Evidence for directional selection at a novel major histocompatibility class I marker in wild common frogs (Rana temporaria) exposed to a viral pathogen (Ranavirus).
Whilst the Major Histocompatibility Complex (MHC) is well characterized in the anuran Xenopus, this region has not previously been studied in another popular model species, the common frog (Rana temporaria). Nor, to date, have there been any studies of MHC in wild amphibian host-pathogen systems. We characterise an MHC class I locus in the common frog, and present primers to amplify both the whole region, and specifically the antigen binding region. As no more than two expressed haplotypes were found in over 400 clones from 66 individuals, it is likely that there is a single class I locus in this species. This finding is consistent with the single class I locus in Xenopus, but contrasts with the multiple loci identified in axolotls, providing evidence that the diversification of MHC class I into multiple loci likely occurred after the Caudata/Anura divergence (approximately 350 million years ago) but before the Ranidae/Pipidae divergence (approximately 230 mya). We use this locus to compare wild populations of common frogs that have been infected with a viral pathogen (Ranavirus) with those that have no history of infection. We demonstrate that certain MHC supertypes are associated with infection status (even after accounting for shared ancestry), and that the diseased populations have more similar supertype frequencies (lower F(ST)) than the uninfected. These patterns were not seen in a suite of putatively neutral microsatellite loci. We interpret this pattern at the MHC locus to indicate that the disease has imposed selection for particular haplotypes, and hence that common frogs may be adapting to the presence of Ranavirus, which currently kills tens of thousands of amphibians in the UK each year.


In vitro studies on cellular and molecular mechanisms of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia: As2O3 induces NB4 cell apoptosis with downregulation of Bcl-2 expression and modulation of PML-RAR alpha/PML proteins.
It has been shown recently in China that arsenic trioxide (As2O3) is a very effective treatment for acute promyelocytic leukemia (APL). APL patients resistant to all-trans retinoic acid (ATRA) and conventional chemotherapy can still respond to AS2O3. In this study, we addressed the possible cellular and molecular mechanisms of this treatment by using NB4 cells as a model. The results show that: (1) As2O3 triggers relatively specific NB4 cell apoptosis at micromolar concentration, as proved by morphology, histogramic related nuclear DNA contents, and DNA gel eletrophoresis. (2) As2O3 does not influence bax, bcl-x, c-myc, and p53 gene expression, but downregulates bcl-2 gene expression at both mRNA and protein levels. (3) As2O3 induces a significant modulation of the PML staining pattern in NB4 cells and HL-60 cells. The micropunctates characteristic of PML-RAR alpha in NB4 cells dissappear after treatment with As2O3, whereas a diffuse PML staining occurs in the perinuclear cytoplasmic region. In addition, a low percentage of untreated NB4 cells exhibits an accumulation of PML positive particles in a compartment of cytoplasm. The percentage of these cells can be significantly increased after As2O3 treatment. A similar PML staining pattern is observed in apoptotic cells. (4) ATRA pretreatment does not influence As2O3-induced apoptosis. These results suggest that induction of cell apoptosis can be one of the mechanisms of the therapeutic effect of As2O3. Moreover, this apoptosis induction occurs independently of the retinoid pathway and may be mediated, at least partly, through the modulation of bcl-2, as well as PML-RAR alpha and/ or PML proteins.


Body temperature regulation during hemodialysis in long-term patients: is it time to change dialysate temperature prescription?
During hemodialysis procedures, changes in the dialysate temperature can raise or lower body temperature because the blood is returned to the patient in thermal equilibrium with the dialysate. Even a dialysate temperature equal to the patient's body temperature as measured from the tympanic membrane, oral cavity, or axilla can result in an increase in the patient's body temperature, leading to cutaneous vasodilation and the potential for cardiovascular instability and hypotension. This deleterious cycle of events can be prevented by suitably adjusting the dialysate temperature. Lowering the dialysate temperature from 37 degrees C to 34-35.5 degrees C has improved the cardiovascular stability of many hemodialysis patients. Continuous monitoring of blood temperature allows the practitioner to make preemptive changes in dialysate temperature because a small change in body temperature can have enormous cardiovascular implications. For example, only 0.3 degrees C to 0.8 degrees C separates the thresholds for skin vasodilation from that for shivering. A suggested improvement in the hemodialysis procedure is to use devices that allow continuous monitoring of arterial and venous blood temperatures and adjust the dialysate temperature automatically, keeping the patient, not the dialysate, isothermic. Less optimal solutions appear to be (1) to monitor arterial and venous temperatures while manually adjusting the dialysate temperature to maintain arterial (and hence body) temperature stability; (2) to monitor peripheral temperatures (oral, tympanic) at regular intervals and adjust dialysate temperature to maintain the body temperature constant; (3) routinely use a dialysate temperature <37.0 degrees C in all patients unless contraindicated.


Add-On Pregnenolone with L-Theanine to Antipsychotic Therapy Relieves Negative and Anxiety Symptoms of Schizophrenia: An 8-Week, Randomized, Double-Blind, Placebo-Controlled Trial.
Pregnenolone (PREG) and L-theanine (LT) have shown ameliorative effects on various schizophrenia symptoms. This is the first study to evaluate the efficacy and safety of augmentation of antipsychotic treatment among patients with chronic schizophrenia or schizoaffective disorder with PREG-LT. Double-blind, placebo-controlled trial of PREG-LT or placebo augmentation was conducted for eight weeks with 40 chronic DSM-IV schizophrenia and schizoaffective disorder patients with suboptimal response to antipsychotics. Oral PREG (50 mg/day) with LT (400 mg/day) or placebo were added to a stable regimen of antipsychotic medication from March 2011 to October 2013. The participants were rated using the Scale for the Assessment of Negative Symptoms (SANS), the Hamilton Scale for Anxiety (HAM-A), and the Positive and Negative Syndrome Scale (PANSS) scales bi-weekly. The decrease of SANS and HAM-A scores were the co-primary outcomes. Secondary outcomes included assessments of general functioning and side effects. Negative symptoms such as blunted affect, alogia, and anhedonia (SANS) were found to be significantly improved with moderate effect sizes among patients who received PREG-LT, in comparison with the placebo group. Add-on PREG-LT also significantly associated with a reduction of anxiety scores such as anxious mood, tension, and cardiovascular symptoms (HAM-A), and elevation of general functioning (GAF). Positive symptoms, antipsychotic agents, concomitant drugs, and illness duration did not associate significantly with effect of PREG-LT augmentation. PREG-LT was well-tolerated. Pregnenolone with L-theanine augmentation may offer a new therapeutic strategy for treatment of negative and anxiety symptoms in schizophrenia and schizoaffective disorder. Further studies are warranted. clinicaltrials.gov Identifier: NCT01831986.


Optimizing patient expectations to improve therapeutic response to medical treatment: A randomized controlled trial of iron infusion therapy.
Patient expectations have the ability to influence health outcomes and have been shown to play an important role as part of the placebo effect to influence the response to medical treatments. Increasing positive expectations have been proposed as an intervention to improve treatment response, although evidence for this to date is limited. We investigated whether a brief 10-min intervention directly targeting patient expectations prior to an iron infusion could enhance expectations and improve treatment response, in terms of patients' reported fatigue. Randomized controlled trial. Forty-three patients diagnosed with iron deficiency anaemia were randomized to a brief expectation intervention or active control group prior to an intravenous iron infusion. Chalder Fatigue Scale scores were assessed prior to randomization and at one and four weeks. The expectation intervention significantly improved patients' expectations about the effectiveness of the intravenous iron infusion, t(21) = -3.95, p = .001. While there were no significant differences between groups in fatigue at the one-week follow-up, fatigue was significantly lower in the intervention group at the four-week follow-up compared to the control group, F(1, 25) = 6.25, p = .019. This was largely influenced by a significant reduction in physical, as opposed to mental fatigue scores. Boosting patients' positive expectations may be an effective way of enhancing patient response to treatment. In particular, targeting patient expectations with a brief intervention prior to medical treatments may result in a greater and longer therapeutic effect.


Developmental analysis of tropomyosin gene expression in embryonic stem cells and mouse embryos.
Tropomyosins (TMs) comprise a family of actin-binding proteins which play an important role in the regulation of contractility in muscle (cardiac, skeletal, and smooth) and nonmuscle cells. Although they are present in all cells, different isoforms are characteristic of specific cell types. In vertebrates, there are four different TM genes (alpha-TM, beta-TM, TM30, and TM4), three of which generate alternatively spliced isoforms. This study defines the expression patterns of these isoforms during murine embryogenesis, using both in vivo and in vitro conditions. The embryonic stem cell culture system, which has been shown to mimic different stages of mouse embryonic development, including the differentiation of primitive organ systems such as the myocardium, is used for our in vitro analysis. Our results demonstrate that several TM isoforms are expressed in specific developmental patterns, often correlated with the differentiation of particular tissues or organs. Surprisingly, other TMs, such as the striated muscle beta-TM and smooth muscle alpha-TM, are expressed constitutively. This study also demonstrates that there is an excellent correlation between the expression patterns of the TM isoforms observed in developing embryonic stem cells and mouse embryos. In addition, a quantitative molecular analysis of TM isoforms was conducted in embryonic, neonatal, and adult cardiac tissue. Our results show for the first time that the alpha- and beta-TM striated muscle transcripts are present in the earliest functional stages of the heart, and these TM isoforms are identical to those present throughout cardiac development.


Primate homologues of rat TGN38: primary structure, expression and functional implications.
cDNAs encoding the human and macaque homologues of rat TGN38 have been cloned and sequenced. The proteins have a highly conserved N terminus (comprising the signal peptide) and C terminus (comprising part of the lumenal domain, the membrane spanning region and cytoplasmic tail) but vary in the other part of the lumenal domain, which contains the repeat region. Whereas rat TGN38 contains 6 tandem repeats of an 8mer, both primate proteins possess 14 tandem repeats of a 14mer sequence. The human protein, like rat TGN38, is localised primarily to the TGN but is present on the cell surface and returns via endosomes. This behaviour is consistent with conservation of the membrane spanning region and the cytoplasmic tail, which contain the retention and retrieval signals, respectively, for localisation in the TGN. The unexpected differences in the lumenal domain can best be rationalised by the fact that both types of repeat domains have most of the properties of mucins. We suggest that TGN38 homologues are mucin-like molecules that regulate membrane traffic to and from the TGN.


