Sodium Phenylbutyrate and Edaravone Abrogate Chronic Restraint Stress-Induced Behavioral Deficits: Implication of Oxido-Nitrosative,Endoplasmic Reticulum Stress Cascade,and Neuroinflammation

Chronic stress exposure can produce deleterious effects on the hippocampus (HC) which eventually leads to cognitive impairment and depression. Endoplasmic reticulum (ER) stress has been reported as one of the major culprits in the development of stress-induced cognitive impairment and depression. We investigated the neuroprotective efficacy of sodium phenylbutyrate (SPB), an ER stress inhibitor, and edaravone, a free radical scavenger, against chronic restraint stress (CRS)-induced cognitive deficits and anxiety- and depressive-like behavior in mice. Adult male Swiss albino mice were restrained for 6 h/day for 28 days and injected (i.p.) with SPB (40 and 120 mg/kg) or edaravone (3 and 10 mg/kg) for the last seven days. After stress cessation, the anxiety- and depressive-like behavior along with spatial learning and memory were examined. Furthermore, oxido-nitrosative stress, proinflammatory cytokines, and gene expression level of ER stress-related genes were assessed in HC and prefrontal cortex (PFC). CRS-exposed mice showed anxiety- and depressive-like behavior, which was significantly improved by SPB and edaravone treatment. In addition, SPB and edaravone treatment significantly alleviated CRS-induced spatial learning and memory impairment. Furthermore, CRS-evoked oxido-nitrosative stress, neuroinflammation, and depletion of Brain-derived neurotrophic factor were significantly ameliorated by SPB and edaravone treatment. We found significant up-regulation of ER stress-related genes in both HC and PFC regions, which were suppressed by SPB and edaravone treatment in CRS mice. Our study provides evidence that SPB and edaravone exerted neuroprotective effects on CRS-induced cognitive deficits and anxiety- and depressive-like behavior, which is possibly coupled with inhibition of oxido-nitrosative stress, neuroinflammation, and ER stress cascade.     

Interference of N-hydroxysuccinimide with bicinchoninic acid protein assay

We report here substantial interference from N-hydroxysuccinimide (NHS) in the bicinchoninic acid (BCA) protein assay. NHS is one of the most commonly used crosslinking agents in bioanalytical sciences, which can lead to serious potential errors in the BCA protein assay based protein estimation if it is present in the protein analyte solution. It was identified to be a reducing substance, which interferes with the BCA protein assay by reducing Cu²⁺ in the BCA working reagent. The absorbance peak and absorbance signal of NHS were very similar to those of bovine serum albumin (BSA), thereby indicating a similar BCA reaction mechanism for NHS and protein. However, the combined absorbance of NHS and BSA was not additive. The time–response measurements of the BCA protein assay showed consistent single-phase kinetics for NHS and gradually decreasing kinetics for BSA. The error in protein estimation due to the presence of NHS was counteracted effectively by plotting additional BCA standard curve for BSA with a fixed concentration of NHS. The difference between the absorbance values of BSA and BSA with a fixed NHS concentration provided the absorbance contributed by NHS, which was then subtracted from the total absorbance of analyte sample to determine the actual absorbance of protein in the analyte sample.     

Assessment of Polycyclic Aromatic Hydrocarbons (PAH) and Heavy Metals in the Vicinity of an Oil Refinery in India

