Microbiome: A New Lease to Microbiology

Indian Journal of Microbiology -     

Elimination of sugarcane grassy shoot disease through apical meristem culture

Elimination of sugarcane grassy shoot disease (SGSD) through apical meristem culture technique for producing clean planting material of sugarcane has been attempted in the present study. The results showed that meristems length of 2 and 3 mm were free from the SGSD pathogen at higher frequency than larger meristem length of 4 mm. However, the frequency of survival of explants during initiation of shoot cultures was higher in larger meristems (60%) in comparison to smaller ones (40%). The micropropagated plantlets raised from meristem culture were confirmed for disease-free by nested polymerase chain reaction (PCR) analysis at monthly interval up to 6 months. This is the first report on the elimination of SGSD phytoplasma through meristem culture in India.     

Rotenone-induced oxidative stress and apoptosis in human liver HepG2 cells

Rotenone, a commonly used pesticide, is well documented to induce selective degeneration in dopaminergic neurons and motor dysfunction. Such rotenone-induced neurodegenration has been primarily suggested through mitochondria-mediated apoptosis and reactive oxygen species (ROS) generation. But the status of rotenone induced changes in liver, the major metabolic site is poorly investigated. Thus, the present investigation was aimed to study the oxidative stress-induced cytotoxicity and apoptotic cell death in human liver cells-HepG2 receiving experimental exposure of rotenone (12.5–250 μM) for 24 h. Rotenone depicted a dose-dependent cytotoxic response in HepG2 cells. These cytotoxic responses were in concurrence with the markers associated with oxidative stress such as an increase in ROS generation and lipid peroxidation as well as a decrease in the glutathione, catalase, and superoxide dismutase levels. The decrease in mitochondrial membrane potential also confirms the impaired mitochondrial activity. The events of cytotoxicity and oxidative stress were found to be associated with up-regulation in the expressions (mRNA and protein) of pro-apoptotic markers viz., p53, Bax, and caspase-3, and down-regulation of anti-apoptotic marker Bcl-2. The data obtain in this study indicate that rotenone-induced cytotoxicity in HepG2 cells via ROS-induced oxidative stress and mitochondria-mediated apoptosis involving p53, Bax/Bcl-2, and caspase-3.     

The effects of disinfectant foam on microbial biofilms

This investigation examined the effects of common aqueous biocides and disinfectant foams derived from them on Pseudomonas aeruginosa biofilms. Biofilms were grown on stainless steel coupons under standardised conditions in a reactor supplemented with low concentrations of organic matter to simulate conditions prevalent in industrial systems. Five-day-old biofilms formed under ambient conditions with continuous agitation demonstrated a low coefficient of variation (5.809%) amongst viable biofilm bacteria from independent trials. Scanning electron microscopy revealed biofilms on coupons with viable biofilm bacteria observed by confocal microscopy. An aqueous solution of a common foaming agent amine oxide (AO) produced negligible effects on bacterial viability in biofilms (p?>?0.05). However, significant biofilm inactivation was noted with aqueous solutions of common biocides (peracetic acid, sodium hypochlorite, sodium ethylenediaminetetraacetic acid) with or without AO (p?<?0.05). Aereation of a mixture of AO with each of these common biocides resulted in significant reductions in the viability of biofilm bacteria (p?<?0.05). In contrast, limited effects were noted by foam devoid of biocides. A relationship between microbial inactivation and the concentration of biocide in foam (ranging from 0.1?–?0.5%) and exposure period were noted (p?<?0.05). Although, lower numbers of viable biofilm bacteria were recovered after treatment with the disinfectant foam than by the cognate aqueous biocide, significant differences between these treatments were not evident (p?>?0.05). In summary, the studies revealed significant biofilm inactivation by biocidal foam prepared with common biocides. Validation of foam disinfectants in controlled trials at manufacturing sites may facilitate developments for clean in place applications. Advantages of foam disinfectants include reductions in the volumes of biocides for industrial disinfection and in their disposal after use.     

