Use of 5-alpha-reductase inhibitors for prostate cancer chemoprevention: American Society of Clinical Oncology/American Urological Association 2008 Clinical Practice Guideline.
PURPOSE To develop an evidence-based guideline on the use of 5-alpha-reductase inhibitors (5-ARIs) for prostate cancer chemoprevention. METHODS The American Society of Clinical Oncology (ASCO) Health Services Committee (HSC), ASCO Cancer Prevention Committee, and the American Urological Association Practice Guidelines Committee jointly convened a Panel of experts, who used the results from a systematic review of the literature to develop evidence-based recommendations on the use of 5-ARIs for prostate cancer chemoprevention. Results The systematic review completed for this guideline identified 15 randomized clinical trials that met the inclusion criteria, nine of which reported prostate cancer period prevalence. CONCLUSION Asymptomatic men with a prostate-specific antigen (PSA) <or= 3.0 ng/mL who are regularly screened with PSA or are anticipating undergoing annual PSA screening for early detection of prostate cancer may benefit from a discussion of both the benefits of 5-ARIs for 7 years for the prevention of prostate cancer and the potential risks (including the possibility of high-grade prostate cancer). Men who are taking 5-ARIs for benign conditions such as lower urinary tract [obstructive] symptoms (LUTS) may benefit from a similar discussion, understanding that the improvement of LUTS relief should be weighed with the potential risks of high-grade prostate cancer from 5-ARIs (although the majority of the Panel members judged the latter risk to be unlikely). A reduction of approximately 50% in PSA by 12 months is expected in men taking a 5-ARI; however, because these changes in PSA may vary across men, and within individual men over time, the Panel cannot recommend a specific cut point to trigger a biopsy for men taking a 5-ARI. No specific cut point or change in PSA has been prospectively validated in men taking a 5-ARI.


Many families may face sharply higher costs if public health insurance for their children is rolled back.
Millions of US children could lose access to public health care coverage if Congress does not renew federal funding for the Children's Health Insurance Program (CHIP), which is set to expire September 30, 2015—the end of the federal fiscal year. Additional cuts in public coverage for children in families with incomes above 133 percent of the federal poverty level are possible if the Affordable Care Act's "maintenance of effort" provisions regarding Medicaid and CHIP are allowed to expire as scheduled in 2019. The potential for a significant rollback of public coverage for children raises important policy questions regarding alternative pathways to affordable and high-quality coverage for low-income children. For many children at risk of losing eligibility for public coverage, the primary alternative pathway to coverage would be through their parents' employer-sponsored insurance, yet relatively little is known about the cost and quality of that coverage. Our estimates, based on data from the Insurance Component of the 2012 and 2013 Medical Expenditure Panel Surveys, show that many families would face sharply higher costs of covering their children. In many cases, the only employer-sponsored coverage available would be a high-deductible plan.


A strategy for high-level expression of soluble and functional human interferon alpha as a GST-fusion protein in E. coli.
Escherichia coli is the most extensively used host for the production of recombinant proteins. However, most of the eukaryotic proteins are typically obtained as insoluble, misfolded inclusion bodies that need solubilization and refolding. To achieve high-level expression of soluble recombinant human interferon alpha (rhIFNalpha) in E. coli, we have first constructed a recombinant expression plasmid (pGEX-hIFNalpha2b), in which we merged the hIFNalpha2b cDNA with the glutathione S-transferase (GST) coding sequence downstream of the tac-inducible promoter. Using this plasmid, we have achieved 70% expression of soluble rhIFNalpha2b as a GST fusion protein using E. coli BL21 strain, under optimized environmental factors such as culture growth temperature and inducer (IPTG) concentration. However, release of the IFN moiety from the fusion protein by thrombin digestion was not optimal. Therefore, we have engineered the expression cassette to optimize the amino acid sequence at the GST-IFN junction and to introduce E. coli preferred codon within the thrombin cleavage site. We have used the engineered plasmid (pGEX-Delta-hIFNalpha2b) and the modified E. coli trxB(-)/gor(-) (Origami) strain to overcome the problem of removing the GST moiety while expressing soluble rhIFNalpha2b. Our results show the production of soluble and functional rhIFNalpha2b at a yield of 100 mg/l, without optimization of any step of the process. The specific biological activity of the purified soluble rhIFNalpha2b was equal to 2.0 x 10(8) IU/mg when compared with the WHO IFNalpha standard. Our data are the first to show that high yield production of soluble and functional rhIFNalpha2b tagged with GST can be achieved in E. coli.


From rolling ball to complete wetting: the dynamic tuning of liquids on nanostructured surfaces.
In this work, for the first time, a dynamic electrical control of the wetting behavior of liquids on nanostructured surfaces, which spans the entire possible range from the superhydrophobic behavior to nearly complete wetting, has been demonstrated. Moreover, this kind of dynamic control was obtained at voltages as low as 22 V. We have demonstrated that the liquid droplet on a nanostructured surface exhibits sharp transitions between three possible wetting states as a function of applied voltage and liquid surface tension. We have examined experimentally and theoretically the nature of these transitions. The reported results provide novel methods of manipulating liquids at the microscale.


Bisphenol A increases hydrogen peroxide generation by thyrocytes both in vivo and in vitro.
Bisphenol A (BPA) is the most common monomer in polycarbonate plastics and an endocrine disruptor. Though some effects of BPA on thyroid hormone (TH) synthesis and action have been described, the impact of this compound on thyroid H2O2 generation remains elusive. H2O2 is a reactive oxygen species (ROS) which could have deleterious effect on thyrocytes if in excess. Therefore, herein we aimed at evaluating the effect of BPA exposition both in vivo and in vitro on H2O2 generation in thyrocytes, besides other essential steps for TH synthesis. Female Wistar rats were treated with vehicle (control) or BPA 40 mg/Kg BW for 15 days, by gavage. We then evaluated thyroid iodide uptake, mediated by sodium-iodide symporter (NIS), thyroperoxidase (TPO) and dual oxidase (DOUX) activities (H2O2 generation). Hydrogen peroxide generation was increased, while iodide uptake and TPO activity were reduced by BPA exposition. We have also incubated the rat thyroid cell line PCCL3 with 10-9 M BPA and evaluated Nis and Duox mRNA levels, besides H2O2 generation. Similar to that found in vivo, BPA treatment also led to increased H2O2 generation in PCCL3. Nis mRNA levels were reduced and Duox2 mRNA levels were increased in BPA-exposed cells. To evaluate the importance of oxidative stress on BPA-induced Nis reduction, PCCL3 was treated with BPA in association to n-acetylcysteine, an antioxidant, which reversed the effect of BPA on Nis. Our data suggest that BPA increases ROS production in thyrocytes, what could lead to oxidative damage thus possibly predisposing to thyroid disease.


A Novel Active Contour Model for MRI Brain Segmentation used in Radiotherapy Treatment Planning.
Brain image segmentation is one of the most important clinical tools used in radiology and radiotherapy. But accurate segmentation is a very difficult task because these images mostly contain noise, inhomogeneities, and sometimes aberrations. The purpose of this study was to introduce a novel, locally statistical active contour model (ACM) for magnetic resonance image segmentation in the presence of intense inhomogeneity with the ability to determine the position of contour and energy diagram. A Gaussian distribution model with different means and variances was used for inhomogeneity, and a moving window was used to map the original image into another domain in which the intensity distributions of inhomogeneous objects were still Gaussian but were better separated. The means of the Gaussian distributions in the transformed domain can be adaptively estimated by multiplying a bias field by the original signal within the window. Then, a statistical energy function is defined for each local region. Also, to evaluate the performance of our method, experiments were conducted on MR images of the brain for segment tumors or normal tissue as visualization and energy functions. In the proposed method, we were able to determine the size and position of the initial contour and to count iterations to have a better segmentation. The energy function for 20 to 430 iterations was calculated. The energy function was reduced by about 5 and 7% after 70 and 430 iterations, respectively. These results showed that, with increasing iterations, the energy function decreased, but it decreased faster during the early iterations, after which it decreased slowly. Also, this method enables us to stop the segmentation based on the threshold that we define for the energy equation. An active contour model based on the energy function is a useful tool for medical image segmentation. The proposed method combined the information about neighboring pixels that belonged to the same class, thereby making it strong to separate the desired objects from the background.


Breast lesion localization: a freehand, interactive MR imaging-guided technique.
To evaluate interactive magnetic resonance (MR) imaging-guided preoperative needle localization and hookwire placement in the noncompressed breast in patients in the prone position. Nineteen MR imaging-guided breast lesion localization procedures were performed in 17 patients aged 38-70 years (mean age, 48 years) by using an open-platform breast coil in either a 1.5-T, closed-bore imager (n = 14) or a 0.5-T, open-bore imager (n = 5). Rapid imaging (fast spin-echo, water-selective fast spin-echo, or water-specific three-point Dixon gradient-echo) was alternated with freehand manipulation of an MR-compatible needle to achieve accurate needle placement. Up to three manipulations of the needle were required during an average of 9 minutes to reach the target lesion. MR imaging findings confirmed the final needle position within 9 mm of the target in all cases. The accuracy of 10 localizations was independently corroborated either at mammography or at ultrasonography. Nine lesions were visible on MR images only. Interactive MR imaging-guided, freehand needle localization is simple, accurate, and requires no special stereotactic equipment. Lesions throughout the breast, including those in the anterior part of the breast and those near the chest wall, which can be inaccessible with standard grid or compression-plate techniques, can be localized. A variety of needle trajectories in addition to the horizontal path are possible, including circumareolar approaches and tangential needle paths designed to avoid puncture of implants.


The 'Russian' influenza in the UK: lessons learned, opportunities missed.
This paper describes British efforts to map the Russian influenza outbreaks of the early 1890s and describe the timing and course of the epidemic waves. Drawing on two surveys conducted by Britain's Local Government Board (LGB), the paper shows how, in a pre-virological era, the board was able to establish that influenza was an intensely infectious disease. Its key observation, however, was that Russian influenza had taken the form of three, and possibly four, distinct waves of infection, with the second wave in the spring of 1891 proving more lethal than the first wave, and the third wave in the winter of 1892 proving almost as lethal again. Most of this mortality was due to excess deaths from respiratory disease, particularly in the middle age ranges, but while these insights could and, arguably, should have aided the public health response, British health authorities preferred to advocate cautious preventive measures that did little to alleviate the pandemic's impact. The policy would prove especially costly in 1918-1919 when the LGB missed the opportunity to provide extra nursing cover for influenza convalescents following the initial summer wave of the 1918 Spanish influenza pandemic.


