Exploring Molecular Approaches in Amyotrophic Lateral Sclerosis: Drug Targets from Clinical and Pre-Clinical Findings.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease (MND) characterized by the death of upper and lower motor neurons (corticospinal tract) in the motor cortex, basal ganglia, brain stem, and spinal cord. The patient experiences the sign and symptoms between 55 to 75 years of age, which include impaired motor movement, difficulty in speaking and swallowing, grip loss, muscle atrophy, spasticity, and sometimes associated with memory and cognitive impairments. Median survival is 3 to 5 years after diagnosis and 5 to 10% of the patients live for more than 10 years. The limited intervention of pharmacologically active compounds, that are used clinically, is majorly associated with the narrow therapeutic index. Pre-clinically established experimental models, where neurotoxin methyl mercury mimics the ALS like behavioural and neurochemical alterations in rodents associated with neuronal mitochondrial dysfunctions and downregulation of adenyl cyclase mediated cAMP/CREB, is the main pathological hallmark for the progression of ALS in central as well in the peripheral nervous system. Despite the considerable investigation into neuroprotection, it still constrains treatment choices to strong care and organization of ALS complications. Therefore, this current review specially targeted the investigation of clinical and pre-clinical features available for ALS to understand the pathogenic mechanisms and to explore the pharmacological interventions associated with the up-regulation of intracellular adenyl cyclase/cAMP/ CREB and activation of mitochondrial-ETC coenzyme-Q10 as a future drug target in the amelioration of ALS mediated motor neuronal dysfunctions.

Auger electron emission initiated by the creation of valence-band holes in graphene by positron annihilation.

Auger processes involving the filling of holes in the valence band are thought to make important contributions to the low-energy photoelectron and secondary electron spectrum from many solids. However, measurements of the energy spectrum and the efficiency with which electrons are emitted in this process remain elusive due to a large unrelated background resulting from primary beam-induced secondary electrons. Here, we report the direct measurement of the energy spectra of electrons emitted from single layer graphene as a result of the decay of deep holes in the valence band. These measurements were made possible by eliminating competing backgrounds by employing low-energy positrons (<1.25 eV) to create valence-band holes by annihilation. Our experimental results, supported by theoretical calculations, indicate that between 80 and 100% of the deep valence-band holes in graphene are filled via an Auger transition.

Association of LPL gene variant and LDL, HDL, VLDL cholesterol and triglyceride levels with ischemic stroke and its subtypes.

Lipoprotein lipase (LPL) plays an important role in lipid metabolism by hydrolyzing triglycerides in chylomicrons and very low density lipoproteins. An increasing number of studies have suggested an association of LPL gene variants with the risk of cardiovascular and cerebrovascular diseases. The aim of this study was to test whether HindIII polymorphism of LPL gene is associated with ischemic stroke and its subtypes as well as plasma lipid levels in a South Indian population from Andhra Pradesh. Five hundred and twenty five ischemic stroke patients and 500 controls were enrolled in this case-control study. The LPL HindIII polymorphism was determined by PCR-RFLP technique and the lipid levels were estimated using commercially available kits. We found significant difference in the genotypic distribution between patients and controls [for HindIII (+/+) vs. HindIII (-/-), χ(2)=4.916; p=0.02; Odds ratio=1.59 (95%CI; 1.054-2.413); HindIII (+/+) vs. HindIII (-/-) and HindIII (+/-), χ(2)=5.25; p=0.02; Odds ratio=1.24 (95%CI; 1.03-1.503)]. A stepwise multiple logistic regression analysis confirmedthese findings. The relationship between HindIII genotypes and plasma levels of HDL, LDL, VLDL and triglycerides was analyzed using ANOVA and further confirmed by Post-hoc analysis. The levels of triglycerides were found to be elevated in individuals bearing HindIII (+/+) genotype in comparison with HindIII (-/-) genotype. HDL levels were found to be significantly reduced and triglyceride levels significantly elevated in HindIII (+/+) genotype in comparison with HindIII (-/-). However, there was no difference in the levels of LDL and VLDL between the two genotypes. Examining the association of LPL gene HindIII polymorphism with stroke subtypes, we found significant association of HindIII polymorphism with Intracranial large artery atherosclerosis [Odds ratio=2.12 955CI (1.656-2.848); p=0.009]. Our results suggest that the HindIII polymorphism of LPL is significantly associated with ischemic stroke risk and elevated levels of plasma triglycerides and reduced HDL levels. Further, this polymorphism is significantly associated with intracranial large artery atherosclerosis which is the most frequent subtype in our region.

C-reactive protein and nitric oxide levels in ischemic stroke and its subtypes: correlation with clinical outcome.

