DRUG UTILISATION STUDY OF STROKE AND OTHER PATIENTS ADMITTED TO GENERAL WARD OF NEUROLOGY UNIT AT QUATERNARY CARE PRIVATE HOSPITAL

Objective: To study the profile of the patient, the pattern of their illness and their drug use patterns attending to general ward of quaternary care hospital.Methods: Prospective observational study of 2 mo was performed at neurology unit of the quaternary care private hospital, India. Patients aged above 20 y with at least one neurological drug in prescription were included to observe their demographic profile, illness pattern and drug use. The data was analysed and summarised as frequency and percentage using microsoft excel and presented as tables.Results: Among 60 patient that were enrolled, more were males (N=33, 55%) than females (N=27, 45%) and within the age group of 60-70 y (N=22, 36.67%). The majority of patients were diagnosed with strokes (N= 48, 80%) where the cerebrovascular accident was most common (N=16, 26.66%). Males (N=29, 60.4%) were more prone to get stroke than females (N=19,39.6%). An average number of drugs per prescription was 5.7, the percentage of antibiotics, generic drugs and injections were 36.6%, 0.05% and 44.14% respectively. A total of 28 different drug classes with 61 different drugs was utilised. Fourteen drug classes had been accounted for 90% of drugs utilisation. Clopidegrol+Aspirin have frequently used the drug.Conclusion: Most people attending neurology unit were elderly. Stroke occupies 1st list for the burden. Average drugs for neurology visit remain high. Wide types of drug classes are utilised in neurological ward

A versatile LC-MS/MS approach for comprehensive, quantitative analysis of central metabolic pathways [version 1; referees: 2 approved]

Liquid chromatography-mass spectrometry (LC-MS/MS) based approaches are widely used for the identification and quantitation of specific metabolites, and are a preferred approach towards analyzing cellular metabolism. Most methods developed come with specific requirements such as unique columns, ion-pairing reagents and pH conditions, and typically allow measurements in a specific pathway alone. Here, we present a single column-based set of methods for simultaneous coverage of multiple pathways, primarily focusing on central carbon, amino acid, and nucleotide metabolism. We further demonstrate the use of this method for quantitative, stable isotope-based metabolic flux experiments, expanding its use beyond steady-state level measurements of metabolites. The expected kinetics of label accumulation pertinent to the pathway under study are presented with some examples. The methods discussed here are broadly applicable, minimize the need for multiple chromatographic resolution methods, and highlight how simple labeling experiments can be valuable in facilitating a comprehensive understanding of the metabolic state of cells

Behavior of a Metabolic Cycling Population at the Single Cell Level as Visualized by Fluorescent Gene Expression Reporters

BACKGROUND: During continuous growth in specific chemostat cultures, budding yeast undergo robust oscillations in oxygen consumption that are accompanied by highly periodic changes in transcript abundance of a majority of genes, in a phenomenon called the Yeast Metabolic Cycle (YMC). This study uses fluorescent reporters of genes specific to different YMC phases in order to visualize this phenomenon and understand the temporal regulation of gene expression at the level of individual cells within the cycling population. METHODOLOGY: Fluorescent gene expression reporters for different phases of the YMC were constructed and stably integrated into the yeast genome. Subsequently, these reporter-expressing yeast were used to visualize YMC dynamics at the individual cell level in cultures grown in a chemostat or in a microfluidics platform under varying glucose concentrations, using fluorescence microscopy and quantitative Western blots. CONCLUSIONS: The behavior of single cells within a metabolic cycling population was visualized using phase-specific fluorescent reporters. The reporters largely recapitulated genome-specified mRNA expression profiles. A significant fraction of the cell population appeared to exhibit basal expression of the reporters, supporting the hypothesis that there are at least two distinct subpopulations of cells within the cycling population. Although approximately half of the cycling population initiated cell division in each permissive window of the YMC, metabolic synchrony of the population was maintained. Using a microfluidics platform we observed that low glucose concentrations appear to be necessary for metabolic cycling. Lastly, we propose that there is a temporal window in the oxidative growth phase of the YMC where the cycling population segregates into at least two subpopulations, one which will enter the cell cycle and one which does not