Plasma profiles of albendazole metabolites after administration of netobimin and albendazole in sheep: effects of parasitism and age.
Netobimin and albendazole were administered to 3-month-old lambs with moderate infections of Nematodirus battus and to comparable parasite-naive lambs. Albendazole sulphoxide and albendazole sulphone concentrations were determined in the plasma of all lambs at frequent intervals after treatment. Both anthelmintic preparations were 100% effective in reducing nematode faecal egg output in the lambs. There were no significant differences in the concentrations of the sulphoxide or sulphone metabolites in parasitized compared with non-parasitized lambs given the same parent anthelmintic. The parasite-naive lambs were subsequently weaned and maintained indoors in conditions designed to preclude nematode parasite infection until they were 9 months old. Netobimin and albendazole were administered again and the plasma profiles of the albendazole sulphoxide and albendazole sulphone metabolites determined. There were no significant differences in the plasma distribution of these metabolites with age of the lambs. The area under the plasma concentration time curve, mean resident time and apparent half-life of the albendazole sulphoxide metabolite was determined following administration of each parent drug and the clearance of the metabolite/systemic availability of parent drug was determined as a marker of the amount of drug available for metabolism. There were no significant differences in pharmacokinetic variables between parasitized and non-parasitized animals nor with the age of the animals.


A simple description of the 3 k cosmic microwave background.
An intuitive model for the expansion of the universe is developed in which special relativity is used to describe events seen by a hypothetical observer in a Lorentz frame of reference. The cosmic microwave background photons he sees are the red-shifted remnants of hot photons emitted from the matter flying rapidly away from him. This special relativistic model, also called the Milne model, represents the extreme case of a Friedmann (general relativistic) universe in the limit of vanishingly small density of matter. The special relativistic model approximates an open universe (one that expands forever) with increasing accuracy as time evolves.


Early-phase GVHD gene expression profile in target versus non-target tissues: kidney, a possible target?
GVHD is a major complication after allo-SCT. In GVHD, some tissues like liver, intestine and skin are infiltrated by donor T cells while others like muscle are not. The mechanism underlying targeted tropism of donor T cells is not fully understood. In the present study, we aim to explore differences in gene expression profile among target versus non-target tissues in a mouse model of GVHD based on chemotherapy conditioning. Expression levels of JAK-signal transducers and activators of transcription (STAT), CXCL1, ICAM1 and STAT3 were increased in the liver and remained unchanged (or decreased) in the muscle and kidney after conditioning. At the start of GVHD the expression levels of CXCL9, ITGb2, SAA3, MARCO, TLR and VCAM1 were significantly higher in the liver or kidney compared with the muscle of GVHD animals. Moreover, biological processes of inflammatory reactions, leukocyte migration, response to bacterium and chemotaxis followed the same pattern. Our data show that both chemotherapy and allogenicity exclusively induce expression of inflammatory genes in target tissues. Moreover, gene expression profile and histopathological findings in the kidney are similar to those observed in the liver of GVHD mice.


A rabbit model of lower eyelid fibrosis.
To create and validate a new model of lower eyelid fibrosis in Dutch-belted rabbits. Five Dutch-belted rabbits were injected with a transcutaneous 1-ml injection of standard 95% ethanol alcohol just inferior to the eyelid margin of one lower eyelid. A control injection of 1 ml of balanced saline solution was given to the opposite eyelid. A small tattoo was placed on the skin overlying the inferior orbital rim and used as a measuring point of reference in relation to the lower eyelid margin. Analysis was twofold: eyelid measurements were made over 8 weeks to determine the presence of eyelid shortening, and a histopathologic analysis was performed. Mean lower eyelid shortening was greater in the ethanol alcohol intervention eyelids than the control group (-3.4 mm +/- 1.67 mm vs. 0.5 mm +/- 0.71 mm, p = 0.01). Histopathologic analysis revealed extensive fibrosis in the ethanol alcohol invention eyelids compared with the control group. Ethanol alcohol induces eyelid fibrosis and lower eyelid shortening. This may be a useful model in the future testing of novel surgical or pharmacologic treatments.


Do inflammatory biomarkers add to the discrimination of cardiovascular disease after allowing for social deprivation? Results from a 10-year cohort study in Glasgow, Scotland.
To assess the additional discriminative value of adding each of five inflammatory biomarkers to the ASSIGN risk score, which includes social deprivation. In this study, 1319 men and women aged 25-64 in the fourth Glasgow MONICA study were followed-up for cardiovascular endpoints. Baseline C-reactive protein, fibrinogen, IL-6, IL-18, and TNFα were related to risk of CVD. The discriminative value of adding each to the ASSIGN score was assessed using area under the receiver operating characteristic (AUC) and relative integrated percentage improvement in classification (RIDI). During a median of 10.5 years, 151 CVD events occurred. After adjusting for ASSIGN variables, each inflammatory marker except IL-18 had a significant (P < 0.05) association with CVD risk. The AUC using ASSIGN [0.799 (95% CI 0.790-0.809)] was improved by the inclusion of C-reactive protein and TNFα [0.805 (95% CI 0.795-0.815); P < 0.03], but not by other combinations. C-reactive protein and TNFα yielded a significant RIDI (IL-6 almost so). C-reactive protein and TNFα together improved the classification of risk by 11% (95% CI, 3-19%) when added to the ASSIGN variables. Some inflammatory biomarkers add moderate discriminative information to the ASSIGN CVD risk score. The clinical utility of this information, cost-effectiveness, and optimization should be assessed in future studies.


Circulating level of lipocalin 2 as a predictor of severity in patients with community-acquired pneumonia.
The aim of this study was to investigate the differential plasma levels of lipocalin 2 (LCN2) and its complex with MMP-9 (where MMP is matrix metalloproteinase) before and after antibiotic treatment in hospitalized adult patients with community-acquired pneumonia (CAP). Plasma LCN2 and LCN2/MMP-9 complex levels were measured in 61 adult patients with CAP and 60 healthy controls using commercial enzyme-linked immunosorbent assay (ELISA). A decrease in the number of white blood cells (WBCs) and neutrophils and decreases in the levels of C-reactive protein (CRP), LCN2, and LCN2/MMP-9 complex were observed after antibiotic treatment. The plasma level of LCN2, but not that of CRP, was correlated with the severity of CAP based on the Pneumonia Severity Index (PSI; r = 0.333, P = 0.009), confusion, urea, respiratory rate and blood pressure (CURB)-65 (r = 0.288, P = 0.024), and Acute Physiology And Chronic Health Evaluation II (APACHE II) scores (r = 0.328, P = 0.010). LCN2 levels were also significantly correlated with LCN2/MMP-9 levels and the numbers of WBCs or neutrophils. Plasma levels of LCN2 and the LCN2/MMP-9 complex can act as adjuvant diagnostic biomarkers for CAP. Plasma LCN2 might play a further role in the clinical assessment of the severity of CAP, which could potentially guide the development of future treatment strategies.


Transducing mechanical force by use of a diffraction grating sensor.
A novel means of transducing mechanical force by using a polymeric-based diffractive grating sensor is presented. The diffraction gratings are successfully fabricated upon poly(dimethyl siloxane) polymer substrates by holographic interference and micromolding. A micromaterial tensile test incorporated into the surface diffraction grating experiment showed that a relationship between the load and the observed diffraction-pattern shift could be obtained. The results show an excellent correlation between the optical measurement and load, with a sensitivity of 0.05 N.


Vascular endothelial growth factor (VEGF) induced proliferation of human fetal derived ciliary epithelium stem cells is mediated by jagged-N cadherin pathway.
The pigmented ciliary epithelium (PCE) of mammalian eye harbors resident population of stem cells that lie in apposition with endothelial cells which release vascular endothelial growth factor (VEGF) that may influence the fate and function of these stem cells in ways that remain unclear. We examined the role of VEGF in proliferation of PCE stem cells and expression of Notch, Jagged, N-Cadherin and β-Catenin which are known to maintain proliferation state of neural stem cells. We cultured human PCE cells obtained from 12-20 weeks old fetal eyes. The neurospheres were analyzed for the proliferation capacity of PCE stem cells in presence of VEGF on 3,6 and 9 day. Real time PCR was used to quantitate the mRNA expression of above mentioned genes on PCE derived neurospheres on 3,6 and 9 day. We found increased number of neurospheres when PCE stem cells were stimulated with VEGF along with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) than EGF and bFGF. BrdU immunostaining was done to analyze the proliferation of CE cells and presence of neural and retinal progenitor markers such as Nestin and Pax6 were also investigated. An increased Notch and Jagged mRNA was observed on 6(th) day in VEGF, EGF and bFGF treated PCE cells as compared to 0,3 and 9 day. A similar pattern was noticed with N-cadherin and β-catenin mRNA levels. These findings may clarify the role of VEGF on PCE stem cell proliferation with possible involvement of Notch, Jagged, N-cadherin and β-Catenin. The data may suggest importance of harvesting 6(th) day neurospheres for transplantation purposes in preclinical investigations pertaining to retinal degenerative diseases, however, additional studies are needed to substantiate the findings.


Cognitive deficit measures in schizophrenia: factor structure and clinical correlates.
The authors aimed to delineate the "common denominators" across a representative number of cognitive measures by administering such measures to a large number of schizophrenic subjects. The authors also aimed to study the consistency of the subjects' performance over a 1-year period and to look into the relationship between cognitive task performance and clinical status. One hundred fifty-seven schizophrenic subjects with a DSM-III-R diagnosis of schizophrenia were assessed under standardized testing conditions on the dichotic listening and shadowing task, the span of apprehension task, the simple reaction time tasks, the critical stimulus duration, and the interstimulus interval in which they would become free from the masking effect. The clinical status of the subjects was assessed during index assessment, and 97 subjects were reassessed at the end of 1 year. Forty-seven subjects were randomly selected for reassessment on the cognitive tasks administered 1 year earlier. The subjects' performance was compared to that of 56 nonschizophrenic patients with nasopharyngeal carcinoma who had undergone radiation therapy. The cognitive task scores were factor analyzed, giving five distinct factors made up of the span of apprehension accuracy scores, simple reaction time scores, shadowing accuracy scores, span of apprehension error scores, and susceptibility to interference scores. At index assessment, the subjects' shadowing accuracy and reaction time were significantly related to their quality of life, social adjustment, and positive symptoms. The subjects' span of apprehension scores were related to their negative symptoms, measured in terms of work and daily life functions. The initial relationship between the subjects' cognitive test scores and their outcome status remained consistent at follow-up. The documented cognitive deficits in schizophrenic patients probably reflect a more enduring trait-like status with correlations with outcome status. These results also indicate that the cognitive measures may be simplified in complexity without a corresponding reduction in their clinical significance.