Studies in different populations have shown that ischemic stroke can trigger an acute phase response resulting in a rise of plasma concentration of C-reactive protein (CRP). However, there are very limited studies on CRP and first ischemic stroke divided into subtypes. High levels of CRP may also be associated with poor outcome. The present study was taken up to investigate the prognostic value of CRP within 24 h of onset of ischemic stroke. Five hundred and eighty one patients with first stroke and 575 age- and sex-matched healthy controls were involved in the study. High-sensitivity C-reactive protein (hsCRP) levels were estimated, and follow-up interviews were conducted with patients at 3, 6, and 12 months post-event to determine stroke outcome. In addition to this plasma, NO( x ) (nitrate and nitrite) was measured to detect the serum NO (an important biomarker of inflammation and oxidative stress) levels in ischemic stroke patients and controls. The relationship between CRP value and poor outcome (>2 on modified Rankin Scale Score and <5 on an extended Glasgow outcome scale) was studied. There was a significant association between elevated levels of CRP and NO with the disease. A stepwise multiple logistic regression analysis confirmed these findings after adjustment for potential confounders [adjusted odds ratio = 2.890, 95% CI (1.603-5.011) with p < 0.01 and adjusted odds ratio = 2.364, 95% CI (1.312-3.998) with p < 0.01 for hsCRP and NO, respectively]. After adjustment of potential confounders, patients with high CRP levels had a significant increased risk of poor outcome [adjusted odds ratio = 3.50, 95% CI (1.312-6.365) and p < 0.001]. Elevated levels of hsCRP associated significantly with all stroke subtypes classified according to Trial of ORG 10172 in Acute Stroke Treatment classification except for lacunar stroke and stroke of other determined etiology. In conclusion, hsCRP and NO levels predict the incidence of ischemic stroke and hsCRP is an independent prognostic factor of poor outcome at 3 months.

Association of tumor necrosis factor-α and matrix metalloproteinase-3 gene variants with stroke.

BACKGROUND AND PURPOSE: There is increasing evidence that the genetic variation in the genes coding for pro-inflammatory markers and matrix metalloproteinase may play an important role in the pathogenesis of various human diseases including stroke. The aim of this study was to evaluate the association of genetic variants within the genes encoding tumor necrosis factor-α (TNF-α) and matrix metalloproteinase-3 (MMP-3), with stroke. METHODS: Five hundred and twenty-five ischemic stroke patients and 500 age- and sex-matched controls were included in this study. We analyzed +488 G/A polymorphism in TNF-α gene and -1612 5A/6A polymorphism in MMP-3 gene. The genotypes were determined by Amplification Refractory Mutation System PCR. The strength of association between genotypes and stroke was measured by the odds ratio with 95% confidence interval (CI) and chi-squared analysis. RESULTS: Allelic and genotypic frequencies of TNF-α G/A polymorphism differed significantly between patients and healthy controls (P < 0.001). A stepwise logistic regression analysis confirmed these findings (P < 0.001). Further, evaluating the association of this polymorphism with stroke subtypes, we found significant association with intracranial large artery atherosclerosis, extracranial large artery atherosclerosis, and stroke of undetermined etiology. As far as MMP-3-1612 5A/6A polymorphism is concerned, there was no significant difference in genotypic distribution and allelic frequency between the patients and healthy controls (P = 0.5 and 0.9, respectively). We tested the gene-gene interaction between TNF-α and MMP-3 genes using the logistic regression model. However, there was no evidence for a gene-gene interaction between TNF-α and MMP-3. CONCLUSION: TNF-α +488 G/A variant is an important risk factor for ischemic stroke in the South Indians from Andhra Pradesh, whereas MMP-3-1612 5A/6A polymorphism is not associated with stroke in the same population.

Interleukin-10-1082 promoter polymorphism and ischemic stroke risk in a South Indian population.

Within the past few years there has been increasing evidence that the genetic variation in the genes coding pro- and anti-inflammatory markers may play an important role in the pathogenesis of various human diseases, including stroke. The aim of the study was to evaluate the association of Interleukin-10 (IL-10)-1082 G/A, promoter polymorphism (rs1800896) with ischemic stroke in a South Indian population from Andhra Pradesh. In this study 480 ischemic stroke patients and 470 age and sex matched healthy controls were included. The ischemic stroke patients were classified according to TOAST classification. The region of interest in the IL-10 gene was amplified by polymerase chain reaction with the use of allele specific oligonucleotide primers flanking the polymorphic region. Association between genotypes and stroke was examined by Odds Ratio (OR) with 95% confidence interval (CI) and Chi-square analysis. Significant difference was observed between the patients and healthy controls, in genotypic distribution as well as allelic frequency (p<0.05). Multiple logistic regression analysis with forward stepwise selection using the potential confounders (sex, age, diabetes, hypertension, smoking and alcoholism) and IL-10 gene variant revealed that -1082 G/A polymorphism in the promoter region of IL-10 gene is significantly [adjusted OR=2.26; 95% C.I. (1.24-4.15), p<0.001] associated with ischemic stroke in the South Indian population from Andhra Pradesh. We found significant association of this polymorphism with stroke of undetermined etiology (p<0.001). Moreover, hypertensive and diabetic individuals bearing A allele of IL-10 gene in high frequency were found to be more predisposed to stroke.

