Genome-wide haplotype association study identifies the FRMD4A gene as a risk locus for Alzheimer's disease.

Recently, several genome-wide association studies (GWASs) have led to the discovery of nine new loci of genetic susceptibility in Alzheimer's disease (AD). However, the landscape of the AD genetic susceptibility is far away to be complete and in addition to single-SNP (single-nucleotide polymorphism) analyses as performed in conventional GWAS, complementary strategies need to be applied to overcome limitations inherent to this type of approaches. We performed a genome-wide haplotype association (GWHA) study in the EADI1 study (n=2025 AD cases and 5328 controls) by applying a sliding-windows approach. After exclusion of loci already known to be involved in AD (APOE, BIN1 and CR1), 91 regions with suggestive haplotype effects were identified. In a second step, we attempted to replicate the best suggestive haplotype associations in the GERAD1 consortium (2820 AD cases and 6356 controls) and observed that 9 of them showed nominal association. In a third step, we tested relevant haplotype associations in a combined analysis of five additional case-control studies (5093 AD cases and 4061 controls). We consistently replicated the association of a haplotype within FRMD4A on Chr.10p13 in all the data set analyzed (OR: 1.68; 95% CI: (1.43-1.96); P=1.1 × 10(-10)). We finally searched for association between SNPs within the FRMD4A locus and Aß plasma concentrations in three independent non-demented populations (n=2579). We reported that polymorphisms were associated with plasma Aß42/Aß40 ratio (best signal, P=5.4 × 10(-7)). In conclusion, combining both GWHA study and a conservative three-stage replication approach, we characterised FRMD4A as a new genetic risk factor of AD.

The evalaution of the foot core system in individuals with plantar heel pain.

OBJECTIVE: To compare foot posture, plantar sensation, plantar fascia thickness, intrinsic foot muscle performance, and abductor hallucis morphology in individuals with and without plantar heel pain (PHP). DESIGN: Cross-Sectional. SETTING: Laboratory. PARTICIPANTS: Sixteen individuals with PHP and sixteen matched healthy participants. MAIN OUTCOME MEASURES: Static foot posture, plantar sensation, plantar fascia thickness, intrinsic foot muscle performance and abductor hallucis morphology were evaluated. Foot posture was assessed with the Foot Posture Index-6. Abductor hallucis morphology and plantar fascia thickness were measured utilizing diagnostic ultrasound. Plantar foot sensation was assessed at the head of the first metatarsal and medial longitudinal arch using Semmes-Weinstein Monofilaments. Intrinsic foot muscle performance was assessed using the intrinsic foot muscle test (IFMT). Mann-Whitney U and independent t-tests were used to examine between group differences. RESULTS: Individuals with PHP exhibited a more pronated foot posture and greater plantar fascia thickness at the proximal insertion compared to healthy controls. Plantar sensation thresholds were higher in the PHP compared to healthy controls at the head of the first metatarsal. There were no group differences in abductor hallucis morphology or IFMT performance. CONCLUSIONS: Individuals with PHP exhibited a more pronated foot posture, thicker plantar fascia, and diminished plantar tactile sensation.

SNP association studies in Alzheimer's disease highlight problems for complex disease analysis.

Genetic linkage and association analyses are two distinct approaches to understanding the genetic etiology of complex disease. Association analysis has become particularly popular in recent times, but the true utility of the strategy remains uncertain. To try to gain better insight into the relevant issues, we have used genetic association analysis to explore the etiology of Alzheimer's disease. Our empirical findings supplement the theoretical debate, illustrating the general doubtfulness of previous positive findings and the limited ability of typical association studies based on candidate genes to discern true medium-sized signals from false positives. Improvements in genotyping technologies and increasing the number of SNPs tested, without sophisticated allowance for all other issues, could simply lead to an unmanageable overload of false-positive signals, themselves obscuring true disease associations.