Inbreeding of artificially bred dairy cattle in the northeastern United States.
Inbreeding coefficients of artificially bred Ayrshire, Guernsey, Holstein, Jersey, and Brown Swiss cows were calculated from relationships between sires and maternal male ancestors. Percentages of inbred cows and average inbreeding coefficients of inbred cows were: Ayrshire, 26 and 11%; Guernsey, 11 and 4%; Holstein, 31 and 1%; Jersey 23 and 2%; and Brown Swiss, 23 and 2%. Percentages of inbred cows and average inbreeding coefficients of all cows increased over time whereas average coefficients of inbred cows decreased over time. These temporal trends may be due in part to having more pedigree information available for cows born in later years. Effects of inbreeding on milk and fat yields of first lactation (2 times milked, 305-day mature equivalent), 48-mo stayability, and first calving interval were estimated with a model including effects for fixed herd-year-seasons, fixed sire and maternal grandsire groups, random sires and maternal grandsires within groups, and random residual. Regression coefficients of milk (kg), fat (kg), 48-mo stayability (proportion of cows surviving to 48 mo of age), and calving interval (days) on inbreeding coefficient (%) were: Ayrshire -27.1, -1.2, -.005, .23; Guernsey -19.3, -.97, -.007, .27; Holstein -21.1, -.78, -.003, .09; Jersey -14.8, -.80, -.002, .63; and Brown Swiss -39.5, -1.36; -.011, .03. There is no cause for concern over current inbreeding, but active inbreeding is not recommended.


Ultra-thin fluorocarbon foils optimise multiscale imaging of three-dimensional native and optically cleared specimens.
In three-dimensional light microscopy, the heterogeneity of the optical density in a specimen ultimately limits the achievable penetration depth and hence the three-dimensional resolution. The most direct approach to reduce aberrations, improve the contrast and achieve an optimal resolution is to minimise the impact of changes of the refractive index along an optical path. Many implementations of light sheet fluorescence microscopy operate with a large chamber filled with an aqueous immersion medium and a further inner container with the specimen embedded in a possibly entirely different non-aqueous medium. In order to minimise the impact of the latter on the optical quality of the images, we use multi-facetted cuvettes fabricated from vacuum-formed ultra-thin fluorocarbon (FEP) foils. The ultra-thin FEP-foil cuvettes have a wall thickness of about 10-12 µm. They are impermeable to liquids, but not to gases, inert, durable, mechanically stable and flexible. Importantly, the usually fragile specimen can remain in the same cuvette from seeding to fixation, clearing and observation, without the need to remove or remount it during any of these steps. We confirm the improved imaging performance of ultra-thin FEP-foil cuvettes with excellent quality images of whole organs such us mouse oocytes, of thick tissue sections from mouse brain and kidney as well as of dense pancreas and liver organoid clusters. Our ultra-thin FEP-foil cuvettes outperform many other sample-mounting techniques in terms of a full separation of the specimen from the immersion medium, compatibility with aqueous and organic clearing media, quick specimen mounting without hydrogel embedding and their applicability for multiple-view imaging and automated image segmentation. Additionally, we show that ultra-thin FEP foil cuvettes are suitable for seeding and growing organoids over a time period of at least ten days. The new cuvettes allow the fixation and staining of specimens inside the holder, preserving the delicate morphology of e.g. fragile, mono-layered three-dimensional organoids.


Complete Circular Genome Sequence and Temperature Independent Adaptation to Anaerobiosis of Listeria weihenstephanensis DSM 24698.
The aim of this study was to analyze the adaptation of the environmental Listeria weihenstephanensis DSM 24698 to anaerobiosis. The complete circular genome sequence of this species is reported and the adaptation of L. weihenstephanensis DSM 24698 to oxygen availability was investigated by global transcriptional analyses via RNAseq at 18 and 34°C. A list of operons was created based on the transcriptional data. Forty-two genes were upregulated anaerobically and 62 genes were downregulated anaerobically. The oxygen dependent gene expression of selected genes was further validated via qPCR. Many of the differentially regulated genes encode metabolic enzymes indicating broad metabolic adaptations with respect to oxygen availability. Genes showing the strongest oxygen-dependent adaption encoded nitrate (narGHJI) and nitrite (nirBD) reductases. Together with the observation that nitrate supported anaerobic growth, these data indicate that L. weihenstephanensis DSM 24698 performs anaerobic nitrate respiration. The wide overlap between the oxygen-dependent transcriptional regulation at 18 and 34°C suggest that temperature does not play a key role in the oxygen-dependent transcriptional regulation of L. weihenstephanensis DSM 24698.


Fiber-laser-based green-pumped picosecond MgO:sPPLT optical parametric oscillator.
We report a stable, high-power, picosecond optical parametric oscillator (OPO) at 160 MHz repetition rate synchronously pumped by a frequency-doubled mode-locked Yb-fiber laser at 532 nm and tunable in the near-infrared, across 874-1008 nm (signal) and 1126-1359 nm (idler). Using a 30-mm-long MgO:sPPLT crystal, the OPO provides average output power up to 780 mW in the signal at 918.58 nm and 600 mW in the idler at 1242 nm. The device operates stably over many days, even close to degeneracy, exhibiting passive long-term power stability better than 1.8% rms in the signal and 2.4% rms in the idler over 2.5 h at a temperature of 55°C. We investigate spectral and temporal characteristics of the signal pulses under different conditions and demonstrate cavity-length tuning enabled by the dispersion properties of MgO:sPPLT. The output signal pulses have a duration of 2.4 ps at 967 nm.


Gametocyte carriage in Plasmodium falciparum-infected travellers.
Gametocytes are the sexual stage of Plasmodium parasites. The determinants of gametocyte carriage have been studied extensively in endemic areas, but have rarely been explored in travellers with malaria. The incidence of gametocytaemia, and factors associated with gametocyte emergence in adult travellers with Plasmodium falciparum malaria was investigated at the Hospital for Tropical Diseases in London. Clinical, parasitological and demographic data for all patients presenting with P. falciparum malaria between January 2001 and December 2011 were extracted from a prospective database. These data were supplemented by manual searches of laboratory records and patient case notes. Seven hundred and seventy three adult patients with laboratory-confirmed P. falciparum malaria were identified. Four hundred and sixty five (60%) were born in a country where malaria is endemic. Patients presented to hospital a median of four days into their illness. The median maximum parasite count was 0.4%. One hundred and ninety six patients (25%) had gametocytes; 94 (12%) on admission, and 102 (13%) developing during treatment. Gametocytaemia on admission was associated with anaemia and a lower maximum parasitaemia. Patients with gametocytes at presentation were less likely to have thrombocytopenia or severe malaria. Patients who developed gametocytes during treatment were more likely to have had parasitaemia of long duration, a high maximum parasitaemia and to have had severe malaria. There was no apparent association between the appearance of gametocytes and treatment regimen. The development of gametocytaemia in travellers with P. falciparum is associated with factors similar to those reported among populations in endemic areas. These data suggest that acquired immunity to malaria is not the only determinant of patterns of gametocyte carriage among patients with the disease.


Effect of insulin on AKR1C3 expression in female adipose tissue: in-vivo and in-vitro study of adipose androgen generation in polycystic ovary syndrome.
Insulin resistance and hyperandrogenism are the cardinal features of polycystic ovary syndrome (PCOS). Women with insulin receptor (INSR) mutations develop severe hyperandrogenism secondary to hyperinsulinaemia. We hypothesised that insulin might drive adipose testosterone generation from androstenedione through aldoketoredutase type 3 (AKR1C3) in women with insulin resistance. Here we assessed the effect of insulin on AKR1C3 activity in vivo and in vitro. Ten women with PCOS, ten healthy controls, and three women with INSR mutations underwent dehydroepiandrosterone (DHEA) challenge; serum androgens were measured every 30 min for 4 h after ingestion of 100 mg DHEA. Additionally, paired subcutaneous and omental fat samples were obtained during abdominal surgery from 38 women. AKR1C3 expression was measured by real-time PCR. Serum steroids and cultured cell media androgens were measured with liquid chromatography tandem mass spectrometry. Women with PCOS had higher androstenedione concentrations than did controls and women with INSR mutations (p=0·01 and p=0·005, respectively). However, area under the curve for testosterone was higher in women with INSR mutations after DHEA than in women with PCOS and controls (874·2 [SE 242] vs 425 [136] and 375·2 [109], p<0·001 for both). AKR1C3 mRNA expression was significantly higher in subcutaneous than in omental adipose tissue (p=0·004). AKR1C3 expression correlated positively with body-mass index in subcutaneous fat (Spearman correlation=0·51, p=0·006). Insulin significantly increased AKR1C3 expression in differentiated subcutaneous adipocytes (p=0·04). Incubation with insulin significantly increased testosterone generation from androstenedione in cultured subcutaneous cell media compared with controls (p<0·001). We have found in-vivo and in-vitro evidence of modulation of AKR1C3 activity by insulin in PCOS and in women with INSR mutations. Insulin seems to drive adipose androgen generation by increasing AKR1C3 activity in female subcutaneous adipose tissue. Selective AKR1C3 inhibition might offer a novel therapeutic target to reduce androgen burden and improve metabolic phenotype in PCOS. Wellcome Trust.


Memory processes in depressed geriatric patients with and without subcortical hyperintensities on MRI.
In this study, 12 patients over age 60 with depression with moderate to severe subcortical hyperintensities (SH) localized to the periventricular white matter were identified by quantitative MRI. Using the California Verbal Learning Test, they were compared with 12 age-, education-, and severity-matched patients with depression with minimal white matter changes on specific aspects of memory performance. Patients with cortical lesions, neurologic or systemic illness affecting cognition, and history of substance abuse were excluded. Patients in the group with high SH showed reduced use of semantic encoding strategies (p < 0.05), reduced learning efficiency (p < 0.05), and a greater discrepancy between free recall and recognition discriminability (p < 0.05) than their low SH counterparts. This pattern of performance on memory tasks is similar to that found in previous studies to be associated with subcortical degenerative disorders such as Huntington's and Parkinson's diseases. Geriatric patients with depression with SH may represent a subgroup with greater subcortical involvement, with associated cognitive and functional decline.


