The Lao Experience in Deploying Influenza A(H1N1)pdm09 Vaccine: Lessons Made Relevant in Preparing for Present Day Pandemic Threats

The Lao PDR, as did most countries of the Mekong Region, embarked on a pandemic vaccine initiative to counter the threat posed by influenza A(H1N1)pdm09. Overall, estimated vaccine coverage of the Lao population was 14%, with uptake in targeted health care workers and pregnant women 99% and 41%, respectively. Adverse Events Following Immunization accounted for only 6% of survey driven, reported vaccination experiences, with no severe consequences or deaths. Public acceptability of the vaccine campaign was high (98%). Challenges to vaccine deployment included: 1) no previous experience in fielding a seasonal influenza vaccine, 2) safety and efficacy concerns, and 3) late arrival of vaccine 10 months into the pandemic. The Lao success in surmounting these hurdles was in large measure attributed to the oversight assigned the National Immunization Program, and national sensitivities in responding to the avian influenza A(H5N1) crisis in the years leading up to the pandemic. The Lao “lessons learned” from pandemic vaccine deployment are made even more relevant four years on, given the many avian influenza strains circulating in the region, all with pandemic potential.     

Identification of a Cryptic Bacterial Promoter in Mouse (mdr1a) P-Glycoprotein cDNA

The efflux transporter P-glycoprotein (P-gp) is an important mediator of various pharmacokinetic parameters, being expressed at numerous physiological barriers and also in multidrug-resistant cancer cells. Molecular cloning of homologous cDNAs is an important tool for the characterization of functional differences in P-gp between species. However, plasmids containing mouse mdr1a cDNA display significant genetic instability during cloning in bacteria, indicating that mdr1a cDNA may be somehow toxic to bacteria, allowing only clones containing mutations that abrogate this toxicity to survive transformation. We demonstrate here the presence of a cryptic promoter in mouse mdr1a cDNA that causes mouse P-gp expression in bacteria. This expression may account for the observed toxicity of mdr1a DNA to bacteria. Sigma 70 binding site analysis and GFP reporter plasmids were used to identify sequences in the first 321 bps of mdr1a cDNA capable of initiating bacterial protein expression. An mdr1a M107L cDNA containing a single residue mutation at the proposed translational start site was shown to allow sub-cloning of mdr1a in E. coli while retaining transport properties similar to wild-type P-gp. This mutant mdr1a cDNA may prove useful for efficient cloning of mdr1a in E. coli.     

Maintenance of Chronic Fatigue Syndrome (CFS) in Young CFS Patients Is Associated with the 5-HTTLPR and SNP rs25531 A > G Genotype

Earlier studies have shown that genetic variability in the SLC6A4 gene encoding the serotonin transporter (5-HTT) may be important for the re-uptake of serotonin (5-HT) in the central nervous system. In the present study we investigated how the 5-HTT genotype i.e. the short (S) versus long (L) 5-HTTLPR allele and the SNP rs25531 A > G affect the physical and psychosocial functioning in patients with chronic fatigue syndrome (CFS). All 120 patients were recruited from The Department of Paediatrics at Oslo University Hospital, Norway, a national referral center for young CFS patients (12–18 years). Main outcomes were number of steps per day obtained by an accelerometer and disability scored by the Functional Disability Inventory (FDI). Patients with the 5-HTT SS or SL_G genotype had a significantly lower number of steps per day than patients with the 5-HTT L_AL_G, SL_A or L_AL_A genotype. Patients with the 5-HTT SS or SL_G genotype also had a significantly higher FDI score than patients with the 5-HTT L_AL_G, SL_A or L_AL_A genotype. Thus, CFS patients with the 5-HTT SS or SL_G genotype had worse 30 weeks outcome than CFS patients with the 5-HTT L_AL_G, SL_A or L_AL_A genotype. The present study suggests that the 5-HTT genotype may be a factor that contributes to maintenance of CFS.     

Climate warming alters effects of management on population viability of threatened species: results from a 30‐year experimental study on a rare orchid

Climate change is expected to influence the viability of populations both directly and indirectly, via species interactions. The effects of large‐scale climate change are also likely to interact with local habitat conditions. Management actions designed to preserve threatened species therefore need to adapt both to the prevailing climate and local conditions. Yet, few studies have separated the direct and indirect effects of climatic variables on the viability of local populations and discussed the implications for optimal management. We used 30 years of demographic data to estimate the simultaneous effects of management practice and among‐year variation in four climatic variables on individual survival, growth and fecundity in one coastal and one inland population of the perennial orchid Dactylorhiza lapponica in Norway. Current management, mowing, is expected to reduce competitive interactions. Statistical models of how climate and management practice influenced vital rates were incorporated into matrix population models to quantify effects on population growth rate. Effects of climate differed between mown and control plots in both populations. In particular, population growth rate increased more strongly with summer temperature in mown plots than in control plots. Population growth rate declined with spring temperature in the inland population, and with precipitation in the coastal population, and the decline was stronger in control plots in both populations. These results illustrate that both direct and indirect effects of climate change are important for population viability and that net effects depend both on local abiotic conditions and on biotic conditions in terms of management practice and intensity of competition. The results also show that effects of management practices influencing competitive interactions can strongly depend on climatic factors. We conclude that interactions between climate and management should be considered to reliably predict future population viability and optimize conservation actions.     

