The mammalian Hsp40 ERdj3 requires its Hsp70 interaction and substrate-binding properties to complement various yeast Hsp40-dependent functions.
Heat shock proteins of 70 kDa (Hsp70s) and their J domain-containing Hsp40 cofactors are highly conserved chaperone pairs that facilitate a large number of cellular processes. The observation that each Hsp70 partners with many J domain-containing proteins (JDPs) has led to the hypothesis that Hsp70 function is dictated by cognate JDPs. If this is true, one might expect highly divergent Hsp70-JDP pairs to be unable to function in vivo. However, we discovered that, when a yeast cytosolic JDP, Ydj1, was targeted to the mammalian endoplasmic reticulum (ER), it interacted with the ER-lumenal Hsp70, BiP, and bound to BiP substrates. Conversely, when a mammalian ER-lumenal JDP, ERdj3, was directed to the yeast cytosol, it rescued the temperature-sensitive growth phenotype of yeast-containing mutant alleles in two cytosolic JDPs, HLJ1 and YDJ1, and activated the ATP hydrolysis rate of Ssa1, the yeast cytosolic Hsp70 that partners with Hlj1 and Ydj1. Surprisingly, ERdj3 mutants that were compromised for substrate binding were unable to rescue the hlj1ydj1 growth defect even though they stimulated the ATPase activity of Ssa1. Yet, J domain mutants of ERdj3 that were defective for interaction with Ssa1 restored the growth of hlj1ydj1 yeast. Taken together, these data suggest that the substrate binding properties of certain JDPs, not simply the formation of unique Hsp70-JDP pairs, are critical to specify in vivo function.


Ultra-Short-Chain PFASs in the Sources of German Drinking Water: Prevalent, Overlooked, Difficult to Remove, and Unregulated.
Per- and polyfluoroalkyl substances (PFASs) have been a focal point of environmental chemistry and chemical regulation in recent years, culminating in a shift from individual PFAS regulation toward a PFAS group regulatory approach in Europe. PFASs are a highly diverse group of substances, and knowledge about this group is still scarce beyond the well-studied, legacy long-chain, and short-chain perfluorocarboxylates (PFCAs) and perfluorosulfonates (PFSAs). Herein, quantitative and semiquantitative data for 43 legacy short-chain and ultra-short-chain PFASs (≤2 perfluorocarbon atoms for PFCAs, ≤3 for PFSAs and other PFASs) in 46 water samples collected from 13 different sources of German drinking water are presented. The PFASs considered include novel compounds like hexafluoroisopropanol, bis(trifluoromethylsulfonyl)imide, and tris(pentafluoroethyl)trifluorophosphate. The ultra-short-chain PFASs trifluoroacetate, perfluoropropanoate, and trifluoromethanesulfonate were ubiquitous and present at the highest concentrations (98% of sum target PFAS concentrations). "PFAS total" parameters like the adsorbable organic fluorine (AOF) and total oxidizable precursor (TOP) assay were found to provide only an incomplete picture of PFAS contamination in these water samples by not capturing these highly prevalent ultra-short-chain PFASs. These ultra-short-chain PFASs represent a major challenge for drinking water production and show that regulation in the form of preventive measures is required to manage them.


Engineering on-chip nanoporous gold material libraries via precision photothermal treatment.
Libraries of nanostructured materials on a single chip are a promising platform for high throughput and combinatorial studies of structure-property relationships in the fields of physics and biology. Nanoporous gold (np-Au), produced by an alloy corrosion process, is a nanostructured material specifically suited for such studies because of its self-similar thermally induced coarsening behavior. However, traditional heat application techniques for the modification of np-Au are bulk processes that cannot be used to generate a library of different pore sizes on a single chip. Here, laser micro-processing offers an attractive solution to this problem by providing a means to apply energy with high spatial and temporal resolution. In the present study we use finite element multiphysics simulations to predict the effects of laser mode (continuous-wave vs. pulsed) and thermal conductivity of the supporting substrate on the local np-Au film temperatures during photothermal annealing. Based on these results we discuss the mechanisms by which the np-Au network is coarsened. Thermal transport simulations predict that continuous-wave mode laser irradiation of np-Au thin films on a silicon substrate supports the widest range of morphologies that can be created through photothermal annealing of np-Au. Using the guidance provided by simulations, we successfully fabricate an on-chip material library consisting of 81 np-Au samples of 9 different morphologies for use in the parallel study of structure-property relationships.


African American church-based HIV testing and linkage to care: assets, challenges and needs.
The US National HIV AIDS strategy promotes the use of faith communities to lessen the burden of HIV in African American communities. One specific strategy presented is the use of these non-traditional venues for HIV testing and co-location of services. African American churches can be at the forefront of this endeavour through the provision of HIV testing and linkage to care. However, there are few interventions to promote the churches' involvement in both HIV testing and linkage to care. We conducted 4 focus groups (n = 39 participants), 4 interviews and 116 surveys in a mixed-methods study to examine the feasibility of a church-based HIV testing and linkage to care intervention in Philadelphia, PA, USA. Our objectives were to examine: (1) available assets, (2) challenges and barriers and (3) needs associated with church-based HIV testing and linkage to care. Analyses revealed several factors of importance, including the role of the church as an access point for testing in low-income neighbourhoods, challenges in openly discussing the relationship between sexuality and HIV, and buy-in among church leadership. These findings can support intervention development and necessitate situating African American church-based HIV testing and linkage to care interventions within a multi-level framework.


A liquid chromatography-mass spectrometry-based metabolome database for tomato.
For the description of the metabolome of an organism, the development of common metabolite databases is of utmost importance. Here we present the Metabolome Tomato Database (MoTo DB), a metabolite database dedicated to liquid chromatography-mass spectrometry (LC-MS)- based metabolomics of tomato fruit (Solanum lycopersicum). A reproducible analytical approach consisting of reversed-phase LC coupled to quadrupole time-of-flight MS and photodiode array detection (PDA) was developed for large-scale detection and identification of mainly semipolar metabolites in plants and for the incorporation of the tomato fruit metabolite data into the MoTo DB. Chromatograms were processed using software tools for mass signal extraction and alignment, and intensity-dependent accurate mass calculation. The detected masses were assigned by matching their accurate mass signals with tomato compounds reported in literature and complemented, as much as possible, by PDA and MS/MS information, as well as by using reference compounds. Several novel compounds not previously reported for tomato fruit were identified in this manner and added to the database. The MoTo DB is available at http://appliedbioinformatics.wur.nl and contains all information so far assembled using this LC-PDA-quadrupole time-of-flight MS platform, including retention times, calculated accurate masses, PDA spectra, MS/MS fragments, and literature references. Unbiased metabolic profiling and comparison of peel and flesh tissues from tomato fruits validated the applicability of the MoTo DB, revealing that all flavonoids and alpha-tomatine were specifically present in the peel, while several other alkaloids and some particular phenylpropanoids were mainly present in the flesh tissue.


A general framework for dose optimization.
Dose optimization is a ubiquitous challenge in clinical practice and includes both pharmacologic and non-pharmacologic interventions. Methods for the statistical assessment of optimum dosing are lacking. We developed a generic framework for dose titration and demonstrated its application in two domains. Optimum warfarin dose was estimated from clinical titration data. In addition, cardiac pacemaker interval optimization was conducted using three conventional techniques. For both data types, optima were obtained from mathematical functions fit to the raw data. The precision of the estimated optima was quantified using bootstrapping. In pacing optimization, the observed precision varied significantly among the techniques, suggesting that impedance cardiography is superior to commonly used echocardiographic methods. The average 95% confidence interval of the estimated optimum warfarin dose was +/-18%, suggesting that titration within this range is of limited utility. By identifying statistically ineffective interventions, objective analysis of optimization data may both improve outcomes and reduce healthcare costs.


A critical period for macromolecular synthesis in long-term heterosynaptic facilitation in Aplysia.
Both long-term and short-term sensitization of the gill and siphon withdrawal reflex in Aplysia involve facilitation of the monosynaptic connections between the sensory and motor neurons. To analyze the relationship between these two forms of synaptic facilitation at the cellular and molecular level, this monosynaptic sensorimotor component of the gill-withdrawal reflex of Aplysia can be reconstituted in dissociated cell culture. Whereas one brief application of 1 microM serotonin produced short-term facilitation in the sensorimotor connection that lasted minutes, five applications over 1.5 hours resulted in long-term facilitation that lasted more than 24 hours. Inhibitors of protein synthesis or RNA synthesis selectively blocked long-term facilitation, but not short-term facilitation, indicating that long-term facilitation requires the expression of gene products not essential for short-term facilitation. Moreover, the inhibitors only blocked long-term facilitation when given during the serotonin applications; the inhibitors did not block the facilitation when given either before or after serotonin application. These results parallel those for behavioral performance in vertebrates and indicate that the critical time window characteristic of the requirement for macromolecular synthesis in long-term heterosynaptic facilitation is not a property of complex circuitry, but an intrinsic characteristic of specific nerve cells and synaptic connections involved in the long-term storage of information.


An up to 16-year prospective study of 304 porcelain veneers.
This study aimed to prospectively analyze the outcomes of 304 feldspathic porcelain veneers prepared by the same operator, in 100 patients, that were in situ for up to 16 years. A total of 304 porcelain veneers on incisors, canines, and premolars in 100 patients completed by one prosthodontist between 1988 and 2003 were sequentially included. Preparations were designed with chamfer margins, incisal reduction, and palatal overlap. At least 80% of each preparation was in enamel. Feldspathic porcelain veneers from refractory dies were etched (hydrofluoric acid), silanated, and cemented (Vision 2, Mirage Dental Systems). Outcomes were expressed as percentages (success, survival, unknown, dead, repair, failure). The results were statistically analyzed using the chi-square test and Kaplan-Meier survival estimation. Statistical significance was set at P < .05. The cumulative survival for veneers was 96% +/- 1% at 5 to 6 years, 93% +/- 2% at 10 to 11 years, 91% +/- 3% at 12 to 13 years, and 73% +/- 16% at 15 to 16 years. The marked drop in survival between 13 and 16 years was the result of the death of 1 patient and the low number of veneers in that period. The cumulative survival was greater when different statistical methods were employed. Sixteen veneers in 14 patients failed. Failed veneers were associated with esthetics (31%), mechanical complications (31%), periodontal support (12.5%), loss of retention >2 (12.5%), caries (6%), and tooth fracture (6%). Statistically significantly fewer veneers survived as the time in situ increased. Feldspathic porcelain veneers, when bonded to enamel substrate, offer a predictable long-term restoration with a low failure rate. The statistical methods used to calculate the cumulative survival can markedly affect the apparent outcome and thus should be clearly defined in outcome studies.


