Plasma very long chain fatty acids in 3,000 peroxisome disease patients and 29,000 controls.
The assay of plasma very long chain fatty acids (VLCFAs), developed in our laboratory in 1981, has become the most widely used procedure for the diagnosis of X-linked adrenoleukodystrophy (X-ALD) and other peroxisomal disorders. We present here our 17 years' experience with this assay. Three VLCFA parameters, the level of hexacosanoic acid (C26:0), the ratio of C26:0 to tetracosanoic acid (C24:0), and of C26:0 to docosanoic acid (C22:0), were measured in 1,097 males (hemizygotes) with X-ALD, 1,282 women heterozygous for this disorder, including 379 obligate heterozygotes, 797 patients with other peroxisomal disorders, and 29,600 control subjects. All X-ALD hemizygotes who had not previously received Lorenzo's oil or a diet with a high erucic acid content had increased VLCFA levels, but the application of a discriminant function based on all three measurements is required to avoid the serious consequences of a false-negative result. VLCFA levels are increased at day of birth, thus providing the potential for neonatal mass screening, are identical in the childhood and adult forms, and do not change with age. Eighty-five percent of obligate heterozygotes had abnormally high VLCFA levels, but a normal result does not exclude carrier status. VLCFA levels were increased in all patients homozygous for Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum's disease, and in patients with deficiencies of peroxisomal acyl-coenzyme A oxidase, bifunctional enzyme, and 3-oxoacyl-coenzyme A thiolase. In these patients the degree of VLCFA excess correlated with clinical severity.


High working memory capacity attenuates the deviation effect but not the changing-state effect: further support for the duplex-mechanism account of auditory distraction.
Serial short-term memory is impaired by background sound, at least when a sound element suddenly deviates from an otherwise repetitive sequence (the deviation effect) and when each sound element in the sequence differs from the preceding one (the changing-state effect). Two competing theories have been proposed to explain these effects: One suggests that both effects are caused by the same mechanism (i.e., attentional resources being depleted by the sound), and the other suggests that the deviation effect is caused by attentional capture and that the changing-state effect is caused by interference between order processes. The present investigation found that working memory capacity predicts susceptibility to the deviation effect, but not to the changing-state effect, both when speech items (Experiment 1) and when tones (Experiment 2) produce the disruption. These results suggest that the two effects are caused by different mechanisms and support the duplex-mechanism account of auditory distraction.


Association of coronary microvascular endothelial dysfunction with vulnerable plaque characteristics in early coronary atherosclerosis.
The aim of this study was to test the hypothesis that coronary microvascular endothelial dysfunction (CMED) is associated with epicardial coronary atherosclerosis. We performed a cross-sectional analysis of a comprehensive invasive assessment of coronary physiology with a focus on endothelium-dependent coronary microvascular function and virtual-histology intravascular ultrasound (VH-IVUS) in a total of 148 consecutive patients with chest pain and angiographically normal coronary arteries or non-obstructive coronary artery disease (CAD). Endothelium-dependent coronary vascular reactivity was evaluated by graded doses of intracoronary acetylcholine (ACh). CMED was defined as a percent increase in coronary blood flow of ≤50% in response to ACh. Patients with CMED (n=87) showed more vulnerable plaque characteristics as compared to those without (n=61); they showed higher plaque burden in association with larger necrotic core volume and higher frequency of imaged arteries containing at least one VH-IVUS-derived thin-capped fibroatheroma (TCFA) (n=22 [25.3%] vs 5 [8.2%], p=0.008). Multivariate logistic regression analysis revealed that CMED was an independent predictor of VH-IVUS-derived TCFA (adjusted odds ratio 2.28 [95% confidence interval: 1.30-4.02], p=0.004). Independently of conventional coronary risk factors, CMED was associated with vulnerable plaque characteristics in patients with non-obstructive CAD.


Smac3, a novel Smac/DIABLO splicing variant, attenuates the stability and apoptosis-inhibiting activity of X-linked inhibitor of apoptosis protein.
X-linked inhibitor of apoptosis protein (XIAP), the most potent member of the inhibitor of apoptosis protein (IAP) family, plays a crucial role in the regulation of apoptosis. XIAP is structurally characterized by three baculovirus IAP repeat (BIR) domains that mediate binding to and inhibition of caspases and a RING domain that confers ubiquitin ligase activity. The caspase inhibitory activity of XIAP can be eliminated by the second mitochondria-derived activator of caspases (Smac)/direct IAP-binding protein with low pI (DIABLO) during apoptosis. Here we report the identification and characterization of a novel isoform of Smac/DIABLO named Smac3, which is generated by alternative splicing of exon 4. Smac3 contains an NH2-terminal mitochondrial targeting sequence required for mitochondrial targeting of Smac3 and an IAP-binding motif essential for Smac3 binding to XIAP. Smac3 is released from mitochondria into the cytosol in response to apoptotic stimuli, where it interacts with the second and third BIR domains of XIAP. Smac3 disrupts processed caspase-9 binding to XIAP, promotes caspase-3 activation, and potentiates apoptosis. Strikingly, Smac3, but not Smac/DIABLO, accelerates XIAP auto-ubiquitination and destruction. Smac3-stimulated XIAP ubiquitination is contingent upon the physical association of XIAP with Smac3 and an intact RING domain of XIAP. Smac3-accelerated XIAP destabilization is, at least in part, attributed to its ability to enhance XIAP ubiquitination. Our study demonstrates that Smac3 is functionally additive to, but independent of, Smac/DIABLO.


In vivo effect of interleukin-1beta and interleukin-1RA on oocyte cytoplasmic maturation, ovulation, and early embryonic development in the mare.
A growing body of evidence suggests that the interleukin-1 system is involved in periovulatory events. Previous work from our lab demonstrated that in the mare, interleukin-1beta (IL-1beta) increases the ovulatory rate of metaphase II oocytes. The present study was conducted to analyze in vivo the effect of IL-1 on oocyte cytoplasmic maturation, ovulation and pregnancy rate. In the present work, IL-1beta (experiment 1, n = 13; experiment 2, n = 25) and interleukin-1RA (IL-1RA; experiment 1, n = 25) were injected intrafollicularly by using the transvaginal ultrasound-guided injection method. Injections were performed on cyclic mares when the diameter of the growing dominant follicle reached 30-34 mm. In experiment 1, mares were inseminated the day of the treatment and all the other day until ovulation. The time of ovulation was determined and a pregnancy diagnosis was performed 14 days after ovulation of the injected follicle. In experiment 2, the cumulus-oocyte complex from each injected follicle was collected by transvaginal ultrasound-guided aspiration 38 h after the intrafollicular injection. Oocyte nuclear stage and oocyte cytoplasmic maturation were assessed by analyzing chromatin configuration, cortical granules migration and mitochondria distribution under a confocal microscope. The results from experiment 1 confirm that an intrafollicular injection of 1 microgram IL-1beta induces ovulation in the mare whereas IL-1RA has no effect at the dose used in the present study. Furthemore, we demonstrated, that in our experimental conditions, IL-1beta and IL-1RA induced a decrease in embryo development. Experiment 2 leads us to observe that IL-1beta is unable to induce cortical granules migration and remodelling of mitochondria, that commonly occurs during oocyte maturation, whereas it acts on nuclear maturation. This result may explain the decrease in embryo development we observed after IL-1beta intrafollicular injection. In conclusion, the present study tends to demonstrate that IL-1beta plays a role in the ovulatory process and may acts on oocyte maturation in the mare, but additional factors are required to complete equine oocyte cytoplasmic maturation to allow embryo development.


Implications of small reductions in diastolic blood pressure for primary prevention.
To estimate the impact of small reductions in the population distribution of diastolic blood pressure (DBP), such as those potentially achievable by population-wide lifestyle modification, on incidence of coronary heart disease (CHD) and stroke. Published data from the Framingham Heart Study, a longitudinal cohort study, and from the National Health and Nutrition Examination Survey II, a national population survey, were used to examine the impact of a population-wide strategy aimed at reducing DBP by an average of 2 mm Hg in a population including normotensive subjects. White men and women aged 35 to 64 years in the United States. Incidence of CHD and stroke, including transient ischemic attacks (TIAs). Data from overviews of observational studies and randomized trials suggest that a 2-mm Hg reduction in DBP would result in a 17% decrease in the prevalence of hypertension as well as a 6% reduction in the risk of CHD and a 15% reduction in risk of stroke and TIAs. From an application of these results to US white men and women aged 35 to 64 years, it is estimated that a successful population intervention alone could reduce CHD incidence more than could medical treatment for all those with a DBP of 95 mm Hg or higher. It could prevent 84% of the number prevented by medical treatment for all those with a DBP of 90 mm Hg or higher. For stroke (including TIAs), a population-wide 2-mm Hg reduction could prevent 93% of events prevented by medical treatment for those with a DBP of 95 mm Hg or higher and 69% of events for treatment for those with a DBP of 90 mm Hg or higher. A combination strategy of both a population reduction in DBP and targeted medical intervention is most effective and could double or triple the impact of medical treatment alone. Adding a population-based intervention to existing levels of hypertension treatment could prevent an estimated additional 67,000 CHD events (6%) and 34,000 stroke and TIA events (13%) annually among all those aged 35 to 64 years in the United States. A small reduction of 2 mm Hg in DBP in the mean of the population distribution, in addition to medical treatment, could have a great public health impact on the number of CHD and stroke events prevented. Whether such DBP reductions can be achieved in the population through lifestyle interventions, in particular through sodium reduction, depends on the results of ongoing primary prevention trials as well as the cooperation of the food industry, government agencies, and health education professionals.


Implementation of surgical debriefing programs in large health systems: an exploratory qualitative analysis.
The role of the "debrief" to address issues related to patient safety and systematic flaws in care is frequently overlooked. In our study, we interview surgical leaders who have developed successful strategies of debriefing within a comprehensive program of quality improvement. Semi-structured interviews of four implementation leaders were performed. The observations, beliefs and strategies of surgical leaders are compared and contrasted. Common themes are identified related to program success and failure. Quality and safety researchers performed, coded and categorized the interviews and coordinated the analysis and interpretation of the results. The authors from the four institutions aided in interpretation and framing of the results. The debriefing programs evaluated were part of comprehensive quality improvement projects. Seven high-level themes and 24 subthemes were identified from the interviews. Themes related to leadership included early engagement, visible ongoing commitment and enforcement. Success appeared to depend upon meaningful and early debriefing feedback. The culture of safety that promoted success included a commitment to open and fair communication and continuous improvement. There were many challenges to the success of debriefing programs. The loss of institutional commitment of resources and personnel was the instigating factor behind the collapse of the program at Michigan. Other areas of potential failure included communication issues and loss of early and meaningful feedback. Leaders of four surgical systems with strong debriefing programs report success using debriefing to improve system performance. These findings are consistent with previously published studies. Success requires commitment of resources, and leadership engagement. The greatest gains may be best achieved by programs that provide meaningful debriefing feedback in an atmosphere dedicated to open communication.


