In vitro folding and oligomerization of a membrane protein. Transition of bacterial porin from random coil to native conformation.

Porin, a channel-forming protein spanning bacterial outer membranes, was denatured in 6 M guanidinium hydrochloride or, alternatively, in sodium dodecyl sulfate at 95 degrees C. Circular dichroism spectra revealed that this protein, which in its native state consist of beta-pleated sheets as the sole detectable secondary structure, is transformed into random coil configuration in the chaotropic agent, or into alpha-helical structure in the detergent. From either state, the mature protein refolds in presence of amphiphilic molecules, attaining full structural and functional competence. As structural criteria, the native trimeric state was assayed by analytical ultracentrifugation, gel electrophoresis in sodium dodecyl sulfate, protease resistance, and circular dichroism spectroscopy. Channel formation in planar lipid bilayers reveals that the refolded protein is also functionally competent. It is concluded that the information required for the complete folding of porin is contained within the primary sequence of the mature polypeptide. The study of rapid refolding clearly reveals that this process occurs in the time range of seconds and that preexisting bilayers are not a prerequisite.     

The eukaryotic replisome tolerates leading‐strand base damage by replicase switching

The high‐fidelity replicative DNA polymerases, Pol ε and Pol δ, are generally thought to be poorly equipped to replicate damaged DNA. Direct and complete replication of a damaged template therefore typically requires the activity of low‐fidelity translesion synthesis (TLS) polymerases. Here we show that a yeast replisome, reconstituted with purified proteins, is inherently tolerant of the common oxidative lesion thymine glycol (Tg). Surprisingly, leading‐strand Tg was bypassed efficiently in the presence and absence of the TLS machinery. Our data reveal that following helicase–polymerase uncoupling a switch from Pol ε, the canonical leading‐strand replicase, to the lagging‐strand replicase Pol δ, facilitates rapid, efficient and error‐free lesion bypass at physiological nucleotide levels. This replicase switch mechanism also promotes bypass of the unrelated oxidative lesion, 8‐oxoguanine. We propose that replicase switching may promote continued leading‐strand synthesis whenever the replisome encounters leading‐strand damage that is bypassed more efficiently by Pol δ than by Pol ε.     

Drivers of reef fish assemblages in an upwelling region from the Eastern Tropical Pacific Ocean

Reef fish assemblages are exposed to a wide range of anthropogenic threats as well as chronic natural disturbances. In upwelling regions, for example, there is a seasonal influx of cool nutrient-rich waters that may shape the structure and composition of reef fish assemblages. Given that climate change may disrupt the natural oceanographic processes by altering the frequency and strength of natural disturbances, understanding how fish assemblages respond to upwelling events is essential to effectively manage reef ecosystems under changing ocean conditions. This study used the baited remote underwater video stations (BRUVS) and the traditional underwater visual census (UVC) to investigate the spatiotemporal patterns of reef fish assemblages in an upwelling region in the North Pacific of Costa Rica. A total of 183 reef fish species from 60 families were recorded, of which 166 species were detected using BRUVS and 122 using UVC. Only 66% of all species were detected using both methods. This study showed that the upwelling had an important role in shaping reef fish assemblages in this region, but there was also a significant interaction between upwelling and location. In addition, other drivers such as habitat complexity and habitat composition had an effect on reef fish abundances and species. To authors’ knowledge, this is the first study in the Eastern Tropical Pacific that combines BRUVS and UVC to monitor reef fish assemblages in an upwelling region, which provides more detailed information to assess the state of reef ecosystems in response to multiple threats and changing ocean conditions.     

Characterization of the Recombinant Exopeptidases PepX and PepN from Lactobacillus helveticus ATCC 12046 Important for Food Protein Hydrolysis

The proline-specific X-prolyl dipeptidyl aminopeptidase (PepX; EC 3.4.14.11) and the general aminopeptidase N (PepN; EC 3.4.11.2) from Lactobacillus helveticus ATCC 12046 were produced recombinantly in E. coli BL21(DE3) via bioreactor cultivation. The maximum enzymatic activity obtained for PepX was 800 µkat_{H-Ala-Pro-pNA} L⁻¹, which is approx. 195-fold higher than values published previously. To the best of our knowledge, PepN was expressed in E. coli at high levels for the first time. The PepN activity reached 1,000 µkat_H-Ala-pNA L⁻¹. After an automated chromatographic purification, both peptidases were biochemically and kinetically characterized in detail. Substrate inhibition of PepN and product inhibition of both PepX and PepN were discovered for the first time. An apo-enzyme of the Zn²⁺-dependent PepN was generated, which could be reactivated by several metal ions in the order of Co²⁺>Zn²⁺>Mn²⁺>Ca²⁺>Mg²⁺. PepX and PepN exhibited a clear synergistic effect in casein hydrolysis studies. Here, the relative degree of hydrolysis (rDH) was increased by approx. 132%. Due to the remarkable temperature stability at 50°C and the complementary substrate specificities of both peptidases, a future application in food protein hydrolysis might be possible.     

A specific miRNA signature in the peripheral blood of glioblastoma patients

The prognosis of patients afflicted by glioblastoma remains poor. Biomarkers for the disease would be desirable in order to allow for an early detection of tumor progression or to indicate rapidly growing tumor subtypes requiring more intensive therapy. In this study, we investigated whether a blood-derived specific miRNA fingerprint can be defined in patients with glioblastoma. To this end, miRNA profiles from the blood of 20 patients with glioblastoma and 20 age- and sex-matched healthy controls were compared. Of 1158 tested miRNAs, 52 were significantly deregulated, as assessed by unadjusted Student's t-test at an alpha level of 0.05. Of these, two candidates, miR-128 (up-regulated) and miR-342-3p (down-regulated), remained significant after correcting for multiple testing by Benjamini-Hochberg adjustment with a p-value of 0.025. The altered expression of these two biomarkers was confirmed in a second cohort of glioblastoma patients and healthy controls by real-time PCR and validated for patients who had received neither radio- nor chemotherapy and for patients who had their glioblastomas resected more than 6 months ago. Moreover, using machine learning, a comprehensive miRNA signature was obtained that allowed for the discrimination between blood samples of glioblastoma patients and healthy controls with an accuracy of 81% [95% confidence interval (CI) 78-84%], specificity of 79% (95% CI 75-83%) and sensitivity of 83% (95% CI 71-85%). In summary, our proof-of-concept study demonstrates that blood-derived glioblastoma-associated characteristic miRNA fingerprints may be suitable biomarkers and warrant further exploration.     

Novel and functional regulatory SNPs in the promoter region of <Emphasis Type="Italic">FOXP3</Emphasis> gene in a Gabonese population

Parasites exert a selection pressure on their hosts and are accountable for driving diversity within gene families and immune gene polymorphisms in a host population. The overwhelming response of regulatory T cells during infectious challenges directs the host immune system to lose the ability to mount parasite specific T cell responses. The underlying idea of this study is that regulatory single nucleotide polymorphism (SNPs) can cause significant changes in gene expression in functional immune genes. We identified and investigated regulatory SNPs in the promoter region of the FOXP3 gene in a group of Gabonese individuals exposed to a variety of parasitic infections. We identified two novel and one promoter variants in 40 individual subjects. We further validated these promoter variants for their allelic gene expression using transient transfection assays. Two promoter variants, −794 (C/G) and the other variant −734/−540 (C/T) revealed a significant higher expression of the reporter gene at basal level in comparison to the major allele. The identification of SNPs that modify function in the promoter regions could provide a basis for studying parasite susceptibility in association studies.