Association of TNF-alpha serum levels and TNFA promoter polymorphisms with risk of myocardial infarction.

Elevated levels of tumor necrosis factor-alpha (TNF-alpha), and presence of polymorphisms of the TNFA gene have been implicated in cardiovascular disease pathogenesis. We explored the relationship between polymorphisms in the TNFA gene (-1031C/T, -863C/A -857T/C, -308G/A, -238G/A), protein levels of TNF-alpha and their association to myocardial infarction (MI) using a sample of 1213 post-MI patients and 1561 healthy controls. MI risk was higher among men with elevated TNF-alpha levels, with the highest compared to the lowest TNF-alpha quartile giving a 70% risk increase (OR [95% CI]: 1.7 [1.1; 2.6]). Obese subjects who also had elevated TNF-alpha levels were at even higher risk for MI (OR [95% CI]: 3.4 [2.1; 5.6]). Higher TNF-alpha levels were seen among smokers (but not among non-smokers) carrying the -857T allele. Furthermore, a rare haplotype occurred more frequently among the cases than the controls. Elevated TNF-alpha levels are associated with increased MI risk. Obese subjects with elevated TNF-a levels, and carriers of polymorphisms in or near TNFA are particularly susceptible to the hazards of smoking, results which may have implications for cardiovascular preventive measures.

Ultra-wetting graphene-based PES ultrafiltration membrane - A novel approach for successful oil-water separation.

Oil pollution in water and separation of oil from water are receiving much attention in recent years due to the growing environmental concerns. Membrane technology is one of the emerging solutions for oil-water separation. However, there is a limitation in using polymeric membrane for oil water separation due to its surface properties (wetting behaviour), thermal and mechanical properties. Here, we have shown a simple method to increase the hydrophilicity of the polyethersulfone (PES) hollow fibre ultrafiltration (UF) membrane by using carboxyl, hydroxyl and amine modified graphene attached poly acrylonitrile-co-maleimide (G-PANCMI). The prepared membranes were characterized for its morphology, water and oil contact angle, liquid entry pressure of oil (LEPoil), water permeability and finally subjected to a continuous 8 h filtration test of oil emulsion in water. The experimental data indicates that the G-PANCMI play an important role in enhancing the hydrophilicity, permeability and selectivity of the PES membrane. The water contact angle (CAw) of the PES membrane is reduced from 63.7 ± 3.8° to 22.6 ± 2.5° which is 64.5% reduction while, the oil contact angle was increased from 43.6 ± 3.5° to 112.5 ± 3.2° which is 158% higher compared to that of the PES membrane. Similarly, the LEPoil increased 350% from 50 ± 10 kPa of the control PES membrane to 175 ± 25 kPa of PES-G-PANCMI membrane. More importantly, the water permeability increased by 43% with >99% selectivity. Based on our findings we believe that the development of PES-G-PANCMI membrane will open up a solution for successful oil-water separation.

Lack of replication of association findings in complex disease: an analysis of 15 polymorphisms in prior candidate genes for sporadic Alzheimer's disease.

There is considerable enthusiasm for the prospect of using common polymorphisms (primarily single nucleotide polymorphisms; SNPs) in candidate genes to unravel the genetics of complex disease. This approach has generated a number of findings of loci which are significantly associated with sporadic Alzheimer's disease (AD). In the present study, a total of 15 genes of interest were chosen from among the previously published reports of significant association in AD. Genotyping was performed on polymorphisms within those genes (14 SNPs and one deletion) using Dynamic Allele Specific Hybridization (DASH) in 204 Swedish patients with sporadic late-onset AD and 186 Swedish control subjects. The genes chosen for analysis were; low-density lipoprotein receptor-related protein (LRP1), angiotensin converting enzyme (DCP1), alpha-2-macroglobulin (A2M), bleomycin hydrolase (BLMH), dihydrolipoyl S-succinyltransferase (DLST), tumour necrosis factor receptor superfamily member 6 (TNFRSF6), nitric oxide synthase (NOS3), presenilin 1 (PSEN1), presenilin 2 (PSEN2), butyrylcholinesterase (BCHE), Fe65 (APBB1), oestrogen receptor alpha (ESR1), cathepsin D (CTSD), methylenetetrahydrofolate reductase (MTHFR), and interleukin 1A (IL1A). We found no strong evidence of association for any of these loci with AD in this population. While the possibility exists that the genes analysed are involved in AD (ie they have weak effects and/or are population specific), results reinforce the need for extensive replication studies if we are to be successful in defining true risk factors in complex diseases.