Native LESA TWIMS-MSI: Spatial, Conformational, and Mass Analysis of Proteins and Protein Complexes.
We have previously demonstrated native liquid extraction surface analysis (LESA) mass spectrometry imaging of small intact proteins in thin tissue sections. We also showed calculation of collision cross sections for specific proteins extracted from discrete locations in tissue by LESA traveling wave ion mobility spectrometry (TWIMS). Here, we demonstrate an integrated native LESA TWIMS mass spectrometry imaging (MSI) workflow, in which ion mobility separation is central to the imaging experiment and which provides spatial, conformational, and mass information on endogenous proteins in a single experiment. The approach was applied to MSI of a thin tissue section of mouse kidney. The results show that the benefits of integration of TWIMS include improved specificity of the ion images and the capacity to calculate collision cross sections for any protein or protein complex detected in any pixel (without a priori knowledge of the presence of the protein).


Quantification of biological aging in young adults.
Antiaging therapies show promise in model organism research. Translation to humans is needed to address the challenges of an aging global population. Interventions to slow human aging will need to be applied to still-young individuals. However, most human aging research examines older adults, many with chronic disease. As a result, little is known about aging in young humans. We studied aging in 954 young humans, the Dunedin Study birth cohort, tracking multiple biomarkers across three time points spanning their third and fourth decades of life. We developed and validated two methods by which aging can be measured in young adults, one cross-sectional and one longitudinal. Our longitudinal measure allows quantification of the pace of coordinated physiological deterioration across multiple organ systems (e.g., pulmonary, periodontal, cardiovascular, renal, hepatic, and immune function). We applied these methods to assess biological aging in young humans who had not yet developed age-related diseases. Young individuals of the same chronological age varied in their "biological aging" (declining integrity of multiple organ systems). Already, before midlife, individuals who were aging more rapidly were less physically able, showed cognitive decline and brain aging, self-reported worse health, and looked older. Measured biological aging in young adults can be used to identify causes of aging and evaluate rejuvenation therapies.


The effect of copper supplementation on red blood cell oxidizability and plasma antioxidants in middle-aged healthy volunteers.
A multicenter European study (FoodCue) was undertaken to provide data on the significance of increased dietary copper as a pro-oxidant or antioxidant in vivo. The present work describes the effect of Cu supplementation on (2,2'-azo-bis(2-amidinopropane) hydrochloride (AAPH)-induced red blood cell oxidation in middle-aged people. Double-blinded copper supplementation was achieved in 26 healthy volunteers (50-70 years) with pills containing 3 mg CuSO(4), 3 mg Cu glycine chelate (CuG) and 6 mg CuG. Each 6 week supplementation period was preceded and followed by 6 weeks of washout (WO) on placebo. The results show significant increases in time necessary to achieve 50% hemolysis (LT(50)) after 3CuSO(4) and 6CuG compared with values after WO periods. Cu supplementation did not increase the levels of (Cu,Zn)SOD activity in red blood cells. Resistance to hemolysis was significantly and positively correlated (r =.30, p <.01) with alpha- and beta-carotene content in the plasma. Together, these data suggest that intake of copper as high as 7 mg/d has no pro-oxidant activity and may rather result in protection of red blood cells against oxidation. The decreased oxidizability of red blood cells did not result from increased (Cu,Zn)SOD activity and may occur through other mechanisms such as changes in membrane antioxidant content.


3D-electron microscopic characterization of interstitial cells in the human bladder upper lamina propria.
To explore the ultrastructure of interstitial cells in the upper lamina propria of the human bladder, to describe the spatial relationships and to investigate cell-cell contacts. Focused ion beam scanning electron microscopy (FIB-SEM), 3-View SEM and confocal laser scanning microscopy were used to analyze the 3D ultrastructure of the upper lamina propria in male and female human bladders. 3View-SEM image stacks as large as 59 × 59 × 17 μm3 (xyz) at a resolution of 16 × 16 × 50 nm3 and high resolution (5 × 5 × 10 nm3 ) FIB-SEM stacks could be analyzed. Interstitial cells with myoid differentiation (mIC) and fibroblast like interstitial cells (fIC) were the major cell types in the upper lamina propria. The flat, sheet-like ICs were oriented strictly parallel to the urothelium. No spindle shaped cells were present. We furthermore identified one branched cell (bIC) with several processes contacting urothelial cells by penetrating the basal membrane. This cell did not make any contacts to other ICs within the upper lamina propria. We found no evidence for the occurrence of telocytes in the upper lamina propria. Comprehensive 3D-ultrastructural analysis of the human bladder confirmed distinct subtypes of interstitial cells. We provide evidence for a foremost unknown direct connection between a branched interstitial cell and urothelial cells of which the functional role has still to be elucidated. 3D-ultrastructure analyses at high resolution are needed to further define the subpopulations of lamina propria cells and cell-cell interactions.


Follicle-stimulating hormone receptor polymorphism affects the outcome of ovulation induction in normogonadotropic (World Health Organization class 2) anovulatory subfertility.
To assess whether an FSH receptor polymorphism (Asn680Ser, rs6166) can affect the outcome of ovulation induction in normogonadotropic (World Health Organization class 2 [WHO2]) anovulatory subfertile women. Prospective, longitudinal, cohort study. University-based fertility unit. A total of 240 consecutive women diagnosed with WHO2 anovulatory subfertility who underwent ovulation induction therapy. Results were replicated in a retrospective cohort of 185 patients with polycystic ovary syndrome (PCOS) (Rotterdam criteria). Ovulation induction using clomiphene citrate (CC) as first-line and exogenous gonadotropins (exFSH) as second-line therapy. Clomiphene-resistant anovulation (CRA), clomiphene failure (CCF), and ongoing pregnancy rate. Genotyped patients (n = 159) were similar to nongenotyped women (n = 81) regarding clinical characteristics and outcomes of ovulation induction. The 680(Ser) allele was associated with CRA. A pooled analysis of both cohorts showed an 89% higher chance of CRA after CC treatment (odds ratio 1.9 [95% confidence interval 1.1-3.3]) in homozygous carriers of the FSH receptor variant (680(Ser/Ser)). A lower chance of ongoing pregnancy (hazard ratio 0.51 [95% confidence interval 0.27-0.98]) was observed among these patients during CC treatment in the prospective cohort. An FSH receptor polymorphism is associated with CRA during treatment with clomiphene citrate. These data may be used to design a treatment algorithm that is more efficacious and better tailored to the individual patient.


QUANTITATIVE SEPARATION OF RADIOACTIVE STEROLS AND BILE ACIDS IN HUMAN FAECES.
A method is described for the quantitative separation of radioactive bile acids and neutral sterols in human faeces on a potassium hydroxide-treated silicic acid column. Results obtained with faecal extracts containing metabolites derived from radioactive cholesterol, cholic acid, and chenodeoxycholic acid show that the efficiency of extraction is at least 96% and that mutual contamination between the bile-acid and sterol fractions is between 0.7 and 3.8%. The method gives better results than one based on separation of the faecal steroids into saponifiable and unsaponifiable material.


Autophagy activation by novel inducers prevents BECN2-mediated drug tolerance to cannabinoids.
Cannabinoids and related drugs generate profound behavioral effects (such as analgesic effects) through activating CNR1 (cannabinoid receptor 1 [brain]). However, repeated cannabinoid administration triggers lysosomal degradation of the receptor and rapid development of drug tolerance, limiting the medical use of marijuana in chronic diseases. The pathogenic mechanisms of cannabinoid tolerance are not fully understood, and little is known about its prevention. Here we show that a protein involved in macroautophagy/autophagy (a conserved lysosomal degradation pathway), BECN2 (beclin 2), mediates cannabinoid tolerance by preventing CNR1 recycling and resensitization after prolonged agonist exposure, and deletion of Becn2 rescues CNR1 activity in mouse brain and conveys resistance to analgesic tolerance to chronic cannabinoids. To target BECN2 therapeutically, we established a competitive recruitment model of BECN2 and identified novel synthetic, natural or physiological stimuli of autophagy that sequester BECN2 from its binding with GPRASP1, a receptor protein for CNR1 degradation. Co-administration of these autophagy inducers effectively restores the level and signaling of brain CNR1 and protects mice from developing tolerance to repeated cannabinoid usage. Overall, our findings demonstrate the functional link among autophagy, receptor signaling and animal behavior regulated by psychoactive drugs, and develop a new strategy to prevent tolerance and improve medical efficacy of cannabinoids by modulating the BECN2 interactome and autophagy activity.


Differential diagnosis of acute meningitis. An analysis of the predictive value of initial observations.
We analyzed data from the records of 422 patients with acute bacterial or viral meningitis. A cerebrospinal fluid (CSF) glucose level less than 1.9 mmol/L, a CSF-blood glucose ratio less than 0.23, a CSF protein level greater than 2.2 g/L, more than 2000 x 10(6)/L CSF leukocytes, or more than 1180 x 10(6)/L CSF polymorphonuclear leukocytes were individual predictors of bacterial infection with 99% certainty or better. Although any one of these tests could rule in bacterial meningitis with high probability, none could rule it out. To better predict whether a patient has bacterial vs viral infection, we developed a logistic multiple regression model using CSF-blood glucose ratio, total polymorphonuclear leukocyte count in CSF, age, and month of onset. This proved highly reliable when validated in an independent test sample, with an area under receiver operating characteristic curve of 0.97. The model should allow physicians to differentiate between acute viral and acute bacterial meningitis with greater accuracy.


Implementing a comprehensive approach to managing faculty roles, rewards, and development in an era of change.
The current environment in which medicine is taught and practiced requires that medical schools pay increased attention to the faculty member's roles, rewards, career development, and productivity. Medical schools must make strategic decisions about the allocation of resources that can nurture their faculties and support the activities in academic and community settings in which faculty are involved. From 1993 to 1995 Allegheny University of the Health Sciences (formerly Medical College of Pennsylvania and Hahnemann University) designed a comprehensive system for the professional development of faculty. This system is based upon expanded categories of faculty academic activity and scholarship. New programs were implemented to reorient faculty toward conducting and documenting the expanded array of scholarly activities. The main characteristics of the new system are the establishment of formally defined performance expectations, the vertical alignment of the individual faculty member's objectives with the department's mission and the school's mission, and an increasing emphasis upon faculty interdependence, accountability, and use of sound business practices. The authors describe these and other aspects of the design of the new system in detail and report initial results and lessons learned from the system's implementation, evaluation, and dissemination throughout the university. The long-term success of this comprehensive professional development program will be assessed over time by observing how this institution advances its mission in a well-planned and cost-effective manner that retains talented, productive, and professionally fulfilled faculty.


