A New Recombinant BCG Vaccine Induces Specific Th17 and Th1 Effector Cells with Higher Protective Efficacy against Tuberculosis

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb) that is a major public health problem. The vaccine used for TB prevention is Mycobacterium bovis bacillus Calmette-Guérin (BCG), which provides variable efficacy in protecting against pulmonary TB among adults. Consequently, several groups have pursued the development of a new vaccine with a superior protective capacity to that of BCG. Here we constructed a new recombinant BCG (rBCG) vaccine expressing a fusion protein (CMX) composed of immune dominant epitopes from Ag85C, MPT51, and HspX and evaluated its immunogenicity and protection in a murine model of infection. The stability of the vaccine in vivo was maintained for up to 20 days post-vaccination. rBCG-CMX was efficiently phagocytized by peritoneal macrophages and induced nitric oxide (NO) production. Following mouse immunization, this vaccine induced a specific immune response in cells from lungs and spleen to the fusion protein and to each of the component recombinant proteins by themselves. Vaccinated mice presented higher amounts of Th1, Th17, and polyfunctional specific T cells. rBCG-CMX vaccination reduced the extension of lung lesions caused by challenge with Mtb as well as the lung bacterial load. In addition, when this vaccine was used in a prime-boost strategy together with rCMX, the lung bacterial load was lower than the result observed by BCG vaccination. This study describes the creation of a new promising vaccine for TB that we hope will be used in further studies to address its safety before proceeding to clinical trials.     

Mortality Caused by Bath Exposure of Zebrafish (Danio rerio) Larvae to Nervous Necrosis Virus Is Limited to the Fourth Day Postfertilization

Nervous necrosis virus (NNV) is a member of the Betanodavirus genus that causes fatal diseases in over 40 species of fish worldwide. Mortality among NNV-infected fish larvae is almost 100%. In order to elucidate the mechanisms responsible for the susceptibility of fish larvae to NNV, we exposed zebrafish larvae to NNV by bath immersion at 2, 4, 6, and 8 days postfertilization (dpf). Here, we demonstrate that developing zebrafish embryos are resistant to NNV at 2 dpf due to the protection afforded by the egg chorion and, to a lesser extent, by the perivitelline fluid. The zebrafish larvae succumbed to NNV infection during a narrow time window around the 4th dpf, while 6- and 8-day-old larvae were much less sensitive, with mortalities of 24% and 28%, respectively.     

Photoinhibition of photosynthesis in intact kiwifruit (Actinidia deliciosa) leaves: Effect of temperature

Photoinhibition of photosynthesis was induced in attached leaves of kiwifruit grown in natural light not exceeding a photon flux density (PFD) of 300 mol·m^-2·s^-1, by exposing them to a PFD of 1500 mol·m^-2·s^-1. The temperature was held constant, between 5 and 35° C, during the exposure to high light. The kinetics of photoinhibition were measured by chlorophyll fluorescence at 77K and the photon yield of photosynthetic O₂ evolution. Photoinhibition occurred at all temperatures but was greatest at low temperatures. Photoinhibition followed pseudo first-order kinetics, as determined by the variable fluorescence (F _v) and photon yield, with the long-term steady-state of photoinhibition strongly dependent on temperature wheareas the observed rate constant was only weakly temperature-dependent. Temperature had little effect on the decrease in the maximum fluorescence (F _m) but the increase in the instantaneous fluorescence (F _o) was significantly affected by low temperatures in particular. These changes in fluorescence indicate that kiwifruit leaves have some capacity to dissipate excessive excitation energy by increasing the rate constant for non-radiative (thermal) energy dissipation although temperature apparently had little effect on this. Direct photoinhibitory damage to the photosystem II reaction centres was evident by the increases in F _o and extreme, irreversible damage occurred at the lower temperatures. This indicates that kiwifruit leaves were most susceptible to photoinhibition at low temperatures because direct damage to the reaction centres was greatest at these temperatures. The results also imply that mechanisms to dissipate excess energy were inadequate to afford any protection from photoinhibition over a wide temperature range in these shade-grown leaves.Abbreviations and symbols fluorescence yield correction coefficient - F _o, F _m, F _v instantaneous, maximum, variable fluorescence - K _D, K _F, K _P, K _T rate constants for non-radiative energy dissipation, fluorescence, photochemistry, energy transfer to photosystem I - PFD photon flux density - PSI, II photosystem I, II - _i photon yield of photosynthesis (incident light)     

