Gitelman syndrome.
Gitelman syndrome (GS), also referred to as familial hypokalemia-hypomagnesemia, is characterized by hypokalemic metabolic alkalosis in combination with significant hypomagnesemia and low urinary calcium excretion. The prevalence is estimated at approximately 1:40,000 and accordingly, the prevalence of heterozygotes is approximately 1% in Caucasian populations, making it one of the most frequent inherited renal tubular disorders. In the majority of cases, symptoms do not appear before the age of six years and the disease is usually diagnosed during adolescence or adulthood. Transient periods of muscle weakness and tetany, sometimes accompanied by abdominal pain, vomiting and fever are often seen in GS patients. Paresthesias, especially in the face, frequently occur. Remarkably, some patients are completely asymptomatic except for the appearance at adult age of chondrocalcinosis that causes swelling, local heat, and tenderness over the affected joints. Blood pressure is lower than that in the general population. Sudden cardiac arrest has been reported occasionally. In general, growth is normal but can be delayed in those GS patients with severe hypokalemia and hypomagnesemia.GS is transmitted as an autosomal recessive trait. Mutations in the solute carrier family12, member 3 gene, SLC12A3, which encodes the thiazide-sensitive NaCl cotransporter (NCC), are found in the majority of GS patients. At present, more than 140 different NCC mutations throughout the whole protein have been identified. In a small minority of GS patients, mutations in the CLCNKB gene, encoding the chloride channel ClC-Kb have been identified.Diagnosis is based on the clinical symptoms and biochemical abnormalities (hypokalemia, metabolic alkalosis, hypomagnesemia and hypocalciuria). Bartter syndrome (especially type III) is the most important genetic disorder to consider in the differential diagnosis of GS. Genetic counseling is important. Antenatal diagnosis for GS is technically feasible but not advised because of the good prognosis in the majority of patients.Most asymptomatic patients with GS remain untreated and undergo ambulatory monitoring, once a year, generally by nephrologists. Lifelong supplementation of magnesium (magnesium-oxide and magnesium-sulfate) is recommended. Cardiac work-up should be offered to screen for risk factors of cardiac arrhythmias. All GS patients are encouraged to maintain a high-sodium and high potassium diet. In general, the long-term prognosis of GS is excellent.


A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing.
Cell type-specific expression of optogenetic molecules allows temporally precise manipulation of targeted neuronal activity. Here we present a toolbox of four knock-in mouse lines engineered for strong, Cre-dependent expression of channelrhodopsins ChR2-tdTomato and ChR2-EYFP, halorhodopsin eNpHR3.0 and archaerhodopsin Arch-ER2. All four transgenes mediated Cre-dependent, robust activation or silencing of cortical pyramidal neurons in vitro and in vivo upon light stimulation, with ChR2-EYFP and Arch-ER2 demonstrating light sensitivity approaching that of in utero or virally transduced neurons. We further show specific photoactivation of parvalbumin-positive interneurons in behaving ChR2-EYFP reporter mice. The robust, consistent and inducible nature of our ChR2 mice represents a significant advance over previous lines, and the Arch-ER2 and eNpHR3.0 mice are to our knowledge the first demonstration of successful conditional transgenic optogenetic silencing. When combined with the hundreds of available Cre driver lines, this optimized toolbox of reporter mice will enable widespread investigations of neural circuit function with unprecedented reliability and accuracy.


The influence of ageing on noise-induced hearing loss.
A lifetime of exposure to noise is likely to have negative effects on the hearing, but the interaction between noise-induced hearing loss (NIHL) and age-related hearing loss is difficult to determine. The most commonly accepted assumption is a simple accumulating effects of noise and ageing on the hearing. However, both a less than additive effect as well as a supraadditive effect has been proposed. Recently an interesting interaction between NIHL and age-related hearing loss has been reported (Gates et al., 2000). NIHL before old age reduces the effects of ageing at noise-associated frequencies, but accelerates the deterioration of hearing in adjacent frequencies. Findings from the longitudinal and cross-sectional gerontological and geriatric population study of 70-year-olds in Gothenburg, Sweden supports these observations. The incidence of tinnitus increases in old age, but not at the same high rate as presbyacusis. According to the gerontological and geriatric population study in Gothenburg tinnitus in old age is related more to hearing loss than to ageing. There are no simple correlations between exposure to noise during the active years and tinnitus in old age.


Roles of birds and bats in early tropical-forest restoration.
Restoration of tropical forest depended in large part on seed dispersal by fruit-eating animals that transported seeds into planted forest patches. We tested effectiveness of dispersal agents as revealed by established recruits of tree and shrub species that bore seeds dispersed by birds, bats, or both. We documented restoration of dispersal processes over the first 76 months of experimental restoration in southern Mexico. Mixed-model repeated-measures randomized-block ANOVAs of seedlings recruited into experimental controls and mixed-species plantings from late-secondary and mature forest indicated that bats and birds played different roles in the first years of a restoration process. Bats dispersed pioneer tree and shrub species to slowly regenerating grassy areas, while birds mediated recruitment of later-successional species into planted stands of trees and to a lesser extent into controls. Of species of pioneer trees and shrubs established in plots, seven were primarily dispersed by birds, three by bats and four by both birds and bats. Of later-successional species recruited past the seedling stage, 13 were of species primarily dispersed by birds, and six were of species dispersed by both birds and bats. No later-successional species primarily dispersed by bats established in control or planted plots. Establishment of recruited seedlings was ten-fold higher under cover of planted trees than in grassy controls. Even pre-reproductive trees drew fruit-eating birds and the seeds that they carried from nearby forest, and provided conditions for establishment of shade-tolerant tree species. Overall, after 76 months of cattle exclusion, 94% of the recruited shrubs and trees in experimental plots were of species that we did not plant.


SBE6: a novel long-range enhancer involved in driving sonic hedgehog expression in neural progenitor cells.
The expression of genes with key roles in development is under very tight spatial and temporal control, mediated by enhancers. A classic example of this is the sonic hedgehog gene (Shh), which plays a pivotal role in the proliferation, differentiation and survival of neural progenitor cells both in vivo and in vitro. Shh expression in the brain is tightly controlled by several known enhancers that have been identified through genetic, genomic and functional assays. Using chromatin profiling during the differentiation of embryonic stem cells to neural progenitor cells, here we report the identification of a novel long-range enhancer for Shh-Shh-brain-enhancer-6 (SBE6)-that is located 100 kb upstream of Shh and that is required for the proper induction of Shh expression during this differentiation programme. This element is capable of driving expression in the vertebrate brain. Our study illustrates how a chromatin-focused approach, coupled to in vivo testing, can be used to identify new cell-type specific cis-regulatory elements, and points to yet further complexity in the control of Shh expression during embryonic brain development.


Enhanced production of reactive oxygen species by gadolinium oxide nanoparticles under core-inner-shell excitation by proton or monochromatic X-ray irradiation: implication of the contribution from the interatomic de-excitation-mediated nanoradiator effect to dose enhancement.
Core-inner-valence ionization of high-Z nanoparticle atomic clusters can de-excite electrons through various interatomic de-excitation processes, thereby leading to the ionization of both directly exposed atoms and adjacent neutral atoms within the nanoparticles, and to an enhancement in photon-electron emission, which is termed the nanoradiator effect. To investigate the nanoradiator-mediated dose enhancement in the radio-sensitizing of high-Z nanoparticles, the production of reactive oxygen species (ROS) was measured in a gadolinium oxide nanoparticle (Gd-oxide NP) solution under core-inner-valence excitation of Gd with either 50 keV monochromatic synchrotron X-rays or 45 MeV protons. This measurement was compared with either a radiation-only control or a gadolinium-chelate magnetic resonance imaging contrast agent solution containing equal amounts of gadolinium as the separate atomic species in which Gd-Gd interatomic de-excitations are absent. Ionization excitations followed by ROS measurements were performed on nanoparticle-loaded cells or aqueous solutions. Both photoexcitation and proton impact produced a dose-dependent enhancement in the production of ROS by a range of factors from 1.6 to 1.94 compared with the radiation-only control. Enhanced production of ROS, by a factor of 1.83, was observed from Gd-oxide NP atomic clusters compared with the Gd-chelate molecule, with a Gd concentration of 48 μg/mL in the core-level photon excitation, or by a factor of 1.82 under a Gd concentration of 12 μg/mL for the proton impact at 10 Gy (p < 0.02). The enhanced production of ROS in the irradiated nanoparticles suggests the potential for additional therapeutic dose enhancements in radiation treatment via the potent Gd-Gd interatomic de-excitation-driven nanoradiator effect.


Red blood cell membrane phosphatidylethanolamine fatty acid content in various forms of retinitis pigmentosa.
In order to test the hypothesis that retinitis pigmentosa (RP) is associated with fatty acid abnormalities within cell membrane phospholipids, red blood cell membrane (RBC) phosphatidylethanolamine (PE) fatty acid content (% of total fatty acids) was measured using high performance liquid chromatography and capillary column gas chromatography in 155 patients from separate families with different genetic types of RP and 101 normal subjects. After controlling for the effects of age and sex, patients with all genetic forms of RP had significantly (P < 0.001) reduced mean RBC PE 22:6 omega 3 (n-3) (docosahexaenoic acid, DHA) content, and significantly (P < 0.001) elevated mean RBC PE dimethyl acetal (DMA) forms of 16:0, 18:0, and 18:1 omega 9 (n-9) as compared with normal subjects. RBC PE content of 22:5 omega 3 (n-3) (a precursor to DHA) and 18:2 omega 6 (n-6) (the major dietary essential fatty acid) were not significantly different in RP than in controls. Analysis by genetic types of RP showed that the mean RBC PE DHA percentages were significantly reduced by 24%, 14%, 30%, and 17%, respectively, in dominant, recessive, X-linked, and isolate forms of RP. The relative amounts of plasmalogens as indicated by DMA forms of 16:0 and 18:0 were significantly (P < 0.01) increased in dominant (by 33% and 25%), recessive (by 36% and 25%), and isolate cases (by 32% and 26%) of RP as compared with normal subjects. No such differences were seen in X-linked cases versus controls. Our data indicate that RBC PE DHA content is decreased in all genetic types of RP patients as compared to control subjects, and that RBC PE plasmalogens are increased in dominant, recessive, and isolate forms of RP. These data raise the possibility that membrane phospholipid fatty acid abnormalities may contribute to the pathogenesis of RP.