Identification of 167 polymorphisms in 88 genes from candidate neurodegeneration pathways.

Catalogs of intra-gene polymorphisms are needed to facilitate wide-ranging candidate gene-based association studies in common complex diseases. With this in mind, we have scanned multiple alignments of expressed sequence tags and of genomic DNA sequences (PCR products from four to eight unrelated individuals) to find polymorphisms in 195 genes putatively involved in neurodegenerative illness (including components of oxidative stress, excitotoxicity, inflammation, apoptosis and aging). This led to the discovery of 167 polymorphisms in 88 genes. These comprised 163 single nucleotide polymorphisms, one insertion/deletion, and three other variations involving more than one base pair. The polymorphisms were distributed in the exons (87), introns (70), and gene flanking regions (10). Of the exonic polymorphisms, 17 would give rise to non-synonymous amino acid substitutions. These findings now provide a valuable resource for association studies in neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease.

Putamen mitochondrial energy metabolism is highly correlated to emotional and intellectual impairment in schizophrenics.

In a recent study, we demonstrated that cytochrome-c oxidase (COX), an indicator of neuronal activity, is increased in several brain regions from chronic, medicated schizophrenics. In the present study, to address the functional significance of those findings, we have measured COX activity in a group of schizophrenics in whom antemortem geriatric measures of motor, intellectual, and emotional impairment had been assessed. COX activity in the putamen was strongly negatively correlated with emotional (r = -.76; p < .005) and intellectual impairment (r = -0.76; p < .005), but not with motor impairment (r = 0.01). No significant correlations could be found in the frontal cortex, thalamus, caudate nucleus, globus pallidus, mesencephalon, or nucleus accumbens. Dopamine D2 receptor density in the putamen, measured with [3H]raclopride, was elevated in schizophrenics as compared to controls, as were Kd values. In contrast to COX activity, D2 receptor binding was moderately, but significantly positively correlated with intellectual impairment (r = 0.64; p < .05) but not with motor impairment. Results expose a unique anomaly in the effects of neuroleptics in terms of increasing neuronal signaling in the putamen, which may underlie a reversal of cognitive deficits in schizophrenics, while at the same time, elevating D2 receptor density that seems to be detrimental.

Mitochondrial function is differentially altered in the basal ganglia of chronic schizophrenics.

In the present study, we have applied a novel strategy involving the postmortem measurement of the mitochondrial respiratory chain enzyme cytochrome-c oxidase (COX; complex IV) to identify regional changes in energy metabolism in the basal ganglia of chronic, medicated schizophrenics. COX activity was decreased in the caudate nucleus but increased in the putamen and nucleus accumbens. An increase in succinate dehydrogenase (complex II) was evident in the putamen and nucleus accumbens, but changes were not seen with NADH dehydrogenase (complex I). An analysis of interregional correlations in energy metabolism revealed several anomalies in the connections between the caudate and putamen and the globus pallidus in schizophrenics. Results provide strong evidence that changes in baseline energy metabolism in specific regions of the basal ganglia may exist in the disease. Based upon the high degree of input it receives from associative cortical areas, results suggest that a defect in the caudate may underlie certain aspects of cognitive decline in schizophrenics. In contrast, an increase in COX in the putamen, which receives extensive projections from the sensorimotor cortex, may reflect an effect of chronic neuroleptic treatment on motor function.