Localization of post-proline cleaving peptidases in Tenebrio molitor larval midgut

Two soluble post-proline cleaving peptidase activities, PPCP1 and PPCP2, were demonstrated in Tenebrio molitor larval midgut with the substrate benzyloxycarbonyl-L-alanyl-L-proline p-nitroanilide. Both activities were serine peptidases. PPCP1 was active in acidic buffers, with maximum activity at pH 5.3, and was located mainly in the more acidic anterior midgut lumen. The dynamics of PPCP1 activity and the total activity of soluble digestive peptidases in the course of food digestion were similar, suggesting that the enzyme participates in protein digestion. PPCP2 is a nondigestive soluble tissue enzyme evenly distributed along the midgut. An increase in the activity of PPCP2 was observed in buffers of pH 5.6-8.6 and was maximal at pH 7.4. The sensitivity of PPCP2 to inhibitors and the effect of pH are similar to prolyl oligopeptidases with a cysteine residue near the substrate binding site.     

Morphology and molecular taxonomy of Evlachovaea-like fungi,and the status of this unusual conidial genus

The entomopathogenic anamorphic genus Evlachovaea was described to differ from other fungi in forming its conidia obliquely to the axis of the conidiogenous cell and with successive conidia having alternate orientations with a zipper- or chevron-like arrangement resulting in flat, ribbon-like chains. Morphological and molecular studies of six Evlachovaea-like isolates baited from Central Brazilian soils using Triatoma infestans (a vector of Chagas disease) and of other entomopathogens with Evlachovaea-like conidiogenesis led to a re-evaluation of the status of this little known fungal genus. The Brazilian isolates formed two distinct groups based on gene sequences for both the internal transcribed spacer (ITS) and translation elongation factor (EF-1α) genes, morphology, and growth patterns; both groups also differed from the type species, Evlachovaea kintrischica. More detailed studies of these fungi indicated that the alternatingly oblique orientations of forming conidia are neither a stable nor invariant character (even on single phialides). Furthermore, the molecular cladistic analysis unambiguously placed the Evlachovaea isolates firmly within the genus Isaria (Hypocreales: Cordycipitaceae). The ITS sequences of E. kintrischica were very similar or even identical to those of Isaria amoenerosea and Isaria cateniobliqua, thereby suggesting that E. kintrischica is a synonym of one of these species, and that the genus Evlachovaea must be treated as a later synonym of Isaria, which must now be recognized to include several highly divergent modes of conidiogenesis. These taxonomic findings are discussed in the context of dramatic changes recently imposed on the nomenclatural standards used to determine the correct names of all pleomorphic fungi.     

Bicarbonate as tracer for assimilated C and homogeneity of 14C and 15N distribution in plants by alternative labeling approaches

Aims

Application of carbon (C) and nitrogen (N) isotopes is an essential tool to study C and N flows in plant-soil-microorganisms systems. When targeting single plants in a community the tracers need to be added via e.g., leaf-labeling or stem-feeding approaches. In this study we: (i) investigated if bicarbonate can be used to introduce ¹⁴C (or ¹³C) into white clover and ryegrass, and (ii) compared the patterns of ¹⁴C and ¹⁵N allocation in white clover and ryegrass to evaluate the homogeneity of tracer distribution after two alternative labeling approaches.

Methods

Perennial ryegrass and white clover were pulse labeled with ¹⁵N urea via leaf-labeling and ¹⁴C either via a ¹⁴CO₂ atm or with ¹⁴C bicarbonate through leaf-labeling. Plants were sampled 4 days after labeling and prepared for bulk isotope analysis and for ¹⁴C imaging to identify plant parts with high and low ¹⁴C activity. Subsequently, plant parts with high and low ¹⁴C activity were separated and analyzed for ¹⁵N enrichment.

Results

Bicarbonate applied by leaf-labeling efficiently introduced ¹⁴C into both white clover and ryegrass, although the ¹⁴C activity in particular for white clover was found predominantly in the labeled leaf. Using ¹⁴C imaging for identification of areas with high (hotspots) and low ¹⁴C activity showed that ¹⁴C was incorporated very heterogeneously both when using bicarbonate and CO₂ as expected when using pulse labeling. Subsequent analysis of ¹⁵N enrichment in plant parts with high and low ¹⁴C activity showed that ¹⁵N also had a heterogeneous distribution (up to two orders of magnitude).