Gender difference in smoking effects on lung function and risk of hospitalization for COPD: results from a Danish longitudinal population study.
Recent findings suggest that females may be more susceptible than males to the deleterious influence of tobacco smoking in developing chronic obstructive pulmonary disease (COPD). This paper studies the interaction of gender and smoking on development of COPD as assessed by lung function and hospital admission. A total of 13,897 subjects, born after 1920, from two population studies, 9,083 from the Copenhagen City Heart Study (CCHS) and 4,814 from the Glostrup Population Studies (GPS), were followed for 7-16 yrs. Data were linked with information on hospital admissions caused by COPD. Based on cross-sectional data, in the CCHS the estimated excess loss of forced expiratory volume in one second (FEV1) per pack-year of smoking was 7.4 mL in female smokers who inhaled and 6.3 mL in male smokers who inhaled. In the GPS, the corresponding excess loss of FEV1 was 10.5 and 8.4 mL in females and males, respectively. Two hundred and eighteen subjects in the CCHS and 23 in the GPS were hospitalized during follow-up. Risk associated with pack-years was higher in females than in males (relative risks (RRs) for 1-20, 20-40 and >40 pack-years were 7.0 (3.5-14.1), 9.8 (4.9-19.6) and 23.3 (10.7-50.9) in females, and 3.2 (1.1-9.1), 5.7 (2.2-14.3) and 8.4 (3.3-21.6) in males) but the interaction term gender x pack-years did not reach significance (p=0.08). Results were similar in the GPS. After adjusting for smoking in more detail, females in both cohorts had an increased risk of hospitalization for COPD compared to males with a RR of 1.5 (1.2-2.1) in the CCHS and 3.6 (1.4-9.0) in the GPS. This was not likely to be caused by a generally increased rate of hospital admission for females. Results were similar when including deaths from COPD as endpoint. In two independent population samples, smoking had greater impact on the lung function of females than males, and after adjusting for smoking females subsequently suffered a higher risk of being admitted to hospital for COPD. Results suggest that adverse effects of smoking on lung function may be greater in females than in males.


A modified protocol with vincristine, topotecan, and cyclophosphamide for recurrent/progressive ewing sarcoma family tumors.
Topotecan has recently been used in the treatment of pediatric cancer. We evaluated our experience with the modified combination of vincristine, topotecan, and cyclophosphamide (VTC) given in 3 days, in children with recurrent Ewing sarcoma. Children received vincristine (1.5 mg/m(2)/1st day), cyclophosphamide (600 mg/m(2)/day × 2 days) + mesna, and topotecan (1 mg/m(2)/day × 3 days) every 21 days. A total of 118 courses of VTC were given to 13 patients. One patient received VTC both at first and at second relapse. Thus, 14 relapse episodes in 13 patients were evaluated. After three courses of VTC chemotherapy (CT), two achieved complete response (CR), five achieved partial response, thus an objective response was attained in 7/14 (50%) episodes. Two patients had stable disease and two patients progressed. In three episodes, CR was achieved by surgery before CT. One of them had a second relapse and attained CR with VTC. Median time from diagnosis to relapse was 23 months (5-45 months). Site of relapse was local in four patients, and metastatic in 10 episodes of nine patients. Seven patients are alive, three with no evidence of disease and four alive with disease; six have died of disease. Local treatment was used in 11 episodes. The toxicity of the VTC combination was limited mainly to the hematopoietic system. In conclusion, the modified VTC protocol in 3 days every 3 weeks seems to be effective and tolerable in children and adolescents with recurrent/progressive Ewing sarcoma.


Genomics, Exometabolomics, and Metabolic Probing Reveal Conserved Proteolytic Metabolism of Thermoflexus hugenholtzii and Three Candidate Species From China and Japan.
Thermoflexus hugenholtzii JAD2T, the only cultured representative of the Chloroflexota order Thermoflexales, is abundant in Great Boiling Spring (GBS), NV, United States, and close relatives inhabit geothermal systems globally. However, no defined medium exists for T. hugenholtzii JAD2T and no single carbon source is known to support its growth, leaving key knowledge gaps in its metabolism and nutritional needs. Here, we report comparative genomic analysis of the draft genome of T. hugenholtzii JAD2T and eight closely related metagenome-assembled genomes (MAGs) from geothermal sites in China, Japan, and the United States, representing "Candidatus Thermoflexus japonica," "Candidatus Thermoflexus tengchongensis," and "Candidatus Thermoflexus sinensis." Genomics was integrated with targeted exometabolomics and 13C metabolic probing of T. hugenholtzii. The Thermoflexus genomes each code for complete central carbon metabolic pathways and an unusually high abundance and diversity of peptidases, particularly Metallo- and Serine peptidase families, along with ABC transporters for peptides and some amino acids. The T. hugenholtzii JAD2T exometabolome provided evidence of extracellular proteolytic activity based on the accumulation of free amino acids. However, several neutral and polar amino acids appear not to be utilized, based on their accumulation in the medium and the lack of annotated transporters. Adenine and adenosine were scavenged, and thymine and nicotinic acid were released, suggesting interdependency with other organisms in situ. Metabolic probing of T. hugenholtzii JAD2T using 13C-labeled compounds provided evidence of oxidation of glucose, pyruvate, cysteine, and citrate, and functioning glycolytic, tricarboxylic acid (TCA), and oxidative pentose-phosphate pathways (PPPs). However, differential use of position-specific 13C-labeled compounds showed that glycolysis and the TCA cycle were uncoupled. Thus, despite the high abundance of Thermoflexus in sediments of some geothermal systems, they appear to be highly focused on chemoorganotrophy, particularly protein degradation, and may interact extensively with other microorganisms in situ.


Early formation of the Moon 4.51 billion years ago.
Establishing the age of the Moon is critical to understanding solar system evolution and the formation of rocky planets, including Earth. However, despite its importance, the age of the Moon has never been accurately determined. We present uranium-lead dating of Apollo 14 zircon fragments that yield highly precise, concordant ages, demonstrating that they are robust against postcrystallization isotopic disturbances. Hafnium isotopic analyses of the same fragments show extremely low initial 176Hf/177Hf ratios corrected for cosmic ray exposure that are near the solar system initial value. Our data indicate differentiation of the lunar crust by 4.51 billion years, indicating the formation of the Moon within the first ~60 million years after the birth of the solar system.


How to estimate the measurement error variance associated with ancestry proportion estimates.
To show how the variance of the measurement error (ME) associated with individual ancestry proportion estimates can be estimated, especially when the number of ancestral populations (k) is greater than 2. We extend existing internal consistency measures to estimate the ME variance, and we compare these estimates with the ME variance estimated by use of the repeated measurement (RM) approach. Both approaches work by dividing the genotyped markers into subsets. We examine the effect of the number of subsets and of the allocation of markers to each subset on the performance of each approach. We used simulated data for all comparisons. Independently of the value of k, the measures of internal reliability provided less biased and more precise estimates of the ME variance than did those obtained with the RM approach. Both methods tend to perform better when a large number of subsets of markers with similar sizes are considered. Our results will facilitate the use of ME correction methods to address the ME problem in individual ancestry proportion estimates. Our method will improve the ability to control for type I error inflation and loss of power in association tests and other genomic research involving ancestry estimates.


Narrow-beam propagation in a two-dimensional scattering medium.
The problem of narrow-beam propagation in a medium with highly anisotropic scattering is considered within radiative transfer theory. A novel solution of the radiative transfer equation within small-angle approximation, accounting for the path length spread, is presented. A new stable numerical algorithm for the simulation of a narrow beam is developed and applied to a narrow beam in a two-dimensional scattering medium. A truly three-dimensional version of the proposed approach is formulated.


[A rare cause of pneumonia: Shewanella putrefaciens].
Shewanella putrefaciens is a gram-negative, non-fermentative, oxidase positive, motile bacillus that produces hydrogen sulphide. It is found widely in the nature especially in marine environments. Although it is accepted as saprophytic, different clinical syndromes, most commonly skin or soft tissue infections, have been associated with S.putrefaciens, mainly in immunocompromised cases and patients with underlying diseases. However, pneumonia cases due to S.putrefaciens are quite limited in the literature. In this report, a case of pneumonia caused by S.putrefaciens was presented. A 43-year-old female patient was admitted to our hospital with the complaints of fever, cough, sputum and weakness. The patient has had brochiectasis since childhood and has used periodical antibiotic therapies due to pneumoniae episodes. She was diagnosed to have pneumonia based on the clinical, radiological and laboratory findings, and empirical antibiotic treatment with ciprofloxacin and ceftazidime combination was initiated. Gram-stained smear of sputum yielded abundant leucocytes and gram-negative bacteria, and the isolate grown in the sputum culture was identified as S.putrefaciens by conventional methods and API 20 NE (BioMerieux, France) system. The isolate was found susceptible to ceftriaxone, ceftazidime, cefepime, ciprofloxacin, piperacillin-tazobactam, cephoperazon-sulbactam, imipenem, amikacin, gentamicin and trimethoprime-sulphametoxazole; whereas resistant to ampicillin, amoxycillin-clavulanate, cefazolin and cefuroxime, by Kirby-Bauer disk diffusion method. According to the antibiogram results, the therapy was changed to ceftriaxone (1 x 2 g, intravenous). The patient was discharged with complete cure after 14 days of therapy. In conclusion, S.putrefaciens should be considered in patients with predisposing factors as an unusual cause of pneumonia and the characteristics such as H2S production and sensitivity to third generation cephalosporins and penicillins should be used to differentiate it from Pseudomonas aeruginosa and prevent the unnecessary use of antipseudomonal antibiotics.


