New insights into the impact of microbiome on horizontal and vertical transmission of a tick-borne pathogen.

BACKGROUND: The impact of host skin microbiome on horizontal transmission of tick-borne pathogens , and of pathogen associated transstadial and transovarial changes in tick microbiome are largely unknown, but are important to control increasingly emerging tick-borne diseases worldwide. METHODS: Focusing on a rickettsiosis pathogen, Rickettsia raoultii, we used R. raoultii-positive and R. raoultii-negative Dermacentor spp. tick colonies to study the involvement of skin microbiota in cutaneous infection with rickettsiae in laboratory mice, and the function of the tick microbiome on maintenance of rickettsiae through all tick developmental stages (eggs, larvae, nymphs, adults) over two generations. RESULTS: We observed changes in the skin bacteria community, such as Chlamydia, not only associated with rickettsial colonization but also with tick feeding on skin. The diversity of skin microbiome differed between paired tick-bitten and un-bitten sites. For vertical transmission, significant differences in the tick microbiota between pathogenic rickettsia-positive and -negative tick chorts was observed across all developmental stages at least over two generations, which appeared to be a common pattern not only for R. raoultii but also for another pathogenic species, Candidatus Rickettsia tarasevichiae. More importantly, bacterial differences were complemented by functional shifts primed for genetic information processing during blood feeding. Specifically, the differences in tick microbiome gene repertoire between pathogenic Rickettsia-positive and -negative progenies were enriched in pathways associated with metabolism and hormone signals during vertical transmission. CONCLUSIONS: We demonstrate that host skin microbiome might be a new factor determining the transmission of rickettsial pathogens through ticks. While pathogenic rickettsiae infect vertebrate hosts during blood-feeding by the tick, they may also manipulate the maturation of the tick through changing the functional potential of its microbiota over the tick's life stages. The findings here might spur the development of new-generation control methods for ticks and tick-borne pathogens. Video Abstract.

Multigene manipulation of photosynthetic carbon metabolism enhances the photosynthetic capacity and biomass yield of cucumber under low-CO2 environment.

Solar greenhouses are important in the vegetable production and widely used for the counter-season production in the world. However, the CO2 consumed by crops for photosynthesis after sunrise is not supplemented and becomes chronically deficient due to the airtight structure of solar greenhouses. Vegetable crops cannot effectively utilize light resources under low-CO2 environment, and this incapability results in reduced photosynthetic efficiency and crop yield. We used cucumber as a model plant and generated several sets of transgenic cucumber plants overexpressing individual genes, including ß-carbonic anhydrase 1 (CsßCA1), ß-carbonic anhydrase 4 (CsßCA4), and sedoheptulose-1,7-bisphosphatase (CsSBP); fructose-1,6-bisphosphate aldolase (CsFBA), and CsßCA1 co-expressing plants; CsßCA4, CsSBP, and CsFBA co-expressing plants (14SF). The results showed that the overexpression of CsßCA1, CsßCA4, and 14SF exhibited higher photosynthetic and biomass yield in transgenic cucumber plants under low-CO2 environment. Further enhancements in photosynthesis and biomass yield were observed in 14SF transgenic plants under low-CO2 environment. The net photosynthesis biomass yield and photosynthetic rate increased by 49% and 79% compared with those of the WT. However, the transgenic cucumbers of overexpressing CsFBA and CsSBP showed insignificant differences in photosynthesis and biomass yield compared with the WT under low-CO2.environment. Photosynthesis, fluorescence parameters, and enzymatic measurements indicated that CsßCA1, CsßCA4, CsSBP, and CsFBA had cumulative effects in photosynthetic carbon assimilation under low-CO2 environment. Co-expression of this four genes (CsßCA1, CsßCA4, CsSBP, and CsFBA) can increase the carboxylation activity of RuBisCO and promote the regeneration of RuBP. As a result, the 14SF transgenic plants showed a higher net photosynthetic rate and biomass yield even under low-CO2environment.These findings demonstrate the possibility of cultivating crops with high photosynthetic efficiency by manipulating genes involved in the photosynthetic carbon assimilation metabolic pathway.

