3D kinematic and dynamic analysis of the front crawl tumble turn in elite male swimmers

The aim of this study was to identify kinematic and dynamic variables related to the best tumble turn times (3mRTT, the turn time from 3-m in to 3-m out, independent variable) in ten elite male swimmers using a three-dimensional (3D) underwater analysis protocol and the Lasso (least absolute shrinkage and selection operator) as statistical method. For each swimmer, the best-time turn was analyzed with five stationary and synchronized underwater cameras. The 3D reconstruction was performed using the Direct Linear Transformation algorithm. An underwater piezoelectric 3D force platform completed the set-up to compute dynamic variables. Data were smoothed by the Savitzky-Golay filtering method. Three variables were considered relevant in the best Lasso model (3mRTT=2.58-0.425 RD+0.204 VPe+0.0046 TD): the head-wall distance where rotation starts (RD), the horizontal speed at the force peak (VPe), and the 3D length of the path covered during the turn (TD). Furthermore, bivariate analysis showed that upper body (CUBei) and lower limb extension indexes at first contact (CLLei) were also linked to the turn time (r=-0.65 and p<0.05 for both variables). Thus the best turn times were associated with a long RD, slower VPe and reduced TD. By an early transverse rotation, male elite swimmers reach the wall with a slightly flexed posture that results in fast extension. These swimmers opt for a movement that is oriented forward and they focus on reducing the distance covered.     

The amino-terminal non-catalytic region of Salmonella typhimurium SigD affects actin organization in yeast and mammalian cells

The internalization of Salmonella into epithelial cells relies on the function of bacterial proteins which are injected into the cell by a specialized type III secretion system. Such bacterial effectors interfere with host cell signalling and induce local cytoskeletal rearrangements. One of such effectors is SigD/SopB, which shares homology with mammalian inositol phosphatases. We made use of the Saccharomyces cerevisiae model for elucidating new aspects of SigD function. Endogenous expression of SigD in yeast caused severe growth inhibition. Surprisingly, sigD alleles mutated in the catalytic site or even deleted for the whole C-terminal phosphatase domain still inhibited yeast growth by inducing loss of actin polarization and precluding the budding process. Accordingly, when expressed in HeLa cells, the same sigD alleles lost the ability of depleting phosphatidylinositol 4,5-bisphosphate from the plasma membrane, but still caused disappearance of actin fibres and loss of adherence. We delineate a region of 25 amino acids (residues 118-142) that is necessary for the effect of SigD on actin in HeLa cells. Our data indicate that SigD exerts a toxic effect linked to its N-terminal region and independent of its phosphatase activity.     

Viability of starved Pasteurella piscicida in seawater monitored by flow cytometry and the effect of antibiotics on its resuscitation

The viability of starved Pasteurella piscicida cells in seawater was analysed by flow cytometry using two fluorescent dyes that stain only living cells. The fluorescent histograms using rhodamine 123 showed that, during the total experimental period, a considerable proportion of cells were able to incorporate this fluorochrome. In addition, the assays employing propidium iodide as dye, demonstrated that the Past. piscicida cells remained viable, maintaining their cellular integrity. Chloramphenicol or ampicillin totally inhibited the resuscitation of non-culturable cells when they were added 8 and 24 h after the incorporation of fresh medium to the microcosms, but had practically no effect after 48 h when cells were fully resuscitated, indicating that active protein and peptidoglycan synthesis were ongoing during all stages of resuscitation.     

Atrazine induced changes in elemental and biochemical composition and nitrate reductase activity in Chlamydomonas reinhardtii

Herbicides play an important role in agricultural practices but the introduction of these compounds into the aquatic environment can have severe consequences for non-target organisms such as microalgae. The ubiquitous green freshwater microalga Chlamydomonas reinhardtii, a model species in all aspects of microalgal physiology, was used to assess the toxicity of atrazine, one of the most widely used herbicides throughout the world. Atrazine acts on photosynthesis and therefore can affect non-target primary producers, such as microalgae.

Growth, dry weight, elemental composition, photosynthetic pigments and protein contents and nitrate reductase activity were studied. After 96 h of exposure to different atrazine concentrations all the parameters studied were affected, but different sensitivities to the herbicide were shown. Nitrate reductase (NR) activity was strongly affected even at an atrazine concentration that did not affect growth (0.1 µM); the lowest concentrations of atrazine assayed (0.1 and 0.25 µM) provoked a > 40% decrease in NR activity and NR decreased > 80% with atrazine concentrations of 0.5 µM. C/N ratio was also affected by all the atrazine concentrations assayed. Nitrate reductase activity and C/N ratio were better indicators of the cellular stress state than data on other biochemical components or growth rate. Among cell parameters assayed, the NR activity stood out as a sensitive cytotoxicity endpoint and the activity of this enzyme can be suggested as a sensitive biomarker of stress induced by atrazine in C. reinhardtii.     

