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Bacterial viruses, that is ‘bacteriophage’ or ‘phage’, can infect and lyse their bacterial hosts, releasing new viral progeny. In addition to the lytic pathway, certain bacteriophage (i.e. ‘temperate’ bacteriophage) can also initiate lysogeny, a latent mode of infection in which the viral genome is integrated into and replicated with the bacterial chromosome. Subsequently, the integrated viral genome, that is the ‘prophage’, can induce and restart the lytic pathway. Here, we explore the relationship among infection mode, ecological context, and viral fitness, in essence asking: when should viruses be temperate? To do so, we use network loop analysis to quantify fitness in terms of network paths through the life history of an infectious pathogen that start and end with infected cells. This analysis reveals that temperate strategies, particularly those with direct benefits to cellular fitness, should be favored at low host abundances. This finding applies to a spectrum of mechanistic models of phage–bacteria dynamics spanning both explicit and implicit representations of intra-cellular infection dynamics. However, the same analysis reveals that temperate strategies, in and of themselves, do not provide an advantage when infection imposes a cost to cellular fitness. Hence, we use evolutionary invasion analysis to explore when temperate phage can invade microbial communities with circulating lytic phage. We find that lytic phage can drive down niche competition amongst microbial cells, facilitating the subsequent invasion of latent strategies that increase cellular resistance and/or immunity to infection by lytic viruses—notably this finding holds even when the prophage comes at a direct fitness cost to cellular reproduction. Altogether, our analysis identifies broad ecological conditions that favor latency and provide a principled framework for exploring the impacts of ecological context on both the short- and long-term benefits of being temperate.

The present study examined microbial substrate utilization by community level physiological profiling and carbon-cycling enzyme assays (cellobiohydrolase, β-xylosidase, and oxidases) in three Japan...

Temperate phage can initiate lysis or lysogeny after infecting a bacterial host. The genetic switch between lysis and lysogeny is mediated by phage regulatory genes as well as host and environmental factors. Recently, a new class of decision switches was identified in phage of the SPbeta group, mediated by the extracellular release of small, phage-encoded peptides termed arbitrium. Arbitrium peptides can be taken up by bacteria prior to infection, modulating the decision switch in the event of a subsequent phage infection. Increasing concentration of arbitrium increases the chance that a phage infection will lead to lysogeny, rather than lysis. Although prior work has centered on the molecular mechanisms of arbitrium-induced switching, here we focus on how selective pressures impact the benefits of plasticity in switching responses. In this work, we examine the possible advantages of near-term adaptation of communication-based decision switches used by the SPbeta-like group. We combine a nonlinear population model with a control theoretic approach to evaluate the relationship between a putative phage reaction norm (i.e., the probability of lysogeny as a function of arbitrium) and the near-term time horizon. We show the adaptive potential of communication-based lysis-lysogeny responses and find that optimal switching between lysis to lysogeny increases near-term fitness compared to fixed responses. We further find that plastic responses are robust to the inclusion of cellular-level stochasticity. These findings provide a principled basis to explore the long-term evolution of phage-encoded decision systems mediated by extracellular decision-signaling molecules, and the feedback between phage reaction norms and ecological context.

Recently, long noncoding RNAs (lncRNAs) are attracting wide attention in the field of cancer research because of its important role in cancer diagnosis and prognosis. But studies on the biological effects and relevant mechanisms of lncRNAs in non‐small cell lung cancer (NSCLC) remain few and need to be enriched. Our study discussed the expression and biological effects of LncRNA NR2F2‐AS1, and further explored its possible molecular mechanisms. As a result, elevated expression of NR2F2‐AS1 was detected in NSCLC tissues and cells and was remarkably associated with the tumor, node, metastasis (TNM) stage and the status of lymphatic metastasis of patients. Down‐regulated NR2F2‐AS1 contributed to the promotion of cell apoptosis and the inhibition of cell proliferation and invasion in A549 and SPC‐A‐1 cells in vivo and vitro. Through bioinformatics analysis, NR2F2‐AS1 functions as a ceRNA directly binding to miR‐320b, BMI1 was a direct target of miR‐320b. Combined with the following cellular experiments, the data showed that NR2F2‐AS1 may influence the NSCLC cell proliferation, invasion and apoptosis through regulating miR‐320b targeting BMI1.

Objective The signalling of the receptor tyrosine kinase Met is critical in promoting trophoblast cell invasion, and the deficiency in HGF/Met signalling is associated with preeclampsia. The semaphorin family member semaphorin4D (sema4D) and its receptor Plexin-B1 have been reported to control tumour cell invasion by coupling with Met. We hypothesized that sema4D/Plexin-B1 may promote trophoblast invasion by activating Met, and downregulation of sema4D/Plexin-B1 may account for the deficiency in Met signalling in preeclamptic placenta. Methods In this study, Met and Erk activation and the expression of sema4D/Plexin-B1 in normal and preeclamptic placentas were comparably measured. The role of sema4D in trophoblast cell invasion and tubulogenesis was examined in vitro using the Transwell invasion assay and tube formation assay in trophoblast-endothelial cell co-culture model. Results Met, sema4D and Plexin-B1 co-localized in various subtypes of human trophoblast cells, including villous trophoblasts and extravillous trophoblasts (EVTs). In early-onset preeclampsia (E-PE) placentas, the phosphorylated Met and Erk as well as sema4D and Plexin-B1 were much lower than those in gestational week-matched preterm-labour (PTL) placentas. In human trophoblast HTR8/SVneo cell line, sema4D could promote Met and Erk phosphorylation as well as enhance trophoblast cell invasion and tubulogenesis with endothelial cells. Moreover, the effect of sema4D on HTR8/SVneo could be blocked by knocking down Met with specific siRNA. Conclusion The crosstalk between sema4D and Met could transactivate Met to promote trophoblast cell invasion and differentiation, and decreased expression of sema4D and Plexin-B1 may be responsible for the deficiency in Met signalling and the development of preeclampsia.