[Assessment of efficacy of acupuncture combined with hyperbaric oxygen therapy for patients with delayed encephalopathy of CO intoxication by magnetic resonance voxel incoherent motion imaging].

OBJECTIVE: To observe the effect of acupuncture plus hyperbaric oxygen (HBO) on cerebral blood perfusion in patients with delayed encephalopathy after carbon monoxide poisoning (DEACMP). METHODS: Twenty-eight patients with DEACMP were randomly divided into acupuncture group (n=14 cases) and control group (n=14 cases). Patients of the acupuncture group were treated by using "Xingnao Kaiqiao" needling technique (specific set of acupuncture points as Yintang ï¼»EX-HN3ï¼½, Shangxing ï¼»GV23ï¼½, Baihui ï¼»GV20ï¼½, Sishenchong ï¼»EX-HN1ï¼½, Fengchi ï¼»GB20ï¼½, Taichong ï¼»LR3ï¼½, etc., and strong stimulation) combined with HBO in an air pressurized tank, and those of the control group treated by simple HBO. The treatment was conducted once a day, 5 days a week for 6 weeks. All the patients underwent head routine magnetic resonance imaging (MRI) and intravoxel incoherent motion imaging(IVIM) scan before and after the treatment. The values of pseudo-diffusion coefficient D (D*) and perfusion fraction (f) of the bilateral semi-oval centers were measured by using MITK software package to calculate the ave-rage fD* value (f×D*). The Barthel index (BI) score was used to assess the patients' daily living ability, and the correlation between parameter values was evaluated by Pearson method. RESULTS: After the treatment, the values of f, fD* and BI scores were significantly increased in both acupuncture group and control groups (P<0.05). The values of the 3 indexes were significantly higher in the acupuncture group than those in the control group (P<0.05). The values of f and fD* were positively correlated with BI score (P<0.05), with the correlation coefficients being 0.822, 0.636 and 0.601, respectively between the fD* and f, BI and f, and BI and fD*. CONCLUSION: Acupuncture combined with HBO can significantly improve the low-perfusion of bilate-ral semi-oval center and daily living ability in patients with DEACMP, being significantly superior to simple HBO therapy in the curative effect.

[Evaluation of the efficacy of acupuncture combined with hyperbaric oxygen on delayed neuropathological sequelae after carbon monoxide poisoning by using mTI-ASL imaging].

OBJECTIVE: Quantitative assessment of white blood flow in semi-oval center of patients with delayed neuropathological sequelae (DNS) after carbon monoxide poisoning treated with acupuncture combined with hyperbaric oxygen (HBO) based on magnetic resonance multi-inversion time arterial spin labeling imaging (mTI-ASL), and to evaluate its efficacy indirectly. METHODS: Twenty-six patients with clinically diagnosed DNS were randomly divided into an observation group (13 cases) and a control group (13 cases). The conventional therapy combined with HBO were given in the control group. In the observation group,on the base of the treatment, Xingnao Kaiqiao acupuncture was applied, the main acupoints were Shuigou (GV 26), Neiguan (PC 6), Baihui (GV 20), Shangxing (GV 23), Yintang (GV 29), Sanyinjiao (SP 6) on the affected side, Sishencong (EX-HN 1), Fenglong (ST 40), Lianquan (CV 23) and Jinjin (EX-HN12) for slurred speech, Jianyu (LI 15), Waiguan (TE 5) and Shousanli (LI 10) for upper limb pain, Huantiao (GB 30), Yanglingquan (GB 34), Yinlingquan (SP 9) for lower limb pain, the treatment was given once every day, 5 days as one course, with an interval of 2 days between the course. The treatment for 6 courses was required. The conventional head MR scan, mTI-ASL and diffusion tensor imaging (DTI) scans before and 1 week after treatment were adopted, Matlab (R2014b), Mricron and Syngo.via software were adopted to measure the cerebral blood flow (CBF) and anisotropy (FA) values of the semi-oval center. The correlation between the parameters was evaluated by Pearson method. And the simple intelligent mental state examination scale (MMSE) was uesd to assess cognitive function. RESULTS: After treatment, the CBF, MMSE scores in both groups and FA values in the observation group were higher than those before treatment (P<0.05). After treatment, the CBF, FA and MMSE scores in the observation group were significantly higher than those in the control group (P<0.05). There was a positive correlation between CBF, FA and MMSE scores (P<0.05), and the correlation between CBF and MMSE was the best (r =0.822). CONCLUSION: Acupuncture combined with hyperbaric oxygen can significantly improved early white matter hypoperfusion and improved cognitive function score in patients with DNS. The curative effect is better than that of hyperbaric oxygen therapy alone. The mTI-ASL imaging can quantitatively evaluate its curative effect.