Similar DNA methylation levels in specific imprinting control regions in children conceived with and without assisted reproductive technology: a cross-sectional study.
While a possible link between assisted reproductive technology (ART) and rare imprinting disorders has been found, it is not clear if this is indicative of subtler disruptions of epigenetic mechanisms. Results from previous studies have been mixed, but some methylation differences have been observed. Children conceived through ART and children conceived spontaneously were recruited for this cross-sectional study. Information about reproductive history, demographic factors, birth characteristics, and infertility treatment was obtained from maternal interview and medical records. Peripheral blood lymphocytes and buccal cell samples were collected from participating children. Methylation analysis was performed on five loci using pyrosequencing. Statistical analysis of methylation differences was performed using linear regression with generalized estimating equations. Results are reported as differences with 95% confidence intervals (CI). A total of 67 ART children and 31 spontaneously conceived (SC) children participated. No significant difference in methylation in lymphocyte samples was observed between groups for any loci. Possible differences were found in buccal cell samples for IGF2 DMR0 (Difference: 2.07; 95% confidence interval (CI): -0.28, 4.42; p = 0.08) and IGF2R (Difference: -2.79; 95% CI: -5.74, 0.16; p = 0.06). Subgroup analysis indicated potential lower methylation in those whose parents used ART for unexplained infertility. Observed differences in methylation between the ART and SC groups were small for all loci in the two sample types examined and no statistical differences were observed. It is still unclear whether or not small differences observed in several studies represent a real difference between groups and if this difference is biologically meaningful. Larger studies with long term follow-up are needed to fully answer these questions.


Principles of cooperation across systems: from human sharing to multicellularity and cancer.
From cells to societies, several general principles arise again and again that facilitate cooperation and suppress conflict. In this study, I describe three general principles of cooperation and how they operate across systems including human sharing, cooperation in animal and insect societies and the massively large-scale cooperation that occurs in our multicellular bodies. The first principle is that of Walk Away: that cooperation is enhanced when individuals can leave uncooperative partners. The second principle is that resource sharing is often based on the need of the recipient (i.e., need-based transfers) rather than on strict account-keeping. And the last principle is that effective scaling up of cooperation requires increasingly sophisticated and costly cheater suppression mechanisms. By comparing how these principles operate across systems, we can better understand the constraints on cooperation. This can facilitate the discovery of novel ways to enhance cooperation and suppress cheating in its many forms, from social exploitation to cancer.


A pilot window-of-opportunity study of preoperative fluvastatin in localized prostate cancer.
Statins inhibit HMG-CoA reductase, the rate-limiting enzyme of the mevalonate pathway. Epidemiological and pre-clinical evidence support an association between statin use and delayed prostate cancer (PCa) progression. Here, we evaluated the effects of neoadjuvant fluvastatin treatment on markers of cell proliferation and apoptosis in men with localized PCa. Thirty-three men were treated daily with 80 mg fluvastatin for 4-12 weeks in a single-arm window-of-opportunity study between diagnosis of localized PCa and radical prostatectomy (RP) (ClinicalTrials.gov: NCT01992042). Percent Ki67 and cleaved Caspase-3 (CC3)-positive cells in tumor tissues were evaluated in 23 patients by immunohistochemistry before and after treatment. Serum and intraprostatic fluvastatin concentrations were quantified by liquid chromatography-mass spectrometry. Baseline characteristics included a median prostate-specific antigen (PSA) level of 6.48 ng/mL (IQR: 4.21-10.33). The median duration of fluvastatin treatment was 49 days (range: 27-102). Median serum low-density lipoprotein levels decreased by 35% after treatment, indicating patient compliance. Median PSA decreased by 12%, but this was not statistically significant in our small cohort. The mean fluvastatin concentration measured in the serum was 0.2 μM (range: 0.0-1.1 μM), and in prostatic tissue was 8.5 nM (range: 0.0-77.0 nM). At these concentrations, fluvastatin induced PCa cell death in vitro in a dose- and time-dependent manner. In patients, fluvastatin treatment did not significantly alter intratumoral Ki67 positivity; however, a median 2.7-fold increase in CC3 positivity (95% CI: 1.9-5.0, p = 0.007) was observed in post-fluvastatin RP tissues compared with matched pre-treatment biopsy controls. In a subset analysis, this increase in CC3 was more pronounced in men on fluvastatin for >50 days. Fluvastatin prior to RP achieves measurable drug concentrations in prostatic tissue and is associated with promising effects on tumor cell apoptosis. These data warrant further investigation into the anti-neoplastic effects of statins in prostate tissue.


Development and validation of a clinical risk score to predict mortality after percutaneous coronary intervention.
To develop and validate a contemporary clinical risk score to predict mortality after percutaneous coronary intervention (PCI). Using data collected from patients undergoing PCI at the South Yorkshire Cardiothoracic Centre, Sheffield, UK, between January 2007 and September 2013, a risk score was developed to predict mortality. Logistic regression was used to evaluate the effect of each variable upon 30-day mortality. A backwards stepwise logistic regression model was then used to build a predictive model. The results were validated both internally and externally with data from Manchester Royal Infirmary, UK. 30-Day mortality status was determined from the UK Office of National Statistics. The development data set comprised 6522 patients from Sheffield. Five risk factors, including cardiogenic shock, procedural urgency, history of renal disease, diabetes mellitus and age, were statistically significant to predict 30-day mortality. The risk score was validated internally on a further 3290 patients from Sheffield and externally on 3230 patients from Manchester. The discrimination of the model was high in the development (C-statistic=0.82, 95% CI 0.79 to 0.85), internal (C-statistic=0.81, 95% CI 0.76 to 0.86) and external (C statistics=0.90, 95% CI 0.87 to 0.93) cohorts. There was no significant difference between observed and predicted mortality in any group. This contemporary risk score reliably predicts 30-day mortality after PCI using a small number of clinical variables obtainable prior to the procedure, without knowledge of the coronary anatomy.


Impact of co-infections and BCG immunisation on immune responses among household contacts of tuberculosis patients in a Ugandan cohort.
Tuberculosis incidence in resource poor countries remains high. We hypothesized that immune modulating co-infections such as helminths, malaria, and HIV increase susceptibility to latent tuberculosis infection (LTBI), thereby contributing to maintaining the tuberculosis epidemic. Adults with sputum-positive tuberculosis (index cases) and their eligible household contacts (HHCs) were recruited to a cohort study between May 2011 and January 2012. HHCs were investigated for helminths, malaria, and HIV at enrolment. HHCs were tested using the QuantiFERON-TB Gold In-Tube (QFN) assay at enrolment and six months later. Overnight whole blood culture supernatants from baseline QFN assays were analyzed for cytokine responses using an 11-plex Luminex assay. Associations between outcomes (LTBI or cytokine responses) and exposures (co-infections and other risk factors) were examined using multivariable logistic and linear regression models. We enrolled 101 index cases and 291 HHCs. Among HHCs, baseline prevalence of helminths was 9% (25/291), malaria 16% (47/291), HIV 6% (16/291), and LTBI 65% (179/277). Adjusting for other risk factors and household clustering, there was no association between LTBI and any co-infection at baseline or at six months: adjusted odds ratio (95% confidence interval (CI); p-value) at baseline for any helminth, 1.01 (0.39-2.66; 0.96); hookworm, 2.81 (0.56-14.14; 0.20); malaria, 1.06 (0.48-2.35; 0.87); HIV, 0.74 (0.22-2.47; 0.63). HHCs with LTBI had elevated cytokine responses to tuberculosis antigens but co-infections had little effect on cytokine responses. Exploring other risk factors, Th1 cytokines among LTBI-positive HHCs with BCG scars were greatly reduced compared to those without scars: (adjusted geometric mean ratio) IFNγ 0.20 (0.09-0.42), <0.0001; IL-2 0.34 (0.20-0.59), <0.0001; and TNFα 0.36 (0.16-0.79), 0.01. We found no evidence that co-infections increase the risk of LTBI, or influence the cytokine response profile among those with LTBI. Prior BCG exposure may reduce Th1 cytokine responses in LTBI.


Conditional LoxP-flanked glucosylceramide synthase allele controlling glycosphingolipid synthesis.
Glycosphingolipids are organizational building blocks of plasma membranes that participate in key cellular functions, such as signaling and cell-to-cell interactions. Glucosylceramide synthase--encoded by the Ugcg gene--controls the first committed step in the major pathway of glycosphingolipid synthesis. Global disruption of the Ugcg gene in mice is lethal during gastrulation. We have now established a Ugcg allele flanked by loxP sites (floxed). When cre recombinase was expressed in the nervous system under control of the nestin promoter, the floxed gene underwent recombination, resulting in a substantial reduction of Ugcg expression and of glycosphingolipid ganglio-series levels. The mice deficient in Ugcg expression in the nervous system show a striking loss of Purkinje cells and abnormal neurologic behavior. The floxed Ugcg allele will facilitate analysis of the function of glycosphingolipids in development, physiology, and in diseases such as diabetes and cancer.


Basal variability in CREB phosphorylation predicts trait-like differences in amygdala-dependent memory.
Much of what is known about the neurobiology of learning and memory comes from studies of the average behavior. In contrast, intersubject differences that emerge within groups are difficult to study systematically and are often excluded from scientific discussion. Nevertheless, population-wide variability is a virtually universal feature of both complex traits, such as intelligence, and hardwired responses, such as defensive behaviors. Here, we use outbred rats to investigate if cAMP response element-binding protein (CREB), a transcription factor that has long been known in experimental settings to be crucial for associative plasticity, participates in natural memory phenotypes. Using a combination of behavioral, biochemical, and viral techniques, we show that a subset of rats with trait-like deficits in aversive memory have basally reduced CREB activity in the lateral amygdala but can be induced to perform at average levels by directly or indirectly enhancing pretraining CREB phosphorylation. These data suggest that endogenous CREB activity in the amygdala may set a critical threshold for plasticity during memory formation.


Nanoscale departures: excess lipid leaving the surface during supported lipid bilayer formation.
The behavior of small liposomes on surfaces of inorganic oxides remains enigmatic. Under appropriate conditions it results in the formation of supported lipid bilayers (SLBs). During this process, some lipids leave the surface (desorb). We were able to visualize this by a combination of time-resolved fluorescence microscopy and fluorescence recovery after photobleaching studies. Our observations also allowed us to analyze the kinetics of bilayer patch growth during the late stages of SLB formation. We found that it entails a balance between desorption of excess lipids and further adsorption of liposomes from solution. These studies were performed with liposomes containing zwitterionic phospholipids (dioleoylphosphatidylcholine alone or a mixture of dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, and cholesterol) on TiO2 in the presence of Ca(2+) but in the absence of other salts.