Background Most species of the Russulaceae are ectomycorrhizal (ECM) fungi, which are widely distributed in different types of forest ecology and drive important ecological and economic functions. Little is known about the composition variation of the Russulaceae fungal community aboveground and in the root and soil during the growing season (June–October) from a Quercus mongolica forest. In this study, we investigated the changes in the composition of the Russulaceae during the growing season of this type of forest in Wudalianchi City, China. Methods To achieve this, the Sanger sequencing method was used to identify the Russulaceae aboveground, and the high-throughput sequencing method was used to analyze the species composition of the Russulaceae in the root and soil. Moreover, we used the Pearson correlation analysis, the redundancy analysis and the multivariate linear regression analysis to analyze which factors significantly affected the composition and distribution of the Russulaceae fungal community. Results A total of 56 species of Russulaceae were detected in the Q. mongolica forest, which included 48 species of Russula, seven species of Lactarius, and one species of Lactifluus. Russula was the dominant group. During the growing season, the sporocarps of Russula appeared earlier than those of Lactarius. The number of species aboveground exhibited a decrease after the increase and were significantly affected by the average monthly air temperature (r = −0.822, p = 0.045), average monthly relative humidity (r = −0.826, p = 0.043), monthly rainfall (r = 0.850, p = 0.032), soil moisture (r = 0.841, p = 0.036) and soil organic matter (r = 0.911, p = 0.012). In the roots and soils under the Q. mongolica forest, the number of species did not show an apparent trend. The number of species from the roots was the largest in September and the lowest in August, while those from the soils were the largest in October and the lowest in June. Both were significantly affected by the average monthly air temperature (r2 = 0.6083, p = 0.040) and monthly rainfall (r2 = 0.6354, p = 0.039). Moreover, the relative abundance of Russula and Lactarius in the roots and soils showed a linear correlation with the relative abundance of the other fungal genera.

Viruses that infect bacteria, i.e., bacteriophage or ‘phage’, are increasingly considered as treatment options for the control and clearance of bacterial infections, particularly as compassionate use therapy for multi-drug resistant infections. In practice, clinical use of phage often involves the application of multiple therapeutic phage, either together or sequentially. However, the selection and timing of therapeutic phage delivery remains largely ad hoc. In this study, we evaluate principles underlying why careful application of multiple phage (i.e., a ‘cocktail’) might lead to therapeutic success in contrast to the failure of single-strain phage therapy to control an infection. First, we use a nonlinear dynamics model of within-host interactions to show that a combination of fast intra-host phage decay, evolution of phage resistance amongst bacteria, and/or compromised immune response might limit the effectiveness of single-strain phage therapy. To resolve these problems, we combine dynamical modeling of phage, bacteria, and host immune cell populations with control-theoretic principles (via optimal control theory) to devise evolutionarily robust phage cocktails and delivery schedules to control the bacterial populations. Our numerical results suggest that optimal administration of single-strain phage therapy may be sufficient for curative outcomes in immunocompetent patients, but may fail in immunodeficient hosts due to phage resistance. We show that optimized treatment with a two-phage cocktail that includes a counter-resistant phage can restore therapeutic efficacy in immunodeficient hosts.

Soil microbial communities and extracellular enzymes have important roles in many terrestrial ecosystem processes and are influenced by climate change drivers. In the present study, the individual and/or interactive effects of climate change drivers on soil microbial communities and extracellular enzyme activities were measured in experimental field plots planted with Pinus densiflora and subjected to air warming (ambient or +3 °C) in combination with precipitation treatments (reduced by 30%, ambient or elevated by 30%). Soil microbial biomass and four extracellular enzyme activities were measured. Additionally, the structure and composition of microbial communities were assessed. Warming increased microbial biomass nitrogen concentration by 22.3% in precipitation control plots and by 17.9% in elevated precipitation plots. Warming lowered the extracellular enzyme activity in precipitation control plots, but increased their activity in elevated precipitation plots. Warming had a differential effect on the composition of bacterial and fungal communities under different precipitation treatments, with significant changes in therelative abundance of Proteobacteria and Acidobacteria. Meanwhile, the alpha diversity index of both bacterial and fungal communities were affected by warming, with variation among the precipitation treatments. Changes in enzyme activities and microbial communities were correlated with shifts in soil environmental conditions (e.g., moisture, temperature, and available nutrients). In conclusion, changes in soil environmental conditions may select for distinct soil microbial communities, further reshaping soil ecosystem processes and functions in a warmer world.

Lipase is one of the most widely used classes of enzymes in biotechnological applications and organic chemistry. Candida rugosa lipases (CRL) can catalyze hydrolysis, esterification and transesterification with high regio-, stereo- and enantio-selectivity. However, thermal inactivation above 45 °C limits CRL's applications. Studies on improving the thermal stability of CRL are often limited by its slow-growing eukaryotic expression host, which is not suitable for large-scale screening. Identification of thermally stable mutants by rational design, regarded as an efficient substitution of experimental efforts, would provide a method for site-directed improvement of CRL. In this study, mutation-induced stability changes in CRL Lip1 were predicted by three rational design methods. Followed by conservative analyses and functional region exclusion, five mutants of a hot-spot, Asp457Phe, Asp457Trp, Asp457Met, Asp457Leu, and Asp457Tyr, were identified and prepared for enzymatic characterization. These five mutants increased the apparent melting temperature of Lip1 from 7.4 °C to 9.3 °C, with the most thermostable mutant, Asp457Phe, exhibiting a 5.5-fold longer half-life at 50 °C and a 10 °C increase in optimum temperature. Furthermore, pH stability of Lip1 was also enhanced due to the introduction of Asp457Phe mutation. The study demonstrates that thermally stable mutants of CRL could be identified with limited experimental efforts using rational design methods.

