Water flux through human aquaporin 1: inhibition by intracellular furosemide and maximal response with high osmotic gradients

This work studies water permeability properties of human aquaporin 1 (hAQP1) expressed in Xenopus laevis oocyte membranes, applying a technique where cellular content is replaced with a known medium, with the possibility of measuring intracellular pressure. Consequences on water transport—produced by well-known anisotonic gradients and by the intracellular effect of probable aquaporin inhibitors—were tested. In this way, the specific intracellular inhibition of hAQP1 by the diuretic drug furosemide was demonstrated. In addition, experiments imposing anisotonic mannitol gradients with a constant ionic strength showed that the relationship between water flux and the applied mannitol gradient deflects from a perfect osmometer response when the gradient is higher than 150 mosmol kg_W⁻¹. These results would indicate that the passage of water molecules through hAQP1 may have a maximum rate. As a whole, this work demonstrates the technical advantage of controlling both intracellular pressure and medium composition in order to study biophysical properties of hAQP1, and contributes information on water channel behavior under osmotic challenges and the discovery of new inhibitors.     

Sawfishes and Other Elasmobranch Assemblages from the Mio-Pliocene of the South Caribbean (Urumaco Sequence,Northwestern Venezuela)

The Urumaco stratigraphic sequence, western Venezuela, preserves a variety of paleoenvironments that include terrestrial, riverine, lacustrine and marine facies. A wide range of fossil vertebrates associated with these facies supports the hypothesis of an estuary in that geographic area connected with a hydrographic system that flowed from western Amazonia up to the Proto-Caribbean Sea during the Miocene. Here the elasmobranch assemblages of the middle Miocene to middle Pliocene section of the Urumaco sequence (Socorro, Urumaco and Codore formations) are described. Based on new findings, we document at least 21 taxa of the Lamniformes, Carcharhiniformes, Myliobatiformes and Rajiformes, and describe a new carcharhiniform species (†Carcharhinus caquetius sp. nov.). Moreover, the Urumaco Formation has a high number of well-preserved fossil Pristis rostra, for which we provide a detailed taxonomic revision, and referral in the context of the global Miocene record of Pristis as well as extant species. Using the habitat preference of the living representatives, we hypothesize that the fossil chondrichthyan assemblages from the Urumaco sequence are evidence for marine shallow waters and estuarine habitats.     

DNA polymerase clamp shows little turnover at established replication sites but sequential de novo assembly at adjacent origin clusters

The spatial and temporal organization of DNA replication was investigated in living cells with a green fluorescent protein fusion to the DNA polymerase clamp PCNA. In situ extractions and photobleaching experiments revealed that PCNA, unlike RPA34, shows little if any turnover at replication sites, suggesting that it remains associated with the replication machinery through multiple rounds of Okazaki fragment synthesis. Photobleaching analyses further showed that the transition from earlier to later replicons occurs by disassembly into a nucleoplasmic pool of rapidly diffusing subcomponents and reassembly at newly activated sites. The fact that these replication sites were de novo assembled in close proximity to earlier ones suggests that activation of neighboring origins may occur by a domino effect possibly involving local changes in chromatin structure and accessibility.     

Homoploid hybrid speciation in a rare endemic Castilleja from Idaho (Castilleja christii,Orobanchaceae)

• Premise of the study: Hybridization is an important evolutionary force in the history of angiosperms; however, there are few examples of stabilized species derived through homoploid hybrid speciation. Homoploid hybrid species are generally detected via the presence of genetic additivity of parental markers, novel ecological and spatial distinctions, and novel morphological traits, all of which may aid in the successful establishment of hybrid species from parental types. Speciation and diversification within the genus Castilleja (Orobanchaceae) has been attributed to high levels of hybridization and polyploidy, though currently there are no examples of homoploid hybrid speciation within the genus. We employed multiple lines of evidence to examine a putative hybrid origin in C. christii, a rare endemic, known only from 80 hectares at the summit of Mt. Harrison (Cassia Co., Idaho). • Methods: We used granule-bound starch synthase II (waxy) sequences and 26 morphological characters to address hybridization between C. christii and widespread congeners C. miniata and/or C. linariifolia in an area of sympatry. Chromosomes of C. christii were also counted for the first time. • Key results: All 230 direct-sequenced C. christii individuals had the additive genomes of both C. miniata and C. linariifolia. Castilleja christii shares traits with both parents but also has floral characters that are unique and transgressive. Cytological counts indicated that all three taxa are diploid. • Conclusions: We conclude that C. christii is a stabilized homoploid hybrid derivative of C. linariifolia and C. miniata and is likely following an independent evolutionary trajectory from its progenitors.     

Effect of semen preparation on casa motility results in cryopreserved bull spermatozoa