St. Louis encephalitis virus possibly transmitted through blood transfusion-Arizona, 2015.
St. Louis encephalitis virus is a mosquito-borne flavivirus that infrequently causes epidemic central nervous system infections. In the United States, blood donors are not screened for St. Louis encephalitis virus infection, and transmission through blood transfusion has not been reported. During September 2015, St. Louis encephalitis virus infection was confirmed in an Arizona kidney transplant recipient. An investigation was initiated to determine the infection source. The patient was interviewed, and medical records were reviewed. To determine the likelihood of mosquito-borne infection, mosquito surveillance data collected at patient and blood donor residences in timeframes consistent with their possible exposure periods were reviewed. To investigate other routes of exposure, organ and blood donor and recipient specimens were obtained and tested for evidence of St. Louis encephalitis virus infection. The patient presented with symptoms of central nervous system infection. Recent St. Louis encephalitis virus infection was serologically confirmed. The organ donor and three other organ recipients showed no laboratory or clinical evidence of St. Louis encephalitis virus infection. Among four donors of blood products received by the patient via transfusion, one donor had a serologically confirmed, recent St. Louis encephalitis virus infection. Exposure to an infected mosquito was unlikely based on the patient's minimal outdoor exposure. In addition, no St. Louis encephalitis virus-infected mosquito pools were identified around the patient's residence. This investigation provides evidence of the first reported possible case of St. Louis encephalitis virus transmission through blood product transfusion. Health care providers and public health professionals should maintain heightened awareness for St. Louis encephalitis virus transmission through blood transfusion in settings where outbreaks are identified.


Safety and efficacy of Favipiravir in moderate to severe SARS-CoV-2 pneumonia.
We examined the safety and efficacy of a treatment protocol containing Favipiravir for the treatment of SARS-CoV-2. We did a multicenter randomized open-labeled clinical trial on moderate to severe cases infections of SARS-CoV-2. Patients with typical ground glass appearance on chest computerized tomography scan (CT scan) and oxygen saturation (SpO2) of less than 93% were enrolled. They were randomly allocated into Favipiravir (1.6 gr loading, 1.8 gr daily) and Lopinavir/Ritonavir (800/200 mg daily) treatment regimens in addition to standard care. In-hospital mortality, ICU admission, intubation, time to clinical recovery, changes in daily SpO2 after 5 min discontinuation of supplemental oxygen, and length of hospital stay were quantified and compared in the two groups. 380 patients were randomly allocated into Favipiravir (193) and Lopinavir/Ritonavir (187) groups in 13 centers. The number of deaths, intubations, and ICU admissions were not significantly different (26, 27, 31 and 21, 17, 25 respectively). Mean hospital stay was also not different (7.9 days [SD = 6] in the Favipiravir and 8.1 [SD = 6.5] days in Lopinavir/Ritonavir groups) (p = 0.61). Time to clinical recovery in the Favipiravir group was similar to Lopinavir/Ritonavir group (HR = 0.94, 95% CI 0.75 - 1.17) and likewise the changes in the daily SpO2 after discontinuation of supplemental oxygen (p = 0.46) CONCLUSION: Adding Favipiravir to the treatment protocol did not reduce the number of ICU admissions or intubations or In-hospital mortality compared to Lopinavir/Ritonavir regimen. It also did not shorten time to clinical recovery and length of hospital stay.


Predicting Self-Reported Illness for Professional Team-Sport Athletes.
To identify contributing factors to the incidence of illness for professional team-sport athletes, using training load (TL), self-reported illness, and well-being data. Thirty-two professional rugby league players (26.0 ± 4.8 y, 99.1 ± 9.6 kg, 1.84 ± 0.06 m) were recruited from the same club. Players participated in prescribed training and responded to a series of questionnaires to determine the presence of self-reported illness and markers of well-being. Internal TL was determined using the session rating of perceived exertion. These data were collected over 29 wk, across the preparatory and competition macrocycles. The predictive models developed recognized increases in internal TL (strain values of >2282 AU, weekly TL >2786 AU, and monotony >0.78 AU) to best predict when athletes are at increased risk of self-reported illness. In addition, a reduction in overall well-being (<7.25 AU) in the presence of increased internal TL, as previously stated, was highlighted as a contributor to self-reported-illness occurrence. These results indicate that self-report data can be successfully used to provide a novel understanding of the interactions between competition-associated stressors experienced by professional team-sport athletes and their susceptibility to illness. This may help coaching staff more effectively monitor players during the season and potentially implement preventive measures to reduce the likelihood of illnesses occurring.


Pre-Columbian mycobacterial genomes reveal seals as a source of New World human tuberculosis.
Modern strains of Mycobacterium tuberculosis from the Americas are closely related to those from Europe, supporting the assumption that human tuberculosis was introduced post-contact. This notion, however, is incompatible with archaeological evidence of pre-contact tuberculosis in the New World. Comparative genomics of modern isolates suggests that M. tuberculosis attained its worldwide distribution following human dispersals out of Africa during the Pleistocene epoch, although this has yet to be confirmed with ancient calibration points. Here we present three 1,000-year-old mycobacterial genomes from Peruvian human skeletons, revealing that a member of the M. tuberculosis complex caused human disease before contact. The ancient strains are distinct from known human-adapted forms and are most closely related to those adapted to seals and sea lions. Two independent dating approaches suggest a most recent common ancestor for the M. tuberculosis complex less than 6,000 years ago, which supports a Holocene dispersal of the disease. Our results implicate sea mammals as having played a role in transmitting the disease to humans across the ocean.


Surface charge tunable catanionic vesicles based on serine-derived surfactants as efficient nanocarriers for the delivery of the anticancer drug doxorubicin.
Self-assembled vesicles composed of amino acid-based cationic/anionic surfactant mixtures show promise as novel effective drug nanocarriers. Here, we report the in vitro performance of vesicles based on cationic (16Ser) and anionic (8-8Ser) serine-based surfactants using a cancer cell model for the delivery of the anticancer drug doxorubicin (DOX). This catanionic mixture yields both negatively (0.20 in the cationic surfactant molar fraction, x16Ser) and positively (x16Ser = 0.58) charged vesicles, hence providing a surface charge tunable system. Low toxicity is confirmed for concentration ranges below 32 μM in both formulations. DOX is successfully encapsulated in the vesicles, resulting in a surface charge switch to negative for the (0.58) system, making both (0.20) and (0.58) DOX-loaded vesicles highly interesting for systemic administration. High uptake by cells was demonstrated using flow cytometry and confocal microscopy. Drug accumulation results in an increase of cell uptake up to 250% and 200% for the (0.20) and (0.58) vesicles, respectively, compared to free DOX and with localizations near the nuclear regions in the cells. The in vitro cytotoxicity studies show that DOX-loaded vesicles induce cell death, confirming the therapeutic potential of the formulations. Furthermore, the efficient accumulation of the drug inside the cell compartments harbors the potential for optimization strategies including phased delivery for prolonged treatment periods or even on-demand release.


The Role of Hydrogen on the Behavior of Intergranular Cracks in Bicrystalline α-Fe Nanowires.
Hydrogen embrittlement (HE) has been extensively studied in bulk materials. However, little is known about the role of H on the plastic deformation and fracture mechanisms of nanoscale materials such as nanowires. In this study, molecular dynamics simulations are employed to study the influence of H segregation on the behavior of intergranular cracks in bicrystalline α-Fe nanowires. The results demonstrate that segregated H atoms have weak embrittling effects on the predicted ductile cracks along the GBs, but favor the cleavage process of intergranular cracks in the theoretically brittle directions. Furthermore, it is revealed that cyclic loading can promote the H accumulation into the GB region ahead of the crack tip and overcome crack trapping, thus inducing a ductile-to-brittle transformation. This information will deepen our understanding on the experimentally-observed H-assisted brittle cleavage failure and have implications for designing new nanocrystalline materials with high resistance to HE.


Variation in the ICAM1 gene is not associated with severe malaria phenotypes.
Evidence from autopsy and in vitro binding studies suggests that adhesion of erythrocytes infected with Plasmodium falciparum to the human host intercellular adhesion molecule (ICAM)-1 receptor is important in the pathogenesis of severe malaria. Previous association studies between polymorphisms in the ICAM1 gene and susceptibility to severe malarial phenotypes have been inconclusive and often contradictory. We performed genetic association studies with 15 single nucleotide polymorphisms (SNPs) around the ICAM1 locus. All SNPs were screened in a family study of 1071 trios from The Gambia, Malawi and Kenya. Two key non-synonymous SNPs with previously reported associations, rs5491 (K56M or 'ICAM-1(Kilifi)') and rs5498 (K469E), were tested in an additional 708 Gambian trios and a case-control study of 4058 individuals. None of the polymorphisms were associated with severe malaria phenotypes. Pooled results across our studies for ICAM-1(Kilifi) were, in severe malaria, odds ratio (OR) 1.02, 95% confidence interval (CI) 0.96-1.09, P=0.54, and cerebral malaria OR 1.07, CI 0.97-1.17, P=0.17. We assess the available epidemiological, population genetic and functional evidence that links ICAM-1(Kilifi) to severe malaria susceptibility.


