Experience of quantity and quality of DNA and RNA extraction from limited pediatric blood samples: A comparative analysis of automated and manual kit-based method.

INTRODUCTION: Optimal DNA and RNA quantity and purity is essential for downstream molecular biology experimentation and to avoid re-processing of sample. Despite availability of different kits and automated systems for nucleic acid isolation there is limited data on their performance evaluation, more so with pediatric blood samples, that are usually compromised in quantity. Hence, we evaluated the performance of automated QIAcube platform using pediatric blood samples in parallel with manual Qiagen extraction kits. MATERIALS AND METHODS: : A total of 500 samples were analyzed based on groups of PBMC and direct blood input. The isolated DNA and RNA were surveyed for quantity and quality tests by spectrophotometric and downstream analysis. RESULTS: : There was no significant difference in the DNA quantity (ng/ul) between manual and automated method based on similar sample input but quality (260/280) was significantly better with the QIAcube platform when direct blood and or PBMCs were used for extraction respectively (1.82 ± 004 Vs. 1.84.002; P-0.000008 and 1.859 ± 005 Vs. 1.843 ± 0.003; P-0.02). Moreover, the standard error mean was low for both quantity and quality in the QIAcube method suggesting uniformity. Comparison of quality assessment by spectrophotometer and qubit fluorimeter showed that QIAcube sheared DNA less (P- 0.038) as compared to manual method (P-0.013). Also, time taken to process the samples in QIAcube was 23% less than the kit-based method. CONCLUSION: Overall analysis of QIAcube platform suggests that it yields more better, uniform, and less-sheared quality of nucleic acid in a relatively less time as compared to manual extraction kits.

Alzheimer's Disease Markers in Aged ApoE-PON1 Deficient Mice.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder associated with aging. Cardiovascular risk factors like hypertension and atherosclerosis increase the risk for AD. Polymorphic alleles of apolipoprotein E (ApoE) are one of the main genetic determinants of AD. OBJECTIVE: Mice, double-knockout (DKO) for ApoE (major cholesterol carrier in brain) and PON1 (paroxonase1, reduces oxidative stress), showed severe age-dependent atherosclerosis of the arteries carrying blood to the brain even on normal diet. This prompted us to investigate the possibility of an AD pathology resulting from the deficiency of ApoE and the induction of oxidative stress. METHODS: Atherosclerotic lesions were quantified by ImageJ. The brain hippocampus of young and old ApoE-PON1 DKO mice and control mice were harvested. RT-PCR analysis was performed for mRNA levels of AD specific markers. Blood levels of S100 calcium-binding protein B (S100B) protein were measured by ELISA. H&E as well as immunostaining was performed to detect amyloid-ß (Aß) plaques and neurofibrillary tangles (NFTs) in brain tissues. Evans blue dye was used to evaluate the vascular permeability and blood-brain barrier (BBB) dysfunction. RESULTS: Results showed that the older DKO mice had severe carotid atherosclerosis, increased mRNA levels of AD markers in brain tissue, and elevated levels of serum S100B protein. Immunological staining confirmed the characteristics of AD. Ex-vivo imaging showed higher levels of Evans blue dye in the ApoE-PON1 DKO mice brain tissues, pointing toward impaired vasculature. CONCLUSION: Aged ApoE-PON1 DKO mice displaying AD specific markers along with Aß plaques, NFTs, and disrupted BBB suggests the animals are suffering from AD.

Phosphorylated Tyr142 ß-catenin localizes to centrosomes and is regulated by Syk.

ß-catenin is a central component of adherent junctions and a key effector of canonical Wnt signaling, in which dephosphorylated Ser/Thr ß-catenin regulates gene transcription. ß-catenin phosphorylation at Tyr142 (PTyr142 ß-catenin), which is induced by receptor and Src family Tyr kinases, represents a previously described ß-catenin switch from adhesive to migratory roles. In addition to classical ß-catenin roles, phosphorylated Ser/Thr ß-catenin and total ß-catenin were involved in centrosomal functions, including mitotic spindle formation and centrosome separation. Here we find that PTyr142 ß-catenin is present in centrosomes in non-transformed and glioblastoma cells and that, in contrast to the Ser/Thr phosphorylated ß-catenin, PTyr142 ß-catenin centrosomal levels drop in mitosis. Furthermore, we show that the inhibitor of Spleen Tyrosine Kinase (Syk) piceatannol decreases centrosomal PTyr142 ß-catenin levels, indicating that Syk regulates centrosome PTyr142 ß-catenin. Our findings suggest that PTyr142 ß-catenin and Syk may regulate centrosomal cohesion. This study highlights the contribution of different phosphorylated ß-catenin forms to the cell and centrosome cycles.

Alzheimer's Disease-Current Status and Future Directions.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder of the brain. The presence of amyloid-beta (Aß) plaques, neurofibrillary tangles (NFTs), loss of neurons, synapses, and altered sensory perceptions, including memory loss, delineate AD. However, the cause of AD is not clearly known. Several genetic and nongenetic factors have been implicated in the disease. Of the genes, the ɛ4 allele of apolipoprotein E is the largest known genetic risk factor of AD. This review article focuses on the various genetic and other predisposing factors that account for AD, pathophysiology of the disease, and the mechanisms by which Aß plaques and NFTs are formed and could affect AD brain. In addition, recent advances and current diagnostics available for AD patients are detailed. As oxidative stress has been implicated in the etiology of the disease, special emphasis is given for nutrition based antioxidant therapies and interventional strategies for reducing/treating AD.