Normalization of cytochrome-c oxidase activity in the rat brain by neuroleptics after chronic treatment with PCP or methamphetamine.

Previous studies, primarily involving the use of positron emission tomography (PET), have contributed to the hypothesis that a state of hypometabolism may underlie schizophrenia. The chronic use of methamphetamine (MAP) or phencyclidine (PCP), both of which have been shown to enhance dopaminergic function in the brain, leads to a psychotic state in man which has prompted the suggestion that these compounds may have utility as models of schizophrenia. In the present study, regional alterations in energy metabolism were examined in the rat brain using cytochrome-c oxidase (COX) and succinate dehydrogenase (SDH) histochemistry following chronic treatment with PCP and MAP. PCP and MAP were administered alone or in the presence of fluphenazine or clozapine to animals for 28 days, after which mitochondrial enzyme activities were estimated. Both PCP and MAP produced profoundly similar decreases in COX activity in a broad spectrum of regions. Most prominent in this regard were the caudate-putamen, nucleus accumbens and septum. No changes were noted in sections stained for SDH activity, suggesting that results were dependent upon neither a generalized mitochondrial dysfunction nor mitochondrial loss. Cell counts and TUNEL histochemistry also failed to reveal any significant differences between control and treated animals, implying that reductions were not a result of cell loss. Both clozapine and fluphenazine offered varying degrees of protection from the effects of PCP and MAP. The results provide evidence which implicates dopaminergic hyperactivity in the finding of reduced energy metabolism in the brains of schizophrenics.

Towards high-throughput genotyping of SNPs by dynamic allele-specific hybridization.

Analysis of single nucleotide polymorphisms (SNPs)--the most common form of variation in the human genome--has become a popular strategy for discovering genes involved in complex diseases such as Alzheimer's disease, obesity and diabetes. It is also widely anticipated that SNPs will play a major role in pharmacogenomics, where the identification of variations in specific genes relevant to drug efficacy, toxicity and metabolism will help to establish optimal therapeutic strategies for individual patients. Reflecting these expectations, many new SNP-related technologies have appeared over the past few years, each with unique advantages, but all with the common goal of simplifying and expediting SNP analysis. We recently introduced a technique termed dynamic allele-specific hybridization (DASH), a convenient method for SNP (and insertion-deletion) genotyping, which is highly applicable to both basic research and clinical diagnostics. Commercial DASH devices are now available, making the technology affordably accessible for all laboratories.

Mitochondrial activity in the mapping of functional brain changes in schizophrenia.

The main contributors to the search for functional brain changes in schizophrenia in the past years have employed imaging techniques such as positron emission tomography (PET), single photon emission computed tomography (SPECT), and magnetic resonance imaging (MRI). Our laboratory has applied a novel strategy involving the post-mortem measurement of the mitochondrial respiratory chain enzyme cytochrome-c-oxidase (COX) to address the question of regional metabolic changes in schizophrenia. This approach is based upon a strong body of evidence which indicates that neuronal COX is highly regulated by the energy demands of the cell and as such represents an endogenous marker of cellular energy metabolism over time. Our original findings indicated that COX activity may be reduced in the striatum and frontal cortex consistent with the concept that a state reduced activity in cortico-striatal circuits may underlie schizophrenia. Subsequent studies from our laboratory on the effects of neuroleptics, PCP, and methamphetamine on animals, have provided additional evidence that a state of dopaminergic overactivity or glutamatergic underactivity produces a hypometabolic state similar to that which is evident in the brains of schizophrenics.

Increased frequency of a new polymorphism in the cell division cycle 2 (cdc2) gene in patients with Alzheimer's disease and frontotemporal dementia.