Human mucosal associated invariant T cells detect bacterially infected cells.
Control of infection with Mycobacterium tuberculosis (Mtb) requires Th1-type immunity, of which CD8+ T cells play a unique role. High frequency Mtb-reactive CD8+ T cells are present in both Mtb-infected and uninfected humans. We show by limiting dilution analysis that nonclassically restricted CD8+ T cells are universally present, but predominate in Mtb-uninfected individuals. Interestingly, these Mtb-reactive cells expressed the Valpha7.2 T-cell receptor (TCR), were restricted by the nonclassical MHC (HLA-Ib) molecule MR1, and were activated in a transporter associated with antigen processing and presentation (TAP) independent manner. These properties are all characteristics of mucosal associated invariant T cells (MAIT), an "innate" T-cell population of previously unknown function. These MAIT cells also detect cells infected with other bacteria. Direct ex vivo analysis demonstrates that Mtb-reactive MAIT cells are decreased in peripheral blood mononuclear cells (PBMCs) from individuals with active tuberculosis, are enriched in human lung, and respond to Mtb-infected MR1-expressing lung epithelial cells. Overall, these findings suggest a generalized role for MAIT cells in the detection of bacterially infected cells, and potentially in the control of bacterial infection.


A comparison of the effectiveness of spinal manipulative therapy for low back pain patients with and without spondylolisthesis.
The effectiveness of spinal manipulative therapy for low back pain is compared between two groups of patients: a small group (25) of patients with lumbar spondylolisthesis and a larger group (260) of patients without spondylolisthesis. This data, which was collected from a previously published study on the effectiveness of manipulation for chronic low back pain, shows that the results of manipulative treatment are not significantly different in those patients with or without lumbar spondylolisthesis.


Major Australian-Antarctic plate reorganization at Hawaiian-Emperor bend time.
A marked bend in the Hawaiian-Emperor seamount chain supposedly resulted from a recent major reorganization of the plate-mantle system there 50 million years ago. Although alternative mantle-driven and plate-shifting hypotheses have been proposed, no contemporaneous circum-Pacific plate events have been identified. We report reconstructions for Australia and Antarctica that reveal a major plate reorganization between 50 and 53 million years ago. Revised Pacific Ocean sea-floor reconstructions suggest that subduction of the Pacific-Izanagi spreading ridge and subsequent Marianas/Tonga-Kermadec subduction initiation may have been the ultimate causes of these events. Thus, these plate reconstructions solve long-standing continental fit problems and improve constraints on the motion between East and West Antarctica and global plate circuit closure.


Increased plasma cathepsin S and trombospondin-1 in patients with acute ST-segment elevation myocardial infarction.
The role of cathepsins in the pathological progression of atherosclerotic lesions in ischem-ic heart disease have been defined in detail more than numerous times. This investigation examined the platelet-specific biomarker trombospondin-1 (TSP-1) and platelet function ex vivo, and compared this with cathepsin S (Cat-S; a biomarker unrelated to platelet activation but also associated this with increased mortality risk) in patients with ST-segment elevation myocardial infarction (STEMI). The STEMI patients were divided into two groups depending on the degree of coronary vessel occlusion: those with closed (n = 90) and open culprit vessel (n = 40). Cat-S and TSP-1 were analyzed before, 1-3 days after and 3 months after percutanous coronary intervention (PCI). During acute STEMI, plasma TSP-1 was significantly elevated in patients with closed cul-prit lesions, but rapidly declined after PCI. In fact, TSP-1 after PCI was significantly lower inpatient samples compared to healthy individuals. In comparison, plasma Cat-S was significantly elevated both before and after PCI. In patients with closed culprit lesions, Cat-S was significantly higher compared to patients with open culprit lesions 3 months after PCI. Although troponin-I were higher (p < 0.01) in patients with closed culprit lesion, there was no correlation with Cat-S and TSP-1. Cat-S but not TSP-1 may be a useful risk biomarker in relation to the severity of STEMI. However, the causality of Cat-S as a predictor for long-term mortality in STEMI remains to be ascertained in future studies.


The RHNumtS compilation: features and bioinformatics approaches to locate and quantify Human NumtS.
To a greater or lesser extent, eukaryotic nuclear genomes contain fragments of their mitochondrial genome counterpart, deriving from the random insertion of damaged mtDNA fragments. NumtS (Nuclear mt Sequences) are not equally abundant in all species, and are redundant and polymorphic in terms of copy number. In population and clinical genetics, it is important to have a complete overview of NumtS quantity and location. Searching PubMed for NumtS or Mitochondrial pseudo-genes yields hundreds of papers reporting Human NumtS compilations produced by in silico or wet-lab approaches. A comparison of published compilations clearly shows significant discrepancies among data, due both to unwise application of Bioinformatics methods and to a not yet correctly assembled nuclear genome. To optimize quantification and location of NumtS, we produced a consensus compilation of Human NumtS by applying various bioinformatics approaches. Location and quantification of NumtS may be achieved by applying database similarity searching methods: we have applied various methods such as Blastn, MegaBlast and BLAT, changing both parameters and database; the results were compared, further analysed and checked against the already published compilations, thus producing the Reference Human Numt Sequences (RHNumtS) compilation. The resulting NumtS total 190. The RHNumtS compilation represents a highly reliable reference basis, which may allow designing a lab protocol to test the actual existence of each NumtS. Here we report preliminary results based on PCR amplification and sequencing on 41 NumtS selected from RHNumtS among those with lower score. In parallel, we are currently designing the RHNumtS database structure for implementation in the HmtDB resource. In the future, the same database will host NumtS compilations from other organisms, but these will be generated only when the nuclear genome of a specific organism has reached a high-quality level of assembly.


Implicit self-stigma in people with mental illness.
People with mental illness often internalize negative stereotypes, resulting in self-stigma and low self-esteem ("People with mental illness are bad and therefore I am bad, too"). Despite strong evidence for self-stigma's negative impact as assessed by self-report measures, it is unclear whether self-stigma operates in an automatic, implicit manner, potentially outside conscious awareness and control. We therefore assessed (i) negative implicit attitudes toward mental illness and (ii) low implicit self-esteem using 2 Brief Implicit Association Tests in 85 people with mental illness. Implicit self-stigma was operationalized as the product of both implicit measures. Explicit self-stigma and quality of life were assessed by self-report. Greater implicit and explicit self-stigma independently predicted lower quality of life after controlling for depressive symptoms, diagnosis, and demographic variables. Our results suggest that implicit self-stigma is a measurable construct and is associated with negative outcomes. Attempts to reduce self-stigma should take implicit processes into account.


Take off your genes and let the doctor have a look: why the Mayo and Myriad decisions have invalidated method claims for genetic diagnostic testing.
Ass'n for Molecular Pathology v. U.S. Patent & Trade Office sent shockwaves through the legal community, when the U.S. District Court for the Southern District of New York rejected a series of patents held by Myriad Genetics, Inc. The court invalidated all of Myriad's compositional patents for human genes and its method patents for diagnosing genetic predispositions to breast cancer. While commentators have discussed the ethical implications of allowing patent rights to human genes in great detail, the Court's ruling on Myriad's method claims went by comparatively unnoticed. The ability to test a patient's genetic profile for predisposition to cancer and other diseases is an incredible achievement in the field of personalized medicine. Whether these tests deserve patent protection is a hotly debated issue that involves weighing the interests of both incentivizing research and making these tests available to the general public. This Comment analyzes the legal framework established by the Supreme Court and U.S. Court of Appeals for the Federal Circuit to decide patent eligibility for genetic diagnostic tests. It concludes that, while the world was spellbound by the ethical quandary of compositional claims on human genes, the recent Supreme Court and Federal Circuit decisions have surreptitiously eliminated genetic diagnostic tests as patentable subject matter under § 101 of the United States Patent Act.


Patients with chronic fatigue syndrome have reduced absolute cortical blood flow.
Prior studies on brain blood flow in chronic fatigue syndrome (CFS) did not find consistent results. This may be because they used single-photon emission computed tomography to measure brain blood flow, which could not measure absolute blood flow. Therefore, the aim of this study was to test the hypothesis that patients with CFS have reduced absolute cerebral blood flow. Xenon-computed tomography blood flow studies were done on 25 CFS patients and seven healthy controls. Analyses were done after stratifying the CFS patients based on the presence or absence of a current psychiatric disorder. Flow was diminished in both groups as follows: patients with no current psychiatric disorders had reduced cortical blood flow in the distribution of both right and left middle cerebral arteries (P<0.05 for both) while those with current psychiatric disorders had reduced blood flow only in the left middle cerebral artery territory (P<0.05). These data indicate that patients with CFS have reduced absolute cortical blood flow in rather broad areas when compared with data from healthy controls and that those devoid of psychopathology had the most reductions in cortical flow. These data support, in part, our earlier findings that patients devoid of psychopathology are the group most at risk of having some of the symptoms of CFS due to brain dysfunction.


Formation and evolution of the nanoparticle environmental corona: The case of Au and humic acid.
Studying the behaviour of nanomaterials after their release into natural water is essential to understand the risk associated to their environmental exposure. In particular, the interaction and adsorption of dissolved organic matter onto nanoparticles strongly influence the behaviour and fate of nanomaterials in natural water systems. We herein study the interaction of Au and Ag nanoparticles and humic acids, the principal component of natural dissolved organic matter. Physicochemical characterization results showed the formation of an organic matter corona, consisting of two layers: a "hard" one, firmly bound to the nanoparticle surface, and a "soft" one, in dynamic equilibrium and, consequently, highly dependent on the media organic matter concentration. The extent of the electro-steric stabilization of the so called environmental corona depends on the size of the supramolecular association of humic acid (which depends on its hydrophilic and lipophilic moieties), the nanoparticle size, the total concentration of organic matter in the media, and the ratio between them. Interestingly, environmental coronas can eventually prevent Ca2+ and Mg2+ induced aggregation at concentrations range present in most of the freshwater bodies. The humic coating formed on top of the Au or control Ag nanoparticles presented a similar profile, but the corrodibility of Ag led to a more natural detachment of the corona. These results were further confirmed by exposing the nanoparticles to a model of natural water and standard mud (LUFA 2.2 dispersion). In the latter case, after several days, nanoparticle sedimentation was observed, which was attributed to interactions with macro organic and inorganic matter (fraction larger than particulate matter).


