Constitutive optimized production of streptokinase in Saccharomyces cerevisiae utilizing glyceraldehyde 3-phosphate dehydrogenase promoter of Pichia pastoris

A novel expression vector constructed from genes of Pichia pastoris was applied for heterologous gene expression in Saccharomyces cerevisiae. Recombinant streptokinase (SK) was synthesized by cloning the region encoding mature SK under the control of glyceraldehyde 3-phosphate dehydrogenase (GAP) promoter of Pichia pastoris in Saccharomyces cerevisiae. SK was intracellularly expressed constitutively, as evidenced by lyticase-nitroanilide and caseinolytic assays. The functional activity was confirmed by plasminogen activation assay and in vitro clot lysis assay. Stability and absence of toxicity to the host with the recombinant expression vector as evidenced by southern analysis and growth profile indicate the application of this expression system for large-scale production of SK. Two-stage statistical approach, Plackett-Burman (PB) design and response surface methodology (RSM) was used for SK production medium optimization. In the first stage, carbon and organic nitrogen sources were qualitatively screened by PB design and in the second stage there was quantitative optimization of four process variables, yeast extract, dextrose, pH, and temperature, by RSM. PB design resulted in dextrose and peptone as best carbon and nitrogen sources for SK production. RSM method, proved as an efficient technique for optimizing process conditions which resulted in 110% increase in SK production, 2352 IU/mL, than for unoptimized conditions.Ravi N. Vellanki, Ravichandra Potumarthi, Kiran K. Doddapaneni, Naveen Anubrolu and Lakshmi N. Mangamoor

Meiotic analysis of the hybrids between cultivated and synthetic tetraploid groundnuts

Groundnut is susceptible to a range of diseases and pests owing to its narrow genetic base. Closely related wild relatives of groundnut are diploid, and crossability between diploid wild relatives and tetraploid cultivated groundnut is difficult. Amphidiploid and autotetraploid groundnuts, which are tetraploids (also called synthetic groundnut), were developed at ICRISAT. Crossability between cultivated tetraploid groundnut and synthetic tetraploid groundnut was a straightforward process. Crosses between cultivated and synthetic tetraploid groundnuts yielded mature seeds. The resultant F1 hybrids were cytologically analysed to study the relationship between the chromosomes/genomes of the parents i.e. between Arachis hypogaea cultivars and synthetic tetraploid groundnut. Meiotic study showed that there was good chromosome pairing between the parental species resulting in high pollen fertility. Thus, development of synthetic groundnut is a feasible way of utilizing important wild species gene pool of Arachis, closely related to cultivated groundnut, for the improvement to the cro

Diabetic ketoacidosis

Diabetic ketoacidosis (DKA) is the most common acute hyperglycaemic emergency in people with diabetes mellitus. A diagnosis of DKA is confirmed when all of the three criteria are present — ‘D’, either elevated blood glucose levels or a family history of diabetes mellitus; ‘K’, the presence of high urinary or blood ketoacids; and ‘A’, a high anion gap metabolic acidosis. Early diagnosis and management are paramount to improve patient outcomes. The mainstays of treatment include restoration of circulating volume, insulin therapy, electrolyte replacement and treatment of any underlying precipitating event. Without optimal treatment, DKA remains a condition with appreciable, although largely preventable, morbidity and mortality. In this Primer, we discuss the epidemiology, pathogenesis, risk factors and diagnosis of DKA and provide practical recommendations for the management of DKA in adults and children

Purification and characterization of an alkaline keratinase from Streptomyces sp.

Abstract not availableRadhika Tatineni, Kiran Kumar Doddapaneni, Ravi Chandra Potumarthi, Ravi Nagaraj Vellanki, Manjusha Thomas Kandathil, Nilima Kolli, Lakshmi Narasu Mangamoor

Kynurenine Metabolism as a Mechanism to Improve Fatigue and Physical Function in Postmenopausal Breast Cancer Survivors Following Resistance Training