HLA class I-mediated stress fiber formation requires ERK1/2 activation in the absence of an increase in intracellular Ca2+ in human aortic endothelial cells.
Following transplantation, HLA class I antibodies targeting donor endothelium stimulate cell proliferation and migration, which contribute to the development of transplant vasculopathy and chronic allograft rejection. Dynamic remodeling of the actin cytoskeleton regulates cell proliferation and migration in endothelial cells (ECs), but the mechanism(s) involved remain incompletely understood. We explored anti-HLA class I antibody-mediated alterations of the cytoskeleton in human aortic ECs (HAECs) and contrasted these findings to thrombin-induced cytoskeleton remodeling. Our results identify two different signaling pathways leading to myosin light chain (MLC) phosphorylation in HAECs. Stimulation of HAECs with thrombin at 1 U/ml induced a robust elevation of intracellular Ca(2+) concentration, increased MLC phosphorylation, and promoted stress fiber formation via MLC kinase (MLCK) and Rho kinase (ROK) in an ERK-independent manner. In contrast, HAECs stimulated with HLA class I antibodies did not promote any detectable change in intracellular Ca(2+) concentration but instead induced MLC phosphorylation and stress fiber assembly via MLCK and ROK in an ERK1/2-dependent manner. Stimulation of HAECs with low-dose thrombin (1 mU/ml) induced signaling cascades that were similar to stimulation with HLA class I antibodies. HLA class I antibodies also stimulated the translocation of mammalian target of rapamycin complex 2 (mTORC2) and ERK1/2 from the cytoplasm to the plasma membrane independently of stress fiber assembly. These findings identify novel roles for HLA class I signaling in ECs and provide new insights into the role of ERK1/2 and mTORC2 in cytoskeleton regulation, which may be important in promoting transplant vasculopathy, tumor angiogenesis, and atherosclerosis.


A STAT4 variant increases liver fibrosis risk in Caucasian patients with chronic hepatitis B.
Host genetic modifiers of the natural history of chronic hepatitis B (CHB) remain poorly understood. Recently, a genome-wide association study (GWAS)-identified polymorphism in the STAT4 gene that contributes to the risk for hepatocellular carcinoma (HCC) was shown to be associated with the full spectrum of hepatitis B virus (HBV) outcomes in Asian patients. However, the functional mechanisms for this effect are unknown and the role of the variant in modulating HBV disease in Caucasians has not been investigated. To determine whether STAT4 genetic variation is associated with liver injury in Caucasian patients with CHB and to investigate potential mechanisms mediating this effect. STAT4 rs7574865 was genotyped in 1085 subjects (830 with CHB and 255 healthy controls). STAT4 expression in liver, PBMCs and NK cells, STAT4 phosphorylation and secretion of interferon-gamma (IFN-γ) according to STAT4 genetic variation was examined. STAT4 rs7574865 genotype was independently associated with hepatic inflammation (OR: 1.42, 95% CI: 1.07-2.06, P = 0.02) and advanced fibrosis (OR: 1.83, 95% CI: 1.19-2.83, P = 0.006). The minor allele frequency of rs7574865 was significantly lower than that in healthy controls. rs7574865 GG risk carriers expressed lower levels of STAT4 in liver, PBMCs and in NK cells, while NK cells from patients with the risk genotype had impaired STAT4 phosphorylation following stimulation with IL-12/IL-18 and a reduction in secretion of IFN-γ. Genetic susceptibility to HBV persistence, hepatic inflammation and fibrosis in Caucasians associates with STAT4 rs7574865 variant. Downstream effects on NK cell function through STAT4 phosphorylation-dependent IFN-γ production likely contribute to these effects.


Ontogeny of aerial righting and wing flapping in juvenile birds.
Mechanisms of aerial righting in juvenile chukar partridge (Alectoris chukar) were studied from hatching to 14 days-post-hatching (dph). Asymmetric movements of the wings were used from 1 to 8 dph to effect progressively more successful righting behaviour via body roll. Following 8 dph, wing motions transitioned to bilaterally symmetric flapping that yielded aerial righting via nose-down pitch, along with substantial increases in vertical force production during descent. Ontogenetically, the use of such wing motions to effect aerial righting precedes both symmetric flapping and a previously documented behaviour in chukar (i.e. wing-assisted incline running) hypothesized to be relevant to incipient flight evolution in birds. These findings highlight the importance of asymmetric wing activation and controlled aerial manoeuvres during bird development and are potentially relevant to understanding the origins of avian flight.


Atomoxetine Enhances Connectivity of Prefrontal Networks in Parkinson's Disease.
Cognitive impairment is common in Parkinson's disease (PD), but often not improved by dopaminergic treatment. New treatment strategies targeting other neurotransmitter deficits are therefore of growing interest. Imaging the brain at rest ('task-free') provides the opportunity to examine the impact of a candidate drug on many of the brain networks that underpin cognition, while minimizing task-related performance confounds. We test this approach using atomoxetine, a selective noradrenaline reuptake inhibitor that modulates the prefrontal cortical activity and can facilitate some executive functions and response inhibition. Thirty-three patients with idiopathic PD underwent task-free fMRI. Patients were scanned twice in a double-blind, placebo-controlled crossover design, following either placebo or 40-mg oral atomoxetine. Seventy-six controls were scanned once without medication to provide normative data. Seed-based correlation analyses were used to measure changes in functional connectivity, with the right inferior frontal gyrus (IFG) a critical region for executive function. Patients on placebo had reduced connectivity relative to controls from right IFG to dorsal anterior cingulate cortex and to left IFG and dorsolateral prefrontal cortex. Atomoxetine increased connectivity from the right IFG to the dorsal anterior cingulate. In addition, the atomoxetine-induced change in connectivity from right IFG to dorsolateral prefrontal cortex was proportional to the change in verbal fluency, a simple index of executive function. The results support the hypothesis that atomoxetine may restore prefrontal networks related to executive functions. We suggest that task-free imaging can support translational pharmacological studies of new drug therapies and provide evidence for engagement of the relevant neurocognitive systems.


A multilocus sequence survey in Arabidopsis thaliana reveals a genome-wide departure from a neutral model of DNA sequence polymorphism.
The simultaneous analysis of multiple genomic loci is a powerful approach to studying the effects of population history and natural selection on patterns of genetic variation of a species. By surveying nucleotide sequence polymorphism at 334 randomly distributed genomic regions in 12 accessions of Arabidopsis thaliana, we examined whether a standard neutral model of nucleotide sequence polymorphism is consistent with observed data. The average nucleotide diversity was 0.0071 for total sites and 0.0083 for silent sites. Although levels of diversity are variable among loci, no correlation with local recombination rate was observed, but polymorphism levels were correlated for physically linked loci (<250 kb). We found that observed distributions of Tajima's D- and D/D(min)- and of Fu and Li's D-, D*- and F-, F*-statistics differed significantly from the expected distributions under a standard neutral model due to an excess of rare polymorphisms and high variances. Observed and expected distributions of Fay and Wu's H were not different, suggesting that demographic processes and not selection at multiple loci are responsible for the deviation from a neutral model. Maximum-likelihood comparisons of alternative demographic models like logistic population growth, glacial refugia, or past bottlenecks did not produce parameter estimates that were more consistent with observed patterns. However, exclusion of highly polymorphic "outlier loci" resulted in a fit to the logistic growth model. Various tests of neutrality revealed a set of candidate loci that may evolve under selection.


Tungstate treatment improves Leydig cell function in streptozotocin-diabetic rats.
Oral administration of sodium tungstate to adult male streptozotocin-diabetic rats for 3 months normalized serum levels of glucose, insulin, luteinizing hormone, and follicle-stimulating hormone. These effects were accompanied by an increase in reproductive performance, which was related to a strong improvement in Leydig cell function markers, such as the recovery of the number of Leydig cells and serum testosterone levels. Moreover, this in vivo recovery was related to a concomitant increase in the cell expression of insulin receptors. Tungstate treatment did not modify Leydig cell function in healthy rats. Furthermore, the addition of tungstate or insulin to the mTLC-1 cell line from Leydig cell origin increased the phosphorylation states of MAP-kinase and glycogen synthase kinase-3. Our results indicate that tungstate treatment in diabetic rats leads to a recovery of reproductive performance by increasing the number of Leydig cells. This increase contributes to the recovery of their functionality, thereby improving the overall function of these cells. We propose that this improvement is caused by the combined effect of the tungstate-induced normalization of insulin glucose and luteinizing hormone serum levels and a direct action of the effector on Leydig cells through modulation of at least MAP-kinase and glycogen synthase kinase-3 activities.


Conformity to traditional Mediterranean diet and cancer incidence: the Greek EPIC cohort.
Adherence to traditional Mediterranean diet (MD) has been reported to be inversely associated with total, as well as cardiovascular, mortality. We have examined the relation between degree of such adherence and incidence of cancer overall in a general population sample of 25 623 participants (10 582 men, 15 041 women) of the Greek segment of the European Prospective Investigation into Cancer and nutrition (EPIC). All subjects completed a validated, interviewer-administered, semi-quantitative food-frequency questionnaire at enrolment. Degree of adherence to the traditional MD was assessed through a 10-point scale (0 minimal; 9 maximal) that incorporated key dietary characteristics. During a median follow-up of 7.9 years and 188 042 total person-years, 851 medically confirmed incident cancer cases (421 men, 430 women) were recorded. Using proportional hazards regression with adjustment for potential confounders, we found that a higher degree of MD adherence was associated with lower overall cancer incidence. A two-point increase in the score corresponded to a 12% reduction in cancer incidence (adjusted hazard ratio, 0.88 (95% confidence interval 0.80, 0.95)). The association was exposure-dependent and stronger among women. This inverse association with MD adherence was considerably stronger than that predicted on the basis of the associations of the individual components of this diet and points to the value of analysing dietary patterns in cancer studies.


Drivers, Beliefs, and Barriers Surrounding Surgical Opioid Prescribing: A Qualitative Study of Surgeons' Opioid Prescribing Habits.
Recent data demonstrate that surgeons overprescribe opioids and vary considerably in the amount of opioids prescribed for common procedures. Limited data exist about why and how surgeons develop certain opioid prescribing habits. We sought to identify surgeons' knowledge, attitudes, and beliefs about opioid prescribing and elicit barriers to guideline-based prescribing. We conducted qualitative semistructured interviews accompanied by demographic surveys at an academic medical center. Surgical residents and faculty members were selected by maximum variation purposive sampling. We used thematic analysis to identify themes associated with opioid prescribing. Twenty surgical residents and twenty-one surgical faculty members were interviewed. Characteristics of individual surgeons, patients, health care teams, practice environments, and the complex interplay between these domains drove prescribing habits. Attending-resident communication about opioid prescribing was extremely limited. Surgeons received little training and feedback about opioid prescribing and were rarely aware of negative long-term consequences, limiting motivation to change prescribing habits. Although surgeons frequently interacted with pain management physicians to comanage patients postoperatively, few involved pain management physicians in preoperative planning. Perceived barriers to guideline-based prescribing included the following: limitations to electronic prescribing, cross-coverage problems, inadequate time for patient education, and impediments to use of nonopioid alternatives. Interventions to improve compliance with opioid prescribing guidelines should include surgeon education and personal feedback. Future interventions should aim to improve attending-resident communication about opioid prescribing, reduce hurdles to electronic prescribing, provide clear pain management plans for cross-covering physicians, assess alternative methods for efficient patient education, and maximize use of nonnarcotic pain medications.