Perturbation of platelet adhesion to endothelial cells by plasminogen activation in vitro.
To investigate whether the endothelium-platelet interactions may be altered by plasminogen activation, cultured human umbilical vein endothelial cells (ECs) were treated with tissue-type plasminogen activator (t-PA) in the presence of plasminogen, and platelet adhesion to ECs was subsequently measured by using a tapered flow chamber. Our results demonstrated that platelets adhered more readily to t-PA treated EC monolayer than to the control monolayer at all shear stress levels tested. This phenomenon was treatment time-dependent and dose-dependent, and it could be blocked by adding plasmin inhibitors, such as epsilon-amino caproic acid and aprotinin. Adherent platelets on t-PA treated EC monolayer underwent more severe shape change than those on the control monolayer. While the extracellular matrix directly treated with t-PA attracted less platelets than the control matrix did, platelet adhesion to the matrix that was produced by t-PA-treated ECs was unaltered. These data suggest that t-PA treatment on ECs compromised antiplatelet-adhesion capability on their apical surface without altering the reactivity of their extracellular matrix towards platelets.


Construct validity, ecological validity and acceptance of self-administered online neuropsychological assessment in adults.
Objective: The goal of this project was to explore the initial psychometric properties (construct and ecological validity) of self-administered online (SAO) neuropsychological assessment (using the www.testmybrain.org platform), compared to traditional testing, in a clinical sample, as well as to evaluate participant acceptance. SAO assessment has the potential to expand the reach of in-person neuropsychological assessment approaches.Method: Counterbalanced, within-subjects design comparing SAO performance to in-person performance in adults with diabetes with and without Chronic Kidney Disease (CKD). Forty-nine participants completed both assessment modalities (type 1 diabetes N = 14, type 2 diabetes N = 35; CKD N = 18).Results: Associations between SAO and analogous in-person tests were adequate to good (r = 0.49-0.66). Association strength between divergent cognitive tests did not differ between SAO versus in-person tests. SAO testing was more strongly associated with age than in-person testing (age R2=0.54 versus 0.23), while prediction of education, HbA1c, and estimated glomerular filtration rate (eGFR) did not differ significantly between test modalities (education R2=0.37 versus 0.30; HbA1c R2=0.20 versus 0.12; eGFR R2 = 0.41 versus 0.33). Associations with measures of everyday functioning were also similar (Functional Activities Questionnaire R2=0.08 versus 0.07; Neuro-QoL R2=0.14 versus 0.16; Diabetes Self-Management Questionnaire R2=0.19 versus 0.19).Conclusions: The selected SAO neuropsychological tests had acceptable construct validity (including divergent, convergent, and criterion-related validity), and similar ecological validity to that of traditional testing. These SAO assessments were acceptable to participants and appear appropriate for use in research applications, although further research is needed to better understand the strengths and weaknesses in other clinical populations.


North Atlantic salinity as a predictor of Sahel rainfall.
Water evaporating from the ocean sustains precipitation on land. This ocean-to-land moisture transport leaves an imprint on sea surface salinity (SSS). Thus, the question arises of whether variations in SSS can provide insight into terrestrial precipitation. This study provides evidence that springtime SSS in the subtropical North Atlantic ocean can be used as a predictor of terrestrial precipitation during the subsequent summer monsoon in Africa. Specifically, increased springtime SSS in the central to eastern subtropical North Atlantic tends to be followed by above-normal monsoon-season precipitation in the African Sahel. In the spring, high SSS is associated with enhanced moisture flux divergence from the subtropical oceans, which converges over the African Sahel and helps to elevate local soil moisture content. From spring to the summer monsoon season, the initial water cycling signal is preserved, amplified, and manifested in excessive precipitation. According to our analysis of currently available soil moisture data sets, this 3-month delay is attributable to a positive coupling between soil moisture, moisture flux convergence, and precipitation in the Sahel. Because of the physical connection between salinity, ocean-to-land moisture transport, and local soil moisture feedback, seasonal forecasts of Sahel precipitation can be improved by incorporating SSS into prediction models. Thus, expanded monitoring of ocean salinity should contribute to more skillful predictions of precipitation in vulnerable subtropical regions, such as the Sahel.


LINE-1 amplification accompanies explosive genome repatterning in rodents.
Transposable elements (TEs) sometimes induce karyotypic changes following recombination, breakage and rearrangement. We used FISH and Southern blot analyses to investigate the amount and distribution of LINE-1 retrotransposons in rodents (genus Taterillus, Muridae, Gerbillinae) that have recently undergone an important genome repatterning. Our results were interpreted in a known phylogenetic framework and clearly showed that LINE-1 elements were greatly amplified and non-randomly distributed in the most rearranged karyotypes. A comparison between FISH and conventional banding patterns provided evidence that LINE-1 insertion sites and chromosome breakpoints were not strongly correlated, thus suggesting that LINE-1 amplification subsequently accompanied Taterillus chromosome evolution. Similar patterns are observed in some cases of genomic stresses (hybrid genomes, cancer and DNA-damaged cells) and usually associated with DNA hypomethylation. We propose that intensively repatterned genomes face transient stress phases during which some epigenetic features, such as DNA methylation, are relaxed, thus allowing TE amplification.


Patterns of self-reported behaviour change associated with receiving voluntary counselling and testing in a longitudinal study from Manicaland, Zimbabwe.
Voluntary counselling and testing (VCT) is promoted as a potential HIV prevention measure. We describe trends in uptake of VCT for HIV, and patterns of subsequent behaviour change associated with receiving VCT in a population-based open cohort in Manicaland, Zimbabwe. The relationship between receipt of VCT and subsequent reported behaviour was analysed using generalized linear models with random effects. At the third survey, 8.6% of participants (1,079/12,533), had previously received VCT. Women who received VCT, both those positive and negative, reduced their reported number of new partners. Among those testing positive, this risk reduction was enhanced with time since testing. Among men, no behavioural risk reduction associated with VCT was observed. Significant increases in consistent condom use, with regular or non-regular partners, following VCT, were not observed. This study suggests that, among women, particularly those who are infected, behavioural risk reduction does occur following VCT.


Fuelling walking and cycling: human powered locomotion is associated with non-negligible greenhouse gas emissions.
Reducing motorized transport and increasing active transport (i.e. transport by walking, cycling and other active modes) may reduce greenhouse gas (GHG) emissions and improve health. But, active modes of transport are not zero emitters. We aimed to quantify GHG emissions from food production required to fuel extra physical activity for walking and cycling. We estimate the emissions (in kgCO2e) per kilometre travelled for walking and cycling from energy intake required to compensate for increased energy expenditure, and data on food-related GHG emissions. We assume that persons who shift from passive modes of transport (e.g. driving) have increased energy expenditure that may be compensated with increased food consumption. The GHG emissions associated with food intake required to fuel a kilometre of walking range between 0.05 kgCO2e/km in the least economically developed countries to 0.26 kgCO2e/km in the most economically developed countries. Emissions for cycling are approximately half those of walking. Emissions from food required for walking and cycling are not negligible in economically developed countries which have high dietary-related emissions. There is high uncertainty about the actual emissions associated with walking and cycling, and high variability based on country economic development. Our study highlights the need to consider emissions from other sectors when estimating net-emissions impacts from transport interventions.


A technique based on laser Doppler flowmetry and photoplethysmography for simultaneously monitoring blood flow at different tissue depths.
The aim of this study was to validate a non-invasive optical probe for simultaneous blood flow measurement at different vascular depths combining three photoplethysmography (PPG) channels and laser Doppler flowmeter (LDF). Wavelengths of the PPG were near-infrared 810 nm with source-to-detector separation of 10 and 25 mm, and green 560 nm with source-to-detector separation of 4 mm. The probe is intended for clinical studies of pressure ulcer aetiology. The probe was placed over the trapezius muscle, and depths from the skin to the trapezius muscle were measured using ultrasound and varied between 3.8 and 23 mm in the 11 subjects included. A provocation procedure inducing a local enhancement of blood flow in the trapezius muscle was used. Blood flows at rest and post-exercise were compared. It can be concluded that this probe is useful as a tool for discriminating between blood flows at different vascular tissue depths. The vascular depths reached for the different channels in this study were at least 23 mm for the near-infrared PPG channel (source-to-detector separation 25 mm), 10-15 mm for the near-infrared PPG channel (separation 10 mm), and shallower than 4 mm for both the green PPG channel (separation 4 mm) and LDF.


Control of daughter cell fates during asymmetric division: interaction of Numb and Notch.
During development of the Drosophila peripheral nervous system, a sensory organ precursor (SOP) cell undergoes rounds of asymmetric divisions to generate four distinct cells of a sensory organ. Numb, a membrane-associated protein, is asymmetrically segregated into one daughter cell during SOP division and acts as an inherited determinant of cell fate. Here, we show that Notch, a transmembrane receptor mediated cell-cell communication, functions as a binary switch in cell fate specification during asymmetric divisions of the SOP and its daughter cells in embryogenesis. Moreover, numb negatively regulates Notch, probably through direct protein-protein interaction that requires the phosphotyrosine-binding (PTB) domain of Numb and either the RAM23 region or the very C-terminal end of Notch. Notch then positively regulates a transcription factor encoded by tramtrack (ttk). This leads to Ttk expression in the daughter cell that does not inherit Numb. Thus, the inherited determinant Numb bestows a bias in the machinery for cell-cell communication to allow the specification of distinct daughter cell fates.


Membrane activity profiling of small molecule B. subtilis growth inhibitors utilizing novel duel-dye fluorescence assay.
Small molecule disruption of the bacterial membrane is both a challenge and interest for drug development. While some avoid membrane activity due to toxicity issues, others are interested in leveraging the effects for new treatments. Existing assays are available for measuring disruption of membrane potential or membrane permeability, two key characteristics of the bacterial membrane, however they are limited in their ability to distinguish between these properties. Here, we demonstrate a high throughput assay for detection and characterization of membrane active compounds. The assay distinguishes the effect of small molecules on either the membrane potential or membrane permeability using the fluorescent dyes TO-PRO-3 iodide and DiOC2(3) without the need for secondary assays. We then applied this assay to a library of 3520 synthetic molecules previously shown to inhibit growth of B. subtilis in order to determine the frequency of membrane activity within such a biologically active library. From the library, we found 249 compounds that demonstrated significant membrane activity, suggesting that synthetic libraries of this kind do not contain a plurality of membrane active molecules.