Recent studies show linkage between Alzheimer's disease (AD) and two loci on chromosome 10. The cell division cycle 2 (cdc2) gene is located close to one of the chromosome 10 markers, and is a candidate gene for AD since it is involved in the pathogenesis of AD. We sequenced coding exons and flanking intronic sequences and the promoter region on the cdc2 gene and found three new single nucleotide polymorphisms (SNPs). We analyzed 272 Caucasian AD cases, 160 controls and 70 cases with frontotemporal dementia (FTD) for these SNPs. Homozygosity for one of the SNPs (Ex6+7I/D) was more frequent in both AD and FTD cases than in controls. In the combined tauopathy (AD and FTD) group the odds ratio (OR) was 1.77 (95% CI 1.19-2.63) for the Ex6+7II genotype. Our findings suggest that the Ex6+7I allele is associated with tauopathies, both AD and FTD.

A histochemical demonstration of altered cytochrome oxidase activity in the rat brain by neuroleptics.

Regional alterations in neuronal functional activity were examined in the rat brain using cytochrome-c oxidase (COX) histochemistry following chronic neuroleptic treatment. Haloperidol, fluphenazine, and clozapine were administered to animals for 28 days after which profiles of COX activity were generated. Significant increases in COX activity were evident in area 2 of the frontal cortex of all treated animals. Clozapine and fluphenazine, but not haloperidol, caused significant increases in COX activity in the caudate nucleus, nucleus accumbens, septum, and pontine nucleus. Statistically significant increases in COX activity were also observed in hippocampal CA2 and CA3 subfields in clozapine treated animals. Results offer support for the concept that neuroleptics achieve their therapeutic effects primarily via an enhancement of brain function in the frontal cortex, but also point to other brain regions which may be involved in the actions of these drugs.

Staurosporine differentiated human SH-SY5Y neuroblastoma cultures exhibit transient apoptosis and trophic factor independence.

The use of chemically differentiated neuroblastoma cells in the study of neuronal function has become a common alternative to primary neuronal cell cultures in recent years, particularly in the area of cell death. Staurosporine, a nonselective protein kinase inhibitor, has been demonstrated to be a particularly strong inducer of differentiation in the SH-SY5Y human neuroblastoma cell line. However, at present, no data exist on the long-term effects of this compound. We have compared the effects of staurosporine with 12-O-tetradecanoyl phorbol-13 acetate and retinoic acid in terms of long-term cell viability and neuronal function in the SH-SY5Y cell line. In the presence of serum, staurosporine-treated cells underwent apoptosis, which ultimately resulted in total cell loss. In contrast, when cultured in defined serum-free medium, a cessation of apoptosis occurred after approximately 1 week, at which point viability could be maintained in excess of 1 month. The addition of aurintricarboxylic acid, which has been demonstrated to prevent apoptosis in a variety of cell models, completely prevented both apoptosis and differentiation in staurosporine-treated cells both under serum-supplemented and serum-free conditions. Apoptosis was not prevented by the protein synthesis inhibitor, cycloheximide. The removal of staurosporine from the culture medium after 3 weeks had no effect on cellular morphology, function, or proliferation, indicating that the attained neuronal phenotype was terminal. Voltage-gated calcium channel sensitivity, used as a measurement of neuronal function, was highest in staurosporine-treated cells. On the basis that apoptosis and neurotrophin independence are hallmarks of the maturation of dorsal root ganglion neurons, results suggest that staurosporine-differentiated SH-SY5Y cells may bear a similar phenotype to that found in vivo. Furthermore, this model may provide for an excellent means of obtaining a stable and homogenous population of postmitotic monoaminergic neurons for investigating neuronal function and differentiation.

A cell culture model of cerebral ischemia as a convenient system to screen for neuroprotective drugs.