Metabolism of heptadecapeptide gastrin in humans studied by region-specific antisera.
The metabolism of synthetic human heptadecapeptide gastrin (G17) in vivo, and in serum in vitro, was studied by radioimmunoassay using region specific antisera, gel filtration, ion exchange chromatography, and high performance liquid chromatography. After infusion of G17 intravenously in normal human volunteers, COOH-terminal and NH2-terminal immunoreactive G17 fragments were generated. At a steady state, approximately 15% of COOH-terminal immunoreactivity was attributable to G14-like material and up to 25% of total NH2-terminal immunoreactivity was attributable to two NH2-terminal fragments; one had the chromatographic properties of 1-13 G17, and the other was less acidic and less hydrophobic. After stopping the infusion of G17, the latter fragments accounted for progressively greater proportions of total gastrin activity. When G17 was incubated in serum in vitro, there was time-dependent and temperature-dependent loss of immunoreactivity, and again COOH-terminal and NH2-terminal immunoreactive fragments were formed. Removal of the NH2-terminal pyroglutamic acid was probably the rate limiting step because synthetic 2-17 G17 was degraded more rapidly in serum (t1/2, 2-3 h) than G17 (t1/2, 3-5 h). EDTA blocked degradation at the COOH-terminus of both 2-17 G17 and G17 but cleavage at the NH2-terminus still occurred, giving rise to a G14-like peptide. The rate of conversion of G17 in serum was not enough to account for the production of fragments in vivo, and it is proposed that these are formed when G17 encounters enzymes on cell surfaces, perhaps during passage through the capillary circulation. The production of these fragments needs to be considered in interpreting studies of the identity, metabolism, and release of gastrin in health and disease.


Rare copy number variation in cerebral palsy.
Recent studies have established the role of rare copy number variants (CNVs) in several neurological disorders but the contribution of rare CNVs to cerebral palsy (CP) is not known. Fifty Caucasian families having children with CP were studied using two microarray designs. Potentially pathogenic, rare (<1% population frequency) CNVs were identified, and their frequency determined, by comparing the CNVs found in cases with 8329 adult controls with no known neurological disorders. Ten of the 50 cases (20%) had rare CNVs of potential relevance to CP; there were a total of 14 CNVs, which were observed in <0.1% (<8/8329) of the control population. Eight inherited from an unaffected mother: a 751-kb deletion including FSCB, a 1.5-Mb duplication of 7q21.13, a 534-kb duplication of 15q11.2, a 446-kb duplication including CTNND2, a 219-kb duplication including MCPH1, a 169-kb duplication of 22q13.33, a 64-kb duplication of MC2R, and a 135-bp exonic deletion of SLC06A1. Three inherited from an unaffected father: a 386-kb deletion of 12p12.2-p12.1, a 234-kb duplication of 10q26.13, and a 4-kb exonic deletion of COPS3. The inheritance was unknown for three CNVs: a 157-bp exonic deletion of ACOX1, a 693-kb duplication of 17q25.3, and a 265-kb duplication of DAAM1. This is the first systematic study of CNVs in CP, and although it did not identify de novo mutations, has shown inherited, rare CNVs involving potentially pathogenic genes and pathways requiring further investigation.


Using mHealth to improve tuberculosis case identification and treatment initiation in South Africa: Results from a pilot study.
Tuberculosis (TB) incidence in South Africa is among the highest globally. Initial loss to follow-up (ILFU), defined as not starting on TB treatment within 28 days of testing positive, is undermining control efforts. We assessed the feasibility, acceptability, and potential of a mHealth application to reduce ILFU. An mHealth application was developed to capture patients TB investigation data, provide results and monitor treatment initiation. This was implemented in two primary health clinics (PHC) in inner-city Johannesburg. Feasibility was assessed by comparing documentation of personal details, specimen results for same individuals during implementation period (paper register and Mhealth application). Effectiveness was assessed by comparing proportion of patients with results within 48 hours, and proportion started on treatment within 28 days of testing TB positive during pre- implementation (paper register) and implementation (mHealth application) periods. In-depth interviews with patients and providers were conducted to assess acceptability of application. Pre-implementation, 457 patients were recorded in paper registers [195 (42.7%) male, median age 34 years (interquartile range IQR (28-40), 45 (10.5%) sputum Xpert positive]. During implementation, 319 patients were recorded in paper register and the mHealth application [131 (41.1%) male, median age 32 years (IQR 27-38), 33 (10.3%) sputum Xpert positive]. The proportion with complete personal details: [mHealth 95.0% versus paper register 94.0%, (p = 0.54)] and proportion with documented results: [mHealth 97.4% versus paper register 97.8%, (p = 0.79)] were not different in the two methods. The proportion of results available within 48 hours: [mHealth 96.8% versus paper register 68.6%), (p <0.001)], and the proportion on treatment within 28 days [mHealth 28/33 (84.8%) versus paper register 30/44 (68.2%), (p = 0.08)] increased during implementation but was not statistically significant. In-depth interviews showed that providers easily integrated the mHealth application into routine TB investigation and patients positively received the delivery of results via text message. Time from sputum collection to TB treatment initiation decreased from 4 days (pre-implementation) to 3 days but was not statistically significant. We demonstrated that implementation of the mHealth application was feasible, acceptable to health care providers and patients, and has potential to reduce the time to TB treatment initiation and ILFU in PHC settings.


Sexual agglutination in budding yeasts: structure, function, and regulation of adhesion glycoproteins.
The sexual agglutinins of the budding yeasts are cell adhesion proteins that promote aggregation of cells during mating. In each yeast species, complementary agglutinins are expressed by cells of opposite mating type that interact to mediate aggregation. Saccharomyces cerevisiae alpha-agglutinin and its analogs from other yeasts are single-subunit glycoproteins that contain N-linked and O-linked oligosaccharides. The N-glycosidase-sensitive carbohydrate is not necessary for activity. The proposed binding domain of alpha-agglutinin has features characteristic of the immunoglobulin fold structures of cell adhesion proteins of higher eukaryotes. The C-terminal region of alpha-agglutinin plays a role in anchoring the glycoprotein to the cell surface. The S. cerevisiae alpha-agglutinin and its analogs from other species contain multiple subunits; one or more binding subunits, which interact with the opposite agglutinin, are disulfide bonded to a core subunit, which mediates cell wall anchorage. The core subunits are composed of 80 to 95% O-linked carbohydrate. The binding subunits have less carbohydrate, and both carbohydrate and peptide play roles in binding. The alpha-agglutinin and alpha-agglutinin genes from S. cerevisiae have been cloned and shown to be regulated by the mating-type locus, MAT, and by pheromone induction. The agglutinins are necessary for mating under conditions that do not promote cell-cell contact. The role of the agglutinins therefore is to promote close interactions between cells of opposite mating type and possibly to facilitate the response to phermone, thus increasing the efficiency of mating. We speculate that they mediate enhanced response to sex pheromones by providing a synapse at the point of cell-cell contact, at which both pheromone secretion and cell fusion occur.


Identification and characterization of an unusual double serine/threonine protein phosphatase 2C in the malaria parasite Plasmodium falciparum.
We have cloned a gene from Plasmodium falciparum with homology to the Mg2+-dependent serine/threonine protein phosphatase 2C (PP2C) family. The predicted coding region is 920 amino acids long, twice the size of other members of this family. We show that this protein can be divided into two halves (Pf2C-1 and Pf2C-2), each a complete phosphatase unit with homology to other phosphatases of this class. To study the function of this PP2C, we have tested the ability of different constructs to complement conditional null mutants of yeast. Our results show that expression of the full-length protein, the first half alone, the second half alone, or a hybrid with the N terminus of the first half and the C terminus of the second half was able to complement the heat shock response defect of a Schizosaccharomyces pombe strain with a PP2C (PTC1) deletion. Recombinant P. falciparum PP2C expressed in Escherichia coli was active in dephosphorylating 32P-labeled casein in an Mg2+- or Mn2+-dependent reaction. Each half alone was also active in recombinant form. Using the two-hybrid system, we have shown that the two halves can interact. Gel filtration assay of P. falciparum protein extracts suggests that full-length PfPP2C is a dimer, and phosphatase activity competition experiments indicate that dimerization of PfPP2C is required for its optimal activity. This unusual phosphatase molecule appears to be composed of four catalytic units on two polypeptide chains.


Differential gene expression during early development in brains of wildtype and biotinidase-deficient mice.
Biotinidase deficiency is an autosomal recessively inherited disorder characterized by neurological and cutaneous abnormalities. Untreated individuals with biotinidase deficiency cannot recycle biotin from biocytin (N-biotinyl-ϵ-lysine), the proteolytic digestion product of protein-bound biotin. Biotin therapy can markedly resolve symptoms, or can prevent the development of symptoms if initiated early. To understand better the pathogenesis of the neurological problems in the disorder in humans, we have compared gene transcription changes during the first week post-birth in the brains of biotinidase-deficient, transgenic, knock-out mice at days 1 and 8 and compared to changes in wildtype mice at the same times. The knockout pups that were not supplemented with unconjugated biotin became symptomatic by day 8 and exhibiting failure to thrive. Wildtype pups remained asymptomatic under the same experimental conditions. We compared all four possible combinations and noted the most significant up- and down-regulated genes in the knockout animals at Day 8 compared to those at Day 1, reflecting the changes in gene expression over the first week of development. These alterations involved neurological development and immunological function pathways and provide some clues to avenues for further research. At this time, these preliminary analyses provide us with limited, but new information. However, with the development of new algorithms and programs examining various mechanisms and pathways in the central nervous system, these analyses may help us to understand better the role of biotinidase and the pathogenesis of biotinidase deficiency.


Intermolecular cascade annulations of N-(arylsulfonyl)acrylamides with dual C(sp3)-H bonds: divergent access to indanes and pyrrolidin-2-ones.
A new divergent intermolecular cascade annulation reaction of N-(arylsulfonyl)acrylamides with dual alkyl C(sp3)-H bonds for producing two types of five-membered rings, indanes and pyrrolidin-2-ones, is described. By using cycloalkanes and common alkanes as a one-carbon unit, an intermolecular [4+1] cascade carboannulation of N-(arylsulfonyl)acrylamides was achieved via a sequence of three C-H bond functionalization/aryl migration/desulfonylation that enables the formation of three C-C bonds and one N-H bond. When the one-carbon unit was changed to cycloalkyl ethers, the alternative intermolecular [4+1] cascade heteroannulation reaction occurred and allowed the construction of two C-C bonds and one C-N bond through dual C-H bond functionalization, aryl migration and desulfonylation cascades.