A disposable acoustofluidic chip for nano/microparticle separation using unidirectional acoustic transducers.
Separation of nano/microparticles based on surface acoustic waves (SAWs) has shown great promise for biological, chemical, and medical applications ranging from sample purification to cancer diagnosis. However, the permanent bonding of a microchannel onto relatively expensive piezoelectric substrates and excitation transducers renders the SAW separation devices non-disposable. This limitation not only requires cumbersome cleaning and increased labor and material costs, but also leads to cross-contamination, preventing their implementation in many biological, chemical, and medical applications. Here, we demonstrate a high-performance, disposable acoustofluidic platform for nano/microparticle separation. Leveraging unidirectional interdigital transducers (IDTs), a hybrid channel design with hard/soft materials, and tilted-angle standing SAWs (taSSAWs), our disposable acoustofluidic devices achieve acoustic radiation forces comparable to those generated by existing permanently bonded, non-disposable devices. Our disposable devices can separate not only microparticles but also nanoparticles. Moreover, they can differentiate bacteria from human red blood cells (RBCs) with a purity of up to 96%. Altogether, we developed a unidirectional IDT-based, disposable acoustofluidic platform for micro/nanoparticle separation that can achieve high separation efficiency, versatility, and biocompatibility.


Dibenzoylmethane exerts metabolic activity through regulation of AMP-activated protein kinase (AMPK)-mediated glucose uptake and adipogenesis pathways.
Dibenzoylmethane (DBM) has been shown to exert a variety of beneficial effects on human health. However, the mechanism of action is poorly understood. In this study, DBM increased phosphorylation of AMP-activated protein kinase (AMPK) and stimulated glucose uptake in a skeletal muscle cell line. Both knockdown of AMPK with siRNA and inhibition with AMPK inhibitor blocked DBM-induced glucose uptake. DBM increased the concentration of intracellular calcium and glucose uptake due to DBM was abolished by STO-609 (a calcium/calmodulin-dependent protein kinase inhibitor). DBM stimulated phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), which was blocked by pretreatment with compound C, an AMPK inhibitor. The expression of glucose transporter type 4 (GLUT4) was increased by DBM. The translocation of GLUT4 to the plasma membrane was also increased by DBM in AMPK dependently. In addition, DBM suppressed weight gain and prevented fat accumulation in the liver and abdomen in mice fed a high-fat diet. In pre-adipocyte cells, DBM decreased the activity of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of fatty acid synthesis. Expression of the adipogenic gene, fatty acid synthase (FAS), was suppressed by DBM in an AMPK-dependent manner. These results showed that the beneficial metabolic effects of DBM might be due to regulation of glucose uptake via AMPK in skeletal muscle and inhibition of adipogenesis in pre-adipocytes.


Common genetic pathways regulate organ-specific infection-related development in the rice blast fungus.
Magnaporthe oryzae is the most important fungal pathogen of rice (Oryza sativa). Under laboratory conditions, it is able to colonize both aerial and underground plant organs using different mechanisms. Here, we characterize an infection-related development in M. oryzae produced on hydrophilic polystyrene (PHIL-PS) and on roots. We show that fungal spores develop preinvasive hyphae (pre-IH) from hyphopodia (root penetration structures) or germ tubes and that pre-IH also enter root cells. Changes in fungal cell wall structure accompanying pre-IH are seen on both artificial and root surfaces. Using characterized mutants, we show that the PMK1 (for pathogenicity mitogen-activated protein kinase 1) pathway is required for pre-IH development. Twenty mutants with altered pre-IH differentiation on PHIL-PS identified from an insertional library of 2885 M. oryzae T-DNA transformants were found to be defective in pathogenicity. The phenotypic analysis of these mutants revealed that appressorium, hyphopodium, and pre-IH formation are genetically linked fungal developmental processes. We further characterized one of these mutants, M1373, which lacked the M. oryzae ortholog of exportin-5/Msn5p (EXP5). Mutants lacking EXP5 were much less virulent on roots, suggesting an important involvement of proteins and/or RNAs transported by EXP5 during M. oryzae root infection.


Nucleotides in 23S rRNA protected by the association of 30S and 50S ribosomal subunits.
We have studied the effect of subunit association on the accessibility of nucleotides in 23S and 5S rRNA. Escherichia coli 50S subunits and 70S ribosomes were subjected to a combination of chemical probes and the sites of attack identified by primer extension. Since the ribose groups and all of the bases were probed, the present study provides a comprehensive map of the nucleotides that are likely to be involved in subunit-subunit interactions. Upon subunit association, the bases of 22 nucleotides and the ribose groups of more than 60 nucleotides are protected in 23S rRNA; no changes are seen in 5S rRNA. Interestingly, the bases of nucleotides A1866, A1891 and A1896, and G2505 become more reactive to chemical probes, indicating localized rearrangement of the structure of the 50S subunit upon association with the 30S subunit. Most of the protected nucleotides are located in four stem-loop structures around positions 715, 890, 1700, and 1920. In free 50S subunits, virtually all of the ribose groups in these four regions are strongly cleaved by hydroxyl radicals, suggesting that these stems protrude from the 50S subunit. When the 30S subunit is bound, most of the ribose groups in the 715, 890, 1700 and 1920 stem-loops are protected, as are many bases in and around the corresponding apical loops. Intriguingly, three of the protected regions of 23S rRNA are known to be linked via tertiary interactions to features of the peptidyl transferase center. Together with the juxtaposition of the subunit-protected regions of 16S rRNA with the small subunit tRNA binding sites, our findings suggest the existence of a communication pathway between the codon-anticodon binding sites of the 30S subunit with the peptidyl transferase center of the 50S subunit via rRNA-rRNA interactions.


Caffeine Boosts Preparatory Attention for Reward-related Stimulus Information.
The intake of caffeine and the prospect of reward have both been associated with increased arousal, enhanced attention, and improved behavioral performance on cognitive tasks, but how they interact to exert these effects is not well understood. To investigate this question, we had participants engage in a two-session cued-reward cognitive task while we recorded their electrical brain activity using scalp electroencephalography. The cue indicated whether monetary reward could be received for fast and accurate responses to a color-word Stroop stimulus that followed. Before each session, participants ingested decaffeinated coffee with either caffeine (3-mg/kg bodyweight) or placebo (3-mg/kg bodyweight lactose). The behavioral results showed that both caffeine and reward-prospect improved response accuracy and speed. In the brain, reward-prospect resulted in an enlarged frontocentral slow wave (contingent negative variation, or CNV) and reduced posterior alpha power (indicating increased cortical activity) before stimulus presentation, both neural markers for preparatory attention. Moreover, the CNV enhancement for reward-prospect trials was considerably more pronounced in the caffeine condition as compared to the placebo condition. These interactive neural enhancements due to caffeine and reward-prospect were mainly visible in preparatory attention activity triggered by the cue (CNV). In addition, some interactive neural enhancements in the processing of the Stroop target stimulus that followed were also observed. The results suggest that caffeine facilitates the neural processes underlying attentional preparation and stimulus processing, especially for task-relevant information.


Tracking of In-111-labeled human umbilical tissue-derived cells (hUTC) in a rat model of cerebral ischemia using SPECT imaging.
In order to increase understanding of how infused cells work, it becomes important to track their initial movement, localization, and engraftment efficiency following transplantation. However, the available in vivo cell tracking techniques are suboptimal. The study objective was to determine the biodistribution of intravenously administered Indium-111 (In-111) oxine labeled human umbilical tissue-derived cells (hUTC) in a rat model of transient middle cerebral occlusion (tMCAo) using single photon emission computed tomography (SPECT). Rats received 3 million In-111 labeled hUTC (i.v.) 48 hrs after tMCAo. Following the administration of either hUTC or equivalent dose of In-111-oxine (18.5 MBq), animals underwent SPECT imaging on days 0, 1, and 3. Radioactivity in various organs as well as in the stroke area and contralateral hemisphere was determined, decay corrected and normalized to the total (whole body including head) radioactivity on day 0. Immunohistochemical analysis was also performed to confirm the beneficial effects of hUTC on vascular and synaptic density, and apoptosis. Most of the radioactivity (43.36±23.07% on day 0) trafficked to the lungs immediately following IV administration of In-111 labeled hUTC (day 0) and decreased drastically to 8.81±7.75 and 4.01±4.52% on days 1 and 3 post-injection, respectively. In contrast, radioactivity measured in the lung of animals that received In-111-oxine alone remained relatively unchanged from day 0 to day 1 (18.38±5.45% at day 0 to 12.59±5.94%) and decreased to 8.34±4.25% on day 3. Significantly higher radioactivity was observed in stroke areas of animals that received In-111 labeled hUTC indicating the presence of cells at the site of injury representing approximately 1% of total administered dose. In addition, there was significant increase in vascular and synaptophysin immunoreactivity in stroke areas of rats that received In-111 labeled hUTC. The present studies showed the tracking of In-111 labeled hUTC to the sites of stroke in a rat model of tMCAo using SPECT. Animals treated with In-111 labeled hUTC showed histological improvements, with higher vascular and synaptic densities observed in the ischemic boundary zone (IBZ).


Elevated risk for squamous cell carcinoma of the conjunctiva among adults with AIDS in the United States.
Squamous cell carcinoma of the conjunctiva (SCCC) has been associated with HIV infection in equatorial Africa, but the evidence for association with HIV in developed countries, where SCCC is rarer, is controversial. We investigated the risk for SCCC and other eye cancers in the updated U.S. HIV/AIDS Cancer Match Registry Study. We calculated standardized incidence ratios (SIRs) to estimate excess risk for SCCC, primary ocular lymphoma, ocular Kaposi sarcoma (KS) and other eye tumors among 491, 048 adults (aged > 15 years or older) with HIV/AIDS diagnosed from 1980 to 2004. We calculated relative proportions (per 10(5)) to gain insight into risk factors. We identified 73 eye cancers (15 SCCC, 35 primary ocular lymphoma, 17 ocular KS and 6 other). Overall SIRs were elevated for SCCC (SIR, 12.2, 95% CI 6.8-20.2), primary ocular lymphoma (21.7, 95% CI 15.1-30.2) and ocular KS (109, 95% CI 63.5-175). Risk for SCCC was elevated regardless of HIV acquisition category, CD4 lymphocyte count and time relative to AIDS-onset. Relative proportions of SCCC risk were highest with age >or=50 (8/10(5)), Hispanic ethnicity (7/10(5)) and residence in regions with high-solar ultraviolet radiation (10/10(5)). We show significantly increased incidence of SCCC among persons with HIV/AIDS in the U.S. The associations with age and geography are in accord with etiological role for ultraviolet radiation in SCCC.


