Ligand-activated Notch undergoes DTX4-mediated ubiquitylation and bilateral endocytosis before ADAM10 processing.
The Notch signaling pathway, which is activated by cell-cell contact, is a major regulator of cell fate decisions. Mammalian Notch1 is present at the cell surface as a heterodimer of the Notch extracellular domain associated with the transmembrane and intracellular domains. After ligand binding, Notch undergoes proteolysis, releasing the Notch intracellular domain (NICD) that regulates gene expression. We monitored the early steps of activation with biochemical analysis, immunofluorescence analysis, and live-cell imaging of Notch1-expressing cells. We found that, upon ligand binding, Notch1 at the cell surface was ubiquitylated by the E3 ubiquitin ligase DTX4. This ubiquitylation event led to the internalization of the Notch1 extracellular domain by the ligand-expressing cell and the internalization of the membrane-anchored fragment of Notch1 and DTX4 by the Notch1-expressing cell, which we referred to as bilateral endocytosis. ADAM10 generates a cleavage product of Notch that is necessary for the formation of the NICD, which has been thought to occur at the cell surface. However, we found that blocking dynamin-mediated endocytosis of Notch1 and DTX4 reduced the colocalization of Notch1 with ADAM10 and the formation of the ADAM10-generated cleavage product of Notch1, suggesting that ADAM10 functions in an intracellular compartment to process Notch. Thus, this study suggests that a specific pool of ADAM10 acts on Notch in an endocytic compartment, rather than at the cell surface.


Optimization of biomass pretreatments using fractional factorial experimental design.
Pretreatments are one of the main bottlenecks for the lignocellulose conversion process and the search for cheaper and effective pretreatment methodologies for each biomass is a complex but fundamental task. Here, we used a 2ν5-1 fractional factorial design (FFD) to optimize five pretreatment variables: milling time, temperature, double treatment, chemical concentration, and pretreatment time in acid-alkali (EA) and acid-organosolv (EO) pretreatments, applied to elephant grass leaves. FFD allowed optimization of the pretreatment conditions using a reduced number of experiments and allowed the identification of secondary interactions between the factors. FFD showed that the temperature can be kept at its lower level and that the first acid step can be eliminated in both pretreatments, without significant losses to enzymatic hydrolysis. EA resulted in the highest release of reducing sugars (maximum of 205 mg/g substrate in comparison to 152 mg/g in EO and 40 mg/g in the untreated sample), using the following conditions in the alkali step: [NaOH] = 4.5% w/v; 85 °C and 100 min after ball milling the sample. The factors statistically significant (P < 0.05) in EA pretreatment were NaOH concentration, which contributes to improved hydrolysis by lignin and silica removal, and the milling time, which has a mechanical effect. For EO samples, the statistically significant factors to improved hydrolysis were ethanol and catalyst concentrations, which are both correlated to higher cellulose amounts in the pretreated substrates. The catalyst is also correlated to lignin removal. The detailed characterization of the main hemicellulosic sugars in the solids after pretreatments revealed their distinct recalcitrance: glucose was typically more recalcitrant than xylose and arabinose, which could be almost completely removed under specific pretreatments. In EA samples, the removal of hemicellulose derivatives was very dependent on the acid step, especially arabinose removal. The results presented herewith contribute to the development of more efficient and viable pretreatments to produce cellulosic ethanol from grass biomasses, saving time, costs and energy. They also facilitate the design of enzymatic cocktails and a more appropriate use of the sugars contained in the pretreatment liquors, by establishing the key recalcitrant polymers in the solids resulting from each processing step.


Pharmacogenetics of anticancer drug sensitivity in pancreatic cancer.
Chemotherapy has produced unsatisfactory results in pancreas cancer and novel approaches, including treatment tailoring by pharmacogenetic analysis and new molecular-targeted drugs, are required. The scarcity of effective therapies may reflect the lack of knowledge about the influence of tumor-related molecular abnormalities on responsiveness to drugs. Advances in the understanding of pancreas cancer biology have been made over the past decade, including the discovery of critical mutations in oncogenes (i.e., K-Ras) as well as the loss of tumor suppressor genes, such as TP53 and p16(INK4). Other studies showed the dysregulation of the expression of proteins involved in the control of cell cycle, proliferation, apoptosis, and invasiveness, such as Bcl-2, Akt, mdm2, and epidermal growth factor receptor. These characteristics might contribute to the aggressive behavior of pancreatic cancer and influence response to treatment. Indeed, the inactivation of p53 may explain the relative resistance to 5-fluorouracil, whereas Bcl-2 overexpression is associated with reduced sensitivity to gemcitabine. However, the future challenge of pancreas cancer chemotherapy relies on the identification of molecular markers that help in the selection of drugs best suited to the individual patient. Recent pharmacogenetic studies focused on genes encoding proteins directly involved in drug activity, showing the role of thymidylate synthase and human equilibrative nucleoside transporter-1 as prognostic factor in 5-fluorouracil- and gemcitabine-treated patients, respectively. Finally, inhibitors of signal transduction and angiogenesis are under extensive investigation, and several prospective trials have been devoted to this area. Pharmacogenetics is likely to play a central role in the personalization of treatment, to stratify patients based on their likelihood of response to both standard agents (i.e., gemcitabine/nucleoside transporters) and targeted treatments (i.e., epidermal growth factor receptor gene mutations and/or amplification and tyrosine kinase inhibitors), Thus, molecular analysis should be implemented in the optimal management of the patient affected by pancreatic adenocarcinoma.


The design and conduct of Project RedDE: A cluster-randomized trial to reduce diagnostic errors in pediatric primary care.
Diagnostic errors contribute to the large burden of healthcare-associated harm experienced by children. Primary care settings involve high diagnostic uncertainty and limited time and information, creating ideal conditions for diagnostic errors. We report on the design and conduct of Project RedDE, a stepped-wedge, cluster-randomized controlled trial of a virtual quality improvement collaborative aimed at reducing diagnostic errors in pediatric primary care. Project RedDE cluster-randomized pediatric primary care practices into one of three groups. Each group participated in a quality improvement collaborative targeting the same three diagnostic errors (missed diagnoses of elevated blood pressure and adolescent depression and delayed diagnoses of abnormal laboratory studies), but in a different sequence. During the quality improvement collaborative, practices worked both independently and collaboratively, leveraging general quality improvement strategies (e.g. process mapping) in addition to error-specific content (e.g. pocket guides for blood pressure norms) delivered during the intervention phase for each error. The quality improvement collaborative intervention included interactive learning sessions and webinars, quality improvement coaching at the team level, and repeated evaluation of failures via root cause analyses. Pragmatic data were collected monthly, submitted to a centralized data aggregator, and returned to the practices in the form of run charts comparing each practice's progress over time to that of the group. The primary analysis used patients as the unit of analysis and compared diagnostic error proportions between the intervention and baseline periods, while secondary analyses evaluated the sustainability of observed reductions in diagnostic errors after the intervention period ended. A total of 43 practices were recruited and randomized into Project RedDE. Eleven practices withdrew before submitting any data, and one practice merged with another participating practice, leaving 31 practices that began work on Project RedDE. All but one of the diverse, national pediatric primary care practices that participated ultimately submitted complete data. Quality improvement collaborative participation was robust, with an average of 63% of practices present on quality improvement collaborative webinars and 85% of practices present for quality improvement collaborative learning sessions. Complete data included 30 months of outcome data for the first diagnostic error worked on, 24 months of outcome data for the second, and 16 months of data for the third. Contamination across study groups was a recurring concern; concerted efforts were made to mitigate this risk. Electronic health records played a large role in teams' success. Project RedDE, a virtual quality improvement collaborative aimed at reducing diagnostic errors in pediatric primary care, successfully recruited and retained a diverse, national group of pediatric primary care practices. The stepped-wedge, cluster-randomized controlled trial design allowed for enhanced scientific efficiency.


