Microbial Biotransformation for the Production of Steroid Medicament

Androstenedione (AD) is a steroid intermediate valuable for the production of steroid medicaments. Microbial biotransformation of phytosterol to produce AD is a well-researched area. However, low substrate solubility of phytosterol in aqueous media and nucleus degradation of AD to androstadienedione (ADD) or 9-hydroxy-AD are the major obstacles for AD production leading to detailed research for optimization of biotransformation process. In this review, microbial transformation of AD with respect to the existing methods of chemical or biochemical synthesis of AD are extensively discussed. This review examines the microbial biotransformation process and limitations for enhanced AD production. Factors affecting the effective biotransformation process to obtain AD are discussed and limitations are highlighted. The main content of this review focuses on the recent and futuristic biotechnological advances and strategies in techniques to enhance AD bioprocess

In Vitro Biotransformation in Drug Discovery

In vitro Biotransformation studies play a crucial role in drug discovery program that determine the fate of the new chemical entities (NCE’s). Enzyme rich matrices such as microsomes, hepatocytes, liver fractions and S9 fractions transform the new chemical entities to different metabolites. Metabolites could be pharmacologically important or toxic. Newly formed metabolites are identified using liquid chromatography interfaced with mass spectrometry. Identification of the biotransformation sites in the new chemical entity helps the medicinal chemists to optimize its structure and develop the NCE as a pharmaceutical drug. Screening pharmaceutical drugs using in vitro biotransformation studies assist in selecting the right new chemical entity for further in vivo studies in animal systems and later in human clinical trials

The spectrum of inherited gray matter Degenerative Brain Disorders (DBD) in children: A single-center study

Objectives: To study the clinical spectrum of inherited gray matter degenerative brain disorders (DBD) in children. Methods: This cross-sectional study evaluated children up to 12 y of age, diagnosed with an inherited gray matter DBD in a tertiary care pediatric hospital between July 2019 and December 2020. Results: A total of 314 children with progressive neuroregression were screened. Of these, 117 children with inherited gray matter DBD were included in the study. The clinic-based prevalence of DBD was 8.2%, and inherited gray matter DBD was 3.1%. The proportion of the inherited gray matter DBD was 37.3% among the overall DBD cases. Children were categorized into three groups based on the age at onset of disease: below 2 years (N = 57, 48.7%), between 2 and 5 years (N = 32, 27.3%), and between 6 and 12 years (N = 28, 23.9%). Based on the predominant cerebral structure involved, gray matter DBD were classified as cerebral gray matter disorders (53%), basal ganglia disorders (34.1%), and cerebellar disorders (12.8%). Overall, the most common disorders were Wilson disease (18%), neuronal ceroid lipofuscinosis (NCL) (17%), and neurodegeneration with brain iron accumulation (NBIA) (16%). The most common gray matter DBD in children <2 years of age were NBIA (n = 11), Rett syndrome (n = 11), and gangliosidoses (n = 10). NCL (n = 14) and ataxia telangiectasia (n = 6) were most common in the age group of 2–5 years. Wilson disease (n = 19) was the most common disorder in the age group of 6–12 years followed by NCL (n = 4) and NBIA (n = 3). Conclusion: Our study highlights the burden and spectrum of gray matter DBD in children. The clinic-based prevalence of DBD was 8.2%, and of inherited gray matter DBD was 3.1%. The proportion of inherited gray matter DBD was 37.3% among the overall DBD cases. Wilson disease, NCL, and NBIA are the most common gray matter DBD in children. Timely diagnosis is important for the prevention of recurrence in subsequent pregnancies

Th2 cytokine mRNA expression.

<p>Th2 cytokines mRNA expression profile analysis of normal and immunized hamsters on days 45 p.c.and 90 p.c were checked by quantitative real-time RT-PCR. The expression of Th2 cytokines viz. IL4 (a,b), IL10 (c,d), TGFß (e,f) was also observed to be ~3.00 folds higher (p<0.001) in the Infected hamsters as compare to vaccinated group as well as control groups. Significance values indicate the difference between the vaccinated group and infected group (***, p<0.001), vaccinated group and BCG group (***, p<0.001), vaccinated group and normal group (*, p<0.05).</p

Parasite burden.

<p>Clinical outcomes following <i>L</i>. <i>donovani</i> challenge in hamsters immunized with rLdSir2RP+BCG. On day 21 after the booster, the hamsters of infected, BCG alone and vaccinated groups were challenged intracardially with 10^<b>7</b> metacyclic promastigotes of <i>L</i>. <i>donovani</i>. Following parameters observed Body weight (a), spleen weight (b), liver weight (c), Parasite burden (no. of amastigotes per 100 cell nuclei) in the spleen(d), liver(e), and bone marrow (f) on days 45 and 60 p.c. Significance values indicate the difference between the vaccinated groups and infected group (***, p<0.001). Data represent mean values standard errors (SE) at the designated time points.</p

Leishmania-specific IgG and its isotypes IgG1 and IgG2 responses.

<p>Serum samples were collected from different groups of hamsters at designated time points and assayed for rLdSir2RP—specific IgG, IgG1, and IgG2 levels by ELISA. Antibody responses in rLdSir2RP vaccinated hamsters in comparison to the unimmunized infected hamsters on days 45, 60 and 90 p.c. The IgG1 were observed to be elevated by 1 to 2 fold in infected control group in comparison to the group of immunized hamsters with rLdSir2RP at the time interval of Days 45(a), d60(b) and d90(c). There was significant elevation of IgG2 level (by 3.5 to 4 folds) in group of hamsters immunized with rLdSir2RP at the time interval of Days 45(a), d60(b)and d90(c). Data are presented as the absorbance at 492 nm and are means ± SE for 3–4 hamsters per group in triplicate wells. Significance values indicate the difference between the vaccinated group and infected group (***, p<0.001).</p

Expression of recombinant LdSir2RP in E. coli Rosetta strain and its purification.

<p>Expression and purification of rLdSir2RP (1A, IB) in prokaryotic cells, Whole Cell Lysate (WCL) of transformed <i>E</i>. <i>coli</i> separated on 12% acrylamide gel and stained with Coomassie blue, M: Molecular wt. markers; Lane 1: WCL before IPTG induction; lane 2: WCL after IPTG (1.0 mM) induction at 37°C. Lane 5: eluted protein (1B). Western blot analysis using anti-rLdSir2RPAb in uninduced WCL, induced WCL and SLD—M: Mol wt marker, Lane 1: uninduced WCL, Lane 2: induced WCL (1C).</p

Cellular responses of rLdSir2RP of L.donovani in vaccinated hamsters.

<p>Nitric oxide production (μM) (a, b, c) by J774A.1 cell line. The J774 A.1 cells were primed with the supernatants of stimulated lymphocytes (3 days with mitogen and 5 days with Ags) of normal, infected and vaccinated hamsters in response to rLdSir2RP, SLD and LPS respectively at 10 μg/ml each. The estimation of NO production was done using Greiss reagent in supernatants collected from macrophage cultures 24 h after incubation and the absorbance of the reaction product was measured at 540 nm. XTT response (d, e, f) of mononuclear cells (lymph nodes) from normal, <i>L</i>. <i>donovani</i> infected and vaccinated hamsters in response to Con A, SLD and rLdSir2RP at 10 μg/ml each Proliferation was represented as mean OD. The data represent the means of triplicate wells ± S.D. Significance values indicate the difference between the SLD and rLdSir2RP stimulation (**, p < 0.01; ***, p<0.001).</p