Conclusion

Bicarbonate can efficiently be used to introduce ¹⁴C or ¹³C into plant via the leaf-labeling method. Both ¹⁴C and ¹⁵N showed heterogeneous distribution in the plant, although the distribution of ¹⁵N was more even than that of ¹⁴C.     

Microbial Growth and Carbon Use Efficiency in the Rhizosphere and Root-Free Soil

Plant-microbial interactions alter C and N balance in the rhizosphere and affect the microbial carbon use efficiency (CUE)–the fundamental characteristic of microbial metabolism. Estimation of CUE in microbial hotspots with high dynamics of activity and changes of microbial physiological state from dormancy to activity is a challenge in soil microbiology. We analyzed respiratory activity, microbial DNA content and CUE by manipulation the C and nutrients availability in the soil under Beta vulgaris. All measurements were done in root-free and rhizosphere soil under steady-state conditions and during microbial growth induced by addition of glucose. Microorganisms in the rhizosphere and root-free soil differed in their CUE dynamics due to varying time delays between respiration burst and DNA increase. Constant CUE in an exponentially-growing microbial community in rhizosphere demonstrated the balanced growth. In contrast, the CUE in the root-free soil increased more than three times at the end of exponential growth and was 1.5 times higher than in the rhizosphere. Plants alter the dynamics of microbial CUE by balancing the catabolic and anabolic processes, which were decoupled in the root-free soil. The effects of N and C availability on CUE in rhizosphere and root-free soil are discussed.     

The impact of stratification by implausible energy reporting status on estimates of diet‐health relationships

The food frequency questionnaire (FFQ) is known to be prone to measurement error. Researchers have suggested excluding implausible energy reporters (IERs) of FFQ total energy when examining the relationship between a health outcome and FFQ‐reported intake to obtain less biased estimates of the effect of the error‐prone measure of exposure; however, the statistical properties of stratifying by IER status have not been studied. Under certain assumptions, including nondifferential error, we show that when stratifying by IER status, the attenuation of the estimated relative risk in the stratified models will be either greater or less in both strata (implausible and plausible reporters) than for the nonstratified model, contrary to the common belief that the attenuation will be less among plausible reporters and greater among IERs. Whether there is more or less attenuation depends on the pairwise correlations between true exposure, observed exposure, and the stratification variable. Thus exclusion of IERs is inadvisable but stratification by IER status can sometimes help. We also address the case of differential error. Examples from the Observing Protein and Energy Nutrition Study and simulations illustrate these results.     

C and N allocation in soil under ryegrass and alfalfa estimated by 13C and 15N labelling

Background and Aims

Below-ground translocated carbon (C) released as rhizodeposits is an important driver for microbial mobilization of nitrogen (N) for plants. We investigated how a limited substrate supply due to reduced photoassimilation alters the allocation of recently assimilated C in plant and soil pools under legume and non-legume species.

Methods

A non-legume (Lolium perenne) and a legume (Medicago sativa) were labelled with ¹⁵N before the plants were clipped or shaded, and labelled twice with ¹³CO₂ thereafter. Ten days after clipping and shading, the ¹⁵N and ¹³C in shoots, roots, soil, dissolved organic nitrogen (DON) and carbon (DOC) and in microbial biomass, as well as the ¹³C in soil CO₂ were analyzed.

Results

After clipping, about 50 % more ¹³C was allocated to regrowing shoots, resulting in a lower translocation to roots compared to the unclipped control. Clipping also reduced the total soil CO₂ efflux under both species and the ¹³C recovery of soil CO₂ under L. perenne. The ¹⁵N recovery increased in the shoots of M. sativa after clipping, because storage compounds were remobilized from the roots and/or the N uptake from the soil increased. After shading, the assimilated ¹³C was preferentially retained in the shoots of both species. This caused a decreased ¹³C recovery in the roots of M. sativa. Similarly, the total soil CO₂ efflux under M. sativa decreased more than 50 % after shading. The ¹⁵N recovery in plant and soil pools showed that shading has no effect on the N uptake and N remobilization for L. perenne, but, the ¹⁵N recovery increased in the shoot of M. sativa.

Conclusions

The experiment showed that the dominating effect on C and N allocation after clipping is the need of C and N for shoot regrowth, whereas the dominating effect after shading is the reduced substrate supply for growth and respiration. Only slight differences could be observed between L. perenne and M. sativa in the C and N distribution after clipping or shading.