[Current state of researches on mechanisms of acupuncture in improvement of hypoxia-ischemic diseases of the central nervous system].

Acupuncture is widely used in the treatment of ischemic diseases of the central nervous system in different clinical stages and has achieved a good clinical effect. The current research showed that acupuncture can improve cerebral blood flow perfusion via increasing blood flow volume, blood flow velocity, the levels of vasomotor substances and reduction of blood viscosity and erythrocyte aggregation index. In recent years, many studies focused on the pathophysiological mechanism of acupuncture in improving cerebral ischemia via triggering the cholinergic vasodilatation, up-regulation of expression of vascular-related proteins and genes，attenuation of inflammatory reaction, etc. Currently, the methods for evaluating the effect of acupuncture are mostly noninvasive functional magnetic resonance imaging in healthy subjects. Future studies should include united selection of acupoints and acupuncture needle manipulations, more reasonable combination of different acupoints, united outcome evaluative standards, better repeatability, employment of big data, etc.

Modification, Biological Evaluation and SAR Studies of Novel 1H-Pyrazol Derivatives Containing N,N'-Disubstituted Urea Moiety as Potential Anti-melanoma Agents.

Malignant melanomas are amongst the most aggressive cancers. BRAF Inhibitors have exhibited therapeutic effects against BRAF-mutant melanoma. In continuation of our earlier studies on anti-melanoma agents based on 1H-pyrazole skeleton, two sets of novel compounds that include 1H-pyrazole-4-amines FA1 - FA13 and corresponding urea derivatives FN1 - FN13 have been synthesized and evaluated for their BRAFV600E inhibitory and antiproliferation activities. Compound FN10 displayed the most potent biological activity against BRAFV600E (IC50 = 0.066 µm) and the A375 human melanoma cell line (GI50 = 0.81 µm), which was comparable to the positive control vemurafenib, and more potent than our previously reported 1H-pyrazole-3-amines and their urea derivatives. The results of SAR studies and molecular docking can guide further optimization and may help to improve potency of these pyrazole-based anti-melanoma agents.

[Effects of naoxintong capsule on the inflammation and prognosis in borderline lesion coronary heart disease patients].

OBJECTIVE: To study the effects of Naoxintong Capsule (NC) on the inflammation and long-term prognosis in the borderline lesion coronary heart disease patients. METHODS: A total of 240 coronary heart disease patients with angina symptoms and accompanied with borderline lesion coronary heart disease (with the diameter stenosis in critical 50% -70%) by means of coronary angiography or multislice computed tomography coronary angiography were recruited. These patients were randomly assigned to the conventional treatment group (including nitrate, beta blockers, anti-platelet, anticoagulation, angiotensin converting enzyme inhibitors, and so on) and the NC treatment group (treated the same way as those for the conventional treatment group and NC). All patients were treated for 12 months. The occurrence of cardiovascular events was observed after treatment. The inflammatory factors in serum [interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and high sensitive C reaction protein (hs-CRP)], matrix metalloproteinases-9 (MMP-9), blood lipids and blood sugar, liver and kidney functions were measured before and after treatment. RESULTS: After 12 months of treatment, the incidence of angina pectoris patients (6.67% vs 15.83%, P < 0.05) and hospitalization due to acute coronary syndrome (ACS) attacks (4.17% vs 10.83%) was significantly lower in the NC treatment group than in the conventional treatment group. There was no statistical difference in the serum levels of IL-6, TNF-alpha, hs-CRP, and MMP-9 between the two groups before treatment (P > 0.05). After 12 months of treatment, serum levels of IL-6, TNF-alpha, hs-CRP, and MMP-9 were significantly lower when compared with before treatment in the same group (P < 0.05). Besides, the serum levels of IL-6, TNF-alpha, hs-CRP, and MMP-9 were significantly lower in the NC group than in the conventional treatment group (P < 0.05). By means of Logistic regression analysis we found that the post-treatment MMP-9 level and IL-6 level were independent risk factors influencing the recurrence of angina pectoris. CONCLUSIONS: NC could alleviate the inflammation. Long-term administration of NC could reduce the recurrence of angina pectoris and decrease the incidence of ACS attack in borderline lesion coronary heart disease patients. The post-treatment MMP-9 level and IL-6 level were independent risk factors influencing the recurrence of angina pectoris.