Role development in health care assistants: the impact of education on practice.
Health care provision has moved towards an approach to patient care that challenges the traditional role boundaries of health care workers. In nursing this has resulted in the delegation of 'nursing care' to health care assistants (HCAs). This study sought to evaluate the impact of a HCA Development Programme on care delivery. Secondly, it sought to understand the preparedness of HCAs to undertake the programme and the new roles set for them. The research method was a qualitative, inductive approach. For part one, data were gathered using a 360-degree approach. Semi-structured interviews were conducted with four HCAs, eight of each of their colleagues and four patients. For part two, 12 HCAs were interviewed. Data were analysed following the principles of thematic analysis. The findings for part one showed positive changes to the HCAs' role, which included skill and knowledge development, increased confidence and initiative and a more holistic approach to care. A range of personal and contextual factors including relationships, hierarchy, staffing, experience, responsibility, patient dependency, attitudes and values affected the development of their role. The HCAs in part two voiced positive and negative views of their role development. Of the 12 HCAs interviewed, eight were prepared to attend the programme, two were undecided, and two were reluctant to attend. The findings indicated that while the HCA Development Programme positively influenced the role of the HCA, there was a need to invest more into preparation for the restructuring of roles.


Antimicrobial resistance among respiratory isolates of Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae in the United States.
A national surveillance study was conducted to determine trends in antimicrobial resistance patterns among three common causes of community-acquired respiratory tract infections. Fifteen participating U.S. medical centers submitted clinically significant isolates of Haemophilus influenzae, Moraxella (Branhamella) catarrhalis, and Streptococcus pneumoniae to two central laboratories for testing with a group of 12 antimicrobial agents. The majority of isolates were recovered from adult males greater than 50 years old. Overall, 84.1% of 378 M. catarrhalis and 16.5% of 564 H. influenzae (29.5% of type b strains; 15.0% of non-type b strains) produced beta-lactamase and were thus resistant to penicillin, ampicillin, and amoxicillin. Resistance in H. influenzae to other agents was 2.1% to tetracycline, 0.7% to trimethoprim-sulfamethoxazole, 1.1% to cefaclor, and 0.2% to cefuroxime and amoxicillin-clavulanate, while the M. catarrhalis isolates yielded very low MICs of these latter drugs. As demonstrated in prior studies, erythromycin showed little activity against H. influenzae. Of 487 S. pneumoniae isolates, 1 (0.2%) was penicillin resistant, while 3.8% were relatively resistant to penicillin, 4.5% were resistant to trimethoprim-sulfamethoxazole, 2.3% were resistant to tetracycline, 1.2% were resistant to chloramphenicol, and 0.2% were resistant to erythromycin. Overall, the lowest resistance rates for these common bacterial respiratory pathogens were noted with amoxicillin-clavulanate, cefuroxime, and cefaclor.


Whey proteins as stabilizers in amorphous solid dispersions.
Whey proteins are extensively used as nutritional supplements but have so far not been investigated as co-formers for amorphous solid dispersions (ASD) to enhance the solubility and dissolution rate of poorly water soluble drugs. In this study, whey protein isolate (WPI) and whey protein hydrolysate (WPH) were each mixed with three poorly water soluble drugs (indomethacin: IND, carvedilol: CAR and furosemide: FUR) and prepared as ASDs at 50% (w/w) drug loading using vibrational ball milling. Subsequently, solid state characteristics, dissolution rate and physical stability of the obtained samples were analyzed. All ASDs showed a significant increase in their glass transition temperatures, as well as faster dissolution rates and higher apparent solubilities compared to both the respective pure crystalline and amorphous drugs. The saturation solubility of the drugs was increased in the presence of the whey proteins, and the investigated ASDs showed supersaturation by attaining higher drug concentrations compared to the respective saturation solubilities. Upon storage, ASDs containing IND were found to be physically stable for at least 27 months, whereas, ASDs containing CAR or FUR were stable for about 8 months and 17 months, respectively. This was a tremendous increase in physical stability compared to the pure amorphous drugs which recrystallized within less than one week. Overall, WPI and WPH proved to be promising co-formers and amorphous stabilizers in ASD formulations.


Antiprion Activity of DB772 and Related Monothiophene- and Furan-Based Analogs in a Persistently Infected Ovine Microglia Culture System.
The transmissible spongiform encephalopathies are fatal neurodegenerative disorders characterized by the misfolding of the native cellular prion protein (PrP(C)) into the accumulating, disease-associated isoform (PrP(Sc)). Despite extensive research into the inhibition of prion accumulation, no effective treatment exists. Previously, we demonstrated the inhibitory activity of DB772, a monocationic phenyl-furan-benzimidazole, against PrP(Sc) accumulation in sheep microglial cells. In an effort to determine the effect of structural substitutions on the antiprion activity of DB772, we employed an in vitro strategy to survey a library of structurally related, monothiophene- and furan-based compounds for improved inhibitory activity. Eighty-nine compounds were screened at 1 μM for effects on cell viability and prion accumulation in a persistently infected ovine microglia culture system. Eleven compounds with activity equivalent to or higher than that of DB772 were identified as preliminary hit compounds. For the preliminary hits, cytotoxicities and antiprion activities were compared to calculate the tissue culture selectivity index. A structure-activity relationship (SAR) analysis was performed to determine molecular components contributing to antiprion activity. To investigate potential mechanisms of inhibition, effects on PrP(C) and PrP(Sc) were examined. While inhibition of total PrP(C) was not observed, the results suggest that a potential target for inhibition at biologically relevant concentrations is through PrP(C) misfolding to PrP(Sc) Further, SAR analysis suggests that two structural elements were associated with micromolar antiprion activity. Taken together, the described data provide a foundation for deeper investigation into untested DB compounds and in the design of effective therapeutics.


Role of GSK3β/α-synuclein axis in methamphetamine-induced neurotoxicity in PC12 cells.
Methamphetamine (METH) is well-known as a potent psychostimulant of abuse worldwide. METH administration can cause neurotoxicity and neurodegenerative injury, which are similar to the two prevalent neurodegenerative disorders Alzheimer's disease (AD) and Parkinson's disease (PD). Recent results suggested that METH exposure increased the level of α-synuclein (α-syn) that could be a possible cause of neurotoxicity. However, the mechanism of METH-induced neurodegeneration remains unclear. This study was aimed at examining the effects of glycogen synthase kinase3β (GSK3β), α-syn, and tau on METH-induced neurotoxicity. Our results indicated that P-GSK3β (Tyr216), P-Tau (Ser396), α-syn, and P-α-syn (Ser129) levels were increased after METH administration in dose- and time-dependent manners. Upon inhibiting the GSK3β activity with LiCl or GSK3β-siRNA, these protein expressions were significantly decreased. We observed that LiCl protected the cells from METH-caused cytotoxicity by weakening the cell morphological damage and preventing cell apoptosis and death. We also found that P-GSK3β colocalized with P-Tau and α-syn by the immunofluorescence method. Further, METH disrupted the cellular autophagy by upregulation of LC3-II and P62 proteins, and the cellular autophagy was restored by LiCl and GSK3β-siRNA. The expressions of the α-syn-specific degradative enzyme glucocerebrosidase (GCase) with its regulator lysosomal integral membrane protein type-2 (LIMP-2) decreased inversely with the doses of METH treatment. The GCase inhibitor conduritol-β-epoxide (CβE) increased the α-syn levels, and LiCl restored GCase and LIMP-2 expressions disrupted by the METH treatment. In summary, we conclude that GSK3β plays key roles in METH-induced neurotoxicity and neurodegenerative injury by promoting abnormal protein phosphorylation and α-syn accumulation, blocking the autophagy-lysosomal degradation pathway, and finally leading to cell apoptosis and death. GSK3β may be a potential target to prevent METH-induced neurodegeneration.


General classes of double phase conjugation.
The double phase-conjugate mirror (DPCM) is a stimulated four-wave mixing effect, mediated by either a chi(3) mechanism or a photorefractive effect. The type of grating that operates in the DPCM can be a transmission or reflection grating, depending on the wave-mixing configuration. Any combination of nonlinearity and grating geometry is possible, so that four general DPCM classes emerge. An analytic solution of the last unsolved class, the photorefractive reflection DPCM, is presented. With these results, general observations concerning the four categories of double phase conjugation are made. An important conclusion is that the type of grating geometry and its associated boundary conditions dictate many of the basic features of the DPCM, regardless of the nonlinearity involved.


Review of ongoing initiatives to improve prescribing efficiency in China; angiotensin receptor blockers as a case history.
Pharmaceutical expenditure is rising by 16% per annum in China and is now 46% of total expenditure. Initiatives to moderate growth include drug pricing regulations and encouraging international non-proprietary name prescribing. However, there is no monitoring of physician prescribing quality and perverse incentives. Assess changes in angiotensin receptor blocker (ARB) utilization and expenditure as more generics become available; compare findings to Europe. Observational retrospective study of ARB utilization and expenditure between 2006 and 2012 in the largest hospital in Chongqing district. Variable and low use of generics versus originators with a maximum of 31% among single ARBs. Similar for fixed dose combinations. Prices typically reduced over time, greatest for generic telmisartan (-54%), mirroring price reductions in some European countries. However, no preferential increase in prescribing of lower cost generics. Accumulated savings of 33 million CNY for this large provider if they adopted European practices. Considerable opportunities to improve prescribing efficiency in China.


Genome sequence of hybrid Vibrio cholerae O1 MJ-1236, B-33, and CIRS101 and comparative genomics with V. cholerae.
The genomes of Vibrio cholerae O1 Matlab variant MJ-1236, Mozambique O1 El Tor variant B33, and altered O1 El Tor CIRS101 were sequenced. All three strains were found to belong to the phylocore group 1 clade of V. cholerae, which includes the 7th-pandemic O1 El Tor and serogroup O139 isolates, despite displaying certain characteristics of the classical biotype. All three strains were found to harbor a hybrid variant of CTXPhi and an integrative conjugative element (ICE), leading to their establishment as successful clinical clones and the displacement of prototypical O1 El Tor. The absence of strain- and group-specific genomic islands, some of which appear to be prophages and phage-like elements, seems to be the most likely factor in the recent establishment of dominance of V. cholerae CIRS101 over the other two hybrid strains.


