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 ß-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.

[Microbial Community Succession in Industrial Composting with Livestock Manure and Peach Branches and Relations with Environmental Factors].

This study sets out to understand the evolution of the microbial community structure in industrial composting with livestock manure and peach branches. Pig manure, peach branches, and decomposed organic fertilizer were used as materials for composting. Changes in physical and chemical indicators and the evolution in the structure of the compost microbial community, determined by high-throughput sequencing, were analyzed. The results of physical and chemical parameters show that the pile reached the high-temperature stage on day 2, and the thermophilic period lasted for 30 days. The changes in total carbon were volatile, and there was an overall decline in the amount of TOC in the whole process of composting; The final content of TN was 20.58 g·kg-1, which was 5.90% lower compared to the initial compost. Alpha analysis indicated that a different microbial community diversity existed at different times during aerobic composting periods. At the bacterial phyla level, Firmicutes and Actinobacteria were the dominant phyla, and the proportion of relative abundance were 79.31%-95.09% and 2.98%-19.70%, respectively, in the entire compost. The relative abundance of Firmicutes and Actinobacteria were 87.36% and 9.66%, respectively, and their respective relative abundances were 79.38% and 19.70% at the end of composting. At the bacterial genus level, the dominant group changed from Clostridium_sensu_stricto_1, Terrisporobacter, and Bacillus to norank_f_Bacillaceae, Bacillus, Oceanbacillus, and Pseudogracilibacillus; Regarding the fungus phyla, the Ascomycota was the dominant phylum. For the fungus genus, the relative abundance of norank_c_Sordariomycetes gradually increased during composting, and finally was predominant group. The redundancy analysis (RDA) showed that the correlation rank between environmental factors and microbial community structure was:pH > NH4+-N > T > TOC > TN, where pH had the greatest impact on the microbial community composition. norank_c_Sordariomycetes, norank_o_Sordariales, and norank_c_Agaricomycetes may be related to the volatilization of ammonium nitrogen.

Use of taxane-containing induction chemotherapy in combination with concurrent chemoradiotherapy in Chinese patients with locally advanced nasopharyngeal carcinoma: a meta-analysis.

PURPOSE: Taxane-containing induction chemotherapy (IC) regimens in combination with concurrent chemoradiotherapy (CCRT) have been compared with non-taxane-containing IC combined with CCRT in randomized controlled trials (RCTs) in Chinese patients with advanced nasopharyngeal carcinoma (NPC). This meta-analysis aimed to systematically evaluate their clinical efficacy and safety profiling in this ethnic population. METHODS: The electronic databases, PubMed, Embase, MEDLINE, and Chinese Biomedical Database, were searched for eligible studies. The outcomes included overall response rate (ORR), 1-year survival rate, and different types of adverse events. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to evaluate the strength of the associations. RESULTS: A total of 12 RCTs (representing 835 patients) were identified. The pooled analysis showed that taxane-containing regimens had a significant improvement in ORR for nasopharyngeal lesion (OR =4.57, 95% CI =1.14-18.30, P=0.032, z=2.15) but not in cervical lymph nodes (OR =1.23, 95% CI =0.65-2.36, P=0.532, z=0.64) and in 1-year survival rates (OR =1.19, 95% CI =0.10-14.82, P=0.893, z=0.13) compared with non-taxane-containing regimens. Regarding the adverse events and toxicities, grade 3-4 leukopenia and neutropenia were significantly different between the two groups (P<0.001) in favor of the non-taxane-containing regimens, but grade 3-4 vomiting was significantly different between the two groups (P<0.005) in favor of the taxane-containing regimens. CONCLUSION: When combined with CCRT, taxane-containing IC regimens may be more efficient for short-term local control in Chinese patients with locally advanced NPC than the non-taxane-containing IC regimens. Moreover, the major toxic effects, which were bone marrow suppression, could be tolerated by majority of patients. More long-term follow-up and high-quality trials of NPC are needed to validate our findings.

[Effect of magnesium deficiency on photosynthetic physiology and triacylglyceride (TAG) accumulation of Chlorella vulgaris].

