Loop-mediated isothermal amplification assay for the detection of Plasmopara viticola infecting grapes

Grapes downy mildew caused by obligate oomycete plant pathogen Plasmopara viticola is a devastating disease worldwide, resulting in significant yield and quality losses. A field survey was conducted in two major grapes cultivated areas of Tamil Nadu for the incidence of grapevine downy mildew. The disease incidence was 43.42%–76.69%, and the highest disease incidence of 76.69% was observed in the Theni district. Totally eight P. viticola isolates were collected from different places in Coimbatore and Theni districts. These isolates were confirmed through microscopic observation and sequencing of COX 2 gene, and the phylogenetic tree was developed to study their phylogenetic relationship among the isolates which shows 97–100% sequence similarity with other P. viticola isolates and less sequence similarity with Plasmopara species. The loop-mediated isothermal amplification (LAMP) assay was developed based on the CesA4 gene sequence of P. viticola. The assay developed was more sensitive as it detected P. viticola genomic DNA up to 20 fmg. LAMP assay specificity was proved by carrying out the assay with genomic DNA extracted from other Oomycetes and fungal plant pathogens. Finally, LAMP assay was validated by testing seventy-eight grapevine leaf samples collected from seven different locations. LAMP assay showed a positive reaction in sixty-two samples tested out of seventy-eight samples tested. Therefore, the LAMP assay described should helpful for early and specific detection of downy mildew pathogen and help in mitigating disease incidence.     

Analysis of Human Blood Plasma Proteome from Ten Healthy Volunteers from Indian Population

Analysis of any mammalian plasma proteome is a challenge, particularly by mass spectrometry, due to the presence of albumin and other abundant proteins which can mask the detection of low abundant proteins. As detection of human plasma proteins is valuable in diagnostics, exploring various workflows with minimal fractionation prior to mass spectral analysis, is required in order to study population diversity involving analysis in a large cohort of samples. Here, we used ‘reference plasma sample’, a pool of plasma from 10 healthy individuals from Indian population in the age group of 25–60 yrs including 5 males and 5 females. The 14 abundant proteins were immunodepleted from plasma and then evaluated by three different workflows for proteome analysis using a nanoflow reverse phase liquid chromatography system coupled to a LTQ Orbitrap Velos mass spectrometer. The analysis of reference plasma sample a) without prefractionation, b) after prefractionation at peptide level by strong cation exchange chromatography and c) after prefractionation at protein level by sodium dodecyl sulfate polyacrylamide gel electrophoresis, led to the identification of 194, 251 and 342 proteins respectively. Together, a comprehensive dataset of 517 unique proteins was achieved from all the three workflows, including 271 proteins with high confidence identified by≥2 unique peptides in any of the workflows or identified by single peptide in any of the two workflows. A total of 70 proteins were common in all the three workflows. Some of the proteins were unique to our study and could be specific to Indian population. The high-confidence dataset obtained from our study may be useful for studying the population diversity, in discovery and validation process for biomarker identification.     

A Phylogenetic Analysis of the L1 Family of Neural Cell Adhesion Molecules

L1-type genes form one of several distinct gene families that encode adhesive proteins, which are predominantly expressed in developing and mature metazoan nervous systems. These proteins have a multitude of different important cellular functions in neuronal and glial cells. L1-type gene products are transmembrane proteins with a characteristic extracellular domain structure consisting of six immunoglobulin and three to five fibronectin type III protein folds. As reported here, L1-type proteins can be identified in most metazoan phyla with the notable exception of Porifera (sponges). This puts the origin of L1-type genes at a point in time when primitive cellular neural networks emerged, approximately 1,200 to 1,500 million years ago. Subsequently, several independent gene duplication events generated multiple paralogous L1-type genes in some phyla, allowing for a considerable diversification of L1 structures and the emergence of new functional features and molecular interactions. One such evolutionary newer feature is the appearance of RGD integrin-binding motifs in some vertebrate L1 family members.     

Statistical Mechanical Approach for Predicting the Transition to Non-B DNA Structures in Supercoiled DNA

Abstract

Supercoiling causes global twist of DNA structure and the supercoiled state has wide influence on conformational transition. A statistical mechanical approach was made for prediction of the transition probability to non-B DNA structures under torsional stress. A conditional partition function was defined as the sum over all possible states of the DNA sequence with basepair 1 and basepair n being in B-form helix and a recurrence formula was developed which expressed the partition function for basepair n with those for less number of pairs. This new definition permits a quick enumeration of every configuration of secondary structures. Energetic parameters of all conformations concerned, involving B-form, interior loop, cruciform and Z-form, were included in the equation. The probability of transition to each non-B conformation could be derived from these conditional partition functions. For treatment of effects of superhelicity, supercoiling energy was considered, and a twist of each conformation was determined to minimize the supercoiling energy. As the twist itself affects the transition probability, the whole scheme of equations was solved by renormalization technique. The present method permits a simultaneous treatment of serveral types of conformations under a common torsional stress.

