Domains of human U4atac snRNA required for U12-dependent splicing in vivo

U4atac snRNA forms a base-paired complex with U6atac snRNA. Both snRNAs are required for the splicing of the minor U12-dependent class of eukaryotic nuclear introns. We have developed a new genetic suppression assay to investigate the in vivo roles of several regions of U4atac snRNA in U12-dependent splicing. We show that both the stem I and stem II regions, which have been proposed to pair with U6atac snRNA, are required for in vivo splicing. Splicing activity also requires U4atac sequences in the 5' stem-loop element that bind a 15.5 kDa protein that also binds to a similar region of U4 snRNA. In contrast, mutations in the region immediately following the stem I interaction region, as well as a deletion of the distal portion of the 3' stem-loop element, were active for splicing. Complete deletion of the 3' stem-loop element abolished in vivo splicing function as did a mutation of the Sm protein binding site. These results show that the in vivo sequence requirements of U4atac snRNA are similar to those described previously for U4 snRNA using in vitro assays and provide experimental support for models of the U4atac/U6atac snRNA interaction.     

Urinary CD133+ Extracellular Vesicles Are Decreased in Kidney Transplanted Patients with Slow Graft Function and Vascular Damage

Extracellular vesicles (EVs) present in the urine are mainly released from cells of the nephron and can therefore provide information on kidney function. We here evaluated the presence of vesicles expressing the progenitor marker CD133 in the urine of normal subjects and of patients undergoing renal transplant. We found that EV expressing CD133 were present in the urine of normal subjects, but not of patients with end stage renal disease. The first day after transplant, urinary CD133⁺ EVs were present at low levels, to increase thereafter (at day 7). Urinary CD133⁺ EVs significantly increased in patients with slow graft function in respect to those with early graft function. In patients with a severe pre-transplant vascular damage of the graft, CD133⁺ EVs did not increase at day 7. At variance, the levels of EVs expressing the renal exosomal marker CD24 did not vary in the urine of patients with end stage renal disease or in transplanted patients in respect to controls. Sorted CD133⁺ EVs were found to express glomerular and proximal tubular markers. These data indicate that urinary CD133⁺ EVs are continuously released during the homeostatic turnover of the nephron and may provide information on its function or regenerative potential.     

Quantitative Ultrasound Spectroscopic Imaging for Characterization of Disease Extent in Prostate Cancer Patients

Three-dimensional quantitative ultrasound spectroscopic imaging of prostate was investigated clinically for the noninvasive detection and extent characterization of disease in cancer patients and compared to whole-mount, whole-gland histopathology of radical prostatectomy specimens. Fifteen patients with prostate cancer underwent a volumetric transrectal ultrasound scan before radical prostatectomy. Conventional-frequency (~ 5 MHz) ultrasound images and radiofrequency data were collected from patients. Normalized power spectra were used as the basis of quantitative ultrasound spectroscopy. Specifically, color-coded parametric maps of 0-MHz intercept, midband fit, and spectral slope were computed and used to characterize prostate tissue in ultrasound images. Areas of cancer were identified in whole-mount histopathology specimens, and disease extent was correlated to that estimated from quantitative ultrasound parametric images. Midband fit and 0-MHz intercept parameters were found to be best associated with the presence of disease as located on histopathology whole-mount sections. Obtained results indicated a correlation between disease extent estimated noninvasively based on midband fit parametric images and that identified histopathologically on prostatectomy specimens, with an r² value of 0.71 (P < .0001). The 0-MHz intercept parameter demonstrated a lower level of correlation with histopathology. Spectral slope parametric maps offered no discrimination of disease. Multiple regression analysis produced a hybrid disease characterization model (r² = 0.764, P < .05), implying that the midband fit biomarker had the greatest correlation with the histopathologic extent of disease. This work demonstrates that quantitative ultrasound spectroscopic imaging can be used for detecting prostate cancer and characterizing disease extent noninvasively, with corresponding gross three-dimensional histopathologic correlation.     