A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation.
Innate immunity relies on the perception of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) located on the host cell's surface. Many plant PRRs are kinases. Here, we report that the Arabidopsis receptor kinase EF-TU RECEPTOR (EFR), which perceives the elf18 peptide derived from bacterial elongation factor Tu, is activated upon ligand binding by phosphorylation on its tyrosine residues. Phosphorylation of a single tyrosine residue, Y836, is required for activation of EFR and downstream immunity to the phytopathogenic bacterium Pseudomonas syringae. A tyrosine phosphatase, HopAO1, secreted by P. syringae, reduces EFR phosphorylation and prevents subsequent immune responses. Thus, host and pathogen compete to take control of PRR tyrosine phosphorylation used to initiate antibacterial immunity.


Fidelity and Promiscuity in an Ant-Plant Mutualism: A Case Study of Triplaris and Pseudomyrmex.
The association between the myrmecophyte Triplaris and ants of the genus Pseudomyrmex is an often-reported example of mutualism but no molecular studies have examined this association to date. In this study, the interspecific relationships of Triplaris were reconstructed using five molecular markers (two chloroplast and three nuclear), and the relationships of the associated Pseudomyrmex using two molecular regions (one mitochondrial and one nuclear). A data set including all known collections of plant hosts and resident ants was also compiled. The pattern of distribution of both organisms reveals that there are varying degrees of host specificity; most ants show broader host usage (promiscuous) but one species (P. dendroicus) is faithful to a single species of Triplaris. In most ant-plant interactions, host usage is not specific at the species level and preferences may result from geographical or ecological sorting. The specificity of P. dendroicus could be based on chemical recognition of the host they were raised on.


Using mass reconstruction along a four-site transect as a method to interpret PM10 in west-central Scotland, United Kingdom.
Concurrent 24-hr samples of particulate matter of median aerodynamic diameter less than 10 microm (PM10) were collected over a 10-day period in August 2000 at four sites along a transect in west-central Scotland, UK (passing from the coast through the city of Glasgow) in line with the prevailing southwesterly wind. Each sample was analyzed for chloride (Cl(-)), nitrate (NO3(-)), sulfate (SO4(2-)), ammonium (NH4(+)), calcium (Ca(2+)), iron (Fe), and organic hydrocarbon material (OHM). The contribution from elemental carbon (EC) was estimated. Sampling days were categorized according to local wind direction, synoptic flow, and air mass back trajectories. Chemical mass balance (CMB) reconstruction of the following PM10 components was derived for each wind direction group and at each transect location: ammonium sulfate ((NH4)2SO4), ammonium nitrate (NH4NO3), sodium chloride (NaCl), gypsum (CaSO4), OHM, EC, soil/surface dusts, and particle-bound water. The results showed that PM10 at the coastal site was dominated by the marine background (NaCl) compared with the urban sites, which were dominated by local primary (EC and soil/resuspension) and secondary sources (NH4NO3, (NH4)2SO4, and OHM). There was evidence of Cl(-) depletion as NaCl aerosol passes over urban areas. There was also evidence of long-range transport of primary PM10 (EC and OHM); for example, at the coastal site from transport from Ireland. The work demonstrates how the general approach of combining mass reconstruction along a transect with other information such as wind/air-mass direction generates insight into the sources contributing to PM10 over a more extended spatial scale than at a single receptor.


Single-operator comparison of early and mid-second-trimester amniocentesis.
We sought to determine whether early amniocentesis is a safe and acceptable method of genetic evaluation in early pregnancy. During the 54-month period from September 1986 to February 1991, 300 consecutive early second-trimester amniocenteses were performed transabdominally at 13-14 weeks' gestation and 567 consecutive mid-second-trimester transabdominal amniocenteses were performed at 16-18 weeks. Group assignment was nonrandomized, interoperator-dependent variables were eliminated, and analysis was performed in one cytogenetics laboratory. The median maternal age and indications for the procedure were similar in both groups. There were no significant differences between the early- and mid-second-trimester amniocenteses in failed sampling, ambiguous results, pregnancy loss from 4 weeks after the procedure to 28 weeks' gestation, preterm birth, or perinatal death rate. Pregnancy loss within 4 weeks of amniocentesis was more frequent in early- than in mid-second-trimester amniocenteses. We conclude that early amniocentesis is a safe and acceptable method of genetic evaluation.


Evaluating the effectiveness of gloves in reducing the hazards of hand-transmitted vibration.
A method of evaluating the effectiveness of gloves in reducing the hazards of hand-transmitted vibration is proposed. The glove isolation effectiveness was calculated from: (a) the measured transmissibility of a glove, (b) the vibration spectrum on the handle of a specific tool (or class of tools), and (c) the frequency weighting indicating the degree to which different frequencies of vibration cause injury. With previously reported tool vibration spectra and glove transmissibilities (from 10-1000 Hz), the method was used to test 10 gloves with 20 different powered tools. The frequency weighting for hand-transmitted vibration advocated in British standard 6842 (1987) and international standard 5349 (1986) greatly influences the apparent isolation effectiveness of gloves. With the frequency weighting, the gloves had little effect on the transmission of vibration to the hand from most of the tools. Only for two or three tools (those dominated by high frequency vibration) did any glove provide useful attenuation. Without the frequency weighting, some gloves showed useful attenuation of the vibration on most powered tools. In view of the uncertain effect of the vibration frequency in the causation of disorders from hand-transmitted vibration, it is provisionally suggested that the wearing of a glove by the user of a particular vibratory tool could be encouraged if the glove reduces the transmission of vibration when it is evaluated without the frequency weighting and does not increase the vibration when it is evaluated with the frequency weighting. A current international standard for the measurement and evaluation of the vibration transmitted by gloves can classify a glove as an antivibration glove when it provides no useful attenuation of vibration, whereas a glove providing useful attenuation of vibration on a specific tool can fail the test.


Reorganization of the host epigenome by a viral oncogene.
Adenovirus small e1a oncoprotein causes ~70% reduction in cellular levels of histone H3 lysine 18 acetylation (H3K18ac). It is unclear, however, where this dramatic reduction occurs genome-wide. ChIP-sequencing revealed that by 24 h after expression, e1a erases 95% of H3K18ac peaks in normal, contact-inhibited fibroblasts and replaces them with one-third as many at new genomic locations. The H3K18ac peaks at promoters and intergenic regions of genes with fibroblast-related functions are eliminated after infection, and new H3K18ac peaks are established at promoters of highly induced genes that regulate cell cycling and at new putative enhancers. Strikingly, the regions bound by the retinoblastoma family of proteins in contact-inhibited fibroblasts gain new peaks of H3K18ac in the e1a-expressing cells, including 55% of RB1-bound loci. In contrast, over half of H3K9ac peaks are similarly distributed before and after infection, independently of RB1. The strategic redistribution of H3K18ac by e1a highlights the importance of this modification for transcriptional activation and cellular transformation as well as functional differences between the RB-family member proteins.


Crosstalk with insulin and dependence on PI3K/Akt/mTOR rather than MAPK pathways in upregulation of basal growth following long-term oestrogen deprivation in three human breast cancer cell lines.
MCF-7, T-47-D, ZR-75-1 human breast cancer cell lines are dependent on oestrogen for growth but can adapt to grow during long-term oestrogen deprivation. This serves as a model for identification of therapeutic targets in endocrine-resistant breast cancer. An overlooked complication of this model is that it involves more than non-addition of oestrogen, and inadequate attention has been given to separating molecular events associated with each of the culture manipulations. Insulin and oestradiol were shown to protect MCF-7 cells against upregulation of basal growth, demonstrating a crosstalk in the growth adaptation process. Increased phosphorylation of p44/42MAPK and c-Raf reflected removal of insulin from the medium and proliferation of all three cell lines was inhibited to a lesser extent by PD98059 and U0126 following long-term oestrogen/insulin withdrawal, demonstrating a reduced dependence on the MAPK pathway. By contrast, long-term oestrogen/insulin deprivation did not alter levels of phosphorylated Akt and did not alter the dose-response of growth inhibition with LY294002 in any of the three cell lines. The IGF1R inhibitor picropodophyllin inhibited growth of all MCF-7 cells but only in the long-term oestrogen/insulin-deprived cells was this paralleled by reduction in phosphorylated p70S6K, a downstream target of mTOR. Long-term oestrogen/insulin-deprived MCF-7 cells had higher levels of phosphorylated p70S6K and developed increased sensitivity to growth inhibition by rapamycin. The greater sensitivity to growth inhibition by rapamycin in all three cell lines following long-term oestrogen/insulin deprivation suggests rapamycin-based therapies might be more effective in breast cancers with acquired oestrogen resistance.


Systematic evaluation of biofilm models for engineering practice: components and critical assumptions.
Biofilm models are valuable tools for the design and evaluation of biofilm-based processes despite several uncertainties including the dynamics and rate of biofilm detachment, concentration gradients external to the biofilm surface, and undefined biofilm reactor model calibration protocol. The present investigation serves to (1) systematically evaluate critical biofilm model assumptions and components and (2) conduct a sensitivity analysis with the aim of identifying parameter subsets for biofilm reactor model calibration. AQUASIM was used to describe submerged-completely mixed combined carbon oxidation and nitrification IFAS and MBBR systems, and tertiary nitrification and denitrification MBBRs. The influence of uncertainties in model parameters on relevant model outputs was determined for simulated scenarios by means of a local sensitivity analysis. To obtain reasonable simulation results for partially penetrated biofilms that accumulated a substantial thickness in the modelled biofilm reactor (e.g. 1,000 microm), an appropriate biofilm discretization was applied to properly model soluble substrate concentration gradients and, consistent with the assumed mechanism for describing biofilm biomass distribution, biofilm biomass spatial variability. The MTBL thickness had a significant impact on model results for each of the modelled reactor configurations. Further research is needed to develop a mathematical description (empirical or otherwise) of the MTBL thickness that is relevant to modern biofilm reactors. No simple recommendations for a generally applicable calibration protocol are provided, but sensitivity analysis has been proven to be a powerful tool for the identification of highly sensitive parameter subsets for biofilm (reactor) model calibration.