Airway hyperresponsiveness is associated with airway remodeling but not inflammation in aging Cav1-/- mice

Background

Airway inflammation and airway remodeling are the key contributors to airway hyperresponsiveness (AHR), a characteristic feature of asthma. Both processes are regulated by Transforming Growth Factor (TGF)-β. Caveolin 1 (Cav1) is a membrane bound protein that binds to a variety of receptor and signaling proteins, including the TGF-β receptors. We hypothesized that caveolin-1 deficiency promotes structural alterations of the airways that develop with age will predispose to an increased response to allergen challenge.

Methods

AHR was measured in Cav1-deficient and wild-type (WT) mice 1 to 12 months of age to examine the role of Cav1 in AHR and the relative contribution of inflammation and airway remodeling. AHR was then measured in Cav1^-/- and WT mice after an ovalbumin-allergen challenge performed at either 2 months of age, when remodeling in Cav1^-/- and WT mice was equivalent, and at 6 months of age, when the Cav1^-/- mice had established airway remodeling.

Results

Cav1^-/- mice developed increased thickness of the subepithelial layer and a correspondingly increased AHR as they aged. In addition, allergen-challenged Cav1^-/- mice had an increase in AHR greater than WT mice that was largely independent of inflammation. Cav1^-/- mice challenged at 6 months of age have decreased AHR compared to those challenged at 2 months with correspondingly decreased BAL IL-4 and IL-5 levels, inflammatory cell counts and percentage of eosinophils. In addition, in response to OVA challenge, the number of goblet cells and α-SMA positive cells in the airways were reduced with age in response to OVA challenge in contrast to an increased collagen deposition further enhanced in absence of Cav1.

Conclusion

A lack of Cav1 contributed to the thickness of the subepithelial layer in mice as they aged resulting in an increase in AHR independent of inflammation, demonstrating the important contribution of airway structural changes to AHR. In addition, age in the Cav1^-/- mice is a contributing factor to airway remodeling in the response to allergen challenge.     

In‐depth characterization of Trichoderma reesei cellobiohydrolase TrCel7A produced in Nicotiana benthamiana reveals limitations of cellulase production in plants by host‐specific post‐translational modifications

Sustainable production of biofuels from lignocellulose feedstocks depends on cheap enzymes for degradation of such biomass. Plants offer a safe and cost‐effective production platform for biopharmaceuticals, vaccines and industrial enzymes boosting biomass conversion to biofuels. Production of intact and functional protein is a prerequisite for large‐scale protein production, and extensive host‐specific post‐translational modifications (PTMs) often affect the catalytic properties and stability of recombinant enzymes. Here we investigated the impact of plant PTMs on enzyme performance and stability of the major cellobiohydrolase TrCel7A from Trichoderma reesei, an industrially relevant enzyme. TrCel7A was produced in Nicotiana benthamiana using a vacuum‐based transient expression technology, and this recombinant enzyme (TrCel7A^rec) was compared with the native fungal enzyme (TrCel7A^nat) in terms of PTMs and catalytic activity on commercial and industrial substrates. We show that the N‐terminal glutamate of TrCel7A^rec was correctly processed by N. benthamiana to a pyroglutamate, critical for protein structure, while the linker region of TrCel7A^rec was vulnerable to proteolytic digestion during protein production due to the absence of O‐mannosylation in the plant host as compared with the native protein. In general, the purified full‐length TrCel7A^rec had 25% lower catalytic activity than TrCel7A^nat and impaired substrate‐binding properties, which can be attributed to larger N‐glycans and lack of O‐glycans in TrCel7A^rec. All in all, our study reveals that the glycosylation machinery of N. benthamiana needs tailoring to optimize the production of efficient cellulases.     

