Production of 9,21-dihydroxy-20-methyl-pregna-4-en-3-one from phytosterols in Mycobacterium neoaurum by modifying multiple genes and improving the intracellular environment

Background Steroid drugs are essential for disease prevention and clinical treatment. However, due to intricated steroid structure, traditional chemical methods are rarely implemented into the whole synthetic process for generating steroid intermediates. Novel steroid drug precursors and their ideal bacterial strains for industrial production have yet to be developed. Among these, 9,21-dihydroxy-20-methyl-pregna-4-en-3-one (9-OH-4-HP) is a novel steroid drug precursor, suitable for the synthesis of corticosteroids. In this study, a combined strategy of blocking Δ1-dehydrogenation and the C19 pathway as well as improving the intracellular environment was investigated to construct an effective 9-OH-4-HP-producing strain. Results The Δ1-dehydrogenation-deficient strain of wild-type Mycobacterium neoaurum DSM 44074 produces 9-OH-4-HP with a molar yield of 4.8%. Hsd4A, encoding a β-hydroxyacyl-CoA dehydrogenase, and fadA5, encoding an acyl-CoA thiolase, were separately knocked out to block the C19 pathway in the Δ1-dehydrogenation-deficient strain. The two engineered strains were able to accumulate 0.59 g L−1 and 0.47 g L−1 9-OH-4-HP from 1 g L−1 phytosterols, respectively. Furthermore, hsd4A and fadA5 were knocked out simultaneously in the Δ1-dehydrogenation-deficient strain. The 9-OH-4-HP production from the Hsd4A and FadA5 deficient strain was 11.9% higher than that of the Hsd4A deficient strain and 40.4% higher than that of the strain with FadA5 deficiency strain, respectively. The purity of 9-OH-4-HP obtained from the Hsd4A and FadA5 deficient strain has reached 94.9%. Subsequently, the catalase katE from Mycobacterium neoaurum and an NADH oxidase, nox, from Bacillus subtilis were overexpressed to improve the intracellular environment, leading to a higher 9-OH-4-HP production. Ultimately, 9-OH-4-HP production reached 3.58 g L−1 from 5 g L−1 phytosterols, and the purity of 9-OH-4-HP improved to 97%. The final 9-OH-4-HP production strain showed the best molar yield of 85.5%, compared with the previous reported strain with 30% molar yield of 9-OH-4-HP. Conclusion KstD, Hsd4A, and FadA5 are key enzymes for phytosterol side-chain degradation in the C19 pathway. Double deletion of hsd4A and fadA5 contributes to the blockage of the C19 pathway. Improving the intracellular environment of Mycobacterium neoaurum during phytosterol bioconversion could accelerate the conversion process and enhance the productivity of target sterol derivatives.

Apigenin Acts as Anti-inflammatory Compound through Reducing IL-1β, IL-6 and TNF-α in LPS-induced Inflammation; in-vivo Study

Background: Consumption of herbal flavonoids instead of chemical drugs has increased in recent years due to fewer side effects and affordability. In this study, the effect of apigenin was investigated on inflammation induced by lipopolysaccharide in male rat's serum by measuring the pro-inflammatory cytokines, i.e., IL-1β, IL-6, and TNF-α.Methods: 90 male Wistar rats weighing 200 ±2 grams were used and divided into control, sham (solvent), and positive control (dexamethasone 15 mg/kg. ip), and 3 experimental groups which received 5, 15 or 30 mg/kg of apigenin, intraperitoneally. In 30 minutes after interventions, lipopolysaccharide (LPS) [30 μg/kg. ip] was injected. Then, at 4, 12- and 24-hour intervals, rats were anesthetized, and blood samples were prepared intracardially. Samples were centrifuged, and serums were separated and stored at -80 ° C. Measurement of IL-1β, IL-6, and TNF-α were conducted by the enzyme-linked immunosorbent assay (ELISA) method. Data were analyzed using the SPSS software version 19.Results: Pre-injection of apigenin at 5 mg/kg dosage were reduced TNF-α and IL-1β levels at 24-hours after LPS injection, compared to control (for both P <0.05). Pre-injection of 15 mg/kg of apigenin was reduced IL-6 level at 24-hours after LPS injection (P <0.05). Pre-injection of 30 mg/kg of apigenin were reduced TNF-α level at 4- (P <0.05), 12- (P <0.01) and 24- (P <0.01) hours, IL-1β level at 24-hours (P <0.01), and IL-6 level at 4- (P <0.05) and 24- (P <0.01) hours after LPS injection.Conclusions: Apigenin reduces proinflammatory cytokines in serum in acute inflammation induction. This impact is close to the dexamethasone effect as an anti-inflammatory steroid drug.

