PURIFICATION AND CHARACTERIZATION OF A PROTEINASE FROM THE PROBIOTIC Lactobacillus rhamnosus OXY

A proteinase produced by the human gastrointestinal isolate Lactobacillus rhamnosus strain OXY was identified and characterized. The prtR2 gene coding for proteinase activity was detected in the examined strain. The PCR primers used were constructed on the basis of the sequence of the prtR2 proteinase gene from Lactobacillus rhamnosus GG. The enzyme was purified by fast protein liquid chromatography (FPLC) using CM-Sepharose Fast Flow and Sephacryl S-300 columns. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the enzyme had a relatively low molecular mass of 60 kD. Protease activity was observed at a pH range from 6.5 to 7.5 with optimum k _cat/K _m values at pH 7.0 and 40°C. Maximum proteolytic activity (59 U mL^?1) was achieved after 48 hr of cultivation. The activity of the enzyme was inhibited only by irreversible inhibitors specific for serine proteinases (PMSF and 3,4-dichloro-isocumarine), suggesting that the enzyme was a serine proteinase. Proteinase activity was increased by Ca²⁺ and Mg²⁺, and inhibited by Cu²⁺, Zn²⁺, Cd²⁺, and Fe^2+.     

Chlamydia pneumoniae infection acts as an endothelial stressor with the potential to initiate the earliest heat shock protein 60-dependent inflammatory stage of atherosclerosis

We identified increased expression and redistribution of the intracellular protein 60-kDa human heat shock protein (hHSP60) (HSPD1) to the cell surface in human endothelial cells subjected to classical atherosclerosis risk factors and subsequent immunologic cross-reactivity against this highly conserved molecule, as key events occurring early in the process of atherosclerosis. The present study aimed at investigating the role of infectious pathogens as stress factors for vascular endothelial cells and, as such, contributors to early atherosclerotic lesion formation. Using primary donor-matched arterial and venous human endothelial cells, we show that infection with Chlamydia pneumoniae leads to marked upregulation and surface expression of hHSP60 and adhesion molecules. Moreover, we provide evidence for an increased susceptibility of arterial endothelial cells for redistribution of hHSP60 to the cellular membrane in response to C. pneumoniae infection as compared to autologous venous endothelial cells. We also show that oxidative stress has a central role to play in endothelial cell activation in response to chlamydial infection. These data provide evidence for a role of C. pneumoniae as a potent primary endothelial stressor for arterial endothelial cells leading to enrichment of hHSP60 on the cellular membrane and, as such, a potential initiator of atherosclerosis.     

Tidal marsh plant responses to elevated CO2, nitrogen fertilization,and sea level rise

Elevated CO₂ and nitrogen (N) addition directly affect plant productivity and the mechanisms that allow tidal marshes to maintain a constant elevation relative to sea level, but it remains unknown how these global change drivers modify marsh plant response to sea level rise. Here we manipulated factorial combinations of CO₂ concentration (two levels), N availability (two levels) and relative sea level (six levels) using in situ mesocosms containing a tidal marsh community composed of a sedge, Schoenoplectus americanus, and a grass, Spartina patens. Our objective is to determine, if elevated CO₂ and N alter the growth and persistence of these plants in coastal ecosystems facing rising sea levels. After two growing seasons, we found that N addition enhanced plant growth particularly at sea levels where plants were most stressed by flooding (114% stimulation in the + 10 cm treatment), and N effects were generally larger in combination with elevated CO₂ (288% stimulation). N fertilization shifted the optimal productivity of S. patens to a higher sea level, but did not confer S. patens an enhanced ability to tolerate sea level rise. S. americanus responded strongly to N only in the higher sea level treatments that excluded S. patens. Interestingly, addition of N, which has been suggested to accelerate marsh loss, may afford some marsh plants, such as the widespread sedge, S. americanus, the enhanced ability to tolerate inundation. However, if chronic N pollution reduces the availability of propagules of S. americanus or other flood‐tolerant species on the landscape scale, this shift in species dominance could render tidal marshes more susceptible to marsh collapse.     

The spatial patterns of directional phenotypic selection

Local adaptation, adaptive population divergence and speciation are often expected to result from populations evolving in response to spatial variation in selection. Yet, we lack a comprehensive understanding of the major features that characterise the spatial patterns of selection, namely the extent of variation among populations in the strength and direction of selection. Here, we analyse a data set of spatially replicated studies of directional phenotypic selection from natural populations. The data set includes 60 studies, consisting of 3937 estimates of selection across an average of five populations. We performed meta‐analyses to explore features characterising spatial variation in directional selection. We found that selection tends to vary mainly in strength and less in direction among populations. Although differences in the direction of selection occur among populations they do so where selection is often weakest, which may limit the potential for ongoing adaptive population divergence. Overall, we also found that spatial variation in selection appears comparable to temporal (annual) variation in selection within populations; however, several deficiencies in available data currently complicate this comparison. We discuss future research needs to further advance our understanding of spatial variation in selection.     