Current status of high dose chemotherapy in breast cancer.
To date, no definite answers are available to the initial question, whether or not HDCT and ASCT can improve quantity or quality of life. Completing the available prospective, randomized phase III studies; using HDCT earlier in the course of treatment; applying drugs that are active breast cancer; comparing the experimental treatment to standard therapy; and using appropriate sample size to detect clinically meaningful and statistically significant differences are all paramount requirements to answer the question successfully. Within the next few years, a number of European [13] and two Canadian Studies [6, 14] that are currently accruing patients, will contribute versus important information regarding the role of HDCT and ABMT for breast cancer.


The Vatican Opinion on Gender Theory.
This article is a reasoned response to the article by Timothy F. Murphy, recently published in the prestigious journal Bioethics, on the supposed opposition between the views of the Catholic Church and what he calls "contemporary science" in relation to certain anthropological issues linked to the gender perspective. To point to "the Vatican" as anchored in an unscientific and anachronistic position, using the term contemporary science to which he attributes a unanimous representation of current scientific thinking on the subject is, in our view, unfounded and completely unacceptable. In his reflection, he does not adequately distinguish between intersex and transgenderism, two clearly different realities with different needs. The author defends the obsolescence of the binary sex/gender model that, in his view, "betrays human sexuality." Furthermore, he does so without providing a plausible justification or a definition of human nature that is able to support the plurality and indeterminacy of sexual conditions, without falling back on untenable dualisms or relativism devoid of scientific objectivity. In our response, we highlight how the dialogue between Faith and Reason, as developed in the recent Magisterium of the Catholic Church, is essential to explain nature, the human being and, in general, all creations. Finally, contemporary science does not provide a monolithic and unquestionable view of the nature of human beings and their sexual identity, as the author claims, with many scientists confirming evidence of a binary human sexuality genetically and phenotypically determined. This paper is a reasoned response to the supposed opposition between the views of the Catholic Church and "contemporary science" in relation to certain anthropological issues linked to the gender perspective.The dialogue between Faith and Reason, as developed in the recent Magisterium of the Catholic Church, is essential to explain nature, the human being and, in general, all creation, against the opinion of those who defend the obsolescence of the binary sex/gender model that, in their view, "betrays human sexuality".


A novel wire mesh "cell" for studying lipid oxidative processes by fourier transform infrared spectroscopy.
A novel infrared (IR) sample handling accessory has been developed to monitor and study oxidation processes of edible oils under moderate temperature conditions by Fourier transform infrared (FT-IR) spectroscopy. A reusable stainless steel mesh IR "cell" was designed and evaluated from the standpoint of mesh size, transmission characteristics, its ability to entrap oil, and techniques to apply sample and normalize path length so as to obtain good quality, reproducible spectra. The concept is to entrap oil within the mesh by means of its inherent surface tension and to take advantage of the high surface area provided by the mesh to facilitate rapid oxidation of the oil by air at ambient or slightly elevated temperatures without having to resort to more extreme temperature conditions to track oxidative changes in real time. Changes taking place in canola oil at room temperature, in the dark and exposed to light, as well as at 50 degrees C are presented to illustrate the performance of the cell in monitoring oxidative changes in real time (e.g., formation of hydroperoxides, loss of cis and formation of trans double bonds). The mesh cell should be useful for comparing the relative performance of antioxidants as well as evaluating the oxidative stability of oils, among other applications.


Dynamic functional connectivity states characterize NREM sleep and wakefulness.
According to recent neuroimaging studies, temporal fluctuations in functional connectivity patterns can be clustered into dynamic functional connectivity (DFC) states and correspond to fluctuations in vigilance. However, whether there consistently exist DFC states associated with wakefulness and sleep stages and what are the characteristics and electrophysiological origin of these states remain unclear. The aims of the current study were to investigate the properties of DFC in different sleep stages and to explore the relationship between the characteristics of DFC and slow-wave activity. We collected both eyes-closed wakefulness and sleep data from 48 healthy young volunteers with simultaneous electroencephalography (EEG) and functional magnetic resonance imaging (fMRI) recordings. EEG data were employed as the gold standard of sleep stage scoring, and DFC states were estimated based on fMRI data. The results demonstrated that DFC states of the fMRI signals consistently corresponded to wakefulness and nonrapid eye movement sleep stages independent of the number of clusters. Furthermore, the mean dwell time of these states significantly correlated with slow-wave activity. The inclusion or omission of regression of the global signal and the selection of parcellation schemes exerted minimal effects on the current findings. These results provide strong evidence that DFC states underlying fMRI signals match the fluctuations of vigilance and suggest a possible electrophysiological source of DFC states corresponding to vigilance states.


The energetics of myocardial stretch. Creatine kinase flux and oxygen consumption in the noncontracting rat heart.
Previous studies have suggested that flux through the creatine kinase reaction is coupled to cardiac performance and to the rate of adenosine triphosphate synthesis in the intact, beating heart. To define the effect of passive myocardial stretch on creatine kinase kinetics, we measured the rate constants and chemical fluxes for both directions of the creatine kinase reaction with the 31P-nuclear magnetic resonance technique of magnetization transfer in isolated, arrested rat hearts at four levels of left ventricular pressure and volume. Adenosine triphosphate synthesis was estimated from oxygen consumption measurements. As left ventricular pressure rose from 0 to 24 mm Hg and oxygen consumption increased by 20%, we observed a twofold increase in the rate constants and fluxes (mean +/- SD, n = 4-7) for the creatine kinase reaction. The forward rate constant increased from 0.33 +/- 0.03 to 0.80 +/- 0.08, and the reverse rate increased from 0.34 +/- 0.11 to 0.74 +/- 0.32/sec. The forward and reverse fluxes for the creatine kinase reaction increased from 12.0 +/- 3.4 to 26.5 +/- 5.8 and from 9.1 +/- 3.4 to 19.1 +/- 3.4 mumol/g dry weight per sec, respectively. At each level of left ventricular pressure, the forward and reverse rate constants were the same. However, as left ventricular pressure increased, the ratio of the forward to the apparent reverse fluxes for the creatine kinase reaction increased. The relationships between the rate constant or flux through the creatine kinase reaction vs. left ventricular pressure were linear. In addition to providing further support for the coupling between creatine kinase and mitochondrial adenosine triphosphate synthesis, these results suggest that flux through the creatine kinase reaction and adenosine triphosphate synthesis increases with myocardial stretch in the intact, noncontracting heart.