// Fuhao Qiao 1, 2 , Jian Fang 1 , Jinfeng Xu 1 , Wenqiu Zhao 1 , Ying Ni 1 , Bufugdi Andreas Akuo 1 , Wei Zhang 1 , Yun Liu 1 , Fangfang Ding 1 , Guanlin Li 3 , Baoguo Liu 4 , Hua Wang 1 and Shihe Shao 1 1 School of Medicine, Jiangsu University, Zhenjiang 212013, Jiangsu, PR China 2 Medical Laboratory, Xintai Hospital of Traditional Chinese Medicine, Xintai 271200, Shandong, PR China 3 School of The Environment and Safety Engineering, Jiangsu University, Zhenjiang 212013, Jiangsu, PR China 4 Nuclear Medicine Laboratory, Taian Jiangong Hospital, Taian 271001, Shandong, PR China Correspondence to: Hua Wang, email: wangjiahan1979@163.com Shihe Shao, email: shaoshihe2006@163.com Keywords: adrenomedullin, autophagy, gastric cancer, RNA interference, signaling pathway Received: August 27, 2016 Accepted: June 13, 2017 Published: June 29, 2017 ABSTRACT Adrenomedullin has been shown to be overexpressed in many tumors, including gastric cancer tumors; however, its mechanism of action remains unclear. In this study, we examined the role of adrenomedullin in the pathogenesis of gastric cancer. Using clinical specimens and immunohistochemistry, we found that the expression levels of adrenomedullin and its receptors are inordinately elevated as compared to the adjacent non-tumor gastric tissues. We used siRNA gene silencing, in BGC-823 gastric cancer cell lines, to target adrenomedullin genes, and found that increased adrenomedullin expression results in the proliferation of tumor cells, tumor invasion, and metastasis. Furthermore, we found that under hypoxic conditions, gastric cancer BGC-823 cells exhibit higher expression levels of adrenomedullin and various other related proteins. Our results indicate the involvement of adrenomedullin in microvessel proliferation and partially in the release of hypoxia in solid tumors. Knockdown of adrenomedullin expression, at the protein level, reduced the levels of phosphoprotein kinase B and B-cell lymphoma 2 but increased the levels of cleaved-caspase3 and Bcl 2 associated x protein (Bax). Therefore, we hypothesized siRNA targeting of adrenomedullin genes inhibits various serine/threonine kinases via a signaling pathway that induces cell apoptosis. SiRNA targeting of adrenomedullin genes and green fluorescent control vectors were used to transfect BGC-823 cells, and western blot analyses were used to detect changes in the rates of autophagy in related proteins using confocal laser scanning microscopy. No significant changes were detected. Therefore, the knockdown of adrenomedullin and its receptors may represent a novel treatment strategy for gastric cancer.

Decidual NK (dNK) cells, identified as CD56brightCD16-CD3-, account for ~70% of lymphocytes within the uterine wall during early pregnancy. Accumulating evidence suggests that tight interactions between placental trophoblasts and dNK cells are critical for trophoblast cell differentiation. However, the underlying mechanism remains to be explored in detail. In the present study, conditioned medium (CM) was collected from cultured primary human dNK cells. Primary cytotrophoblasts (CTBs) or the human trophoblast cell line HTR8/SVneo was treated with dNK-CM and co-cultured with human umbilical vein endothelial cells (HUVECs) in a three-dimensional Matrigel scaffold, and the formation of tube structures was dynamically monitored with live cell imaging. Trophoblast invasion was analyzed with a transwell invasion assay. The data demonstrated that the treatment of HTR8/SVneo cells or CTBs with dNK-CM remarkably promoted trophoblast invasion and tube formation in the presence of HUVECs. The epithelial marker E-cadherin was reduced, while the expression of endothelial markers NCAM, VE-cadherin and integrin β1 was significantly promoted in the HTR8/SVneo cells upon treatment with dNK-CM. Antibody blocking experiments revealed that the dNK cells promoted trophoblast invasion through the production of IL-8 and HGF, and they induced trophoblast differentiation toward endothelial phenotype by producing VEGF-C and HGF. These results provide new evidence to clarify the finely tuned interactions between trophoblasts and dNK cells at the maternal-fetal interface.