An inhalation model of allergic fungal asthma: Aspergillus fumigatus-induced inflammation and remodeling in allergic airway disease.
The ability to accurately mimic normal processes for sensitization and allergen challenge in an experimental animal model are useful in that they allow researchers to critically manipulate the complex interactions of multiple cell types. In the context of the allergic lung, multiple cell types form complex cellular networks and function to regulate a variety of temporal and spatial changes. Mouse models of allergic airway disease have proven to be highly useful for dissecting these complex interactions, particularly in addressing remodeling of the allergic airway in chronic asthma. Until we can better represent the normal processes that initiate and perpetuate asthma, our understanding of the mechanisms of tissue injury leading to chronic remodeling of the airways and effective therapeutic strategies to treat this disease will remain limited. It was with this goal in mind that we set about devising an inhalational model of Aspergillus fumigatus-induced fungal asthma in a murine experimental system.


Diagnostic triage and the role of natriuretic peptide testing and echocardiography for suspected heart failure: an appropriateness ratings evaluation by UK GPs.
Some UK GPs are acquiring access to natriuretic peptide (NP) testing or echocardiography as diagnostic tests for heart failure. This study developed appropriateness ratings for the diagnostic application of these tests in routine general practice. To develop appropriateness ratings for the diagnostic application of NP testing or echocardiography for heart failure in general practice. An appropriateness ratings evaluation in UK general practice. Four presenting symptoms (cough, bilateral ankle swelling, dyspnoea, fatigue), three levels of risk of cardiovascular disease (low, intermediate, high), and dichotomous categorisations of cardiovascular/chest examination and electrocardiogram result, were used to create 540 appropriateness scenarios for patients in whom NP testing or echocardiography might be considered. These were rated by a 10-person expert panel, consisting of GPs and GPs with specialist interests in cardiology, in a two-round RAND Appropriateness Method. Onward referral for NP testing or echocardiography was rated as an appropriate next step in 217 (40.2%) of the 540 scenarios; in 194 (35.9%) it was rated inappropriate. The ratings also show where NP testing or echocardiography were ranked as equivalent next steps and when one test was seen as the more appropriate than the other. NP testing should be the routine test for suspected heart failure where referral for diagnostic testing is considered appropriate. An abnormal electrocardiogram status makes referral to echocardiography an accompanying, or more appropriate, next step alongside NP testing, especially in the presence of dyspnoea. Abnormal NP testing should subsequently be followed up with referral for echocardiography.


High precision, localized proton gradients and fluxes generated by a microelectrode device induce differential growth behaviors of pollen tubes.
Pollen tubes are tip-growing plant cells that deliver the sperm cells to the ovules for double fertilization of the egg cell and the endosperm. Various directional cues can trigger the reorientation of pollen tube growth direction on their passage through the female tissues. Among the external stimuli, protons serve an important, regulatory role in the control of pollen tube growth. The generation of local guidance cues has been challenging when investigating the mechanisms of perception and processing of such directional triggers in pollen tubes. Here, we developed and characterized a microelectrode device to generate a local proton gradient and proton flux through water electrolysis. We confirmed that the cytoplasmic pH of pollen tubes varied with environmental pH change. Depending on the position of the pollen tube tip relative to the proton gradient, we observed alterations in the growth behavior, such as bursting at the tip, change in growth direction, or complete growth arrest. Bursting and growth arrest support the hypothesis that changes in the extracellular H+ concentration may interfere with cell wall integrity and actin polymerization at the growing tip. A change in growth direction for some pollen tubes implies that they can perceive the local proton gradient and respond to it. We also showed that the growth rate is directly correlated with the extracellular pH in the tip region. Our microelectrode approach provides a simple method to generate protons and investigate their effect on plant cell growth.


[Darwin or Lamarck? Understanding the ocular surface and its normal or abnormal differentiation in order to cure ocular surface destruction with corneal opacification].
According to the World Health Organization, 5.1% of blindnesses or visual impairments are related to corneal opacification. Cornea is a transparent tissue placed in front of the color of the eye. Its transparency is mandatory for vision. The ocular surface is a functional unit including the cornea and all the elements involved in maintaining its transparency i.e., the eyelids, the conjunctiva, the lymphoid tissue of the conjunctiva, the limbus, the lacrymal glands and the tear film. The destruction of the ocular surface is a disease caused by : traumatisms, infections, chronic inflammations, cancers, toxics, unknown causes or congenital abnormalities. The treatment of the ocular surface destruction requires a global strategy including all the elements that are involved in its physiology. The microenvironnement of the ocular surface must first be restored, i.e., the lids, the conjunctiva, the limbus and the structures that secrete the different layers of the tear film. In a second step, the transparency of the cornea can be reconstructed. A corneal graft performed in a healthy ocular surface microenvironnement will have a better survival rate. To achieve these goals, a thorough understanding of the renewal of the epitheliums and the role of the epithelial stem cells are mandatory.


Toxicological assessment of cigarette ingredients.
Ingredients have been used in industrial manufacture of tobacco products since the early part of the 20th century. However, unlike other consumer goods, until now no regulatory authority has determined how tobacco ingredients should be assessed. Although there is currently no consensus on how added cigarette ingredients should be evaluated, this paper reviews some of the institutional guidance alongside published literature with a view to determining if there is a generally accepted approach in the absence of any strict regulation. Our aim was to review the recommendations, to compare them to the working practices as demonstrated from published studies, and to draw conclusions on currently used methodologies for testing ingredients added to cigarettes. The extent of testing is discussed in the light of practical and theoretical constraints and an example of an industry testing program is presented.


Polyphenols from green tea inhibit the growth of melanoma cells through inhibition of class I histone deacetylases and induction of DNA damage.
Melanoma is the leading cause of skin cancer-related deaths. We have examined the effect of green tea polyphenols (GTPs), a natural mixture of epicatechin monomers, on melanoma cancer cell growth and the molecular mechanism underlying these effects using different human melanoma cell lines as an in vitro model. Treatment of melanoma cell lines (A375, Hs294t, SK-Mel28 and SK-Mel119) with GTPs significantly inhibited the cell viability as well as colony formation ability of melanoma cells in a dose-dependent manner. These effects of GTPs were associated with a significant inhibition of histone deacetylase (HDAC) activity, reduction in the levels of class I HDAC proteins, enhancement of histone acetyltransferase (HAT) activity and induction of DNA damage, as detected by Comet assay, in melanoma cells. GTPs-induced decrease in the levels of class I HDAC proteins is mediated through proteasomal degradation. Valproic acid, an inhibitor of HDACs, exhibited a similar pattern of reduced viability and induction of death of melanoma cells. Treatment of A375 and Hs294t cells with GTPs resulted in a decrease in the levels of cyclins and cyclin dependent kinases of G1 phase of cell cycle whereas upregulated the levels of tumor suppressor proteins (Cip1/WAF1/p21, p16 and p53).


Exposing Hidden Alternative Backbone Conformations in X-ray Crystallography Using qFit.
Proteins must move between different conformations of their native ensemble to perform their functions. Crystal structures obtained from high-resolution X-ray diffraction data reflect this heterogeneity as a spatial and temporal conformational average. Although movement between natively populated alternative conformations can be critical for characterizing molecular mechanisms, it is challenging to identify these conformations within electron density maps. Alternative side chain conformations are generally well separated into distinct rotameric conformations, but alternative backbone conformations can overlap at several atomic positions. Our model building program qFit uses mixed integer quadratic programming (MIQP) to evaluate an extremely large number of combinations of sidechain conformers and backbone fragments to locally explain the electron density. Here, we describe two major modeling enhancements to qFit: peptide flips and alternative glycine conformations. We find that peptide flips fall into four stereotypical clusters and are enriched in glycine residues at the n+1 position. The potential for insights uncovered by new peptide flips and glycine conformations is exemplified by HIV protease, where different inhibitors are associated with peptide flips in the "flap" regions adjacent to the inhibitor binding site. Our results paint a picture of peptide flips as conformational switches, often enabled by glycine flexibility, that result in dramatic local rearrangements. Our results furthermore demonstrate the power of large-scale computational analysis to provide new insights into conformational heterogeneity. Overall, improved modeling of backbone heterogeneity with high-resolution X-ray data will connect dynamics to the structure-function relationship and help drive new design strategies for inhibitors of biomedically important systems.


A critical role for the transient receptor potential channel type 6 in human platelet activation.
While calcium signaling is known to play vital roles in platelet function, the mechanisms underlying its receptor-operated calcium entry component (ROCE) remain poorly understood. It has been proposed, but never proven in platelets, that the canonical transient receptor potential channel-6 (TRPC6) mediates ROCE. Nonetheless, we have previously shown that the mouse TRPC6 regulates hemostasis, thrombogenesis by regulating platelet aggregation. In the present studies, we used a pharmacological approach to characterize the role of TRPC6 in human platelet biology. Thus, interestingly, we observed that a TRPC6 inhibitor exerted significant inhibitory effects on human platelet aggregation in a thromboxane receptor (TPR)-selective manner; no additional inhibition was observed in the presence of the calcium chelator BAPTA. This inhibitor also significantly inhibited human platelet secretion (dense and alpha granules), integrin IIb-IIIa, Akt and ERK phosphorylation, again, in a TPR-selective manner; no effects were observed in response to ADP receptor stimulation. Furthermore, there was a causal relationship between these inhibitory effects, and the capacity of the TRPC6 inhibitor to abrogate elevation in intracellular calcium, that was again found to be TPR-specific. This effect was not found to be due to antagonism of TPR, as the TRPC6 inhibitor did not displace the radiolabeled antagonist [3H]SQ29,548 from its binding sites. Finally, our studies also revealed that TRPC6 regulates human clot retraction, as well as physiological hemostasis and thrombus formation, in mice. Taken together, our findings demonstrate, for the first time, that TRPC6 directly regulates TPR-dependent ROCE and platelet function. Moreover, these data highlight TRPC6 as a novel promising therapeutic strategy for managing thrombotic disorders.