Statistical downscaling with spatial misalignment: Application to wildland fire PM2.5 concentration forecasting.
Fine particulate matter, PM2.5, has been documented to have adverse health effects and wildland fires are a major contributor to PM2.5 air pollution in the US. Forecasters use numerical models to predict PM2.5 concentrations to warn the public of impending health risk. Statistical methods are needed to calibrate the numerical model forecast using monitor data to reduce bias and quantify uncertainty. Typical model calibration techniques do not allow for errors due to misalignment of geographic locations. We propose a spatiotemporal downscaling methodology that uses image registration techniques to identify the spatial misalignment and accounts for and corrects the bias produced by such warping. Our model is fitted in a Bayesian framework to provide uncertainty quantification of the misalignment and other sources of error. We apply this method to different simulated data sets and show enhanced performance of the method in presence of spatial misalignment. Finally, we apply the method to a large fire in Washington state and show that the proposed method provides more realistic uncertainty quantification than standard methods.


FAAH genetic variation enhances fronto-amygdala function in mouse and human.
Cross-species studies enable rapid translational discovery and produce the broadest impact when both mechanism and phenotype are consistent across organisms. We developed a knock-in mouse that biologically recapitulates a common human mutation in the gene for fatty acid amide hydrolase (FAAH) (C385A; rs324420), the primary catabolic enzyme for the endocannabinoid anandamide. This common polymorphism impacts the expression and activity of FAAH, thereby increasing anandamide levels. Here, we show that the genetic knock-in mouse and human variant allele carriers exhibit parallel alterations in biochemisty, neurocircuitry and behaviour. Specifically, there is reduced FAAH expression associated with the variant allele that selectively enhances fronto-amygdala connectivity and fear extinction learning, and decreases anxiety-like behaviours. These results suggest a gain of function in fear regulation and may indicate for whom and for what anxiety symptoms FAAH inhibitors or exposure-based therapies will be most efficacious, bridging an important translational gap between the mouse and human.


Advances in classification of EEG signals via evolving fuzzy classifiers and dependant multiple HMMs.
Two novel approaches to the problem of brain signals (electroencephalogram (EEG)) classification are introduced in the paper. The first method is based on a modular probabilistic network architecture that employs multiple dependant hidden Markov models (DM-HMM-D) on the input features (channels). The second method, eClass, is based on an on-line evolvable fuzzy rule base of EEG signal prototypes that represent each class and take into consideration the spatial proximity between input signals. Both approaches use supervised learning but differ in their mode of operation. eClass is designed recursively, on-line, and has an evolvable structure, while DM-HMM-D is trained off-line, in a block-based mode, and has a fixed architecture. Both methods have been extensively tested on real EEG data that is recorded during several experimental sessions involving a single female subject who is exposed to mild pain induced by a laser beam. Experimental results illustrate the viability of the proposed approaches and their potential in solving similar classification problems.


Histone-like protein: a novel method for measuring stress in fish.
We assessed the effect of chronic stress using a group of potent, broad-spectrum antimicrobial polypeptides, called histone-like proteins (HLPs), which appear to be an important component of non-specific immunity in channel catfish Ictalurus punctatus skin. An enzyme-linked immunosorbent assay (ELISA) was developed to measure the predominant HLP (HLP-1) in channel catfish skin. Catfish were then exposed to a chronic stress consisting of overcrowding and elevated ammonia. Healthy unstressed fish had consistently high HLP-1 levels, but fish that had been stressed for 1 wk had significantly depressed HLP-1 levels; HLP-1 levels declined further in fish stressed for 3 or 4 wk. The time-dependent decline in HLP-1 levels was not accompanied by any gross signs of disease. In contrast to HLP-1 levels, antibacterial activity in the skin was significantly greater in fish stressed for 1 wk compared with unstressed fish; in addition, antibacterial activity was the same in fish that were unstressed or stressed for 3 or 4 wk. This suggests that other antibiotics besides HLP-1 may be induced in the skin, especially during early stages of stress, that may compensate for depressed HLP-1 levels. Our results indicate that chronic stress has a significant suppressive effect on HLP-1 levels in channel catfish skin. The reduction of HLP-1 in the absence of clinical signs of disease, combined with evidence that its levels are not affected by the acute stressors of capture or sampling, suggests that HLP levels may be a promising indicator for monitoring fish health.


[Informed consent for genetic information management in Colombian law].
Deciphering the human genome has allowed the development of new health care ways which have been generically referred to as genomic medicine. However, this new knowledge is not devoid of risks related to the access and use of genetic information, involving not only the individual, but also the biological family, ethnic group and community to which he/she belong to. This affects both individual and collective rights, which must be protected by law. This manuscript reviews the Colombian law, specifically in terms of the access and use of genetic information for diagnostic and treatment purposes, focusing on the informed consent process. The concept of genetic information, the risks and benefits associated with genetic data management and the description and critical analysis of the present Colombian regulations were thus reviewed. As a conclusion, the juridical framework of Colombia shows deficiencies regarding the protection of both individual and collective rights linked to the access and use of genetic information.


Incidence of metallo-beta-lactamase-producing Pseudomonas aeruginosa in diabetes and cancer patients.
Metallo-beta-lactamase (MBL)-producing Pseudomonas aeruginosa strains have been reported to be an important cause of nosocomial infections. There is not enough information from India regarding their prevalence in diabetic and cancer patients. The present study was undertaken over a period of one year from January to December 2006 to study the incidence of MBL P. aeruginosa and the clinical outcome in diabetes and cancer patients admitted to S.L. Raheja Hospital, Mumbai. Two hundred and thirty isolates of P. aeruginosa were obtained from different samples of patients. These isolates were subjected to susceptibility testing to anti-pseudomonal drugs as per CLSI guidelines. They were further screened for the production of MBL by disc potentiation testing using EDTA-impregnated imipenem and meropenem discs. Of the 230 isolates of P. aeruginosa, 60 (26%) isolates were found resistant to carbapenems (both imipenem and meropenem) and 33 (14.3%) were found to be MBL producers. Of the 33 MBL-producing isolates, 24 (72.7%) were diabetic patients, six (18.1%) were cancer patients and three (9%) patients had both diabetes and cancer. Five (15.1%) patients responded to the combination therapy of colistin, piperacillin with tazobactam and amikacin, while 28 (84.8%) patients responded to the combination therapy of amikacin, piperacillin with tazobactam and gatifloxacin. Thus, the rapid dissemination of MBL producers is worrisome and necessitates the implementation of not just surveillance studies but also proper and judicious selection of antibiotics, especially carbapenems.


Worldwide Population Structure, Long-Term Demography, and Local Adaptation of Helicobacter pylori.
Helicobacter pylori is an important human pathogen associated with serious gastric diseases. Owing to its medical importance and close relationship with its human host, understanding genomic patterns of global and local adaptation in H. pylori may be of particular significance for both clinical and evolutionary studies. Here we present the first such whole genome analysis of 60 globally distributed strains, from which we inferred worldwide population structure and demographic history and shed light on interesting global and local events of positive selection, with particular emphasis on the evolution of San-associated lineages. Our results indicate a more ancient origin for the association of humans and H. pylori than previously thought. We identify several important perspectives for future clinical research on candidate selected regions that include both previously characterized genes (e.g., transcription elongation factor NusA and tumor necrosis factor alpha-inducing protein Tipα) and hitherto unknown functional genes.


Insect-microbe mutualism without vertical transmission: a stinkbug acquires a beneficial gut symbiont from the environment every generation.
The broad-headed bug Riptortus clavatus (Heteroptera: Alydidae) possesses a number of crypts at a posterior midgut region, which house a dense population of a bacterial symbiont belonging to the genus Burkholderia. Although the symbiont is highly prevalent (95 to 100%) in the host populations, the symbiont phylogeny did not reflect the host systematics at all. In order to understand the mechanisms underlying the promiscuous host-symbiont relationship despite the specific and prevalent association, we investigated the transmission mode and the fitness effects of the Burkholderia symbiont in R. clavatus. Inspection of eggs and a series of rearing experiments revealed that the symbiont is not vertically transmitted but is environmentally acquired by nymphal insects. The Burkholderia symbiont was present in the soil of the insect habitat, and a culture strain of the symbiont was successfully isolated from the insect midgut. Rearing experiments by using sterilized soybean bottles demonstrated that the cultured symbiont is able to establish a normal and efficient infection in the host insect, and the symbiont infection significantly improves the host fitness. These results indicated that R. clavatus postnatally acquires symbiont of a beneficial nature from the environment every generation, uncovering a previously unknown pathway through which a highly specific insect-microbe association is maintained. We suggest that the stinkbug-Burkholderia relationship may be regarded as an insect analogue of the well-known symbioses between plants and soil-associated microbes, such as legume-Rhizobium and alder-Frankia relationships, and we discuss the evolutionary relevance of the mutualistic but promiscuous insect-microbe association.


A comparison of measles and canine distemper virus polypeptides.
The polypeptides induced by canine distemper virus (CDV) strains have been characterized by polyacrylamide slab gel electrophoresis of infected cell lysates labelled with 35S-methionine, 14C-amino acids, 3H-glucosamine and 3H-mannose, or 32P-orthophosphate. Seven virus-induced polypeptides have been asigned the following nomenclature and mol. wt.: a large polypeptide L (180,000); a large glycoprotein G (77,000); a nucleocapsid-associated protein P (73,000); the nucleocapsid protein N (60,000); the smaller glycoprotein F0 (59,000); a membrane protein M (35,000) and a small polypeptide S (15,000). During pulse-chase experiments with 3H-glucosamine and 14C-amino acids the intensity of the F0 band decreases and that of the F1 and F2 bands increases; the H polypeptide band becomes more diffuse and the S-protein disappears. The N- and P- but not the M-proteins have been found to be phosphorylated. The polypeptide pattern of the Onderstepoort strain of CDV has been compared with that of two other CDV and with 17 measles and subactue sclerosing panencephalitis (SSPE) strains. Differences in the mobilities of various polypeptides have been observed between CDV and measles and SSPE strains; however, the only consistent difference is the mol. wt. of the M-protein of CDV strains which is smaller by 2000 than that of MV and this may be a biochemical marker to distinguish CDV from measles and SSPE virus strains.