In this study, we evaluated the performance of three blood culture systems in a Chinese tertiary-care hospital. Samples of simulated bacteremia were prepared using 10 mL of fresh blood from healthy humans and bacterial suspensions of known cell density. Portions of the specimens were treated with an antibiotic or antifungal drug at specified concentrations to simulate antibacterial drug treatment. We analyzed three blood culture systems: BACTEC Plus, BacT/Alert, and VersaTREK. Both time-to-detection (TTD) of 10 types of bacteria and five types of yeasts in samples without antibiotic treatment and positive detection rate of samples treated with different concentrations of antibiotic or antifungal drugs were compared among the culture systems. We also retrospectively analyzed the use of the culture systems in our hospital from 2015 to 2018. In the simulated study, in the absence of antibiotics, the VersaTREK REDOX 1 displayed the shortest TTD for Pseudomonas aeruginosa, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, Candida albicans, and Candida glabrata (P < 0.001). Among the anaerobically cultured samples, BACTEC lytic/10 anaerobic/F displayed the shortest TTD for Escherichia coli, S. aureus, Enterococcus faecalis, S. pneumoniae, Bacteroides fragilis, and Bacteroides vulgatus (P < 0.001). Comparatively, BacT/Alert FA/FN showed no advantages. In antibiotic-treated samples, overall recovery rates for the BACTEC, BacT/Alert, and VersaTREK systems were, were 70.2, 43.7, and 27.4%, respectively. BACTEC facilitated higher recovery rate than the other two systems (P < 0.001). In antifungal treatment, the overall recovery rates for the BACTEC, BacT/Alert, and VersaTREK systems were 93.9, 98.3, and 69.4%, respectively. BACTEC Plus showed a recovery rate comparable to that of BacT/Alert (P = 0.835), and the recovery rate of both these systems was higher than that of VersaTREK (P < 0.001). The TTD values and positive rates determined in the retrospective study were consistent with those obtained in the simulated study. The combination of BACTEC PLUS Aerobic/F and BACTEC lytic/10 anaerobic/F culture systems displayed the best clinical performance. Furthermore, the BacT/Alert FAN culture system was found to be more resistant to antifungal drugs and levofloxacin, whereas the VersaTREK system is considered more suitable for primary blood cultures without antibiotic supplementation.

In this study, biodiesel production from Chinese tallow kernel oil catalyzed by Candida rugosa lipase (CRL) in ionic liquid was investigated. Biodiesel was prepared from Chinese tallow kernel oil. Nineteen kinds of ionic liquids were screened for CRL to enhance its catalysis ability for biodiesel synthesis. Interestingly, [Hmim][PF6], not only help to simplify the downstream processing, but also boost the efficiency of CRL from about 35% to 95.4%. In addition, the biodiesel mixed with various blending ratios of fossil diesel was used to fuel the conventional diesel engine to examine fuel and emission properties. Most of fuel characteristics of the biodiesel met the standards set by EN and ASTM. Its output power was found to be close to pure diesel. The emission products, including hydrocarbons (HC, 52.7% lower) and carbon monoxide (CO, 48.6% lower), decreased substantially with the exception of oxynitride (NOx) emission, which displayed a 12.8% increase. The results suggest that biodiesel production from woody oil catalyzed by CRL in [Hmim][PF6], is feasible in future application with a promising prospect.

The prevailing paradigm in ecological studies of viruses and their microbial hosts is that the reproductive success of viruses depends on the proliferation of the ‘predator’, that is, the virus particle. Yet, viruses are obligate intracellular parasites, and the virus genome—the actual unit of selection—can persist and proliferate from one cell generation to the next without lysis or the production of new virus particles. Here, we propose a theoretical framework to quantify the invasion fitness of viruses using an epidemiological cell-centric metric that focuses on the proliferation of viral genomes inside cells instead of virus particles outside cells. This cell-centric metric enables direct comparison of viral strategies characterized by obligate killing of hosts (e.g. via lysis), persistence of viral genomes inside hosts (e.g. via lysogeny), and strategies along a continuum between these extremes (e.g. via chronic infections). As a result, we can identify environmental drivers, life history traits, and key feedbacks that govern variation in viral propagation in nonlinear population models. For example, we identify threshold conditions given relatively low densities of susceptible cells and relatively high growth rates of infected cells in which lysogenic and other chronic strategies have higher potential viral reproduction than lytic strategies. Altogether, the theoretical framework helps unify the ongoing study of eco-evolutionary drivers of viral strategies in natural environments.

International audience; AbstractKey messageThinning increased microbial biomass but did not alter enzyme activities in the soil ofPinus densifloraSieb. et Zucc. forests in South Korea. This effect of thinning was larger under a relatively heavy thinning intensity, but there was divergence in the magnitude between sites.ContextThe balance between microbial biomass accumulation and enzymatic C and N assimilation determines the level of bio-available C and N. However, the effects of thinning on these parameters remain contradictory and unconfirmed.AimsThe effects of thinning intensity on microbial biomass and enzyme activity were assessed in the soil of Pinus densiflora Sieb. et Zucc. forests in South Korea.MethodsUn-thinned control and 15 and 30% basal area thinning treatments were applied to two 51- to 60-year-old P. densiflora forests with different management histories, topographies, rainfall amounts, and soils. Seven years after thinning, microbial biomass and activities of N-acetyl-glucosaminidase, β-glucosidase, cellobiohydrolase, β-xylosidase, phenol oxidase, and peroxidase were measured before and after seasonally concentrated rains and at 0–10 cm depth.ResultsMicrobial biomass was generally highest under the 30% basal area thinning and lowest under the control, and was positively correlated to total soil C and N. The increase in microbial biomass was lower at the site displaying sandier, drier, and more acidic soils and retaining smaller amounts of thinning residue. Conversely, thinning had no significant effect on activities of all enzymes at both sites in both periods.ConclusionThinning can promote accumulation of microbial biomass without significant change in enzyme activities participating in the assimilation of C and N. This effect of thinning tended to increase with thinning intensity but differed in magnitude between sites.

Air warming (TC: control; TW: +3 °C) and precipitation manipulation (PC: control; PD: −30%; PI: +30%) were established to examine effects of these treatments on fine root production (FRP), fine root mortality (FRM), and total root (coarse and fine root) biomass in 33- to 59-month-old Pinus densiflora Sieb. et Zucc. seedlings for two years. We hypothesized that warming and altered precipitation would affect the growth, death, and biomass of fine roots by changing soil temperature and soil water availability. Mean annual FRP and total root biomass were significantly altered by only precipitation manipulation: they were 29.3% (during the two-year period) and 69.0% (after the entire two years) higher, respectively, in PD plots than in PC plots, respectively. In contrast, only warming had a significant effect on mean annual FRM, being 13.2% lower in TW plots than TC plots during the two-year period. Meanwhile, fine root biomass was affected negatively and simultaneously by both soil temperature and soil moisture. It seemed that fine root dynamics have changed so that they maintain their systems in response to the altered soil temperature and moisture. The current study adds significant knowledge for understanding the fine root dynamics of P. densiflora seedlings under altered temperature and precipitation regimes.