Improving the Production of 9,21-di​hydroxy-​20-​methyl-pregna-​4-​en-​3-​one from Phytosterols in Mycobacterium Neoaurum by Modifying Multiple Genes and Improving the Intracellular Environment

BackgroundSteroid drugs are particularly important for disease prevention and clinical treatment. However, traditional chemical methods are rarely implemented during the whole synthetic process to generate steroid intermediates due to the intricate steroid structure. Novel steroid drug precursors and their ideal bacterial strains for industrial production have yet to be developed. Among these, 9-OH-4-HP is a potential steroid drug precursor for the synthesis of corticosteroids. In this study, a combined strategy of blocking Δ1-dehydrogenation and the C19 pathway as well as improving the intracellular environment was investigated to construct an effective 9-OH-4-HP-producing strain.ResultsA Δ1-dehydrogenation-deficient strain of wild-type Mycobacterium neoaurum DSM 44074 produces 9-OH-4-HP with a molar yield of 4.8%. hsd4A, encoding a β-hydroxyacyl-CoA dehydrogenase, and fadA5 encoding an acyl-COA thiolase, were separately knocked out to block the C19 pathway in the Δ1-dehydrogenation-deficient strain. The two engineered strains could accumulate 0.59 g L-1 and 0.47 g L-1 9-OH-4-HP from 1 g L-1 phytosterols. Furthermore, hsd4A and fadA5 were knocked out simultaneously in the Δ1-dehydrogenation-deficient strain. The 9-OH-4-HP production from the Hsd4A and FadA5 double-deficient strain was 11.9% higher than that of the Hsd4A -deficient strain and 40.4% higher than that of the strain with FadA5 deficiency, and its selectivity reached 94.9%. Subsequently, the catalase katE from Mycobacterium and an NADH oxidase, nox, from Bacillus subtilis were overexpressed to improve the intracellular environment. Ultimately, 9-OH-4-HP production reached 3.58 g L-1 from 5 g L-1 phytosterols, and the selectivity of 9-OH-4-HP improved to 97%.Conclusionhsd4A and fadA5 are key enzymes in the C19 pathway for phytosterol side chain degradation. Deletion of hsd4A and fadA5 could almost entirely block the C19 pathway. Improving the intracellular environment of Mycobacterium during phytosterol bioconversion could accelerate the conversion process and enhance the productivity of target sterol derivatives.

Awareness on Use of Steroid Drugs among Football Players

Steroids are man-made chemicals that resemble hormones that are naturally made in the human body. The main role of steroids in sports is to enhance performance and reduce inflammation. The aim of the present study is to assess the awareness of the use of steroid drugs among football players and the ill effects of steroid drug intake. A Cross-sectional study was done on 100 football players and veterans registered with the State Football Association. A pretested structured questionnaire assessing the awareness on the use of steroid drugs was the survey instrument. Both male and female football players and veterans within the age group 18-50 years were selected after obtaining informed consent. The sampling method used was a simple random sampling method. The questionnaire comprised of 10 open-ended questions and were administered through Google forms. 76% of the participants responded that they use steroid drugs at least once and 24% responded that they never used steroid drugs. According to the above survey, we conclude that there is a high rate of usage of steroid drugs among football players.

Concurrence of ocular infection with Demodex folliculorum

Background The ectoparasite Demodex spp. is the most common human parasite detected in skin lesions such as rosacea, lichen, and keratosis. It is also an etiological factor in blepharitis. As Demodex spp. are involved in the transmission of pathogens that can play a key role in the pathogenesis of demodecosis, the aim was to assess the concurrence of Demodex folliculorum and bacterial infections. Methods The study involved 232 patients, including 128 patients infected with Demodex folliculorum and 104 non-infected patients. All patients underwent ophthalmological examination. The material for microbiological tests was collected from the conjunctival sac. Samples were plated on basic microbiological media and then incubated. Strains were identified based on morphological evaluation of the colonies on the media and preparations stained by the Gram method. Results Physiological flora was found in all patients infected with D. folliculorum and 9 (8.7%) participants from the control group. Only in patients infected with D. folliculorum we isolated Staphylococcus aureus (9 patients, 7%), Acinetobacter baumannii (one patient, 0.8%), Streptococcus pneumoniae (one patient, 0.8%), Gram-negative bacteria (one patient, 0.8%), and Bacillus spp. (one patient, 0.8%) in the conjunctival sac. Conclusions Patients infected with Demodex spp. should also undergo microbiological examination of conjunctival swabs. The treatment of each patient should be individualized, adapted to the clinical condition, and in cases of bacterial co-infection, an antibiotic and/or a topical steroid drug should be additionally prescribed. Furthermore, daily hygiene of the eyelid margins should be recommended.

EFFECT OF THE NEW STEROID DRUG GESTOBUTANOIL FOR HORMONE REPLACEMENT THERAPY OF GESTAGENOUS INSUFFICIENCY ON THE DEVELOPMENT OF FETUS AND OFFSPRING IN EXPERIMENTS ON RATS

A study of the embryo- and fetotoxic effects registered in the antenatal and postnatal periods of development after the intragastric administration of Gestobutanoil in two doses (equitherapeutic and 10 times higher) has been performed. It has been found that the introduction of gestagen Gestobutanoil in both doses did not affect the body weight of pregnant animals, which continued to increase progressively during pregnancy. The embryotropic effect of gestagen was revealed to be dose-dependent. The introduction of gestagen in a dose of 0.25 mg / kg (calculated equitherapeutic dose) had no adverse effect on the development of the embryo and fetus of rats. The gestagen in a dose of 2.5 mg / kg (10 equitherapeutic dose) had a negative effect on the course of pregnancy, which has been manifested by a statistically significant increase in preimplantation death and total fetal mortality, as well as a statistically significant decrease in the number of fetuses. It has been shown that intragastric administration of Gestobutanoil in doses of 0.25 and 2.5 mg / kg to female rats from the 6th to the 19th days of pregnancy did not adversely affect the early development of the first generation offspring (postnatal mortality, body mass dynamics, and development of rats’ sensory-motor reflexes).