Visceral Adipose Tissue Inflammatory Factors (TNF-Alpha, SOCS3) in Gestational Diabetes (GDM): Epigenetics as a Clue in GDM Pathophysiology.
Gestational diabetes (GDM) is among the most challenging diseases in westernized countries, affecting mother and child, immediately and in later life. Obesity is a major risk factor for GDM. However, the impact visceral obesity and related epigenetics play for GDM etiopathogenesis have hardly been considered so far. Our recent findings within the prospective 'EaCH' cohort study of women with GDM or normal glucose tolerance (NGT), showed the role, critical factors of insulin resistance (i.e., adiponectin, insulin receptor) may have for GDM pathophysiology with epigenetically modified expression in subcutaneous (SAT) and visceral (VAT) adipose tissues. Here we investigated the expression and promoter methylation of key inflammatory candidates, tumor necrosis factor-alpha (TNF-α) and suppressor of cytokine signaling 3 (SOCS3) in maternal adipose tissues collected during caesarian section (GDM, n = 19; NGT, n = 22). The mRNA expression of TNF-α and SOCS3 was significantly increased in VAT, but not in SAT, of GDM patients vs. NGT, accompanied by specific alterations of respective promoter methylation patterns. In conclusion, we propose a critical role of VAT and visceral obesity for the pathogenesis of GDM, with epigenetic alterations of the expression of inflammatory factors as a potential factor.


Outcome of Ventriculosubgaleal Shunt in the Management of Infectious and Non-infectious Hydrocephalus in Pre-term Infants.
Hydrocephalus in premature infants is an onerous disease. In such situations, choosing the best option for cerebrospinal fluid (CSF) diversion is difficult. Ventriculosubgaleal shunt is an effective method of temporary CSF diversion in such situations. In this retrospective study, we compare the outcome of ventriculosubgaleal shunt in premature infants with hydrocephalus of infectious and noninfectious etiology. All premature children with hydrocephalus secondary to various etiologies who underwent ventriculosubgaleal shunt were studied. The participants were grouped into two depending upon the etiology of hydrocephalus: Group 1 (infectious) and Group 2 (non-infectious). The primary outcome was a successful conversion to ventriculoperitoneal shunt (VPS) and the secondary outcome was mortality. Data were entered into statistical software SPSS version 16 and appropriate statistical analysis was performed to conclude any statistical significance between groups. The study included 16 infants among whom 9 were in the infectious group and 7 in the non-infectious group. Primary end point of conversion to VPS was achieved in 55.5% of patients in group 1 and 85.7% in group 2. The secondary end point, i.e., mortality was observed in 44.4% of patients in group 1 and 14.2% in group 2. The average duration during which this was achieved was 40 days (range 20-60 days) in group 1 and 25 days (range 20-30 days) in group 2. Ventriculosubgaleal shunt is a safe and effective procedure in infants awaiting definitive VPS for hydrocephalus of infectious as well as noninfectious origin. There was no statistical difference in the rate of successful conversion to a permanent VPS from ventriculosubgaleal shunt in hydrocephalus of either etiologies. Complications and time for successful conversion were more in postmeningitic hydrocephalus.


Human Papillomavirus Type16- L1 VLP Production in Insect Cells.
Infection by high-risk papillomavirus is regarded as the major risk factor in the development of cervical cancer. Recombinant DNA technology allows expression of the L1 major capsid protein of HPV in different expression systems, which has intrinsic capacity to self-assemble into viral-like particles (VLP). VLPS are non-infectious, highly immunogenic and can elicit neutralizing antibodies. VLP-based HPV vaccines can prevent persistent HPV infections and cervical cancer. In this study recombinant HPV-16 L1 protein was produced in Sf9 insect cells and VLP formation was confirmed. Complete HPV-16 L1 gene was inserted into pFast HTa plasmid and transformed into DH10BAC Escherichia coli containing bacmid and helper plasmid. The recombinant Bacmid colonies turned to white and non-recombinant colonies harboring L1 gene remained blue in the presence of X-gal and IPTG in colony selection strategy. To confirm the recombinant bacmid production, PCR was applied using specific L1 primers. To produce recombinant baculovirus, the recombinant bacmid DNA was extracted and transfected into Sf9 cells using Cellfectin. The expression of L1 in Sf9 cells was identified through SDS-PAGE and western blot analysis using specific L1 monoclonal antibody. Self-assembled HPV16L-VLPs in Sf9 cells was confirmed by electron microscopy. The recombinant protein L1 was predominantly ~60 KD in SDS-PAGE with distinct immunoreactivity in western blot analysis and formed VLPS as confirmed by electron microscopy. Application of recombinant baculovirus containing HPV-16 L1 gene will certainly prove to be a constructive tool in production of VLPs for prophylactic vaccine development as well as diagnostic tests.


Different patterns of chronic hypoxia lead to hierarchical adaptive mechanisms in goldfish metabolism.
Some hypoxia-tolerant species, such as goldfish, experience intermittent and severe hypoxia in their natural habitat, causing them to develop multiple physiological adaptations. However, in fish, the metabolic impact of regular hypoxic exposure on swimming performance in normoxia is less well understood. Therefore, we experimentally tested whether chronic exposure to constant (30 days at 10% air saturation) or intermittent hypoxia (3 h in normoxia and 21 h in hypoxia, 5 days a week) would result in similar metabolic and swimming performance benefits after reoxygenation. Moreover, half of the normoxic and intermittent hypoxic fish were put on a 20-day normoxic training regime. After these treatments, metabolic rate (standard and maximum metabolic rates: SMR and MMR) and swimming performance [critical swimming speed (Ucrit) and cost of transport (COT)] were assessed. In addition, enzyme activities [citrate synthase (CS), cytochrome c oxidase (COX) and lactate dehydrogenase (LDH)] and mitochondrial respiration were examined in red muscle fibres. We found that acclimation to constant hypoxia resulted in (1) metabolic suppression (-45% SMR and -27% MMR), (2) increased anaerobic capacity (+117% LDH), (3) improved swimming performance (+80% Ucrit, -71% COT) and (4) no changes at the mitochondrial level. Conversely, the enhancement of swimming performance was reduced following acclimation to intermittent hypoxia (+45% Ucrit, -41% COT), with a 55% decrease in aerobic scope, despite a significant increase in oxidative metabolism (+201% COX, +49% CS). This study demonstrates that constant hypoxia leads to the greatest benefit in swimming performance and that mitochondrial metabolic adjustments only provide minor help in coping with hypoxia.


Correct biological timing in Arabidopsis requires multiple light-signaling pathways.
Circadian oscillators provide rhythmic temporal cues for a range of biological processes in plants and animals, enabling anticipation of the day/night cycle and enhancing fitness-associated traits. We have used engineering models to understand the control principles of a plant's response to seasonal variation. We show that the seasonal changes in the timing of circadian outputs require light regulation via feed-forward loops, combining rapid light-signaling pathways with entrained circadian oscillators. Linear time-invariant models of circadian rhythms were computed for 3,503 circadian-regulated genes and for the concentration of cytosolic-free calcium to quantify the magnitude and timing of regulation by circadian oscillators and light-signaling pathways. Bioinformatic and experimental analysis show that rapid light-induced regulation of circadian outputs is associated with seasonal rephasing of the output rhythm. We identify that external coincidence is required for rephasing of multiple output rhythms, and is therefore important in general phase control in addition to specific photoperiod-dependent processes such as flowering and hypocotyl elongation. Our findings uncover a fundamental design principle of circadian regulation, and identify the importance of rapid light-signaling pathways in temporal control.


Predictors of recurrence, early treatment failure and death from Staphylococcus aureus bacteraemia: Observational analyses within the ARREST trial.
Adjunctive rifampicin did not reduce failure/recurrence/death as a composite endpoint in the ARREST trial of Staphylococcus aureus bacteraemia, but did reduce recurrences. We investigated clinically-defined 14-day treatment failure, and recurrence and S. aureus-attributed/unattributed mortality by 12-weeks to further define their predictors. A post-hoc exploratory analysis using competing risks models was conducted to identify sub-groups which might benefit from rifampicin. A points-based recurrence risk score was developed and used to compare rifampicin's benefits. Recurrence was strongly associated with liver and renal failure, diabetes and immune-suppressive drugs (p < 0.005); in contrast, failure and S. aureus-attributed mortality were associated with older age and higher neutrophil counts. Higher SOFA scores predicted mortality; higher Charlson scores and deep-seated initial infection focus predicted failure. Unexpectedly, recurrence risk increased with increasing BMI in placebo (p = 0.04) but not rifampicin (p = 0.60) participants (pheterogeneity = 0.06). A persistent focus was judged the primary reason for recurrence in 23(74%). A 5-factor risk score based on BMI, Immunosuppression, Renal disease, Diabetes, Liver disease (BIRDL) strongly predicted recurrence (p < 0.001). Rifampicin reduces recurrences overall; those with greatest absolute risk reductions were identified using a simple risk score. Source control and adequate duration of antibiotic therapy remain essential to prevent recurrence and improve outcomes.


Specific real-time PCR for simultaneous detection and identification of Legionella pneumophila serogroup 1 in water and clinical samples.
Legionella pneumophila, a bacterium that replicates within aquatic amoebae and persists in the environment as a free-living microbe, is the causative agent of Legionnaires' disease. Among the many Legionella species described, L. pneumophila is associated with 90% of human disease, and within the 15 serogroups (Sg), L. pneumophila Sg1 causes more than 84% of Legionnaires' disease worldwide. Thus, rapid and specific identification of L. pneumophila Sg1 is of the utmost importance for evaluation of the contamination of collective water systems and the risk posed. Previously we had shown that about 20 kb of the 33-kb locus carrying the genes coding for the proteins involved in lipopolysaccharide biosynthesis (LPS gene cluster) by L. pneumophila was highly specific for Sg1 strains and that three genes (lpp0831, wzm, and wzt) may serve as genetic markers. Here we report the sequencing and comparative analyses of this specific region of the LPS gene cluster in L. pneumophila Sg6, -10, -12, -13, and -14. Indeed, the wzm and wzt genes were present only in the Sg1 LPS gene cluster, which showed a very specific gene content with respect to the other five serogroups investigated. Based on this observation, we designed primers and developed a classical and a real-time PCR method for the detection and simultaneous identification of L. pneumophila Sg1 in clinical and environmental isolates. Evaluation of the selected primers with 454 Legionella and 38 non-Legionella strains demonstrated 100% specificity. Sensitivity, specificity, and predictive values were further evaluated with 209 DNA extracts from water samples of hospital water supply systems and with 96 respiratory specimens. The results showed that the newly developed quantitative Sg1-specific PCR method is a highly specific and efficient tool for the surveillance and rapid detection of high-risk L. pneumophila Sg1 in water and clinical samples.


