Production of sesquiterpene patchoulol in mitochondrion-engineered Saccharomyces cerevisiae.

Patchoulol is a natural sesquiterpene, which is widely used in perfumes and cosmetics. In the work, the mitochondria of S. cerevisiae were engineered for patchoulol production. The patchoulol titer of mitochondria-compartmentalized strain (1.79 mg/L) was 2.71-fold higher than that of control strain (0.66 mg/L) using genome-integrated patchoulol synthase, indicating that mitochondria compartmentation resulted in higher concentration of FPP (farnesyl pyrophosphate) precursor for patchoulol production. Moreover, when fused FPP synthase and patchoulol synthase was overexpressed in the strain with a mitochondria-localized DMAPP (dimethylallyl diphosphate) pathway, the production of patchoulol increased significantly to 19.24 mg/L, indicating more precursors were provided for patchoulol production. Nevertheless, the introduction of excess foreign proteins into mitochondria might cause a certain stress on mitochondria and showed a negative effect on the growth of yeast cells, which could hinder the expression of foreign pathways and reduce the patchoulol production. In conclusion, mitochondria-engineered yeast cells showed important potential for the enhanced biosynthesis of patchoulol, and further engineering could be considered based on the present work.

Bacterial community in saline farmland soil on the Tibetan plateau: responding to salinization while resisting extreme environments.

BACKGROUND: Salinization damages the health of soil systems and reduces crop yields. Responses of microbial communities to salinized soils and their functional maintenance under high salt stress are valuable scientific problems. Meanwhile, the microbial community of the salinized soil in the plateau environment is less understood. Here, we applied metagenomics technology to reveal the structure and function of microorganisms in salinized soil of the Tibetan Plateau. RESULTS: The diversity of composition and function of microbial community in saline soil have changed significantly. The abundances of chemoautotrophic and acidophilic bacteria comprising Rhodanobacter, Acidobacterium, Candidatus Nitrosotalea, and Candidatus Koribacter were significantly higher in saline soil. The potential degradation of organic carbon in the saline soil, as well as the production of NO and N2O via denitrification, and the production of sulfate by sulfur oxidation were significantly higher than the non-saline soil. Both types of soils were rich in genes encoding resistance to environmental stresses (i.e., cold, ultraviolet light, and hypoxia in Tibetan Plateau). The resistance of the soil microbial communities to the saline environment is based on the absorption of K+ as the main mechanism, with cross-protection proteins and absorption buffer molecules as auxiliary mechanisms in our study area. Network analysis showed that functional group comprising chemoautotrophic and acidophilic bacteria had significant positive correlations with electrical conductivity and total sulfur, and significant negative correlations with the total organic carbon, pH, and available nitrogen. The soil moisture, pH, and electrical conductivity are likely to affect the bacterial carbon, nitrogen, and sulfur cycles. CONCLUSIONS: These results indicate that the specific environment of the Tibetan Plateau and salinization jointly shape the structure and function of the soil bacterial community, and that the bacterial communities respond to complex and harsh living conditions. In addition, environmental feedback probably exacerbates greenhouse gas emissions and accelerates the reduction in the soil pH. This study will provide insights into the microbial responses to soil salinization and the potential ecological risks in the special plateau environment.

EphA2 antagonism alleviates LPS-induced acute lung injury via Nrf2/HO-1, TLR4/MyD88 and RhoA/ROCK pathways.

Eph receptor tyrosine kinases have a wide range of biological functions and have gradually been recognized increasingly as key regulators of inflammation and injury diseases. Although previous studies suggested that EphA2 receptor may be involved in the regulation of inflammation and vascular permeability in injured lung, the detailed effects of EphA2 on LPS-induced acute lung injury (ALI) are still inadequate and the underlying mechanism remains poorly understood. In this study, we detected the effects of EphA2 antagonism on inflammation, pulmonary vascular permeability and oxidative stress in LPS-induced ALI and investigate the potential mechanism. Our results showed that EphA2 antagonism markedly inhibited the cytokines release and inflammatory cells infiltration in BALF, prevented the LPS-induced elevations of MPO activity and MDA level in lung tissues. Our study also found that EphA2 antagonism significantly decreased the wet/dry ratios, reduced the Evans blue albumin extravasation in lung tissues and obviously alleviated the LPS-induced increment of pulmonary vascular permeability. Mechanistically, EphA2 antagonism significantly increased the activation of Nrf2 along with its target antioxidant enzyme HO-1 and inhibited the expressions of TLR4/MyD88 in lung tissues and A549 alveolar epithelial cells. Furthermore, EphA2 antagonism dramatically inhibited the LPS-evoked activations of RhoA/ROCK in lung tissues. In conclusion, our data indicate that EphA2 receptor plays an essential role in LPS-induced ALI and EphA2 antagonism has protective effects against LPS-induced ALI via Nrf2/HO-1, TLR4/MyD88 and RhoA/ROCK pathways. These results suggest that antagonism of EphA2 may be an effective therapeutic strategy for the treatment of ALI.