Iron(II) complexes of 2,6-di(1H-pyrazol-3-yl)-pyridine derivatives with hydrogen bonding and sterically bulky substituents.
Syntheses of 2,6-di(5-aminopyrazol-3-yl)pyridine (L(1)), 2,6-di(5-tertbutylcarboxamidopyrazol-3-yl)pyridine (L(2)), 2,6-di(5-tertbutylpyrazol-3-yl)pyridine (L(3)), 2-(5-tertbutylpyrazol-3-yl)-6-(5-methylpyrazol-3-yl)pyridine (L(4)) and 2-(5-tertbutylpyrazol-3-yl)-6-(5-aminopyrazol-3-yl)pyridine (L(5)) are reported. Iron complex salts of the first four ligands were crystallographically characterised. The structures exhibit intermolecular hydrogen bonding between the cations and the anions and/or solvent, leading to a fluorite (flu) net, a 1D ladder structure, and a homochiral self-penetrating helical network related to the (10,3)-a (srs) topology. All the complexes are high-spin in the crystal, and bulk samples are also fully or predominantly high-spin at room temperature and below although two of the dried materials exhibit partial spin-state transitions on cooling.


Age-related differences in distractibility and response to methylphenidate in monkeys.
Increased susceptibility to distraction is a symptom of normal aging and several clinical syndromes, including Alzheimer's disease and attention deficit disorders. In the present study, aged and young adult macaques were well-trained to perform an automated delayed matching-to-sample (DMTS) task which assesses both attention and short-term memory. On 19% of all trials, a task-relevant distracting stimulus was presented during either the initial 1 or 3 s of delay intervals (early onset) or the final 1 or 3 s of delay intervals (late onset). In aged monkeys, both early and late onset distractors lasting 1 or 3 s impaired delayed recall on trials with the shortest delay intervals, but did not affect accuracy on trials with long delay intervals. In contrast, young adult monkeys were impaired only by the presence of an early onset distractor lasting 3 s. Impairment was selective for only those trials with the shortest delay intervals. Late onset distractors were relatively ineffective in producing distractibility in young adult animals. Methylphenidate (MPH; 0.005-1.0 mg/kg) failed to reduce distractibility in aged monkeys, producing locomotor abnormalities and hypophagia at doses ranging from 0.25 to 1.0 mg/kg. In young adult monkeys, however, distractibility was significantly attenuated by administration of the 0.125 mg/kg dose. Habituation to the distracting stimulus (under saline conditions) was assessed throughout the study and was not evident at any time point of testing. These data indicate that attention and recall after brief delays are impaired following exposure to a task-relevant distracting stimulus in both aged and young adult monkeys, but that aged monkeys are more susceptible to distraction and do not receive significant benefit from MPH administration.


School interventions after the Joplin tornado.
To qualitatively describe interventions by schools to meet children's needs after the May 2011 Joplin, Missouri tornado. Qualitative exploratory study conducted six months after the tornado. Key informant interviews with school staff (teachers, psychologists, guidance counselor, nurse, principal), public health official, and physicians. After the tornado, school staff immediately worked to contact every enrolled child to provide assistance and coordinate recovery services. Despite severe damage to half of the city's schools, the decision was made to reopen schools at the earliest possible time to provide a safe, reassuring environment and additional services. An expanded summer school session emphasized child safety and emotional wellbeing. The 2011-2012 school year began on time, less than three months after the disaster, using temporary facilities. Displaced children were bused to their usual schools regardless of their new temporary residence locations. In just-in-time training sessions, teachers developed strategies to support students and staff experiencing anxiety or depression. Certified counselors conducted school-based, small-group counseling for students. Selective referrals were made to community mental health providers for children with greatest needs. Evidence from Joplin adds to a small body of empirical experience demonstrating the important contribution of schools to postdisaster community recovery. Despite timely and proactive services, many families and children struggled after the tornado. Improvements in the effectiveness of postdisaster interventions at schools will follow from future scientific evidence on optimal approaches.


Accelerated evolution of PAK3- and PIM1-like kinase gene families in the zebra finch, Taeniopygia guttata.
Genes encoding protein kinases tend to evolve slowly over evolutionary time, and only rarely do they appear as recent duplications in sequenced vertebrate genomes. Consequently, it was a surprise to find two families of kinase genes that have greatly and recently expanded in the zebra finch (Taeniopygia guttata) lineage. In contrast to other amniotic genomes (including chicken) that harbor only single copies of p21-activated serine/threonine kinase 3 (PAK3) and proviral integration site 1 (PIM1) genes, the zebra finch genome appeared at first to additionally contain 67 PAK3-like (PAK3L) and 51 PIM1-like (PIM1L) protein kinase genes. An exhaustive analysis of these gene models, however, revealed most to be incomplete, owing to the absence of terminal exons. After reprediction, 31 PAK3L genes and 10 PIM1L genes remain, and all but three are predicted, from the retention of functional sites and open reading frames, to be enzymatically active. PAK3L, but not PIM1L, gene sequences show evidence of recurrent episodes of positive selection, concentrated within structures spatially adjacent to N- and C-terminal protein regions that have been discarded from zebra finch PAK3L genes. At least seven zebra finch PAK3L genes were observed to be expressed in testis, whereas two sequences were found transcribed in the brain, one broadly including the song nuclei and the other in the ventricular zone and in cells resembling Bergmann's glia in the cerebellar Purkinje cell layer. Two PIM1L sequences were also observed to be expressed with broad distributions in the zebra finch brain, one in both the ventricular zone and the cerebellum and apparently associated with glial cells and the other showing neuronal cell expression and marked enrichment in midbrain/thalamic nuclei. These expression patterns do not correlate with zebra finch-specific features such as vocal learning. Nevertheless, our results show how ancient and conserved intracellular signaling molecules can be co-opted, following duplication, thereby resulting in lineage-specific functions, presumably affecting the zebra finch testis and brain.


Structure and Function of the Nuclear Pore Complex Cytoplasmic mRNA Export Platform.
The last steps in mRNA export and remodeling are performed by the Nup82 complex, a large conserved assembly at the cytoplasmic face of the nuclear pore complex (NPC). By integrating diverse structural data, we have determined the molecular architecture of the native Nup82 complex at subnanometer precision. The complex consists of two compositionally identical multiprotein subunits that adopt different configurations. The Nup82 complex fits into the NPC through the outer ring Nup84 complex. Our map shows that this entire 14-MDa Nup82-Nup84 complex assembly positions the cytoplasmic mRNA export factor docking sites and messenger ribonucleoprotein (mRNP) remodeling machinery right over the NPC's central channel rather than on distal cytoplasmic filaments, as previously supposed. We suggest that this configuration efficiently captures and remodels exporting mRNP particles immediately upon reaching the cytoplasmic side of the NPC.


