Transcriptomics and Proteomics Approach for the Identification of Altered Blood microRNAs and Plasma Proteins in Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder caused by the selective destruction of dopaminergic neurons (DA-nergic). Clinically, PD is diagnosed based on developing signs and symptoms. A neurological and physical examination and sometimes medical and family history also help in the diagnosis of PD. However, most of these features are visible when more than 80% of the dopaminergic neurons have degenerated. An understanding of the selective degeneration process at the cellular and molecular level and the development of new biomarkers are required for effective PD management. Several studies have been carried out using a selected set of miRNAs/ mRNAs and proteins to develop biomarkers of PD; however, an unbiased and combined miRNA-protein profiling study was required to identify the markers of progressive and selected degeneration of dopaminergic neurons in PD patients. In the present study, we have carried out global protein profiling through LC-MS/MS and miRNA profiling by using a "brain-specific" miRNA array panel of 112 miRNAs in PD patients and healthy controls to find the unprejudiced group of proteins and miRNAs that are deregulating in PD. In the whole blood samples of PD patients compared to healthy controls, the expression of 23 miRNAs and 289 proteins was significantly increased, whereas the expression of 4 miRNAs and 132 proteins was considerably downregulated. Network analysis, functional enrichment, annotation, and analysis of miRNA-protein interactions were also performed as part of the bioinformatics investigation of the discovered miRNAs and proteins revealing several pathways that lead to PD development and pathogenesis. Based on the analysis of miRNA and protein profiling, we have identified four miRNAs (hsa-miR-186-5p, miR-29b, miR-139 & has-miR-150-5p) and four proteins (YWHAZ, PSMA4, HYOU1, & SERPINA1), which can be targeted for the development of new biomarkers of PD. In vitro studies have identified the role of miR-186-5p in regulating the levels of the YWHAZ/YWHAB & CALM2 gene, which has shown maximum downregulation in PD patients and is known for its role in neuroprotection from apoptotic cell death & calcium regulation. In conclusion, our research has identified a group of miRNA-proteins that can be developed as PD biomarkers; however, future studies on the release of these miRNAs and proteins in extracellular vesicles circulating in the blood of PD patients can further validate these as specific biomarkers of PD.

Interaction of cytochrome P450s with environmental risk factors increases their expression and risk to head and neck cancer.

To investigate the role of interaction of tobacco metabolizing polymorphic cytochrome P450s (CYPs) and glutathione S-transferase M1 (GSTM1) with environmental risk factors in modifying the susceptibility to head and neck squamous cell carcinoma (HNSCC), a case-control study with 1250 proven cases of HNSCC and equal number of healthy controls was planned. A small but significant increase in the risk to HNSCC (1-2 fold) in the cases with variant genotypes of CYPs (1A1 or 1B1 or 2E1) increased several folds (up to 13 fold) in regular tobacco or alcohol users. This several fold increase in risk could be due to more than multiplicative interaction observed between the risk genotypes of CYPs and tobacco or alcohol. A synergistic effect was also observed between tobacco as well as alcohol users among cases with risk genotypes of CYPs and GSTM1 that resulted in a further increase in risk (up to 29 fold) to HNSCC. Interestingly, the increase in the risk in tobacco users among cases with variant genotypes of CYPs or a combination of CYPs & GSTM1 (-) was associated with a higher mRNA expression of CYPs when compared to nontobacco users in controls with wild type of genotypes of CYPs & GSTM1. The data suggest that the interaction of genetic and environmental risk factors leads to increased expression of CYPs which may increase the levels of tobacco-derived carcinogens thereby modifying the risk to HNSCC.

Identification of Altered Blood MicroRNAs and Plasma Proteins in a Rat Model of Parkinson's Disease.