Molecular Basis of Glycosaminoglycan Heparin Binding to the Chemokine CXCL1 Dimer

Glycosaminoglycan (GAG)-bound and soluble chemokine gradients in the vasculature and extracellular matrix mediate neutrophil recruitment to the site of microbial infection and sterile injury in the host tissue. However, the molecular principles by which chemokine-GAG interactions orchestrate these gradients are poorly understood. This, in part, can be directly attributed to the complex interrelationship between the chemokine monomer-dimer equilibrium and binding geometry and affinities that are also intimately linked to GAG length. To address some of this missing knowledge, we have characterized the structural basis of heparin binding to the murine CXCL1 dimer. CXCL1 is a neutrophil-activating chemokine and exists as both monomers and dimers (K_d = 36 μm). To avoid interference from monomer-GAG interactions, we designed a trapped dimer (dCXCL1) by introducing a disulfide bridge across the dimer interface. We characterized the binding of GAG heparin octasaccharide to dCXCL1 using solution NMR spectroscopy. Our studies show that octasaccharide binds orthogonally to the interhelical axis and spans the dimer interface and that heparin binding enhances the structural integrity of the C-terminal helical residues and stability of the dimer. We generated a quadruple mutant (H20A/K22A/K62A/K66A) on the basis of the binding data and observed that this mutant failed to bind heparin octasaccharide, validating our structural model. We propose that the stability enhancement of dimers upon GAG binding regulates in vivo neutrophil trafficking by increasing the lifetime of “active” chemokines, and that this structural knowledge could be exploited for designing inhibitors that disrupt chemokine-GAG interactions and neutrophil homing to the target tissue.     

Dietary supplementation with resveratrol and/or docosahexaenoic acid alters hippocampal gene expression in adult C57Bl/6 mice

The hippocampus is an important brain structure for multiple cognitive functions, including memory formation. It is particularly sensitive to insults, such as stress, ischemia, and aging; all of these can affect hippocampal and therefore cognitive function. To understand the potential of diet for the preservation of hippocampal function, we investigated the effects of dietary supplementation with resveratrol (RES) or docosahexaenoic acid (DHA), or their combination, on hippocampal gene expression in adult C57BL/6 mice. Animals in the supplemented group received either 50 mg/kg/day of RES or DHA, while the combination group received 50 mg/kg/day of each supplement. Dietary supplements were mixed with the AIN93G diet, and supplementation lasted 6 weeks. The control group received AIN93G diet alone for the same period. At the end of the experiment, the hippocampi were processed for genome-wide gene expression and pathway analyses. Most of the genes that were significantly altered were associated with inflammatory responses as determined by pathway analysis. RES-supplemented animals showed decreased expression of IL-6 (P=.001), MAPKapk2 (P=.015), and increased expression for PI3KR2 (P=.034) and Wnt7a (P=.004) expression. DHA-supplemented animals showed a decreased IL-6 (P=.003) and an increased Wnt7a (P=.003) expression. Animals on the combination diet showed a decreased IL-6 (P=.005) and Apolipoprotien E (ApoE) (P=.035) expression. Our findings demonstrate that hippocampal gene expression is significantly altered by all three dietary supplementation regimes. Moreover, our analysis indicates that RES and DHA likely exert their beneficial effects through antiinflammatory mechanisms.     

Circadian Variation in Circulating Levels of Melatonin,Testosterone and Estradiol-17β in a Tropical Freshwater Catfish,Clarias batrachus,during Different Phases of the Annual Reproductive Cycle

In the present investigation, daily variations in plasma levels of melatonin, testosterone and estradiol-17β were analysed in the tropical freshwater catfish, Clarias batrachus during early and late-preparatory, pre-spawning, late-spawning and post-spawning phases of its annual reproductive cycle. Plasma melatonin levels exhibited a significant circadian rhythm during all the reproductive phases but peaks were invariably at night. The amplitude of melatonin was high during the early-preparatory phase and low in the pre-spawning phase. Testosterone also displayed significant rhythm during all the studied phases except post spawning phase. Estradiol-17β revealed diurnal rhythm only during preparatory and pre-spawning phases. The peak in the levels of plasma testosterone and estradiol-17β were recorded during the photophase. During the late-preparatory phase, the rhythm of testosterone coincided with the rhythm of estradiol-17β.