Coffee and tea consumption in the Scottish Heart Health Study follow up: conflicting relations with coronary risk factors, coronary disease, and all cause mortality.
To relate habitual (cups per day) tea and coffee consumption to conventional coronary risk factors and subsequent risk of coronary heart disease and death. Cohort study. Nationwide random population study. Over 11,000 men and women aged 40-59 who took part in the Scottish Heart Health Study lifestyle and risk factor survey in 1984-87. Participants were followed up to the end of 1993, an average of 7.7 years, for all cause mortality, coronary death, or any major coronary event (death, non-fatal infarction or coronary artery surgery). Cox's proportional hazards regression model was used to estimate the hazard in consumers of tea and coffee relative to the zero consumption group, both before and after correction for other factors. Coffee and tea consumption showed a strong inverse relation. For many conventional risk factors, coffee showed a weak, but beneficial, gradient with increasing consumption, whereas increasing tea consumption showed the reverse. Increasing coffee consumption was associated with beneficial effects for mortality and coronary morbidity, whereas tea showed the opposite. Adjusting for age and social class had some effect in reducing associations. Multiple adjustment for other risk factors removed the associations for tea and most of those for coffee although there was a residual benefit of coffee consumption in avoiding heart disease among men. The epidemiological differences shown in this study occurred despite the pharmacological similarities between tea and coffee. Either they differ more than is realised, or they identify contrasting associated lifestyle and health risks, for which this multiple adjustment was inadequate.


Spinal anesthesia mediates improved early function and pain relief following surgical repair of ankle fractures.
To our knowledge, no study to date has compared the use of spinal and general anesthesia in patients undergoing operative fixation of an unstable ankle fracture. The purpose of this study was to assess the effects of anesthesia type on postoperative pain and function in a large cohort of patients. Between October 2000 and November 2006, 501 patients who underwent surgical fixation of an unstable ankle fracture were followed prospectively. Patients receiving spinal anesthesia were compared with a cohort who received general anesthesia. All patients were evaluated at three, six, and twelve months postoperatively with use of standardized, validated general and limb-specific outcome instruments. Standard and multivariable analyses comparing outcomes at these intervals were performed. Four hundred and sixty-six patients (93%) who had been followed for a minimum of one year met the inclusion criteria. Compared with the general anesthesia group, the spinal anesthesia group had a greater mean age (p = 0.005), higher classification on the American Society of Anesthesiologists system (p = 0.03), and a greater number of patients with diabetes (p = 0.02). There was no difference in sex distribution between the groups. At three months, patients who received spinal anesthesia had significantly better pain scores (p = 0.03) and total scores on the American Orthopaedic Foot and Ankle Society outcome instrument (p = 0.02). At six months, patients in the spinal anesthesia group continued to have better pain scores (p = 0.04), but there was no longer a difference in total scores (p = 0.06). At twelve months, no difference was detected between the groups in terms of functional or pain scores. There was no difference in complication rates between the groups. Patients who undergo fixation of an ankle fracture under spinal anesthesia seem to experience less pain and have better function in the early postoperative period. We recommend that, unless there is a specific contraindication, patients should be offered spinal anesthesia when undergoing operative fixation of an ankle fracture.


Transmission of respiratory viruses when using public ground transport: A rapid review to inform public health recommendations during the COVID-19 pandemic.
In response to the COVID-19 pandemic, numerous countries worldwide declared national states of emergency and implemented interventions to minimise the risk of transmission among the public. Evidence was needed to inform strategies for limiting COVID-19 transmission on public transport. On 20 March 2020, we searched MEDLINE, CENTRAL, Web of Science and the World Health Organization's database of 'Global research on coronavirus disease (COVID-19)' to conduct a rapid review on interventions that reduce viral transmission on public ground transport. After screening 74 records, we identified 4 eligible studies. These studies suggest an increased risk of viral transmission with public transportation use that may be reduced with improved ventilation. International and national guidelines suggest the following strategies: keep the public informed, stay at home when sick, and minimise public transport use. Where use is unavoidable, environmental control, respiratory etiquette and hand hygiene are recommended, while a risk-based approach needs to guide the use of non-medical masks.


Low birthweight infants and total parenteral nutrition immediately after birth. II. Randomised study of biochemical tolerance of intravenous glucose, amino acids, and lipid.
This randomised study aimed to compare the biochemical tolerance of three parenteral regimens administered during the first 48 hours of life. Twenty nine infants were randomised to either: (a) glucose 10%; (b) glucose 10%/amino acids; (c) glucose 10%/amino acids/lipid. Blood samples for plasma amino acid profiles, cholesterol, and triglyceride concentrations were taken on arrival in the neonatal unit and again between 36 and 48 hours of life. Arterial or capillary blood gas analysis and blood glucose estimates were performed routinely during the first 48 hours of life. There was a sharp decline in plasma amino acid concentrations in the group following (a) compared with the two groups following (b) and (c) regimens. In all groups plasma triglyceride and cholesterol were not significantly different before and after 48 hours of lipid infusion. Peak mean (SE) bilirubin concentrations (203 (12) v 181 (19) v 220 (20) mumol/l) and the need for phototherapy (nine v eight v five infants) were similar for each of the groups. Hypoglycaemia occurred most frequently during the (b) regimen and least commonly in the (c) group. There are potential health gains from giving parenteral nutrition to low birthweight infants immediately after birth, and this study indicates that restriction of nutritional intake immediately after birth in preterm infants may cause significant metabolic disturbance. This can be prevented by starting a regimen of intravenous amino acids and lipid immediately after birth.


Structural Modification of Lipopolysaccharide Conferred by mcr-1 in Gram-Negative ESKAPE Pathogens.
mcr-1 was initially reported as the first plasmid-mediated colistin resistance gene in clinical isolates of Escherichia coli and Klebsiella pneumoniae in China and has subsequently been identified worldwide in various species of the family Enterobacteriaceaemcr-1 encodes a phosphoethanolamine transferase, and its expression has been shown to generate phosphoethanolamine-modified bis-phosphorylated hexa-acylated lipid A in E. coli Here, we investigated the effects of mcr-1 on colistin susceptibility and on lipopolysaccharide structures in laboratory and clinical strains of the Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, K. pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens, which are often treated clinically by colistin. The effects of mcr-1 on colistin resistance were determined using MIC assays of laboratory and clinical strains of E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa Lipid A structural changes resulting from MCR-1 were analyzed by mass spectrometry. The introduction of mcr-1 led to colistin resistance in E. coli, K. pneumoniae, and A. baumannii but only moderately reduced susceptibility in P. aeruginosa Phosphoethanolamine modification of lipid A was observed consistently for all four species. These findings highlight the risk of colistin resistance as a consequence of mcr-1 expression among ESKAPE pathogens, especially in K. pneumoniae and A. baumannii Furthermore, the observation that lipid A structures were modified despite only modest increases in colistin MICs in some instances suggests more sophisticated surveillance methods may need to be developed to track the dissemination of mcr-1 or plasmid-mediated phosphoethanolamine transferases in general.


High serum ferritin is associated with worse outcome of patients with decompensated cirrhosis.
Studies in patients with decompensated cirrhosis showed a correlation between serum ferritin levels and patients' prognosis. Besides, red blood cell distribution width (RDW) and mean platelet volume (MPV) have been associated with the severity of hepatic function. The aim of this study was to evaluate the prognostic impact of serum ferritin and RDW/MPV in the outcome [survival, death, or liver transplantation (LT)] of patients with stable decompensated cirrhosis. Consecutive adult patients with stable decompensated cirrhosis admitted to our department between September 2010 and February 2016 were included. Serum ferritin, RDW and MPV were recorded in every patient. They were followed up and their outcome (alive, death, or LT) was evaluated. 192 consecutive patients with stable decompensated cirrhosis (142 men, age 54.2±12 years); at the end of follow up [12 (range: 1-64) months] 62 patients remained alive and 130 died or underwent LT. In multivariate analysis, serum ferritin (HR 1.001, 95%CI 1.00-1.002, P=0.005) and GFR (HR 0.96, 95%CI 0.92-0.99, P=0.035) were the only independent factors significantly associated with the outcome. Ferritin had low discriminative ability (AUC: 0.61) to the outcome yielding a sensitivity and specificity of 85.3% and 44.2%, respectively, at the best cut-off point (>55 ng/mL), while patients with ferritin >55 ng/mL (n=145) had a worse outcome compared to those with ferritin ≤55 ng/mL (n=47) (log rank P=0.001). RDW and MPV were not associated with the outcome. High serum ferritin, but not RDW/MPV, is associated with worse outcome in patients with established decompensated cirrhosis. However, further studies are needed to elucidate better this issue.


An implementation evaluation of a policy aiming to improve financial access to maternal health care in Djibo district, Burkina Faso.
To bring down its high maternal mortality ratio, Burkina Faso adopted a national health policy in 2007 that designed to boost the assisted delivery rate and improving quality of emergency obstetrical and neonatal care. The cost of transportation from health centres to district hospitals is paid by the policy. The worst-off are exempted from all fees. The objectives of this paper are to analyze perceptions of this policy by health workers, assess how this health policy was implemented at the district level, identify difficulties faced during implementation, and highlight interactional factors that have an influence on the implementation process. A multiple site case study was conducted at 6 health centres in the district of Djibo in Burkina Faso. The following sources of data were used: 1) district documents (n = 23); 2) key interviews with district health managers (n = 10), health workers (n = 16), traditional birth attendants (n = 7), and community management committees (n = 11); 3) non-participant observations in health centres; 4) focus groups in communities (n = 62); 5) a feedback session on the findings with 20 health staff members. All the activities were implemented as planned except for completely subsidizing the worst-off, and some activities such as surveys for patients and the quality assurance service team aiming to improve quality of care. District health managers and health workers perceived difficulties in implementing this policy because of the lack of clarity on some topics in the guidelines. Entering the data into an electronic database and the long delay in reimbursing transportation costs were the principal challenges perceived by implementers. Interactional factors such as relations between providers and patients and between health workers and communities were raised. These factors have an influence on the implementation process. Strained relations between the groups involved may reduce the effectiveness of the policy. Implementation analysis in the context of improving financial access to health care in African countries is still scarce, especially at the micro level. The strained relations of the providers with patients and the communities may have an influence on the implementation process and on the effects of this health policy. Therefore, power relations between actors of the health system and the community should be taken into consideration. More studies are needed to better understand the influence of power relations on the implementation process in low-income countries.