[Interleukin-8 monoclonal antibody attenuates smooth muscle cell proliferation and balloon inflation-induced abdominal aorta stenosis in rabbits].

OBJECTIVE: To observe the effects of interleukin-8 monoclonal antibody on smooth muscle cell proliferation and balloon inflation-induced abdominal aorta stenosis in rabbits. METHODS: Thirty-six New Zealand white rabbits were randomly assigned to balloon inflation group (group A, n = 12), interleukin-8 monoclonal antibody pre-treated rabbits (2 mg/kg for 3 days before balloon inflation, group B, n = 12) and sham-operated control group (group C, n = 12). Peripheral blood was collected before experiment and at 4 h, 1, 3, 7, 14, and 28 days post balloon inflation or sham operation and the levels of IL-8 were measured by enzyme linked immunosorbent assay (ELISA). The ratio of positive and negative masculine cells in the high power microscopic field was determined in proliferating cell nuclear antigen (PCNA) stained slide. Histopathologic examination was performed in abdominal aorta and luminal area, intima and tunica media area were measured. RESULTS: Plasma interleukin-8 began to rise at 4 h and peaked at 1 day and remained increased up to 28 days after balloon inflation in rabbits of group A, plasma interleukin-8 level in group A was significantly higher than in group B and C at 4 h and thereafter post operation. The ratio of positive and negative masculine cells was significantly increased in group A compared to group C and was significantly lower in group B than in group A. Abdominal aorta stenosis, luminal area, intima and tunica media area were significantly reduced in group B than in group A. Correlation analysis indicated that there were positive relations between plasma IL-8 level and intima thickness, area of intima and tunica media, respectively (r = 0.894, 0.783, 0.801, 0.912, all P < 0.01). CONCLUSIONS: Plasma IL-8 level is increased in this abdominal aorta stenosis model and is positively correlated to the severity of abdominal aorta stenosis. IL-8 monoclonal antibody could significantly reduce abdominal aorta stenosis in this abdominal aorta stenosis model.

Development of a highly efficient gene targeting system allowing rapid genetic manipulations in Penicillium decumbens.

Penicillium decumbens is an important industrial filamentous fungus and has been widely used in biorefinery due to its high production of cellulase and hemicellulase. However, molecular engineering has still rarely been applied for strain improvement in P. decumbens. It has been proven that gene targeting manipulation in many filamentous fungi is hampered by nonhomologous end-joining (NHEJ) pathway. To improve gene targeting efficiency in P. decumbens, the putative pku70 encoding the Ku70 homologue involved in the NHEJ pathway was identified and deleted. The Deltapku70 strain showed no apparent defect in vegetative growth, conidiation, and cellulase production, and displayed similar sensitivity to chemical agents of hygromycin B, ethyl methane sulfonate, and H2O2 at different concentrations compared with the wild-type strain. The effect of the absence of pku70 on gene targeting was tested by disruption of creA encoding a putative carbon catabolite repressor and xlnR encoding a putative transcriptional activator. Efficiency of gene targeting for both genes was 100% in the Deltapku70 strain, compared with the low efficiency in the wild-type recipient. Furthermore, the integration types for three single targeting cassettes and the cotransformation of two independent targeting cassettes were primarily investigated in P. decumbens. The highly efficient gene targeting system established in this study will open the way to large-scale functional genomic analysis in P. decumbens and contribute to the study of the mechanism of lignocellulose degradation by P. decumbens.