Genetic structure of four plasmids found in Acinetobacter baumannii isolate D36 belonging to lineage 2 of global clone 1.
Four plasmids ranging in size from 4.7 to 44.7 kb found in the extensively antibiotic resistant Acinetobacter baumannii isolate D36 that belongs to lineage 2 of global clone 1 were examined. D36 includes two cryptic plasmids and two carrying antibiotic resistance genes. The smallest plasmid pD36-1 (4.7 kb) carries no resistance genes but includes mobA and mobC mobilisation genes related to those found in pRAY* (pD36-2, 6,078 bp) that also carries the aadB gentamicin, kanamycin and tobramycin resistance gene cassette. These two plasmids do not encode a Rep protein. Plasmid pRAY* was found to be mobilised at high frequency by the large conjugative plasmid pA297-3 but a pRAY* derivative lacking the mobA and mobC genes was not. The two larger plasmids, pD36-3 and pD36-4, encode Rep_3 family proteins (Pfam1051). The cryptic plasmid pD36-3 (6.2 kb) has RepAci1 and pD36-4 (44.7 kb) encodes two novel Rep_3 family proteins suggesting a co-integrate. Plasmid pD36-4 includes the sul2 sulfonamide resistance gene, the aphA1a kanamycin/neomycin resistance gene in Tn4352::ISAba1 and a mer module in a hybrid Tn501/Tn1696 transposon conferring resistance to mercuric ions. New examples of dif modules flanked by pdif sites (XerC-XerD binding sites) that are part of many A. baumannii plasmids were also identified in pD36-3 and pD36-4 which carry three and two dif modules, respectively. Homologs of three dif modules, the sup sulphate permease module in pD36-3, and of the abkAB toxin-antitoxin module and the orf module in pD36-4, were found in different contexts in diverse Acinetobacter plasmids, consistent with module mobility. A novel insertion sequence named ISAba32 found next to the pdif site in the abkAB dif module is related to members of the ISAjo2 group which also are associated with the pdif sites of dif modules. Plasmids found in D36 were also found in some other members of GC1 lineage 2.


Biofilm Formation As a Response to Ecological Competition.
Bacteria form dense surface-associated communities known as biofilms that are central to their persistence and how they affect us. Biofilm formation is commonly viewed as a cooperative enterprise, where strains and species work together for a common goal. Here we explore an alternative model: biofilm formation is a response to ecological competition. We co-cultured a diverse collection of natural isolates of the opportunistic pathogen Pseudomonas aeruginosa and studied the effect on biofilm formation. We show that strain mixing reliably increases biofilm formation compared to unmixed conditions. Importantly, strain mixing leads to strong competition: one strain dominates and largely excludes the other from the biofilm. Furthermore, we show that pyocins, narrow-spectrum antibiotics made by other P. aeruginosa strains, can stimulate biofilm formation by increasing the attachment of cells. Side-by-side comparisons using microfluidic assays suggest that the increase in biofilm occurs due to a general response to cellular damage: a comparable biofilm response occurs for pyocins that disrupt membranes as for commercial antibiotics that damage DNA, inhibit protein synthesis or transcription. Our data show that bacteria increase biofilm formation in response to ecological competition that is detected by antibiotic stress. This is inconsistent with the idea that sub-lethal concentrations of antibiotics are cooperative signals that coordinate microbial communities, as is often concluded. Instead, our work is consistent with competition sensing where low-levels of antibiotics are used to detect and respond to the competing genotypes that produce them.


Implementation and evaluation of telehealth tools and technologies.
In June 2009, the National Center for Research Resources (NCRR), National Institutes of Health (NIH), convened a conference of experts to discuss future directions for research in addressing healthcare disparities through the use of telehealth technologies. As part of this conference, a panel was convened to review the status of current efforts to assess, implement, and evaluate telehealth technologies, and to recommend future directions for research. The panel members provided a series of practical recommendations to those who are contemplating establishing a telehealth service, as well as recommendations to the NIH on future funding for telehealth research. The recommendations to the NIH focused on three broad areas of concern: (1) technology assessment, (2) evaluation, and (3) technical assistance, education, and dissemination. The panel members emphasized the need for NIH to support research in areas that have been seriously underfunded in the past, including but not limited to primary care research, multisite collaborative telehealth studies, nonphysician telehealth services, and methodological development to develop a "gold standard" for telehealth studies.


High treatment success in children treated for multidrug-resistant tuberculosis: an observational cohort study.
Few studies have described the management of multidrug-resistant (MDR) tuberculosis (TB) in children and evidence-based guidance on management is lacking. We describe the presentation, treatment and outcome in children treated for severe and non-severe MDR-TB in Cape Town, South Africa. We conducted an observational cohort study of all children (<15 years) treated for MDR-TB if routinely initiated on treatment between January 2009 and December 2010. Treatment was based on local standard of care, based on international guidelines. Data were collected through family interviews and folder review. Standardised definitions were used for diagnosis, severity of disease, adverse events and outcome. Of 149 children started on MDR-TB treatment, the median age was 36 months (IQR 16-66), 32 (22%; of 146 tested) had HIV infection and 59 (40%) had a confirmed diagnosis. Ninety-four (66%) children were treated with an injectable drug and the median total duration of treatment was 13 months (IQR 11-18). Thirty-six (24%) children were cured, 101 (68%) probably cured, 1 (1%) was transferred out, 8 (5%) were lost to follow-up and 3 (2%) died. Children with severe disease were older (54 months (IQR 18-142) vs 31.5 months (IQR 17.5-53.5); p=0.012), more commonly had HIV infection (OR 6.25; 95% CI 2.50 to 15.6; p<0.001) and were more likely to die (p=0.008). A confirmed diagnosis of MDR-TB is not possible in all cases but this should not impede the treatment of MDR-TB in children. More than 90% of children with MDR-TB can be successfully treated. Non-severe disease could be successfully treated with reduced treatment duration.


Absorption linear dichroism measured directly on a single light-harvesting system: the role of disorder in chlorosomes of green photosynthetic bacteria.
Chlorosomes are light-harvesting antennae of photosynthetic bacteria containing large numbers of self-aggregated bacteriochlorophyll (BChl) molecules. They have developed unique photophysical properties that enable them to absorb light and transfer the excitation energy with very high efficiency. However, the molecular-level organization, that produces the photophysical properties of BChl molecules in the aggregates, is still not fully understood. One of the reasons is heterogeneity in the chlorosome structure which gives rise to a hierarchy of structural and energy disorder. In this report, we for the first time directly measure absorption linear dichroism (LD) on individual, isolated chlorosomes. Together with fluorescence-detected three-dimensional LD, these experiments reveal a large amount of disorder on the single-chlorosome level in the form of distributions of LD observables in chlorosomes from wild-type bacterium Chlorobaculum tepidum . Fluorescence spectral parameters, such as peak wavelength and bandwidth, are measures of the aggregate excitonic properties. These parameters obtained on individual chlorosomes are uncorrelated with the observed LD distributions and indicate that the observed disorder is due to inner structural disorder along the chlorosome long axis. The excitonic disorder that is also present is not manifested in the LD distributions. Limiting values of the LD parameter distributions, which are relatively free of the effect of structural disorder, define a range of angles at which the excitonic dipole moment is oriented with respect to the surface of the two-dimensional aggregate of BChl molecules. Experiments on chlorosomes of a triple mutant of Chlorobaculum tepidum show that the mutant chlorosomes have significantly less inner structural disorder and higher symmetry, compatible with a model of well-ordered concentric cylinders. Different values of the transition dipole moment orientations are consistent with a different molecular level organization of BChl's in the mutant and wild-type chlorosomes.


Spectral response of low-loss optical waveguides.
The attainment of 20-dB/km attenuation in experimental single-mode glass optical waveguides has spurred interest in their use for optical communications. The primary wavelength region of interest is in the red or near-infrared region of the spectrum. In this work independent attenuation measurements from 600 nm to 1060 nm have been made on low-loss waveguides and bulk cladding glass, using both laser and scanning-prism monochromator sources. Three bands were observed in the waveguides, at 725 nm, 875 nm, and 950 nm, and identified as due to OH in the glass. Absorptions too small to be precisely measured in the bulk glass measured in them. are seen to be exceedingly important in the waveguides and easily measured in them.


Testosterone inhibits adipogenic differentiation in 3T3-L1 cells: nuclear translocation of androgen receptor complex with beta-catenin and T-cell factor 4 may bypass canonical Wnt signaling to down-regulate adipogenic transcription factors.
Testosterone supplementation in men decreases fat mass; however, the mechanisms by which it inhibits fat mass are unknown. We hypothesized that testosterone inhibits adipogenic differentiation of preadipocytes by activation of androgen receptor (AR)/beta-catenin interaction and subsequent translocation of this complex to the nucleus thereby bypassing canonical Wnt signaling. We tested this hypothesis in 3T3-L1 cells that differentiate to form fat cells in adipogenic medium. We found that these cells express AR and that testosterone and dihydrotestosterone dose-dependently inhibited adipogenic differentiation as analyzed by Oil Red O staining and down-regulation of CCAAT/enhancer binding protein-alpha and -delta and peroxisome proliferator-activated receptor-gamma2 protein and mRNA. These inhibitory effects of androgens were partially blocked by flutamide or bicalutamide. Androgen treatment was associated with nuclear translocation of beta-catenin and AR. Immunoprecipitation studies demonstrated association of beta-catenin with AR and T-cell factor 4 (TCF4) in the presence of androgens. Transfection of TCF4 cDNA inhibited adipogenic differentiation, whereas a dominant negative TCF4 cDNA construct induced adipogenesis and blocked testosterone's inhibitory effects. Our gene array analysis indicates that testosterone treatment led to activation of some Wnt target genes. Expression of constitutively activated AR fused with VP-16 did not inhibit the expression of CCAAT/enhancer binding protein-alpha in the absence of androgens. Testosterone and dihydrotestosterone inhibit adipocyte differentiation in vitro through an AR-mediated nuclear translocation of beta-catenin and activation of downstream Wnt signaling. These data provide evidence for a regulatory role for androgens in inhibiting adipogenic differentiation and a mechanistic explanation consistent with the observed reduction in fat mass in men treated with androgens.


Iturin levels on wheat spikes linked to biological control of Fusarium head blight by Bacillus amyloliquefaciens.
The TrigoCor strain of Bacillus amyloliquefaciens provides consistent control against Fusarium head blight of wheat in controlled settings but there is a lack of disease and deoxynivalenol suppression in field settings. Since production of antifungal compounds is thought to be the main mode of action of TrigoCor control, we quantified levels of a key family of antifungal metabolites, iturins, as well as monitored Bacillus populations on wheat spikes over 14 days post-application in both the greenhouse and the field. We found that initial iturin levels on spikes in the greenhouse were three times greater than on spikes in the field, but that by 3 days post-application, iturin levels were equivalent and very low in both settings. We also determined that iturins declined rapidly over a 3-day post-application period on wheat spikes in both environments, despite the presence of significant Bacillus populations. Greenhouse trials and antibiosis tests indicated that the lower iturin levels on wheat spikes in the field could be a major factor limiting disease control in field settings. Future efforts to improve Bacillus disease control on wheat spikes and in the phyllosphere of various plants should focus on maintaining higher levels of iturins over critical infection periods.