Parkinson's disease (PD) is the age-related neurological disorder characterized by the degeneration of dopamine (DA) neurons in the substantia nigra pars compacta (SNpc). PD is based on motor deficits which start to appear when up to 80% of the DA neurons of SNpc have been lost. Effective management of PD requires the development of novel biomarkers. Therefore, the present study aimed to characterize biomarkers of PD using miRNomics, proteomics, and bioinformatics approaches. Rats exposed to rotenone (2.5 mg/kg b.wt) for 2 months were used as an animal model to identify the unbiased set of miRNAs and proteins deregulated in blood samples. OpenArray, a real-time PCR-based array, is used for high-throughput profiling of miRNAs, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to carry out the global protein profiling. Systematic bioinformatics analysis of miRNAs and proteins was also performed, including annotation, functional classification and functional enrichment, network analysis, and miRNA-protein interaction analysis. Expression of 19 miRNAs and 96 proteins was significantly upregulated in the blood, while 22 proteins were significantly downregulated in blood samples of rotenone-exposed rats. In silico pathway analysis of deregulated proteins and miRNAs in rotenone-exposed rats has identified multiple pathways leading to PD. In summary, we have identified a set of miRNAs (miR-144, miR-96, and miR-29a) and proteins (PLP1, TUBB4A, and TUBA1C), which can be used as a potential biomarker of PD, while further validation required large human population studies.

Studies on Regulation of Global Protein Profile and Cellular Bioenergetics of Differentiating SH-SY5Y Cells.

The SH-SY5Y cells differentiated by sequential exposure of retinoic acid (RA) and brain-derived neurotrophic growth factor (BDNF) are a well-employed cellular model for studying the mechanistic aspects of neural development and neurodegeneration. Earlier studies from our lab have identified dramatic upregulation (77 miRNAs) and downregulation (17 miRNAs) of miRNAs in SH-SY5Y cells differentiated with successive exposure of RA + BDNF and demonstrated the essential role of increased levels of P53 proteins in coping with the differentiation-induced changes in protein levels. In continuation to our earlier studies, we have performed unbiased LC-MS/MS global protein profiling of naïve and differentiated SH-SY5Y cells and analyzed the identified proteins in reference to miRNAs identified in our earlier studies to identify the cellular events regulated by both identified miRNAs and proteins. Analysis of LC-MS/MS data has shown a significant increase and decrease in levels of 215 and 163 proteins, respectively, in differentiated SH-SY5Y cells. Integrative analysis of miRNA identified in our previous studies and protein identified in the present study is carried out to discover novel miRNA-protein regulatory modules to elucidate miRNA-protein regulatory relationships of differentiating neurons. In silico network analysis of miRNAs and proteins deregulated upon SH-SY5Y differentiation identified cell cycle, synapse formation, axonogenesis, differentiation, neuron projection, and neurotransmission, as the topmost involved pathways. Further, measuring mitochondrial dynamics and cellular bioenergetics using qPCR and Seahorse XFp Flux Analyzer, respectively, showed that differentiated cells possess increased mitochondrial dynamics and OCR relative to undifferentiated cells. In summary, our studies have identified a novel set of proteins deregulated during neuronal differentiation and establish the role of miRNAs identified in earlier studies in the regulation of proteins identified by LC-MS/MS-based global profiling of differentiating neurons, which will help in future studies related to neural development and neurodegeneration.

Polymorphism in cytochrome P4502A6 reduces the risk to head and neck cancer and modifies the treatment outcome.

The present case-control study consisting of 1300 cases of head and neck squamous cell carcinoma (HNSCC) and the equal number of controls aimed to investigate the association of functionally important polymorphisms in cytochrome P4502A6 (CYP2A6*1B, CYP2A6*4C, CYP2A6*9-rs28399433) with HNSCC and the treatment response in cases receiving a combination of chemotherapy/radiotherapy (CT/RT). A significant decrease in risk to HNSCC was observed in the cases with deletion (CYP2A6*4B and CYP2A6*4C) or reduced activity genotypes (CYP2A6*9) of CYP2A6. This risk to HNSCC was further reduced significantly in tobacco users among the cases when compared to nontobacco users among the cases. The risk was also reduced to a slightly greater extent in alcohol users among the cases when compared to nonalcohol users among the cases. In contrast with decreased risk to HNSCC, almost half of the cases with variant genotypes of CYP2A6 (CYP2A6*1A/*4C+*1B/*4C+*4C/*4C and *9/*9) did not respond to the treatment. Likewise, the survival rate in cases receiving the treatment, after 55 months of follow-up was significantly lower in cases with deletion (6.3%) or reduced activity (11.9%) allele than in the cases with common alleles (41%). The present study has shown that CYP2A6 polymorphism significantly reduces the risk to HNSCC. Our data further suggested that CYP2A6 polymorphism may worsen the treatment outcome in the cases receiving CT/RT.