Formation of the statolith in the ctenophore Mnemiopsis leidyi.
The aboral sensory organ (apical organ) of ctenophores contains a statocyst with a single large statolith. The statolith comprises living cells (lithocytes), each containing a large membrane-bound concretion. The statolith is supported on the distal ends of four compound motile mechanoresponsive cilia (balancers) which control the beat frequencies of the eight locomotory comb rows, and thereby the orientation of animals to gravity. In Mnemiopsis leidyi and Pleurobrachia pileus, lithocytes arise in the thickened epithelial floor of the apical organ on opposite sides along the tentacular plane. Lithocytes progressively differentiate and migrate toward the apical surface where they bud off next to the bases of the balancers. New lithocytes are transported up the balancers by ciliary surface motility to form the statolith (Noda, 2013). The statolith has a superellipsoidal shape due to the rectangular arrangement of the four balancers and the addition of new lithocytes to its ends via the balancers. The size of the statolith increases with animal size, starting at the highest rate of growth in younger stages and gradually decreasing in larger animals. The total number of developing lithocytes in the epithelial floor increases rapidly in smaller animals and reaches a plateau range in larger animals. Lithocytes are therefore produced continually throughout life for enlargement of the statolith and possibly for turnover and replacement of existing lithocytes. The dome cilia enclosing the statocyst were observed to propagate slow, low-ampitude waves distally. The dome cilia may act as an undulating screen to prevent foreign objects in the seawater from being transported non-specifically up the balancers to make a defective statolith.


Absence of undergraduate medical students from psychiatric training: A study from central India.
Psychiatric problem is as common as among the 25% of the general population and 15% patients attending specialty clinic. Majority of the cases are dealt by primary care physician at the first contact, but there is only 2 weeks of clinical posting and 20 h of theory classes during MBBS course. Earlier studies have reported low attendance during the posting and theory classes. There are few studies on this aspect. On this background, the present study was planned. One hundred 8th semester students of MBBS from two medical colleges (Group A government aided college and Group B from Private college under autonomous university) from the same locality of Central Maharashtra were selected for the study after due approval by the Institutional Ethical Committee. Questionnaire was prepared by the psychiatric faculty of both institutes and administered to the individual students. The data were analyzed using suitable statistical methods (Chi-square test and t-test). Only 27% of the students in Group A attended 25%-30% of clinical posting, whereas none of them attended a single theory class. While in Group B, 1/3rd of students attended 30%-40% of clinical posting and 20%-25% of theory class. No separate examination in the subject (88%-94%) and adjustment of attendance in the other major subjects (64-86%) were cited as main reasons for nonattendance among the majority of the students in both the groups. Strict adherence to the attendance by the institutional authority, increase in the training hours and separate examination in the subject at undergraduate level may help in improving the attendance and change in attitude of the students toward psychiatry.


Effects of NO donors and synthase agonists on endothelial cell uptake of L-Arg and superoxide production.
It is commonly believed that the activity of NO synthase (NOS) solely controls NO production from its substrates, L-Arg and O(2). The Michaelis-Menten constant (K(m)) of NOS for L-Arg is in the micromolar range; cellular levels of L-Arg are much higher. However, evidence strongly suggests that cellular supply of L-Arg may become limiting and lead to reduced NO and increased superoxide anion (O(-)(2)*) formation, promoting cardiovascular dysfunction. Uptake of L-Arg into cells occurs primarily (approximately 85%) through the actions of a Na(+)-independent, carrier-mediated transporter (system y(+)). We have examined the effects of NOS agonists (substance P, bradykinin, and ACh) and NO donors (S-nitroso-N-acetyl-penicillamine and dipropylenetriamine NONOate) on transport of L-Arg into bovine aortic endothelial cells (BAEC). Our results demonstrate that NOS agonists increase y(+) transporter activity. A rapidly acting NO donor initially increases L-Arg uptake; however, after longer exposure, L-Arg uptake is suppressed. Exposure of BAEC without L-Arg to substance P and a Ca(2+) ionophore (A-23187) increased O(-)(2)* formation, which was blocked with concurrent presence of L-Arg or the NOS antagonist N(omega)-nitro-L-arginine methyl ester. We conclude that factors including NO itself control y(+) transport function and the production of NO and O(-)(2)*.


The globalization of epidemiology: introductory remarks.
We are all living in the era of globalization, and like it or not, it is going to change the way we practice epidemiology, the kinds of questions we ask, and the methods we use to answer them. Increasingly, pubic health problems are being shifted from rich countries to poor countries and from rich to poor populations within Western countries. There is increasing interest and concern about the situation in non-Western populations on the part of Western epidemiologists, with regards to collaborative research, skills transfer, and 'volunteerism' to enable the 'benefits' of Western approaches to epidemiology to be shared by the non-Western world. However, most existing collaborations benefit Western epidemiologists rather than the countries in which the research is conducted. Even when research in non-Western populations is conducted as a genuine collaboration, it can too often 'export failure' from the West. On the other hand, non-Western epidemiologists are increasingly developing new and innovative approaches to health research that are more appropriate to the global public health issues they are addressing. These include recognition of the importance of context and the importance of diversity and local knowledge, and a problem-based approach to addressing the major public health problems using appropriate technology. These debates formed the background for a plenary session on 'International Epidemiology and International Health' at the recent International Epidemiological Association (IEA) meeting in Montreal, and the papers from this session are presented here. The development of a truly global epidemiology can not only better address the public health problems in non-Western populations, but can shed light on the current limitations of epidemiology in addressing the major public health problems in the West.


Neuropilin-1 Associated Molecules in the Blood Distinguish Poor Prognosis Breast Cancer: A Cross-Sectional Study.
Circulating plasma and peripheral blood mononuclear (PBMCs) cells provide an informative snapshot of the systemic physiological state. Moreover, they provide a non-invasively accessible compartment to identify biomarkers for personalized medicine in advanced breast cancer. The role of Neuropilin-1 (NRP-1) and its interacting molecules in breast tumor tissue was correlated with cancer progression; however, the clinical impact of their systemic levels was not extensively evaluated. In this cross-sectional study, we found that circulating and tumor tissue expression of NRP-1 and circulating placental growth factor (PlGF) increase in advanced nodal and metastatic breast cancer compared with locally advanced disease. Tumor tissue expression of NRP-1 and PlGF is also upregulated in triple negative breast cancer (TNBC) compared to other subtypes. Conversely, in PBMCs, NRP-1 and its interacting molecules SEMA4A and SNAI1 are significantly downregulated in breast cancer patients compared to healthy controls, indicating a protective role. Moreover, we report differential PBMC expression profiles that correlate inversely with disease stage (SEMA4A, SNAI1, PLXNA1 and VEGFR3) and can differentiate between the TNBC and non-TNBC tumor subtypes (VEGFR3 and PLXNA1). This work supports the importance of NRP-1-associated molecules in circulation to characterize poor prognosis breast cancer and emphasizes on their role as favorable drug targets.


Exceptionally long CDR3H are not isotype restricted in bovine immunoglobulins.
Exceptionally long third complementarity determining regions of the heavy chain (CDR3H) were previously described as a specificity of bovine IgG and IgM immunoglobulins. In addition, the genomic organization of the immunoglobulin heavy chain locus remains to be elucidated with a special focus on the number of variable segments (IGHV). By analyzing the variable regions according to the isotype-specific PCR using cDNA-PCR, we were able to prove the existence of exceptional long CDR3H in all bovine isotypes. The corresponding sequences of three distinct amplicons were grouped according to the length of the CDR3H. Sequences of CDR3H possessed 5 to 10, 12 to 31 or at least 48 amino acid residues. Long and mid-length CDR3H were composed of mainly hydrophilic amino acid residues, while short CDR3H also contained hydrophobic amino acid residues. All sequences with long CDR3H were related to the germline variable segment 10. Using the current genome assembly, Bos taurus NCBI build 6.1, the genomic organization of the bovine immunoglobulin heavy-chain locus was analyzed. A main locus was investigated on BTA21. Exons coding for variable, diversity, and joining segments, as well as for the constant regions of different isotypes, were also localized on BTA7, BTA8, and BTA20. Together with the information from unplaced contigs, 36 IGHV were detected of which 13 are putatively functional. Phylogenetic analysis revealed two bovine IGHV families (boVH1, boVH2). Thus, the existence of the two bovine families suggested was demonstrated, where boVH1 comprises all functional segments. This study substantially improves the understanding of the generation of immunoglobulin diversity in cattle.


DsTRD: Danshen Transcriptional Resource Database.
Salvia miltiorrhiza has been comprehensively studied as a medicinal model plant. However, research progress on this species is significantly hindered by its unavailable genome sequences and limited number of expressed sequence tags in the National Center for Biotechnology Information database. Thus, a transcript database must be developed to assist researchers to browse, search, and align sequences for gene cloning and functional analysis in S. miltiorrhiza. In this study, the Danshen Transcriptional Resource Database (DsTRD) was built using 76,531 transcribed sequences assembled from 12 RNA-Seq transcriptomes. Among these 12 RNA-seq data, ten were downloaded from NCBI database. The remaining two were enced on the Hiseq2000 platform using the stem and hairy-root of S. miltiorrhiza. The transcripts were annotated as protein-coding RNAs, long non-coding RNAs, microRNA precursors, and phased secondary small-interfering RNA genes through several bioinformatics methods. The tissue expression levels for each transcript were also calculated and presented in terms of RNA-Seq data. Overall, DsTRD facilitates browsing and searching for sequences and functional annotations of S. miltiorrhiza. DsTRD is freely available at http://bi.sky.zstu.edu.cn/DsTRD/home.php.