ERCC1 function in nuclear excision and interstrand crosslink repair pathways is mediated exclusively by the ERCC1-202 isoform

ERCC1 (excision repair cross-complementation group 1) plays essential roles in the removal of DNA intrastrand crosslinks by nucleotide excision repair, and that of DNA interstrand crosslinks by the Fanconi anemia (FA) pathway and homology-directed repair processes (HDR). The function of ERCC1 thus impacts on the DNA damage response (DDR), particularly in anticancer therapy when DNA damaging agents are employed. ERCC1 expression has been proposed as a predictive biomarker of the response to platinum-based therapy. However, the assessment of ERCC1 expression in clinical samples is complicated by the existence of 4 functionally distinct protein isoforms, which differently impact on DDR. Here, we explored the functional competence of each ERCC1 protein isoform and obtained evidence that the 202 isoform is the sole one endowed with ERCC1 activity in DNA repair pathways. The ERCC1 isoform 202 interacts with RPA, XPA, and XPF, and XPF stability requires expression of the ERCC1 202 isoform (but none of the 3 others). ERCC1-deficient non-small cell lung cancer cells show abnormal mitosis, a phenotype reminiscent of the FA phenotype that can be rescued by isoform 202 only. Finally, we could not observe any dominant-negative interaction between ERCC1 isoforms. These data suggest that the selective assessment of the ERCC1 isoform 202 in clinical samples should accurately reflect the DDR-related activity of the gene and hence constitute a useful biomarker for customizing anticancer therapies.     

Europium Nanoparticle-Based High Performing Immunoassay for the Screening of Treponemal Antibodies

Treponema pallidum subspecies pallidum (Tp) is the causative agent of syphilis which mainly spreads through sexual contact, blood transfusion and perinatal route. In order to curtail the spread of the infection and to clinically manage the disease, timely, accurate and reliable diagnosis is very important. We have developed an immunoassay for the detection of treponemal antibodies in human serum or plasma samples. In vivo biotinylated and non-biotinylated versions of the recombinant antigen were designed by the fusion of three Tp-specific antigens namely Tp15, Tp17 and Tp47. These fusion antigens were expressed in E. coli and purified using single-step metal affinity chromatography. Biotinylated fusion antigen immobilized on streptavidin coated plate was used to capture the treponemal antibodies and the non-biotinylated antigen coated on europium nanoparticles was used as tracer. Assays with two different incubation times of 10 min and 1 h were developed, and following the incubation the europium fluorescence was measured using time-resolved fluorometry. The developed time-resolved fluorometric (TRF) immunoassays were evaluated with in-house and commercial serum/plasma sample panels. For well-established treponemal antibodies positive or negative samples, the sensitivity of TRF immunoassay with 10 min incubation time was 97.4%, and of TRF immunoassay with 1 h incubation time was 98.7%, and the specificities of both the TRF immunoassays were 99.2%. For the samples with discordant results with the reference assays, both the TRF immunoassays showed better specificity than the Enzygnost syphilis enzyme immunoassay as a screening test. The two different incubation times did not have any significant effect on the signal to cutoff (S/Co) ratios obtained with the two immunoassays (p = 0.06). Our results indicate that the developed immunoassay with a short incubation time of 10 min has the potential to be used in clinical laboratories and in blood-bank settings as a screening test for treponemal antibodies.     

Heterotrophic Compensation: A Possible Mechanism for Resilience of Coral Reefs to Global Warming or a Sign of Prolonged Stress?

Thermally induced bleaching has caused a global decline in corals and the frequency of such bleaching events will increase. Thermal bleaching severely disrupts the trophic behaviour of the coral holobiont, reducing the photosynthetically derived energy available to the coral host. In the short term this reduction in energy transfer from endosymbiotic algae results in an energy deficit for the coral host. If the bleaching event is short-lived then the coral may survive this energy deficit by depleting its lipid reserves, or by increasing heterotrophic energy acquisition. We show for the first time that the coral animal is capable of increasing the amount of heterotrophic carbon incorporated into its tissues for almost a year following bleaching. This prolonged heterotrophic compensation could be a sign of resilience or prolonged stress. If the heterotrophic compensation is in fact an acclimatization response, then this physiological response could act as a buffer from future bleaching by providing sufficient heterotrophic energy to compensate for photoautotrophic energy losses during bleaching, and potentially minimizing the effect of subsequent elevated temperature stresses. However, if the elevated incorporation of zooplankton is a sign that the effects of bleaching continue to be stressful on the holobiont, even after 11 months of recovery, then this physiological response would indicate that complete coral recovery requires more than 11 months to achieve. If coral bleaching becomes an annual global phenomenon by mid-century, then present temporal refugia will not be sufficient to allow coral colonies to recover between bleaching events and coral reefs will become increasingly less resilient to future climate change. If, however, increasing their sequestration of zooplankton-derived nutrition into their tissues over prolonged periods of time is a compensating mechanism, the impacts of annual bleaching may be reduced. Thus, some coral species may be better equipped to face repeated bleaching stress than previously thought.