Unexplained pulmonary hypertension in elderly patients.
Idiopathic pulmonary arterial hypertension (IPAH) preferentially affects young women. However, a subset of patients with IPAH is elderly. Our objective was to compare elderly (age >/= 65 years) vs younger persons with unexplained pulmonary hypertension (PH) and a presumptive diagnosis of IPAH. Clinical, echocardiographic, hemodynamic, and survival data were collected on consecutive patients with suspected IPAH after evaluation in a large tertiary center PH clinic. Of 197 patients (mean age +/- SD, 52 +/- 16 years; 80% female), 48 patients (24%) were elderly. Elderly and younger patients had similar symptom severity, systolic pulmonary artery (PA) pressure (82.7 +/- 20.3 mm Hg vs 86.9 +/- 18.8 mm Hg, respectively; p = 0.21), and severity of right ventricular enlargement and dysfunction. Elderly patients had higher pulmonary capillary wedge pressure (PCWP) [15.3 +/- 7.3 mm Hg vs 11.1 +/- 5.3 mm Hg; p < 0.0001] and more frequently failed (56%) to meet hemodynamic criteria for IPAH (PH with PCWP < 15 mm Hg) than did younger patients (19%). Elderly patients also had higher systemic systolic (p < 0.0001) and pulse (p < 0.0001) pressures and more cardiovascular disease. Among those patients with normal PCWP, elderly patients had worse survival than young patients (p = 0.007). Among those patients with elevated PCWP, elderly patients had lower PA pressures (p = 0.04) and better survival (p = 0.02). Elderly patients with clinically suspected IPAH often fail to meet hemodynamic criteria for IPAH due to elevated PCWP. Studies to define the proper diagnostic strategy and the safety and efficacy of pulmonary vasodilators in elderly patients with unexplained PH are needed.


Bioinformatics challenges in the proteomic analysis of human plasma.
Mass spectrometry has become the method of choice for studying proteins in complex mixtures in a qualitative and quantitative fashion. The application of mass spectrometry-based proteomics analyses on plasma has correspondingly been established as an important method for disease-associated biomarker discovery and validation. Yet despite being a readily available human sample, plasma poses several important challenges to the proteomics researcher. With a focus on bioinformatics aspects, this chapter will discuss the problems involved in analyzing plasma proteomics data, along with the scope of solutions available through specialised tools and sophisticated analysis methods.


Characterization and biological role of the O-polysaccharide gene cluster of Yersinia enterocolitica serotype O:9.
Yersinia enterocolitica serotype O:9 is a gram-negative enteropathogen that infects animals and humans. The role of lipopolysaccharide (LPS) in Y. enterocolitica O:9 pathogenesis, however, remains unclear. The O:9 LPS consists of lipid A to which is linked the inner core oligosaccharide, serving as an attachment site for both the outer core (OC) hexasaccharide and the O-polysaccharide (OPS; a homopolymer of N-formylperosamine). In this work, we cloned the OPS gene cluster of O:9 and identified 12 genes organized into four operons upstream of the gnd gene. Ten genes were predicted to encode glycosyltransferases, the ATP-binding cassette polysaccharide translocators, or enzymes required for the biosynthesis of GDP-N-formylperosamine. The two remaining genes within the OPS gene cluster, galF and galU, were not ascribed a clear function in OPS biosynthesis; however, the latter gene appeared to be essential for O:9. The biological functions of O:9 OPS and OC were studied using isogenic mutants lacking one or both of these LPS parts. We showed that OPS and OC confer resistance to human complement and polymyxin B; the OPS effect on polymyxin B resistance could be observed only in the absence of OC.


Global increase and geographic convergence in antibiotic consumption between 2000 and 2015.
Tracking antibiotic consumption patterns over time and across countries could inform policies to optimize antibiotic prescribing and minimize antibiotic resistance, such as setting and enforcing per capita consumption targets or aiding investments in alternatives to antibiotics. In this study, we analyzed the trends and drivers of antibiotic consumption from 2000 to 2015 in 76 countries and projected total global antibiotic consumption through 2030. Between 2000 and 2015, antibiotic consumption, expressed in defined daily doses (DDD), increased 65% (21.1-34.8 billion DDDs), and the antibiotic consumption rate increased 39% (11.3-15.7 DDDs per 1,000 inhabitants per day). The increase was driven by low- and middle-income countries (LMICs), where rising consumption was correlated with gross domestic product per capita (GDPPC) growth (P = 0.004). In high-income countries (HICs), although overall consumption increased modestly, DDDs per 1,000 inhabitants per day fell 4%, and there was no correlation with GDPPC. Of particular concern was the rapid increase in the use of last-resort compounds, both in HICs and LMICs, such as glycylcyclines, oxazolidinones, carbapenems, and polymyxins. Projections of global antibiotic consumption in 2030, assuming no policy changes, were up to 200% higher than the 42 billion DDDs estimated in 2015. Although antibiotic consumption rates in most LMICs remain lower than in HICs despite higher bacterial disease burden, consumption in LMICs is rapidly converging to rates similar to HICs. Reducing global consumption is critical for reducing the threat of antibiotic resistance, but reduction efforts must balance access limitations in LMICs and take account of local and global resistance patterns.


Preventing Compulsory Admission to Psychiatric Inpatient Care: Perceived Coercion, Empowerment, and Self-Reported Mental Health Functioning after 12 Months of Preventive Monitoring.
To evaluate the effects of a preventive monitoring program targeted to reduce compulsory rehospitalization and perceived coercion in patients with severe mental disorder. We analyze patient outcomes in terms of perceived coercion, empowerment, and self-reported mental health functioning at 12 months. The program consists of individualized psychoeducation, crisis cards and, after discharge from the psychiatric hospital, a 24-month preventive monitoring. In total, 238 psychiatric inpatients who had had compulsory admission(s) during the past 24 months were included in the trial. T1-assessment 12 months after baseline was achieved for 182 patients. Study participants reported lower levels of perceived coercion, negative pressures, and process exclusion, a higher level of optimism, and a lesser degree of distress due to symptoms, interpersonal relations, and social role functioning (significant time effects). However, improvements were not confined to the intervention group, but seen also in the treatment-as-usual group (no significant group or interaction effects). Altered perceptions were linked to older age, shorter illness duration, female sex, non-psychotic disorder, and compulsory hospitalization not due to risk of harm to others. Our findings suggest that changes in the subjective perspective were fueled primarily by participation in this study rather than by having received the specific intervention. The study contributes to a better understanding of the interaction between "objective" measures (compulsory readmissions) and patients' perceptions and highlights the need for treatment approaches promoting empowerment in individuals with a history of involuntary psychiatric hospitalizations.


Vascular responsiveness to serotonin metabolites in mineralocorticoid hypertension.
This study characterizes vascular responsiveness to serotonin and its metabolites and to several monoamines that are structurally related to serotonin in deoxycorticosterone acetate (DOCA)-salt hypertension. Mesenteric arteries from normotensive and hypertensive rats were excised and cut into helical strips for isometric force recording. Dose-response curves to serotonin in arteries from hypertensive rats were shifted significantly to the left compared with those in arteries from normotensive rats (ED25: DOCA-treated = 2.4 X 10(-8) M; control = 17.1 X 10(-8) M). Contractile responses to 5-hydroxyindole acetic acid and 5-hydroxytryptophol were greater in mesenteric arteries from hypertensive rats, whereas reactivity to 5-methoxytryptamine and melatonin in arteries from hypertensive rats did not differ from that in arteries from normotensive rats. Mesenteric arteries from both rat groups were unresponsive to the serotonin metabolite N-acetylserotonin. Contractile responses to 5,6-dihydroxytryptamine and 6-hydroxytryptamine were greater in mesenteric arteries from hypertensive rats, whereas responsiveness to 3-hydroxytryptamine in hypertensive arteries did not differ from normotensive values. Contractile responses to serotonin and its metabolites and to the structurally related monoamines were inhibited by the serotonergic antagonist ketanserin. These results demonstrate that vascular sensitivity to serotonin is increased in DOCA-hypertensive rats. Based on the experiments with serotonin metabolites and with other monoamines, the increased responsiveness to these compounds appears to be related to the structural location of hydroxyl and amine moieties.


Paternal age effect mutations and selfish spermatogonial selection: causes and consequences for human disease.
Advanced paternal age has been associated with an increased risk for spontaneous congenital disorders and common complex diseases (such as some cancers, schizophrenia, and autism), but the mechanisms that mediate this effect have been poorly understood. A small group of disorders, including Apert syndrome (caused by FGFR2 mutations), achondroplasia, and thanatophoric dysplasia (FGFR3), and Costello syndrome (HRAS), which we collectively term "paternal age effect" (PAE) disorders, provides a good model to study the biological and molecular basis of this phenomenon. Recent evidence from direct quantification of PAE mutations in sperm and testes suggests that the common factor in the paternal age effect lies in the dysregulation of spermatogonial cell behavior, an effect mediated molecularly through the growth factor receptor-RAS signal transduction pathway. The data show that PAE mutations, although arising rarely, are positively selected and expand clonally in normal testes through a process akin to oncogenesis. This clonal expansion, which is likely to take place in the testes of all men, leads to the relative enrichment of mutant sperm over time-explaining the observed paternal age effect associated with these disorders-and in rare cases to the formation of testicular tumors. As regulation of RAS and other mediators of cellular proliferation and survival is important in many different biological contexts, for example during tumorigenesis, organ homeostasis and neurogenesis, the consequences of selfish mutations that hijack this process within the testis are likely to extend far beyond congenital skeletal disorders to include complex diseases, such as neurocognitive disorders and cancer predisposition.


Use of amino acids in refractive surgery.
To assess the influence of amino acid supplements on corneal fibroblast activity during corneal healing in patients with chronic ulcers, as well as after refractive surgery when delayed re-epithelialization had been observed in the fellow eye. Studies were conducted in vitro on eye bank eyes, and in vivo. In vitro cultures of rabbit corneal fibroblasts were enriched with progressively higher concentrations of amino acids. The human eye bank cornea study involved corneal epithelium removal, with corneas kept for 7 days in standard storage medium, either simple or enriched with amino acids. Clinical studies included treatment of two groups: Group 1 included six eyes of six patients with chronic epithelial defects, resistant to usual treatment; Group 2 included 21 eyes of 21 patients with delayed re-epithelialization after PRK in the first eye (12 +/- 3 days). Group 1 was treated for 1 month, and Group 2 received treatment preoperatively and postoperatively after surgery in the fellow eye until re-epithelialization was achieved. All patients received 13 amino acids and Vitamin C in three tablets, three times per day. Cultures of rabbit corneal fibroblasts showed that progressively increasing amino acid supplements led to an increase in the percentage of fibroblasts. De-epithelialized human eye bank corneas incubated with amino acids showed thicker and better organized re-epithelialization when compared to corneas incubated in simple standard storage media. In the clinical study, five eyes in Group 1 showed substantial improvement, one eyes remained unchanged. All eyes in Group 2 showed complete re-epithelialization within 60 hours after surgery. Results suggest improvement of re-epithelialization when an increase of serum and tear film amino acids is obtained through oral administration.