Identification and characterization of a novel gene, TrCCD1, and its possible function in hyphal growth and conidiospore development of Trichoderma reesei.

To investigate genes with essential functions during hyphal growth or sporulation in the asexual filamentous fungus Trichoderma reesei, we screened a collection of T-DNA insertion mutants and identified the genomic integration events. Two mutants with abnormal phenotypes, named as ccdO and ccdP, were found to have independent T-DNA insertions into a putative TrCCD1 gene locus, the product of which has significant homology to carotenoid cleavage dioxygenases (CCDs). Compared to the parental strain, both mutants tended to produce slow-growing hyphae and had a more than 50% reduction in colony growth rate. Simultaneously, the hyphae of the growing mutants formed wilting tip while the parental strain elongated straightly. To the effect of the TrCCD1 mutation on the conidiospore development, less spores were formed in the mutants than in the parental strain. In addition, disruption of TrCCD1 resulted in another phenotype characterized by a remarkable enhancement in the total carotenoid content. When the wild-type TrCCD1 gene was reintroduced into the ccd mutants, the abnormal phenotypes were rescued. These results suggest that TrCCD1 is involved in carotenoid metabolism and likely required for hyphal growth and conidiospore development in filamentous fungi T. reesei.

Purification and characterization of the cold-active alkaline protease from marine cold-adaptive Penicillium chrysogenum FS010.

An extracellular cold-active alkaline serine protease from Penicillium chrysogenum FS010 has been purified. The purification procedure involved: ammonium sulfate precipitation, DEAE ion-exchange chromatography and sephadex G-100 gel chromatography. SDS-PAGE of the purified enzyme indicated a molecular weight of 41,000 +/- 1,000 Da. The protease is stable in a pH range of 7.0-9.0 and has a maximum activity at pH 9.0. Compared with other industrial proteases, the enzyme shows a high hydrolytic activities at lower temperatures and a high sensitivity at a temperature over 50 degrees C. The isoelectric point of the enzyme is approximate to 6.0. Enzymatic activity is enhanced by the addition of divalent cations such as Mg(2+) and Ca(2+) and inhibited by addition of Cu(2+)and Co(2+). PMSF and DFP are its specific inhibitors. The application of the cold-active alkaline protease is extremely extensive, and widely used in detergents, feed, food, leather and many other industries.

Metformin inhibits nuclear factor kappaB activation and decreases serum high-sensitivity C-reactive protein level in experimental atherogenesis of rabbits.

Previous studies demonstrated that metformin has obvious antiatherogenic properties, but the exact mechanism remains unclear. Therefore, we established an atherosclerotic rabbit model in order to investigate the potential effects of metformin on transcription factor nuclear factor kappaB (NF-kappaB) and serum high-sensitivity C-reactive protein (hs-CRP) level, which had been regarded as proatherogenic factors. New Zealand rabbits were randomly divided into three groups: a control group (n = 8), an atherosclerotic group (AS group, n = 8), and a metformin treatment group (Met group, n = 8). The experimental atherosclerotic rabbit model was successfully established at the end of the 8th week. From the 9th week, rabbits in the Met group were administered with 150 mg/kg metformin daily by gavage. Blood samples were collected at days 0 and 8, and at 16 weeks to detect the level of blood lipid and serum glucose. At the end of the experiment, blood samples were withdrawn for determining serum hs-CRP. Aortic samples were harvested for histomorphometric analysis. Immunohistochemistry and Western blotting were used to detect the expression of NF-kappaB subunit p65 in nuclear extracts and phosphorylation of inhibitor of nuclear factor kappaB (IkappaB) in cytoplasmic extracts. An experimental atherosclerotic rabbit model was successfully established. The expression of nuclear NF-kappaB subunit p65 and cytoplasmic phosphorylation of IkappaB protein in the vessel wall was enhanced (P < 0.01, respectively) in the AS group, and serum hs-CRP level was significantly increased in the AS group compared with the control group (3.90 +/- 0.25 mg/l versus 1.36 +/- 0.14 mg/l, P < 0.01). Treatment with metformin significantly attenuated the progression of aortic atherosclerosis. In the Met group, there was a marked reduction in nuclear NF-kappaB subunit p65 and cytoplasmic phosphorylation of IkappaB protein expression (P < 0.01). Serum hs-CRP concentration was also significantly decreased (3.20 +/- 0.20 mg/l versus 3.90 +/- 0.25 mg/l, P < 0.05). Metformin inhibits the phosphorylation of IkappaB and the activation of NF-kappaB in the vessel wall of experimental atherogenesis of rabbits, as well as decreasing the serum level of hs-CRP, thus suggesting that metformin has vascular anti-inflammatory properties, which may be one of its antiatherogenic mechanisms.