Plasma Hepcidin Levels in Healthy Children from Chandigarh, Northern India.

Hepcidin is a key molecule involved in iron homeostasis. We measured hepcidin levels in 50 healthy children from Chandigarh, Northern India for establishing normal ranges. Hepcidin ranges (19.96-36.6 ng/mL; 0-2 years) and (9.54-36.15 ng/mL; 2-6 years) with mean (SD) of 32.5 (4.84) ng/mL, and 31.13 (6.62) ng/mL respectively were noted in study participants. The mean (SD) and ranges for plasma hepcidin in boys and girls in the study was 31.01 (6.71) ng/mL (9.54-36.6 ng/mL) and 32.7 (4.14) ng/mL (19-36.2 ng/mL), respectively.

Nuclear phosphorylated Y142 ß-catenin accumulates in astrocytomas and glioblastomas and regulates cell invasion.

Glioblastoma multiforme (GBM) is a fast growing brain tumor characterized by extensive infiltration into the surrounding tissue and one of the most aggressive cancers. GBM is the most common glioma (originating from glial-derived cells) that either evolves from a low grade astrocytoma or appears de novo. Wnt/ß-catenin and Hepatocyte Growth Factor (HGF)/c-Met signaling are hyperactive in human gliomas, where they regulate cell proliferation, migration and stem cell behavior. We previously demonstrated that ß-catenin is phosphorylated at Y142 by recombinant c-Met kinase and downstream of HGF signaling in neurons. Here we studied phosphoY142 (PY142) ß-catenin and dephospho S/T ß-catenin (a classical Wnt transducer) in glioma biopsies, GBM cell lines and biopsy-derived glioma cell cultures. We found that PY142 ß-catenin mainly localizes in the nucleus and signals through transcriptional activation in GBM cells. Tissue microarray analysis confirmed strong nuclear PY142 ß-catenin immunostaining in astrocytoma and GBM biopsies. By contrast, active ß-catenin showed nuclear localization only in GBM samples. Western blot analysis of tumor biopsies further indicated that PY142 and active ß-catenin accumulate independently, correlating with the expression of Snail/Slug (an epithelial-mesenchymal transition marker) and Cyclin-D1 (a regulator of cell cycle progression), respectively, in high grade astrocytomas and GBMs. Moreover, GBM cells stimulated with HGF showed increasing levels of PY142 ß-catenin and Snail/Slug. Importantly, the expression of mutant Y142F ß-catenin decreased cell detachment and invasion induced by HGF in GBM cell lines and biopsy-derived cell cultures. Our results identify PY142 ß-catenin as a nuclear ß-catenin signaling form that downregulates adhesion and promotes GBM cell invasion.

Chemokines induce axon outgrowth downstream of Hepatocyte Growth Factor and TCF/ß-catenin signaling.

Axon morphogenesis is a complex process regulated by a variety of secreted molecules, including morphogens and growth factors, resulting in the establishment of the neuronal circuitry. Our previous work demonstrated that growth factors [Neurotrophins (NT) and Hepatocyte Growth Factor (HGF)] signal through ß-catenin during axon morphogenesis. HGF signaling promotes axon outgrowth and branching by inducing ß-catenin phosphorylation at Y142 and transcriptional regulation of T-Cell Factor (TCF) target genes. Here, we asked which genes are regulated by HGF signaling during axon morphogenesis. An array screening indicated that HGF signaling elevates the expression of chemokines of the CC and CXC families. In line with this, CCL7, CCL20, and CXCL2 significantly increase axon outgrowth in hippocampal neurons. Experiments using blocking antibodies and chemokine receptor antagonists demonstrate that chemokines act downstream of HGF signaling during axon morphogenesis. In addition, qPCR data demonstrates that CXCL2 and CCL5 expression is stimulated by HGF through Met/b-catenin/TCF pathway. These results identify CC family members and CXCL2 chemokines as novel regulators of axon morphogenesis downstream of HGF signaling.

ß-Catenin Signalling in Glioblastoma Multiforme and Glioma-Initiating Cells.

Glioblastoma multiforme (GBM) is a commonly occurring brain tumor with a poor prognosis. GBM can develop both "de novo" or evolve from a previous astrocytoma and is characterized by high proliferation and infiltration into the surrounding tissue. Following treatment (surgery, radiotherapy, and chemotherapy), tumors often reappear. Glioma-initiating cells (GICs) have been identified in GBM and are thought to be responsible for tumors initiation, their continued growth, and recurrence. ß-catenin, a component of the cell-cell adhesion complex and of the canonical Wnt pathway, regulates proliferation, adhesion, and migration in different cell types. ß-catenin and components of the Wnt canonical pathway are commonly overexpressed in GBM. Here, we review previous work on the role of Wnt/ß-catenin signalling in glioma initiation, proliferation, and invasion. Understanding the molecular mechanisms regulating GIC biology and glioma progression may help in identifying novel therapeutic targets for GBM treatment.