Host-pathogen interactions made transparent with the zebrafish model.
The zebrafish holds much promise as a high-throughput drug screening model for immune-related diseases, including inflammatory and infectious diseases and cancer. This is due to the excellent possibilities for in vivo imaging in combination with advanced tools for genomic and large scale mutant analysis. The context of the embryo's developing immune system makes it possible to study the contribution of different immune cell types to disease progression. Furthermore, due to the temporal separation of innate immunity from adaptive responses, zebrafish embryos and larvae are particularly useful for dissecting the innate host factors involved in pathology. Recent studies have underscored the remarkable similarity of the zebrafish and human immune systems, which is important for biomedical applications. This review is focused on the use of zebrafish as a model for infectious diseases, with emphasis on bacterial pathogens. Following a brief overview of the zebrafish immune system and the tools and methods used to study host-pathogen interactions in zebrafish, we discuss the current knowledge on receptors and downstream signaling components that are involved in the zebrafish embryo's innate immune response. We summarize recent insights gained from the use of bacterial infection models, particularly the Mycobacterium marinum model that illustrate the potential of the zebrafish model for high-throughput antimicrobial drug screening.


Factors associated with rifampin resistance in staphylococcal periprosthetic joint infections (PJI): a matched case-control study.
Rifampin combination therapy plays an important role in the management of staphylococcal periprosthetic joint infection (PJI). However, the emergence of rifampin resistance is a feared complication. We retrospectively analysed predetermined potential risk factors in patients with rifampin-resistant staphylococcal PJI in a multicentre case-control study. Cases (n = 48) were defined as PJI caused by rifampin-resistant staphylococci. Rifampin-susceptible controls (n = 48) were matched for microorganism and type of prosthetic joint. Uni- and multivariable conditional logistic regression analyses were performed to estimate odds ratios (OR) with 95 % confidence intervals (95 % CI). Forty-eight cases (31 men; median age 67 years; age range 39-88 years) with hip- (n = 29), knee- (n = 13), elbow- (n = 4), shoulder- (n = 1) or ankle-PJI (n = 1) were enrolled in the study. Staphylococcus aureus and coagulase-negative staphylococci were isolated in ten and 38 episodes, respectively. Most of the cases (n = 44, 92 %) had a previous PJI, and 93 % (n = 41) of these had been treated with rifampin. There was an independent association of emergence of rifampin resistance with male sex (OR 3.6, 95 % CI 1.2-11), ≥ 3 previous surgical revisions (OR 4.7, 95 % CI 1.6-14.2), PJI treatment with high initial bacterial load (inadequate surgical debridement, <2 weeks of intravenous treatment of the combination medication; OR 4.9, 95 % CI 1.6-15) and inadequate rifampin therapy (OR 5.4, 95 % CI 1.2-25). Based on our results, extensive surgical debridement and adequate antibiotic therapy are needed to prevent the emergence of rifampin resistance.


Adsorption of Basic Yellow 28 from aqueous solutions with clinoptilolite and amberlite.
The objective of this study was to investigate the adsorption of Basic Yellow 28 that is a cationic dye on clinoptilolite and amberlite XAD-4. Both equilibrium and batch rate adsorption in aqueous solutions of the dyestuff were investigated. Adsorption rate data were analysed using the pseudo-first order kinetic model of Lagergren and the pseudo-second order model to determine adsorption rate constants at 20, 30 and 40 degrees C. The adsorption equilibrium data were analysed using various adsorption isotherm models and the results have shown that adsorption behaviour of Basic Yellow 28 by clinoptilolite and amberlite could be described by either Langmuir or Freundlich models. Langmuir adsorption isotherm constants corresponding to adsorption capacity, Q(0), were found to be 59.6, 52.9 and 56.7 mg/g for clinoptilolite at 20, 30 and 40 degrees C, respectively. Lower adsorption capacities for Basic Yellow 28 on amberlite were obtained. The increase of adsorption rate constants with an increase in temperature for BY 28 adsorption on amberlite indicated chemisorption with dissociation and increased availability of sites due to higher penetration of adsorbing molecules into the pores.


Prokaryotic metabolic activity and community structure in Antarctic continental shelf sediments.
The prokaryote community activity and structural characteristics within marine sediment sampled across a continental shelf area located off eastern Antarctica (66 degrees S, 143 degrees E; depth range, 709 to 964 m) were studied. Correlations were found between microbial biomass and aminopeptidase and chitinase rates, which were used as proxies for microbial activity. Biomass and activity were maximal within the 0- to 3-cm depth range and declined rapidly with sediment depths below 5 cm. Most-probable-number counting using a dilute carbohydrate-containing medium recovered 1.7 to 3.8% of the sediment total bacterial count, with mostly facultatively anaerobic psychrophiles cultured. The median optimal growth temperature for the sediment isolates was 15 degrees C. Many of the isolates identified belonged to genera characteristic of deep-sea habitats, although most appear to be novel species. Phospholipid fatty acid (PLFA) and isoprenoid glycerol dialkyl glycerol tetraether analyses indicated that the samples contained lipid components typical of marine sediments, with profiles varying little between samples at the same depth; however, significant differences in PLFA profiles were found between depths of 0 to 1 cm and 13 to 15 cm, reflecting the presence of a different microbial community. Denaturing gradient gel electrophoresis (DGGE) analysis of amplified bacterial 16S rRNA genes revealed that between samples and across sediment core depths of 1 to 4 cm, the community structure appeared homogenous; however, principal-component analysis of DGGE patterns revealed that at greater sediment depths, successional shifts in community structure were evident. Sequencing of DGGE bands and rRNA probe hybridization analysis revealed that the major community members belonged to delta proteobacteria, putative sulfide oxidizers of the gamma proteobacteria, Flavobacteria, Planctomycetales, and Archaea. rRNA hybridization analyses also indicated that these groups were present at similar levels in the top layer across the shelf region.


Virulence and biodegradation potential of dynamic microbial communities associated with decaying Cladophora in Great Lakes.
Cladophora mats that accumulate and decompose along shorelines of the Great Lakes create potential threats to the health of humans and wildlife. The decaying algae create a low oxygen and redox potential environment favoring growth and persistence of anaerobic microbial populations, including Clostridium botulinum, the causal agent of botulism in humans, birds, and other wildlife. In addition to the diverse population of microbes, a dynamic chemical environment is generated, which involves production of numerous organic and inorganic substances, many of which are believed to be toxic to the sand and aquatic biotic communities. In this study, we used 16S-rDNA-based-amplicon sequencing and microfluidic-based quantitative PCR approaches to characterize the bacterial community structure and the abundances of human pathogens associated with Cladophora at different stages (up to 90days) of algal decay in laboratory microcosms. Oxygen levels were largely depleted after a few hours of incubation. As Cladophora decayed, the algal microbial biodiversity decreased within 24h, and the mat transitioned from an aerobic to anaerobic environment. There were increasing abundances of enteric and pathogenic bacteria during decomposition of Cladophora, including Acinetobacter, Enterobacter, Kluyvera, Cedecea, and others. In contrast, there were no or very few sequences (<0.07%) assigned to such groups in fresh Cladophora samples. Principal coordinate analysis indicated that the bacterial community structure was dynamic and changed significantly with decay time. Knowledge of microbial communities and chemical composition of decaying algal mats is critical to our further understanding of the role that Cladophora plays in a beach ecosystem's structure and function, including the algal role in trophic interactions. Based on these findings, public and environmental health concerns should be considered when decaying Cladophora mats accumulate Great Lakes shorelines.


Inequalities in microbial contamination of drinking water supplies in urban areas: the case of Lilongwe, Malawi.
Over past decades strategies for improving access to drinking water in cities of the Global South have mainly focused on increasing coverage, while water quality has often been overlooked. This paper focuses on drinking water quality in the centralized water supply network of Lilongwe, the capital of Malawi. It shows how microbial contamination of drinking water is unequally distributed to consumers in low-income (unplanned areas) and higher-income neighbourhoods (planned areas). Microbial contamination and residual disinfectant concentration were measured in 170 water samples collected from in-house taps in high-income areas and from kiosks and water storage facilities in low-income areas between November 2014 and January 2015. Faecal contamination (Escherichia coli) was detected in 10% of the 40 samples collected from planned areas, in 59% of the 64 samples collected from kiosks in the unplanned areas and in 75% of the 32 samples of water stored at household level. Differences in water quality in planned and unplanned areas were found to be statistically significant at p < 0.05. Finally, the paper shows how the inequalities in microbial contamination of drinking water are produced by decisions both on the development of the water supply infrastructure and on how this is operated and maintained.


Persecutory ideation and insomnia: findings from the second British National Survey of Psychiatric Morbidity.
It is clinically and theoretically plausible that insomnia contributes to the development and maintenance of paranoid fears. The primary aim of the study was to establish in a large sample whether insomnia and paranoia are associated more strongly than by chance. Cross-sectional data on paranoia, insomnia, anxiety, worry, depression, irritability, and cannabis use were obtained from the second British National Survey of Psychiatric Morbidity, a general population survey of adults aged 16-74 years living in Great Britain (N = 8580). It was found that insomnia was associated with an approximately two to threefold increase in paranoid thinking. Paranoia and insomnia were both strongly associated with the presence of anxiety, worry, depression, irritability and cannabis use. In a path analysis the association of paranoia and insomnia was partially explained by the affective symptoms, and, to a much lesser degree, cannabis use. The results are consistent with recent developments in the cognitive understanding of persecutory delusions, in which insomnia, negative affect, and substance use are identified as key factors. Longitudinal studies of insomnia and paranoia, and tests of the effects of sleep interventions on levels of paranoia, are now required to examine causality.