Potential gains in life expectancy by reducing inequality of lifespans in Denmark: an international comparison and cause-of-death analysis.
Reducing lifespan inequality is increasingly recognized as a health policy objective. Whereas lifespan inequality declined with rising longevity in most developed countries, Danish life expectancy stagnated between 1975 and 1995 for females and progressed slowly for males. It is unknown how Danish lifespan inequality changed, which causes of death drove these developments, and where the opportunities for further improvements lie now. We present an analytical strategy based on cause-by-age decompositions to simultaneously analyze changes in Danish life expectancy and lifespan inequality from 1960 to 2014, as well as current Swedish-Danish differences. Stagnation in Danish life expectancy coincided with a shorter period of stagnation in lifespan inequality (1975-1990). The stagnation in life expectancy was mainly driven by increases in cancer and non-infectious respiratory mortality at higher ages (-.63 years) offsetting a reduction in cardiovascular and infant mortality (+ 1.52 years). Lifespan inequality stagnated because most causes of death did not show compression over the time period. Both these observations were consistent with higher smoking-related mortality in Danes born in 1919-1939. After 1995, life expectancy and lifespan equality increased in lockstep, but still lag behind Sweden, mainly due to infant mortality and cancer. Since 1960, Danish improvements in life expectancy and lifespan equality were halted by smoking-related mortality in those born 1919-1939, while also reductions in old-age cardiovascular mortality held back lifespan equality. The comparison with Sweden suggests that Denmark can reduce inequality in lifespans and increase life expectancy through a consistent policy target: reducing cancer and infant mortality.


A Model to Investigate Single-Strand DNA Responses in G1 Human Cells via a Telomere-Targeted, Nuclease-Deficient CRISPR-Cas9 System.
DNA replication stress has the potential to compromise genomic stability and, therefore, cells have developed elaborate mechanisms to detect and resolve problems that may arise during DNA replication. The presence of single-stranded DNA (ssDNA) is often associated with DNA replication stress and serves as a signal for both checkpoint and repair responses. In this study, we exploited a CRISPR-Cas9 system to induce regions of ssDNA in the genome. Specifically, single-guide RNAs bearing sequence complementarity to human telomeric repeats, were used to target nuclease-deficient Cas9 (dCas9) to telomeres. Such targeting was associated with the formation of DNA-RNA hybrids, leaving one telomeric DNA strand single-stranded. This ssDNA then recruited DNA repair and checkpoint proteins, such as RPA, ATRIP, BLM and Rad51, at the telomeres. Interestingly, targeting of all these proteins to telomeric ssDNA was observed even in cells that were in the G1 phase of the cell cycle. Therefore, this system has the potential to serve as a platform for further investigation of DNA replication stress responses at specific loci in the human genome and in all phases of the cell cycle.


Using literature-based discovery to identify novel therapeutic approaches.
We present a promising in silico paradigm called literature-based discovery (LBD) and describe its potential to identify novel pharmacologic approaches to treating diseases. The goal of LBD is to generate novel hypotheses by analyzing the vast biomedical literature. Additional knowledge resources, such as ontologies and specialized databases, are often used to supplement the published literature. MEDLINE, the largest and most important biomedical bibliographic database, is the most common source for exploiting LBD. There are two variants of LBD, open discovery and closed discovery. With open discovery we can, for example, try to find a novel therapeutic approach for a given disease, or find new therapeutic applications for an existing drug. With closed discovery we can find an explanation for a relationship between two concepts. For example, if we already have a hypothesis that a particular drug is useful for a particular disease, with closed discovery we can identify the mechanisms through which the drug could have a therapeutic effect on the disease. We briefly describe the methodology behind LBD and then discuss in more detail currently available LBD tools; we also mention in passing some of those no longer available. Next we present several examples in which LBD has been exploited for identifying novel therapeutic approaches. In conclusion, LBD is a powerful paradigm with considerable potential to complement more traditional drug discovery methods, especially for drug target discovery and for existing drug relabeling.


What is being measured, and by whom? Facilitation of communication on technical measures amongst competent authorities in the implementation of the European Union Broiler Directive (2007/43/EC).
The European Union (EU) Broiler Directive (2007/43/EC) is unique amongst current EU Directives, which address animal welfare, in that it uses outcome data collected at abattoirs and on farm to monitor on-farm broiler welfare and vary the maximum permitted stocking density on farm. In this study, we describe how, by bringing together personnel from the competent authorities in 22 member states (MSs) who have responsibility for implementing the Directive, and engaging in exchange of information and technical methods regarding the Broiler Directive, it has been possible to identify differences in approach with regard to 'what data is being collected, and by whom' across EU MSs. Online questionnaires and workshop exercises enabled us to identify priority areas for knowledge transfer and training. For example, foot pad dermatitis, hock burn, dead on arrival and total rejections (birds rejected as unfit for human consumption by the meat inspection staff at slaughter) were identified by the MSs as measures of medium-to-low priority in terms of knowledge transfer because there are assessment methods for these conditions that are already well accepted by competent authorities. On the other hand, breast lesions, cellulitis, emaciation, joint lesions, respiratory problems, scratches, wing fractures and a number of environmental measures were identified as having high priority in terms of knowledge transfer. The study identified that there is significant variability in the stage of implementation between MSs, and responses from the participating MSs indicated that sharing of guidance and technical information between MSs may be of value in the future set-up process for those MSs engaged in implementation of the Directive.


The Australian Imaging, Biomarkers and Lifestyle (AIBL) study of aging: methodology and baseline characteristics of 1112 individuals recruited for a longitudinal study of Alzheimer's disease.
The Australian Imaging, Biomarkers and Lifestyle (AIBL) flagship study of aging aimed to recruit 1000 individuals aged over 60 to assist with prospective research into Alzheimer's disease (AD). This paper describes the recruitment of the cohort and gives information about the study methodology, baseline demography, diagnoses, medical comorbidities, medication use, and cognitive function of the participants. Volunteers underwent a screening interview, had comprehensive cognitive testing, gave 80 ml of blood, and completed health and lifestyle questionnaires. One quarter of the sample also underwent amyloid PET brain imaging with Pittsburgh compound B (PiB PET) and MRI brain imaging, and a subgroup of 10% had ActiGraph activity monitoring and body composition scanning. A total of 1166 volunteers were recruited, 54 of whom were excluded from further study due to comorbid disorders which could affect cognition or because of withdrawal of consent. Participants with AD (211) had neuropsychological profiles which were consistent with AD, and were more impaired than participants with mild cognitive impairment (133) or healthy controls (768), who performed within expected norms for age on neuropsychological testing. PiB PET scans were performed on 287 participants, 100 had DEXA scans and 91 participated in ActiGraph monitoring. The participants comprising the AIBL cohort represent a group of highly motivated and well-characterized individuals who represent a unique resource for the study of AD. They will be reassessed at 18-month intervals in order to determine the predictive utility of various biomarkers, cognitive parameters and lifestyle factors as indicators of AD, and as predictors of future cognitive decline.


Virtual reality: emerging role of simulation training in vascular access.
Evolving new technologies in vascular access mandate increased attention to patient safety; an often overlooked yet valuable training tool is simulation. For the end-stage renal disease patient, simulation tools are effective for all aspects of creating access for peritoneal dialysis and hemodialysis. Based on aviation principles, known as crew resource management, we place equal emphasis on team training as individual training to improve interactions between team members and systems, cumulating in improved safety. Simulation allows for environmental control and standardized procedures, letting the trainee practice and correct mistakes without harm to patients, compared with traditional patient-based training. Vascular access simulators range from suture devices, to pressurized tunneled conduits for needle cannulation, to computer-based interventional simulators. Simulation training includes simulated case learning, root cause analysis of adverse outcomes, and continual update and refinement of concepts. Implementation of effective human to complex systems interaction in end-stage renal disease patients involves a change in institutional culture. Three concepts discussed in this article are as follows: (1) the need for user-friendly systems and technology to enhance performance, (2) the necessity for members to both train and work together as a team, and (3) the team assigned to use the system must test and practice it to a proficient level before safely using the system on patients.


A two-compartment bone tumor model to investigate interactions between healthy and tumor cells.
We produced a novel three-dimensional (3D) bone tumor model (BTM) to study the interactions between healthy and tumor cells in a tumor microenvironment, the migration tendency of the tumor cells, and the efficacy of an anticancer drug, Doxorubicin, on the cancer cells. The model consisted of two compartments: (a) a healthy bone tissue mimic, made of poly(lactic acid-co-glycolic acid) (PLGA)/beta-tricalcium phosphate (β-TCP) sponge seeded with human fetal osteoblastic cells (hFOB) and human umbilical vein endothelial cells (HUVECs), and (b) a tumor mimic, made of lyophilized collagen sponge seeded with human osteosarcoma cells (Saos-2). The tumor mimic component was placed into a central cavity created in the healthy bone mimic and together they constituted the complete 3D bone tumor model (3D-BTM). The porosities of both sponges were higher than 85% and the diameters of the pores were 199 ± 52 μm for the PLGA/TCP and 50-150 μm for the collagen scaffolds. The compression Young's modulus of the PLGA/TCP and the collagen sponges were determined to be 4.76 MPa and 140 kPa, respectively. Cell proliferation, morphology, calcium phosphate forming capacity and alkaline phosphatase production were studied separately on both the healthy and tumor mimics. All cells demonstrated cellular extensions and spread well in porous scaffolds indicating good cell-material interactions. Confocal microscopy analysis showed direct contact between the cells present in different parts of the 3D-BTM. Migration of HUVECs from the healthy bone mimic to the tumor compartment was confirmed by the increase in the levels of angiogenic factors vascular endothelial growth factor, basic fibroblast growth factor, and interleukin 8 in the tumor component. Doxorubicin (2.7 μg.ml-1) administered to the 3D-BTM caused a seven-fold decrease in the cell number after 24 h of interaction with the anticancer drug. Caspase-3 enzyme activity assay results demonstrated apoptosis of the osteosarcoma cells. This novel 3D-BTM has a high potential for use in studying the metastatic capabilities of cancer cells, and in determining the effective drug types and combinations for personalized treatments.


