Creation and application of immortalized bait libraries for targeted enrichment and next-generation sequencing

Since the introduction of next-generation sequencing, several techniques have been developed to selectively enrich and sequence specific parts of the genome at high coverage. These techniques include enzymatic methods employing molecular inversion probes, PCR based approaches, hybrid capture, and in-solution capture. In-solution capture employs RNA probes transcribed from a pool of DNA template oligos designed to match regions of interest to specifically bind and enrich genomic DNA fragments. This method is highly efficient, especially if genomic target regions are large in size or quantity. Diverse in-solution capture kits are available, but are costly when large sample numbers need to be analyzed. Here we present a cost-effective strategy for the design of custom DNA libraries, their transcription into RNA libraries, and application for in-solution capture. We show the efficacy by comparing the method to a commercial kit and further demonstrate that emulsion PCR can be used for bias free amplification and virtual immortalization of DNA template libraries

Screening of human gene promoter activities using transfected-cell arrays

Promoters are the best characterized transcriptional regulatory sequences in complex genomes because of their predictable location immediately upstream of transcription start sites. Despite a substantial body of literature describing transcriptional promoters, the identification of true start sites for all human transcripts is far from complete. The same is true of the key structural and functional elements responsible for promoter action in different cell types. In order to identify elements responsible for promoter activity, we applied transfected-cell array technology to functionally evaluate promoters for genes involved in inflammatory bowel disease. Seventy-four promoters were examined by reverse transfection of a promoter-fluorescent reporter constructs into a human embryonic kidney cell line (HEK293T). Sixteen (21.6%) promoters were found to be active in HEK293 T cells. Correlations between promoter activity and endogenous transcript level were calculated, and 75% of active promoters were found to be associated with transcriptional activity of their gene counterparts. These results provide experimental evidence of promoter activity, which may aid in understanding the regulation of gene expression. Moreover, this is the first large-scale functional study of regulatory sequences to use a high-throughput transfected-cell array technique

Evaluation of a Previously Suggested Plasma Biomarker Panel to Identify Alzheimer's Disease

There is an urgent need for biomarkers in plasma to identify Alzheimer's disease (AD). It has previously been shown that a signature of 18 plasma proteins can identify AD during pre-dementia and dementia stages (Ray et al, Nature Medicine, 2007). We quantified the same 18 proteins in plasma from 174 controls, 142 patients with AD, and 88 patients with other dementias. Only three of these proteins (EGF, PDG-BB and MIP-1δ) differed significantly in plasma between controls and AD. The 18 proteins could classify patients with AD from controls with low diagnostic precision (area under the ROC curve was 63%). Moreover, they could not distinguish AD from other dementias. In conclusion, independent validation of results is important in explorative biomarker studies

Alzheimer's disease - input of vitamin D with mEmantine assay (AD-IDEA trial): study protocol for a randomized controlled trial

BACKGROUND: Current treatments for Alzheimer\u27s disease and related disorders (ADRD) are symptomatic and can only temporarily slow down ADRD. Future possibilities of care rely on multi-target drugs therapies that address simultaneously several pathophysiological processes leading to neurodegeneration. We hypothesized that the combination of memantine with vitamin D could be neuroprotective in ADRD, thereby limiting neuronal loss and cognitive decline. The aim of this trial is to compare the effect after 24 weeks of the oral intake of vitamin D3 (cholecalciferol) with the effect of a placebo on the change of cognitive performance in patients suffering from moderate ADRD and receiving memantine. METHODS: The AD-IDEA Trial is a unicentre, double-blind, randomized, placebo-controlled, intent-to-treat, superiority trial. Patients aged 60 years and older presenting with moderate ADRD (i.e., Mini-Mental State Examination [MMSE] score between 10-20), hypovitaminosis D (i.e., serum 25-hydroxyvitamin D [25OHD] < 30 ng/mL), normocalcemia (i.e., serum calcium < 2.65 mmol/L) and receiving no antidementia treatment at time of inclusion are being recruited. All participants receive memantine 20 mg once daily -titrated in 5 mg increments over 4 weeks- and each one is randomized to one of the two treatment options: either cholecalciferol (one 100,000 IU drinking vial every 4 weeks) or placebo (administered at the same pace). One hundred and twenty participants are being recruited and treatment continues for 24 weeks. Primary outcome measure is change in cognitive performance using Alzheimer\u27s Disease Assessment Scale-cognition score. Secondary outcomes are changes in other cognitive scores (MMSE, Frontal Assessment Battery, Trail Making Test parts A and B), change in functional performance (Activities of Daily Living scale, and 4-item Instrumental Activities of Daily Living scale), posture and gait (Timed Up & Go, Five Time Sit-to-Stand, spatio-temporal analysis of walking), as well as the between-groups comparison of compliance to treatment and tolerance. These outcomes are assessed at baseline, 12 and 24 weeks, together with the serum concentrations of 25OHD, calcium and parathyroid hormone. DISCUSSION: The combination of memantine plus vitamin D may represent a new multi-target therapeutic class for the treatment of ADRD. The AD-IDEA Trial seeks to provide evidence on its efficacy in limiting cognitive and functional declines in ADRD. TRIAL REGISTRATION: ClinicalTrials.gov number, NCT01409694