Expression of Antimicrobial Peptides and Cytokines in Human Omentum Following Abdominal Surgery.

Introduction Omentum can secrete out biological agents like different growth factors, cytokines, and antimicrobial peptides. The aim of our study was to determine the expression of antimicrobial peptides and cytokines in human omentum tissue and its response to intra-abdominal infection. Methodology Omentum tissue was obtained from 60 patients: control (n=20) and cases (n=40). mRNA expression of antimicrobial peptides (LL-37, HBD-1, HBD-2, HNP1-3) and cytokines (TNF- α, IL-8, IL-10, IL1ß) was evaluated using Real-Time PCR. Protein quantification was done by Immunoblotting and ELISA. Results Significantly higher expression of antimicrobial peptides (LL-37, HBD-1, HBD-2, HNP1-3) and cytokines (TNF- α, IL-8, IL-10, IL1ß) was observed in cases as compared to control at both the transcriptional and translational level (p<0.0001). Conclusion Omentum governs a population of antimicrobial peptides with potent immunologic functions. The expression of antimicrobial peptides and cytokines is inducible and increases with the severity of infection. Omentum is thus an immunologically active and adaptable organ but its complete regulatory mechanism is still elusive.

An update on the diagnostic biomarkers for asthma.

Asthma is a respiratory disorder accounts for ~339 million cases per annum. The initial diagnosis of asthma relies on the symptomatic identification of characters, such as wheeze, shortness of breath, chest tightness, and cough. The presence of two or more of these symptoms may be considered as indicative of asthma. The asthma-diagnostic also involves spirometry test before and after inhaling a bronchodilator like albuterol. Because asthma pathophysiology involves participation of immune system, the cytokines play an important role. The review discusses various molecules that are or may be used as biomarkers for the asthma diagnosis.

Waste candle soot derived carbon nanoparticles: A competent alternative for the management of Helicoverpaarmigera.

Helicoverpaarmigera (Lepidoptera: Noctuidae) is considered as one of the foremost pests of global agriculture. This pest is contemplated for substantial economic loss apart from the socio-economic and environmental costs associated with its control. Farmers adopt several strategies for the control of this pest but the cost associated with these strategies is always a big question. This is the first time when waste-candle soot (CS) derived carbon nanoparticles (CNPs) are explored for the putative toxicity to H. armigera. In the present study, the entomotoxic effects of CNPs on H. armigera were investigated and compared with that of commercially available multi-walled carbon nanotubes (MWCNTs). Larvae fed on both the nanomaterials exhibited significant weight reduction and enhanced levels of antioxidant enzymes. Moths developed from the treated larvae exhibit very poor egg-laying capacity and poor egg hatchability. However, these entomotoxic effects were found more noticeable in larvae and moths fed on CNPs that eventually led to the complete cessation of the population build-up of H. armigera. These findings advocate the candidature of CNPs as a cost-effective alternative for efficient control of H. armigera in pest management programs.

A combined microRNA and proteome profiling to investigate the effect of ZnO nanoparticles on neuronal cells.