Low 25-hydroxyvitamin D levels and low bone density assessed by quantitative ultrasonometry in a cohort of postmenopausal Italian nuns.
This study was aimed at evaluating the effect of clothing style on bone mass and fractures in 70 postmenopausal nuns residing in a monastery in Naples. Sixty healthy women matched for age, body mass index, and menopausal status were enrolled as controls. Each participant underwent measurement by quantitative ultrasonometry (QUS) using a DBM Sonic Bone Profiler (IGEA S.p.A., Carpi, Modena, Italy) at proximal phalanges, responded to questionnaires regarding lifestyle, calcium intake, medical history, including clinical fragility fractures, and was submitted to routine biochemical assessment. A significant reduction in ultrasonometric parameters of bone mass was found in nuns compared with controls (p from 0.007 to <0.0001). 25-hydroxyvitamin D (25-OH vit D) levels were reduced by more than 50% in nuns (9.8 ± 4.2 vs 23.5 ± 5.7 nmol/L; p < 0.0001), whereas their estimated daily calcium intake was higher (1.004 ± 0.23 vs 0.721 ± 0.25 g of controls; p = 0.0007). Age at menopause was significantly lower in nuns' group (p = 0.016). Incidence of fractures was higher in nuns (39% vs 10%; p = 0.0029), and the best predictors of fractures were age at menopause (odds ratio [OR]: 1.12; 95% confidence interval [CI]: 1.01-1.30), amplitude-dependent speed of sound T-score (OR: 1.15; 95% CI: 1.03-1.63), and bone transmission time T-score (OR: 1.30; 95% CI: 1.15-1.81). This study documented low 25-OH vit D levels, increased frequency of clinical fractures, and low bone mass detected by QUS in Southern Italian nuns.


An early requirement for maternal FoxH1 during zebrafish gastrulation.
The Forkhead Box H1 (FoxH1) protein is a co-transcription factor recruited by phosphorylated Smad2 downstream of several TGFbetas, including Nodal-related proteins. We have reassessed the function of zebrafish FoxH1 using antisense morpholino oligonucleotides (MOs). MOs targeting translation of foxH1 disrupt embryonic epiboly movements during gastrulation and cause death on the first day of development. The FoxH1 morphant phenotype is much more severe than that of zebrafish carrying foxh1/schmalspur (sur) DNA-binding domain mutations, FoxH1 splice-blocking morphants or other Nodal pathway mutants, and it cannot be altered by concomitant perturbations in Nodal signaling. Apart from disrupting epiboly, FoxH1 MO treatment disrupts convergence and internalization movements. Late gastrula-stage FoxH1 morphants exhibit delayed mesoderm and endoderm marker gene expression and failed patterning of the central nervous system. Probing FoxH1 morphant RNA by microarray, we identified a cohort of five keratin genes--cyt1, cyt2, krt4, krt8 and krt18--that are normally transcribed in the embryo's enveloping layer (EVL) and which have significantly reduced expression in FoxH1-depleted embryos. Simultaneously disrupting these keratins with a mixture of MOs reproduces the FoxH1 morphant phenotype. Our studies thus point to an essential role for maternal FoxH1 and downstream keratins during gastrulation that is epistatic to Nodal signaling.


Cell wall proteins of group B Streptococcus and low incidence of neonatal disease in southern Israel.
To determine the maternal group B Streptococcus (GBS) prevalence of carriage and serotype distribution and the neonatal disease incidence to formulate a policy for treatment and prevention regarding GBS diseases in southern Israel. A prospective study was conducted between January and October 2000. Cultures were obtained from 681 healthy, pregnant women and processed as recommended. Samples were cultured on blood-agar plates with and without added gentamicin. GBS was identified by beta-hemolysis and a positive CAMP test and confirmed by agglutination with specific antiserum. Serotyping was done by the Lancefield precipitin method using monospecific antisera to polysaccharides Ia, Ib and II-VIII and surface proteins C, R and X. Carriage prevalence of 12.3% and neonatal disease incidence of 0.095/1,000 live births were documented. Surface proteins C and R were found in 85.7% of positive cases. Serotypes Ia (17.8%), Ib (10.7%), II (27.4%), III (20.2%) and V (14.3%) were distributed as previously reported from developed countries. Developing a pentavalent vaccine based on serotypes Ia, Ib, II, III and V in conjugation to a GBS cell wall protein transporter, such as C or R, has theoretical advantages in the southern Israeli population over vaccines that use foreign proteins.


Does influenza vaccination increase consultations, corticosteroid prescriptions, or exacerbations in subjects with asthma or chronic obstructive pulmonary disease?
Concern over the safety of influenza vaccination in individuals with obstructive airways disease has contributed to suboptimal rates of vaccine uptake in this group. We investigated the safety of influenza vaccine in older people with asthma or chronic obstructive pulmonary disease (COPD) in a cohort from the UK General Practice Research Database (GPRD). A population based cohort study of 12,000 individuals with asthma or COPD from 432 general practices was conducted. Incidence rate ratios (IRR) were calculated for asthma or COPD diagnoses, prescriptions for oral corticosteroids, and acute exacerbations on the day of vaccination and on days 1-2 and 3-14 after vaccination compared with other time periods in the influenza season. The IRRs for asthma or COPD diagnoses and oral corticosteroid prescriptions were increased on the day of vaccination (for example, the IRR for oral corticosteroid prescriptions for subjects with asthma during the 1992-3 influenza season was 8.24 (95% confidence interval 5.54 to 12.26)). However, there was no consistent increase in the IRR of any of the outcomes on days 1-2 or 3-14 after vaccination, and most of these IRRs were close to 1. Rates of exacerbation were low and showed no consistent statistically significant increase during any risk periods. Older people with asthma or COPD commonly have diagnoses recorded or prescriptions for oral corticosteroids given on the day of influenza vaccination, but there is no increased risk of adverse acute outcomes in the first 2 weeks after vaccination. Our findings strongly suggest that influenza vaccination is safe in this population.


Pertussis toxin sensitive endothelin-1 coupling to inositol phosphate formation via a GTP-binding protein: comparison in SHR and WKY cultured aortic smooth muscle cells.
The effects of pertussis toxin on endothelin-1 and noradrenaline coupling to inositol phosphate (IP) formation was investigated in cultured aortic smooth muscle cells from 14 week SHR and WKY rats. Endothelin-1 (10(-6) M) stimulated IP formation was decreased in cells from SHR compared to WKY (WKY 1117 +/- 157, SHR 668 +/- 85% of basal). Pre-incubation with pertussis toxin produced a significant and similar reduction in endothelin stimulated IP production in both SHR (54% reduction) and WKY (55%). However, the observed reduction in endothelin-1 stimulated IP accumulation was still apparent in SHR when compared to WKY. Pertussis toxin preincubation followed by removal of extracellular calcium reduced further the endothelin responses by similar amounts in SHR and WKY cells, but SHR stimulated IP formation remained significantly decreased compared to WKY. The extent of pertussis toxin ADP-ribosylation of Gi alpha was similar in both SHR and WKY cells. Endothelin-1 produced a reduction in the extent of ADP-ribosylation of Gi alpha and this was of similar magnitude in both SHR and WKY cell membranes. In contrast, noradrenaline stimulated IP formation was unaffected by pertussis toxin pre-incubation. It was concluded that SHR cells do not appear to have an alteration in endothelin-1 activated, pertussis toxin sensitive G-protein coupling to IP formation or in the dependence of inositol phosphate formation on extracellular calcium.


Adsorption of carbon dioxide, methane, and their mixtures in porous carbons: effect of surface chemistry, water content, and pore disorder.
The adsorption of carbon dioxide, methane, and their mixtures in nanoporous carbons in the presence of water is studied using experiments and molecular simulations. Both the experimental and numerical samples contain polar groups that account for their partially hydrophilicity. For small amounts of adsorbed water, although the shape of the adsorption isotherms remain similar, both the molecular simulations and experiments show a slight decrease in the CO2 and CH4 adsorption amounts. For large amounts of adsorbed water, the experimental data suggest the formation of methane or carbon dioxide clathrates in agreement with previous work. In contrast, the molecular simulations do not account for the formation of such clathrates. Another important difference between the simulated and experimental data concerns the number of water molecules that desorb upon increasing the pressure of carbon dioxide and methane. Although the experimental data indicate that water remains adsorbed upon carbon dioxide and methane adsorption, the molecular simulations suggest that 40 to 75% of the initial amount of adsorbed water desorbs with carbon dioxide or methane pressure. Such discrepancies show that differences between the simulated and experimental samples are crucial to account for the rich phase behavior of confined water-gas systems. Our simulations for carbon dioxide-methane coadsorption in the presence of water suggest that the pore filling is not affected by the presence of water and that adsorbed solution theory can be applied for pressures as high as 15 MPa.


Evaluation of osteogenic cell culture and osteogenic/peripheral blood mononuclear human cell co-culture on modified titanium surfaces.
This study aimed to determine the effect of a bioactive ceramic coating on titanium in the nanothickness range on human osteogenic cells, peripheral blood mononuclear cells (PBMC) and on osteogenic cells co-cultured with PBMC without exogenous stimuli. Cell viability, proliferation, adhesion, cytokine release (IL1β, TGFβ1, IL10 and IL17) and intracellular stain for osteopontin and alkaline phosphatase were assessed. Morphologic evaluation showed smaller and less spread cell aspects in co-culture relative to osteogenic cell culture. Cell viability, proliferation and adhesion kinetics were differently influenced by surface texture/chemistry in culture versus co-culture. Cytokine release was also influenced by the interaction between mononuclear and osteogenic cells (mediators released by mononuclear cells acted on osteogenic cells and vice versa). In general, 'multi-cell type' interactions played a more remarkable role than the surface roughness or chemistry utilized on the in vitro cellular events related to initial stages of bone formation.


Guiding the surgical gesture using an electro-tactile stimulus array on the tongue: a feasibility study.
Under conventional "open-" surgery, the physician has to take care of the patient, interact with other clinicians and check several monitoring devices. Nowadays, the computer assisted surgery proposes to integrate 3-D cameras in the operating theatre in order to assist the surgeon in performing minimally invasive surgical punctures. The cameras localize the needle and the computer guides the surgeon towards an intracorporeal clinically defined target. A visualization system (screen) is employed to provide the surgeon with indirect visual spatial information about the intracorporeal positions of the needle. The present work proposes to use another sensory modality to guide the surgeon, thus keeping the visual modality fully dedicated to the surgical gesture. For this, the sensory substitution paradigm using the Bach-y-Rita's "Tongue Display Unit" (TDU) is exploited to provide to the surgeon information of the position tool. The TDU device is composed of a 6 x 6 matrix of electrodes transmitting electrotactile information on the tongue surface. The underlying idea consists in transmitting information about the deviation of the needle movement with regard to a preplanned "optimal" trajectory. We present an experiment assessing the guidance effectiveness of an intracorporeal puncture under TDU guidance with respect to the performance evidenced under a usual visual guidance system.