A Gas chromatographic (GLPC) model for the sense of smell. Variation of olfactory sensitivity with conditions of stimulation

Computer simulation of an olfactory detector has been developed using a chemical kinetic scheme originally proposed by McNab and Koshland for bacterial chemotaxis. This model describes response as a function of two opposed reactions, both of which are activated by odorant. One reaction turns on response, while its opponent shuts it off. Net response to various stimulus profiles is compared to psychophysical experiments, with particular attention paid to simulating magnitude estimation and odor adaptation results. Effects of the access route to this detector are evaluated. Transport of odorant molecules is treated as having two sequential steps: step (i), airborne odorant is carried parallel to a retentive layer (mucus) into the detector region; step (ii), molecules diffuse through the retentive layer to the detector. Step (i) is represented as analogous to GLPC on an open tubular column. Each step has a characteristic time constant, which is proportional to (distance)²/diffusion coefficient. Response to highly volatile odorants tends to be limited by step (ii), while odorants of low volatility approach the step (i) limit. Sensitivity at both limits is attenuated by increasing the thickness of the retentive layer, but sensitivity at the step (i) limit is also affected by changes in air passageway and airflow characteristics. This picture can be used to explain variations in women's sensitivity to odorants of low volatility with the menstrual cycle, while their detection of volatile odorants fluctuates to a much lesser extent.     

Carbonic anhydrase inhibitors. Inhibition of cytosolic isozyme XIII with aromatic and heterocyclic sulfonamides: a novel target for the drug design

The inhibition of the newly discovered cytosolic carbonic anhydrase isozyme XIII (CA XIII) has been investigated with a series of aromatic and heterocyclic sulfonamides, including some of the clinically used derivatives, such as acetazolamide, methazolamide, dichlorophenamide, dorzolamide, and valdecoxib. Inhibition data for the physiologically relevant isozymes I and II (cytosolic forms) and the tumor associated isozyme IX (transmembrane) were also provided for comparison. A very interesting and unusual inhibition profile against CA XIII with these sulfonamides has been observed. The clinically used compounds (except valdecoxib, which was a weak CA XIII inhibitor) potently inhibit CA XIII, with Ki's in the range of 17-23 nM, whereas sulfanilamide, halogenated sulfanilamides, homosulfanilamide, 4-aminoethylbenzenesulfonamide, and orthanilamide were slightly less effective, with Ki's in the range of 32-56 nM. Several low nanomolar (Ki's in the range of 1.3-2.4 nM) CA XIII inhibitors have also been detected, all of them belonging to the sulfanilyl-sulfonamide type of inhibitors, of which aminobenzolamide is the best known representative. Because CA XIII is an active isozyme predominantly expressed in salivary glands, kidney, brain, lung, gut, uterus, and testis, where it probably plays an important role in pH regulation, its inhibition by sulfonamides may lead to novel therapeutic applications for this class of pharmacological agents.     

How Much Is Too Little to Detect Impacts? A Case Study of a Nuclear Power Plant

Several approaches have been proposed to assess impacts on natural assemblages. Ideally, the potentially impacted site and multiple reference sites are sampled through time, before and after the impact. Often, however, the lack of information regarding the potential overall impact, the lack of knowledge about the environment in many regions worldwide, budgets constraints and the increasing dimensions of human activities compromise the reliability of the impact assessment. We evaluated the impact, if any, and its extent of a nuclear power plant effluent on sessile epibiota assemblages using a suitable and feasible sampling design with no ‘before’ data and budget and logistic constraints. Assemblages were sampled at multiple times and at increasing distances from the point of the discharge of the effluent. There was a clear and localized effect of the power plant effluent (up to 100 m from the point of the discharge). However, depending on the time of the year, the impact reaches up to 600 m. We found a significantly lower richness of taxa in the Effluent site when compared to other sites. Furthermore, at all times, the variability of assemblages near the discharge was also smaller than in other sites. Although the sampling design used here (in particular the number of replicates) did not allow an unambiguously evaluation of the full extent of the impact in relation to its intensity and temporal variability, the multiple temporal and spatial scales used allowed the detection of some differences in the intensity of the impact, depending on the time of sampling. Our findings greatly contribute to increase the knowledge on the effects of multiple stressors caused by the effluent of a power plant and also have important implications for management strategies and conservation ecology, in general.     