Diseases associated with Abelson-related gene (also called ABL2) include leukemia; furthermore, previous researches have studied the expressions and functions of ABL2 in different types of malignancies and found that it plays an important role in almost all kinds of cancers. Nevertheless, the mechanism of ABL2 in gastric cancer (GC) remains vague. In the present study, the level of ABL2 in human GC tissues was detected by immunohistochemistry. Also, the GC cell lines MGC-803 and BGC-823 were selected to stably knock down and overexpress the level of ABL2 by corresponding lentiviral vectors. Puromycin was used to maintain the stable low expression of ABL2 MGC-803 cells compared with control cells; what is more, the high expression of ABL2 BGC-823 cells was also obtained. Based on it, we detected the proteins associated with apoptosis, such as Bcl-2 family and caspase family by western blotting. The most appropriate concentration of puromycin to kill GC cells is 1 µg/mL; then, we obtained the corresponding stable cell lines. Furthermore, we found that high level of ABL2 in BGC-823 cells increased the expression of Bcl-XL, total PARP, and caspase3, while decreased the level of cleaved caspase3 and cleaved caspase9. Consistent results are received in MGC-803 cells. In addition, ABL2 overexpression led to the protein related with Ras/Erk and PI3K/AKT signaling pathway increased; also, we found that the major proteins play a significant role in it. All the data showed that high expression of ABL2 suppresses apoptosis through Ras/Erk and PI3K/AKT signaling pathway in GC cell lines.

Industrialization of enzyme-catalyzed biodiesel production has been hindered by low conversion efficiency and long reaction time. As a representative example, Rhizopus oryzae lipase (ROL) exhibited a poor performance in biodiesel production. However, higher yields and shorter reaction time could be achieved by screening different lipases that could synergically catalyze the reaction with ROL. The results showed that Novozym 435 combined with ROL displayed the best performance, with a biodiesel yield that was 30% higher than that of ROL alone in 30 h. Hence, the use of a synergic strategy was investigated systematically. Under optimized conditions, the biodiesel yield was as high as 98.3% and the reaction time was successfully shortened from 60 h to 21 h. Moreover, the combined lipases retained a yield of about 80% after 20 cycles in a solvent-free system. In addition to ROL, the performance of other lipases whose regioselectivity were similar to ROL was improved when they were combined with Novozym 435. In conclusion, synergic catalysis is a promising strategy for enhancing biodiesel yield and minimizing reaction time for ROL.

Rhizomucor miehei lipase (RML), as a kind of eukaryotic protein catalyst, plays an important role in the food, organic chemical, and biofuel industries. However, RML retains its catalytic activity below 50°C, which limits its industrial applications at higher temperatures. Soluble expression of this eukaryotic protein in Escherichia coli not only helps to screen for thermostable mutants quickly but also provides the opportunity to develop rapid and effective ways to enhance the thermal stability of eukaryotic proteins. Therefore, in this study, RML was engineered using multiple computational design methods, followed by filtration via conservation analysis and functional region assessment. We successfully obtained a limited screening library (only 36 candidates) to validate thermostable single point mutants, among which 24 of the candidates showed higher thermostability and 13 point mutations resulted in an apparent melting temperature ( T m app ) of at least 1°C higher. Furthermore, both of the two disulfide bonds predicted from four rational-design algorithms were further introduced and found to stabilize RML. The most stable mutant, with T18K/T22I/E230I/S56C-N63C/V189C-D238C mutations, exhibited a 14.3°C-higher T m app and a 12.5-fold increase in half-life at 70°C. The catalytic efficiency of the engineered lipase was 39% higher than that of the wild type. The results demonstrate that rationally designed point mutations and disulfide bonds can effectively reduce the number of screened clones to enhance the thermostability of RML. IMPORTANCE R. miehei lipase, whose structure is well established, can be widely applied in diverse chemical processes. Soluble expression of R. miehei lipase in E. coli provides an opportunity to explore efficient methods for enhancing eukaryotic protein thermostability. This study highlights a strategy that combines computational algorithms to predict single point mutations and disulfide bonds in RML without losing catalytic activity. Through this strategy, an RML variant with greatly enhanced thermostability was obtained. This study provides a competitive alternative for wild-type RML in practical applications and further a rapid and effective strategy for thermostability engineering.

Although coarse woody debris (CWD) is important for soil functioning, the mechanism which affects soil properties beneath CWD are unclear. Here, initial changes in microbial and soil properties were studied using homogenous CWD samples in eight Korean red pine (Pinus densiflora Sieb. et Zucc.) forests. For each forest, CWD samples (diameter: 11.1 ± 0.1 cm; length: 10.2 ± 0.0 cm) from similarly aged Korean red pine trees were laid on the mineral soil surface from May to June, 2016, and soils were sampled at points beneath CWD and at a distance of 1 m from the CWD after 1 year. Soils beneath the CWD had higher moisture but lower inorganic nitrogen (N) and a higher microbial biomass C (carbon)/N ratio than those sampled 1 m from the CWD. No differences in total C and N, labile C, pH, and C substrate utilization between the soils were significant. The difference in inorganic N between the soils decreased with increasing CWD decomposition, whereas that for microbial biomass fraction in total C and N increased correspondingly. Our results showed that soil microbial affinity for retaining N might become higher than that for retaining C under the presence of CWD, which possibly alters N availability and generates a spatial heterogeneity in forest soils.