Metabolic Adaptation of Mycobacterium neoaurum ATCC 25795 in the Catabolism of Sterols for Producing Important Steroid Intermediates.

To understand the adaptation of Mycobacterium neoaurum ATCC25795 ( Mn) in sterol catabolism and steroid production, we used integrated transcriptome and proteome analysis to identify the biochemical pathways utilized in this process. Metabolic alterations during sterol catabolism center on propionyl-CoA pools. Generally, enhanced pathways for metabolizing propionyl-CoA were found in Mn, which were tightly coordinated with cell-envelope biosynthesis. The cells responded to sterol substrates and toxic steroid products by changing the composition of the cell envelope. This adaptive mechanism allowed Mn to use minimally water-soluble sterol as a carbon source. Several putative efflux proteins were found to be induced in Mn. They probably transported products to the extracellular environment, protecting the cells against high intracellular levels of toxic intermediates, inhibition of which also influenced sterol uptake. The work provided various targets for rational engineering of robust Mn with powerful sterol-uptake capacity and strong tolerance to toxic products for the steroid industry.

Integrated Transcriptome and Proteome Studies Reveal the Underlying Mechanisms for Sterol Catabolism and Steroid Production in Mycobacterium neoaurum.

Integrated transcriptome and proteome studies were performed to investigate sterol biotransformation in wild-type Mycobacterium neoaurum ATCC 25795 ( Mn) and the mutant strains producing steroid intermediates. Transcriptome and proteome studies indicated that several metabolic activities were noticeably dynamic, including cholesterol degradation, central carbon metabolism, cell envelope biosynthesis, glycerol metabolism, and transport. Interestingly, a poor overall correlation between mRNA and translation profiles was found, which might contribute to the metabolic adaptation in cholesterol catabolism. A gene cluster covering 111 genes was discovered to encode for cholesterol catabolism in Mn. Generally, transcription and/or translation of the genes in KstR1 regulon was upregulated, and the induction of genes in KstR2 regulon was not as significant as that of KstR1 regulon. Several induced genes showing potential roles for cholesterol catabolism were found. Further identification of these genes and investigation of the correlation among key metabolic activities could help for the development of efficient steroid-producing strains.

The utility of using the bispectral index-Vista for detecting cross-clamping decline in cerebral blood flow velocity.

BACKGROUND: Patients undergoing carotid endarterectomy for extracranial internal carotid artery stenosis are at risk of cerebral ischemia/hypoperfusion. Criterion recommended by European and American committees to determine whether to place a shunt consisted of a decline in transcranial Doppler ultrasonography-measured middle cerebral artery blood flow velocity (MCBFV) to < 30% to 40% of intraoperative preclamp value. OBJECTIVE: To assess the discriminative power of the bispectral index (BIS)-Vista monitor for detecting a 40% decline in MCBFV with cross-clamping. METHODS: In 20 patients undergoing carotid endarterectomy under remifentanil/propofol anesthesia, BIS-Vista data, MCBFV, and pulsatility index from bilaterally mounted BIS-Vista and transcranial Doppler monitors were continuously recorded. RESULTS: Coefficient of determination revealed good correlation (r = 0.763) between ipsilateral BIS-Vista and MCBFV after cross-clamping. BIS-Vista exhibited a high discriminative power of 0.850 (95% confidence interval, 0.455-0.966) area under the receiver-operating characteristic curve in detecting an ipsilateral 40% MCBFV decline. Two-way analysis of variance (location by time) suggests that BIS-Vista exhibited a global decline; ie, both BIS-Vistas declined when 1 carotid on either side was clamped because there was no significant interhemispheric difference (P = .112) in mean BIS-Vista values over time. CONCLUSION: Although we demonstrated good correlation and high discriminative power of the BIS-Vista monitor in depicting a MCBFV decline that could serve as indicator of decline in cerebral activity, BIS-Vista cannot be considered a reliable indicator of cerebral ischemia/hypoperfusion that could replace transcranial Doppler monitoring to determine whether a shunt is to be placed.