Aggregation cultures of rat brain were exposed to a combination of anoxia and hypoglycaemia for 30 minutes. Thereafter, the release of lactate dehydrogenase into the cell culture medium was monitored up to 4 days as a measure of cell damage after the ischemic insult. Some cultures were treated with different concentrations of deprenyl or tolcapone, selective inhibitors of monoamine oxidase B and catechol-O-methyltransferase, respectively. After 1 day in culture, the release of lactate dehydrogenase was significantly reduced in cultures treated with deprenyl (at 1 nM. 100 nM, and 10 microM), as well as in cultures treated with 1 nM or 100 nM tolcapone; 10 microM of tolcapone, on the other hand, resulted in a toxic effect on the cell aggregates. No differences in the release of lactate dehydrogenase into the medium was observed in the aggregates treated with drugs as compared with the control cultures after 2 or 4 days post-ischemia.

Nanofiber based triple layer hydro-philic/-phobic membrane--a solution for pore wetting in membrane distillation.

The innovative design and synthesis of nanofiber based hydro-philic/phobic membranes with a thin hydro-phobic nanofiber layer on the top and a thin hydrophilic nanofiber layer on the bottom of the conventional casted micro-porous layer which opens up a solution for membrane pore wetting and improves the pure water flux in membrane distillation.

Self-cleaning Metal Organic Framework (MOF) based ultra filtration membranes--a solution to bio-fouling in membrane separation processes.

Bio-fouling is a serious problem in many membrane-based separation processes for water and wastewater treatment. Current state of the art methods to overcome this are to modify the membranes with either hydrophilic additives or with an antibacterial compound. In this study, we propose and practise a novel concept to prevent bio-fouling by developing a killing and self-cleaning membrane surface incorporating antibacterial silver nanoparticles and highly hydrophilic negatively charged carboxylic and amine functional groups. The innovative surface chemistry helps to reduce the contact angle of the novel membrane by at least a 48% and increase the pure water flux by 39.4% compared to the control membrane. The flux drop for the novel membrane is also lower (16.3% of the initial flux) than the control membrane (55.3% of the initial flux) during the long term experiments with protein solution. Moreover, the novel membrane continues to exhibit inhibition to microbes even after 1320 min of protein filtration. Synthesis of self-cleaning ultrafiltration membrane with long lasting properties opens up a viable solution for bio-fouling in ultrafiltration application for wastewater purification.

Neuroleptic-induced mitochondrial enzyme alterations in the rat brain.

For years, it has been known that neuroleptics have the capacity to interfere with the mitochondrial respiratory chain in vitro. We report that haloperidol and fluphenazine, classical neuroleptics, cause a generalized reduction in the activity of NADH: ubiquinone oxidoreductase (complex I) in the rat brain in vivo, an effect that was not observed with the atypical neuroleptic, clozapine. MPTP, which bears significant structural similarities with haloperidol, also demonstrated a significant reduction in complex I activity after low-dose, chronic administration. Interestingly, an increase in the activity of cytochrome-c oxidase (complex IV), probably reflecting enhanced functional neuronal activity, was observed in the frontal cortex of all chronically treated animals, an effect that is unlikely to result from compensation for the inhibition of complex I. Results suggest that previous findings, in which a reduction in the activity of cytochrome-c oxidase was observed in postmortem brain samples from schizophrenics, are not dependent on treatment with neuroleptics.

A novel screen for nuclear mitochondrial gene associations with Parkinson's disease.

Genetic factors play an important role in the aetiology of Parkinson's disease (PD). We have screened nuclear genes encoding subunits of mitochondrial complex I for associations between single nucleotide polymorphisms (SNPs) and PD. Abnormal functioning of complex I is well documented in human PD. Moreover, toxicological inhibition of complex I can lead to parkinsonism in animals. Thus, commonly occurring variants in these genes could potentially influence complex I function and the risk of developing PD. A sub-set of 70 potential SNPs in 31 nuclear complex I genes were selected and association analysis was performed on 306 PD patients plus 321 unaffected control subjects. Genotyping was performed using the DASH method. There was no evidence that the examined SNPs were significant genetic risk factors for PD, although this initial screen could not exclude the possibility that other disease-influencing variations exist within these genes.