Zinc oxide nanoparticles (ZnO NPs) are one of the most broadly used engineered nanomaterials. The toxicity potential of ZnO NPs has been explored in several studies; however, its neurotoxicity, especially its molecular mechanism, has not been studied in depth. In this study, we have used a cellular model of neuronal differentiation (nerve growth factor differentiated PC12 cells) to compare the effect of ZnO NPs exposure on neuronal (differentiated or mature neurons) and non-neuronal (undifferentiated) cells. Our studies have shown that the noncytotoxic concentration of ZnO NPs causes neurite shortening and degeneration in differentiated PC12 cells. Brain-specific microRNA (miRNA) array and liquid chromatography with tandem mass spectrometry (LC-MS/MS) are used to carry out profiling of miRNAs and proteins in PC12 cells exposed with ZnO NPs. Exposure of ZnO NPs produced significant deregulation of a higher number of miRNAs (15) and proteins (267) in neuronal cells in comparison to miRNAs (8) and proteins (207) of non-neuronal cells (8). In silico pathway analysis of miRNAs and proteins deregulated in ZnO NPs exposed differentiated PC12 cells have shown pathways leading to neurodegenerative diseases and mitochondrial dysfunctions are primarily targeted pathways. Further, a bioenergetics study carried out using Seahorse XFp metabolic flux analyzer has confirmed the involvement of mitochondrial dysfunctions in ZnO NPs exposed differentiated PC12 cells. In conclusion, differentiated PC12 cells (neuronal) were found more vulnerable than undifferentiated (non-neuronal PC12 cells) toward the exposure of ZnO NPs and deregulation of miRNAs and mitochondrial dysfunctions play a significant role in its toxicity.

Interaction of glutathione-s-transferase genotypes with environmental risk factors in determining susceptibility to head and neck cancer and treatment response and survival outcome.

The present case-control study aimed to investigate the role of interaction of glutathione-s-transferase (GST) genotypes with environmental risk factors in determining susceptibility to head and neck squamous cell carcinoma (HNSCC) involving 1,250 cases and equal number of healthy controls. An increase in the risk of HNSCC and its subsites (larynx, pharynx, and oral cavity) was observed among the cases with null genotypes of GSTM1 (odds ratio [OR] = 1.87) or GSTT1 (OR = 1.39) while reduced risk (OR = 0.81) was observed the cases with variant genotype of GSTP1. Tobacco use in the form of smoking or chewing interacted multiplicatively with GSTM1 or GSTT1 to increase the risk several folds (3-10 folds) in HNSCC and its subsites. Alcohol use also increased the risk (2-3 folds) to HNSCC and its subsites in cases with null or variant genotypes of GSTs, though this risk was of lesser magnitude when compared to the tobacco users. A synergistic effect of both, tobacco smoking and alcohol drinking, led to several folds (25-folds) increased risk to HNSCC among the cases with null genotype of GSTM1 and GSTT1 when compared to nonsmokers and nondrinkers with wild genotype of GSTM1 and GSTT1 in controls. Furthermore, cases with variant genotypes of GSTP1 (Val/Val) showed superior treatment response with improved survival rate and lower risk of death when compared to the patients with wild type genotype (Ile/Ile). The data suggest that though polymorphism in GSTs may be a modest risk factor for determining HNSCC risk, gene-environment interactions significantly modify the susceptibility to HNSCC by several folds.

Prospective evaluation of XRCC-1 Arg194Trp polymorphism as bio-predictor for clinical outcome in locally advanced laryngeal cancer undergoing cisplatin-based chemoradiation.

BACKGROUND: To determine X-ray repair cross-complementing 1 gene (XRCC-1) Arg194Trp polymorphism as bio-predictor for clinical outcome in advanced laryngeal squamous cell carcinoma undergoing cisplatin-based chemoradiation (CRT). METHODS: A total of 150 patients were enrolled in this prospective study. XRCC-1 Arg194Trp genotyping categorized patients as wild (C/C) and polymorphic (C/T or T/T). The primary endpoint was to assess acute radiation-induced toxicity (ARIT). RESULTS: A significant correlation of skin (P- .04) and oral mucosal ARIT (P- .01) was noticed in the XRCC-1 polymorphic variant. A higher treatment response was noted in the polymorphic variant, and it shows a trend toward significance (P- .08). With 33 months of median follow-up, 2-year progression-free survival (PFS) and overall survival (OS) of wild vs polymorphic variant were 34.6% vs 46.9% (P- .066) and 50.6% vs 62.2% (P- .12). CONCLUSION: XRCC-1 polymorphic variants have significantly higher grade of >2 ARIT and may have improved trend for treatment response and PFS.

Validation of gene expression profiles of candidate genes using low density array in peripheral blood of tobacco consuming head and neck cancer patients and auto/taxi drivers with preneoplastic lesions.

