Novel adjuvant for immunization against tuberculosis: DNA vaccine expressing <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> antigen 85A and interleukin-15 fusion product elicits strong immune responses in mice

Objectives

To investigate the potential of interleukin (IL)-15 as a novel adjuvant for Mycobacterium tuberculosis (Mtb) antigen 85A (Ag85A) vaccine.

Results

C57BL/6 mice were intramuscularly immunized three times with a plasmid expressing the Ag85A-IL-15 fusion protein (pcDNA3.1-Ag85A-IL-15), with the empty pcDNA3.1 vector and the pcDNA3.1-Ag85A as control. Mice vaccinated with pcDNA3.1-Ag85A-IL-15 generated more secretory IgA (sIgA) into their lung (209 ± 21 μg/ml) and acquired an enhanced serum IgG response to Ag85A. IgG2a/IgG1 ratios were upregulated, natural killer cell activity was augmented and Ag85A-specific splenic T cell proliferation was enhanced in these mice as well. Vaccination with pcDNA3.1-Ag85A-IL-15 promoted the polarization of CD4⁺ T cells towards a Th1 type in the spleen, and significantly upregulated the serum level of interferon (IFN)-γ (458 ± 98 pg/ml), a typical Th1 cytokine. IFN-γ-expressing CD8⁺ cells were also increased in the spleen after pcDNA3.1-Ag85A-IL-15 immunization.

Conclusions

A superior immune type I response in mice vaccinated with plasmid Ag85A-IL-15 has been achieved.

     

Electron microscopic study of mouse embryonic stem cell-derived cardiomyocytes

Differentiation of embryonic stem cell (ESC)-derived embryoid bodies (EBs) is a heterogeneous process. ESCs can differentiate in vitro into different cell types including beating cardiomyocytes. The main aim of the present study was to develop an improved preparation method for scanning electron microscopic study of ESC-derived cardiac bundles and to investigate the fine structural characteristics of mouse ESCs-derived cardiomyocytes using electron microscopy. The mouse ESCs differentiation was induced by EBs’ development through hanging drop, suspension and plating stages. Cardiomyocytes appeared in the EBs’ outgrowth as beating clusters that grew in size and formed thick branching bundles gradually. Cardiac bundles showed cross striation even when they were observed under an inverted microscope. They showed a positive immunostaining for cardiac troponin I and α-actinin. Transmission and scanning electron microscopy (TEM & SEM) were used to study the structural characteristics of ESC-derived cardiomyocytes. Three weeks after plating, differentiated EBs showed a superficial layer of compact fibrous ECM that made detailed observation of cardiac bundles impossible. We tried several preparation methods to remove unwanted cells and fibers, and finally we revealed the branching bundles of cardiomyocytes. In TEM study, most cardiomyocytes showed parallel arrays of myofibrils with a mature sarcomeric organization marked by H-bands, M-lines and numerous T-tubules. Cardiomyocytes were connected to each other by intercalated discs composed of numerous gap junctions and fascia adherences.     

Disturbances to great bustards (<Emphasis Type="Italic">Otis tarda</Emphasis>) in central Spain: human activities,bird responses and management implications

We investigated the effects of human activities on the behaviour of great bustards (Otis tarda) in a Special Protection Area in central Spain. We recorded 532 disturbances, at a rate of 0.93 disturbances per hour, a high value compared to other studies. Escape (flight/running) was observed more often than alert. Flight was more frequent than running. Car traffic and walkers were the main sources of disturbance. Motorcyclists, dogs, helicopters and aeroplanes were also harmful in relation to their abundance and time of permanence. Farming and shepherding produced few disturbances and usually did not cause a flight response. These activities are thus considered compatible with the conservation of the great bustards. Hunting caused an increase in the frequency of disturbance on weekends and holidays with respect to working days. We propose access restrictions to car traffic and helicopters/airplanes and hunting limitations in those areas more frequently used by the species. Pablo Sastre and Carlos Ponce Cabas contributed equally to this work.     

Precise Reconstruction of Veins and Bile Ducts in Rat Liver Transplantation

Rat orthotopic liver transplantation (ROLT) remains a technically demanding procedure, especially regarding the reconstruction of the suprahepatic vena cava (SHVC). In this study, a new microsuture technique was developed for anastomosis of the SHVC, and a special single-groove cuff and blade-cut stent were introduced. With these modified techniques, we aimed to make a precise anastomosis of the SHVC and to provide optimal cuffs and stents for the reconstruction of the veins and bile ducts. According to different microsuture techniques for the SHVC and different types of cuffs and stents, three ROLT groups were created to compare the operation times and prognoses. Sham operations were performed as controls in the fourth group. The time expenditures with each step were compared among the transplantation groups. Biochemical parameters were tested at the end of a 1-month observation period. The short- and long-term survival rates of the transplantation groups were recorded and compared. Our new microsuture technique was faster than the conventional continuous suture technique for SHVC anastomosis (P < 0.05). The use of a single-groove cuff for reconstruction of the portal vein and the infrahepatic vena cava shortened the anastomotic time (P < 0.05). The use of blade-cut stents resulted in fewer biliary complications and better survival over the short and long terms (P < 0.05). Our new microsuture technique and the single-groove cuffs proved to be a precise method for venous reconstruction which shortened the anhepatic time and the anastomotic time significantly. The blade-cut stents apparently reduced the incidence of biliary complications. In summary, with this precise microsuture technique and delicate cuffs and stents, excellent long-term survival can be achieved easily and stably for ROLT.     