Using nuclear morphometry to predict the need for treatment among men with low grade, low stage prostate cancer enrolled in a program of expectant management with curative intent.
We assessed the use of quantitative clinical and pathologic information to predict which patients would eventually require treatment for prostate cancer (CaP) in an expectant management (EM) cohort. We identified 75 men having prostate cancer with favorable initial biopsy characteristics; 30 developed an unfavorable biopsy (Gleason grade >6, >2 cores with cancer, >50% of a core with cancer, or a palpable nodule) requiring treatment and 45 maintained favorable biopsies throughout a median follow-up of 2.7 years. Demographic, clinical data and quantitative tissue histomorphometry determined by digital image analysis were analyzed. Logistic regression (LR) modeling generated a quantitative nuclear grade (QNG) signature based on the enrollment biopsy for differentiation of Favorable and Unfavorable groups using a variable LR selection criteria of P(z)<0.05. The QNG signature utilized 12 nuclear morphometric descriptors (NMDs) and had an area under the receiver operator characteristic curve (ROC-AUC) of 87% with a sensitivity of 82%, specificity of 70% and accuracy of 75%. A multivariable LR model combining QNG signature with clinical and pathological variables yielded an AUC-ROC of 88% and a sensitivity of 81%, specificity of 78% and accuracy of 79%. A LR model using prostate volume, PSA density, and number of pre-diagnosis biopsies resulted in an AUC-ROC of 68% and a sensitivity of 85%, specificity of 37% and accuracy of 56%. QNG using EM prostate biopsies improves the predictive accuracy of LR models based on traditional clinicopathologic variables in determining which patients will ultimately develop an unfavorable biopsy. Our QNG-based model must be rigorously, prospectively validated prior to use in the clinical arena.


A 20 nm spin Hall nano-oscillator.
Spin Hall nano-oscillators (SHNOs) are an emerging class of pure spin current driven microwave signal generators. Through the fabrication of 20 nm nano-constrictions in Pt/NiFe bilayers, we demonstrate that SHNOs can be scaled down to truly nanoscopic dimensions, with the added benefit of ultra-low operating currents and improved power conversion efficiency. The lateral confinement leads to a strong shape anisotropy field as well as an additional demagnetizing field whose reduction with increasing auto-oscillation amplitude can yield a positive current tunability contrary to the negative tunability commonly observed for localized excitations in extended magnetic layers. Micromagnetic simulations corroborate the experimental findings and suggest that the active magnetodynamic area resides up to 100 nm outside of the nano-constriction.


Effect of the Glucagon-like Peptide-1 Analogue Exenatide Extended Release in Cats with Newly Diagnosed Diabetes Mellitus.
Exenatide extended release (ER) is a glucagon-like peptide-1 analogue that increases insulin secretion, inhibits glucagon secretion and induces satiation in humans with type 2 diabetes mellitus. The use of exenatide ER is safe and stimulates insulin secretion in healthy cats. The objective of this study is to assess the safety of exenatide ER and its effect on body weight, remission and metabolic control in newly diagnosed diabetic cats receiving insulin and a low-carbohydrate diet. Thirty client-owned cats. Prospective placebo-controlled clinical trial. Cats were treated with exenatide ER or 0.9% saline, administered SC, once weekly. Both groups received insulin glargine and a low-carbohydrate diet. Exenatide ER was administered for 16 weeks, or in cats that achieved remission it was given for 4 weeks after discontinuing insulin treatment. Nonparametric tests were used for statistical analysis. Cats in the exenatide ER and placebo groups had transient adverse signs including decreased appetite (60% vs. 20%, respectively, P = .06) and vomiting (53% vs. 40%, respectively, P = .715). Body weight increased significantly in the placebo group (P = .002), but not in cats receiving exenatide ER. Cats on exenatide ER achieved remission or good metabolic control in 40% or 89%, respectively, whereas in control cats percentages were 20% or 58% (P = .427 and P = .178, respectively). Exenatide ER is safe in diabetic cats and does not result in weight gain. Our pilot study suggests that, should there be an additional clinically relevant beneficial effect of exenatide ER in insulin-treated cats on rate of remission and good metabolic control, it would likely approximate 20% and 30%, respectively.


A workplace breast cancer screening program. Costs and components.
Screening for breast cancer can result in early detection of malignancies and lives saved. Many employers now offer periodic screening as an employee health benefit, and some have established screening programs in the workplace. This study was performed to identify the employer costs of breast cancer screening in the workplace, referrals for suspicious findings, and initial treatment of malignant disease. Additionally, the costs for these same services, had they been obtained outside of a workplace screening program, were estimated. Data on program components and associated costs for an established employer based breast cancer screening program were obtained. These costs were compared to those among a hypothetical cohort of women not enrolled in the workplace screening program. From 1989 through 1995, 1,416 women participated in the program. Nearly 2,500 screening mammograms and approximately 2,773 clinical breast examinations were performed, resulting in 292 referrals to physicians outside of the program for additional diagnostic procedures and treatment as needed. These referrals resulted in the detection of 12 malignancies: 8 Stage I; 3 Stage II; and 1 Stage III. Mammographic and clinical breast examination screening cost $249,041; referrals resulting in benign disease or no detectable disease cost $185,002; and referrals resulting in malignant disease, followed by initial treatment, cost $148,530. Therefore, the total cost was $582,573. Approximately 47% of the cost of referrals and initial treatment were due to employee lost productivity. Total cost in the hypothetical cohort was $1,067,948 under the assumptions that all women received screening outside of the workplace, and that the same number of malignancies were detected at the same stage as in the workplace program. These findings indicate referrals resulting in detection of benign disease or no disease accounted for a substantial proportion of the total cost of the program. In addition, employee lost productivity accounted for almost 50% of the cost of all referrals and initial treatment. Workplace screening is a relatively efficient approach for early detection of breast cancer when compared to off site screening or no screening. The efficiency could be improved with a reduction in the number and cost of unnecessary referrals.


A blind deconvolution approach to ultrasound imaging.
In this paper, a single-input multiple-output (SIMO) channel model is introduced for the deconvolution process of ultrasound imaging; the ultrasound pulse is the single system input and tissue reflectivity functions are the channel impulse responses. A sparse regularized blind deconvolution model is developed by projecting the tissue reflectivity functions onto the null space of a cross-relation matrix and projecting the ultrasound pulse onto a low-resolution space. In this way, the computational load is greatly reduced and the estimation accuracy can be improved because the proposed deconvolution model contains fewer variables. Subsequently, an alternating direction method of multipliers (ADMM) algorithm is introduced to efficiently solve the proposed blind deconvolution problem. Finally, the performance of the proposed blind deconvolution method is examined using both computer-simulated data and practical in vitro and in vivo data. The results show a great improvement in the quality of ultrasound images in terms of signal-to-noise ratio and spatial resolution gain.


Optimal inequalities for state-independent contextuality.
Contextuality is a natural generalization of nonlocality which does not need composite systems or spacelike separation and offers a wider spectrum of interesting phenomena. Most notably, in quantum mechanics there exist scenarios where the contextual behavior is independent of the quantum state. We show that the quest for an optimal inequality separating quantum from classical noncontextual correlations in a state-independent manner admits an exact solution, as it can be formulated as a linear program. We introduce the noncontextuality polytope as a generalization of the locality polytope and apply our method to identify two different tight optimal inequalities for the most fundamental quantum scenario with state-independent contextuality.


Socioeconomic status, antioxidant micronutrients, and correlates of oxidative damage: the Coronary Artery Risk Development in Young Adults (CARDIA) study.
To examine whether socioeconomic status (SES) (education, occupation, income), is associated both cross sectionally and prospectively with circulating concentrations of a) two correlates of oxidative damage, F(2)-isoprostanes (F(2)-IsoPs) and gamma-glutamyltransferase (GGT); and b) antioxidant nutrients (ascorbic acid and carotenoids). We also examine whether the proposed associations are mediated by smoking, alcohol consumption, and depression. Risk for chronic disease increases with decreasing SES. One pathway by which low SES might influence disease risk is by promoting oxidative stress. Data from 1278 participants in the Coronary Artery Risk Development in Young Adults (CARDIA) study were used to examine the association of SES with oxidation correlates and antioxidant nutrients. Education, occupation, health behaviors, and body mass index (BMI) were assessed during Years 0, 10, and 15 of the study; income and depression were evaluated at Years 10 and 15. F(2)-isoprostanes were measured at Year 15, gamma-glutamyltransferase (GGT) at Years 0 and 10, carotenoids at Years 0 and 15, and ascorbic acid at Years 10 and 15. Cross sectionally, oxidation correlates decreased and antioxidant nutrients increased with increasing SES, estimated in several ways, independent of age, sex, race, and BMI. Prospectively, lower Year 0 education and occupation predicted greater increases in GGT and greater decreases in carotenoids over 10 to 15 years. Prospective associations of Year 0 SES with Year 15 carotenoids were independent of Year 15 SES. Smoking, drinking, and depression symptoms partially mediated these effects. Circulating oxidation correlates increase and antioxidant nutrients decrease with decreasing SES, both cross sectionally and prospectively.