Mechanisms of improvement of left ventricle remodeling by trans-planting two kinds of autologous bone marrow stem cells in pigs.

BACKGROUND: The necrosis of a large number of myocardial cells after acute myocardial infarction (AMI) results in a decrease of cardiac function and ventricle remodeling. Stem cell transplantation could improve cardiac function after AMI, but the involving mechanisms have not been completely understood. The present study aimed to investigate the effects of transplantation of autologous bone marrow mononuclear cells (BM-MNC) and mesenchymal stem cells (MSCs) via the coronary artery on the ventricle remodeling after AMI as well as the mechanisms of the effects of transplantation of different stem cells on ventricle remodeling. METHODS: A total of 36 male pigs were enrolled in this study, which were divided into 4 groups: control group, simple infarct model group, BM-MNC transplantation group, and MSCs transplantation group. At 90 minutes when a miniature porcine model with AMI was established, transplantation of autologous BM-MNC ((4.7 +/- 1.7) x 10(7)) and MSCs ((6.2 +/- 1.6) x 10(5)) was performed in the coronary artery via a catheter. Ultrasound, electron microscope, immunohistochemical examination and real time reverse transcriptase-polymerase chain reaction were used respectively to observe cardiac functions, counts of blood vessels of cardiac muscle, cardiac muscle nuclear factor (NF)-kappaB, myocardial cell apoptosis, and the expression of the mRNA of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in cardiac muscles. Multivariate Logistic regression was used to analyze the correlation factors of left ventricular end-diastolic diameter (EDD). RESULTS: The number of blood vessels in the infarct zone and around its border in the BM-MNC transplantation group was more than those in the infarct model group and MSCs group (P = 0.0001) and there was less myocardial cell apoptosis in the stem cell transplantation group than that in the infarct model group (all P < 0.01). The positive rate of NF-kappaB in the stem cell transplantation group was lower than that in the infarct model group (P = 0.001). The gene expression of VEGF in the infarct border zone of the BM-MNC group was higher than that in the MSCs group (P = 0.0001). The gene expression of bFGF in the infarct border zone in the MSCs transplantation group was higher than that in the infarct model group and the BM-MNC group (P = 0.0001). Left ventricular ejection fraction was inversely proportional to the apoptotic rate of myocardial cells and cardiac muscle NF-kappaB but positively correlated with the number of blood vessels and the expression of VEGF and bFGF in the infarct zone and infarct border zone. The Multivariate Logistic regression analysis on the factors influencing the left ventricular end-diastolic diameter after stem cell transplantation showed that the expression of VEGF mRNA in the cardiac muscles in the infarct zone, the number of apoptotic myocardial cells and the expression of NF-kappaB in the infarct border zone were independent factors for predicting the inhibitory effect on the dilation of left ventricular EDD after stem cell transplantation. CONCLUSIONS: Transplantation of autologous BM-MNC and MSCs in pigs can improve the condition of left ventricular remodeling and recover the cardiac functions after AMI. The improvement of cardiac functions is related to the increase of blood vessels, the increased expression of VEGF and bFGF, the reduction of myocardial cell apoptosis, and the decrease of NF-kappaB level in cardiac muscle tissues after stem cell transplantation.

Expression of ethylene-forming enzyme (EFE) of Pseudomonas syringae pv. glycinea in Trichoderma viride.

