Enhanced production of cordycepin in Ophiocordyceps sinensis using growth supplements under submerged conditions.

Cordycepin is a crucial bioactive compound produced by the fungus Cordyceps spp. Its therapeutic potential has been recognized for a wide range of biological properties such as anticancer, anti-diabetic, antidepressant, antioxidant, immunomodulation, etc. Moreover, its human random clinical trials depicted a promising anti-inflammatory activity that reduced the airway inflammation remarkably in asthmatic patients. But its overexploitation and low production of cordycepin in naturally growing biomass are insufficient to meet its existing market demand for its therapeutic use. Therefore, strategies for enhancement of cordycepin production in Cordyceps spp. are warranted. However, specifically, wild type Ophiocordyceps sinensis possesses a very low content of cordycepin and has restricted growth in natural mycelial biomass. To overcome these limitations, this study attempted to enhance cordycepin production in its mycelial biomass in vitro under submerged conditions by adding various growth supplements. The effect of these growth supplements was evaluated by reversed-phase high-performance liquid chromatography (RP-HPLC) which demonstrated that among nucleosides- hypoxanthine and adenosine; amino acids-glycine and glutamine; plant hormones- 1-naphthaleneacetic acid (NAA) and 3-indoleacetic acid (IAA); vitamin-thiamine (B1) from each group of growth supplements yielded a higher amount of cordycepin with 466.48 ±â€¯3.88, 380.23 ±â€¯1.78, 434.97 ±â€¯2.32, 269.78 ±â€¯2.92, 227.61 ±â€¯2.34, 226.02 ±â€¯1.69 and 185.26 ±â€¯2.35 mg/L respectively as compared to control with 13.66 ±â€¯0.64 mg/L. Further, at the transcriptional level, quantitative real time-polymerase chain reaction (qRT-PCR) analysis of genes associated with metabolism and cordycepin biosynthesis depicted significant upregulation of major downstream genes- NT5E, RNR, purA, and ADEK which corroborated well with RP-HPLC analysis. Taken together, the present study identified growth supplements as potential precursors to activate the cordycepin biosynthesis pathway leading to improved cordycepin production in O. sinensis.

Dissociation of neonatal and adult mice brain for simultaneous analysis of microglia, astrocytes and infiltrating lymphocytes by flow cytometry.

The technical difficulty to isolate microglia, astrocytes and infiltrating immune cells from mouse brain is nowadays a limiting factor in the study of neuroinflammation. Brain isolation requirements are cell-type and animal-age dependent, but current brain dissociation procedures are poorly standardized. This lack of comprehensive studies hampers the selection of optimized methodologies. Thus, we present here a comparative analysis of dissociation methods and Percoll-based separation to identify the most efficient procedure for the combined isolation of healthy microglia, astrocytes and infiltrated leukocytes; distinguishing neonatal and adult mouse brain. Gentle mechanical dissociation and DNase I incubation was supplemented with papain or collagenase II. Dispase II digestion was also used alone or in combination. In addition, cell separation efficiency of 30 % and 30-70 % Percoll gradients was compared. In these experiments, cell yield and integrity of freshly dissociated cells was measured by flow cytometry. We found that papain digestion in combination with dispase II followed by 30 % Percoll separation is the most balanced method to obtain a mixture of microglia, astrocytes and infiltrated immune cells; while addition of dispase II was not an advantage for neonatal brain. These dissociation conditions allowed flow cytometry detection of a slight glial activation triggered by sublethal LPS injection. In conclusion, the enzymes and Percoll density gradients tested here affected differently resting microglia, activated microglia/macrophages, astrocytes and infiltrated lymphocytes. Also, newborn and adult brain showed contrasting reactions to digestion. Our study highlights the strength of flow cytometry for the simultaneous analysis of neuroimmune cell populations once extraction is optimized.

Vitamin C acts as a hepatoprotectant in carbofuran treated rat liver slices in vitro.

Carbamates, most commonly used pesticides in agricultural practices, have been reported to produce free radicals causing deleterious effects in animals. The present study was designed to assess the carbofuran induced oxidative stress in rat liver slices in vitro and also to evaluate protective role of vitamin C by incubating them in Krebs-Ringer HEPES Buffer (KRHB) containing incubation media (Williams medium E (WME) supplemented with glucose and antibiotics) with different concentrations of carbofuran. The results demonstrated that carbofuran caused significant increase in lipid peroxidation and inhibition in the activity of hepatic superoxide dismutase (SOD) in concentration dependent manner. The data with incubation medium reflected that carbofuran at lowest concentration caused an increase in SOD activity followed by its inhibition at higher concentration. Carbofuran treatment caused inhibition in the activity of catalase in liver slices and WME incubation medium. Pre-incubation of liver slices and the WME media with vitamin C restored the values of biochemical indices tested. The results indicated that carbofuran might induce oxidative stress in hepatocytes. The pretreatment with vitamin C may offer hepatoprotection from toxicity of pesticide at low concentration only.

Loading Dose only versus Standard Dose Magnesium Sulfate Seizure Prophylaxis in Severe Pre-eclamptic Women.

INTRODUCTION: Magnesium sulfate is the drug of choice for prevention of seizures in the pre-eclamptic woman. There is no agreement in the published randomized trials regarding the optimal time to initiate magnesium sulfate, the dose to use (both loading and maintenance) as well as the duration of therapy. The objective of this study is to determine whether magnesium sulfate prophylaxis is needed for up to 24 hours postpartum in all patients with severe pre-eclampsia for the prevention of seizure. METHODS: It is a randomized controlled trial conducted on 60 pregnant women with severe preeclampsia randomized into standard dose and loading dose only regimen. RESULTS: Out of 30 cases in each group 1 (3.3%) patient in standard regimen and 2 (6.7%) patients in loading dose only developed seizure. The occurrence of seizure is not significant statistically. In both regimens, there was no maternal mortality. Total of 3 patients needed MICU care and 12 patient developed maternal complications. MgSO4 toxicities were seen only in standard dose regimen that is in 17 (56.7%) of the patients. The median number of IM injections of MgSO4 received in standard dose regimen was 8±2.176. In standard dose regimen 73.3 percent baby were alive whereas in case of loading dose only regimen 93.3 percent of baby were alive after 48 hours of delivery. CONCLUSIONS: Single dose of magnesium sulfate is equally effective as standard dose regimen in terms of seizure prophylaxis in severe pre eclamptic women, with added advantage of reduced maternal toxicity and better neonatal outcome.

A single metabolite production by Escherichia coli BW25113 and its pflA.cra mutant cultivated under microaerobic conditions using glycerol or glucose as a carbon source.

Abundant, low prices and a highly reduced nature make glycerol to be an ideal feedstock for the production of reduced biochemicals and biofuels. Escherichia coli has been paid much attention as the platform of microbial cell factories due to its high growth rate (giving higher metabolite production rate) and the capability of utilizing a wide range of carbon sources. However, one of the drawbacks of using E. coli as a platform is its mixed metabolite formation under anaerobic conditions. In the present study, it was shown that ethanol could be exclusively produced from glycerol by the wild type E. coli, while d-lactic acid could be exclusively produced from glucose by pflA.cra mutant, where the glucose uptake rate could be increased by this mutant as compared to the wild type strain. It was also shown that the growth rate is significantly reduced in pflA.cra mutant for the case of using glycerol as a carbon source due to redox imbalance. The metabolic regulation mechanisms behind the fermentation characteristic were clarified to some extent.