Preparation of connexin43‐integrated giant Liposomes by a baculovirus expression–liposome fusion method

Connexin‐43 (Cx43) containing giant liposomes (GL) were prepared by a baculovirus expression–liposome fusion method. Recombinant budded viruses expressing Cx43 were prepared and then fused with GLs containing DOPG/DOPC at pH 4.5. Connexon formation on the GL membrane was observed by transmission electron microscope. Hydrophilic fluorescent dye transfers were observed through a Cx43‐mediated pathway not only between Sf9 (Spodoptera frugiperda) cells with Cx43 but also from giant Cx43 liposomes to Cx43‐expressing U2OS cells (human osteosarcoma cell). The functional connexin‐containing liposome is expected to be useful for cellular cytosolic delivery systems. The original orientation and function of Cx43 was maintained after integration into the liposomes. The liposome fusion method will create new opportunities as a tool for analysis of channel membrane proteins. Biotechnol. Bioeng. 2010;107: 836–843. © 2010 Wiley Periodicals, Inc.     

Population dynamics of Bifidobacterium species in human feces during raffinose administration monitored by fluorescence in situ hybridization-flow cytometry

The population dynamics of bifidobacteria in human feces during raffinose administration were investigated at the species level by using fluorescence in situ hybridization (FISH) coupled with flow cytometry (FCM) analysis. Although double-staining FISH-FCM using both fluorescein isothiocyanate (FITC) and indodicarbocyanine (Cy5) as labeling dyes for fecal samples has been reported, the analysis was interfered with by strong autofluorescence at the FITC fluorescence region because of the presence of autofluorescence particles/debris in the fecal samples. We circumvented this problem by using only Cy5 fluorescent dye in the FISH-FCM analysis. Thirteen subjects received 2 g of raffinose twice a day for 4 weeks. Fecal samples were collected, and the bifidobacterial populations were monitored using the established FISH-FCM method. The results showed an increase in bifidobacteria from about 12.5% of total bacteria in the prefeeding period to about 28.7 and 37.2% after the 2-week and 4-week feeding periods, respectively. Bifidobacterium adolescentis, the Bifidobacterium catenulatum group, and Bifidobacterium longum were the major species, in that order, at the prefeeding period, and these bacteria were found to increase nearly in parallel during the raffinose administration. During the feeding periods, indigenous bifidobacterial populations became more diverse, such that minor species in human adults, such as Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium dentium, and Bifidobacterium angulatum, proliferated. Four weeks after raffinose administration was stopped, the proportion of each major bifidobacterial species, as well as that of total bifidobacteria, returned to approximately the original values for the prefeeding period, whereas that of each minor species appeared to differ considerably from its original value. To the best of our knowledge, these results provide the first clear demonstration of the population dynamics of indigenous bifidobacteria at the species level in response to raffinose administration.     

Studies on Haematococcus pluvialis for improved production of astaxanthin by mutagenesis

Cultures of Haematococcus pluvialis were exposed to mutagens like u.v. and EMS (ethyl methanesulphonate). The results showed that the survival rate decreased with the increase in u.v. exposure time and increase in EMS concentration. These mutants were further screened using inhibitors of the carotenoid biosynthetic pathway viz. diphenylamine (15–90 M), nicotine (160–320 M) and compactin (1.5–3.0 M). The mutants thus obtained showed early enhanced (2.2–3.2-fold) astaxanthin accumulation and also exhibited higher lycopene cyclase activity.     

Preclinical and clinical developments for combination treatment of influenza

Antiviral drugs are an important measure of control for influenza in the population, particularly for those that are severely ill or hospitalised. The neuraminidase inhibitor (NAI) class of drugs, including oseltamivir, have been the standard of care (SOC) for severe influenza illness for many years. The approval of drugs with novel mechanisms of action, such as baloxavir marboxil, is important and broadens potential treatment options for combination therapy. The use of antiviral treatments in combination for influenza is of interest; one potential benefit of this treatment strategy is that the combination of drugs with different mechanisms of action may lower the selection of resistance due to treatment. In addition, combination therapy may become an important treatment option to improve patient outcomes in those with severe illness due to influenza or those that are immunocompromised. Clinical trials increasingly evaluate drug combinations in a range of patient cohorts. Here, we summarise preclinical and clinical advances in combination therapy for the treatment of influenza with reference to immunocompromised animal models and clinical data in hospitalised patient cohorts where available. There is a wide array of drug categories in development that have also been tested in combination. Therefore, in this review, we have included polymerase inhibitors, monoclonal antibodies (mAbs), host-targeted therapies, and adjunctive therapies. Combination treatment regimens should be carefully evaluated to determine whether they provide an added benefit relative to effectiveness of monotherapy and in a variety of patient cohorts, particularly, if there is a greater chance of an adverse outcome. Safe and effective treatment of influenza is important not only for seasonal influenza infection, but also if a pandemic strain was to emerge.     

