structural changes only after induction of experimental diabetes; no such changes were observed in AR-TG heterozygotes or nontransgenic mice with or without experimental diabetes induction. We observed that nondiabetic AR-TG mice did not show lens structural changes even though they had lenticular sorbitol levels almost as high as the diabetic AR-TG lenses that showed early signs of cataract. Over-expression of AR led to increases in the ratio of activated to total levels of extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal (JNK1/2), which are known to be involved in cell growth and apoptosis, respectively. After diabetes induction, AR-TG but not WT controls had decreased levels of phosphorylated as well as total ERK1/2 and JNK1/2 compared to their nondiabetic counterparts. These results indicate that high AR expression in the context of hyperglycemia and insulin deficiency may constitute a risk factor that could predispose the lens to disturbances in signaling through the ERK and JNK pathways and thereby alter the balance of cell growth and apoptosis that is critical to lens transparency and homeostasis.

Effects of CEES and LPS synergistically stimulate oxidative stress inactivates OGG1 signaling in macrophage cells.

2-chloroethyl ethyl sulphide (CEES), a monofunctional analogue of sulfur mustard, is a strong vesicant and an alkylating chemical warfare agent. We studied the molecular mechanism of oxidative stress triggered signaling cascades in murine macrophages exposed to CEES with lipopolysaccharide (LPS). Exposure of CEES with specific dose of LPS stimulates oxidative stress caused increasing level of intracellular ROS and RNS, decreased antioxidant enzymes, increasing bimolecular damage, reduced cell viability, and cell cycle arrest. Synergistic exposure of CEES and LPS provoked significant increase in phosphorylation of MAPKs, Akt, tuberin, that down regulate OGG1 expression and 8-OHdG accumulations. Treatment with Akt and ERK1/2 inhibitors, the cells with constitutively active inhibiting activity of Akt and ERK1/2MAPK significant reduce CEES and LPS challenge tuberin but not the OGG1. In addition, the N-acetylcysteine inhibited ROS/RNS generation, elevation of antioxidants level, expression of ERK1/2, Akt, tuberin phosphorylation, resulted in deceased 8-OHdG accumulation and upregulation of OGG1 protein expression suggesting no involvement of Akt and ERK1/2MAPK pathways after CEES and LPS challenge. Collectively, our results indicate that exposure of CEES and LPS induces oxidative stress and the activation of tuberin, and 8-OHdG accumulation via upstream signaling pathways including Akt and ERK1/2MAPK pathway in macrophages but not the down regulation of OGG1.

Hyperglycemia-induced oxidative stress induces apoptosis by inhibiting PI3-kinase/Akt and ERK1/2 MAPK mediated signaling pathway causing downregulation of 8-oxoG-DNA glycosylase levels in glial cells.

Glial cells are very important for normal brain function and alterations in their activity due to hyperglycemia, could contribute to diabetes-related cognitive dysfunction. Oxidative insults often cause rapid changes in almost all cells including glial cells. However, pathophysiologic mechanisms that lead to diabetic complications are not completely elucidated. Therefore, we examined whether elevated glucose levels directly or indirectly disrupt antioxidant defense mechanisms causing alterations in signaling pathways, cell cycle dysregulation, and reactive oxygen/nitrogen species-mediated apoptosis in glial cells. Findings of this study demonstrated that exposure of glial cells to high glucose markedly induces cellular and molecular injuries, as evidenced by elevated levels of reactive oxygen/nitrogen species, biomolecules damage, cell cycle dysregulation, decrease in antioxidant enzymes, and decrease in cell viability. Pretreatment of cells with N-acetyl-L-cysteine reduced high glucose-induced cytotoxicity by increasing the levels of antioxidant enzymes, and decreasing the number of apoptotic cells. Further, at molecular level high glucose treatment resulted in a significant increase in phosphorylation of Akt, MAPKs, tuberin, down regulation of 8-oxoG-DNA glycosylase and increase in 8-hydroxydeoxyguanosine accumulations. Pretreatment of cells with N-acetyl-L-cysteine, phosphatidylinositol3-kinase/Akt and ERK1/2 inhibitors completely abolished the apoptotic effects of high glucose. Moreover, N-acetyl-L-cysteine significantly inhibited reactive oxygen/nitrogen species generation, elevated antioxidants levels, inhibited Akt, ERK1/2, tuberin phosphorylation, decreased 8-hydroxydeoxyguanosine accumulation and upregulated 8-oxoG-DNA glycosylase expression. Our results demonstrate that high glucose induces apoptosis and inhibits proliferation of glial cells, which may be mediated by the phosphorylation of tuberin, down regulation of 8-oxoG-DNA glycosylase and 8-hydroxydeoxyguanosine accumulation via activation of Akt and ERK1/2MAPK pathways.