Multiple TORC1-Associated Proteins Regulate Nitrogen Starvation-Dependent Cellular Differentiation in Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae undergoes differentiation into filamentous-like forms and invades the growth medium as a foraging response to nutrient and environmental stresses. These developmental responses are under the downstream control of effectors regulated by the cAMP/PKA and MAPK pathways. However, the upstream sensors and signals that induce filamentous growth through these signaling pathways are not fully understood. Herein, through a biochemical purification of the yeast TORC1 (Target of Rapamycin Complex 1), we identify several proteins implicated in yeast filamentous growth that directly associate with the TORC1 and investigate their roles in nitrogen starvation-dependent or independent differentiation in yeast.We isolated the endogenous TORC1 by purifying tagged, endogenous Kog1p, and identified associated proteins by mass spectrometry. We established invasive and pseudohyphal growth conditions in two S. cerevisiae genetic backgrounds (Σ1278b and CEN.PK). Using wild type and mutant strains from these genetic backgrounds, we investigated the roles of TORC1 and associated proteins in nitrogen starvation-dependent diploid pseudohyphal growth as well as nitrogen starvation-independent haploid invasive growth.We show that several proteins identified as associated with the TORC1 are important for nitrogen starvation-dependent diploid pseudohyphal growth. In contrast, invasive growth due to other nutritional stresses was generally not affected in mutant strains of these TORC1-associated proteins. Our studies suggest a role for TORC1 in yeast differentiation upon nitrogen starvation. Our studies also suggest the CEN.PK strain background of S. cerevisiae may be particularly useful for investigations of nitrogen starvation-induced diploid pseudohyphal growth

Eggs of the mosquito Aedes aegypti survive desiccation by rewiring their polyamine and lipid metabolism.

Upon water loss, some organisms pause their life cycles and escape death. While widespread in microbes, this is less common in animals. Aedes mosquitoes are vectors for viral diseases. Aedes eggs can survive dry environments, but molecular and cellular principles enabling egg survival through desiccation remain unknown. In this report, we find that Aedes aegypti eggs, in contrast to Anopheles stephensi, survive desiccation by acquiring desiccation tolerance at a late developmental stage. We uncover unique proteome and metabolic state changes in Aedes embryos during desiccation that reflect reduced central carbon metabolism, rewiring towards polyamine production, and enhanced lipid utilisation for energy and polyamine synthesis. Using inhibitors targeting these processes in blood-fed mosquitoes that lay eggs, we infer a two-step process of desiccation tolerance in Aedes eggs. The metabolic rewiring towards lipid breakdown and dependent polyamine accumulation confers resistance to desiccation. Furthermore, rapid lipid breakdown is required to fuel energetic requirements upon water reentry to enable larval hatching and survival upon rehydration. This study is fundamental to understanding Aedes embryo survival and in controlling the spread of these mosquitoes

Thiol trapping and metabolic redistribution of sulfur metabolites enable cells to overcome cysteine overload

Cysteine is an essential requirement in living organisms. However, due to its reactive thiol side chain, elevated levels of intracellular cysteine can be toxic and therefore need to be rapidly eliminated from the cellular milieu. In mammals and many other organisms, excess cysteine is believed to be primarily eliminated by the cysteine dioxygenase dependent oxidative degradation of cysteine, followed by the removal of the oxidative products. However, other mechanisms of tackling excess cysteine are also likely to exist, but have not thus far been explored. In this study, we use Saccharomyces cerevisiae, which naturally lacks a cysteine dioxygenase, to investigate mechanisms for tackling cysteine overload. Overexpressing the high affinity cysteine transporter, YCT1, enabled yeast cells to rapidly accumulate high levels of intracellular cysteine. Using targeted metabolite analysis, we observe that cysteine is initially rapidly interconverted to non-reactive cystine in vivo. A time course revealed that cells systematically convert excess cysteine to inert thiol forms; initially to cystine, and subsequently to cystathionine, S-Adenosyl-L-homocysteine (SAH) and S-Adenosyl L-methionine (SAM), in addition to eventually accumulating glutathione (GSH) and polyamines. Microarray based gene expression studies revealed the upregulation of arginine/ornithine biosynthesis a few hours after the cysteine overload, and suggest that the non-toxic, non-reactive thiol based metabolic products are eventually utilized for amino acid and polyamine biogenesis, thereby enabling cell growth. Thus, cells can handle potentially toxic amounts of cysteine by a combination of thiol trapping, metabolic redistribution to non-reactive thiols and subsequent consumption for anabolism