This pilot examines whether resistance training (RT) can induce changes in kynurenine (KYN) metabolism, which may contribute to improved physical function in breast cancer survivors (BCSs). Thirty-six BCSs (63.2 ± 1.1 years) underwent assessments of physical function and visual analog scale (100 cm) fatigue and quality of life before and after 12 weeks of RT (N = 22) or non-exercise control (CBCT©: Cognitively Based Compassion Training, N = 10). Blood was collected before and after interventions for assessment of KYN, kynurenic acid (KYNA), and peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α). At baseline, the women were moderately fatigued (mean score: 46 cm) and at risk of poor functional mobility. A group*time interaction was observed for all measures of strength with improvements (~25–35%) following RT (p’s < 0.01), but not CBCT. Time effects were observed for fatigue (−36%) and quality of life (5%) (p’s < 0.01), where both groups improved in a similar manner. A group*time interaction was observed for KYN (p = 0.02) and PGC-1α (p < 0.05), with KYN decreasing and PGC-1α increasing following RT and the opposite following CBCT. These changes resulted in KYN/KYNA decreasing 34% post-RT, but increasing 21% following CBCT. These data support RT as a therapeutic intervention to counteract the long-term side effect of fatigue and physical dysfunction in BCSs. Additionally, the results suggest that this effect may be mediated through the activation of PGC-1α leading to alterations in KYN metabolism

Metabolic Regulation of the Epigenome Drives Lethal Infantile Ependymoma

Posterior fossa A (PFA) ependymomas are lethal malignancies of the hindbrain in infants and toddlers. Lacking highly recurrent somatic mutations, PFA ependymomas are proposed to be epigenetically driven tumors for which model systems are lacking. Here we demonstrate that PFA ependymomas are maintained under hypoxia, associated with restricted availability of specific metabolites to diminish histone methylation, and increase histone demethylation and acetylation at histone 3 lysine 27 (H3K27). PFA ependymomas initiate from a cell lineage in the first trimester of human development that resides in restricted oxygen. Unlike other ependymomas, transient exposure of PFA cells to ambient oxygen induces irreversible cellular toxicity. PFA tumors exhibit a low basal level of H3K27me3, and, paradoxically, inhibition of H3K27 methylation specifically disrupts PFA tumor growth. Targeting metabolism and/or the epigenome presents a unique opportunity for rational therapy for infants with PFA ependymoma. Hypoxia reprograms the cellular metabolome and epigenome to promote growth of the most lethal ependymomas

Smac mimetic promotes glioblastoma cancer stem-like cell differentiation by activating NF-κB

Recently, a broader role of inhibitor of apoptosis (IAP) proteins besides their antiapoptotic functions has been described. Therefore, we investigated the effect of non-toxic concentrations of the small-molecule Smac mimetic BV6, which antagonizes IAP proteins, on differentiation of cancer stem-like cells (CSLCs) derived from primary glioblastoma (GBM) specimens. Here, we identify a novel function of BV6 in regulating differentiation of GBM CSLCs by activating NF-κB. BV6 at non-lethal doses stimulates morphological changes associated with the differentiation of GBM CSLCs. BV6 increases transcriptional activity, mRNA and protein levels of the astrocytic marker GFAP without altering expression of the neuronal marker β-III-tubulin, indicating that BV6 induces astrocytic differentiation of GBM CSLCs. Molecular studies reveal that BV6 triggers processing of the NF-κB subunit p100 to p52, nuclear translocation of p52 and p50 and increased NF-κB DNA-binding. Intriguingly, inhibition of NF-κB by overexpression of dominant-negative IκBα super-repressor (IκBα-SR) blocks the BV6-stimulated increase in GFAP and differentiation. Interestingly, this BV6-stimulated differentiation is associated with reduced expression of stemness markers such as CD133, Nanog and Sox2 in GBM CSLCs. In contrast, BV6 does not alter cell morphology, differentiation and expression of stemness markers in non-malignant neural stem cells. Importantly, BV6 treatment reduces clonogenicity of GBM CSLCs in vitro and in vivo, suppresses their tumorigenicity in orthotopic and subcutaneous mouse models and significantly increases the survival of mice. By identifying a novel role of BV6 in promoting differentiation of GBM CSLCs, these findings provide new insights into Smac mimetic-regulated non-apoptotic functions with important implications for targeting GBM CSLCs