The changes in the contents of free amino acids in hemolymph of Anopheles stephenst were determined by automatic amino acid analyzer.The changes in hemolymph protein were determined by ultraviolet absorption method.Free amino acids in hemolymph of infected mosquitoes were compared with those in noninfected mosquitoes.At 4 days after blood meal,6 kinds of amino acids decreased markedly,and 5 kinds of amino acids increased markedly; at 7 days after blood meal,4 kinds of amino decreased markedly,while 7 kinds of amino accids increased markedly; at 11 days after cids blood meal,9 kinds of amino acids decreased markedly,and 4 kinds of amino acids increased remarkably.The protein concentration of infected mosquitoes was higher than that of noninfected ones.

Objective To establish a sensitive,valid and rapid high performance liquid chromatography-trap mass spectrometry (HPLC-MS) method for determining carbocisteine concentration in human plasma. Methods Plasma samples were pretreated by 100 mL/L perchloric acid to remove the interference protein. The supernatant was separated by reverse phase HPLC,electrospray MS was used to detect carbocisteine in plasma samples,and selective ion method of multiple reaction modes was used to determine the concentration of carbocisteine. Results The limit of detection was 0.01 ?g/mL and the linear range was 0.2-200.0 ?g/mL. The recovery rate was within the range of 89.2%-105.6%. Conclusion The proposed method could be used to determine carbocisteine in biological samples.

Ethanol tolerance is related to the expression of multiple genes, and genome-based engineering approaches are much more efficient than manipulation of single genes. In this study, ultraviolet (UV) mutagenesis, dielectric barrier discharge (DBD) air plasma mutagenesis, and artificial transcription factor (ATF) technology were adopted to treat an industrial yeast strain S. cerevisiae Sc4126 to obtain mutants with improved ethanol tolerance. Mutants with high ethanol tolerance were obtained, and the ratio of positive mutants was compared. Among the three approaches, the rate of positive mutation obtained by ATF technology was 10- to 100-folds of that of the two other methods, with highest genetic stability, suggesting the ATF technology promising for rapid alteration of phenotypes of industry yeast strains for efficient ethanol fermentation.
Adaptation, Physiological ; drug effects ; Drug Resistance, Fungal ; genetics ; Ethanol ; pharmacology ; Fungal Proteins ; genetics ; metabolism ; Industrial Microbiology ; methods ; Mutagenesis ; Saccharomyces cerevisiae ; drug effects ; genetics ; growth & development

BACKGROUND: Activated by Ca2+, phospholipase A2 will aggravate the influx of Ca2+ or the release of intracellular Ca2+, and then forms a vicious circle, which results in a continuous increase in free calcium level and leads to server injury in neural cells.OBJECTIVE: To discuss the protective effects of nimodipine on acute ischemic brain injury caused by activation of phospholipase A2.DESIGN: A completely randomized controlled trial.SETTING: Intensive Care Unit (ICU) of Children's Hospital, Chongqing Medical University.MATERIALS: From January 2001 to October 2003, it was completed at the ICU of Children' s Hospital, Chongqing Medical University. Thirty male rats were selected and divided into sham operation group, ischemia group and nimodipine treated group randomly, with 10 rats in each group.METHODS: In sham operation group, the right common carotid artery was identified by blunt dissection without ligation under anesthesia in rats. In ischemia group, at 30 minutes before cerebral ischemia, 2 mL saline was injected intraperitoneally. In nimodipine treated group, at 30 minutes before cerebral ischemia, 0.2 g/L nimodipine (2 mg/kg) was injected intraperitoneally. In all the three groups, the duration between ischemia and decollation was 120 minutes. Rats were decollated under anesthesia and their brains were taken out to assess the activity of phospholipase A2, the free calcium level in brain cells, the brain water content and the changes in mRNA levels of type Ⅱ phospholipase A2 (secretive phospholipase A2) and type Ⅳ phospholipase A2 (cytoplasmic phospholipase A2) in brain tissue.pholipase A2) and type Ⅱ phospholipase A2 (cytoplasmic phospholipase A2)in brain tissue were measured in rats in all the groups.pholipsse A2 in brain tissue: In ischemia group and nimodipine treated group, the activity of phospholipase A2 were higher than that in sham operation group [(57.8 ±7.2),(42.5±6.1), (17.1±5.3)%, P＜ 0.05-0.01], and it was a litter lower in nimodipine brain cells: It was higher in nimodipine treated group and ischemia group than that in sham operation group [(775.8±105.5), (497.2±45.9), (103.8±10.3) μmol/L,P ＜ 0.05-0.01], and it was lower in nimodipine group than in ischemia group (P ＜ 0.01).that in sham operation group [(82.9±0.5), (80.0±1.1), (72.1±0.01)%, P ＜ 0.05-0.01], and it was lower in nimodipine treated group than that in ischemia group (Ppase A2 could be detected in brain tissue. And the mRNA level of type Ⅱ phospholipase A2 in brain tissue was very low. At 120 minutes after ischemia, mRNA of type Ⅱ phospholipase A2 was detectable and the expression of type Ⅱ phospholipase A2 was increased. Compared to ischemia group, the expression of type Ⅱ phospholipase A2 was not decreased in nimodipine treated group while the expression of type Ⅱ phospholipase A2 was decreased.CONCLUSION: Nimodipine is capable of decreasing the free calcium level in brain cells, the activity of phospholipase A2 in brain tissue and the brain water content after ischemia. However, it cannot significantly inhibit the expressions of type Ⅱ phospholipase A2 and type Ⅱ phospholipase A2 after cerebral ischemia.