In this study, hydrophobic macroporous resin NKA was employed as matrix for immobilization of free Rhizopus oryzae lipase (ROL). The performance of the immobilized ROL was significantly enhanced. The recovery activity was up to 1,293.78 % and the specific activity increased to 152,914 U/g-protein, which was 46-fold higher than that of the free lipase. Moreover, the immobilized lipase showed higher thermostability and better pH-resistance than its free counterpart. Additionally, three different nonaqueous modification strategies (including bioimprinting, lecithin coating, and lyophilization protection) were further utilized to improve the performance of the immobilized lipase. The corresponding enhancements were 33.68 %, 31.98 %, and 99.86 %. When these modifications were combined together, the activity improved 209.51 %. In order to confirm its practical application, the modified ROL was used to biorefine rapeseed oil deodorizer distillate (RODD) for biodiesel production. The highest conversion yield reached 98.23 %, much close to that (97.46 %) of Novozym 435. The results suggest that the prepared lipase in this study is a promising biocatalyst with high stability, efficiency and operational reusability.

To examine the effect of exopolysaccharides (EPS) on the microstructure and textural and rheological properties of yoghurt, purified EPS were added to buffalo milk to make yoghurts. The results showed that the viscosity, water-holding capacity, hardness, and microstructure of yoghurt were distinctly affected by the level of addition of EPS. With the increasing amount of supplemented EPS, the viscosity of buffalo yoghurt showed a downward trend with the increasing shear velocity, and an increasing degree of recovery of viscosity with the decrease in shear velocity, indicating that the steric barrier formed between the EPS and protein enhanced the viscoelastic properties. The microstructure showed more dense interspaced voids with EPS addition.

As the decomposition of lignocellulosic compounds is a rate-limiting stage in the nutrient mineralization from organic matters, elucidation of the changes in soil enzyme activity can provide insight into the nutrient dynamics and ecosystem functioning. The current study aimed to assess the effect of thinning intensities on soil conditions. Un-thinned control, 20 % thinning, and 30 % thinning treatments were applied to a Larix kaempferi forest, and total carbon and nitrogen, total carbon to total nitrogen ratio, extractable nutrients (inorganic nitrogen, phosphorus, calcium, magnesium, potassium), and enzyme activities (acid phosphatase, β-glucosidase, β-xylosidase, β-glucosaminidase) were investigated. Total carbon and nitrogen concentrations were significantly increased in the 30 % thinning treatment, whereas both the 20 and 30 % thinning treatments did not change total carbon to total nitrogen ratio. Inorganic nitrogen and extractable calcium and magnesium concentrations were significantly increased in the 20 % thinning treatment; however, no significant changes were found for extractable phosphorus and potassium concentrations either in the 20 or the 30 % thinning treatment. However, the applied thinning intensities had no significant influences on acid phosphatase, β-glucosidase, β-xylosidase, and β-glucosaminidase activities. These results indicated that thinning can elevate soil organic matter quantity and nutrient availability, and different thinning intensities may affect extractable soil nutrients inconsistently. The results also demonstrated that such inconsistent patterns in extractable nutrient concentrations after thinning might not be fully explained by the shifts in the enzyme-mediated nutrient mineralization.

The aim of this study was to investigate the short-term effects of warming treatment and precipitation manipulation on the growth, photosynthetic rate, and chlorophyll content of Pinus densiflora seedlings based on a climate change scenario in Korea. Two year old P. densiflora seedlings were planted in a nursery in April 2013. The air temperature of warmed plots (W) was set to increase by 3 °C compared to control plots (C) using an infrared heater in May 2013. The three precipitation manipulations consisted of decreased precipitation using transparent panels (–30%; P–), increased precipitation using pumps and drip irrigation (+30%; P+), and a control (0%; P0). Root collar diameter and seedling height of P. densiflora were measured in April and October 2013. Net photosynthetic rate and total chlorophyll contents were measured from June to October 2013. Warming treatment increased the growth of root collar diameter, and the interaction effect of the warming treatment and precipitation manipulation on it was also significant. In contrast, no significant effects were found for the growth of seedling height. These results demonstrate that warming treatment might affect the growth of root collar diameter and seedling height differently. It was also found that WP– treatment decreased net photosynthetic rates. This pattern might be due to the soil moisture availability, as soil moisture content was lowest in the WP– treatment and net photosynthetic rate was elevated with increasing soil moisture content. Meanwhile, warming treatment increased the total chlorophyll content. The results suggest that chlorophyll synthesis was promoted by a temperature increase in the leaves. The current study, which simulated a climate change environment in an open field, provides important information for the prediction of the ecophysiological effects of changes in temperature and precipitation on P. densiflora seedlings in Korea. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) that is funded by the Ministry of Education (2013R1A1A2012242) and by the Korea Forest Service (S111115L030100). Scopus

In the present study, a surface-active enzyme, lipase was immobilized in polyethyleneimine (PEI) microcapsules and then modified with oxidized multiwall carbon nanotubes (MWCNTs). The resulting lipase microcapsules exhibited higher activity and stability, since the activity of microcapsules was 21.9 folds than that of the free counterpart. Numerous interfaces which were created in polycondensation enhanced the performance of lipases. Illustrated by confocal laser scanning microscope (CLSM), it was found that microcapsules, whose barrier properties against molecules with diameter >4.6 nm, were with a semipermeable and porous membrane structure. The lipases preferred to locate in the wall of the microcapsules. The oxidized multiwall carbon nanotubes (MWCNTs) were further added to modify microcapsules, resulting in even higher activity. The nanocomposites were examined by scanning electron microscope (SEM) and zeta-potential analyzer. The results indicated the superior catalytic performances were attributed to the augmented interface and decreased positive charge. Finally, the MWCNTs modified microcapsules were utilized in producing biodiesel with a 97.15% yield and retained nearly 90% yield after running 10 cycles. This approach of microcapsulation will be highly beneficial for preparing various bio-active microcapsules with excellent catalytic performance.