A 4-dimethylaminobenzoate-functionalized Ti6-oxo cluster with a narrow band gap and enhanced photoelectrochemical activity: a combined experimental and computational study.
Organic donor-π-bridge-acceptor (D-π-A) dyes with arylamines as an electron donor have been widely used as photosensitizers for dye-sensitized solar cells (DSSCs). However, titanium-oxo clusters (TOCs) functionalized with this kind of D-π-A structured dye-molecule have rarely been explored. In the present study, the 4-dimethylaminobenzoate-functionalized titanium-oxo cluster [Ti6(μ3-O)6(OiPr)6(DMABA)6]·2C6H5CH3 (DMABA = 4-dimethylaminobenzoate) was synthesized and structurally characterized by single-crystal X-ray diffraction. For comparison, two other Ti6-oxo clusters, namely [Ti6(μ3-O)6(OiPr)6(AD)6] (AD = 1-adamantanecarboxylate) and [Ti6(μ3-O)2(μ2-O)(μ2-OiPr)4(OiPr)10(DMM)2] (DMM = dimethylmalonate), were also studied. The DMABA-functionalized cluster exhibits a remarkably reduced band gap of ∼2.5 eV and much enhanced photocurrent response in comparison with the other two clusters. The electronic structures and electronic transitions of the clusters were studied by DFT and TDDFT calculations. The computational results suggest that the low-energy transitions of the DMABA-functionalized cluster have a substantial charge-transfer character arising from the DMABA → {Ti6} cluster core ligand-to-core charge transfer (LCCT), along with the DMABA-based intra-ligand charge transfer (ILCT). These low-energy charge transfer transitions provide efficient electron injection pathways for photon-to-electron conversion.


Association of circulating metabolites in plasma or serum and risk of stroke: Meta-analysis from seven prospective cohorts.
To conduct a comprehensive analysis of circulating metabolites and incident stroke in large prospective population-based settings. We investigated the association of metabolites with risk of stroke in seven prospective cohort studies including 1,791 incident stroke events among 38,797 participants in whom circulating metabolites were measured by Nuclear Magnetic Resonance (1H-NMR) technology. The relationship between metabolites and stroke was assessed using Cox proportional hazards regression models. The analyses were performed considering all incident stroke events and ischemic and hemorrhagic events separately. The analyses revealed ten significant metabolite associations. Amino acid histidine (hazard ratio (HR) per standard deviation (SD) = 0.90, 95% confidence interval (CI): 0.85, 0.94; P = 4.45×10-5), glycolysis-related metabolite pyruvate (HR per SD = 1.09, 95% CI: 1.04, 1.14; P = 7.45×10-4), acute phase reaction marker glycoprotein acetyls (HR per SD = 1.09, 95% CI: 1.03, 1.15; P = 1.27×10-3), cholesterol in high-density lipoprotein (HDL) 2 and several other lipoprotein particles were associated with risk of stroke. When focusing on incident ischemic stroke, a significant association was observed with phenylalanine (HR per SD = 1.12, 95% CI: 1.05, 1.19; P = 4.13×10-4) and total and free cholesterol in large HDL particles. We found association of amino acids, glycolysis-related metabolites, acute phase reaction markers, and several lipoprotein subfractions with the risk of stroke. These findings support the potential of metabolomics to provide new insights into the metabolic changes preceding stroke.


Nonmedical prescription opioid use and illegal drug use: initiation trajectory and related risks among people who use illegal drugs in Vancouver, Canada.
We investigated the prevalence of and risk factors associated with initiating nonmedical prescription opioid use (NMPOU) before and after illegal drugs using data from two linked cohort studies of street youth and adults who use illegal drugs in Vancouver, Canada. All participants who attended a study visit between 2013 and 2016 were eligible for the primary analyses. Among 512 youth and 833 adult participants, the prevalence of NMPOU was extremely high (88% among street youth; 90% among adults), and over one-third of those who reported engaging in NMPOU had initiated NMPOU before illegal drug use (vs. transitioning from illegal drugs to NMPOU). Participants who reported either transitioning to or from NMPOU had higher risk profiles, particularly related to substance use, when compared with those who reported never engaging in NMPOU. Sub-analyses restricted to only those who engaged in NMPOU found few statistically significant differences between those who initiated NMPOU prior to illegal drugs versus those who initiated illegal drugs prior to NMPOU. Findings suggest that among people who use illegal drugs, early NMPOU trajectories do not appear to critically shape future patterns and practices.


High-speed two-dimensional laser scanner based on Bragg gratings stored in photothermorefractive glass.
A high-speed free-space wavelength-multiplexed optical scanner with high-speed wavelength selection coupled with narrowband volume Bragg gratings stored in photothermorefractive (PTR) glass is reported. The proposed scanner with no moving parts has a modular design with a wide angular scan range, accurate beam pointing, low scanner insertion loss, and two-dimensional beam scan capabilities. We present a complete analysis and design procedure for storing multiple tilted Bragg-grating structures in a single PTR glass volume (for normal incidence) in an optimal fashion. Because the scanner design is modular, many PTR glass volumes (each having multiple tilted Bragg-grating structures) can be stacked together, providing an efficient throughput with operations in both the visible and the infrared (IR) regions. A proof-of-concept experimental study is conducted with four Bragg gratings in independent PTR glass plates, and both visible and IR region scanner operations are demonstrated.


Comparison of multi-drug injection versus placebo after hallux valgus surgery.
Hallux valgus surgery is followed by a significant amount of postoperative pain. Local multi-drug injection can be an option for pain control, but few clinical studies exist. Between May 2008 and July 2009, 30 consecutive patients (60 feet) received simultaneous bilateral proximal osteotomies for the correction of hallux valgus deformities. Each patient received local infiltration of the test solution made with ropivacaine, morphine, ketorolac, and epinephrine on one foot and same amount of normal saline on the other foot. The test side and the control side were randomly selected and both the patient and the surgeon remained blinded until the end of the data collection. The multi-drug injection resulted in significantly less pain at 4 hours after the operation through the night of the first postoperative day. The difference in visual analogue scale (VAS) between the two sides was most significant at 8 hours after the operation, and then gradually decreased through the first and second postoperative day. Mean satisfaction VAS was significantly higher on the injection side (8.2+/-0.9) compared to the control side (6.2+/-1.9, p<0.0001). Local multi-drug injection was easy to perform and safe and effective in reducing pain and enhancing patient satisfaction after hallux valgus surgery.


INTEREST IN CD2, a global patient-centred study of long-term cervical dystonia treatment with botulinum toxin.
Longitudinal cohort studies provide important information about the clinical effectiveness of an intervention in the routine clinical setting, and are an opportunity to understand how a population presents for treatment and is managed. INTEREST IN CD2 (NCT01753349) is a prospective, international, 3-year, longitudinal, observational study following the course of adult idiopathic cervical dystonia (CD) treated with botulinum neurotoxin type A (BoNT-A). The primary objective is to document long-term patient satisfaction with BoNT-A treatment. Here we report baseline data. This analysis includes 1036 subjects (67.4% of subjects were female; mean age was 54.7 years old; mean TWSTRS Total score was 31.7). BoNT-A injections were usually given in line with BoNT-A prescribing information. The most commonly injected muscles were splenius capitis (87.3%), sternocleidomastoid (82.6%), trapezius (64.3%), levator scapulae (40.9%) and semispinalis capitis (26.9%); 35.5% of subjects were injected using a guidance technique. Most subjects (87.8%) had been previously treated with BoNT-A (median interval between last pre-study injection and study baseline was 4 months); of these 84.8% reported satisfaction with BoNT-A treatment at peak effect during their previous treatment cycle and 51.5% remained satisfied at the end of the treatment. Analyses by geographical region revealed heterogeneity in the clinical characteristics and BoNT-A injection practice of CD subjects presenting for routine treatment. These baseline analyses provide sizeable data regarding the epidemiology and clinical presentation of CD, and demonstrate an international heterogeneity of clinical practice. Future longitudinal analyses of the full 3-year study will explore how these factors impact treatment satisfaction.