The efe gene encoding an ethylene-forming enzyme from Pseudomonas syringae pv. glycinea has been expressed for the first time under the control of Trichoderma reesei cbh1 promoter in Trichoderma viride. Reverse transcription polymerase chain reaction analysis showed that transformant Y2 produced mRNA of the efe gene. Southern blot analysis showed that there was one copy of efe gene which was integrated into the chromosomal DNA of T. viride. Ethylene production by transformant Y2 was efficiently induced by cellulose, while very low level of ethylene was produced when sodium carboxymethyl cellulose or lactose was used as carbon source. Peptone exerted a much greater stimulatory effect on ethylene production. A high level of ethylene was produced when transformant Y2 was cultured in solid fermentation medium containing wheat straw, indicating that plant wastes could be directly converted to ethylene by the recombinant filamentous fungus.

Agrobacterium-mediated transformation (AMT) of Trichoderma reesei as an efficient tool for random insertional mutagenesis.

Filamentous fungus Trichoderma reesei QM9414 was successfully transformed with Agrobacterium tumefaciens AGL-1 for random integration of transforming DNA (T-DNA). Co-cultivation of T. reesei conidia or protoplasts with A. tumefaciens in the presence of acetosyringone resulted in the formation of hygromycin B-resistant fungal colonies with high transformation frequency. Nine randomly selected resistant clones were proved to be stable through mitotic cell division. The integration of the hph gene into T. reesei genome was determined by PCR and dot blot analysis. Transgenic T. reesei strains were analyzed using TAIL-PCR for their T-DNA contents. The results showed that T-DNA inserts occurred evidently by fusing DNA at T-DNA borders via random recombination, which suggests that Agrobacterium-mediated transformation is a potentially powerful tool towards tagged mutagenesis and gene transfer technology for T. reesei.

Novel cold-adaptive Penicillium strain FS010 secreting thermo-labile xylanase isolated from Yellow Sea.

A novel cold-adaptive xylanolytic Penicillium strain FS010 was isolated from Yellow sea sediments. The marine fungus grew well from 4 to 20 degrees; a lower (0 degrees) or higher (37 degrees) temperature limits its growth. The strain was identified as Penicillium chrysogenum. Compared with mesophilic P. chrysogenum, the cold-adaptive fungus secreted the cold-active xylanase (XYL) showing high hydrolytic activities at low temperature (2-15 degrees) and high sensitivity to high temperature (>50 degrees). The XYL gene was isolated from the cold-adaptive P. chrysogenum FS010 and designated as xyl. The deduced amino acid sequence of the protein encoded by xyl showed high homology with the sequence of glycoside hydrolase family 10. The gene was subcloned into an expression vector pGEX-4T-1 and the encoded protein was overexpressed as a fusion protein with glutathione-S-transferase in Escherichia coli BL21. The expression product was purified and subjected to enzymatic characterization. The optimal temperature and pH for recombinant XYL was 25 degrees and 5.5, respectively. Recombinant XYL showed nearly 80% of its maximal activity at 4 degrees and was active in the pH range 3.0-9.5.

[Cloning and expression of endoglucanase of marine cold-adapted bacteria Pseudoalteromonas sp. MB-1].

The cold-adapted gram-negative rod bacterium MB-1 which could secret cellulase was screened from mud of the bottom of the Huanghai. According to the sequence of 16S rDNA, this bacterium screened was identified as one species of Pseudoalteromonas and was named as Pseudoalteromonas sp. MB-1. The gene celA encoding cold-adapted endogluanase was cloned and then jointed to pGEX-4T-1 to construct expression plasmid pGEX-celA which was expressed in E. coli BL21. Analysis to the supernatant of E. coli sonicate revealed that the concentration of GST-CelA was about 78.5 mg/L. Properties of the fusion enzyme of GST-CelA including the optimum temperature at 35 degrees C and the optimum pH about 7.2, showed that this fusion enzyme still belonged to cold-adapted enzyme and neutral enzyme. The result lays solid base for the fundamental theory and application research on cold-adapted cellulase from Pseudoalteromonas sp. MB-1.

