Supplementation of critical amino acids improves glycerol and lactose uptake and enhances recombinant protein production in Escherichia coli.

BACKGROUND: Lactose-based induction strategy in E. coli cultivation has several advantages over IPTG as it is cheap, does not impart metabolic stress to cells, and is non-toxic to cells. However, complexity of lactose as an inducer limits its application in fed-batch cultivation. A mixed glycerol-lactose based induction strategy is generally opted during fed-batch cultivation of E. coli. However, slow growth of E. coli in glycerol and lactose results in slower induction of heterologous protein. MAIN METHODS AND MAJOR RESULTS: In this study, initially we have demonstrated supplementation of critical amino acids (AAs) improves uptake rate of glycerol and lactose in wildtype E. coli BL21(DE3) in defined medium. A feeding strategy of mixed glycerol-lactose feed along with supplement of critical AAs enhances recombinant production of pramlintide multimer (rPramlintide). High cell density cultivation of E. coli using mixed glycerol-lactose feed and critical AAs supplement resulted in final cell density of 52.2 ± 0.90 g L-1  and rPramlintide titer of 7.8 g L-1 . RT-qPCR analysis of genes involved in glycerol and lactose metabolism of recombinant culture showed upregulation with AAs supplementation. CONCLUSIONS AND IMPLICATIONS: We hypothesize that supplementation of critical AAs serves dual purpose: (i) faster assimilation of carbon sources, and (ii) combating metabolic stress arises due to AAs starvation. The substrate uptake and gene expression profiles demonstrate that AAs addition enhances glycerol and lactose assimilation due to overall improvement in their metabolism governed by global regulators of carbon metabolism.

Amino acid supplementation for enhancing recombinant protein production in E. coli.

Recombinant protein production (RPP) in Escherichia coli (E. coli) often induces metabolic burden to the cells that compromise their overall growth and productivity. Amino acid starvation due to RPP is a major contributor of the metabolic burden on the cells and induces global stress response known as a stringent-like response. In this study, the effect of amino acid supplementation in a chemically defined medium on cellular growth and recombinant pramlintide production was investigated. Based on the consumption profile, few amino acids were categorized as growth-promoting (GP1) and protein production promoting (GP2). Feeding strategies of GP1 and GP2 were tested in shake flasks followed by scale up into the bioreactor. A 40% increase in the recombinant pramlintide (rPramlintide) production (protein concentration of 3.09 ± 0.12 g/L and yield of 227.69 ± 19.72 mg pramlintide per gram dry cell weight) was realized. Furthermore, transcriptomics data indicated the downregulation of several genes associated with global stress response and genes involved in amino acid biosynthesis in test culture, supported by proteomics analysis. These results signify that the external supply of critical amino acids decreases cellular stress during RPP and improves process productivity.

Management of early years of simple and mucopurulent chronic bronchitis with pre-defined Homoeopathic medicines ­ a Prospective Observational Study with 2-Years Follow-Up

Background Simple and mucopurulent chronic bronchitis (SMCB) is characterized by recurrent mucoid or mucopurulent expectoration in absence of localized suppurative disease. This observational open label study was undertaken to evaluate the effects of homeopathic medicine in SMCB. Methods 1902 patients were screened from 07 centres out of which 1305 were excluded. 597 patients were enrolled as per the inclusion and exclusion criteria. A total of 14 pre-defined homeopathic medicines were shortlisted for prescription after repertorizing the pathological symptoms of SMCB. Outcomes were assessed through chronic bronchitis symptom scale (CBSS) and FEV1/ FVC ratio with spirometry for over a period of two years. Appearance of any change (relief/ worse)/ status quo was immediately followed by placebo/ change in dilution/ change in remedy. Statistical analysis was done using SPSS version 20. Results: 532 patients were analyzed based on the intention to treat principle using last observation carry forward method. Mean CBS score reduced from 29.86±4.5 at baseline to 12.33±7.6 at completion of 2 years. Repeated measures ANOVA, at time points 0 (baseline), 3, 6, 9, 12 ,15, 18, 21 and 24 months, showed significant reduction in CBS scores [Wilk's Lambda 0.104, F=564, df 524; p=00001]. The FEV1 and FEV1/FVC was maintained within normal limits. 86% prescriptions included Lycopodium, Arsenicum album, Pulsatilla, Phosphorus, Stannum metallicum, Calcarea carbonica, Silicea, Bryonia alba. Conclusion: The result suggests effectiveness of homeopathic treatment in early years of SCMB patients. Controlled trials are warranted. (AU)

Management of distress during climacteric years by homeopathic therapy.

OBJECTIVES: The purpose of this study was to ascertain the usefulness of homeopathic therapy in the management of distressing symptoms encountered during climacteric years in women (primary objective) and also the changes brought about in the levels of follicle-stimulating hormone (FSH) and lipid profile in these women after homeopathic treatment (secondary objective). MATERIALS AND METHODS: An open, multicenter, prospective, observational study was carried out to ascertain the usefulness of homeopathic treatment in distress during climacteric years (DDCY). Patients were enrolled from the general outpatient department of the six Institutes/Units of Central Council for Research in Homoeopathy (CCRH) and were required to complete a follow-up period of 1 year as per the protocol designed by the CCRH. A uniform questionnaire assessing 15 predefined symptoms of menopause was adopted, with assessment of each symptom at every visit. Levels of serum FSH and lipid profile were monitored at entry and at completion. Effect size of the study was also calculated. CARA Software was used for repertorization of the presenting symptoms of menopause along with the characteristic attributes of each patient to arrive at a simillimum. The selected medicine was prescribed in a single dose as per the homeopathic principles. The assessment of the results was made through statistical analysis using the Wilcoxon signed rank test on Statistical Package for Social Sciences (SPSS) comparing symptom score at entry and completion of 1 year of treatment and t test for analyzing improvement in laboratory findings. RESULTS: Homeopathic therapy was found to be useful in relieving menopausal distressing symptoms such as hot flashes, night sweats, anxiety, palpitation, depression, insomnia, and so on. Influence on serum levels of FSH, high-density lipoprotein, and low-density lipoprotein was not significant but serum levels of cholesterol, triglycerides, and very-low-density lipoprotein decreased significantly. Effect size of the study was found to be large. The medicines found to be most frequently indicated and useful were Sepia, Lachesis, Calcarea carb., Lycopodium, and Sulphur. CONCLUSIONS: This study proves the usefulness of homeopathic medicines in relieving DDCY.