The purpose of this study was to develop a multiplex touchdown PCR to identify three waterborne pathogens in water samples. Specific primers based on VP1332, inv and ipaH gene sequences were designed for V. parahaemolyticus, Y. enterocolitca and Shigella, respectively. By optimizing reaction conditions, assays obtained high specifity, sensitivity and repeatability. The detection limit can be as low as 0.005ng/μl V. parahaemolyticus and 0.5ng/μl Y. enterocolitca and 0.005ng/μl of Shigella respectively. The detection process was about 3~4 hours, saving a lot of time compared with microbiological culture method that required 2~3 days. The system could be useful for the detection of multiple pathogens in water samples to protect public health.

This study investigated the contribution of termites to mass loss of dead wood ( Macaranga bancana , Elateriospermum tapos , and Dillenia beccariana ) in a lowland tropical rainforest, Brunei Darussalam. Mesh bag method was used to exclude termites, and the mass remaining was monitored after 3, 7, 13, and 23 months. C/N ratio of the samples was analyzed after 13 and 23 months. Initial wood density was 0.63, 0.92, and 1.02 g/cm 3 for M. bancana , E. tapos , and D. beccariana , respectively, and the termite contribution to mass loss was an average (range) of 13.05±5.68 (4.17-29.59%), 3.48±1.13 (2.20-6.49), and 3.40±1.92% (0.74-10.78), respectively. Until 7 months, termites contributed highly to mass loss, given the low initial wood density, and interaction effect of species and treatment was significant. After 7 months, the contribution decreased in M. bancana and E. tapos , whereas it increased consistently in D. beccariana . The interaction effect was not significant, whereas differences in C/N ratio among the species were significant, with a lower C/N ratio in M. bancana and E. tapos than in D. beccariana . After 23 months, the differences in C/N ratio were not significant, and ants were present at 40% of control samples in M. bancana and E. tapos . Our results suggest that the contribution of termites to mass loss varies by dead wood species and is temporally variable. Initial wood traits could affect the termite feeding in the beginning, however, termites thereafter could forage in response to the varying C/N ratio among species and predators.

The high affinity binding of the anthrax protective antigen (PA) to one of its receptors, capillary morphogenesis protein 2 (CMG2), is essential for the intoxication process of anthrax toxin. To acquire novel research tools to study the PA–CMG2 interaction, we generated several anti-CMG2 monoclonal antibodies (MAbs). We demonstrated that one of the MAbs, 4B5, could inhibit PA–CMG2 binding and could also protect the sensitive cells against an anthrax lethal toxin challenge. We identified the epitope recognized by 4B5 and confirmed that the key residues of the epitope were the residues 119 YI–LK 125 of CMG2. Based on our results, we propose that 4B5 binds to the E122 pocket of CMG2 and interrupts the interaction between the pocket and the PA 2β3–2β4 loop. To our knowledge, this is the first report to illustrate that an anti-CMG2 antibody could inhibit the PA–CMG2 interaction and therefore interfere with the intoxication of anthrax toxin.

The major aim of present study is to develop and evaluate chimeric virus-like particles (VLPs) displaying a neutralizing epitope of anthrax protective antigen (PA) as a potential vaccine against anthrax. The truncated hepatitis B virus core (HBc) protein (aa 1-144) was used as a carrier, and the 2beta2-2beta3 loop of the PA domain 2 (aa 302-325) which has been shown contains a dominant neutralizing epitope was inserted into the major immunodominant region (MIR) of the HBc. The recombinant protein HBc-N144-PA-loop2 was expressed in Escherichia coli, and was able to form HBc-like particles confirmed by electron microscopy. The immunogenicity of these chimeric particles was evaluated in mice and guinea pigs. In mice the HBc-N144-PA-loop2 was able to induce PA-epitope specific antibodies; in guinea pigs it was able to induce PA-epitope specific antibodies and anthrax toxin-neutralizing antibodies regardless of whether alum adjuvant was used or not, and was able to partially protect the immunized guinea pigs against virulent anthrax spores challenge. This study suggests chimeric HBc particles carrying a neutralizing epitope of PA can induce protective immunity against Bacillus anthracis.