Depletion of serum zinc in ischemic stroke patients.

The pathogenesis of a number of diseases like cardiovascular diseases, cancer and neurological disorders, has been associated with changes in the balance of certain trace elements. In this study we aimed at investigating the levels of trace elements like calcium, copper, iron and zinc, in ischemic stroke patients in comparison with healthy controls. Serum samples were collected from 256 ischemic stroke patients and 180 healthy, age and sex matched controls. Trace element levels were detected using commercially available kits and an Auto-Analyzer (ChemWell 2910, Awareness Technology, US). The concentrations of calcium, copper and iron were not significantly different in patients when compared to healthy controls. The concentration of zinc was significantly lower in stroke patients (P = 0.001) as compared to normal subjects. To conclude, patients with acute ischemic stroke have reduced levels of serum zinc. Zinc may represent an independent risk factor for stroke and therefore a possible target for prevention. Additional studies are needed to further examine the role of zinc in the pathogenesis of stroke.

Estrogen receptor α genetic variants and the risk of stroke in a South Indian population from Andhra Pradesh.

BACKGROUND: Stroke is a complex disease caused by combination of multiple risk factors. Recent findings have suggested that stroke has a strong genetic component. Evidence suggests that variations in the estrogen receptor α (ESR1) gene may influence stroke risk. AIMS: The present study was carried out to investigate the role of ESR1 gene polymorphisms [PvuII (rs 2234693) and XbaI (rs 9340799)] with stroke in a South Indian population from Andhra Pradesh. The relationship between ESR1 genotypes with estradiol levels was also investigated in pre- and postmenopausal women. METHODS: Four hundred patients with ischemic stroke and three hundred and eighty subjects were enrolled in this case-control study. Ischemic stroke subtypes were classified according to TOAST (Trial of Org 10172 in Acute Stroke Treatment) classification. The ESR1 PvuII and XbaI genotypes were determined by PCR-RFLP method. Serum estradiol was measured by ELISA. RESULTS: In case of PvuII polymorphism statistically significant difference was observed in the genotypic and allelic frequencies between patients and controls (joint analysis of men and women) (p=0.003 and 0.004 respectively). However, the XbaI genotypes and alleles did not show an association with stroke in the study population. When the analysis was carried out separately for men and women, the PvuII polymorphism did not show significant association with stroke in men; women showed a significant association. Further when women were grouped in to premenopausal and postmenopausal, the premenopausal group did not show a significant association with the polymorphism but significant association with stroke was found in postmenopausal women. A stepwise multiple logistic regression analysis confirmed these findings. Women with pp genotype had low estradiol levels in comparison with PP genotypic individuals (p<0.05). Further evaluating the association of this polymorphism with stroke subtypes, we found significant association of PvuII polymorphism with extracranial atherosclerosis, lacunar and cardioembolic stroke. CONCLUSION: In conclusion our results suggest the PvuII gene polymorphism is significantly associated with stroke in postmenopausal women in a South Indian population from Andhra Pradesh. The pp genotypes have average 17ß estradiol levels which are significantly low in comparison with PP genotypes. Therefore postmenopausal women with a high frequency of pp genotype are more predisposed to ischemic stroke. However, this is a preliminary study and the results need to be confirmed in a larger cohort.

Association of the -344C/T aldosterone synthase (CYP11B2) gene variant with hypertension and stroke.

