SIRT1 Limits Adipocyte Hyperplasia through c-Myc Inhibition

The expansion of fat mass in the obese state is due to increased adipocyte hypertrophy and hyperplasia. The molecular mechanism that drives adipocyte hyperplasia remains unknown. The NAD⁺-dependent protein deacetylase sirtuin 1 (SIRT1), a key regulator of mammalian metabolism, maintains proper metabolic functions in many tissues, counteracting obesity. Here we report that differentiated adipocytes are hyperplastic when SIRT1 is knocked down stably in mouse 3T3-L1 preadipocytes. This phenotype is associated with dysregulated adipocyte metabolism and enhanced inflammation. We also demonstrate that SIRT1 is a key regulator of proliferation in preadipocytes. Quantitative proteomics reveal that the c-Myc pathway is altered to drive enhanced proliferation in SIRT1-silenced 3T3-L1 cells. Moreover, c-Myc is hyperacetylated, levels of p27 are reduced, and cyclin-dependent kinase 2 (CDK2) is activated upon SIRT1 reduction. Remarkably, differentiating SIRT1-silenced preadipocytes exhibit enhanced mitotic clonal expansion accompanied by reduced levels of p27 as well as elevated levels of CCAAT/enhancer-binding protein β (C/EBPβ) and c-Myc, which is also hyperacetylated. c-Myc activation and enhanced proliferation phenotype are also found to be SIRT1-dependent in proliferating mouse embryonic fibroblasts and differentiating human SW872 preadipocytes. Reducing both SIRT1 and c-Myc expression in 3T3-L1 cells simultaneously does not induce the adipocyte hyperplasia phenotype, confirming that SIRT1 controls adipocyte hyperplasia through c-Myc regulation. A better understanding of the molecular mechanisms of adipocyte hyperplasia will open new avenues toward understanding obesity.     

Mechanisms of nitric oxide crosstalk with reactive oxygen species scavenging enzymes during abiotic stress tolerance in plants

Nitric oxide (NO) acts in a concentration and redox-dependent manner to counteract oxidative stress either by directly acting as an antioxidant through scavenging reactive oxygen species (ROS), such as superoxide anions (O₂^?*), to form peroxynitrite (ONOO^?) or by acting as a signaling molecule, thereby altering gene expression. NO can interact with different metal centres in proteins, such as heme-iron, zinc–sulfur clusters, iron–sulfur clusters, and copper, resulting in the formation of a stable metal–nitrosyl complex or production of varied biochemical signals, which ultimately leads to modification of protein structure/function. The thiols (ferrous iron–thiol complex and nitrosothiols) are also involved in the metabolism and mobilization of NO. Thiols bind to NO and transport it to the site of action whereas nitrosothiols release NO after intercellular diffusion and uptake into the target cells. S-nitrosoglutathione (GSNO) also has the ability to transnitrosylate proteins. It is an NO˙ reservoir and a long-distance signaling molecule. Tyrosine nitration of proteins has been suggested as a biomarker of nitrosative stress as it can lead to either activation or inhibition of target proteins. The exact molecular mechanism(s) by which exogenous and endogenously generated NO (or reactive nitrogen species) modulate the induction of various genes affecting redox homeostasis, are being extensively investigated currently by various research groups. Present review provides an in-depth analysis of the mechanisms by which NO interacts with and modulates the activity of various ROS scavenging enzymes, particularly accompanying ROS generation in plants in response to varied abiotic stress.     

Genetic diversity analysis of maize lines using AFLP and TE-based molecular marker systems

Maize has been domesticated in diverse environments ranging from low latitudes in tropical countries to high latitudes in Canada. Because maize breeding programs primarily focus on hybrid vigor by selectively crossing inbred lines to maximize recombination, we collected a diverse array of commercial hybrid and inbred lines from southern Asia, China, and Canada and analyzed them by amplified length fragment polymorphism (AFLP), sequence-specific amplified polymorphism (SSAP), and CACTA-transposon display (TD) analyses. Cluster analyses using these molecular marker systems clearly differentiated these maize lines into three groups: southern Asian lines, northern Asian lines, and Canadian lines. However, principal coordinate analysis (PCoA) based on Nei’s distances grouped them into two groups: Asian and Canadian lines. Thus, groupings by cluster dendrograms and PCoA showed that geographic origin was a more dominant factor than growing seasonal differences resulting from different latitudes. The overall genetic diversity (Ht) was found to be high (more than 80 % molecular variations) among the maize lines by all three of the marker systems.     