Construction of highly efficient E. coli expression systems containing low oxygen induced promoter and partition region.

A series of high-copy-number Escherichia coli expression vectors equipped with an oxygen-sensitive promoter P(vgb) of Vitreoscilla hemoglobin (encoded by the vgb gene) were constructed and characterized. Plasmid pKVp containing P(vgb) was inducible by low oxygen tension, while plasmid pKVpP containing a partition (par) region from plasmid pSC101 ligated to P(vgb) provided inheritable stability for the vectors in the absence of ampicillin. Plasmid pKVpV had the Vitreoscilla hemoglobin operon vgb ligated to P(vgb), while a construct containing P(vgb), the vgb operon and a par region constituted plasmid pKVpPV. Shake-flask studies demonstrated that plasmids pKVpV and pKVpPV expressed higher levels of Vitreoscilla hemoglobin under low aeration condition (5% air saturation in water) compared with the levels observed under strong aeration (20% air saturation in water). Introduction of either the enhanced green fluorescent protein (eGFP) gene egfp or the toluene dioxygenase (TDO) gene tod into either pKVpV (P(vgb), vgb operon) or pKVpPV (P(vgb), vgb operon, par) slightly attenuated (approximately 30%) the strong expression of VHb under low aeration. However, all displayed approximately a three-fold increase versus that observed for strong aeration. Recombinant E. coli harboring either pKVp-E (P(vgb), egfp) or pKVpP-E (P(vgb), par, egfp) displayed at least a two-fold increase in eGFP expression under conditions of low aeration and absence of antibiotic, compared with that under strong aeration after 24 h of cultivation. Strong expression of TDO was also observed using low aeration in recombinant E. coli harboring pKVpPV-T (P(vgb), vgb operon, par, tod) or pKVpP-T (P(vgb), par, tod). Plasmids containing the par region were stable over 100 generations. These results indicate that the novel expression system combining plasmid stability over the cell growth phase and a promoter inducible by low oxygen tension will be very useful for high-density production of foreign proteins.

Novel cellulase profile of Trichoderma reesei strains constructed by cbh1 gene replacement with eg3 gene expression cassette.

To construct strains of the filamentous fungus Trichoderma reesei with low cellobiohydrolases while high endoglucanase activity, the P(cbh1)-eg3-T(cbh1) cassette was constructed and the coding sequence of the cellobiohydrolase I (CBHI) gene was replaced with the coding sequence of the eg3 gene by homologous recombination. Disruption of the cbh1 gene was confirmed by PCR, Southern dot blot and Western hybridization analysis in two transformants denoted as L13 and L29. The filter paper-hydrolyzing activity of strain L29 was 60% of the parent strain Rut C30, and the CMCase activity was increased by 33%. This relatively modest increase suggested that the eg3 cDNA under the control of the cbh1 promoter was not efficiently transcribed as the wild type cbhl gene. However our results confirmed that homologous recombination could be used to construct strains of the filamentous fungus Trichoderma reesei with novel cellulase profile. Such strains are of interest from the basic science perspective and also have potential in ustrial applications.

Cloning and sequence analysis of a mitochondrial gene cluster encoding cytochrome C oxidase subunit III from Trichoderma pseudokoningii.

A mitochondrial gene cluster encoding cytochrome c oxidase subunit III (COX3), an ORF (called ORF250) similar to NADH dehydrogenase subunit VI (ND6), ten tRNA molecules, partial rRNA small subunit and rRNA large subunit from Trichoderma pseudokoningii S38 was cloned and sequenced. These genes are tandemly clustered on the mitochondrial genome of Trichoderma pseudokoningii S38. Phylogenetic analysis showed that cytochrome C oxidase subunits III exhibited high degree of similarity to sequences from Hypocrea jecorina, Verticillium lecanii, Podospora anserine, Neurospora crassa and Magnaporthe grisea (99, 90, 84, 82 and 79% identity, respectively). Prediction of transmembrane helices revealed that COX3 was a transmembrane protein. Northern dot blot analysis showed that the cytochrome c oxidase subunits III gene we had cloned is actively transcribed in the T. pseudokoningii mitochondria.