Antibiotics versus topical antiseptics for chronic suppurative otitis media.
Chronic suppurative otitis media (CSOM), sometimes referred to as chronic otitis media (COM), is a chronic inflammation and infection of the middle ear and mastoid cavity, characterised by ear discharge (otorrhoea) through a perforated tympanic membrane. The predominant symptoms of CSOM are ear discharge and hearing loss. Antibiotics and antiseptics kill or inhibit the micro-organisms that may be responsible for the infection. Antibiotics can be applied topically or administered systemically via the oral or injection route. Antiseptics are always directly applied to the ear (topically). To assess the effectiveness of antibiotics versus antiseptics for people with chronic suppurative otitis media (CSOM). The Cochrane ENT Information Specialist searched the Cochrane ENT Register; Central Register of Controlled Trials (CENTRAL; 2019, Issue 4, via the Cochrane Register of Studies); Ovid MEDLINE; Ovid Embase; CINAHL; Web of Science; ClinicalTrials.gov; ICTRP and additional sources for published and unpublished trials. The date of the search was 1 April 2019. We included randomised controlled trials (RCTs) with at least a one-week follow-up involving patients (adults and children) who had chronic ear discharge of unknown cause or CSOM, where ear discharge had continued for more than two weeks. The intervention was any single, or combination of, antibiotic agent, whether applied topically (without steroids) or systemically. The comparison was any single, or combination of, topical antiseptic agent, applied as ear drops, powders or irrigations, or as part of an aural toileting procedure. Two comparisons were topical antiseptics compared to: a) topical antibiotics or b) systemic antibiotics. Within each comparison we separated where both groups of patients had received topical antibiotic a) alone or with aural toilet and b) on top of background treatment (such as systemic antibiotics). We used the standard Cochrane methodological procedures. We used GRADE to assess the certainty of the evidence for each outcome. Our primary outcomes were: resolution of ear discharge or 'dry ear' (whether otoscopically confirmed or not), measured at between one week and up to two weeks, two weeks to up to four weeks, and after four weeks; health-related quality of life using a validated instrument; and ear pain (otalgia) or discomfort or local irritation. Secondary outcomes included hearing, serious complications and ototoxicity measured in several ways. We identified seven studies (935 participants) across four comparisons with antibiotics compared against acetic acid, aluminium acetate, boric acid and povidone-iodine. None of the included studies reported the outcomes of quality of life or serious complications. A. Topical antiseptic (acetic acid) versus topical antibiotics (quinolones or aminoglycosides) It is very uncertain if there is a difference in resolution of ear discharge with acetic acid compared with aminoglycosides at one to two weeks (risk ratio (RR) 0.88, 95% confidence interval (CI) 0.72 to 1.08; 1 study; 100 participants; very low-certainty evidence). No study reported results for ear discharge after four weeks. It was very uncertain if there was more ear pain, discomfort or local irritation with acetic acid or topical antibiotics due to the low numbers of participants reporting events (RR 0.16, 95% CI 0.02 to 1.34; 2 RCTs; 189 participants; very low-certainty evidence). No differences between groups were reported narratively for hearing (quinolones) or suspected ototoxicity (aminoglycosides) (very low-certainty evidence). B. Topical antiseptic (aluminium acetate) versus topical antibiotics No results for the one study comparing topical antibiotics with aluminium acetate could be used in the review. C. Topical antiseptic (boric acid) versus topical antibiotics (quinolones) One study reported more participants with resolution of ear discharge when using topical antibiotics (quinolones) compared with boric acid ear drops at between one to two weeks (risk ratio (RR) 1.56, 95% confidence interval (CI) 1.27 to 1.92; 1 study; 409 participants; moderate-certainty evidence). This means that one additional person will have resolution of ear discharge for every five people receiving topical antibiotics (compared with boric acid) at two weeks. No study reported results for ear discharge after four weeks. There was a bigger improvement in hearing in the topical antibiotic group compared to the topical antiseptic group (mean difference (MD) 2.79 decibels (dB), 95% CI 0.48 to 5.10; 1 study; 390 participants; low-certainty evidence) but this difference may not be clinically significant. There may be more ear pain, discomfort or irritation with boric acid compared with quinolones (RR 0.56, 95% CI 0.32 to 0.98; 2 studies; 510 participants; low-certainty evidence). Suspected ototoxicity was not reported. D. Topical antiseptic (povidone-iodine) versus topical antibiotics (quinolones) It is uncertain if there is a difference between quinolones and povidone-iodine with respect to resolution of ear discharge at one to two weeks (RR 1.02, 95% CI 0.82 to 1.26; 1 RCT, 39 participants; very low-certainty evidence). The study reported qualitatively that there were no differences between the groups for hearing and no patients developed ototoxic effects (very low-certainty evidence). No results for resolution of ear discharge beyond four weeks, or ear pain, discomfort or irritation, were reported. E. Topical antiseptic (acetic acid) + aural toileting versus topical + systemic antibiotics (quinolones) One study reported that participants receiving topical and oral antibiotics had less resolution of ear discharge compared with acetic acid ear drops and aural toileting (suction clearance every two days) at one month (RR 0.69, 95% CI 0.53 to 0.90; 100 participants). The study did not report results for resolution of ear discharge at between one to two weeks, ear pain, discomfort or irritation, hearing or suspected ototoxicity. Treatment of CSOM with topical antibiotics (quinolones) probably results in an increase in resolution of ear discharge compared with boric acid at up to two weeks. There was limited evidence for the efficacy of other topical antibiotics or topical antiseptics and so we are unable to draw conclusions. Adverse events were not well reported.


Percutaneous vertebroplasty for osteoporotic vertebral compression fracture.
Percutaneous vertebroplasty is widely used to treat acute and subacute painful osteoporotic vertebral fractures although recent placebo-controlled trials have questioned its value. To synthesise the available evidence regarding the benefits and harms of vertebroplasty for treatment of osteoporotic vertebral fractures. We searched CENTRAL, MEDLINE and EMBASE up to November 2014. We also reviewed reference lists of review articles, trials and trial registries to identify any other potentially relevant trials. We included randomised and quasi-randomised controlled trials (RCTs) including adults with painful osteoporotic vertebral fractures of any duration and comparing vertebroplasty with placebo (sham), usual care, or any other intervention. As it is least prone to bias, vertebroplasty compared with placebo was the primary comparison. Major outcomes were mean overall pain, disability, disease-specific and overall health-related quality of life, patient-reported treatment success, new symptomatic vertebral fractures and number of other serious adverse events. At least two review authors independently selected trials for inclusion, extracted data, performed 'Risk of bias' assessment and assessed the quality of the body of evidence for the main outcomes using GRADE. Eleven RCTs and one quasi-RCT conducted in various countries were included. Two trials compared vertebroplasty with placebo (209 randomised participants), six compared vertebroplasty with usual care (566 randomised participants) and four compared vertebroplasty with kyphoplasty (545 randomised participants). Trial size varied from 34 to 404 participants, most participants were female, mean age ranged between 63.3 and 80 years, and mean symptom duration varied from a week to more than six months.Both placebo-controlled trials were judged to be at low overall risk of bias while other included trials were generally considered to be at high risk of bias across a range of criteria, most seriously due to lack of participant and study personnel blinding.Compared with placebo, there was moderate quality evidence based upon two trials that vertebroplasty provides no demonstrable benefits with respect to pain, disability, disease-specific or overall quality of life or treatment success. At one month, mean pain (on a scale 0 to 10, higher scores indicate more pain) was 5 points with placebo and 0.7 points better (1.5 better to 0.15 worse) with vertebroplasty, an absolute pain reduction of 7% (15% better to 1.5% worse) and relative reduction of 10% (21% better to 2% worse) (two trials, 201 participants). At one month, mean disability measured by the Roland Morris Disability Questionnaire (scale range 0 to 23, higher scores indicate worse disability) was 13.6 points in the placebo group and 1.1 points better (2.9 better to 0.8 worse) in the vertebroplasty group, absolute improvement in disability 4.8% (12.8% better to 3.3% worse), relative change 6.3% better (17.0% better to 4.4% worse) (two trials, 201 participants).At one month, disease-specific quality of life measured by the QUALEFFO (scale 0 to 100, higher scores indicating worse quality of life) was 2.4 points in the placebo group and 0.40 points worse (4.58 better to 5.38 worse) in the vertebroplasty group, absolute change: 0.4% worse (5% worse to 5% better), relative change 0.7% worse (9% worse to 8% better (based upon one trial, 73 participants). At one month overall quality of life measured by the EQ5D (0 = death to 1 = perfect health, higher scores indicate greater quality of life at one month was 0.27 points in the placebo group and 0.05 points better (0.01 worse to 0.11 better) in the vertebroplasty group, absolute improvement in quality of life 5% (1% worse to 11% better), relative change 18% better (4% worse to 39% better) (two trials, 201 participants). Based upon one trial (78 participants) at one month, 9/40 (or 225 per 1000) people perceived that treatment was successful in the placebo group compared with 12/38 (or 315 per 1000; range 150 to 664) in the vertebroplasty group, RR 1.40 (95% CI 0.67 to 2.95), absolute risk difference 9% more reported success (11% fewer to 29% more); relative change 40% more reported success (33% fewer to 195% more).Based upon moderate quality evidence from three trials (one placebo, two usual care, 281 participants) with up to 12 months follow-up, we are uncertain whether or not vertebroplasty increases the risk of new symptomatic vertebral fractures (28/143 observed in the vertebroplasty group compared with 19/138 in the control group; RR 1.47 (95% CI 0.39 to 5.50).Similary, based upon moderate quality evidence from two placebo-controlled trials (209 participants), we are uncertain about the exact risk of other adverse events (3/106 were observed in the vertebroplasty group compared with 3/103 in the placebo group; RR 1.01 (95% CI 0.21 to 4.85)). Notably, serious adverse events reported with vertebroplasty included osteomyelitis, cord compression, thecal sac injury and respiratory failure.Our subgroup analyses provided limited evidence that the effects did not differ according to duration of pain ≤ 6 weeks versus > 6 weeks. Including data from the six trials that compared vertebroplasty with usual care in a sensitivity analyses inconsistently altered the primary results, with all combined analyses displaying substantial to considerable heterogeneity. Based upon moderate quality evidence, our review does not support a role for vertebroplasty for treating osteoporotic vertebral fractures in routine practice. We found no demonstrable clinically important benefits compared with a sham procedure and subgroup analyses indicated that results did not differ according to duration of pain ≤ 6 weeks versus > 6 weeks. Sensitivity analyses confirmed that open trials comparing vertebroplasty with usual care are likely to have overestimated any benefit of vertebroplasty. Correcting for these biases would likely drive any benefits observed with vertebroplasty towards the null, in keeping with findings from the placebo-controlled trials.Numerous serious adverse events have been observed following vertebroplasty. However due to the small number of events, we cannot be certain about whether or not vertebroplasty results in a clinically important increased risk of new symptomatic vertebral fractures and/or other serious adverse events. Patients should be informed about both the lack of high quality evidence supporting benefit of vertebroplasty and its potential for harm.


Use and misuse of nitrogen in agriculture: the German story.
Nitrogen (N) fertilization in agriculture has been discussed controversially in Germany for almost two centuries. The agronomist Carl Sprengel, who published his theory on the mineral nutrition of plants in 1828, advocated the use of mineral N fertilizers. Chemist Justus von Liebig, on the other hand, vehemently denied around 1850 the need for N fertilization. Although it soon became evident that Sprengel was right and Liebig was wrong, not much synthetic N fertilizer was used in German agriculture until around 1915, when the Haber-Bosch technique enabled the commercial production of NH3. The use of N fertilizers since then has grown, especially since 1950. To increase agricultural productivity, German governments have promoted, directly and indirectly, the use of N in crop and in animal production. Unfortunately, it was overlooked that N surpluses in agriculture increased rapidly; around 1980 they amounted yearly to more than 100 kg ha(-1). The extensive use of N in agriculture is causing environmental damage and is contributing substantially to the external costs of present agriculture. The main N compounds that affect the environment are N2O, NH3, and NO3. These compounds are considered to contribute one third to the external costs of agriculture. Additionally, the high rate of human intake of animal proteins and lipids has adversely affected the health of the country's population. Fundamental corrections in German farm policy appear inevitable.