TaqMan Low-Density Array (TLDA) based Real-Time PCR (RT-PCR) of selected genes showed increased expression of polycyclic aromatic hydrocarbons (PAHs) metabolizing cytochrome P450s (CYPs), glutathione S-transferases (GSTs) and associated transcription factors in biopsy and peripheral blood samples isolated from head and neck squamous cell carcinoma (HNSCC) patients when compared to the controls. The genes involved in DNA repair, signal transduction pathway, EMT pathway, apoptosis, and cell adhesion/motility were found to be altered in both peripheral blood and biopsy samples of HNSCC patients. Transcription profiles in blood isolated from auto/taxi drivers, with pre-neoplastic lesions and history of tobacco use, also showed similar alterations. The present TLDA data thus demonstrates that low-density array of selected genes in peripheral blood has the potential to be used as a surrogate for providing insight into cancer progression pathways and possibly as an early biomarker for monitoring tobacco induced HNSCC.

Could excision repair cross-complementing group-1 mRNA expression from peripheral blood lymphocytes predict locoregional failure with cisplatin chemoradiation for locally advanced laryngeal cancer?

AIM: The association of excision repair cross-complementing 1 mRNA (ERCC-1 mRNA) expression with the outcome has been reported with immunohistochemistry (IHC) using tumor tissue in head and neck cancer. We evaluated ERCC-1 mRNA expression by reverse transcription polymerase chain reaction (RT-PCR) from peripheral blood lymphocytes (PBLs) as bio-predictor of locoregional failure (LRF) to chemoradiation (CRT) for locally advanced laryngeal squamous cell cancer (LALSCC). METHODS: A total of 107 male patients with LALSCC were enrolled in this prospective study. ERCC-1 mRNA expression by PBLs was determined by RT-PCR. Definitive CRT was delivered with 35 mg/m2 weekly cisplatin. Response Evaluation Criteria in Solid Tumor 1.1 (RECIST 1.1) were used in evaluating treatment response. The primary objective was to assess LRF. The influence of patient characteristics, treatment response, weekly cisplatin cycles, ERCC mRNA expression was determined for LRF, progression-free survival (PFS) and overall survival (OS). RESULTS: A total of 98 patients completed definitive CRT. The median value of 2-ΔΔCT ERCC-1 mRNA expression was 3.9; based on which it was categorized as low and high. Correlation of ERCC-1 expression with treatment response was insignificant (P- .38). With a median follow-up of 33 months; 2-year LRF, PFS, and OS was 63.3%, 34.7% and 79.4%. The 2-year LRF, PFS and OS for low versus high expression were 53.1% versus 73.5% (P-value = 0.036), 44.9% versus 24.4% (P-value = 0.047) and 81.6% versus 77.2% (P-value = 0.33), respectively. In multivariate analysis, ERCC-1 expression, T-stage, N-stage and tumor subsite are predictive factors for LRF; T-stage and nodal recurrence for OS; stage and treatment response for PFS. CONCLUSION: LALSCC patient with ERCC-1 mRNA low expression was associated with lower LRF rate, and improved PFS.

Antibacterial spectrum of human omentum and differential expression of beta defensins.

BACKGROUND: Human ß defensins (hBD1 and hBD2) are cationic, cysteine-rich peptides and form an integral part of the mammalian innate immune system. hBD1 is constitutively expressed in epithelial cells, whereas hBD2 increases in response to bacterial infection. Human omentum is known for its anti-inflammatory properties and also possesses an antibacterial activity of its own. We hypothesized that antimicrobial peptides, ß defensins, may govern host defense mechanism in the microbe-rich environment of the peritoneal cavity. Therefore, we analyzed the expression of hBD1 and hBD2 in omentum tissue in vivo and also studied the antibacterial activity of omentum against common pathogens. METHODOLOGY: Omentum tissues were obtained from 30 patients (15 cases and 15 controls). Real-time polymerase chain reaction (PCR) was used to evaluate the mRNA expression of hBD1 and hBD2. Protein quantification was done using Western blotting technique. Antibacterial susceptibility was performed to check the antibacterial activity of omentum. RESULT: Significantly higher expression of hBD2 was observed in cases compared to controls at both the transcriptional and translational levels. In comparison with an array of antibiotics, activated omentum also showed antibacterial property even at lower concentration of its extract. CONCLUSION: Omentum directly responds to bacterial infection, which may be due to differential expression of hBD1 and hBD2 in human omental tissue. These peptides (hBD1 and hBD2) may be an ideal candidate for novel antibiotic class with a broad-spectrum activity.