Petroleum products are one of the major sources of energy for industry and daily life. Growth of the petroleum industry and shipping of petroleum products has resulted in the pollution. Populations living in the vicinity of oil refinery waste sites may be at greater risk of potential exposure to polycyclic aromatic hydrocarbons (PAH) through inhalation, ingestion, and direct contact with contaminated media. PAH have often been found to coexist with environmental pollutants including heavy metals due to similar pollution sources. The levels and distribution patterns of Σ16 PAH (sum of the 16 PAH) and heavy metals (lead, copper, nickel, cobalt, and chromium) were determined in soil and sediment in the vicinity (5 km radius) of an oil refinery in India. Concentrations of Σ16 PAH in the soils and sediments were found to be 60.36 and 241.23 ppm, respectively. Higher amount of PAH in sediments as compared to soil is due to low water solubility of PAH, settled in the bottom of aquatic bodies. The levels of lead, copper, nickel, cobalt, and chromium (total) in soil were 12.52, 13.52, 18.78, 4.84, and 8.29 ppm, while the concentrations of these metals in sediments were 16.38, 47.88, 50.15, 7.07, and 13.25 ppm, respectively. Molecular diagnostics indices of PAH (Ratio of Phenanthrene/Anthracene, Fluranthene/Pyrene) calculated for soil and sediment samples indicate that the oil refinery environment is contaminated with PAH from petrogenic as well as pyrolytic origin and heavy vehicular traffic on the Agra- Delhi National highway. Sixteen PAH priority pollutants were detected in the United States in entire samples collected near oil refinery areas and concentrations of Σ16 PAH in soil was found to be 1.20 times higher than the threshold value for PAH in soil by ICRCL (Inter-Departmental Committee on the Redevelopment of Contaminated Land). This concentration could lead to disastrous consequences for the biotic and abiotic components of the ecosystem and may affect the soil quality, thus impairing plant growth and its bioaccumulation in food chain.     

Investigation on the role of p53 codon 72 polymorphism and interactions with tobacco, betel quid, and alcohol in susceptibility to cancers in a high-risk population from North East India

The association of TP53 codon 72 polymorphism with cancer susceptibility remains uncertain and varies with ethnicity. Northeast India represents a geographically, culturally, and ethnically isolated population. The area reports high rate of tobacco usage in a variety of ways of consumption, compared with the rest of Indian population. A total of 411 cancer patients (161 lung, 134 gastric, and 116 oral) and 282 normal controls from the ethnic population were analyzed for p53 codon 72 polymorphism by polymerase chain reaction-restriction fragment length polymorphism. No significant difference in genotypic distribution of p53 between cases and controls was observed. Results suggested betel quid chewing as a major risk factor for all the three cancers (odds ratio [OR]=3.54, confidence interval [CI]=2.01-6.25, p<0.001; OR=1.74, CI=1.04-2.92, p=0.03; and OR=1.85, CI=1.02-3.33, p=0.04 for lung, gastric, and oral cancers, respectively). Tobacco smoking was associated with risk of lung and oral cancers (OR=1.88, CI=1.11-3.19, p=0.01 and OR=1.68, CI=1.00-2.81, p=0.04). Interactions between p53 genotypes and risk factors were analyzed to look for gene-environment interactions. Interaction of smoking and p53 genotype was significant only for oral cancer. Interactions of betel quid with p53 genotypes in lung cancer showed significant increase for all the three genotypes, indicating a major role of betel quid (OR=5.90, CI=1.67-20.81, p=0.006; OR=5.44, CI=1.67-17.75, p=0.005; and OR=5.84, CI=1.70-19.97, p=0.005 for Arg/Arg, Arg/Pro, and Pro/Pro, respectively). In conclusion, high incidence of these cancers in northeast India might be an outcome of risk habits; further, tissue- and carcinogen-specific risk modification by p53 gene is probable.     

N-terminal myristoylation alters the calcium binding pathways in neuronal calcium sensor-1

Neuronal calcium sensor-1 (NCS-1) interacts with many membranes and cytosolic proteins, both in a Ca²⁺-dependent and in a Ca₂₊-independent manner, and its physiological role is governed by its N-terminal myristoylation. To understand the role of myristoylation in altering Ca²⁺ response and other basic biophysical properties, we have characterized the Ca²⁺ filling pathways in both myristoylated (myr) and non-myristoylated (non-myr) forms of NCS-1. We have observed that Ca²⁺ binds simultaneously to all three active EF-hands in non-myr NCS-1, whereas in the case of myr NCS-1, the process is sequential, where the second EF-hand is filled first, followed by the third and fourth EF-hands. In the case of myr NCS-1, the observed sequential Ca²⁺ binding process becomes more prominent in the presence of Mg²⁺. Besides, the analysis of ¹⁵N-relaxation data reveals that non-myr NCS-1 is more dynamic than myr NCS-1. The overall molecular tumbling correlation time increases by approximately 20% upon myristoylation. Comparing the apo forms of non-myr NCS-1 and myr NCS-1, we found the possibility of existence of some substates, which are structurally closer to the holo form of the protein. There are more such substates in the case of non-myr NCS-1 than in the case of the myr NCS-1, suggesting that the former accesses larger volumes of conformational substates compared with the latter. Further, the study reveals that the possibility of Ca²⁺ binding simultaneously to different parts of the protein is more favourable in non-myr NCS-1 than in myr NCS-1.     