Stroke is a complex disease caused by combination of multiple risk factors. Recent findings have suggested that stroke has a significant genetic component. Various types of genetic polymorphisms have been suggested to contribute to the risk of stroke. Gene polymorphisms of renin-angiontensin aldosterone system (RAAS) have been suggested to be risk factors for hypertension, cardiovascular diseases and stroke. In the present case-control study we investigated the association of -344C/T (rs1799998) [corrected] polymorphism in the promoter region of the human aldosterone (CYP11B2) gene with genetic predisposition to hypertension, ischemic stroke and stroke subtypes classified according to TOAST (Trial of Org 10172 in Acute Stroke Treatment) classification. Four hundred and three stroke patients (hypertensives:normotensives=219:184) and three hundred and ninety four, sex and age matched healthy controls (hypertensives:normotensives=118:276) were involved in the study. The region of interest in the CYP11B2 gene was amplified by polymerase chain reaction and genotypes determined by subjecting the PCR products to restriction digestion by the enzyme HaeIII. Significant difference was observed in the genotypic distribution and allelic frequency between the stroke patients and healthy controls. TT genotype and T allele associated significantly with hypertension and stroke (p<0.000 in hypertension and p=0.000 in case of stroke). A stepwise logistic regression analysis confirmed these findings. To establish that this polymorphism is associated with stroke independent of hypertension, we compared stroke patients without hypertension with normotensive controls. Significant difference was observed in genotypic distribution and allelic frequency between the two groups (p=0.000). Further evaluating the association of this polymorphism with stroke subtypes we found significant associations with intracranial large artery atherosclerosis, lacunar stroke and cardioembolic stroke (p=0.000 in each case). In conclusion our study suggests that -344T allele of CYP11B2 gene is an important risk factor for hypertension and ischemic stroke. However, this is a preliminary study and the results need to be confirmed in a larger cohort.

VNTR polymorphism in intron 4 of the eNOS gene and the risk of ischemic stroke in a South Indian population.

Ischemic stroke is a leading cause of death throughout the world. An increasing number of studies have suggested that genetic factors are important in the stroke risk. The aim of our study was to investigate whether the Variable Number of Tandem Repeats (VNTR) polymorphism in intron 4 of the endothelial nitric oxide synthase (eNOS) gene is associated with ischemic stroke in a South Indian population. 357 patients and 283 controls were enrolled in this case-control study. The ischemic stroke patients were classified according to TOAST classification. The eNOS gene polymorphism was determined by polymerase chain reaction-polyacrylamide gel electrophoresis. The genotypes were confirmed by sequencing the PCR products. There were significant differences in the genotype and allele frequencies of eNOS polymorphism between the patients with ischemic stroke and healthy controls (p=0.000). Multiple logistic regression analysis with forward stepwise selection using the potential confounders (sex, age, diabetes, hypertension, smoking and alcoholism) and eNOS gene variant revealed that the VNTR polymorphism in intron 4 of the eNOS gene is significantly [adjusted odds ratio=6.23, 95%CI (4.30-9.29), p=0.000] associated with ischemic stroke in the South Indian population from Andhra Pradesh. We did not find significant association of this polymorphism with any specific stroke subtype. Further hypertensives bearing 4a allele in high frequency are more predisposed to stroke.

Nanoporous TiO2 and WO3 films by anodization of titanium and tungsten substrates: influence of process variables on morphology and photoelectrochemical response.

The photoelectrochemical response of nanoporous films, obtained by anodization of Ti and W substrates in a variety of corrosive media and at preselected voltages in the range from 10 to 60 V, was studied. The as-deposited films were subjected to thermal anneal and characterized by scanning electron microscopy and X-ray diffraction. Along with the anodization media developed by previous authors, the effect of poly(ethylene glycol) (PEG 400) or D-mannitol as a modifier to the NH4F electrolyte and glycerol addition to the oxalic acid electrolyte was studied for TiO2 and WO3, respectively. In general, intermediate anodization voltages and film growth times yielded excellent-quality photoelectrochemical response for both TiO2 and WO3 as assessed by linear-sweep photovoltammetry and photoaction spectra. The photooxidation of water and formate species was used as reaction probes to assess the photoresponse quality of the nanoporous oxide semiconductor films. In the presence of formate as an electron donor, the incident photon to electron conversion efficiency (IPCE) ranged from approximately 130% to approximately 200% for both TiO2 and WO3 depending on the film preparation protocol. The best photoactive films were obtained from poly(ethylene glycol) (PEG 400) containing NH4F for TiO2 and from aqueous NaF for WO3.

Overoxidized poly(pyrrole-co-[3-(pyrrol-1-yl)-propanesulfonate])-coated platinum electrodes for selective detection of catecholamine neurotransmitters.

The cation-exchange and anion-exclusion properties of the poly¿pyrrole-co-[3-(pyrrol-1-yl)propanesulfonate]¿-(PPy-PS) copolymer are exploited for imparting higher selectivity to measurements of primary neurotransmitters in the presence of ascorbic acid. Such incorporation of ionizable sulfonated groups in the pyrrole ring prior to its electropolymerization leads to effective rejection of the anionic ascorbate species and preferential collection of the cationic dopamine and norepinephrine. Overoxidized PPy-PS films thus offer better discrimination against ascorbic acid than Nafion or overoxidized polypyrrole coatings. Experimental variables influencing the permselective behavior of the PPy-PS layer, including the electropolymerization time and solution pH, were explored. The selectivity and sensitivity improvements associated with the increased electrostatic character of overoxidized polypyrrole films hold promise for neurochemical electrochemical studies.