Scientific and budgetary trade-offs between morphological and molecular methods for deep-sea biodiversity assessment.
Deep-sea biodiversity, a source of critical ecological functions and ecosystem services, is increasingly subject to the threat of disturbance from existing practices (e.g., fishing, waste disposal, oil and gas extraction) as well as emerging industries such as deep-seabed mining. Current scientific tools may not be adequate for monitoring and assessing subsequent changes to biodiversity. In this paper, we evaluate the scientific and budgetary trade-offs associated with morphology-based taxonomy and metabarcoding approaches to biodiversity surveys in the context of nascent deep-seabed mining for polymetallic nodules in the Clarion-Clipperton Zone, the area of most intense interest. For the dominant taxa of benthic meiofauna, we discuss the types of information produced by these methods and use cost-effectiveness analysis to compare their abilities to yield biological and ecological data for use in environmental assessment and management. On the basis of our evaluation, morphology-based taxonomy is less cost-effective than metabarcoding but offers scientific advantages, such as the generation of density, biomass, and size structure data. Approaches that combine the two methods during the environmental assessment phase of commercial activities may facilitate future biodiversity monitoring and assessment for deep-seabed mining and for other activities in remote deep-sea habitats, for which taxonomic data and expertise are limited. Integr Environ Assess Manag 2022;18:655-663. © 2021 SETAC.


Toward health status insurance.
This paper advances a new concept in health insurance. Health status insurance is a health insurance whose primary aim is to improve health status and decrease inequality in health within the covered population. Redistribution and control of cost is a secondary aim, closely integrated with the health status aim. Health status insurance differs from conventional health insurance in four respects: 1) the health status and health risks of enrollees are assessed and individual plans are developed jointly by the providers and enrollees to raise their health status to a given level; 2) interventions are not limited to the patients, but they extend to the patients' physical and social environments; 3) supplementary income or help in organizing are provided to enrollees who do not have an income sufficient to maintain their health or who need help to overcome local environmental situations adverse to their health, respectively; and, 4) the health status insurance organization actively champions national policies to change the social environment for the improvement of health. The concept of health status insurance is applicable to national health insurance systems, large private health insurance systems, or, national health service (when the responsible governmental agency becomes the health status insurer). Three modalities of implementation are presented. The readiness of society and governments to adopt health status insurance is discussed from a historical perspective.


Comprehensive candidate gene study highlights UGT1A and BNC2 as new genes determining continuous skin color variation in Europeans.
Natural variation in human skin pigmentation is primarily due to genetic causes rooted in recent evolutionary history. Genetic variants associated with human skin pigmentation confer risk of skin cancer and may provide useful information in forensic investigations. Almost all previous gene-mapping studies of human skin pigmentation were based on categorical skin color information known to oversimplify the continuous nature of human skin coloration. We digitally quantified skin color into hue and saturation dimensions for 5,860 Dutch Europeans based on high-resolution skin photographs. We then tested an extensive list of 14,185 single nucleotide polymorphisms in 281 candidate genes potentially involved in human skin pigmentation for association with quantitative skin color phenotypes. Confirmatory association was revealed for several known skin color genes including HERC2, MC1R, IRF4, TYR, OCA2, and ASIP. We identified two new skin color genes: genetic variants in UGT1A were significantly associated with hue and variants in BNC2 were significantly associated with saturation. Overall, digital quantification of human skin color allowed detecting new skin color genes. The variants identified in this study may also contribute to the risk of skin cancer. Our findings are also important for predicting skin color in forensic investigations.


Implementing intensive insulin therapy: development and audit of the Bath insulin protocol.
Intensive insulin therapy to control blood glucose has been found to reduce mortality among critically ill patients in a surgical intensive care unit, though a simple prescriptive insulin infusion protocol to achieve this has not been published previously. This study documents the development and routine use of a simple prescriptive intravenous insulin infusion protocol for critically ill patients and compares the results with previous practice. During development the protocol was optimized and practical issues of implementation addressed. The optimized protocol was then used for all ICU admissions, and a prospectively defined retrospective chart audit performed for the first month of use. Results were compared with a similar time period the previous year. In September 2002, 27 admissions were started on the protocol. Blood glucose for the time on the protocol had a median value of 6.2 (IQR 5.9-7.1) mmol/l compared with 9.2 (IQR 8.1-10.2) mmol/l for those on insulin in 2001. Blood glucose for the whole ICU stay for those on the protocol in 2002 had a median value of 6.6 (IQR 6.0-7.4) mmol/l compared with 8.6 (IQR 8.0-9.4) mmol/l in 2001. Blood glucose for all ICU patients in 2002 had a median value of 6.5 (IQR 6.0-7.3) mmol/l compared with 7.2 (IQR 6.3-8.3) mmol/l in 2001. Three blood glucose recordings were less than 2.2 mmol/l in September 2002. This study provides initial effectiveness and safety data for the Bath Insulin Protocol Further audits in a larger patient population are now needed.


Postural Orthostatic Tachycardia With Chronic Fatigue After HPV Vaccination as Part of the "Autoimmune/Auto-inflammatory Syndrome Induced by Adjuvants": Case Report and Literature Review.
We report the case of a 14-year-old girl who developed postural orthostatic tachycardia syndrome (POTS) with chronic fatigue 2 months following Gardasil vaccination. The patient suffered from persistent headaches, dizziness, recurrent syncope, poor motor coordination, weakness, fatigue, myalgias, numbness, tachycardia, dyspnea, visual disturbances, phonophobia, cognitive impairment, insomnia, gastrointestinal disturbances, and a weight loss of 20 pounds. The psychiatric evaluation ruled out the possibility that her symptoms were psychogenic or related to anxiety disorders. Furthermore, the patient tested positive for ANA (1:1280), lupus anticoagulant, and antiphospholipid. On clinical examination she presented livedo reticularis and was diagnosed with Raynaud's syndrome. This case fulfills the criteria for the autoimmune/auto-inflammatory syndrome induced by adjuvants (ASIA). Because human papillomavirus vaccination is universally recommended to teenagers and because POTS frequently results in long-term disabilities (as was the case in our patient), a thorough follow-up of patients who present with relevant complaints after vaccination is strongly recommended.


Latent Membrane Protein 1 as a molecular adjuvant for single-cycle lentiviral vaccines.
Molecular adjuvants are a promising method to enhance virus-specific immune responses and protect against HIV-1 infection. Immune activation by ligands for receptors such as CD40 can induce dendritic cell activation and maturation. Here we explore the incorporation of two CD40 mimics, Epstein Barr Virus gene LMP1 or an LMP1-CD40 chimera, into a strain of SIV that was engineered to be limited to a single cycle of infection. Full length LMP1 or the chimeric protein LMP1-CD40 was cloned into the nef-locus of single-cycle SIV. Human and Macaque monocyte derived macrophages and DC were infected with these viruses. Infected cells were analyzed for activation surface markers by flow cytometry. Cells were also analyzed for secretion of pro-inflammatory cytokines IL-1β, IL-6, IL-8, IL-12p70 and TNF by cytometric bead array. Overall, single-cycle SIV expressing LMP1 and LMP1-CD40 produced a broad and potent T(H)1-biased immune response in human as well as rhesus macaque macrophages and DC when compared with control virus. Single-cycle SIV-LMP1 also enhanced antigen presentation by lentiviral vector vaccines, suggesting that LMP1-mediated immune activation may enhance lentiviral vector vaccines against HIV-1.


Revisiting the role of fat mass in the life extension induced by caloric restriction.
One of the most robust observations in the biology of aging is that caloric restriction (CR) extends life in a variety of species. Although CR results in a severalfold decrease in fat mass (FM), the role of fat on life extension was considered to be minimal. Two main reasons accounted for this belief. First, although increased FM is associated with changes in substrate oxidation and in glucose homeostasis, in part through the effects of free fatty acids (FFA) and glycerol, several studies have suggested that longevity is determined independent of FM. Second, CR has systemic effects on a range of functions including neurological, endocrine, reproductive, immunological and antineoplastic, none of which have been historically linked to fat. In the last few years, an explosion of evidence has demonstrated that fat tissue is a very active endocrine gland which secretes a variety of peptides (such as leptin and plasminogen activating inhibitor-1), cytokines (such as tumor necrosis factor), and complement factors (such as D, C3, and B). This is in addition to the presence of substrates, such as glycerol and FFA, which are stored and released by fat cells and are known to have a major role in hepatic and peripheral glucose metabolism. We propose that many of the systemic effects of CR can now be explained by the chronic effects related to decreased plasma levels of peptides, cytokines, complement factors, and substrates. In fact, all of the benefits of CR on the neuroendocrine system and those related to the improvement in glucose homeostasis can be attributed to decrease in adipose cells and their products. Other evidence from epidemiological data in human obesity supports the role of fat mass and its body distribution as a risk factor for morbidity and mortality in humans due to impaired glucose metabolism (similar to rodents), for cancer (similar to rodents), and for the development of atherosclerotic vascular disease (in humans). If all or most of the life-extending benefits of CR can be attributed to decreased fat stores, the expression of specific candidate proteins may be explored and manipulated in the search for the most powerful adipose-dependent signals that modulate life expectancy.


The MBP fusion protein restores the activity of the first phosphatase domain of CD45.
CD45 is a receptor-like protein tyrosine phosphatase critically involved in the regulation of initial effector functions in B- and T-cells. The protein comprises two phosphatase (PTP) domains in its cytoplasmic region. However, whether each PTP domain has enzyme activity by itself or whether both domains are required to build up a functional enzyme is unclear. We have studied different constructions of human CD45 comprising the two PTP domains, both separately and as a single protein, fused to maltose-binding protein (MBP). In apparent contrast with previous studies, we show that the first PTP domain of CD45 (when fused to MBP) may be a viable phosphatase in the absence of the second domain. Phosphatase activity resides in the monomeric form of the protein and is lost after proteolytic cleavage of the fusion partner, indicating that MBP specifically activates the first PTP domain. Furthermore, changes in the optimal pH for activity with respect to wild-type CD45 suggest that protein-protein interactions involving residues in the neighbourhood of the catalytic site mediate enzyme activation.