Objective To analyze the etiology of fever of unknown origin (FUO).Methods A total of 372 patients with FUO who hospitalized in Capital Medical University Affiliated Beijing Friendship Hospital were retrospectively analyzed from January 2003 to August 2013.All the patients were divided into two groups:group A (January 2003-December 2007) and group B (January 2008-August 2013).Diagnosis rate,duration of hospitalization (days) and time to diagnosis between the two groups were artificially compared.Results Of the 372 FUO cases,336 were positively diagnosed with a diagnosis rate of 90.3％.Infectious diseases were still the primary causes of FUO (60.2％),including 72 cases (32.1％) of tuberculosis.Connective tissue diseases accounted for 12.9％ of the FUO cases,malignancies were 8.3％,and miscellaneous diseases were 8.9％.Yet thirty six patients (9.7％) could not be confirmed until they were discharged from hospital.The duration of fever in patients with malignancies was longer than that with infectious diseases [60.0 (30.0,90.0) days vs 30.0 (20.0,60.0) days,P =0.003].Time to diagnosis of connective tissue disease and malignancies was longer than infectious diseases [(12.0 (7.3,18.8) days and 11.0 (7.0,18.0) vs 5.0 (3.0,8.0) days,both P values =0.000].The duration of hospitalization in group A was longer than that of group B [17.0(12.0,30.0) days vs 14.0(10.0,20.0) days,P =0.000].The diagnosis rate and time to diagnosis of group A were similar with those of group B.The proportion of connective tissue diseases in group A was higher than group B(18.1％ vs 9.2％,x2 =6.201,P =0.013).The proportion of infectious disease,malignancies and miscellaneous diseases was not significantly different between the two groups.Conclusions Infectious diseases are the major causes of FUO,and the most common cause is tuberculosis.Connective tissue diseases and malignancies are the second and third causes of FUO.The duration of fever and time to diagnosis are significantly different between the different origins.

BACKGROUND:Oligodendrocytes are mostly differentiated from oligodendrocyte precursor cel s. A suitable medium and cel seeding density have a significant impact on the process of the isolation of oligodendrocyte precursor cel s to obtain oligodendrocytes. OBJECTIVE:To explore the optimization of oligodendrocyte culture conditions. METHODS:Oligodendrocyte precursor cel s isolated from the newborn rats 48 hours after birth were cultured in DMEM/high glucose medium or DMEM/F12 medium using seeding densities of 2×104 cel s/cm2, 4×104 cel s/cm2, 8×104 cel s/cm2, 16×104 cel s/cm2, 32×104 cel s/cm2, and 64×104 cel s/cm2, respectively. Oligodendrocyte precursor cel s were induced to differentiate into oligodendrocytes at 72 hours after cel adhesion. Morphology of differentiated oligodendrocyte precursor cel s were observed under a light microscope, and the differentiation results were identified by immunofluorescence staining after 7-day induced differentiation. RESULTS AND CONCLUSION:Morphology of oligodendrocyte precursor cel s were recognized when cultured in DMEM/high glucose medium or DMEM/F12 medium using seeding densities of 2×104 cel s/cm2, 4×104 cel s/cm2, and 8×104 cel s/cm2, respectively. Immunofluorescence staining showed that myelin basic protein-positive cel s were found after 7-day induced differentiation, and the positive cel number were 16.40±3.30, 49.95±2.33, and 76.95±4.86 in DMEM/F12 medium, and 12.65±2.53, 32.10±1.17, and 54.05±1.56 in DMEM/high glucose medium (P<0.05). These findings indicate that DMEM/F12 medium is more suitable for culturing oligodendrocyte precursor cel s compared with DMEM/high glucose medium to some extent. The number of differentiated oligodendrocytes was gradual y increased with the enhanced seeding density of oligodendrocyte precursor cel s, and the seeding densities from 4×104 to 8×104 cel s/cm2 were appropriate for the observation of cel morphology.