Junxia Zhang,1,* Qiang Zhou,2,* Ge Gao,3,* Yanfen Wang,1 Zhihui Fang,1 Guanlin Li,4 Mengfei Yu,5 Lingfei Kong,6 Ying Xing,3 Xiaoqun Gao11Department of Anatomy, Basic Medical College, Zhengzhou University, Henan, People’s Republic of China; 2Department of Pathology, Children’s Hospital of Zhengzhou City, Henan, People’s Republic of China; 3Department of Physiology, Basic Medical College, Zhengzhou University, Henan, People’s Republic of China; 4Department of Laboratory, The First Affiliated Hospital of Zhengzhou University, Henan, People’s Republic of China; 5Department of Pharmacy, Zhengzhou Central Hospital, Zhengzhou University, Henan, People’s Republic of China; 6Department of Pathology, Henan Provincial People’s Hospital, Henan, People’s Republic of China *These authors contributed equally to this workAbstract: Glioblastoma is one of the most common malignant tumors in the nervous system in both adult and pediatric patients. Studies suggest that abnormal activation of receptor tyrosine kinases contributes to pathological development of glioblastoma. However, current therapies targeting tyrosine kinase receptors have poor therapeutic outcomes. Here, we examined anticancer effects of ponatinib, a multi-targeted tyrosine kinase inhibitor, on glioblastoma cells both in the U87MG cell line and in the mouse xenograft model. We showed that ponatinib treatment reduced cell viability and induced cell apoptosis in a dose-dependent manner in U87MG cells. In addition, ponatinib suppressed migration and invasion of U87MG cells effectively. Furthermore, ponatinib-treated tumors showed an obvious reduction of tumor volume and an increase of apoptosis as compared with vehicle-treated tumors in the mouse xenograft model. These findings support a potential application of ponatinib as a chemotherapeutic option against glioblastoma cells.Keywords: cancer therapy, glioma, glioblastoma multiforme

Lethal factor (LF), a virulence factor of Bacillus anthracis, plays key roles in anthrax pathogenesis and host-pathogen interactions. The detailed mechanisms by which LF contributes to infection are still under investigation. While these studies require pure, homogeneous and reliable LF preparations, most methods reported for production of recombinant LF (rLF) in B. anthracis or Escherichia coli either are complicated or add extra residues to the protein. In this work, we modified our previous method by codon optimization and chromatograph workflow refinement and developed an improved strategy for efficient production of rLF from the periplasm of E. coli. We were able to obtain fully functional rLF with a purity above 95% and with a considerable yield of 5 mg/L. The preparation was characterized by SDS–PAGE, Western blot, and N-terminal sequencing, and the activity was validated by intoxication of macrophages and Fischer 344 rats. Our final product is suitable for most research involving drug development and mechanism analysis of anthrax pathogenesis.

Plant disease identification based on image processing could quickly and accurately provide useful information for the prediction and control of plant diseases. In this study, 21 color features, 4 shape features and 25 texture features were extracted from the images of two kinds wheat diseases (wheat stripe rust and wheat leaf rust) and two kinds of grape diseases (grape downy mildew and grape powdery mildew), principal component analysis (PCA) was performed for reducing dimensions in feature data processing, and then neural networks including backpropagation (BP) networks, radial basis function (RBF) neural networks, generalized regression networks (GRNNs) and probabilistic neural networks (PNNs) were used as the classifiers to identify wheat diseases and grape diseases, respectively. The results showed that these neural networks could be used for image recognition of these diseases based on reducing dimensions using PCA and acceptable fitting accuracies and prediction accuracies could be obtained. For the two kinds of wheat diseases, the optimal recognition result was obtained when image recognition was conducted based on PCA and BP networks, and the fitting accuracy and the prediction accuracy were both 100%. For the two kinds of grape diseases, the optimal recognition results were obtained when GRNNs and PNNs were used as the classifiers after reducing the dimensions of feature data with PCA, and the prediction accuracies were 94.29% with the fitting accuracies equal to 100%.

The pandemic 2009 H1N1 influenza virus broke out in North America and spread rapidly throughout the world. The type I interferon (IFN) response represents one of the first lines of defense against influenza virus infections. In this study, the protective potential of human exogenous IFN-ω against pandemic 2009 A (H1N1) influenza virus was assessed both in vitro and in guinea pigs. The viral loads of pandemic 2009 A (H1N1) influenza virus strains A/California/04/2009 and A/Beijing/501/2009 were reduced by up to 5000-fold in Caco-2 cells by the addition of human IFN-ω. With daily intranasal treatment with human IFN-ω the viral load of pandemic 2009 A (H1N1) influenza virus strain A/California/04/2009 decreased by 1000-fold in lung tissues of guinea pigs. These results provide strong support for the application of human IFN-ω pretreatment to human influenza control.

The whole encoding sequence for Yersinia pestis LcrV antigen was cloned into pET-32a(+) and expressed in Escherichia coli BL21 (DE3). The LcrV was high level expressed in the E. coli cytoplasm in a completely soluble form. Recombinant LcrV could be purified from the supernatant of the bacteria lysate after chromatography using a combination of Phenyl-Sepharose F F, DEAE-Sepharose F F and Hiload™ Superdex™ 75. The final yield of approximately 3 g of purified rLcrV from 42 L bioreactor containing 25 L LB medium was obtained. High-titer IgG directed against rLcrV was detected positive after immunization on the BALB/c mice. The results presented here exhibit the ability to generate multi-gram quantities of non-tagged rLcrV from E. coli that can be used for the development of vaccine for preventing plague.

Anthrax toxin consists of three proteins, protective antigen (PA), lethal factor, and edema factor. PA is the major component in the current anthrax vaccine, but the antigenic epitopes on it are not well-defined. We generated a pool of toxin-neutralizing anti-PA monoclonal antibodies (MAbs) to analyze the neutralizing epitopes of PA. Nine toxin-neutralizing MAbs obtained were found bound to three different domains of PA respectively, among which three MAbs with the strongest toxin-neutralizing activity recognized the same epitope within domain 2. This epitope was fine mapped to the chymotrypsin-sensitive site, (312)SFFD(315), in the 2beta(2)-2beta(3) loop of PA, using phage-displayed random peptide libraries and mutation analysis. The result demonstrated for the first time that the 2beta(2)-2beta(3) loop, which is involved in the transition of PA oligomers from prepore to pore, contains a dominant neutralizing epitope. This work contributes to the immunological and functional analysis of PA and offers perspective for the development of a new epitope vaccine against anthrax.