PalmTraits 1.0, a species-level functional trait database of palms worldwide.
Plant traits are critical to plant form and function -including growth, survival and reproduction- and therefore shape fundamental aspects of population and ecosystem dynamics as well as ecosystem services. Here, we present a global species-level compilation of key functional traits for palms (Arecaceae), a plant family with keystone importance in tropical and subtropical ecosystems. We derived measurements of essential functional traits for all (>2500) palm species from key sources such as monographs, books, other scientific publications, as well as herbarium collections. This includes traits related to growth form, stems, armature, leaves and fruits. Although many species are still lacking trait information, the standardized and global coverage of the data set will be important for supporting future studies in tropical ecology, rainforest evolution, paleoecology, biogeography, macroecology, macroevolution, global change biology and conservation. Potential uses are comparative eco-evolutionary studies, ecological research on community dynamics, plant-animal interactions and ecosystem functioning, studies on plant-based ecosystem services, as well as conservation science concerned with the loss and restoration of functional diversity in a changing world.


Cellular pathways for transport and efflux of ascorbate and dehydroascorbate.
The mechanisms allowing the cellular transport of ascorbic acid represent a primary aspect for the understanding of the roles played by this vitamin in pathophysiology. Considerable research effort has been spent in the field, on several animal models and different cell types. Several mechanisms have been described to date, mediating the movements of different redox forms of ascorbic acid across cell membranes. Vitamin C can enter cells both in its reduced and oxidized form, ascorbic acid (AA) and dehydroascorbate (DHA), utilizing respectively sodium-dependent transporters (SVCT) or glucose transporters (GLUT). Modulation of SVCT expression and function has been described by cytokines, steroids and post-translational protein modification. Cellular uptake of DHA is followed by its intracellular reduction to AA by several enzymatic and non-enzymatic systems. Efflux of vitamin C has been also described in a number of cell types and different pathophysiological functions were proposed for this phenomenon, in dependence of the cell model studied. Cellular efflux of AA is mediated through volume-sensitive (VSOAC) and Ca(2+)-dependent anion channels, gap-junction hemichannels, exocytosis of secretory vesicles and possibly through homo- and hetero-exchange systems at the plasma membrane level. Altogether, available data suggest that cellular efflux of ascorbic acid - besides its uptake - should be taken into account when evaluating the cellular homeostasis and functions of this important vitamin.


Serotonin 1A Receptors on Astrocytes as a Potential Target for the Treatment of Parkinson's Disease.
Astrocytes are the most abundant neuron-supporting glial cells in the central nervous system. The neuroprotective role of astrocytes has been demonstrated in various neurological disorders such as amyotrophic lateral sclerosis, spinal cord injury, stroke and Parkinson's disease (PD). Astrocyte dysfunction or loss-of-astrocytes increases the susceptibility of neurons to cell death, while astrocyte transplantation in animal studies has therapeutic advantage. We reported recently that stimulation of serotonin 1A (5-HT1A) receptors on astrocytes promoted astrocyte proliferation and upregulated antioxidative molecules to act as a neuroprotectant in parkinsonian mice. PD is a progressive neurodegenerative disease with motor symptoms such as tremor, bradykinesia, rigidity and postural instability, that are based on selective loss of nigrostriatal dopaminergic neurons, and with non-motor symptoms such as orthostatic hypotension and constipation based on peripheral neurodegeneration. Although dopaminergic therapy for managing the motor disability associated with PD is being assessed at present, the main challenge remains the development of neuroprotective or disease- modifying treatments. Therefore, it is desirable to find treatments that can reduce the progression of dopaminergic cell death. In this article, we summarize first the neuroprotective properties of astrocytes targeting certain molecules related to PD. Next, we review neuroprotective effects induced by stimulation of 5-HT1A receptors on astrocytes. The review discusses new promising therapeutic strategies based on neuroprotection against oxidative stress and prevention of dopaminergic neurodegeneration.


Dystrophin-glycoprotein complex and vinculin-talin-integrin system in human adult cardiac muscle.
Costameres were identified, for the first time, in skeletal and cardiac muscle, as regions associated with the sarcolemma, consisting of densely clustered patches of vinculin; they have many characteristics common to the cell-extracellular matrix-type of adherens junctions. Costameres are considered 'proteic machinery' and they appear to comprise two protein complexes, the dystrophin-glycoprotein complex (DGC) and the vinculin-talin-integrin system. In comparison to skeletal muscle, few studies have focused on cardiac muscle regarding these two complexes, and study is generally relative to dystrophin or to cardiac diseases, such as cardiomyopathies. However, insufficient data are available on these proteins in healthy human cardiomyocytes. For this reason, we performed an immunohistochemical study using human cardiac muscle fibers, in order to define the real distribution and the spatial relationship between the proteins in these two complexes. Our data showed a real costameric distribution of DGC and of the vinculin-talin-integrin system; all tested proteins were present in T-tubule and in intercalated disks. Moreover, our data demonstrated that all tested proteins of DGC colocalized with each other, as all tested components of the vinculin-talin-integrin system, and that all tested proteins of DGC colocalized with all tested proteins of the vinculin-talin-integrin system. Finally, all tested proteins of the two complexes were localized in the region of the sarcolemma over the I band, in 100% of our observations. The present study, for the first time, analyzed the majority of proteins of DGC and of the vinculin-talin-integrin system in cardiac muscle fibers, and it confirmed that DGC and the vinculin-talin-integrin system have a role in the transduction of mechanical force to the extracellular matrix. Finally it attributed a key role in the regulation of action potential duration to cardiac myocytes.


Vessel health and preservation: An integrative review.
To describe and synthesise current knowledge on the maintenance and preservation of vessels in patients who need the placement of a vascular access device. To administer drugs, blood or intravenous fluids, nurses or doctors insert a peripheral vascular access device on the arm using the traditional approach. This approach implies that devices are blindly inserted until flow is satisfactory and all possible sites have been exhausted. A proactive approach would ensure at the outset that the best device is used for each patient, eliminating repeated attempts at cannulation. An integrative review was conducted using data recorded until July 2017. Searches were conducted in PubMed, Cochrane Library, CINAHL and Scopus. A modified version of Cooper's five-stage method and the PRISMA guidelines were used to perform the integrative review. Nine papers were included in this review. The patients were active participants in a proactive approach to vessel health and preservation. The involvement of each healthcare professional in vessel health and preservation improves outcomes and expands the use of a proactive approach to vascular device management. Because nurses are directly involved in the use of such devices and support patients during the decision-making process, they should take the lead in the use of the proactive approach. Despite the many documented advantages of the proactive approach to preserving vessels in many settings and healthcare systems, it has not been widely tested. Future research is needed to guarantee high-quality vessel health and preservation care, thus contributing to the development and dissemination of the proactive approach. The proactive approach preserves vessels for future needs, improves the delivery of the treatment plan and reduces length of stay, costs, risk of infection, complications and pain perceived by patients. This approach also ensures better use of nurses' time and vascular access device material.


Using an atlas of gene regulation across 44 human tissues to inform complex disease- and trait-associated variation.
We apply integrative approaches to expression quantitative loci (eQTLs) from 44 tissues from the Genotype-Tissue Expression project and genome-wide association study data. About 60% of known trait-associated loci are in linkage disequilibrium with a cis-eQTL, over half of which were not found in previous large-scale whole blood studies. Applying polygenic analyses to metabolic, cardiovascular, anthropometric, autoimmune, and neurodegenerative traits, we find that eQTLs are significantly enriched for trait associations in relevant pathogenic tissues and explain a substantial proportion of the heritability (40-80%). For most traits, tissue-shared eQTLs underlie a greater proportion of trait associations, although tissue-specific eQTLs have a greater contribution to some traits, such as blood pressure. By integrating information from biological pathways with eQTL target genes and applying a gene-based approach, we validate previously implicated causal genes and pathways, and propose new variant and gene associations for several complex traits, which we replicate in the UK BioBank and BioVU.


EXPRESS: F-actin links Epac-PKC signaling to purinergic P2X3 receptors sensitization in dorsal root ganglia following inflammation.
Sensitization of purinergic P2X3 receptors (P2X3Rs) contributes to the production of exaggerated nociceptive responses following inflammatory injury. We showed previously that prostaglandin E2 (PGE2) potentiates P2X3R-mediated ATP currents in dorsal root ganglion neurons isolated from both control and complete Freund's adjuvant-induced inflamed rats. PGE2 potentiation of ATP currents depends only on PKA signaling in control neurons, but it depends on both PKA and PKC signaling in inflamed neurons. We further found that inflammation evokes an increase in exchange proteins directly activated by cAMP (Epacs) in dorsal root ganglions. This increase promotes the activation of PKC to produce a much enhanced PGE2 effect on ATP currents and to elicit Epac-dependent flinch nocifensive behavioral responses in complete Freund's adjuvant rats. The link between Epac-PKC signaling and P2X3R sensitization remains unexplored. Here, we show that the activation of Epacs promotes the expression of phosphorylated PKC and leads to an increase in the cytoskeleton, F-actin, expression at the cell perimeter. Depolymerization of F-actin blocks PGE2-enhanced ATP currents and inhibits P2X3R-mediated nocifensive responses after inflammation. Thus, F-actin is dynamically involved in the Epac-PKC-dependent P2X3R sensitization. Furthermore, Epacs induce a PKC-dependent increase in the membrane expression of P2X3Rs. This increase is abolished by F-actin depolymerization, suggesting that F-actin mediates Epac-PKC signaling of P2X3R membrane expression. Thus, after inflammation, an Epac-PKC dependent increase in F-actin in dorsal root ganglion neurons enhances the membrane expression of P2X3Rs to bring about sensitization of P2X3Rs and abnormal pain behaviors.


Phospholipid headgroup dynamics in DOPG-d5-cytochrome c complexes as revealed by 2H and 31P NMR: the effects of a peripheral protein on collective lipid fluctuations.
The dynamics of the glycerol headgroup of dioleoylphosphatidylglycerol (DOPG) in hydrated bilayers were studied by 2H and 31P NMR spectroscopy, and the effects of binding a peripheral protein, cytochrome c, were evaluated. The fast headgroup segmental motions (tau c, 10(-10)-(-13) s) of DOPG in fully hydrated bilayers were not affected upon binding of cytochrome c, as evaluated by the spin-lattice (T1) relaxation of deuterons in the DOPG glycerol headgroup. In contrast, the spin-spin (T2e) relaxation is strongly affected, indicating that slow cooperative bilayer motions (tau c, 10(-3)-10(-6) s) are enhanced upon the interaction with cytochrome c, 2H and 31P NMR spectral lineshape analysis reveal details of the nature of these motions. The importance of these effects are discussed in terms of a possible mechanism for modulating membrane-associated processes.