As an excellent biological resource, Chlorella has wide applications for production of biofuel, bioactive substances and water environment restoration. Therefore, it is very important to understand the photosynthetic physiology characteristics of Chlorella. Magnesium ions play an important role in the growth of microalgae, not only the central atom of chlorophyll, but also the cofactor of some key enzyme in the metabolic pathway. A laboratory study was conducted to evaluate the effects of magnesium deficiency on several photosynthetic and physiological parameters and the triacylglyceride (TAG) accumulation of the green alga, Chlorella vulgaris, in the photoautotrophic culture process. Chlorella vulgaris biomass, protein, chlorophyll a and chlorophyll b contents decreased by 20%, 43.96%, 27.52% and 28.07% in response to magnesium deficiency, while the total oil content increased by 19.60%. Moreover, magnesium deficiency decreased the maximal photochemical efficiency F(v)/F(m) by 22.54%, but increased the non-photochemical quenching parameters qN. Our results indicated the decline of chlorophyll caused by magnesium, which affected the photosynthesis efficiency, lead to the growth inhibition of Chlorella vulgaris and affected the protein synthesis and increased the triacylglyceride (TAG) accumulation.

Pre-endurance training prevents acute alcoholic liver injury in rats through the regulation of damaged mitochondria accumulation and mitophagy balance.

AIM: The aim of this study is to investigate the effect of pre-endurance training on the prevention of alcohol-induced acute hepatic injury and on hepatic mitophagy. METHODS: Forty-eight male Sprague-Dawley rats were randomly divided into four groups: (1) control group, (2) 12-week exercise training group, (3) 5-day alcohol intake group, and (4) 12-week exercise training plus 5-day alcohol intake group. The rats were examined to determine the following: BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), hypoxia-inducible factor-1α (HIF-1α), cytochrome P450 2E1 (CYP2E1), alcohol dehydrogenase (ADH), microtubule-associated protein 1 light chain 3 (LC3II), Beclin1 mRNA and protein expressions, mitochondrial reactive oxygen species (ROS) production, mitochondrial thiobarbituric acid-reactive substances (TBARS) level, aconitase and ATP synthase activities, mitochondrial inner membrane potential, NADH/NAD(+) ratio, triglyceride (TG), the number of mtDNA and mitochondrial respiration functions in liver tissue, and serum ALT and AST. RESULTS: Pre-endurance training attenuated acute alcohol treatment-induced increase in mitochondrial TBARS, ROS production, NADH/NAD(+) ratio, state 4 respiration rate, TG, serum ALT and AST, as well as BNIP3, HIF-1α, LC3II, and Beclin 1 mRNA and protein levels, however, CYP2E1 and ADH mRNA and protein levels unchanged. Meanwhile, it attenuated the acute alcohol intake-induced decrease in aconitase activity, inner mitochondrial membrane potential (Δψ), ATP synthase activity, state 3 respiration rate, respiratory control ratio, and the number of mtDNA. CONCLUSION: Pre-endurance training can decrease acute alcohol intake-induced damaged mitochondria accumulation and reduced acute alcohol intake-induced mitophagy, which built a new balance between mitophagy and damaged mitochondria accumulation.

[Effect of abamectin insecticide on the microbial community in broccoli phyllosphere].

The indigenous microbial communities within the plant phyllosphere are highly diverse and include many different species of bacteria, filamentous fungi, yeasts and algae which play important ecological roles. This study was the first attempt to assess the impact of abamectin treatments on microbial communities of broccoli phyllosphere using two culture-independent techniques of phospholipid fatty acid analysis (PLFA) and terminal restriction fragment length polymorphism (T-RFLP). Results showed that low concentration of abamectin treatments did not affect the microbial biomass and microbial community structure of broccoli phyllosphere significantly. However, high concentration of abamectin treatments significantly change the microbial community structure including a decrease of total and bacterial biomass, and a decrease in the ratio of Gram-positive bacteria to Gram-negative bacteria, but did not change the fungal biomass. Moreover, PLFA suggested that the number of unsaturated and cyclopropane phospholipid fatty acids (PLFAs: 16: 1omega9t, 18: 1omega7, cy17:0 and cy19:0) increased with high concentration abamectin treatment, while the saturated PLFAs i15:0, a15:0, i16:0 and a17:0 decreased. The appearance terminal restriction fragments (T-RFs: 58, 96, 236 and 420 bp) indicated that some bacteria might play a significant role in abamectin degradation in broccoli phylosphere, while the disappeared T-RFs (51, 89, 99, 338, 66, 223 and 482 bp) implied some other bacteria might potentially serve as microbial indicator of abamectin exposure.

[Modern pharmacodynamics research and clinic application of huangqintang].