Quantitative analysis of hyperpolarized 129Xe ventilation imaging in healthy volunteers and subjects with chronic obstructive pulmonary disease.
In this study, hyperpolarized (129) Xe MR ventilation and (1) H anatomical images were obtained from three subject groups: young healthy volunteers (HVs), subjects with chronic obstructive pulmonary disease (COPD) and age-matched controls (AMCs). Ventilation images were quantified by two methods: an expert reader-based ventilation defect score percentage (VDS%) and a semi-automated segmentation-based ventilation defect percentage (VDP). Reader-based values were assigned by two experienced radiologists and resolved by consensus. In the semi-automated analysis, (1) H anatomical images and (129) Xe ventilation images were both segmented following registration to obtain the thoracic cavity volume and ventilated volume, respectively, which were then expressed as a ratio to obtain the VDP. Ventilation images were also characterized by generating signal intensity histograms from voxels within the thoracic cavity volume, and heterogeneity was analyzed using the coefficient of variation (CV). The reader-based VDS% correlated strongly with the semi-automatically generated VDP (r = 0.97, p < 0.0001) and with CV (r = 0.82, p < 0.0001). Both (129) Xe ventilation defect scoring metrics readily separated the three groups from one another and correlated significantly with the forced expiratory volume in 1 s (FEV1 ) (VDS%: r = -0.78, p = 0.0002; VDP: r = -0.79, p = 0.0003; CV: r = -0.66, p = 0.0059) and other pulmonary function tests. In the healthy subject groups (HVs and AMCs), the prevalence of ventilation defects also increased with age (VDS%: r = 0.61, p = 0.0002; VDP: r = 0.63, p = 0.0002). Moreover, ventilation histograms and their associated CVs distinguished between subjects with COPD with similar ventilation defect scores, but visibly different ventilation patterns.


Predicted distribution of the glass sponge Vazella pourtalesi on the Scotian Shelf and its persistence in the face of climatic variability.
Emerald Basin on the Scotian Shelf off Nova Scotia, Canada, is home to a globally unique aggregation of the glass sponge Vazella pourtalesi, first documented in the region in 1889. In 2009, Fisheries and Oceans Canada (DFO) implemented two Sponge Conservation Areas to protect these sponge grounds from bottom fishing activities. Together, the two conservation areas encompass 259 km2. In order to ascertain the degree to which the sponge grounds remain unprotected, we modelled the presence probability and predicted range distribution of V. pourtalesi on the Scotian Shelf using random forest modelling on presence-absence records. With a high degree of accuracy the random forest model predicted the highest probability of occurrence of V. pourtalesi in the inner basins on the central Scotian Shelf, with lower probabilities at the shelf break and in the Fundian and Northeast Channels. Bottom temperature was the most important determinant of its distribution in the model. Although the two DFO Sponge Conservation Areas protect some of the more significant concentrations of V. pourtalesi, much of its predicted distribution remains unprotected (over 99%). Examination of the hydrographic conditions in Emerald Basin revealed that the V. pourtalesi sponge grounds are associated with a warmer and more saline water mass compared to the surrounding shelf. Reconstruction of historical bottom temperature and salinity in Emerald Basin revealed strong multi-decadal variability, with average bottom temperatures varying by 8°C. We show that this species has persisted in the face of this climatic variability, possibly indicating how it will respond to future climate change.


marA locus causes decreased expression of OmpF porin in multiple-antibiotic-resistant (Mar) mutants of Escherichia coli.
Mar (multiple antibiotic resistant) mutants of Escherichia coli express chromosomally mediated resistance to a variety of structurally unrelated hydrophilic and hydrophobic antibiotics. Insertion of transposon Tn5 into the marA locus at min 34.05 on the chromosome completely reverses the Mar phenotype (A. M. George and S. B. Levy, J. Bacteriol. 155:531-540, 1983). We found that among changes in the outer membrane of Mar mutants, porin OmpF was greatly reduced, although Mar mutants were more resistant than cells lacking only OmpF. Transduction of the marA region from a Mar strain, but not a wild-type strain, led to loss of OmpF. P1 transduction of marA::Tn5 into a Mar mutant partially restored OmpF levels. Therefore, OmpF reduction required a mutation in the marA region. Mar mutants of an ompF-lacZ operon fusion strain expressed 50 to 75% of the beta-galactosidase activity of the isogenic non-Mar parental strain, while Mar mutants of a protein fusion strain expressed less than 10% of the enzyme activity in the non-Mar strain. These changes were completely reversed by insertion of marA::Tn5. The responsiveness of OmpF-LacZ to osmolarity and temperature changes was similar in Mar and wild-type strains. Although some transcriptional control may have been present, OmpF reduction appeared to occur primarily by a posttranscriptional mechanism. The steady-state levels of ompF mRNA were twofold lower and the mRNA was five times less stable in the Mar mutant than in the wild-type strain. Expression of micF, which lowers ompF mRNA levels, was elevated in Mar strains, as revealed by a micF-lacZ fusion. Studies with strains deleted for the micF locus showed that the marA-dependent reduction of OmpF required an intact micF locus. Our findings suggest that the marA locus directly or indirectly increases micF expression, causing a posttranscriptional decrease in ompF mRNA and reduced amounts of OmpF.


Sensitivity of wheat and rice to low levels of atmospheric ethylene.
Ethylene (C2H4) gas is produced throughout the life cycle of plants and can accumulate in closed growth chambers to levels 100 times higher than in outside environments. Elevated atmospheric C2H4 can cause a variety of abnormal responses, but the sensitivity to elevated C2H4 is not well characterized. We evaluated the C2H4 sensitivity of wheat (Triticum aestivum L.) and rice (Oryza sativa L.) in five studies. The first three studies compared the effects of continuous C2H4 levels ranging from 0 to 1000 nmol mol-1 (ppb) in a growth chamber throughout the life cycle of the plants. A short-term 1000 nmol mol-1 treatment was included in which exposure was stopped at anthesis. Yield was reduced by 36% in wheat and 63% in rice at 50 nmol mol-1 and both species were virtually sterile when continuously exposed to 1000 nmol mol-1. However, the yield reductions were much less with exposure that stopped at anthesis, suggesting the detrimental effect of C2H4 on yield was greatest around the time of seed set. Two additional studies evaluated the differential sensitivity of two wheat cultivars (Super Dwarf and USU-Apogee) to 50 nmol mol-1 C2H4 at three CO2 levels [350, 1200, 5000 micromoles mol-1 (ppm)] in a greenhouse. Yield of USU-Apogee was not significantly reduced by C2H4 but the yield of Super Dwarf was reduced by 60%. Elevated CO2 did not influence the sensitivity to C2H4. A difference in the C2H4 sensitivity of USU-Apogee between greenhouse and growth chamber trials suggests that C2H4 sensitivity is dependent on the environment. Collectively, the data suggest that relatively low levels of C2H4 could induce anomalous plant responses by accumulation in greenhouses and growth chambers with inadequate ventilation. The data also suggest that C2H4 sensitivity can be reduced by both genetic and environmental manipulations.


Somaesthetic disturbances in fibromyalgia are exaggerated by sensory motor conflict: implications for chronicity of the disease?
Conflict between sensory-motor central nervous processing generates somaesthetic disturbances, including pain, in healthy volunteers (HVs). Such conflict has been proposed as a potential cause of pain that occurs in the absence of injury or when the pain response is disproportionate to the injury. Fibromyalgia (FMS) exemplifies the former state. We hypothesized that the artificial generation of such conflict would exacerbate somaesthetic perceptions including pain in FMS greater than in HVs. Twenty-nine adults with FMS took part in an established task that generates varied degrees of sensory-motor conflict during congruent/incongruent limb movements. A qualitative methodology recorded any changes in sensory experience. Data generated were compared with age and gender-matched HV data. Twenty-six subjects (89.7%) with FMS reported changes in sensory perception at some stage in the protocol in addition to, or worse than, baseline compared with 14 (48%) of HVs. All stages of the protocol generated a higher frequency of report in the FMS population than that of the maximum report in the HVs population. New perceptions included disorientation, pain, perceived changes in temperature, limb weight or body image. Our findings support the hypothesis that motor-sensory conflict can exacerbate pain and sensory perceptions in those with FMS to a greater extent than in HVs.


Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray.
Diffuse large B-cell lymphoma (DLBCL) can be divided into prognostically important subgroups with germinal center B-cell-like (GCB), activated B-cell-like (ABC), and type 3 gene expression profiles using a cDNA microarray. Tissue microarray (TMA) blocks were created from 152 cases of DLBCL, 142 of which had been successfully evaluated by cDNA microarray (75 GCB, 41 ABC, and 26 type 3). Sections were stained with antibodies to CD10, bcl-6, MUM1, FOXP1, cyclin D2, and bcl-2. Expression of bcl-6 (P <.001) or CD10 (P =.019) was associated with better overall survival (OS), whereas expression of MUM1 (P =.009) or cyclin D2 (P <.001) was associated with worse OS. Cases were subclassified using CD10, bcl-6, and MUM1 expression, and 64 cases (42%) were considered GCB and 88 cases (58%) non-GCB. The 5-year OS for the GCB group was 76% compared with only 34% for the non-GCB group (P <.001), which is similar to that reported using the cDNA microarray. Bcl-2 and cyclin D2 were adverse predictors in the non-GCB group. In multivariate analysis, a high International Prognostic Index score (3-5) and the non-GCB phenotype were independent adverse predictors (P <.0001). In summary, immunostains can be used to determine the GCB and non-GCB subtypes of DLBCL and predict survival similar to the cDNA microarray.


Influence of the concentrations of liposomes and a submicron emulsion on the rheological properties of a topical gel.
Two different kinds of liposomes and a submicron emulsion were added to a topical hydrogel consisting of alginates. The rheological characteristics of the gels after various dilutions were investigated. The various preparations studied exhibited a similar rheological behaviour. No important interactions were observed by mixing the hydrogel with liposomes or a submicron emulsion. The viscoelastic properties and gel state were preserved, only a decrease in consistency was measured.