BIS-vista occipital montage in patients undergoing neurosurgical procedures during propofol-remifentanil anesthesia.

BACKGROUND: Neurosurgical procedures that require a frontal approach could be an impediment for a successful Bispectral Index (BIS) frontal sensor placement. The aim of this study was to explore the utility of using the new BIS-Vista monitor (Aspect Medical Systems, Newton, MA) for occipital sensor placement in the patients undergoing brain neurosurgical procedures during propofol-remifentanil anesthesia. METHODS: Two BIS Quatro sensors (Aspect Medical Systems, Newton, MA) mounted on the occipital and frontal regions were connected to two BIS-Vista monitors at three anesthesia states: before induction, during anesthesia maintenance, and recovery. RESULTS: There were significant differences before induction (P = 0.0002) and at anesthesia maintenance (P = 0.0014) between mean +/- SD occipital (83.4 +/- 4.8, 66.7 +/- 7.2) and frontal (93.1 +/- 3.4, 56.9 +/- 9.1) BIS-Vista values. During anesthesia recovery, there was no difference (P = 0.7421) between occipital (54.6 +/- 9.3) and frontal (53.1 +/- 7.3) BIS-Vista values. Bland and Altman analysis revealed a BIS-Vista negative-bias (limits of agreement) of -9.7 (+1.1, -20.5) before anesthesia induction, +9.8 positive-bias (+22.8, -1.7) during anesthesia maintenance, and -0.9 bias (+10.9, -12.8) during anesthesia recovery. CONCLUSION: We demonstrated that not only the regional limits of agreement are too wide to allow data of the two montages to be used interchangeably but also the variation is a function of anesthetic depth. However, keeping in mind a relatively consistent BIS-Vista -10 bias before induction and +10 bias during anesthesia maintenance with limits of agreement of approximately +/-11 BIS units, approximately double the clinically acceptable less than 10 BIS units level of agreement, BIS-Vista off-label occipital montage might be helpful in following a trend of propofol-remifentanil anesthesia in individual cases where frontal access is particularly difficult.

[Exploration and application by combining human patient simulator drivers of critical and emergency events with micro-division teaching during training primary residents].

OBJECTIVE: To explore the feasibility of combining human patient simulator (HPS) drivers with micro-division teaching during primary resident training by George Miller' medical education step-wised principle of goal and competence. METHOD: The 20 residents from department of anesthesia who are less than 3 years in clinical training were randomized into two Groups. The all residents in Group T received HPS training, and no HPS training in Group N. In simulation system, we designed 8 programmed critical and emergency events. We disassembled and quantified the programmed design and training process with Micro-division teaching principle. The training mode was run by test-feedback-self-analysis-instructor guide-summarize-re-practice-retest. The feedback assessment from all residents were collected after finishing HPS training. RESULTS: The training score in Group T was much higher than Group N (P < 0.05). CONCLUSIONS: The training mode with HPS is an accessory teaching means because it can improve clinical thinking and skill training of primary resident.

Bispectral index monitoring of a narcolepsy-cataplexy episode during regional anesthesia.