Resource plasticity-driven carbon-nitrogen budgeting enables specialization and division of labor in a clonal community

Previously, we found that in glucose-limited Saccharomyces cerevisiae colonies, metabolic constraints drive cells into groups exhibiting gluconeogenic or glycolytic states. In that study, threshold amounts of trehalose - a limiting, produced carbon-resource, controls the emergence and self-organization of cells exhibiting the glycolytic state, serving as a carbon source that fuels glycolysis (Varahan et al., 2019). We now discover that the plasticity of use of a non-limiting resource, aspartate, controls both resource production and the emergence of heterogeneous cell states, based on differential metabolic budgeting. In gluconeogenic cells, aspartate is a carbon source for trehalose production, while in glycolytic cells using trehalose for carbon, aspartate is predominantly a nitrogen source for nucleotide synthesis. This metabolic plasticity of aspartate enables carbon-nitrogen budgeting, thereby driving the biochemical self-organization of distinct cell states. Through this organization, cells in each state exhibit true division of labor, providing growth/survival advantages for the whole community

Genome-scale reconstruction of Gcn4/ATF4 networks driving a growth program.

Growth and starvation are considered opposite ends of a spectrum. To sustain growth, cells use coordinated gene expression programs and manage biomolecule supply in order to match the demands of metabolism and translation. Global growth programs complement increased ribosomal biogenesis with sufficient carbon metabolism, amino acid and nucleotide biosynthesis. How these resources are collectively managed is a fundamental question. The role of the Gcn4/ATF4 transcription factor has been best studied in contexts where cells encounter amino acid starvation. However, high Gcn4 activity has been observed in contexts of rapid cell proliferation, and the roles of Gcn4 in such growth contexts are unclear. Here, using a methionine-induced growth program in yeast, we show that Gcn4/ATF4 is the fulcrum that maintains metabolic supply in order to sustain translation outputs. By integrating matched transcriptome and ChIP-Seq analysis, we decipher genome-wide direct and indirect roles for Gcn4 in this growth program. Genes that enable metabolic precursor biosynthesis indispensably require Gcn4; contrastingly ribosomal genes are partly repressed by Gcn4. Gcn4 directly binds promoter-regions and transcribes a subset of metabolic genes, particularly driving lysine and arginine biosynthesis. Gcn4 also globally represses lysine and arginine enriched transcripts, which include genes encoding the translation machinery. The Gcn4 dependent lysine and arginine supply thereby maintains the synthesis of the translation machinery. This is required to maintain translation capacity. Gcn4 consequently enables metabolic-precursor supply to bolster protein synthesis, and drive a growth program. Thus, we illustrate how growth and starvation outcomes are both controlled using the same Gcn4 transcriptional outputs that function in distinct contexts

LCâUV and LCâMS evaluation of stress degradation behavior of desvenlafaxine

The objective of current study was to develop a validated specific stability indicating reversed-phase liquid chromatographic method for the quantitative determination of desvenlafaxine in bulk sample and pharmaceutical dosage form in the presence of degradation products. Forced degradation studies were performed on bulk sample of desvenlafaxine as per ICH prescribed stress conditions using acid, base, oxidative and photolytic degradation to show the stability indicating power of the method. Significant degradation was observed under acidic stress condition and the degradation product formed was identified by LCâMS and a degradation pathway for drug has been proposed. Successful separation of drug from degradation products formed under stress conditions was achieved on a SymmetryShield column C18 (5 μm, 250 mmÃ4.6 mm, i.d.) using the mobile phase consisting of a mixture of 0.2% (v/v) triethylamine in ammonium acetate (0.05 M; pH 6.5) and methanol using isocratic gradient. Keywords: Reversed-phase liquid chromatography, Liquid chromatography mass spectrometer (LC/MS), Desvenlafaxine, Stress studies, Validation, Degradatio