FTY720 attenuates excitotoxicity and neuroinflammation.
FTY720 (fingolimod, Gilenya™), a structural analog of sphingosine-1-phosphate (S1P), is the first oral drug approved for treatment the relapsing-remitting form of multiple sclerosis (MS), and its efficacy has been related to induced lymphopenia and consequent immunosuppression via modulation of S1P1 receptors (S1P1R). However, due to its lipophilic nature, FTY720 crosses the blood brain barrier (BBB) and could act directly on neural cells. In this study, we investigated the effectiveness of FTY720 as a neuroprotective agent using in vitro and in vivo models of excitotoxic neuronal death and examined if FTY720 exerts a direct action on neurons, or/and an indirect modulation of inflammation-mediated neurodegeneration as a possible mechanism of neuroprotection. Primary neuronal and organotypic cortical cultures were treated with N-methyl-D-aspartic acid (NMDA) to induce excitotoxic cell death (measured by lactate dehydrogenase (LDH) assay or propidium iodide uptake, respectively). The effects of FTY720 treatment (10, 100 and 1,000 nM) on neuronal survival were examined. As an in vivo model of neuronal death and inflammation, we used intracerebroventricular (icv) administration of kainic acid (KA; 0.5 μg/2 μl) in Sprague-Dawley rats. FTY720 was applied icv (1 μg/2 μl), together with KA, plus intraperitoneally (ip; 1 mg/kg) 24 h before, and daily, until sacrifice 3 days after icv. Rats were evaluated for neurological score, neuronal loss in CA3 hippocampal region and activation of microglia at the lesion site. In addition, we tested FTY720 as a modulator of microglia responses using microglial cell cultures activated with lipopolysaccharide (LPS) and its effects in stress signalling pathways using western blotting for p38 and JNK1/2 mitogen-activated protein kinases (MAPKs). FTY720 was able to reduce excitotoxic neuronal death in vitro. Moreover, in vivo repeated FTY720 administration attenuated KA-induced neurodegeneration and microgliosis at the CA3 lesion site. Furthermore, FTY720 negatively modulates p38 MAPK in LPS-activated microglia, whereas it had no effect on JNK1/2 activation. These data support a role for FTY720 as a neuroprotective agent against excitotoxin-induced neuronal death and as a negative modulator of neuroinflammation by targeting the p38 MAPK stress signalling pathway in microglia.


Sleep and circadian rhythm disturbances: multiple genes and multiple phenotypes.
Sleep is regulated by two broad mechanisms: the circadian system, which generates 24-h rhythms of sleep propensity and a wake-dependent homeostatic sleep process whereby sleep pressure increases during wake and dissipates during sleep. These, in turn, regulate multiple brain structures and neurotransmitter systems. In view of the complexity of sleep it is not surprising that there is considerable variation between individuals in both sleep timing and propensity. Furthermore, marked abnormalities in sleep are commonly encountered in psychiatric and neurodegenerative disorders. Teasing apart the genetic versus environmental contributions to normal and abnormal sleep is complex. Here we attempt to summarise what recent progress has been made, and what will be needed in the future to gain a more complete understanding of this fundamental aspect of physiology.


Multiple genomic signatures of selection in goats and sheep indigenous to a hot arid environment.
Goats and sheep are versatile domesticates that have been integrated into diverse environments and production systems. Natural and artificial selection have shaped the variation in the two species, but natural selection has played the major role among indigenous flocks. To investigate signals of natural selection, we analyzed genotype data generated using the caprine and ovine 50K SNP BeadChips from Barki goats and sheep that are indigenous to a hot arid environment in Egypt's Coastal Zone of the Western Desert. We identify several candidate regions under selection that spanned 119 genes. A majority of the genes were involved in multiple signaling and signal transduction pathways in a wide variety of cellular and biochemical processes. In particular, selection signatures spanning several genes that directly or indirectly influenced traits for adaptation to hot arid environments, such as thermo-tolerance (melanogenesis) (FGF2, GNAI3, PLCB1), body size and development (BMP2, BMP4, GJA3, GJB2), energy and digestive metabolism (MYH, TRHDE, ALDH1A3), and nervous and autoimmune response (GRIA1, IL2, IL7, IL21, IL1R1) were identified. We also identified eight common candidate genes under selection in the two species and a shared selection signature that spanned a conserved syntenic segment to bovine chromosome 12 on caprine and ovine chromosomes 12 and 10, respectively, providing, most likely, the evidence for selection in a common environment in two different but closely related species. Our study highlights the importance of indigenous livestock as model organisms for investigating selection sweeps and genome-wide association mapping.


Web-based physiotherapy for people with axial spondyloarthritis (WEBPASS) - a study protocol.
Evidence suggests people with axial spondyloarthritis (axial SpA) should exercise up to five times per week but lack of time, symptoms, cost and distance are barriers to regular exercise in axial SpA. Personalised exercise programmes delivered via the internet might support people with axial SpA to reach these exercise targets. The aim of this study is to investigate the effect of, and adherence to, a 12 month personalised web-based physiotherapy programme for people with axial SpA. Fifty people with axial SpA will be recruited to this prospective, interventional cohort study. Each participant will be assessed by a physiotherapist and an individualised exercise programme set up on www.webbasedphysio.com . Participants will be asked to complete their programme five times per week for 12 months. With the exception of adherence, data will be collected at baseline, 6 and 12 months. The primary outcome measure is adherence to the exercise programme over each four week cycle (20 sessions maximum per cycle) and over the 12 months. Secondary measures include function (BASFI), disease activity (BASDAI), work impairment (WPAI:SpA), quality of life (ASQoL, EQ5D), attitude to exercise (EMI-2, EAQ), spinal mobility (BASMI), physical activity and the six minute walk test. Participants will also be interviewed to explore their adherence, or otherwise, to the intervention. This study will determine the adherence and key clinical outcomes of a targeted web-based physiotherapy programme for axial SpA. This data will inform clinical practice and the development and implementation of similar programmes. ClinicalTrials.gov: NCT02666313 , 20th January 2016.