The epigenome of evolving Drosophila neo-sex chromosomes: dosage compensation and heterochromatin formation.
Sex chromosomes originated from autosomes but have evolved a highly specialized chromatin structure. Drosophila Y chromosomes are composed entirely of silent heterochromatin, while male X chromosomes have highly accessible chromatin and are hypertranscribed as a result of dosage compensation. Here, we dissect the molecular mechanisms and functional pressures driving heterochromatin formation and dosage compensation of the recently formed neo-sex chromosomes of Drosophila miranda. We show that the onset of heterochromatin formation on the neo-Y is triggered by an accumulation of repetitive DNA. The neo-X has evolved partial dosage compensation and we find that diverse mutational paths have been utilized to establish several dozen novel binding consensus motifs for the dosage compensation complex on the neo-X, including simple point mutations at pre-binding sites, insertion and deletion mutations, microsatellite expansions, or tandem amplification of weak binding sites. Spreading of these silencing or activating chromatin modifications to adjacent regions results in massive mis-expression of neo-sex linked genes, and little correspondence between functionality of genes and their silencing on the neo-Y or dosage compensation on the neo-X. Intriguingly, the genomic regions being targeted by the dosage compensation complex on the neo-X and those becoming heterochromatic on the neo-Y show little overlap, possibly reflecting different propensities along the ancestral chromosome that formed the sex chromosome to adopt active or repressive chromatin configurations. Our findings have broad implications for current models of sex chromosome evolution, and demonstrate how mechanistic constraints can limit evolutionary adaptations. Our study also highlights how evolution can follow predictable genetic trajectories, by repeatedly acquiring the same 21-bp consensus motif for recruitment of the dosage compensation complex, yet utilizing a diverse array of random mutational changes to attain the same phenotypic outcome.


Environmental and socioeconomic impacts of utilizing waste for biochar in rural areas in Indonesia--a systems perspective.
Biochar is the product of incomplete combustion (pyrolysis) of organic material. In rural areas, it can be used as a soil amendment to increase soil fertility. Fuel-constrained villagers may however prefer to use biochar briquettes as a higher-value fuel for cooking over applying it to soils. A systems-oriented analysis using life cycle assessment (LCA) and cost benefit analysis (CBA) was conducted to analyze these two alternative uses of biochar, applying the study to a rural village system in Indonesia. The results showed soil amendment for enhanced agricultural production to be the preferential choice with a positive benefit to the baseline scenario of -26 ecopoints (LCA) and -173 USD (CBA) annually pr. household. In this case, the positive effects of carbon sequestration to the soil and the economic value of the increased agricultural production outweighed the negative environmental impacts from biochar production and the related production costs. Use of biochar in briquettes for cooking fuel yielded negative net effects in both the LCA and CBA (85 ecopoints and 176 USD), even when positive health effects from reduced indoor air pollution were included. The main reasons for this are that emissions during biochar production are not compensated by carbon sequestration and that briquette making is labor-intensive. The results emphasize the importance of investigating and documenting the carbon storage effect and the agricultural benefit in biochar production-utilization systems for a sustainable use. Further research focus on efficient production is necessary due to the large environmental impact of biochar production. In addition, biochar should continue to be used in those soils where the agricultural effect is most beneficial.


Equivocal Evidence for Colony Level Stress Effects on Bumble Bee Pollination Services.
Climate change poses a threat to global food security with extreme heat events causing drought and direct damage to crop plants. However, by altering behavioural or physiological responses of insects, extreme heat events may also affect pollination services on which many crops are dependent. Such effects may potentially be exacerbated by other environmental stresses, such as exposure to widely used agro-chemicals. To determine whether environmental stressors interact to affect pollination services, we carried out field cage experiments on the buff-tailed bumble bee (Bombus terrestris). Using a Bayesian approach, we assessed whether heat stress (colonies maintained at an ambient temperature of 25 °C or 31 °C) and insecticide exposure (5 ng g-1 of the neonicotinoid insecticide clothianidin) could induce behavioural changes that affected pollination of faba bean (Vicia faba). Only the bumble bee colonies and not the plants were exposed to the environmental stress treatments. Bean plants exposed to heat-stressed bumble bee colonies (31 °C) had a lower proportional pod set compared to colonies maintained at 25 °C. There was also weak evidence that heat stressed colonies caused lower total bean weight. Bee exposure to clothianidin was found to have no clear effect on plant yields, either individually or as part of an interaction. We identified no effect of either colony stressor on bumble bee foraging behaviours. Our results suggest that extreme heat stress at the colony level may impact on pollination services. However, as the effect for other key yield parameters was weaker (e.g. bean yields), our results are not conclusive. Overall, our study highlights the need for further research on how environmental stress affects behavioural interactions in plant-pollinator systems that could impact on crop yields.


Monitoring Deuterium Uptake in Single Bacterial Cells via Two-Dimensional Raman Correlation Spectroscopy.
Raman-stable isotope labeling using heavy water (Raman-D2O) is attracting great interest as a fast technique with various applications ranging from the identification of pathogens in medical samples to the determination of microbial activity in the environment. Despite its widespread applications, little is known about the fundamental processes of hydrogen-deuterium (H/D) exchange, which are crucial for understanding molecular interactions in microorganisms. By combining two-dimensional (2D) correlation spectroscopy and Raman deuterium labeling, we have investigated H/D exchange in bacterial cells under time dependence. Most C-H stretching signals decreased in intensity over time, prior to the formation of the C-D stretching vibration signals. The intensity of the C-D signal gradually increased over time, and the shape of the C-D signal was more uniform after longer incubation times. Deuterium uptake showed high variability between the bacterial genera and mainly led to an observable labeling of methylene and methyl groups. Thus, the C-D signal encompassed a combination of symmetric and antisymmetric CD2 and CD3 stretching vibrations, depending on the bacterial genera. The present study allowed for the determination of the sequential order of deuterium incorporation into the functional groups of proteins, lipids, and nucleic acids and hence understanding the process of biomolecule synthesis and the growth strategies of different bacterial taxa. We present the combination of Raman-D2O labeling and 2D correlation spectroscopy as a promising approach to gain a fundamental understanding of molecular interactions in biological systems.


Temperature-Dependent Rheological and Viscoelastic Investigation of a Poly(2-methyl-2-oxazoline)-b-poly(2-iso-butyl-2-oxazoline)-b-poly(2-methyl-2-oxazoline)-Based Thermogelling Hydrogel.
The synthesis and characterization of an ABA triblock copolymer based on hydrophilic poly(2-methyl-2-oxazoline) (pMeOx) blocks A and a modestly hydrophobic poly(2-iso-butyl-2-oxazoline) (piBuOx) block B is described. Aqueous polymer solutions were prepared at different concentrations (1-20 wt %) and their thermogelling capability using visual observation was investigated at different temperatures ranging from 5 to 80 °C. As only a 20 wt % solution was found to undergo thermogelation, this concentration was investigated in more detail regarding its temperature-dependent viscoelastic profile utilizing various modes (strain or temperature sweep). The prepared hydrogels from this particular ABA triblock copolymer have interesting rheological and viscoelastic properties, such as reversible thermogelling and shear thinning, and may be used as bioink, which was supported by its very low cytotoxicity and initial printing experiments using the hydrogels. However, the soft character and low yield stress of the gels do not allow real 3D printing at this point.


Hapten-specific T cell responses to 4-hydroxy-3-nitrophenyl acetyl.
The primary anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibody response is known to have a heteroclitic fine specificity, i.e., anti-NP antibodies bind (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP) with greater affinity than NP itself. Past studies of NP-specific DTH responses and NP-specific T cell-mediated suppression have demonstrated sharing of fine specificity patterns and idiotypic structure between receptors on NP-specific T cells and anti-NP antibodies. We now analyze the fine specificity of NP-specific cutaneous sensitivity (CS) reactions to NP-O-succinimide (NP-O-Su) and NIP-O-succinimide (NIP-O-Su). The specificity of these responses is shown to be controlled by genes in the Igh gene complex. Cross-reactive CS responses induced by NP-O-Su elicited by NIP-O-Su were observed in strains of mice possessing the Igh-1b allotype but not in strains bearing the Igh-1c or Igh-1j allotypes. The CS reactivity could be adoptively transferred to naive recipients, and the ability of transfer CS reactivity was T cell dependent. In contrast to the genetic requirement for I-A region homology to adoptively transfer DTH reactions, compatibility at either the H-2K, H-21, or H-2D regions was sufficient to transfer NP-specific CS reactivity to naive recipients. Furthermore, in contrast to DTH responses, cyclophosphamide pretreatment was not required to induce CS responsiveness. Thus, the specificity of NP-O-Su-induced CS responses is controlled by both H-2- and Igh-linked genes.


Serum Vascular Endothelial Growth Factor Levels in the IVAN Trial; Relationships with Drug, Dosing, and Systemic Serious Adverse Events.
To describe serum vascular endothelial growth factor (sVEGF) in patients with neovascular age-related macular degeneration (nAMD) receiving anti-VEGF agents and associations between sVEGF and systemic serious adverse events (SSAEs). Exploratory analyses of a randomized controlled trial that enrolled 610 participants with nAMD and compared 2 anti-VEGF antibodies, ranibizumab and bevacizumab, and 2 treatment regimens, monthly vs. discontinuous, with 2 years' follow-up. Adults aged 50+ years with treatment-naïve nAMD and a visual acuity of ≥25 letters (Snellen equivalent 20/320) in the affected eye. Intravitreal injection of anti-VEGF antibodies. sVEGF and occurrence of SSAE, with particular interest in arteriothromboembolic events (ATE) and immunologically mediated events (IME). On average, sVEGF (measured at months 0, 1, 11, 12, 23, and 24) decreased from a geometric mean of 168 pg/mL at baseline to 64 pg/mL at month 24. The decrease was greater with bevacizumab than with ranibizumab and was dependent on time since last treatment; at month 24 sVEGF was 11% lower with bevacizumab if treated ≥3 months previously, 51% lower if treated 2 months previously, and 76% lower if treated the previous month, compared with ranibizumab. The hazard of experiencing an ATE increased with age (hazard ratio [HR] = 2.01; 95% confidence interval [CI] = 1.32-3.05; P = 0.001) and higher sVEGF (HR = 1.16; 95% CI = 1.03-1.30, per 100 unit rise in sVEGF; P = 0.013). There was no association between sVEGF and the hazard of an IME (HR = 1.01; 95% CI = 0.76-1.33; P = 0.942); however, the hazard of an IME was significantly increased by treatment with bevacizumab compared with ranibizumab (HR = 3.53; 95% CI = 1.35-9.22; P = 0.010). The hazard of an "other SSAE" (not categorized as ATE or IME) increased with age (HR 1.51, 95% CI 1.14-2.01, P = 0.005) and decreased if an injection had been administered within the previous month (HR = 0.68; 95% CI = 0.45-1.03; P = 0.069). The decrease in sVEGF is greater with bevacizumab than with ranibizumab, but this difference is eliminated when treatment is withheld for 3 months. Higher sVEGF increased the hazard of an ATE and bevacizumab increases the hazard of an IME compared with ranibizumab.