Effects of caffeine on cytoplasmic free Ca2+ concentration in pancreatic beta-cells are mediated by interaction with ATP-sensitive K+ channels and L-type voltage-gated Ca2+ channels but not the ryanodine receptor.
In the pancreatic beta-cell, an increase in the cytoplasmic free Ca2+ concentration ([Ca2+]i) by caffeine is believed to indicate mobilization of Ca2+ from intracellular stores, through activation of a ryanodine receptor-like channel. It is not known whether other mechanisms, as well, underlie caffeine-induced changes in [Ca2+]i. We studied the effects of caffeine on [Ca2+]i by using dual-wavelength excitation microfluorimetry in fura-2-loaded beta-cells. In the presence of a non-stimulatory concentration of glucose, caffeine (10-50 mM) consistently increased [Ca2+]i. The effect was completely blocked by omission of extracellular Ca2+ and by blockers of the L-type voltage-gated Ca2+ channel, such as D-600 or nifedipine. Depletion of agonist-sensitive intracellular Ca2+ pools by thapsigargin did not inhibit the stimulatory effect of caffeine on [Ca2+]i. Moreover, this effect of caffeine was not due to an increase in cyclic AMP, since forskolin and 3-isobutyl-1-methylxanthine (IBMX) failed to raise [Ca2+]i in unstimulated beta-cells. In beta-cells, glucose and sulphonylureas increase [Ca2+]i by causing closure of ATP-sensitive K+ channels (KATP channels). Caffeine also caused inhibition of KATP channel activity, as measured in excised inside-out patches. Accordingly, caffeine (> 10 mM) induced insulin release from beta-cells in the presence of a non-stimulatory concentration of glucose (3 mM). Hence, membrane depolarization and opening of voltage-gated L-type Ca2+ channels were the underlying mechanisms whereby the xanthine drug increased [Ca2+]i and induced insulin release. Paradoxically, in glucose-stimulated beta-cells, caffeine (> 10 mM) lowered [Ca2+]i. This effect was due to the fact that caffeine reduced depolarization-induced whole-cell Ca2+ current through the L-type voltage-gated Ca2+ channel in a dose-dependent manner. Lower concentrations of caffeine (2.5-5.0 mM), when added after glucose-stimulated increase in [Ca2+]i, induced fast oscillations in [Ca2+]i. The latter effect was likely to be attributable to the cyclic AMP-elevating action of caffeine, leading to phosphorylation of voltage-gated Ca2+ channels. Hence, in beta-cells, caffeine-induced changes in [Ca2+]i are not due to any interaction with intracellular Ca2+ pools. In these cells, a direct interference with KATP channel- and L-type voltage-gated Ca(2+)-channel activity is the underlying mechanism by which caffeine increases or decreases [Ca2+]i.


Rapid RHD Zygosity Determination Using Digital PCR.
Paternal zygosity testing is used for determining homo- or hemizygosity of RHD in pregnancies that are at a risk of hemolytic disease of the fetus and newborn. At present, this is achieved by using real-time PCR or the Rhesus box PCR, which can be difficult to interpret and unreliable, particularly for black African populations. DNA samples extracted from 53 blood donors were analyzed using 2 multiplex reactions for RHD-specific targets against a reference (AGO1)2 to determine gene dosage by digital PCR. Results were compared with serological data, and the correct genotype for 2 discordant results was determined by long-range PCR (LR-PCR), next-generation sequencing, and conventional Sanger sequencing. The results showed clear and reliable determination of RHD zygosity using digital PCR and revealed that 4 samples did not match the serologically predicted genotype. Sanger sequencing and long-range PCR followed by next-generation sequencing revealed that the correct genotypes for samples 729M and 351D, which were serologically typed as R1R2 (DCe/DcE), were R2r' (DcE/dCe) for 729M and R1r″ (DCe/dcE), R0ry (Dce/dCE), or RZr (DCE/dce) for 351D, in concordance with the digital PCR data. Digital PCR provides a highly accurate method to rapidly define blood group zygosity and has clinical application in the analysis of Rh phenotyped or genotyped samples. The vast majority of current blood group genotyping platforms are not designed to define zygosity, and thus, this technique may be used to define paternal RH zygosity in pregnancies that are at a risk of hemolytic disease of the fetus and newborn and can distinguish between homo- and hemizygous RHD-positive individuals.


Connecting proximate mechanisms and evolutionary patterns: pituitary gland size and mammalian life history.
At the proximate level, hormones are known to play a critical role in influencing the life history of mammals, including humans. The pituitary gland is directly responsible for producing several hormones, including those related to growth and reproduction. Although we have a basic understanding of how hormones affect life history characteristics, we still have little knowledge of this relationship in an evolutionary context. We used data from 129 mammal species representing 14 orders to investigate the relationship between pituitary gland size and life history variation. Because pituitary gland size should be related to hormone production and action, we predicted that species with relatively large pituitaries should be associated with fast life histories, especially increased foetal and post-natal growth rates. Phylogenetic analyses revealed that total pituitary size and the size of the anterior lobe of the pituitary significantly predicted a life history axis that was correlated with several traits including body mass, and foetal and post-natal growth rates. Additional models directly examining the association between relative pituitary size and growth rates produced concordant results. We also found that relative pituitary size variation across mammals was best explained by an Ornstein-Uhlenbeck model of evolution, suggesting an important role of stabilizing selection. Our results support the idea that the size of the pituitary is linked to life history variation through evolutionary time. This pattern is likely due to mediating hormone levels but additional work is needed. We suggest that future investigations incorporating endocrine gland size may be critical for understanding life history evolution.


The Phenotypic and Genetic Underpinnings of Flower Size in Polemoniaceae.
Corolla length is a labile flower feature and has strong implications for pollinator success. However, the phenotypic and genetic bases of corolla elongation are not well known, largely due to a lack of good candidate genes for potential genetic exploration and functional work. We investigate both the cellular phenotypic differences in corolla length, as well as the genetic control of this trait, in Saltugilia (Polemoniaceae). Taxa in this clade exhibit a large range of flower sizes and differ dramatically in pollinator guilds. Flowers of each species were collected from multiple individuals during four stages of flower development to ascertain if cell number or cell size is more important in determining flower size. In Saltugilia, increased flower size during development appears to be driven more by cell size than cell number. Differences in flower size between species are governed by both cell size and cell number, with the large-flowered S. splendens subsp. grantii having nearly twice as many cells as the small-flowered species. Fully mature flowers of all taxa contain jigsaw cells similar to cells seen in sepals and leaves; however, these cells are not typically found in the developing flowers of most species. The proportion of this cell type in mature flowers appears to have substantial implications, comprising 17-68% of the overall flower size. To identify candidate genes responsible for differences in cell area and cell type, transcriptomes were generated for two individuals of the species with the smallest (S. australis) and largest (S. splendens subsp. grantii) flowers across the same four developmental stages visualized with confocal microscopy. Analyses identified genes associated with cell wall formation that are up-regulated in the mature flower stage compared to mid-stage flowers (75% of mature size). This developmental change is associated with the origin of jigsaw cells in the corolla tube of mature flowers. Further comparisons between mature flowers in the two species revealed 354 transcripts that are up-regulated in the large-flowered S. splendens subsp. grantii compared to the small-flowered S. australis. These results are likely broadly applicable to Polemoniaceae, a clade of nearly 400 species, with extensive variation in floral form and shape.


Rural Residence Predicts Lower Cardiac Mortality and Better Mental Health Outcomes.
Previous studies have found higher rates of heart disease and worse mental health outcomes among individuals residing in rural areas. To our knowledge, no research has used county-level data to measure the effect of "ruralness" (the degree to which a county is rural) on heart disease and mentally unhealthy days while controlling for other sociodemographic factors. This study analyzes the effect of ruralness on heart disease death rates and the average number of mentally unhealthy days on a county-level. Linear regressions were performed using county-level data to analyze the effect of "Ruralness" on heart disease death rates and mental unhealthiness while controlling for confounding variables. Geographic analysis was also used. Higher rural-urban continuum codes predict lower rates of cardiac mortality (β = -.075 deaths per 100,000 people/continuum code, t = -4.36, P <.001) and fewer mentally unhealthy days (β = -.265 monthly mentally unhealthy days/continuum code, t = -16.45, P < .001). Being from a rural area correlates with lower rates of heart disease death and mental unhealthiness after controlling for sociodemographic confounders. This adds nuance to the previously reported trend of heart disease being more prevalent in rural areas.


Cellular toxicity associated with exposure to perfluorinated carboxylates (PFCAs) and their metabolic precursors.
The biotransformation of fluorotelomer based compounds yields saturated and unsaturated fluorotelomer aldehydes (FTALs and FTUALs, respectively) and carboxylic acids (FTCAs and FTUCAs, respectively) as intermediate metabolites that subsequently transform to perfluorinated carboxylic acids (PFCAs). Previous studies have demonstrated that the FTCAs and FTUCAs are 1 to 5 orders of magnitude more toxic than PFCAs after exposure to aquatic organisms. Additionally, FTUALs have demonstrated reactivity with proteins, which may be associated with toxicity through the inhibition of protein function. The purpose of this study was to carry out a comprehensive assessment of the relative toxicity between PFCAs and their intermediate precursor metabolites: the FTALs, FTUALs, FTCAs, and FTUCAs. Analytes were separately incubated with human liver epithelial (THLE-2) cells to assess how varying the functional group and the fluorinated chain length affects cell viability. For each analyte, dose-response EC50 values were calculated. The EC50 values for FTUCAs and FTCAs were similar, with values ranging from 22 ± 9 and 24 ± 9 μM for the 10:2 congeners to 1004 ± 20 and 1004 ± 24 μM for the 4:2 congeners, respectively. The EC50 values for the PFCAs ranged from 65 ± 41 (PFDA) to 1361 ± 146 (PFBA) μM. The range of toxicity between PFCAs and their acid precursors were similar. However, the comparative toxicity between the 6:2 and 8:2 congeners and their corresponding PFCA had toxicity thresholds that varied depending on the functional headgroup, where FTUALs ≥ FTALs > FTUCAs ≥ FTCAs > PFCAs. For all PFCAs and acid precursors, toxicity depended on the length of the fluorinated chain, where the longer chain lengths yielded greater bioaccumulation and enhanced toxicity, results which agreed with those previously reported. By contrast, FTALs and FTUALs were the most toxic of all the analytes examined, where toxicity was enhanced at shorter chain lengths, with EC50 values of 7 ± 1 μM (6:2 FTUAL) and 8.6 ± 0.8 μM (6:2 FTAL). DNA adducts were not detectable for the aldehyde precursors, using a quantitative long-range PCR method. Our data provide the first evidence that aldehyde intermediates have demonstrated toxicity in cellular systems that is more significant than PFCAs and their corresponding acid intermediates.