Exocytosis in peptidergic nerve terminals exhibits two calcium-sensitive phases during pulsatile calcium entry.
The link between electrical activity, Ca2+ entry through voltage-gated channels, and transmitter or hormone secretion is a central issue in neurobiology. In peptidergic nerve terminals of the mammalian neurohypophysis (NHP), secretion is elicited by patterned bursts of action potentials (APs). All parameters of the bursts are important to elicit efficient secretion, including AP frequency, AP broadening, burst duration, and interburst interval (Leng, 1988). We have studied Ca(2+)-secretion coupling of peptide-containing large dense-core vesicles (LDCV) in isolated rat NHP terminals. Ca2+ influx through voltage-gated Ca2+ channels was elicited and recorded by the whole-cell patch-clamp technique. Exocytosis was monitored on line with high temporal resolution by the capacitance detection technique (Neher and Marty, 1982). AP bursts were simulated by depolarizing pulse trains that mimic pulsatile submembrane Ca2+ elevations predicted for physiological stimuli. The characteristic capacitance response (delta Cm) to a train of depolarizing pulses was triphasic. It consisted of a threshold phase during which early pulses did not elicit secretion, a subsequent secretory phase during which Cm increases were coupled to depolarizing pulses, and a fatigued or inactivated state during which additional Ca2+ entry was ineffective. Both the threshold phase and secretory phase were correlated with the integrals of Ca2+ current. Ca2+ chelators affect both the threshold and secretory phase at submillimolar concentrations. Thus, a "shell" rather than "microdomain" model of Ca2+ elevation is appropriate for analyzing Ca(2+)-secretion coupling in NHP terminals (Nowycky and Pinter, 1993). We propose a two-step model, with a ca(2+)-dependent preparatory step followed by a final exocytotic step that is coupled to active Ca2+ influx. The results suggest that under physiological conditions, APs early in a burst prepare an NHP terminal for secretion, but later APs actually trigger exocytosis. Since NHP terminals do not possess a readily releasable pool of vesicles that require only a single Ca2+ step for exocytosis as seen in chromaffin cells (Neher and Zucker, 1993) and melanotrophs (Thomas et al, 1993a), Ca(2+)-secretion coupling mechanisms may be heterologous even within a single class of vesicles.


The prevalence of DSM-III-R diagnoses in a national sample of Dutch adolescents.
We estimated the 6-month prevalence of psychiatric disorders among Dutch adolescents, using standardized, internationally available, and replicable assessment procedures, and assessed sex differences and comorbidity of diagnoses. In phase 1, the parent, self-report, and teacher versions of the Child Behavior Checklist screened a sample representative of 13- to 18-year-olds from the Dutch general population. In phase 2, the parent (P) and child (C) versions of the Diagnostic Interview Schedule for Children (DISC) provided DSM-III-R diagnoses for a selected subsample of 780 subjects. The prevalence of any disorder was 21.5% for the DISC-C and 21.8% for the DISC-P. There was little overlap between subjects identified as having a disorder by the DISC-P and the DISC-C; only 4% met the criteria for any disorder on both. The most common disorders were simple phobia, social phobia, and conduct disorder. The most frequent comorbid diagnoses were anxiety and mood disorders. Although prevalences of more than 21% for DISC-C- and DISC-P-derived diagnoses seem high, many adolescents with DSM-III-R diagnoses functioned quite well. The prevalence of any DSM-III-R diagnosis based on the DISC-C or DISC-P, in combination with the criterion for a definite case, was 7.9%.


Association of lipoprotein(a), insulin resistance, and reproductive hormones in a multiethnic cohort of pre- and perimenopausal women (The SWAN Study).
Because evidence suggests lipoprotein (Lp(a)) may be an important cardiovascular risk factor in women, we evaluated whether reproductive hormones may influence Lp(a) concentrations and insulin resistance in a large multicenter study of women transitioning to postmenopause. Data were taken from the Study of Women's Health Across the Nation (SWAN), a prospective multiethnic study of menopausal transition (1,368 Caucasians, 808 African-Americans, 220 Chinese, 216 Hispanic, and 251 Japanese). Blood was assayed for Lp(a), follicle stimulating hormone, estradiol (E2), testosterone, and sex hormone binding globulin (free estradiol index and free androgen index), thyroid stimulating hormone, and glucose and insulin (to calculate insulin resistance). African-American women had twofold higher mean Lp(a) values than women of the other 4 race/ethnic groups, adjusted for body mass index (BMI). Lp(a) was modestly correlated with E2 (r = 0.10) in women without self-reported diagnosed heart disease but not in women with self-reported heart disease. Lp(a) was positively associated with log insulin resistance in women without self-reported heart disease but not in women with self-reported heart disease, an association that was not significant after adjusting for ethnicity, BMI, smoking, age, and site. Race/ethnicity, particularly being African-American, accounted for most of the explained Lp(a) variations. Lp(a) was very modestly associated (r = -0.04) with insulin resistance after adjusting for ethnicity and BMI, and this association was not modified by reproductive hormones, including androgens.


Structural and biochemical characterization of the bilin lyase CpcS from Thermosynechococcus elongatus.
Cyanobacterial phycobiliproteins have evolved to capture light energy over most of the visible spectrum due to their bilin chromophores, which are linear tetrapyrroles that have been covalently attached by enzymes called bilin lyases. We report here the crystal structure of a bilin lyase of the CpcS family from Thermosynechococcus elongatus (TeCpcS-III). TeCpcS-III is a 10-stranded β barrel with two alpha helices and belongs to the lipocalin structural family. TeCpcS-III catalyzes both cognate as well as noncognate bilin attachment to a variety of phycobiliprotein subunits. TeCpcS-III ligates phycocyanobilin, phycoerythrobilin, and phytochromobilin to the alpha and beta subunits of allophycocyanin and to the beta subunit of phycocyanin at the Cys82-equivalent position in all cases. The active form of TeCpcS-III is a dimer, which is consistent with the structure observed in the crystal. With the use of the UnaG protein and its association with bilirubin as a guide, a model for the association between the native substrate, phycocyanobilin, and TeCpcS was produced.


Capturing intraoperative process deviations using a direct observational approach: the glitch method.
To develop a sensitive, reliable tool for enumerating and evaluating technical process imperfections during surgical operations. Prospective cohort study with direct observation. Operating theatres on five sites in three National Health Service Trusts. Staff taking part in elective and emergency surgical procedures in orthopaedics, trauma, vascular and plastic surgery; including anaesthetists, surgeons, nurses and operating department practitioners. Reliability and validity of the glitch count method; frequency, type, temporal pattern and rate of glitches in relation to site and surgical specialty. The glitch count has construct and face validity, and category agreement between observers is good (κ=0.7). Redundancy between pairs of observers significantly improves the sensitivity over a single observation. In total, 429 operations were observed and 5742 glitches were recorded (mean 14 per operation, range 0-83). Specialty-specific glitch rates varied from 6.9 to 8.3/h of operating (ns). The distribution of glitch categories was strikingly similar across specialties, with distractions the commonest type in all cases. The difference in glitch rate between specialty teams operating at different sites was larger than that between specialties (range 6.3-10.5/h, p<0.001). Forty per cent of glitches occurred in the first quarter of an operation, and only 10% occurred in the final quarter. The glitch method allows collection of a rich dataset suitable for analysing the changes following interventions to improve process safety, and appears reliable and sensitive. Glitches occur more frequently in the early stages of an operation. Hospital environment, culture and work systems may influence the operative process more strongly than the specialty.