Recognition of the Helical Structure of β-1,4-Galactan by a New Family of Carbohydrate-binding Modules

The microbial enzymes that depolymerize plant cell wall polysaccharides, ultimately promoting energy liberation and carbon recycling, are typically complex in their modularity and often contain carbohydrate-binding modules (CBMs). Here, through analysis of an unknown module from a Thermotoga maritima endo-β-1,4-galactanase, we identify a new family of CBMs that are most frequently found appended to proteins with β-1,4-galactanase activity. Polysaccharide microarray screening, immunofluorescence microscopy, and biochemical analysis of the isolated module demonstrate the specificity of the module, here called TmCBM61, for β-1,4-linked galactose-containing ligands, making it the founding member of family CBM61. The ultra-high resolution x-ray crystal structures of TmCBM61 (0.95 and 1.4 Å resolution) in complex with β-1,4-galactotriose reveal the molecular basis of the specificity of the CBM for β-1,4-galactan. Analysis of these structures provides insight into the recognition of an unexpected helical galactan conformation through a mode of binding that resembles the recognition of starch.     

<Emphasis Type="Italic">In Vitro</Emphasis> Characterization of Chitosan Gels for Buccal Delivery of Celecoxib: Influence of a Penetration Enhancer

Celecoxib (Cx) shows high efficacy in the treatment of osteoarthritis and rheumatoid arthritis as a result of its high specificity for COX-2, without gastrolesivity or interference with platelet function at therapeutic concentrations. Besides of anti-inflammatory effects, Cx also has a potential role for oral cancer chemoprevention. For these conditions, oral administration in long-term treatment is a concern due to its systemic side effects. However, local application at the site of injury (e.g., buccal inflammation conditions or chemoprevention of oral cancer) is a promising way to reduce its toxicity. In this study, the in vitro characterization of mucoadhesive chitosan (CHT) gels associated to Azone® was assessed to explore the potential buccal mucosal administration of Cx in this tissue. Rheological properties of gels were analyzed by a rheometer with cone-plate geometry. In vitro Cx release and permeability studies used artificial membranes and pig cheek mucosa, respectively. Mucoadhesion were measured with a universal test machine. CHT gels (3.0%) containing 2.0% or 3.0% Az showed more appropriate characteristics compared to the others: pH values, rheology, higher amount of Cx retained in the mucosa, and minimal permeation through mucosa, besides the highest mucoadhesion values, ideal for buccal application. Moreover, the flux (J) and amounts of drug released decreased with increased CHT and Az concentrations. CHT gels (3.0%) associated with 2.0% or 3.0% Az may be considered potential delivery systems for buccal administration of Cx.     

Differential protein expression in endothelial cells exposed to serum from patients with acute graft-vs-host disease,depending on steroid response

Graft-versus-host disease (GVHD) is a complication of allogeneic haematopoietic cell transplantation. Endothelial injury is crucial as pathophysiological substrate for GVHD. GVHD first-line treatment is high-dose corticosteroids, although some patients are steroid-refractory. Through the present study, we compared the endothelial proteomic profiles in response to serum from steroid-refractory acute GVHD (SR-aGVHD) and steroid-sensitive acute GVHD (SS-aGVHD) patients. Blood samples from SR-aGVHD (n = 4) and SS-aGVHD (n = 8) patients were collected at aGVHD diagnosis. Endothelial cell cultures were exposed (48 h) to patients' serum. Protein extraction and proteomic analysis were performed. Differences were statistically evaluated by multivariate analysis. Forty-four proteins contributed to separate all samples into the two study groups, among which 15 participated significantly (p < 0.05), 10 exhibiting a fold change >1.2. Differentially expressed proteins were mainly associated with oxidative phosphorylation (Cytochrome C oxidase subunit 6B1, CX6B1), inflammation and angiogenesis (Apolipoprotein D, APOD), cell survival (Rapamycin-insensitive companion of mTOR, RICTR), and oxidative stress (Riboflavin kinase, RIFK). This pilot study used a novel approach to distinguish the aGVHD response to steroid treatment. The proteins differentially expressed could constitute potential biomarkers for steroid-treatment response. These findings signify a step forward to identify the mechanisms of response to steroids, of high clinical relevance considering the SR-aGVHD elevated mortality.     

Verotoxin-producing Escherichia coli (VTEC), enteropathogenic E. coli (EPEC) and necrotoxigenic E. coli (NTEC) isolated from healthy cattle in Spain

AIMS: To determine the prevalence and characteristics of verotoxigenic Escherichia coli (VTEC), enteropathogenic E. coli (EPEC) and necrotoxigenic E. coli (NTEC) in healthy cattle. METHODS AND RESULTS: Faecal samples from 412 healthy cattle were screened for the presence of VTEC, EPEC and NTEC. Four isolates from each sample were studied. VTEC, EPEC and NTEC were isolated in 8.7%, 8.2% and 9.9% of the animals, respectively. VTEC and NTEC were isolated more frequently from calves and heifers than from adults. Seventy (4.2%), 69 (4.2%) and 74 (4.5%) of the 1648 E. coli isolates were VTEC, EPEC and NTEC, respectively. Seventeen (24.3%) of the VTEC strains were eae-positive. Thirty-six (51.4%) of VTEC strains belonged to E. coli serogroups associated with haemorrhagic colitis and haemolytic uraemic syndrome in humans. The serogroups most prevalent among the EPEC strains were O10, O26, O71, O145 and O156. CONCLUSIONS: Healthy cattle are a reservoir of VTEC, EPEC and NTEC. SIGNIFICANCE AND IMPACT OF THE STUDY: Although most of the VTEC strains were eae-negative, a high percentage of VTEC strains belonged to serogroups associated with severe disease in humans.