To collect the researching documents of recent years about the pharmacodynamics research and modem clinic application of Huangqintang and its relative prescription, and to discuss the current general situation of the pharmacodynamics research and clinic application. Huangqintang has the pharmacodynamics effect such as resist bacteria and inflammation, adjust immune system, ease pain, protect the liver, lower the index of aminotransferase, anti-cancer and virus, lower the physical temperature, and calm down the emotion. The original and relative prescription can be commonly used in curing intestines disease such as diarrhea, acute enteritis and gastritis, ameba diarrhea, etc. It can also be used in internal medicine, gynecology, dermatology, etc.

A novel acidophile community populating waste ore deposits at an acid mine drainage site.

Waste ore samples (pH 3.0) were collected at an acid mine drainage (AMD) site in Anhui, China. The present acidophilic microbial community in the waste ore was studied with 16S rRNA gene clone library and denaturing gradient gel electrophoresis (DGGE). Eighteen different clones were identified and affiliated with Actinobacteria, low G + C Gram-positives, Thermomicrobia, Acidobacteria, Proteobacteria, candidate division TM7, and Planctomycetes. Phylogenetic analysis of 16S rRNA gene sequences revealed a diversity of acidophiles in the samples that were mostly novel. It is unexpected that the moderately thermophilic acidophiles were abundant in the acidic ecosystem and may play a great role in the generation of AMD. The result of DGGE was consistent with that of clone library analysis. These findings help in the better understanding of the generation mechanism of AMD and in developing a more efficient method to control AMD.

[Clinic utilization of Guizhi decoction in modern times].

To discuss the general situation of the clinic utilization of Guizhi decoction in modem times. Look up the literature about the clinic utilization research of guizhi decoction in recent years, then to analyze and coordinate the useful informations. Guizhi decoction is widely used in clinic areas. The range of Guizhi decoction' treatment involves circulation, immunity, procreation, endocrine, digestion, nerve, etc. and covers various clinic illness such as internal medicine, surgery, gynaecology and obstetrics, pediatrics, ophthalmology and otorhinolaryngology, stomatology, etc.

[Pharmacodynamics research of Guizhi decoction in modern times].

To discuss the present general situation of the pharmacodynamics research of Guizhi decoction. Look up the literature about the research of pharmacodynamics experiments of Guizhi decoction in recent years, then to analyze and coordinate the useful information. Guizhi decoction has the bilateral accommodation for testing animals'suboriferous, body temperature, blood pressure, peristalsis and immune function. And it has the pharmacodynamics effect in resisting bacteria, virus, pain and allergy, reducing the blood sugar, increasing the cardiac muscle blood flux, improving the digestion and transmition of stomach and intestines, and easing up spasmus. It can also prevent the arthritis, and cure the cervical spondylosis and chronic pancreatitis.

[Biodiversity of acidophiles in the sediment at an acid mine drainage site].

A sediment sample was collected at an acid mine drainage site in Anhui. The acidophiles composition and community structure in the sediment was studied with 16S rDNA clone library and denaturing gradient gel electrophoresis (DGGE). The results indicated that the acidophiles in the sediment were novel compared with the data in GenBank. The acidophiles affiliated with Acidobacteria, beta/gamma-Proteobacteria, delta-Proteobacteria, Nitrospira, Candidate Division TM7, low G + C Gram-positive. delta-Proteobacteria were major group in this acidic ecosystem. The research of aicdophiles community in sediment can help us develop high efficient method to treat acid mine drainage (AMD).

[Stereotactic microelectrode-guided posteroventral pallidotomy for Parkinson's disease].

OBJECTIVE: To assess postoperative effects of microelectrode-guided posteroventral pallidotomy (PVP) for Parkinson's disease. METHODS: Intraoperative microelectrode recordings and microstimulation were used to explore the globus pallidus to performance of posteroventral pallidotomy in 48 patients with Parkinson's disease (47 unilateral and 1 bilateral). Assessment was made at baseline preoperatively and at 6 months intervals postoperatively, with unified Parkinson's disease rating scale (UPDRS). RESULTS: All patients were significantly improved on the limbs contralateral to the lesion side 6 - 34 months after operation (mean 24 months). The improvement was seen in the 'on' or 'off' state: UPDRS scores with patients on levodopa were improved by an average of 28.7%, while off medication scores showed reductions (47.6%) at 24 months. There were no deaths and no visual complications, but there were 4 patients (8.3%) of a delayed contralateral limbs dystonia after pallidotomy. CONCLUSIONS: The techniques of microelectrode recording and microstimulation indicate the location of the internal capsule and optic tract, which allow easy identification of these structure and facilitate PVP target in conjunction with radiofrequency microelectrode stimulation.