Consumer Perspectives on Gambling Harm Minimisation Measures in an Australian Jurisdiction.
This paper investigates consumer perspectives of implemented and proposed gambling harm minimisation measures taken from a geographically stratified survey of adult residents in Tasmania, Australia. Electronic gaming machine (EGM) gamblers were asked whether current and proposed EGM harm minimisation measures impacted on their actual or anticipated gambling expenditure and enjoyment. Participants were analysed based on their endorsement of Problem Gambling Severity Index criteria (scores 0-27), and categorised as non-problem gamblers (score 0), low-risk gamblers (scores 1-2), and moderate/problem gamblers (scores 3+). Specifically, we wanted to identify harm minimisation policies that resulted in the lowest decreases in enjoyment for non-problem gamblers and the highest decreases in expenditure for moderate/problem gamblers. Regarding current policies, the lowest decrease in enjoyment for non-problem gamblers was the ban on Automatic Teller Machines (ATMs) in EGM venues (0.2 %) while the highest decrease in expenditure for moderate/problem gamblers was the reduction in maximum lines (46.9 %). For the proposed measures, the lowest decrease in enjoyment for non-problem gamblers was visible clocks (1.2 %) while the highest decrease in expenditure for moderate/problem gamblers was reducing cash withdrawals (36.3 %). These results suggest universal EGM harm minimisation measures can differentially target non-problem and moderate/problem gamblers.


The PD-L1- and IL6-mediated dampening of the IL27/STAT1 anticancer responses are prevented by α-PD-L1 or α-IL6 antibodies.
Interleukin-27 (IL27) is a type-I cytokine of the IL6/IL12 family and is predominantly secreted by activated macrophages and dendritic cells. We show that IL27 induces STAT factor phosphorylation in cancerous cell lines of different tissue origin. IL27 leads to STAT1 phosphorylation and recapitulates an IFN-γ-like response in the microarray analyses, with up-regulation of genes involved in antiviral defense, antigen presentation, and immune suppression. Like IFN-γ, IL27 leads to an up-regulation of TAP2 and MHC-I proteins, which mediate increased tumor immune clearance. However, both cytokines also upregulate proteins such as PD-L1 (CD274) and IDO-1, which are associated with immune escape of cancer. Interestingly, differential expression of these genes was observed within the different cell lines and when comparing IL27 to IFN-γ. In coculture experiments of hepatocellular carcinoma (HCC) cells with peripheral blood mononuclear cells, pre-treatment of the HCC cells with IL27 resulted in lowered IL2 production by anti-CD3/-CD28 activated T-lymphocytes. Addition of anti-PD-L1 antibody, however, restored IL2 secretion. The levels of other TH 1 cytokines were also enhanced or restored upon administration of anti-PD-L1. In addition, we show that the suppression of IL27 signaling by IL6-type cytokine pre-stimulation-mimicking a situation occurring, for example, in IL6-secreting tumors or in tumor inflammation-induced cachexia-can be antagonized by antibodies against IL6-type cytokines or their receptors. Therapeutically, the antitumor effects of IL27 (mediated, e.g., by increased antigen presentation) might thus be increased by combining IL27 with blocking antibodies against PD-L1 or/and IL6-type cytokines.


Value-based differential pricing: efficient prices for drugs in a global context.
This paper analyzes pharmaceutical pricing between and within countries to achieve second-best static and dynamic efficiency. We distinguish countries with and without universal insurance, because insurance undermines patients' price sensitivity, potentially leading to prices above second-best efficient levels. In countries with universal insurance, if each payer unilaterally sets an incremental cost-effectiveness ratio (ICER) threshold based on its citizens' willingness-to-pay for health; manufacturers price to that ICER threshold; and payers limit reimbursement to patients for whom a drug is cost-effective at that price and ICER, then the resulting price levels and use within each country and price differentials across countries are roughly consistent with second-best static and dynamic efficiency. These value-based prices are expected to differ cross-nationally with per capita income and be broadly consistent with Ramsey optimal prices. Countries without comprehensive insurance avoid its distorting effects on prices but also lack financial protection and affordability for the poor. Improving pricing efficiency in these self-pay countries includes improving regulation and consumer information about product quality and enabling firms to price discriminate within and between countries.


Thiourea derivatives as chelating agents for bioconjugation of rhenium and technetium.
Potential tetradentate thiocarbamoylbenzamidine derivatives H4L have been synthesized from the corresponding benzimidoyl chlorides and triglycine. They are suitable chelating agents for the oxidotechnetium(v) and oxidorhenium(v) cores and form stable, neutral [MO(HL)] complexes with an equatorial SN3 coordination sphere and an additional, uncoordinated carboxylic group, which can be used for bioconjugation. Representatives of the rhenium and 99Tc products have been isolated and analyzed with spectroscopic methods and X-ray diffraction. Bioconjugates of these complexes with angiotensin-II have been synthesized and structurally characterized. Analogous 99mTc complexes have been produced and tested in vitro and in vivo. The experiments confirm a considerable stability for the [99mTc(HL)] product as well as for its bioconjugate and recommend this class of compounds for further bioconjugation studies towards clinical applications.


Low enzymatic activity haplotypes of the human catechol-O-methyltransferase gene: enrichment for marker SNPs.
Catechol-O-methyltransferase (COMT) is an enzyme that plays a key role in the modulation of catechol-dependent functions such as cognition, cardiovascular function, and pain processing. Three common haplotypes of the human COMT gene, divergent in two synonymous and one nonsynonymous (val(158)met) position, designated as low (LPS), average (APS), and high pain sensitive (HPS), are associated with experimental pain sensitivity and risk of developing chronic musculoskeletal pain conditions. APS and HPS haplotypes produce significant functional effects, coding for 3- and 20-fold reductions in COMT enzymatic activity, respectively. In the present study, we investigated whether additional minor single nucleotide polymorphisms (SNPs), accruing in 1 to 5% of the population, situated in the COMT transcript region contribute to haplotype-dependent enzymatic activity. Computer analysis of COMT ESTs showed that one synonymous minor SNP (rs769224) is linked to the APS haplotype and three minor SNPs (two synonymous: rs6267, rs740602 and one nonsynonymous: rs8192488) are linked to the HPS haplotype. Results from in silico and in vitro experiments revealed that inclusion of allelic variants of these minor SNPs in APS or HPS haplotypes did not modify COMT function at the level of mRNA folding, RNA transcription, protein translation, or enzymatic activity. These data suggest that neutral variants are carried with APS and HPS haplotypes, while the high activity LPS haplotype displays less linked variation. Thus, both minor synonymous and nonsynonymous SNPs in the coding region are markers of functional APS and HPS haplotypes rather than independent contributors to COMT activity.


Use of digitalis glycosides to identify the mechanisms of amantadine transport by renal tubules.
The mechanism(s) for uptake of organic cations by renal cortical tubules was (were) examined further. Renal cortical tubules were purified from rat kidneys by a Percoll gradient centrifugation technique. Bicarbonate buffer (Krebs-Henseleit, KHS) conditions were altered, and chemical modulators were used which affect the activity of the basolateral Na+/K+-ATPase. Renal tubule uptake of the achiral organic cation amantadine was determined. The cardiac glycosides digoxin and acetylstrophanthidin and ouabagenin did not alter amantadine uptake by either proximal or distal tubule fragments in KHS. However, ouabain inhibited proximal tubule amantadine uptake in a dose-dependent manner with lower potency than distal tubule amantadine uptake in KHS. Ouabain did not inhibit amantadine tubule uptake in phosphate buffer. However, inhibition of amantadine uptake by ouabain returned in a time-dependent manner upon addition of bicarbonate to the phosphate buffer. Low extracellular sodium or potassium did not alter amantadine uptake by proximal tubules. Hypokalemic and hypokalemic/ hyponatremic conditions decreased the inhibitory potency of ouabain for amantadine uptake by proximal tubules. For distal tubules, both hyponatremic and hypokalemic conditions, alone and together, decreased the inhibitory potency of ouabain, but did not affect amantadine uptake in the absence of ouabain. Hypochloremic conditions decreased affinity for amantadine uptake by distal, but not proximal tubules. No change in maximal transport capacity for amantadine uptake was observed under hypochloremic conditions for either tubule fragment. These studies challenge the widely accepted concept of Na+/ K+-adenosine triphosphatase activity and maintenance of the basolateral membrane potential as rate-limiting steps for the energy-dependent renal tubule uptake of organic cations. Furthermore, these studies suggest a mechanism for ouabain inhibition of organic cation renal tubule uptake that may not involve the Na+/K+-adenosine triphosphatase and may be possibly bicarbonate-dependent.