Real-time loop-mediated isothermal amplification assay for rapid and sensitive detection of anthrax spores in spiked soil and talcum powder

Loop-mediated isothermal amplification (LAMP) assay is a powerful and innovative gene amplification technique that specifically amplifies the target gene under isothermal conditions with a high degree of sensitivity, rapidity and specificity. The major advantage of the LAMP assay is monitoring of amplified products without the requirement of any sophisticated equipment. In the present study a real time LAMP assay was employed for rapid and real time detection of Bacillus anthracis spores spiked in 0.1 g of soil and talcum powder ranging from 2 to 10⁷ spores. DNA was isolated from spiked soil and talcum powder using PBS containing 1% Triton X-100, and heat treatment. Isolated DNA was used as template for LAMP and PCR. LAMP amplification was obtained in 60 min under isothermal condition at 63°C by employing a set of six primers targeting the pag gene of B. anthracis. The detection limit of LAMP assay in soil and talcum powder was found to be as low as 5 spores, compared to 10³ spores and 10⁴ spores by PCR in talcum powder and soil, respectively. The findings suggest that LAMP is a more rapid and sensitive assay than PCR for detecting anthrax spores, additionally the methodology to prepare DNA from spiked samples is simple, rapid and cost effective.     

Genome-resolved metagenomics to study co-occurrence patterns and intraspecific heterogeneity among plant pathogen metapopulations

Assessment of pathogen diversity in agricultural fields is essential for informing management decisions and the development of resistant plant varieties. However, many population genomic studies have relied on culture-based approaches that do not provide quantitative assessment of pathogen populations at the field-level or the associated host microbiome. Here, we applied whole-genome shotgun sequencing of microbial DNA extracted directly from the washings of pooled leaf samples, collected from individual tomato and pepper fields in Alabama that displayed the classical symptoms of bacterial spot disease caused by Xanthomonas spp. Our results revealed that while the occurrence of both X. perforans and X. euvesicatoria within fields was limited, evidence of co-occurrence of up to three distinct X. perforans genotypes was obtained in 7 of 10 tomato fields sampled. These population dynamics were accompanied by the corresponding type 3 secreted effector repertoires associated with the co-occurring X. perforans genotypes, indicating that metapopulation structure within fields should be considered when assessing the adaptive potential of X. perforans. Finally, analysis of microbial community composition revealed that co-occurrence of the bacterial spot pathogens Pseudomonas cichorii and Xanthomonas spp. is common in Alabama fields and provided evidence for the non-random association of several other human and plant opportunists.     

Transformation to telephonic genetic counselling during SARS-CoV-2 pandemic: challenges and suggested protocol in Indian scenario

To combat the dreaded diseases in rice like bacterial blight (BB) and blast, host plant resistance has been advocated as the most suitable and sustainable method. Through the present study, we have successfully incorporated three major BB resistance genes, namely Xa21, xa13 and xa5 into NLR3449, a high yielding, blast resistant, fine-grain type, popular rice variety through marker-assisted backcross breeding. Foreground selection was carried out using polymerase chain reaction based, gene-specific markers, namely pTA248 (Xa21), xa13prom (xa13) and xa5FM (xa5) at each generation of backcrossing, while 127 polymorphic SSR markers spanning on 12 chromosomes were used for background selection and backcrossing was limited to two rounds. At BC₂F₁ generation, a single plant (NLR-87-10) with 89.9% recovery, possessing all the three BB resistance genes was forwarded to BC₂F₂ generation. A solitary BC₂F₂ plant, namely NLR-87-10-106 possessing all the three resistance genes and 96% genome recovery was identified and advanced through selfing until BC₂F₄ generation by adopting pedigree-method of selection. Three best BC₂F₄ lines, possessing high level of resistance against BB and blast, and equivalent or superior to NLR 34449 in terms of yield, grain quality and agro-morphological traits were identified and advanced for multi-location trials.

     

Proteomic profiling of metformin effects in 3T3-L1 adipocytes by SILAC-based quantification

Metformin is a common and generally the first medication prescribed for treatment of type 2 diabetes. Its mechanism involves affecting pathways that regulate glucose and lipid metabolism in metabolic cells such as that of muscle and liver cells. In spite of various studies exploring its effects, the proteome changes in adipocytes in response to metformin remains poorly understood. In this study, we performed stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomic profiling to study the effects of metformin specifically on 3T3-L1 adipocytes. We define proteins that exhibited altered levels with metformin treatment, 400 of them showing statistically significant changes in our study. Our results suggest that metformin affects not only the PPAR signaling pathway, as well as glucose and lipid metabolism, but also protein folding, endoplasmic reticulum stress, negative regulation of appetite, and one-carbon folate metabolism in adipocytes. This proteomic investigation provides important insight into effects of metformin in adipocytes.