A set of energetic parameters of DNA secondary structures has been chosen for calculation. Some DNA sequences were submitted to the calculation, and all the sequences that we submitted gave stable convergence. Some of them have been investigated the critical supercoil density for the transition to non-B DNA structures. Even though the reliability of the set of parameters was not enough, the prediction of secondary structure transition showed good agreement with reported observation. Hence, the present algorithm can estimate the probability of local conformational change of DNA under a given supercoil density, and also be employed to predict some specific sequences in which conformational change is sensitive to superhelicity.     

The combined effect of acetylation and glycation on the chaperone and anti-apoptotic functions of human α-crystallin

N^ε-acetylation occurs on select lysine residues in α-crystallin of the human lens and alters its chaperone function. In this study, we investigated the effect of N^ε-acetylation on advanced glycation end product (AGE) formation and consequences of the combined N^ε-acetylation and AGE formation on the function of α-crystallin. Immunoprecipitation experiments revealed that N^ε-acetylation of lysine residues and AGE formation co-occurs in both αA- and αB-crystallin of the human lens. Prior acetylation of αA- and αB-crystallin with acetic anhydride (Ac₂O) before glycation with methylglyoxal (MGO) resulted in significant inhibition of the synthesis of two AGEs, hydroimidazolone (HI) and argpyrimidine. Similarly, synthesis of ascorbate-derived AGEs, pentosidine and N^ε-carboxymethyl lysine (CML), was inhibited in both proteins by prior acetylation. In all cases, inhibition of AGE synthesis was positively related to the degree of acetylation. While prior acetylation further increased the chaperone activity of MGO-glycated αA-crystallin, it inhibited the loss of chaperone activity by ascorbate-glycation in both proteins. BioPORTER-mediated transfer of αA- and αB-crystallin into CHO cells resulted in significant protection against hyperthermia-induced apoptosis. This effect was enhanced in acetylated and MGO-modified αA- and αB-crystallin. Caspase-3 activity was reduced in α-crystallin transferred cells. Glycation of acetylated proteins with either MGO or ascorbate produced no significant change in the anti-apoptotic function. Collectively, these data demonstrate that lysine acetylation and AGE formation can occur concurrently in α-crystallin of human lens, and that lysine acetylation improves anti-apoptotic function of α-crystallin and prevents ascorbate-mediated loss of chaperone function.     

A Simulation Optimization Approach to Epidemic Forecasting

Reliable forecasts of influenza can aid in the control of both seasonal and pandemic outbreaks. We introduce a simulation optimization (SIMOP) approach for forecasting the influenza epidemic curve. This study represents the final step of a project aimed at using a combination of simulation, classification, statistical and optimization techniques to forecast the epidemic curve and infer underlying model parameters during an influenza outbreak. The SIMOP procedure combines an individual-based model and the Nelder-Mead simplex optimization method. The method is used to forecast epidemics simulated over synthetic social networks representing Montgomery County in Virginia, Miami, Seattle and surrounding metropolitan regions. The results are presented for the first four weeks. Depending on the synthetic network, the peak time could be predicted within a 95% CI as early as seven weeks before the actual peak. The peak infected and total infected were also accurately forecasted for Montgomery County in Virginia within the forecasting period. Forecasting of the epidemic curve for both seasonal and pandemic influenza outbreaks is a complex problem, however this is a preliminary step and the results suggest that more can be achieved in this area.     

Sestrin2 regulates monocyte activation through AMPK-mTOR nexus under high-glucose and dyslipidemic conditions

The vicious cycle between hyperinsulinemia and insulin resistance results in the progression of atherosclerosis in the vessel wall. The complex interaction between hyperglycemia and lipoprotein abnormalities promotes the development of atherogenesis. In the early phase of atherosclerosis, macrophage-derived foam cells play an important role in vascular remodeling. Mechanistic target of rapamycin (mTOR) signaling pathway has been identified to play an essential role in the initiation, progression, and complication of atherosclerosis. Recently sestrin2, an antioxidant, was shown to modulate TOR activity and thereby regulating glucose and lipid metabolism. But the role of sestrin2 in monocyte activation is still not clearly understood. Hence, this study is focussed on investigating the role of sestrin2 in monocyte activation under hyperglycemic and dyslipidemic conditions. High-glucose and oxidized low-density lipoprotein (LDL) treatments mediated proinflammatory cytokine production (M1) with a concomitant decrease in the anti-inflammatory cytokine (M2) levels in human monocytic THP1 cells. Both glucose and oxidized LDL (OxLDL) in a dose and time-dependent manner increased the mTOR activation with a marked reduction in the levels of pAMPK and sestrin2 expression. Both high-glucose and OxLDL treatment increased foam cell formation and adhesion of THP1 cells to endothelial cells. Experiments employing activator or inhibitor of adenosine monophosphate kinase (AMPK) as well as overexpression or silencing of sestrin2 indicated that high-glucose mediated monocyte polarization and adhesion of monocytes to the endothelial cells were appeared to be programmed via sestrin2-AMPK-mTOR nexus. Our results evidently suggest that sestrin2 plays a major role in regulating monocyte activation via the AMPK–mTOR-pathway under diabetic and dyslipidemic conditions and also AMPK regulates sestrin2 in a feedback mechanism.