[Betamethasone and aqueous extract of Arctium lappa for treating angiostrongyliasis].
Angiostrongylus costaricensis is a parasite that causes abdominal angiostrongyliasis in humans. The treatment for it includes the use of anti-inflammatory drugs, despite the lack of studies to justify this approach. The objective of this paper was to evaluate the effect of betamethasone and Arctium lappa on the evolution of intestinal lesions induced by this parasite. Adult male Swiss mice were used, distributed into four groups: infected and treated with betamethasone; infected and treated with Arctium lappa; infected and not treated; and control group. The treatments were started on the 15th day after infection and continued for 15 days. The presence of eosinophilic infiltration and granuloma was evaluated (1-mild; 2-moderate; 3-severe). Betamethasone allowed the lesions to evolve into more severe forms, while the extract did not interfere with disease progression. The substances applied were ineffective for protection against the lesions induced by Angiostrongylus costaricensis in mice. These findings discourage the use of betamethasone and Arctium lappa for humans affected by abdominal angiostrongyliasis.


MUC1 Vaccines, Comprised of Glycosylated or Non-Glycosylated Peptides or Tumor-Derived MUC1, Can Circumvent Immunoediting to Control Tumor Growth in MUC1 Transgenic Mice.
It remains challenging to produce decisive vaccines against MUC1, a tumor-associated antigen widely expressed by pancreas, breast and other tumors. Employing clinically relevant mouse models, we ruled out such causes as irreversible T-cell tolerance, inadequate avidity, and failure of T-cells to recognize aberrantly glycosylated tumor MUC1. Instead, every tested MUC1 preparation, even non-glycosylated synthetic 9mer peptides, induced interferon gamma-producing CD4(+) and CD8(+) T-cells that recognized glycosylated variants including tumor-associated MUC1. Vaccination with synthetic peptides conferred protection as long as vaccination was repeated post tumor challenge. Failure to revaccinate post challenge was associated with down-regulated tumor MUC1 and MHC molecules. Surprisingly, direct admixture of MUC1-expressing tumor with MUC1-hyperimmune T-cells could not prevent tumor outgrowth or MUC1 immunoediting, whereas ex vivo activation of the hyperimmune T-cells prior to tumor admixture rendered them curative. Therefore, surrogate T-cell preactivation outside the tumor bed, either in culture or by repetitive vaccination, can overcome tumor escape.


Protein tyrosine phosphatase receptor type O (Ptpro) regulates cerebellar formation during zebrafish development through modulating Fgf signaling.
Protein activities controlled by receptor protein tyrosine phosphatases (RPTPs) play comparably important roles in transducing cell surface signals into the cytoplasm by protein tyrosine kinases. Previous studies showed that several RPTPs are involved in neuronal generation, migration, and axon guidance in Drosophila, and the vertebrate hippocampus, retina, and developing limbs. However, whether the protein tyrosine phosphatase type O (ptpro), one kind of RPTP, participates in regulating vertebrate brain development is largely unknown. We isolated the zebrafish ptpro gene and found that its transcripts are primarily expressed in the embryonic and adult central nervous system. Depletion of zebrafish embryonic Ptpro by antisense morpholino oligonucleotide knockdown resulted in prominent defects in the forebrain and cerebellum, and the injected larvae died on the 4th day post-fertilization (dpf). We further investigated the function of ptpro in cerebellar development and found that the expression of ephrin-A5b (efnA5b), a Fgf signaling induced cerebellum patterning factor, was decreased while the expression of dusp6, a negative-feedback gene of Fgf signaling in the midbrain-hindbrain boundary region, was notably induced in ptpro morphants. Further analyses demonstrated that cerebellar defects of ptpro morphants were partially rescued by inhibiting Fgf signaling. Moreover, Ptpro physically interacted with the Fgf receptor 1a (Fgfr1a) and dephosphorylated Fgfr1a in a dose-dependant manner. Therefore, our findings demonstrate that Ptpro activity is required for patterning the zebrafish embryonic brain. Specifically, Ptpro regulates cerebellar formation during zebrafish development through modulating Fgf signaling.


Aminopyridines for the treatment of neurologic disorders.
To identify the different indications for the treatment of neurologic disorders with the potassium channel blockers 4-aminopyridine (4-AP) and 3,4-diaminopyridine (3,4-DAP). 4-AP is an effective symptomatic treatment for downbeat nystagmus (DBN), episodic ataxia type 2 (EA2) (5-10 mg TID), and impaired gait in multiple sclerosis (MS) (10 mg BID). 3,4-DAP (5 mg/d-20 mg TID) improves symptoms in Lambert-Eaton myasthenic syndrome (LEMS) (randomized placebo-controlled trials for all 4 entities). 4-AP may also be effective in cerebellar gait ataxia of different etiologies (2 case series), upbeat nystagmus, and limb ataxia in MS (single cases). In the recommended dosages, they are well tolerated. The assumed mode of action is a blockade of mainly Kv1.5: in DBN, this increases the excitability of Purkinje cells (PC), and in EA2, restores the precision of resting discharge of PC. In MS, 4-AP improves the conduction of action potentials in demyelinated axons, and in LEMS, 3,4-DAP facilitates the transmission at the neuromuscular endplate by prolonging the action potential duration. There is sufficient evidence that APs are indicated for the symptomatic treatment of DBN, EA2, gait ataxia due to MS and cerebellar disorders, and LEMS with a reasonable risk-benefit profile.


Search for Subsolar-Mass Binaries in the First Half of Advanced LIGO's and Advanced Virgo's Third Observing Run.
We report on a search for compact binary coalescences where at least one binary component has a mass between 0.2 M_{⊙} and 1.0 M_{⊙} in Advanced LIGO and Advanced Virgo data collected between 1 April 2019 1500 UTC and 1 October 2019 1500 UTC. We extend our previous analyses in two main ways: we include data from the Virgo detector and we allow for more unequal mass systems, with mass ratio q≥0.1. We do not report any gravitational-wave candidates. The most significant trigger has a false alarm rate of 0.14 yr^{-1}. This implies an upper limit on the merger rate of subsolar binaries in the range [220-24200] Gpc^{-3} yr^{-1}, depending on the chirp mass of the binary. We use this upper limit to derive astrophysical constraints on two phenomenological models that could produce subsolar-mass compact objects. One is an isotropic distribution of equal-mass primordial black holes. Using this model, we find that the fraction of dark matter in primordial black holes in the mass range 0.2 M_{⊙}<m_{PBH}<1.0 M_{⊙} is f_{PBH}≡Ω_{PBH}/Ω_{DM}≲6%. This improves existing constraints on primordial black hole abundance by a factor of ∼3. The other is a dissipative dark matter model, in which fermionic dark matter can collapse and form black holes. The upper limit on the fraction of dark matter black holes depends on the minimum mass of the black holes that can be formed: the most constraining result is obtained at M_{min}=1 M_{⊙}, where f_{DBH}≡Ω_{DBH}/Ω_{DM}≲0.003%. These are the first constraints placed on dissipative dark models by subsolar-mass analyses.


Multiple pathways for homologous recombination in Saccharomyces cerevisiae.
The genes in the RAD52 epistasis group of Saccharomyces cerevisiae are necessary for most mitotic and meiotic recombination events. Using an intrachromosomal inverted-repeat assay, we previously demonstrated that mitotic recombination of this substrate is dependent upon the RAD52 gene. In the present study the requirement for other genes in this epistasis group for recombination of inverted repeats has been analyzed, and double and triple mutant strains were examined for their epistatic relationships. The majority of recombination events are mediated by a RAD51-dependent pathway, where the RAD54, RAD55 and RAD57 genes function downstream of RAD51. Cells mutated in RAD55 or RAD57 as well as double mutants are cold-sensitive for inverted-repeat recombination, whereas a rad51 rad55 rad57 triple mutant is not. The RAD1 gene is not required for inverted-repeat recombination but is able to process spontaneous DNA lesions to produce recombinant products in the absence of RAD51. Furthermore, there is still considerably more recombination in rad1 rad51 mutants than in rad52 mutants, indicating the presence of another, as yet unidentified, recombination pathway.


Structural changes in a Schiff base molecular assembly initiated by scanning tunneling microscopy tip.
We report the controlled self-organization and switching of newly designed Schiff base (E)-4-((4-(phenylethynyl) benzylidene) amino) benzenethiol (EPBB) molecules on a Au (111) surface at room temperature. Scanning tunneling microscopy and spectroscopy (STM/STS) were used to image and analyze the conformational changes of the EPBB molecules. The conformational change of the molecules was induced by using the STM tip while increasing the tunneling current. The switching of a domain or island of molecules was shown to be induced by the STM tip during scanning. Unambiguous fingerprints of the switching mechanism were observed via STM/STS measurements. Surface-enhanced Raman scattering was employed, to control and identify quantitatively the switching mechanism of molecules in a monolayer. Density functional theory calculations were also performed in order to understand the microscopic details of the switching mechanism. These calculations revealed that the molecular switching behavior stemmed from the strong interaction of the EPBB molecules with the STM tip. Our approach to controlling intermolecular mechanics provides a path towards the bottom-up assembly of more sophisticated molecular machines.


Contrast in atomically resolved EF-SCEM imaging.
Energy-filtered scanning confocal electron microscopy (EF-SCEM) is a technique that uses the reduced depth of field of an aberration-corrected transmission electron microscope to provide three-dimensional (3D) compositional information. Using a silicon sample in the <110> orientation, we show that EF-SCEM image data can be recorded that shows lattice resolution in the plane perpendicular to the incident beam direction. The confocal effect is demonstrated through the reduction of the mean intensity as the confocal plane is displaced from the sample mid-plane, unlike optical sectioning in high-angle annular dark-field scanning transmission electron microscopy (STEM). Simulations of the EF-SCEM data show agreement with the experimental data, and allow the interpretability of the data to be explored. The effects of channelling, absorption and delocalisation complicate the quantitative and qualitative interpretation of the data, highlighting the need for matching to simulations. Finally the effects of the finite detector pin-hole aperture size are explored, and we show that the EF-SCEM contrast in the plane perpendicular to the beam direction starts to resemble that of a STEM spectrum imaging experiment as the aperture size increases.