Predatory Dinosaurs from the Sahara and Late Cretaceous Faunal Differentiation
Late Cretaceous (Cenomanian) fossils discovered in the Kem Kem region of Morocco include large predatory dinosaurs that inhabited Africa as it drifted into geographic isolation. One, represented by a skull approximately 1.6 meters in length, is an advanced allosauroid referable to the African genus Carcharodontosaurus. Another, represented by a partial skeleton with slender proportions, is a new basal coelurosaur closely resembling the Egyptian genus Bahariasaurus. Comparisons with Cretaceous theropods from other continents reveal a previously unrecognized global radiation of carcharodontosaurid predators. Substantial geographic differentiation of dinosaurian faunas in response to continental drift appears to have arisen abruptly at the beginning of the Late Cretaceous.


Bifurcation Analysis of Reaction Diffusion Systems on Arbitrary Surfaces.
In this paper, we present computational techniques to investigate the effect of surface geometry on biological pattern formation. In particular, we study two-component, nonlinear reaction-diffusion (RD) systems on arbitrary surfaces. We build on standard techniques for linear and nonlinear analysis of RD systems and extend them to operate on large-scale meshes for arbitrary surfaces. In particular, we use spectral techniques for a linear stability analysis to characterise and directly compose patterns emerging from homogeneities. We develop an implementation using surface finite element methods and a numerical eigenanalysis of the Laplace-Beltrami operator on surface meshes. In addition, we describe a technique to explore solutions of the nonlinear RD equations using numerical continuation. Here, we present a multiresolution approach that allows us to trace solution branches of the nonlinear equations efficiently even for large-scale meshes. Finally, we demonstrate the working of our framework for two RD systems with applications in biological pattern formation: a Brusselator model that has been used to model pattern development on growing plant tips, and a chemotactic model for the formation of skin pigmentation patterns. While these models have been used previously on simple geometries, our framework allows us to study the impact of arbitrary geometries on emerging patterns.


An epigenetic switch is crucial for spermatogonia to exit the undifferentiated state toward a Kit-positive identity.
Epigenetic modifications influence gene expression and chromatin remodeling. In embryonic pluripotent stem cells, these epigenetic modifications have been extensively characterized; by contrast, the epigenetic events of tissue-specific stem cells are poorly understood. Here, we define a new epigenetic shift that is crucial for differentiation of murine spermatogonia toward meiosis. We have exploited a property of incomplete cytokinesis, which causes male germ cells to form aligned chains of characteristic lengths, as they divide and differentiate. These chains revealed the stage of spermatogenesis, so the epigenetic differences of various stages could be characterized. Single, paired and medium chain-length spermatogonia not expressing Kit (a marker of differentiating spermatogonia) showed no expression of Dnmt3a2 and Dnmt3b (two de novo DNA methyltransferases); they also lacked the transcriptionally repressive histone modification H3K9me2. By contrast, spermatogonia consisting of ~8-16 chained cells with Kit expression dramatically upregulated Dnmt3a2/3b expression and also displayed increased H3K9me2 modification. To explore the function of these epigenetic changes in spermatogonia in vivo, the DNA methylation machinery was destabilized by ectopic Dnmt3b expression or Np95 ablation. Forced Dnmt3b expression induced expression of Kit; whereas ablation of Np95, which is essential for maintaining DNA methylation, interfered with differentiation and viability only after spermatogonia become Kit positive. These data suggest that the epigenetic status of spermatogonia shifts dramatically during the Kit-negative to Kit-positive transition. This shift might serve as a switch that determines whether spermatogonia self-renew or differentiate.


Antimicrobial catheters for reduction of symptomatic urinary tract infection in adults requiring short-term catheterisation in hospital: a multicentre randomised controlled trial.
Catheter-associated urinary tract infection (CAUTI) is a major preventable cause of harm for patients in hospital. We aimed to establish whether short-term routine use of antimicrobial catheters reduced risk of CAUTI compared with standard polytetrafluoroethylene (PTFE) catheterisation. In our parallel, three group, multicentre, randomised controlled superiority trial, we enrolled adults (aged ≥16 years) requiring short-term (≤14 days) catheterisation at 24 hospitals in the UK. Participants were randomly allocated 1:1:1 with a remote computer allocation to receive a silver alloy-coated catheter, a nitrofural-impregnated catheter, or a PTFE-coated catheter (control group). Patients undergoing unplanned catheterisation were also included and consent for participation was obtained retrospectively. Participants and trial staff were unmasked to treatment assignment. Data were collected by trial staff and by patient-reported questionnaires for 6 weeks after randomisation. The primary outcome was incidence of symptomatic urinary tract infection for which an antibiotic was prescribed by 6 weeks. We postulated that a 3·3% absolute reduction in CAUTI would represent sufficient benefit to recommend routine use of antimicrobial catheters. This study is registered, number ISRCTN75198618. 708 (10%) of 7102 randomly allocated participants were not catheterised, did not confirm consent, or withdrew, and were not included in the primary analyses. Compared with 271 (12·6%) of 2144 participants in the control group, 263 (12·5%) of 2097 participants allocated a silver alloy catheter had the primary outcome (difference -0·1% [95% CI -2·4 to 2·2]), as did 228 (10·6%) of 2153 participants allocated a nitrofural catheter (-2·1% [-4·2 to 0·1]). Rates of catheter-related discomfort were higher in the nitrofural group than they were in the other groups. Silver alloy-coated catheters were not effective for reduction of incidence of symptomatic CAUTI. The reduction we noted in CAUTI associated with nitrofural-impregnated catheters was less than that regarded as clinically important. Routine use of antimicrobial-impregnated catheters is not supported by this trial. UK National Institute for Health Research Health Technology Assessment Programme.


Probing the limits of the molecular replacement method: the case of Trypanosoma brucei phosphoglycerate kinase.
It is of general interest to explore the limits of the molecular replacement method. Described here are the specifics of a successful molecular replacement structure determination in a difficult case: the X-ray crystal structure of a Trypanosoma brucei phosphoglycerate kinase (PGK) ternary complex. This ternary complex crystallizes with four 45 kDa subunits in the asymmetric unit, whereas the available search models were all monomers consisting of two distinct domains. Initial molecular replacement attempts using complete subunits were unsuccessful. Attributing this failure to a presumed change in the relative orientations of the N- and C-terminal phosphoglycerate kinase domains, a second attempt was made using each domain as an independent search model. In this way, two N-terminal and then two C-terminal domains could be oriented and positioned in the unit cell. On the basis of this result, a new search model containing both domains in the correct mutual orientation was created and used to identify the two remaining phosphoglycerate kinase subunits. The ability to successfully orient and position an N-terminal domain containing 8.4% of the scattering mass in the asymmetric unit was the key to this structure determination. Further investigations show that a truncated version of this search model containing 5.8% of the scattering mass would have been sufficient for this purpose. A retrospective analysis suggests that the effectiveness of this probe is enhanced by structural conservation, retained temperature factors and a disparity in the degree of order among the various subunits in the T. brucei PGK asymmetric unit. Based on these observations, 231 well ordered Calpha atoms were selected from a single refined T. brucei PGK subunit and it was demonstrated that this collection of atoms, representing just 1.6% of the scattering mass, could be correctly oriented and positioned in the unit cell.


A nanobody suite for yeast scaffold nucleoporins provides details of the nuclear pore complex structure.
Nuclear pore complexes (NPCs) are the main conduits for molecular exchange across the nuclear envelope. The NPC is a modular assembly of ~500 individual proteins, called nucleoporins or nups. Most scaffolding nups are organized in two multimeric subcomplexes, the Nup84 or Y complex and the Nic96 or inner ring complex. Working in S. cerevisiae, and to study the assembly of these two essential subcomplexes, we here develop a set of twelve nanobodies that recognize seven constituent nucleoporins of the Y and Nic96 complexes. These nanobodies all bind specifically and with high affinity. We present structures of several nup-nanobody complexes, revealing their binding sites. Additionally, constitutive expression of the nanobody suite in S. cerevisiae detect accessible and obstructed surfaces of the Y complex and Nic96 within the NPC. Overall, this suite of nanobodies provides a unique and versatile toolkit for the study of the NPC.


In vitro-selected RNA cleaving DNA enzymes from a combinatorial library are potent inhibitors of HIV-1 gene expression.
Selective inactivation of a target gene by antisense mechanisms is an important biological tool to delineate specific functions of the gene product. Approaches mediated by ribozymes and RNA-cleaving DNA enzymes (DNA enzymes) are more attractive because of their ability to catalytically cleave the target RNA. DNA enzymes have recently gained a lot of importance because they are short DNA molecules with simple structures that are expected to be stable to the nucleases present inside a mammalian cell. We have designed a strategy to identify accessible cleavage sites in HIV-1 gag RNA from a pool of random DNA enzymes, and for isolation of DNA enzymes. A pool of random sequences (all 29 nucleotides long) that contained the earlier-identified 10-23 catalytic motif were tested for their ability to cleave the target RNA. When the pool of random DNA enzymes was targeted to cleave between any A and U nucleotides, DNA enzyme 1836 was identified. Although several DNA enzymes were identified using a pool of DNA enzymes that was completely randomized with respect to its substrate-binding properties, DNA enzyme-1810 was selected for further characterization. Both DNA enzymes showed target-specific cleavage activities in the presence of Mg(2+) only. When introduced into a mammalian cell, they showed interference with HIV-1-specific gene expression. This strategy could be applied for the selection of desired target sites in any target RNA.


Liver-expressed antimicrobial peptide 2 elevation contributes to age-associated cognitive decline.
Elderly individuals frequently report cognitive decline, while various studies indicate hippocampal functional declines with advancing age. Hippocampal function is influenced by ghrelin through hippocampus-expressed growth hormone secretagogue receptor (GHSR). Liver-expressed antimicrobial peptide 2 (LEAP2) is an endogenous GHSR antagonist that attenuates ghrelin signaling. Here, we measured plasma ghrelin and LEAP2 levels in a cohort of cognitively normal individuals older than 60 and found that LEAP2 increased with age while ghrelin (also referred to in literature as "acyl-ghrelin") marginally declined. In this cohort, plasma LEAP2/ghrelin molar ratios were inversely associated with Mini-Mental State Examination scores. Studies in mice showed an age-dependent inverse relationship between plasma LEAP2/ghrelin molar ratio and hippocampal lesions. In aged mice, restoration of the LEAP2/ghrelin balance to youth-associated levels with lentiviral shRNA Leap2 downregulation improved cognitive performance and mitigated various age-related hippocampal deficiencies such as CA1 region synaptic loss, declines in neurogenesis, and neuroinflammation. Our data collectively suggest that LEAP2/ghrelin molar ratio elevation may adversely affect hippocampal function and, consequently, cognitive performance; thus, it may serve as a biomarker of age-related cognitive decline. Moreover, targeting LEAP2 and ghrelin in a manner that lowers the plasma LEAP2/ghrelin molar ratio could benefit cognitive performance in elderly individuals for rejuvenation of memory.