Elevated Stearoyl-CoA Desaturase in Brains of Patients with Alzheimer's Disease

The molecular bases of Alzheimer's disease (AD) remain unclear. We used a lipidomic approach to identify lipid abnormalities in the brains of subjects with AD (N = 37) compared to age-matched controls (N = 17). The analyses revealed statistically detectable elevations in levels of non-esterified monounsaturated fatty acids (MUFAs) and mead acid (20:3n-9) in mid-frontal cortex, temporal cortex and hippocampus of AD patients. Further studies showed that brain mRNAs encoding for isoforms of the rate-limiting enzyme in MUFAs biosynthesis, stearoyl-CoA desaturase (SCD-1, SCD-5a and SCD-5b), were elevated in subjects with AD. The monounsaturated/saturated fatty acid ratio (‘desaturation index’) – displayed a strong negative correlation with measures of cognition: the Mini Mental State Examination test (r = −0.80; P = 0.0001) and the Boston Naming test (r = −0.57; P = 0.0071). Our results reveal a previously unrecognized role for the lipogenic enzyme SCD in AD

IL-6-174 G/C and -572 C/G Polymorphisms and Risk of Alzheimer’s Disease

Associations between interleukin 6 (IL-6) polymorphisms and Alzheimer’s disease (AD) remain controversial and ambiguous. The aim of this meta-analysis is to explore more precise estimations for the relationship between IL-6-174 G/C and -572 C/G polymorphisms and risk for AD. Electronic searches for all publications in databases PubMed and EMBASE were conducted on the associations between IL-6 polymorphisms and risk for AD until January 2012. Odds ratio (OR) and 95% confidence intervals (CIs) were calculated using fixed and random effects models. Twenty-seven studies were included with a total of 19,135 individuals, involving 6,632 AD patients and 12,503 controls. For IL-6-174 G/C polymorphism, the combined results showed significant differences in recessive model (CC vs. CG+GG: OR = 0.65, 95%CI = 0.52–0.82). As regards IL-6-572 C/G polymorphism, significant associations were shown in dominant model (CG+GG vs. CC: OR  = 0.73, 95% CI = 0.62–0.86) and in additive model (GG vs. CC, OR  = 0.66, 95% CI = 0.46–0.96). In conclusion, genotype CC of IL-6-174 G/C and genotype GG plus GC of IL-6-572 C/G could decrease the risk of AD

Transient Focal Cerebral Ischemia/Reperfusion Induces Early and Chronic Axonal Changes in Rats: Its Importance for the Risk of Alzheimer's Disease

The dementia of Alzheimer's type and brain ischemia are known to increase at comparable rates with age. Recent advances suggest that cerebral ischemia may contribute to the pathogenesis of Alzheimer's disease (AD), however, the neuropathological relationship between these two disorders is largely unclear. It has been demonstrated that axonopathy, mainly manifesting as impairment of axonal transport and swelling of the axon and varicosity, is a prominent feature in AD and may play an important role in the neuropathological mechanisms in AD. In this study, we investigated the early and chronic changes of the axons of neurons in the different brain areas (cortex, hippocampus and striatum) using in vivo tracing technique and grading analysis method in a rat model of transient focal cerebral ischemia/reperfusion (middle cerebral artery occlusion, MCAO). In addition, the relationship between the changes of axons and the expression of β-amyloid 42 (Aβ42) and hyperphosphorylated Tau, which have been considered as the key neuropathological processes of AD, was analyzed by combining tracing technique with immunohistochemistry or western blotting. Subsequently, we found that transient cerebral ischemia/reperfusion produced obvious swelling of the axons and varicosities, from 6 hours after transient cerebral ischemia/reperfusion even up to 4 weeks. We could not observe Aβ plaques or overexpression of Aβ42 in the ischemic brain areas, however, the site-specific hyperphosphorylated Tau could be detected in the ischemic cortex. These results suggest that transient cerebral ischemia/reperfusion induce early and chronic axonal changes, which may be an important mechanism affecting the clinical outcome and possibly contributing to the development of AD after stroke

Orally Administrated Cinnamon Extract Reduces β-Amyloid Oligomerization and Corrects Cognitive Impairment in Alzheimer's Disease Animal Models

An increasing body of evidence indicates that accumulation of soluble oligomeric assemblies of β-amyloid polypeptide (Aβ) play a key role in Alzheimer's disease (AD) pathology. Specifically, 56 kDa oligomeric species were shown to be correlated with impaired cognitive function in AD model mice. Several reports have documented the inhibition of Aβ plaque formation by compounds from natural sources. Yet, evidence for the ability of common edible elements to modulate Aβ oligomerization remains an unmet challenge. Here we identify a natural substance, based on cinnamon extract (CEppt), which markedly inhibits the formation of toxic Aβ oligomers and prevents the toxicity of Aβ on neuronal PC12 cells. When administered to an AD fly model, CEppt rectified their reduced longevity, fully recovered their locomotion defects and totally abolished tetrameric species of Aβ in their brain. Furthermore, oral administration of CEppt to an aggressive AD transgenic mice model led to marked decrease in 56 kDa Aβ oligomers, reduction of plaques and improvement in cognitive behavior. Our results present a novel prophylactic approach for inhibition of toxic oligomeric Aβ species formation in AD through the utilization of a compound that is currently in use in human diet