Biofuels are renewable and environmentally friendly, but high production cost makes them economically not competitive, and the development of robust strains is thus one of the prerequisites. In this article, strain improvement studies based on the information from systems biology studies are reviewed, with a focus on their applications on stress tolerance improvement. Furthermore, the contribution of systems biology, synthetic biology and metabolic engineering in strain development for biofuel production is discussed, with an expectation for developing more robust strains for biofuel production.
Biofuels ; Genetic Engineering ; methods ; Industrial Microbiology ; methods ; trends ; Lignin ; metabolism ; Saccharomyces cerevisiae ; genetics ; metabolism ; physiology ; Synthetic Biology ; methods ; Systems Biology ; methods

In order to save energy consumption for the downstream processes, consecutive very-high-gravity batch fermentation was developed for ethanol production with the self-flocculating yeast Saccharomyces cerevisiae flo. The fermentation system exhibited a high ethanol productivity of 8.2 g/(L x h) with average ethanol concentration around 120 g/L. However, deterioration of the sedimentation performance of yeast flocs was observed as the consecutive fermentation process was prolonged, which significantly extended the time required for yeast flocs to separate from fermentation broth, and exaggerated the inhibition of high ethanol concentration on the yeast flocs, making them quickly lost viability and the fermentation system interrupted after 11 consecutive batches. Experimental results illustrated that decrease of the size of yeast flocs was the main reason, which could be prevented by stimulating the propagation of the yeast flocs. Thus, yeast was purged from the fermentation system at the end of each batch, and the concentration of yeast flocs within the fermentor was maintained at a relatively low level to stimulate their propagation. Although the ethanol productivity was decreased to 4.0 g/(L x h), the size of yeast flocs was stabilized after 10 consecutive batches and maintained for another 14 batches without further decrease, indicating the fermentation system could be operated reliably.
Ethanol ; analysis ; metabolism ; Fermentation ; Flocculation ; Industrial Microbiology ; economics ; methods ; Saccharomyces cerevisiae ; growth & development ; metabolism

Directed evolution of transcription factors can be employed to effectively improve the phenotypes which are controlled by multiple genetic loci. In this study, we used error-prone PCR for the directed evolution of SPT3, which is the component of yeast Spt-Ada-Gcn5-acetyltransferase (SAGA) complex responsible for the transcription of stress-related genes, and studied its effect on the improvement of ethanol tolerance. Mutant library was constructed by ligating the error-prone PCR products with a modified pYES2.0 plasmid, and the expression plasmids were subsequently transformed to yeast industrial strain Saccharomyces cerevisiae 4126. One mutant strain M25 showing superior growth in presence of 10% ethanol was selected. M25 produced 11.7% more ethanol than the control strain harboring the empty vector when 125 g/L glucose was used as substrate. This study revealed that SPT3 is an important transcription factor for the metabolic engineering of yeast ethanol tolerance.
Directed Molecular Evolution ; methods ; Drug Resistance, Fungal ; Drug Tolerance ; Ethanol ; metabolism ; pharmacology ; Industrial Microbiology ; methods ; Saccharomyces cerevisiae ; drug effects ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; genetics ; Trans-Activators ; genetics ; Transcription Factors ; genetics

Improvement of stress tolerance to various adverse environmental conditions (such as toxic products, high temperature) of the industrial microorganisms is important for industrial applications. Ethanol produced by yeast fermentation is inhibitory to both yeast cell growth and metabolisms, and consequently is one of the key stress elements of brewer's yeast. Research on the biochemical and molecular mechanism of the tolerance of yeast can provide basis for breeding of yeast strain with improved ethanol tolerance. In recent years, employing global gene transcriptional analysis and functional analysis, new knowledge on the biochemical and molecular mechanisms of yeast ethanol tolerance has been accumulated, and novel genes and biochemical parameters related to ethanol tolerance have been revealed. Based on these studies, the overexpression and/or disruption of the related genes have successfully resulted in the breeding of new yeast strains with improved ethanol tolerance. This paper reviewed the recent research progress on the molecular mechanism of yeast ethanol tolerance, as well as the genetic engineering manipulations to improve yeast ethanol tolerance. The studies reviewed here not only deepened our knowledge on yeast ethanol tolerance, but also provided basis for more efficient bioconversion for bio-energy production.
Drug Tolerance ; genetics ; Ethanol ; metabolism ; pharmacology ; Fermentation ; Genetic Engineering ; methods ; Industrial Microbiology ; methods ; Saccharomyces cerevisiae ; drug effects ; genetics ; Saccharomyces cerevisiae Proteins ; genetics