Structural analysis of protein tyrosine phosphatase 1B reveals potentially druggable allosteric binding sites

Catalytic proteins such as human protein tyrosine phosphatase 1B (PTP1B), with conserved and highly polar active sites, warrant the discovery of druggable nonactive sites, such as allosteric sites, and potentially, therapeutic small molecules that can bind to these sites. Catalyzing the dephosphorylation of numerous substrates, PTP1B is physiologically important in intracellular signal transduction pathways in diverse cell types and tissues. Aberrant PTP1B is associated with obesity, diabetes, cancers, and neurodegenerative disorders. Utilizing clustering methods (based on root mean square deviation, principal component analysis, nonnegative matrix factorization, and independent component analysis), we have examined multiple PTP1B structures. Using the resulting representative structures in different conformational states, we determined consensus clustroids and used them to identify both known and novel binding sites, some of which are potentially allosteric. We report several lead compounds that could potentially bind to the novel PTP1B binding sites and can be further optimized. Considering the possibility for drug repurposing, we discovered homologous binding sites in other proteins, with ligands that could potentially bind to the novel PTP1B binding sites.     

A Peptide Based Pro-drug Disrupts Alzheimer’s Amyloid into Non-toxic Species and Reduces Aβ Induced Toxicity In Vitro

Aggregation of Amyloid β (Aβ) in the interneuronal spaces is a major etiopathological factor for onset and progression of Alzheimer’s disease (AD). Since the mechanism of aggregation is not fully understood, control and modulation of the aggregation process is a challenging task. Although, several strategies were developed for the past few decades, yet there is no proper therapeutics available. Herein, we report a peptide based pro-drug, termed as a conformational Pro-Drug peptide (PDp), which disrupts existing Aβ fibrils, but does not produce toxic soluble oligomers, through a series of spontaneous chemical reactions resulting in in situ generation of β-sheet destabilizing factors. Furthermore, PDp reduces Aβ mediated toxicity examined on an in vitro model consisting of the human neuroblastoma SH-SY5Y cells. PDp also disrupts fibrils originated from AD affected human cerebrospinal fluid. These findings will help to understand the process of amyloidogenesis better and also indicate a novel approach for therapeutically important peptide design.     

Performance comparison of four exome capture systems for deep sequencing

Background

Recent developments in deep (next-generation) sequencing technologies are significantly impacting medical research. The global analysis of protein coding regions in genomes of interest by whole exome sequencing is a widely used application. Many technologies for exome capture are commercially available; here we compare the performance of four of them: NimbleGen’s SeqCap EZ v3.0, Agilent’s SureSelect v4.0, Illumina’s TruSeq Exome, and Illumina’s Nextera Exome, all applied to the same human tumor DNA sample.

Results

Each capture technology was evaluated for its coverage of different exome databases, target coverage efficiency, GC bias, sensitivity in single nucleotide variant detection, sensitivity in small indel detection, and technical reproducibility. In general, all technologies performed well; however, our data demonstrated small, but consistent differences between the four capture technologies. Illumina technologies cover more bases in coding and untranslated regions. Furthermore, whereas most of the technologies provide reduced coverage in regions with low or high GC content, the Nextera technology tends to bias towards target regions with high GC content.

Conclusions

We show key differences in performance between the four technologies. Our data should help researchers who are planning exome sequencing to select appropriate exome capture technology for their particular application.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-449) contains supplementary material, which is available to authorized users.