Co-expression of P450 BM3 and glucose dehydrogenase by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> and its application in an NADPH-dependent indigo production system

P450 BM3 mutant can catalyze indole to indoxyl, and indoxyl can dimerize to form indigo. But the reaction catalyzed by P450 BM3 requires NADPH, as coenzyme regeneration is very important in this system. As we know, when glucose dehydrogenase oxidizes glucose to glucolactone, NADH or NADPH can be formed, which can contribute to NADPH regeneration in the reaction catalyzed by P450 BM3. In this paper, a recombinant Escherichia coli BL21 (pET28a (+)-P450 BM3-gdh0310) was constructed to co-express both P450 BM3 gene and glucose dehydrogenase (GDH) gene. To improve the expression level of P450 BM3 and GDH in E. coli and to avoid the complex and low-efficiency refolding operation in the purification procedure, the expression conditions were optimized. Under the optimized conditions, the maximum P450 BM3 and GDH activities amounted to 8173.13 and 0.045 U/mg protein, respectively. Then bioconversion of indole to indigo was carried out by adding indole and glucose to the culture after improved expression level was obtained under optimized conditions, and 2.9 mM (760.6 mg/L) indigo was formed with an initial indole concentration of 5 mM.     

Isolation and characterization of a marine algicidal bacterium against the harmful raphidophyceae <Emphasis Type="Italic">Chattonella marina</Emphasis>

A bacterial strain named AB-4 showing algicidal activity against Chattonella marina was isolated from coastal water of ULjin, Republic of Korea. The isolated strain was identified as Bacillus sp. by culture morphology, biochemical reactions, and homology research based on 16S rDNA. The bacterial culture led to the lysis of algal cells, suggesting that the isolated strain produced a latent algal-lytic compound. Amongst changes in algicidal activity by different culture filtrate volumes, the 10% (100 μl/ml) concentration showed the biggest change in algicidal activity; there, estimated algicidal activity was 95%. The swimming movements of Chattonella marina cells were inhibited because of treatment of the bacterial culture; subsequently, Chattonella marina cells became swollen and rounded. With longer exposure time, algal cells were disrupted and cellular components lost their integrity and decomposed. The released algicide(s) were heat-tolerant and stable in pH variations, except pH 3, 4, and 5. Culture filtrate of Bacillus sp. AB-4 was toxic against harmful algae bloom (HAB) species and nontoxic against livefood organisms. Bacillus sp. AB-4 showed comparatively strong activity against Akashiwo sanguinea, Fibriocapsa japonica, Heterosigma akashiwo, and Scrippsiella trochoidea. These results suggest that the algicidal activity of Bacillus sp. AB-4 is potentially useful for controlling outbreaks of Chattonella marina.     

含度量误差的黑龙江省主要树种生物量相容性模型

基于516株样木的生物量数据,采用非线性度量误差模型理论和方法,构建了黑龙江省15个主要树种(组)总生物量与地上、地下、树干、树冠、树枝、树叶6个分项生物量以及分项生物量间的相容性生物量模型,分别选出各树种总生物量和各分项生物量的最优模型,采用比值函数分级联合控制方程组构建了以总生物量为基础的相容性模型,并采用对数变换对总生物量模型消除异方差,采用加权回归对各分项生物量模型消除异方差.结果表明:本文所建的15个树种(组)相容性生物量模型中,总生物量的预估精度最高,达到90％以上;其次是地上部分生物量和树干生物量,预估精度在87.5％以上;地下部分、树冠、树枝和树叶生物量的预估精度相对较低,但绝大多数树种(组)的预估精度在80％以上;所有树种(组)总生物量、地上部分生物量、树干生物量模型的模拟效率(EF)值达0.9以上,绝大多数树种(组)的地下部分、树冠、树枝、树叶生物量模型的EF值在0.8以上.     

寄主植物对棉铃虫越冬蛹抗寒能力的影响

通过对滞育蛹过冷却点的测定 ,初步明确寄主对棉铃虫Helicoverpaarmigera的越冬抗寒性有影响 ,幼虫取食棉花时产生的滞育蛹的过冷却点低于取食玉米的滞育蛹的过冷却点 ,即前者的抗寒能力高于后者 ;但用Bt棉喂养后的棉铃虫滞育蛹抗寒能力下降     

Biosynthesis of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid from lignocellulosic biomass

d-lactic acid is a versatile and important industrial chemical that can be applied in the synthesis of thermal-resistant poly-lactic acid. Biosynthesis of d-lactic acid can be achieved by a variety of microorganisms, including lactic acid bacteria, yeast, and fungi; however, the final product yield, optical purity, and the utilization of both glucose and xylose are restricted. Consequently, engineered microbial systems are essential to attain high titer, productivity, and complete utilization of sugars. Herein, we critically evaluate the promising wild-type microorganisms, as well as genetically modified microorganisms to produce enantiomerically pure d-lactic acid, particularly from renewable lignocellulosic biomass. In addition, innovative bioreactor operation, metabolic flux analysis, and recent genetic engineering methods for targeted microbial d-lactic acid synthesis will be discussed.