Non-Photochemical Quenching Capacity in Arabidopsis thaliana Affects Herbivore Behaviour

Under natural conditions, plants have to cope with numerous stresses, including light-stress and herbivory. This raises intriguing questions regarding possible trade-offs between stress defences and growth. As part of a program designed to address these questions we have compared herbivory defences and damage in wild type Arabidopsis thaliana and two “photoprotection genotypes”, npq4 and oePsbS, which respectively lack and overexpress PsbS (a protein that plays a key role in qE-type non-photochemical quenching). In dual-choice feeding experiments both a specialist (Plutella xylostella) and a generalist (Spodoptera littoralis) insect herbivore preferred plants that expressed PsbS most strongly. In contrast, although both herbivores survived equally well on each of the genotypes, for oviposition female P. xylostella adults preferred plants that expressed PsbS least strongly. However, there were no significant differences between the genotypes in levels of the 10 most prominent glucosinolates; key substances in the Arabidopsis anti-herbivore chemical defence arsenal. After transfer from a growth chamber to the field we detected significant differences in the genotypes’ metabolomic profiles at all tested time points, using GC-MS, but no consistent “metabolic signature” for the lack of PsbS. These findings suggest that the observed differences in herbivore preferences were due to differences in the primary metabolism of the plants rather than their contents of typical “defence compounds”. A potentially significant factor is that superoxide accumulated most rapidly and to the highest levels under high light conditions in npq4 mutants. This could trigger changes in planta that are sensed by herbivores either directly or indirectly, following its dismutation to H₂O₂.     

Characterization of a myosin VII MyTH/FERM domain

A group of closely related myosins is characterized by the presence of at least one MyTH/FERM (myosin tail homology; band 4.1, ezrin, radixin, moesin) domain in their C-terminal tails. This domain interacts with a variety of binding partners, and mutations in either the MyTH4 or the FERM domain of myosin VII and myosin XV result in deafness, highlighting the functional importance of each domain. The N-terminal MyTH/FERM region of Dictyostelium myosin VII (M7) has been isolated as a first step toward gaining insight into the function of this domain and its interaction with binding partners. The M7 MyTH4/FERM domain (MF1) binds to both actin and microtubules in vitro, with dissociation constants of 13.7 and 1.7 μM, respectively. Gel filtration and UV spectroscopy reveal that MF1 exists as a monomer in solution and forms a well-folded, compact conformation with a high degree of secondary structure. These results indicate that MF1 forms an integrated structural domain that serves to couple actin filaments and microtubules in specific regions of the cytoskeleton.     

Effects of herbivory on competition intensity in two arctic-alpine tundra communities with different productivity

The effects of long-term (11 yr) exclusion of vertebrate herbivores on competition intensity and plant community structure were studied using manipulative field experiments in two arctic-alpine plant communities with contrasting productivity: an unproductive snowbed and a considerably more productive tall herb meadow. In the snowbed, the exclusion of herbivorous mammals resulted in a significant increase in the biomasses of vascular plants and cryptogams, whereas no corresponding response was observed on the tall herb meadow. The intensity of competition, measured with a neighbour removal experiment, did not differ significantly between three of the four habitat×treatment combinations – snowbed exclosures, meadow exclosures and open meadow plots – but was significantly lower on open snowbed plots. Our results thus suggest that the low competition intensity in the unproductive snowbed is caused by herbivorous mammals, which tend to depress plant biomass in relatively unproductive habitats. When herbivorous mammals have been excluded for a sufficiently long time to allow the build-up of plant biomass even in unproductive habitats, between-habitat differences in competition intensity disappear.     

The Hyplip2 locus causes hypertriglyceridemia by decreased clearance of triglycerides

The Hyplip2 congenic mouse strain contains part of chromosome 15 from MRL/MpJ on the BALB/cJ background. Hyplip2 mice show increased plasma levels of cholesterol and predominantly triglycerides (TGs) and are susceptible to diet-induced atherosclerosis. This study aimed at elucidation of the mechanism(s) explaining the hypertriglyceridemia. Hypertriglyceridemia can result from increased intestinal or hepatic TG production and/or by decreased LPL-mediated TG clearance. The intestinal TG absorption and chylomicron formation were studied after intravenous injection of Triton WR1339 and an intragastric load of olive oil containing glycerol tri[(3)H]oleate. No difference was found in intestinal TG absorption. Moreover, the hepatic VLDL-TG production rate and VLDL particle production, after injection of Triton WR1339, were also not affected. To investigate the LPL-mediated TG clearance, mice were injected intravenously with glycerol tri[(3)H]oleate-labeled VLDL-like emulsion particles. In Hyplip2 mice, the particles were cleared at a decreased rate (half-life of 25 +/- 6 vs. 11 +/- 2 min; P < 0.05) concomitant with a decreased uptake of emulsion TG-derived (3)H-labeled fatty acids by the liver and white adipose tissue. The increased plasma TG levels in Hyplip2 mice do not result from an enhanced intestinal absorption or increased hepatic VLDL production but are caused by decreased LPL-mediated TG clearance.