Narcolepsy or Gélineau syndrome is an extremely incapacitating chronic sleep disorder of unknown etiology that is characterized by uncontrollable attacks of deep sleep and is typically associated with cataplexy sudden loss of muscle tone. The Bispectral Index (BIS), an electroencephalographic-derived cerebral monitor, used for monitoring the effects of anesthetic/hypnotic drugs was shown to correlate to various conditions that could influence the eletroencephalogram. We assessed the utility of using BIS for monitoring a possible narcolepsy-cataplexy episode and whether a distinctive BIS profile might offer an early warning of an impending narcoleptic/cataplectic spell. We recorded both hemispheres, using two synchronized BIS-XP monitors, during a narcolepsy-cataplexy episode in a 57-yr-old male patient undergoing lower limb surgery under femoral nerve block regional anesthesia. The patient went through three stages: first a prodromal "intermittent low-vigilance" phase interrupted by high electromyographic activity. This was followed by a second "continuous low-vigilance" phase of BIS around 75 with low electromyographic activity, ending with a third "nonresponsive vigilance" phase of a full-blown narcolepsy-cataplexy episode of BIS around 45 with complete loss of muscle power. The purpose of presenting this report is to emphasize the fact that narcoleptic patients can still run the risk of loss of consciousness with atonia under regional anesthesia, and such an undesirable complication cannot be under-estimated. BIS monitoring is a simple method that could offer an early warning of an imminent episode, with its associated hazards, in patients with narcolepsy-cataplexy undergoing surgery under regional anesthesia.

[A case-control study on the risk factors for attempted suicide in patients with major depression].

OBJECTIVE: To understand the environmental risk factors on attempted suicide in patients with major depression, and to study the interaction between factors as single nucleotide polymorphism(SNP) of TPH2 gene rs7305115 associated to attempted suicide in major depression. METHODS: Paired case-control study on 215 suicide attempters with major depression (92 male, 123 female) and molecular biological techniques were used to study the relation between TPH2 gene rs7305115 SNP,interrelated environmental factors and the rate of attempted suicide. Controls were paired with cases according to the same gender, similar age (no more than 3 years) and from the same district. RESULTS: There were remarkably significant differences in gene types and gene frequency between case and control groups (P < 0.001). Data from multivariate conditional logistic regression model analysis showed that hopelessness, negative life-events and family history of suicide were relationship of attempted suicide in patients with major depression with OR values as 0.33 (95% CI: 0.22-0.99), 7.68 (95% CI: 5.79-13.74), 6.64 (95% CI: 2.48-11.04), 2.98 (95% CI: 1.17-5.04) respectively. There was no first level interaction between any of the two risk factors. CONCLUSION: Results from the study supported the idea that hopelessness, negative life-events and family history of suicide were risk factors of attempted suicide in major deprbssion while TPH2 gene rs7305115 A/A might be the protective factor.

[Preparation of recombinant HIV-TATm-survivin (T34A) protein and its pro-apoptosis activity to cancer cells in vitro].

As a novel member of the IAP (Inhibitor of apoptosis protein) family, survivin was observed to be expressed in most human cancerous cells. Fusion protein TATm-survivin (T34A) has drawn considerable attention because it is a potential anti-tumor protein that can be transduced into cancer cell with the help of HIV-TAT domain. In this study, the cDNA encoding survivin was cloned by RT-PCR from human breast cancer cell lines B-Cap-37. An expression vector of pRSET-B-HIV-tatm-survivin (T34A) was constructed by PCR after survivin (T34A) was mutated by site-directed mutagenesis. Subsequently, the resultant plasmid was transformed into E. coli BL21 (DE3). Recombinant HIV-TATm-Survivin (T34A) protein was expressed efficiently with 0.5mM IPTG as inducer, reaching a yield of 650mg/liter (as inclusion body) in fermentation culture. The inclusion bodies were solubilized, refolded and purified to a purity of 96% by ion exchange chromatograghy and size-exclusion chromatography. Remarkable effects of the purified recombinant HIV-TATm-Survivin (T34A) on the morphology of cell line SW1990 and B-Cap-37 were observed after being administrated for 4h. MTT assay showed recombinant HIV-TATm-survivin (T34A) protein could inhibit significantly cell proliferation of SW1990 and B-Cap-37 and SSMC-7721 in vitro. Apoptosis rate and cell circle of SW1990 and B-Cap-37 that had been treated with target protein (final concentration 30 microg/mL) were detected with flow cytometry. Results revealed that more than 65% cancer cells were arrested at G1 phase. The study suggested that TATm-survivin (T34A) protein was a hopeful protein drug in the treatment of cancers by facilitating apoptosis of cancer cells. Key words recombinant HIV-TATm-Survivin (T34A), expression and purification, pro-apoptosis bioactivity, SW1990 and B-Cap-37 cancer cell lines