Individual Differences in Foraging Strategies of Parasitic Sabre-Tooth Blennies

Originally, evolutionary game theory typically predicted that optimal behaviour in a given situation is uniform or bimodal. However, the growing evidence that animals behave more variably while individuals may differ consistently in their behaviour, has led to the development of models that predict a distribution of strategies. Here we support the importance of such models in a study on a coral reef fish host–parasite system. Parasitic blennies (Plagiotremus sp.) regularly attack other fishes to bite off scales and mucus. Individuals of some victim species react to being bitten with punishing the parasite through aggressive chasing. Our field observations and laboratory experiments show that individual blennies differ markedly in how they incorporate being punished into their foraging decisions. We discuss how these differences may affect the payoff structure and hence the net effect of punishment on punishers and on the appearance of a public good for look-alikes.     

Dynamics of a Cytokine Storm

Six volunteers experienced severe inflammatory response during the Phase I clinical trial of a monoclonal antibody that was designed to stimulate a regulatory T cell response. Soon after the trial began, each volunteer experienced a “cytokine storm”, a dramatic increase in cytokine concentrations. The monoclonal antibody, TGN1412, raised serum concentrations of both pro- and anti-inflammatory cytokines το very hiγh values during the first day, while lymphocyte and monocyte concentrations plummeted. Because the subjects were healthy and had no prior indications of immune deficiency, this event provided an unusual opportunity to study the dynamic interactions of cytokines and other measured parameters. Here, the response histories of nine cytokines have been modeled by a set of linear ordinary differential equations. A general search procedure identifies parameters of the model, whose response fits the data well during the five-day measurement period. The eighteenth-order model reveals plausible cause-and-effect relationships among the cytokines, showing how each cytokine induces or inhibits other cytokines. It suggests that perturbations in IL2, IL8, and IL10 have the most significant inductive effect, while IFN-γ and IL12 have the greatest inhibiting effect on other cytokine concentrations. Although TNF-α is a major pro-inflammatory factor, IFN-γ and three other cytokines have faster initial and median response to TGN1412 infusion. Principal-component analysis of the data reveals three clusters of similar cytokine responses: [TNF-α, IL1, IL10], [IFN-γ, IL2, IL4, IL8, and IL12], and [IL6]. IL1, IL6, IL10, and TNF-α have the highest degree of variability in response to uncertain initial conditions, exogenous effects, and parameter estimates. This study illuminates details of a cytokine storm event, and it demonstrates the value of linear modeling for interpreting complex, coupled biological system dynamics from empirical data.     

Identifying factors likely to influence compliance with diagnostic imaging guideline recommendations for spine disorders among chiropractors in North America: a focus group study using the Theoretical Domains Framework

ABSTRACT: BACKGROUND: The Theoretical Domains Framework (TDF) was developed to investigate determinants of specific clinical behaviors and inform the design of interventions to change professional behavior. This framework was used to explore the beliefs of chiropractors in an American Provider Network and two Canadian provinces about their adherence to evidence-based recommendations for spine radiography for uncomplicated back pain. The primary objective of the study was to identify chiropractors' beliefs about managing uncomplicated back pain without x-rays and to explore barriers and facilitators to implementing evidence-based recommendations on lumbar spine x-rays. A secondary objective was to compare chiropractors in the United States and Canada on their beliefs regarding the use of spine x-rays. METHODS: Six focus groups exploring beliefs about managing back pain without x-rays were conducted with a purposive sample. The interview guide was based upon the TDF. Focus groups were digitally recorded, transcribed verbatim, and analyzed by two independent assessors using thematic content analysis based on the TDF. RESULTS: Five domains were identified as likely relevant. Key beliefs within these domains included the following: conflicting comments about the potential consequences of not ordering x-rays (risk of missing a pathology, avoiding adverse treatment effects, risks of litigation, determining the treatment plan, and using x-ray-driven techniques contrasted with perceived benefits of minimizing patient radiation exposure and reducing costs; beliefs about consequences); beliefs regarding professional autonomy, professional credibility, lack of standardization, and agreement with guidelines widely varied (social/professional role & identity); the influence of formal training, colleagues, and patients also appeared to be important factors (social influences); conflicting comments regarding levels of confidence and comfort in managing patients without x-rays (belief about capabilities); and guideline awareness and agreements (knowledge). CONCLUSIONS: Chiropractors' use of diagnostic imaging appears to be influenced by a number of factors. Five key domains may be important considering the presence of conflicting beliefs, evidence of strong beliefs likely to impact the behavior of interest, and high frequency of beliefs. The results will inform the development of a theory-based survey to help identify potential targets for behavioral-change strategies.