Computer-assisted sperm analyzers (CASA) have become the standard tool for evaluating sperm motility and kinetic patterns because they provide objective data for thousands of sperm tracks. However, these devices are not ready-to-use and standardization of analytical practices is a fundamental requirement. In this study, we evaluated the effects of some settings, such as frame rate and frames per field, chamber and time of analysis, and samples preparations, including thawing temperature, sperm sample concentration, and media used for dilution, on the kinetic results of bovine frozen-thawed semen using a CASA. In Experiment 1, the frame rate (30-60 frame/s) significantly affected motility parameters, whereas the number of frames per field (30 or 45) did not seem to affect sperm kinetics. In Experiment 2, the thawing protocol affects sperm motility and kinetic parameters. Sperm sample concentration significantly limited the opportunity to perform the analysis and the kinetic results. A concentration of 100 and 50 × 10⁶ sperm/mL limited the device's ability to perform the analysis or gave wrong results, whereas 5, 10, 20, and 30 × 10⁶ sperm/mL concentrations allowed the analysis to be performed, but with different results (Experiment 3). The medium used for the dilution of the sample, which is fundamental for a correct sperm head detection, affects sperm motility results (Experiment 4). In this study, Makler and Leja chambers were used to perform the semen analysis with CASA devices. The chamber used significantly affected motility results (Experiment 5). The time between chamber loading and analysis affected sperm velocities, regardless of chamber used. Based on results recorded in this study, we propose that the CASA evaluation of motility of bovine frozen-thawed semen using Hamilton-Thorne IVOS 12.3 should be performed using a frame rate of 60 frame/s and 30 frames per field. Semen should be diluted at least at 20 × 10⁶ sperm/mL using PBS. Furthermore, it is necessary to consider the type of chamber used and perform the analysis within 1 or 2 min, regardless of the chamber used.     

Oxidative damage and anti-oxidant capacity in two migratory bird species at a stop-over site

We quantified in the garden warbler (Sylvia borin) and the barn swallow (Hirundo rustica), two long-distance migratory songbirds, the early oxidative damage (ROMs) and plasma anti-oxidant capacity (OXY) variation of individuals caught at a stop-over site after a sustained flight across the sea, during spring migration. Our main goal was to quantify the oxidative damage and anti-oxidant capacity variation in these two migratory species in relation to fat and muscle stores. The birds were sampled in Ponza, a small island along the migratory route of these species. The levels of ROMs and OXY did not show any differences between the two species and in general were higher in individuals with higher fat and protein stores. Nevertheless, the balance between ROMs and OXY was better in individuals in good condition. These patterns were similar in both species. No sex differences emerged for both ROMs and OXY in the barn swallow, the only species that could be sexed. Both markers of oxidative stress did not show any significant variation across a 30-min restrained experiment. These data are the first of this kind in wild birds in a migratory context and suggest that individuals in better condition are exposed to lower oxidative stress, providing an indirect evidence of the oxidative cost caused by prolonged flights.     

Cytomegalovirus Infection in Inflammatory Bowel Disease Is Not Associated with Worsening of Intestinal Inflammatory Activity

Background

Cytomegalovirus is highly prevalent virus and usually occurs in immunocompromised patients. The pathophysiology and treatment of inflammatory bowel disease often induce a state of immunosuppression. Because this, there are still doubts and controversies about the relationship between inflammatory bowel disease and cytomegalovirus.

Aim

Evaluate the frequency of cytomegalovirus in patients with inflammatory bowel disease and identify correlations.

Methods

Patients with inflammatory bowel disease underwent an interview, review of records and collection of blood and fecal samples. The search for cytomegalovirus was performed by IgG and IgM blood serology, by real-time PCR in the blood and by qualitative PCR in feces. Results were correlated with red blood cell levels, C-reactive protein levels, erythrocyte sedimentation rates and fecal calprotectin levels for each patient.

Results

Among the 400 eligible patients, 249 had Crohn''s disease, and 151 had ulcerative colitis. In the group of Crohn''s disease, 67 of the patients had moderate or severe disease, but 126 patients presented with active disease, based on the evaluation of the fecal calprotectin. In patients with ulcerative colitis, only 21 patients had moderate disease, but 76 patients presented with active disease, based on the evaluation of the fecal calprotectin. A large majority of patients had positive CMV IgG. Overall, 10 patients had positive CMV IgM, and 9 patients had a positive qualitative detection of CMV DNA by PCR in the feces. All 400 patients returned negative results after the quantitative detection of CMV DNA in blood by real-time PCR. Analyzing the 19 patients with active infections, we only found that such an association occurred with the use of combined therapy (anti-TNF-alpha + azathioprine)

Conclusion

The findings show that latent cytomegalovirus infections are frequent and active cytomegalovirus infection is rare. We did not find any association between an active infection of CMV and inflammatory bowel disease activity.     

The Population Decline and Extinction of Darwin’s Frogs

Darwin’s frogs (Rhinoderma darwinii and R. rufum) are two species of mouth-brooding frogs from Chile and Argentina. Here, we present evidence on the extent of declines, current distribution and conservation status of Rhinoderma spp.; including information on abundance, habitat and threats to extant Darwin’s frog populations. All known archived Rhinoderma specimens were examined in museums in North America, Europe and South America. Extensive surveys were carried out throughout the historical ranges of R. rufum and R. darwinii from 2008 to 2012. Literature review and location data of 2,244 archived specimens were used to develop historical distribution maps for Rhinoderma spp. Based on records of sightings, optimal linear estimation was used to estimate whether R. rufum can be considered extinct. No extant R. rufum was found and our modelling inferred that this species became extinct in 1982 (95% CI, 1980–2000). Rhinoderma darwinii was found in 36 sites. All populations were within native forest and abundance was highest in Chiloé Island, when compared with Coast, Andes and South populations. Estimated population size and density (five populations) averaged 33.2 frogs/population (range, 10.2–56.3) and 14.9 frogs/100 m² (range, 5.3–74.1), respectively. Our results provide further evidence that R. rufum is extinct and indicate that R. darwinii has declined to a much greater degree than previously recognised. Although this species can still be found across a large part of its historical range, remaining populations are small and severely fragmented. Conservation efforts for R. darwinii should be stepped up and the species re-classified as Endangered.