Massive horizontal gene transfer in bdelloid rotifers.
Horizontal gene transfer in metazoans has been documented in only a few species and is usually associated with endosymbiosis or parasitism. By contrast, in bdelloid rotifers we found many genes that appear to have originated in bacteria, fungi, and plants, concentrated in telomeric regions along with diverse mobile genetic elements. Bdelloid proximal gene-rich regions, however, appeared to lack foreign genes, thereby resembling those of model metazoan organisms. Some of the foreign genes were defective, whereas others were intact and transcribed; some of the latter contained functional spliceosomal introns. One such gene, apparently of bacterial origin, was overexpressed in Escherichia coli and yielded an active enzyme. The capture and functional assimilation of exogenous genes may represent an important force in bdelloid evolution.


Genomic instability after targeted irradiation of human lymphocytes: evidence for inter-individual differences under bystander conditions.
Environmental (222)radon exposure is a human health concern, and many studies demonstrate that very low doses of high LET alpha-particle irradiation initiate deleterious genetic consequences in both irradiated and non-irradiated bystander cells. One consequence, radiation-induced genomic instability (RIGI), is a hallmark of tumorigenesis and is often assessed by measuring delayed chromosomal aberrations. We utilised a technique that facilitates transient immobilization of primary lymphocytes for targeted microbeam irradiation and have reported that environmentally relevant doses, e.g. a single (3)He(2+) particle traversal to a single cell, are sufficient to induce RIGI. Herein we sought to determine differences in radiation response in lymphocytes isolated from five healthy male donors. Primary lymphocytes were irradiated with a single particle per cell nucleus. We found evidence for inter-individual variation in radiation response (RIGI, measured as delayed chromosome aberrations). Although this was not highly significant, it was possibly masked by high levels of intra-individual variation. While there are many studies showing a link between genetic predisposition and RIGI, there are few studies linking genetic background with bystander effects in normal human lymphocytes. In an attempt to investigate inter-individual variation in the induction of bystander effects, primary lymphocytes were irradiated with a single particle under conditions where fractions of the population were traversed. We showed a marked genotype-dependent bystander response in one donor after exposure to 15% of the population. The findings may also be regarded as a radiation-induced genotype-dependent bystander effect triggering an instability phenotype.


Type of closure prevents microbial contamination of cosmetics during consumer use.
The dispensing closure used for containers plays an important role in protecting cosmetics from in-use microbial contamination. This hypothesis was tested by aseptically packing unpreserved shampoo and skin lotion into containers with three different closure types which provided various degrees of protection against consumer and environmental microbial insults. Shampoo was packed in containers with slit-cap (n = 25), flip-cap (n = 25), or screw-cap (n = 28) closures. Skin lotion was packed in containers with pump-top (n = 21), flip-cap (n = 18), or screw-cap (n = 21) closures. The products were then used by volunteers under actual in-use conditions for 3 (shampoo) or 2 (skin lotion) weeks. After use, the products were evaluated for microbial contamination by using standard methods for enumeration and identification. The standard screw-cap closure provided only minimal protection against microbial contamination of both the shampoo (29% contamination incidence) and the skin lotion (71%). The slit-cap closure on the shampoo container and the flip-cap closure on the skin lotion container provided slightly enhanced degrees of protection (21 and 39% contamination incidence, respectively). The greatest amount of protection (i.e., lowest contamination incidence) was provided by the flip-cap closure for the shampoo container (0%) and the pump-top closure for the skin lotion container (10%). As a result, closure type plays an important role in protecting poorly preserved products from in-use microbial contamination.


Healthy Public Policy Options to Promote Physical Activity for Chronic Disease Prevention: Understanding Canadian Policy Influencer and General Public Preferences.
Attitudes and beliefs of policy influencers and the general public toward physical activity policy may support or impede population-level action, requiring improved understanding of aggregate preferences toward policies that promote physical activity. In 2016, the Chronic Disease Prevention Survey was administered to a census sample of policy influencers (n = 302) and a stratified random sample of the public (n = 2400) in Alberta and Québec. Using net favorable percentages and the Nuffield Council on Bioethics' intervention ladder framework to guide analysis, the authors examined support for evidence-based healthy public policies to increase physical activity levels. Less intrusive policy options (ie, policies that are not always the most impactful) tended to have higher levels of support than policies that eliminated choice. However, there was support for certain types of policies affecting influential determinants of physical activity such as the built environment (ie, provided they enabled rather than restricted choice) and school settings (ie, focusing on children and youth). Overall, the general public indicated stronger levels of support for more physical activity policy options than policy influencers. The authors' findings may be useful for health advocates in identifying support for evidence-based healthy public policies affecting more influential determinants of physical activity.


Application of the matched nested case-control design to the secondary analysis of trial data.
A nested case-control study is an efficient design that can be embedded within an existing cohort study or randomised trial. It has a number of advantages compared to the conventional case-control design, and has the potential to answer important research questions using untapped prospectively collected data. We demonstrate the utility of the matched nested case-control design by applying it to a secondary analysis of the Abnormal Doppler Enteral Prescription Trial. We investigated the role of milk feed type and changes in milk feed type in the development of necrotising enterocolitis in a group of 398 high risk growth-restricted preterm infants. Using matching, we were able to generate a comparable sample of controls selected from the same population as the cases. In contrast to the standard case-control design, exposure status was ascertained prior to the outcome event occurring and the comparison between the cases and matched controls could be made at the point at which the event occurred. This enabled us to reliably investigate the temporal relationship between feed type and necrotising enterocolitis. A matched nested case-control study can be used to identify credible associations in a secondary analysis of clinical trial data where the exposure of interest was not randomised, and has several advantages over a standard case-control design. This method offers the potential to make reliable inferences in scenarios where it would be unethical or impractical to perform a randomised clinical trial.


Characterization of the Microbiome at the World's Largest Potable Water Reuse Facility.
Conventional water resources are not sufficient in many regions to meet the needs of growing populations. Due to cyclical weather cycles, drought, and climate change, water stress has increased worldwide including in Southern California, which serves as a model for regions that integrate reuse of wastewater for both potable and non-potable use. The Orange County Water District (OCWD) Advanced Water Purification Facility (AWPF) is a highly engineered system designed to treat and produce up to 100 million gallons per day (MGD) of purified water from a municipal wastewater source for potable reuse. Routine facility microbial water quality analysis is limited to standard indicators at this and similar facilities. Given recent advances in high throughput DNA sequencing techniques, complete microbial profiling of communities in water samples is now possible. By using 16S/18S rRNA gene sequencing, metagenomic and metatranscriptomic sequencing coupled to a highly accurate identification method along with 16S rRNA gene qPCR, we describe a detailed view of the total microbial community throughout the facility. The total bacterial load of the water at stages of the treatment train ranged from 3.02 × 106 copies in source, unchlorinated wastewater feed to 5.49 × 101 copies of 16S rRNA gene/mL after treatment (consisting of microfiltration, reverse osmosis, and ultraviolet/advanced oxidation). Microbial diversity and load decreased by several orders of magnitude after microfiltration and reverse osmosis treatment, falling to almost non-detectable levels that more closely resembled controls of molecular grade laboratory water than the biomass detected in the source water. The presence of antibiotic resistance genes and viruses was also greatly reduced. Overall, system design performance was achieved, and comprehensive microbial community analysis was found to enable a more complete characterization of the water/wastewater microbial signature.


Parental comprehension and satisfaction in informed consent in paediatric clinical trials: a prospective study on childhood leukaemia.
To evaluate the extent to which parents are satisfied with and understand the information they are given when their consent is sought for their child to participate in a phase III randomised clinical trial and the reasons for their decision. The authors carried out a prospective study. The authors included all parents whose consent was sought for their child to participate in the FRALLE 2000A protocol (acute lymphoblastic leukaemia) at two centres. The parents were questioned twice by a qualified psychologist using a semidirected interview, 1 and 6 months after consent was sought. 43 first interviews were carried out. All the parents declared they were satisfied with the explanations provided by the physician. 35 (81%) parents felt that the information provided with the request for consent was appropriate. Eight (19%) parents did not realise that their child had been included in a research protocol. 16 (39%) parents did not understand the concept of randomisation. Half the parents could explain neither the aim of the clinical trial nor the potential benefit of inclusion to their child. Only one third of the parents were aware that they had an alternative. The principal factor underlying their decision, as stated by 29 parents (67%), was confidence in the medical team. The parents signed consent forms without having fully understood all the elements specific to the experimental protocol. Rather, the parents based their decision on their confidence in the medical team, even when their child's life was at risk.


[Health and disease among Panará (Kreen-Akarôre) Indians in Central Brazil after twenty-five years of contact with our world, with an emphasis on tuberculosis].
The Panará, who had previously lived in isolation from Brazilian national society in the Amazon forest, were first contacted in 1973. Two years later they were moved to another area in Central Brazil. During this same period they were reduced to 82 members, the survivors of a population of 400 to 500 in the mid-1960s. In 1995 they returned to a small area in their old territory still not occupied by outsiders. There, three years later, a health survey showed a presumed diagnosis of tuberculosis in 15 individuals out of a population of 181. Further tests in the town of Colider, based on clinical data and chest X-rays, confirmed the diagnosis in 10 Panará (6 children under 10 years of age and 4 adults from 40 to 50 years old). BCG scars were present in the entire population. The nutritional status of Panará children was better than that of other indigenous groups in the Amazon region. The following measures were introduced for Tb control: a) treatment follow-up in the village, under direct supervision by both a nurse and the local indigenous health worker; b) compliance with defined criteria for ending treatment; c) periodic control of contacts and non-contacts; c) and establishment of a reference system with the health services in Colider.


Luteinizing hormone stimulates the formation of inositol trisphosphate and cyclic AMP in rat granulosa cells. Evidence for phospholipase C generated second messengers in the action of luteinizing hormone.
The following studies were conducted to determine whether luteinizing hormone (LH), a hormone which increases cellular levels of cyclic AMP, also provokes increases in 'second messengers' derived from inositol lipid metabolism (i.e. inositol phosphates and diacylglycerol). Rat granulosa cells isolated from mature Graafian follicles were prelabelled for 3 h with myo-[2-3H]inositol. LH provoked rapid (5 min) and sustained (up to 60 min) increases in the levels of inositol mono-, bis, and trisphosphates (IP, IP2 and IP3, respectively). Time course studies revealed that IP3 was formed more rapidly than IP2 and IP following LH treatment. The response to LH was concentration-dependent with maximal increases at LH concentrations of 1 microgram/ml. LiCl (2-40 mM) enhanced the LH-provoked accumulation of all [3H]inositol phosphates, presumably by inhibiting the action of inositol phosphate phosphatases. The effectiveness of LH, however, was dependent on the concentration of lithium employed; maximal increases in IP were observed at 10 mM-LiCl, whereas maximal increases in IP2 and IP3 were observed at 20 mM- and 40 mM-LiCl, respectively. The stimulatory effects of LH on inositol phosphate and progesterone accumulation were also compared with changes in cyclic nucleotide levels. LH rapidly increased levels of inositol phosphates, progesterone and cyclic AMP, but transiently reduced levels of cyclic GMP. These results demonstrate that LH increases both cyclic AMP and inositol trisphosphate (and presumably diacylglycerol) in rat granulosa cells. Our findings suggest that two messenger systems exist to mediate the action of LH in granulosa cells.