A Data-Driven Mathematical Model of CA-MRSA Transmission among Age Groups: Evaluating the Effect of Control Interventions

Community associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has become a major cause of skin and soft tissue infections (SSTIs) in the US. We developed an age-structured compartmental model to study the spread of CA-MRSA at the population level and assess the effect of control intervention strategies. We used Monte-Carlo Markov Chain (MCMC) techniques to parameterize our model using monthly time series data on SSTIs incidence in children (≤19 years) during January 2004 -December 2006 in Maricopa County, Arizona. Our model-based forecast for the period January 2007–December 2008 also provided a good fit to data. We also carried out an uncertainty and sensitivity analysis on the control reproduction number, which we estimated at 1.3 (95% CI [1.2,1.4]) based on the model fit to data. Using our calibrated model, we evaluated the effect of typical intervention strategies namely reducing the contact rate of infected individuals owing to awareness of infection and decolonization strategies targeting symptomatic infected individuals on both and the long-term disease dynamics. We also evaluated the impact of hypothetical decolonization strategies targeting asymptomatic colonized individuals. We found that strategies focused on infected individuals were not capable of achieving disease control when implemented alone or in combination. In contrast, our results suggest that decolonization strategies targeting the pediatric population colonized with CA-MRSA have the potential of achieving disease elimination.     

Vesicular stomatitis virus-based H5N1 avian influenza vaccines induce potent cross-clade neutralizing antibodies in rhesus macaques

We analyzed the ability of a vaccine vector based on vesicular stomatitis virus (VSV) to induce a neutralizing antibody (NAb) response to avian influenza viruses (AIVs) in rhesus macaques. Animals vaccinated with vectors expressing either strain A/Hong Kong/156/1997 or strain A/Vietnam/1203/2004 H5 hemagglutinin (HA) were able to generate robust NAb responses. The ability of the vectors to induce NAbs against homologous and heterologous AIVs after a single dose was dependent upon the HA antigen incorporated into the VSV vaccine. The vectors expressing strain A/Vietnam/1203/2004 H5 HA were superior to those expressing strain A/Hong Kong/156/1997 HA at inducing cross-clade NAbs.     

Impact of elevated temperatures on greenhouse gas emissions in rice systems: interaction with straw incorporation studied in a growth chamber experiment

Aims

Two pot experiments in a “walk-in” growth chamber with controlled day and night temperatures were conducted to investigate the influence of elevated temperatures along with rice straw incorporation on methane (CH₄) and nitrous oxide (N₂O) emissions as well as rice yield.

Methods

Three temperature regimes–29/25, 32/25, and 35/30 °C (Exp. I) and 29/22, 32/25, and 35/28 °C (Exp. II), representing daily maxima/minima were used in the study. Two amounts of rice straw (0 and 6 t ha^?1) were applied with four replications in each temperature regime. CH₄ and N₂O emissions as well as soil redox potential (Eh) were monitored weekly throughout the rice-growing period.

Results

Elevated temperatures increased CH₄ emission rates, with a more pronounced effect from flowering to maturity. The increase in emissions was further enhanced by incorporation of rice straw. A decrease in soil Eh to <?100 mV and CH₄ emissions was observed early in rice straw–incorporated pots while the soil without straw did not reach negative Eh levels (Exp. I) or showed a delayed decrease (Exp. II). Moreover, soil with high organic C (Exp. II) had higher CH₄ emissions. In contrast to CH₄ emissions, N₂O emissions were negligible during the rice-growing season. The global warming potential (GWP) was highest at high temperature with rice straw incorporation compared with low temperature without rice straw. On the other hand, the high temperature significantly increased spikelet sterility and reduced grain yield (p?<?0.05).

Conclusions

Elevated temperature increased GWP while decreased rice yield. This suggests that global warming may result in a double negative effect: higher emissions and lower yields.     

Mammalian Adaptation in the PB2 Gene of Avian H5N1 Influenza Virus

The substitution of glutamic acid (E) for lysine (K) at position 627 of the PB2 protein of avian H5N1 viruses has been identified as a virulence and host range determinant for infection of mammals. Here, we report that the E-to-K host-adaptive mutation in the PB2 gene appeared from day 4 and 5 along the respiratory tracts of mice and was complete by day 6 postinoculation. This mutation correlated with efficient replication of the virus in mice.     

Homology modeling and docking studies of FabH (β-ketoacyl-ACP synthase III) enzyme involved in type II fatty acid biosynthesis of Chlorella variabilis: a potential algal feedstock for biofuel production

The concept of using microalgae as an alternative renewable source of biofuel has gained much importance in recent years. However, its commercial feasibility is still an area of concern for researchers. Unraveling the fatty acid metabolic pathway and understanding structural features of various key enzymes regulating the process will provide valuable insights to target microalgae for augmented oil content. FabH (β-ketoacyl-acyl carrier protein synthase; KAS III) is a condensing enzyme catalyzing the initial elongation step of type II fatty acid biosynthetic process and acyl carrier protein (ACP) facilitates the shuttling of the fatty acyl intermediates to the active site of the respective enzymes in the pathway. In the present study, a reliable three-dimensional structure of FabH from Chlorella variabilis, an oleaginous green microalga was modeled and subsequently the key residues involved in substrate binding were determined by employing protein–protein docking and molecular dynamics (MD) simulation protocols. The FabH-ACP complex having the lowest docking energy score showed the binding of ACP to the electropositive FabH surface with strong hydrogen bond interactions. The MD simulation results indicated that the substrate-complexed FabH adopted a more stable conformation than the free enzyme. Further, the FabH structure retained its stability throughout the simulation although noticeable displacements were observed in the loop regions. Molecular simulation studies suggested the importance of crucial hydrogen bonding of the conserved Arg⁹¹ of FabH with Glu⁵³ and Asp⁵⁶ of ACP for exhibiting high affinity between the enzyme and substrate. The molecular modeling results are consistent with available experimental results on the flexibility of FabH and the present study provides first in silico insights into the structural and dynamical aspect of catalytic mechanism of FabH, which could be used for further site-specific mutagenic experiments to develop engineered high oil-yielding microalgal strains for biofuel production.