Identification of functional regions on the tail of Acanthamoeba myosin-II using recombinant fusion proteins. I. High resolution epitope mapping and characterization of monoclonal antibody binding sites.
We used a series of COOH-terminally deleted recombinant myosin molecules to map precisely the binding sites of 22 monoclonal antibodies along the tail of Acanthamoeba myosin-II. These antibodies bind to 14 distinguishable epitopes, some separated by less than 10 amino acids. The positions of the binding sites visualized by electron microscopy agree only approximately with the physical positions of these sites on the alpha-helical coiled-coil tail. On the other hand, the epitope map agrees precisely with competitive binding studies: all antibodies that share an epitope compete with each other for binding to myosin. Antibodies with adjacent epitopes can compete with each other at linear distances up to 5 or 6 nm, and many antibodies that bind 3-7-nm apart can enhance the binding of each other to myosin. Most of the antibodies that bind to the distal 37 nm of the tail disrupt assembly of octameric minifilaments and, depending upon the exact location of the binding site, stop assembly at specific steps yielding, for example, monomers, antiparallel dimers, parallel dimers or antiparallel tetramers. The effects of these antibodies on assembly identify sites on the tail that are required for individual steps in minifilament assembly. Experiments on the assembly of truncated myosin-II tails have revealed a complementary group of sites that participate in the assembly reactions (Sinard, J.H., D.L. Rimm, and T.D. Pollard. 1990. J. Cell Biol. 111:2417-2426). Antibodies that bind to the distal tail but do not affect assembly appear to have a low affinity for myosin-II. Antibodies that bind to the proximal 50 nm of the tail do not inhibit the assembly of minifilaments. Many antibodies that bind to the tail of myosin-II, even some that have no obvious effect on minifilament assembly, can inhibit the actomyosin ATPase activity and the contraction of an actin gel formed in crude extracts. An antibody that binds between amino acids 1447 and 1467 inhibits the phosphorylation of serine residues distal to residue 1483.


SynPharm: A Guide to PHARMACOLOGY Database Tool for Designing Drug Control into Engineered Proteins.
A major challenge in synthetic biology, particularly for mammalian systems, is the inclusion of adequate external control for the synthetic system activities. Control at the transcriptional level can be achieved by adaptation of bacterial repressor-operator systems (e.g., TetR), but altering the activity of a protein by controlling transcription is indirect and for longer half-life mRNAs, decreasing activity this way can be inconveniently slow. Where possible, direct modulation of protein activity by soluble ligands has many advantages, including rapid action. Decades of drug discovery and pharmacological research have uncovered detailed information on the interactions between large numbers of small molecules and their primary protein targets (as well as off-target secondary interactions), many of which have been well studied in mammals, including humans. In principle, this accumulated knowledge would be a powerful resource for synthetic biology. Here, we present SynPharm, a tool that draws together information from the pharmacological database GtoPdb and the structural database, PDB, to help synthetic biologists identify ligand-binding domains of natural proteins. Consequently, as sequence cassettes, these may be suitable for building into engineered proteins to confer small-molecule modulation on them. The tool has ancillary utilities which include assessing contact changes among different ligands in the same protein, predicting possible effects of genetic variants on binding residues, and insights into ligand cross-reactivity among species.


Continuous decline in mortality from coronary heart disease in Japan despite a continuous and marked rise in total cholesterol: Japanese experience after the Seven Countries Study.
The Seven Countries Study in the 1960s showed very low mortality from coronary heart disease (CHD) in Japan, which was attributed to very low levels of total cholesterol. Studies of migrant Japanese to the USA in the 1970s documented increase in CHD rates, thus CHD mortality in Japan was expected to increase as their lifestyle became Westernized, yet CHD mortality has continued to decline since 1970. This study describes trends in CHD mortality and its risk factors since 1980 in Japan, contrasting those in other selected developed countries. We selected Australia, Canada, France, Japan, Spain, Sweden, the UK and the USA. CHD mortality between 1980 and 2007 was obtained from WHO Statistical Information System. National data on traditional risk factors during the same period were obtained from literature and national surveys. Age-adjusted CHD mortality continuously declined between 1980 and 2007 in all these countries. The decline was accompanied by a constant fall in total cholesterol except Japan where total cholesterol continuously rose. In the birth cohort of individuals currently aged 50-69 years, levels of total cholesterol have been higher in Japan than in the USA, yet CHD mortality in Japan remained the lowest: >67% lower in men and > 75% lower in women compared with the USA. The direction and magnitude of changes in other risk factors were generally similar between Japan and the other countries. Decline in CHD mortality despite a continuous rise in total cholesterol is unique. The observation may suggest some protective factors unique to Japanese.


In vitro inhibition of angiogenesis by antibodies directed against the 37kDa/67kDa laminin receptor.
The 37kDa/67kDa laminin receptor (LRP/LR) is a central receptor mediating interactions between tumour cells and the basement membrane and is thereby a key player in adhesion and invasion, essential processes in metastatic cancer. To affect continued tumour growth, tumours induce angiogenesis for the constant delivery of nutrients and oxygen. This study aims to determine the blocking effect of the anti-LRP/LR specific antibody, W3 on the angiogenic potential of HUVE (human umbilical vein endothelial) cells. Flow cytometric analysis revealed that 97% of HUVE cells display cell surface LRP/LR. An angiogenesis assay was conducted employing HUVE cells seeded on the basement membrane reconstituent Matrigel™ supplemented with the pro-angiogenic factor vascular endothelial growth factor (VEGF). Post 18h incubation at 37°C tubular structures, namely tube lengths were assessed. Treatment of established tubular structures with 100 µg/ml anti-LRP/LR specific antibody completely blocked angiogenesis. Our findings suggest a central role of the 37kDa/67kDa LRP/LR in tube formation and recommends anti-LRP/LR specific antibodies as potential therapeutic tools for treatment of tumour angiogenesis.


Qualitative differences between traditional and rural-longitudinal medical student OSCE performance.
To ensure adequate observation, supervision, and mentoring of trainees, long-term preceptorships or apprenticeships are being reestablished in medical education. Equivalence in academic performance has been demonstrated between longitudinal students in the Rural Physician Associate Program (RPAP), who spend 9 months in a rural community during their third year of medical school, and their peers who complete their clerkships at different hospitals and clinics (traditional). We qualitatively reviewed the end of session Objective Structured Clinical Examination (OSCE) for both groups and compared their performances. The high and low performers on four OSCE scenarios (cough, dysuria in a teen, preventive care in an older male, medication reconciliation) for two cohorts of students: longitudinal (n=47) and traditional primary care clerkship students (n=60) were selected for review. These 16 videotapes were reviewed independently by three researchers. The themes and subthemes were discussed over four meetings. Both high and low scoring longitudinal students demonstrated more consistent use of rapport building skills. Longitudinal students appeared to have an effective pattern in their patient interactions and were more rehearsed at explaining preventive care recommendations such as the pros and cons of the prostate-specific antigen (PSA) test. Traditional students displayed a more complete mastery of the adolescent interview and followed a mnemonic taught during lecture. Qualitative assessment of OSCE data reveals information not captured in the quantitative scores. In this study, longitudinal students demonstrated better mastery of rapport building and content knowledge and had an effective routine to their patient encounters not evident in the traditional students' scenarios.


Carbohydrate loading and metabolism during exercise in men and women.
During endurance exercise at approximately 65% maximal O2 consumption, women oxidize more lipids, and therefore decrease carbohydrate and protein oxidation, compared with men (L.J. Tarnopolsky, M.A. Tarnopolsky, S.A. Atkinson, and J.D. MacDougall. J. Appl. Physiol. 68: 302-308, 1990; S.M. Phillips, S.A. Atkinson, M.A. Tarnopolsky, and J.D. MacDougall. J. Appl. Physiol. 75: 2134-2141, 1993). The main purpose of this study was to examine the ability of similarly trained male (n = 7) and female (n = 8) endurance athletes to increase muscle glycogen concentrations in response to an increase in dietary carbohydrate from 55-60 to 75% of energy intake for a period of 4 days (carbohydrate loading). In addition, we sought to examine whether gender differences existed in metabolism during submaximal endurance cycling at 75% peak O2 consumption (VO2 peak) for 60 min. The men increased muscle glycogen concentration by 41% in response to the dietary manipulation and had a corresponding increase in performance time during an 85% VO2 peak trial (45%), whereas the women did not increase glycogen concentration (0%) or performance time (5%). The women oxidized significantly more lipid and less carbohydrate and protein compared with the men during exercise at 75% VO2-peak. We conclude that women did not increase muscle glycogen in response to the 4-day regimen of carbohydrate loading described. In addition, these data support previous observations of greater lipid and lower carbohydrate and protein oxidation by women vs. men during submaximal endurance exercise.


Long-term programming of enhanced aggression by peripuberty stress in female rats.
Human literature has linked adverse early life experiences with an increased risk to develop violent behaviors in both boys and girls. We have previously shown that male rats submitted to stress during the peripuberty period display as adults abnormal aggressive behavior against both male intruders and female partners. In the present study, we examined whether the same stress protocol would affect the development of aggressive behaviors in female rats. We evaluated the behavior of these peripuberty stressed female rats when confronted, at adulthood, with either female or male intruders, and during their cohabitation with male partners. Given that estrus cycle influences mood and aggressive behaviors, female aggressive behavior was assessed at different estrus cycle phases: estrus and diestrus, and during pregnancy and lactancy. Additionally, we evaluated postpartum plasma levels of vasopressin, oxytocin and corticosterone, hormones associated with aggression and the regulation of social behavior. Compared to control females, females submitted to stressful events during puberty exhibited higher and more sustained rates of aggression during adulthood independently on the estrus cycle or the sex of the intruder, and they had higher levels of plasma vasopressin. Significant correlations between plasma levels of vasopressin and corticosterone and aggressive behavior were also found. Strikingly, our results showed opposite intragroup correlations suggesting a different role of these hormones on aggression depending on life experiences. We provide here an animal model, devoid of cultural influences strongly supporting a role for biological factors in the development of aggressive behaviors following exposure to stressful events at puberty in females.