Correction to: Effect of Gestational Exposure of Cypermethrin on Postnatal Development of Brain Cytochrome P450 2D1 and 3A1 and Neurotransmitter Receptors.

The original version of this article unfortunately contained errors in Fig. 4a. Representative image of b-actin of brain region were copied incorrectly during the preparation of the figures.

Correction to: Plasma Fibrinogen Is a Natural Deterrent to Amyloid ß-Induced Platelet Activation and Neuronal Toxicity.

Following publication of the original article [1], the author reported an error in Figure 1. The correct version of Figure 1 is as follows.

A proteomic approach to investigate enhanced responsiveness in rechallenged adult rats prenatally exposed to lindane.

Proteomic analysis was carried out in substantia nigra (SNi) and hippocampus (Hi) isolated from rat offspring born to mothers exposed to lindane (orally; 0.25 mg/kg) from gestation day 5 (GD5) to GD 21 and subsequently rechallenged (orally; 2.5 mg/kg X 21 days) at adulthood (12 weeks). 2D gel electrophoresis revealed no significant differences in the expression of proteins in brain regions isolated from prenatally exposed offspring at adulthood. Significantly greater magnitude of alterations was observed in the expression of proteins related to mitochondrial and energy metabolism, ubiquitin-proteasome pathway, structural and axonal growth leading to increased oxidative stress in Hi and SNi isolated from rechallenged offspring when compared to control offspring treated postnatally with lindane. Western blotting and DNA laddering showed a greater magnitude of increase in apoptosis in the Hi and SNi of rechallenged offspring. Ultrastructural analysis demonstrated disrupted mitochondrial integrity, synaptic disruption and necrotic structures in the brain region of rechallenged offspring. Neurobehavioral studies also demonstrated a greater magnitude of alterations in cognitive and motor functions in rechallenged rats. The data suggest that prenatal exposure of lindane induces persistent molecular changes in the nervous system of offspring which are unmasked leading to neurodegeneration following rechallenge at adulthood.

Correction to: Prenatal Exposure of Cypermethrin Induces Similar Alterations in Xenobiotic-Metabolizing Cytochrome P450s and Rate-Limiting Enzymes of Neurotransmitter Synthesis in Brain Regions of Rat Offsprings During Postnatal Development.

The original version of this article unfortunately contained an error at Fig. 10.

Similarities in mRNA expression of peripheral blood drug metabolizing enzymes and cancer marker genes with biopsy samples of head and neck cancer patients.

Purpose: To develop peripheral blood mRNA expression profiles of drug metabolizing enzymes (DMEs) as a surrogate to monitor tobacco induced head and neck squamous cell carcinoma (HNSCC), attempts were made to investigate (i) similarities in alterations with the cancer marker genes in biopsy samples and (ii) if alterations similar to that seen in biopsy samples are reflected in peripheral blood. Methods: Total RNA from eight soft gingival tissues and eight biopsy samples of HNSCC patients and total DNA and RNA from blood of healthy controls (n = 150) and HNSCC patients (n = 150) was processed for expression and genotyping studies. Blood from patients receiving chemo-radiotherapy was processed for follow-up study. Results: qRT-PCR revealed significant increase in mRNA expression of DMEs in biopsy and blood samples of HNSCC patients when compared to controls. Similar alterations were observed in cancer marker genes in these samples. Patients with variant genotypes of DMEs showed greater magnitude of alterations in mRNA expression when compared to wild type controls. Responders of chemo-radiotherapy showed significant decline in induction of mRNA expression of DMEs and cancer marker genes Conclusions: The data suggest that peripheral blood expression profiles could be used to monitor tobacco-induced HNSCC as well as the treatment response.