Arginase-II negatively regulates renal aquaporin-2 and water reabsorption.
Type-II l-arginine:ureahydrolase, arginase-II (Arg-II), is abundantly expressed in the kidney. The physiologic role played by Arg-II in the kidney remains unknown. Herein, we report that in mice that are deficient in Arg-II (Arg-II-/-), total and membrane-associated aquaporin-2 (AQP2) protein levels were significantly higher compared with wild-type (WT) controls. Water deprivation enhanced Arg-II expression, AQP2 levels, and membrane association in collecting ducts. Effects of water deprivation on AQP2 were stronger in Arg-II-/- mice than in WT mice. Accordingly, a decrease in urine volume and an increase in urine osmolality under water deprivation were more pronounced in Arg-II-/- mice than in WT mice, which correlated with a weaker increase in plasma osmolality in Arg-II-/- mice. There was no difference in vasopressin release under water deprivation conditions between either genotype of mice. Although total AQP2 and phosphorylated AQP2-S256 levels (mediated by PKA) in kidneys under water deprivation conditions were significantly higher in Arg-II-/- mice compared with WT animals, there is no difference in the ratio of AQP2-S256:AQP2. In cultured mouse collecting duct principal mCCDcl1 cells, expression of both Arg-II and AQP2 were enhanced by the vasopressin type 2 receptor agonist, desamino- d-arginine vasopressin (dDAVP). Silencing Arg-II enhanced the expression and membrane association of AQP2 by dDAVP without influencing cAMP levels. In conclusion, in vivo and in vitro experiments demonstrate that Arg-II negatively regulates AQP2 and the urine-concentrating capability in kidneys via a mechanism that is not associated with the modulation of the cAMP pathway.-Huang, J., Montani, J.-P., Verrey, F., Feraille, E., Ming, X.-F., Yang, Z. Arginase-II negatively regulates renal aquaporin-2 and water reabsorption.


The relationship between asthma, asthma control and economic outcomes in the United States.
Asthma, a serious chronic lung disease affecting approximately 26 million Americans, remains clinical and economic burdens on the healthcare system. Although associations between uncontrolled asthma and poor health outcomes is known, the extent of this impact of uncontrolled asthma on economic outcomes in the United States (US) is unknown. We sought to determine the relationship between asthma, asthma control and economic outcomes in the US. The 2008-2010 Medical Expenditure Panel Surveys were used to estimate the impact of uncontrolled asthma (asthma-related emergency department [ED] visit, use of >3 canisters of quick-relief inhaler in past 3 months or asthma attack in past 12 months) on medical expenditures, utilization and productivity. Estimates were generated using multivariate regression controlling for sociodemographics and comorbidity. Medical expenditures attributable to asthma were up to $4423 greater for those with markers of uncontrolled asthma compared with those who did not have asthma. Frequency of hospital discharges were up to 4.6-fold greater for those with uncontrolled asthma than those without asthma (p < 0.01), while all others with asthma did not have significantly more discharges. ED visits were up to 1.8-fold greater for those with uncontrolled asthma compared with those without asthma (p < 0.01). Productivity was significantly (p < 0.01) decreased (more likely to be unemployed, more days absent from work and more activity limitations) for those with uncontrolled asthma. In recent national data, individuals with asthma and markers of uncontrolled asthma had higher medical expenditures, greater utilization and decreased productivity.


The kallikrein-kinin system in blood pressure homeostasis.
1. The acute effects of the kallikrein inhibitor aprotinin (498 ki.u./min), and the kininase II inhibitor SQ 14,225 (250 MICROGRAM), GIVEN INTRAVEnously during saralasin-induced angiotensin blockade, were studied in conscious sham-operated rats and rats with benign and malignant two-kidney, one-clip Goldblatt hypertension during dietary sodium restriction. 2. The blood pressure of conscious sham-operated rats increased significantly in response to aprotinin. It remained unchanged after SQ 14,225 in contrast to the significant vasodepressor effect seen when SQ 14,225 was given to the same rats under surgical stress and pentobarbital anaesthesia. 3. Benignly hypertensive rats showed a consistent vasopressor response to aprotinin and a marked vasodepressor response to SQ 14,225. The effects of both inhibitors were markedly and significantly blunted in malignantly hypertensive rats. 4. Our demonstration that two agents with known opposite actions on the kallikrein-kinin system produced predictable and opposite effects on blood pressure may indicate that this system is involved in the homeostatic regulation of blood pressure. It may play an important antihypertensive role in benign two-kidney, one-clip Goldblatt hypertension, a role which might be impaired in malignant hypertension.


A mortality study of employees of the nuclear industry in Oak Ridge, Tennessee.
An analysis was conducted of 27,982 deaths among 106,020 persons employed at four Federal nuclear plants in Oak Ridge, Tennessee, between 1943 and 1985. The main objectives were to extend the evaluation of the health effects of employment in the nuclear industry in Oak Ridge to include most workers who were omitted from earlier studies, to compare the mortality experience of workers among the facilities, to address methodological problems that occur when individuals employed at more than one facility are included in the analysis, and to conduct dose-response analyses for those individuals with potential exposure to external radiation. All-cause mortality and all-cancer mortality were in close agreement with national rates. The only notable excesses occurred for white males for lung cancer [standardized mortality ratio (SMR) = 1.18, 1,849 deaths] and non-malignant respiratory disease (SMR = 1.12, 1,568 deaths). A more detailed analysis revealed substantial differences in death rates among workers at the Oak Ridge plants. Evaluation of internally adjusted log SMRs using Poisson regression showed that workers employed only at Tennessee Eastman Corporation or K-25 and at multiple facilities had higher death rates than similar workers employed only at X-10 or Y-12, and that the differences were primarily due to non-cancer causes. Analysis of selected cancer causes for white males indicated large differences among the workers at the different facilities for lung cancer, leukemia and other lymphatic cancer. Dose-response analyses for external penetrating radiation were limited to a subcohort of 28,347 white males employed at X-10 or Y-12. Their collective recorded dose equivalent was 376 Sv. There was a strong "healthy worker effect" in this subcohort-all-cause SMR = 0.80 (4,786 deaths) and all-cancer SMR = 0.87 (1,134 deaths). Variables included in the analyses were age, birth cohort, a measure of socioeconomic status, length of employment, internal radiation exposure potential and facility. For external radiation dose with a 10-year lag, the excess relative risk was 0.31 per Sv (95% CI = -0.16, 1.01) for all causes and 1.45 per Sv (95% CI = 0.15, 3.48) for all cancer. The estimated excess relative risk for leukemia was negative but imprecisely determined. A preliminary dose adjustment procedure was developed to compensate for missing dose but not other dosimetry errors. Results of the analyses using the adjusted doses suggest that the effect of missing dose is an upward bias in dose-response coefficients and test statistics.


Genomic organization, promoter region analysis, and chromosome localization of the mouse bcl-x gene.
The bcl-x gene, a bcl-2 family member, is highly regulated during lymphoid development, and its expression modulates apoptosis in lymphoid and other cell populations. Several forms of bcl-x mRNAs with different biologic functions have been described in rodents and humans. In this study, we have determined the organization and promoter region of the mouse bcl-x gene in an effort to understand the molecular basis for the different bcl-x mRNA species identified in tissues. We show that mouse bcl-x maps to the distal mouse chromosome 2 at approximately 89 cM, and exhibits a three-exon structure with an untranslated first exon and a facultative first intron. The coding region of bcl-xL is generated by the juncture of exons II and III through a splicing reaction, whereas bcl-xS is generated by an alternatively utilized donor splice site located within exon II. Analysis of multiple cDNAs and primer extension experiments revealed major transcription initiation sites in brain and thymus within a GC-rich region, with multiple Sp1-binding motifs located upstream of exon I. Another promoter was mapped to a 57-bp region localized upstream of the translation initiation codon by transfection of reporter constructs into FL5.12 and K562 cell lines. The remarkable similarity between the genomic regions of bcl-2 and bcl-x suggests that these genes have evolved from a common ancestral gene or through gene duplication.


The effect of interferon-gamma, interleukin-4 and immunoglobulin receptor cross-linking of monocytes on allergen-specific T-cell response.
Modulation of the proliferative responses of an allergen-specific human Th2 cell line by cytokine-treated monocytes was examined. The response of this cell line to the specific allergen, Amb a V (from short ragweed pollen), increased following the addition of interleukin-1 beta (IL-1 beta). However, in the presence of exogenous interferon-gamma (IFN-gamma), there was greater than 40% reduction in the responsiveness of these T cells. The addition of IL-1 beta did not reverse the inhibitory effect of IFN-gamma. To determine the primary target cell type for IFN-gamma, autologous monocytes were pretreated with IL-4, IFN-gamma, or medium alone, and used as antigen-presenting cells (APC). We showed that the responses of T cells to Amb a V were significantly down-regulated in the presence of autologous monocytes pretreated with IFN-gamma, but not for monocytes pretreated with IL-4. Similar inhibitory effect of IFN-gamma was confirmed using a human T-cell line specific for a ragweed allergen, Amb a I, and a human T-cell clone raised against ragweed extract. Cross-linking of CD23 (Fc epsilon RII) on monocytes pretreated with IFN-gamma increased this inhibitory effect in an additive fashion, but, in the absence of IFN-gamma treatment, such cross-linking had no effect. These inhibitory effects were not due to alterations in the surface expression of HLA-DR on the monocytes, and the addition of exogenous IL-1 beta was unable to reverse these effects. In similar experiments, cross-linking of CD64 (Fc gamma R) on monocytes showed no significant effects. In conclusion, IFN-gamma is important in regulating the function of monocytes involved in Th2 cell responses to allergens. IL-4 treatment, as well as cross-linking of FcR of monocytes, have no direct effect on such response.