Ultrasound-mediated delivery and distribution of polymeric nanoparticles in the normal brain parenchyma of a metastatic brain tumour model.
The treatment of brain diseases is hindered by the blood-brain barrier (BBB) preventing most drugs from entering the brain. Focused ultrasound (FUS) with microbubbles can open the BBB safely and reversibly. Systemic drug injection might induce toxicity, but encapsulation into nanoparticles reduces accumulation in normal tissue. Here we used a novel platform based on poly(2-ethyl-butyl cyanoacrylate) nanoparticle-stabilized microbubbles to permeabilize the BBB in a melanoma brain metastasis model. With a dual-frequency ultrasound transducer generating FUS at 1.1 MHz and 7.8 MHz, we opened the BBB using nanoparticle-microbubbles and low-frequency FUS, and applied high-frequency FUS to generate acoustic radiation force and push nanoparticles through the extracellular matrix. Using confocal microscopy and image analysis, we quantified nanoparticle extravasation and distribution in the brain parenchyma. We also evaluated haemorrhage, as well as the expression of P-glycoprotein, a key BBB component. FUS and microbubbles distributed nanoparticles in the brain parenchyma, and the distribution depended on the extent of BBB opening. The results from acoustic radiation force were not conclusive, but in a few animals some effect could be detected. P-glycoprotein was not significantly altered immediately after sonication. In summary, FUS with our nanoparticle-stabilized microbubbles can achieve accumulation and displacement of nanoparticles in the brain parenchyma.


Development and clinical validation of the Genedrive point-of-care test for qualitative detection of hepatitis C virus.
Recently approved direct acting antivirals provide transformative therapies for chronic hepatitis C virus (HCV) infection. The major clinical challenge remains to identify the undiagnosed patients worldwide, many of whom live in low-income and middle-income countries, where access to nucleic acid testing remains limited. The aim of this study was to develop and validate a point-of-care (PoC) assay for the qualitative detection of HCV RNA. We developed a PoC assay for the qualitative detection of HCV RNA on the PCR Genedrive instrument. We validated the Genedrive HCV assay through a case-control study comparing results with those obtained with the Abbott RealTime HCV test. The PoC assay identified all major HCV genotypes, with a limit of detection of 2362 IU/mL (95% CI 1966 to 2788). Using 422 patients chronically infected with HCV and 503 controls negative for anti-HCV and HCV RNA, the Genedrive HCV assay showed 98.6% sensitivity (95% CI 96.9% to 99.5%) and 100% specificity (95% CI 99.3% to 100%) to detect HCV. In addition, melting peak ratiometric analysis demonstrated proof-of-principle for semiquantification of HCV. The test was further validated in a real clinical setting in a resource-limited country. We report a rapid, simple, portable and accurate PoC molecular test for HCV, with sensitivity and specificity that fulfils the recent FIND/WHO Target Product Profile for HCV decentralised testing in low-income and middle-income countries. This Genedrive HCV assay may positively impact the continuum of HCV care from screening to cure by supporting real-time treatment decisions. NCT02992184 .


Individual and Work Factors Associated with Psychosocial Health of Registered Nurses During the Covid-19 Pandemic: A Mixed Methods Study.
To describe the initial influence of the Covid-19 pandemic on U.S. nurses' psychosocial health, and to identify factors associated with poor psychosocial health outcomes. We conducted a convergent (QUAN+qual) mixed methods study. From June to August 2020, we administered surveys (N = 629) and conducted semi-structured interviews (N = 34) among nurses working across healthcare settings in 18 states. We developed separate multivariable logistic regression models for three psychosocial outcomes (anxiety, depressive symptoms, insomnia). We used content analysis to process and analyze qualitative data, and integrated results in the final analysis step. Nurses reported high rates of depressive symptoms (22%), anxiety (52%), and insomnia (55%). Disturbances to sleep were both a contributing factor to, and an outcome of, poor psychosocial health. Evidence-based interventions addressing work stress and sleep, and proactive monitoring of nurses' psychosocial health by employers are urgently needed.


Rhizobium meliloti elicits transient expression of the early nodulin gene ENOD12 in the differentiating root epidermis of transgenic alfalfa.
To study the molecular responses of the host legume during early stages of the symbiotic interaction with Rhizobium, we have cloned and characterized the infection-related early nodulin gene MtENOD12 from Medicago truncatula. In situ hybridization experiments have shown that, within the indeterminate Medicago nodule, transcription of the MtENOD12 gene begins in cell layers of meristematic origin that lie ahead of the infection zone, suggesting that these cells are undergoing preparation for bacterial infection. Histochemical analysis of transgenic alfalfa plants that express an MtENOD12 promoter-beta-glucuronidase gene fusion has confirmed this result and further revealed that MtENOD12 gene transcription occurs as early as 3 to 6 hr following inoculation with R. meliloti in a zone of differentiating root epidermal cells which lies close to the growing root tip. It is likely that this transient, nodulation (nod) gene-dependent activation of the ENOD12 gene also corresponds to the preparation of the plant for bacterial infection. We anticipate that this extremely precocious response to Rhizobium will provide a valuable molecular marker for studying early signal exchange between the two symbiotic organisms.


Risk factors for statin-associated rhabdomyolysis.
To identify and characterize risk factors for rhabdomyolysis in patients prescribed statin monotherapy or statin plus fibrate therapy. A nested case-control study was conducted within a cohort of 252,460 new users of lipid-lowering medications across 11 geographically dispersed U.S. health plans. Twenty-one cases of rhabdomyolysis confirmed by medical record review were compared to 200 individually matched controls without rhabdomyolysis. A conditional logistic regression model was applied to evaluate the effects of age, gender, comorbidities, concurrent medication use, dosage, and duration of statin use on the development of rhabdomyolysis. Statin users 65 years of age and older have four times the risk of hospitalization for rhabdomyolysis than those under age 65 (odds ratio (OR) = 4.36, 95% confidence interval (CI): 1.5,14.1). We also observed a joint effect of high statin dosage and renal disease (p = 0.022). When these two variables were added to the model with age, we obtained an OR of 5.73 for dosage (95%CI: 0.63, 52.6) and 6.26 for renal disease (95%CI: 0.46, 63.38). Although not statistically significant, we did observe a greater than twofold increase in risk for rhabdomyolysis among females (OR = 2.53, 95%CI: 0.91, 7.32). Findings of this study indicate that older age is a risk factor for rhabdomyolysis among statin users. Although the evidence is not as strong, high statin dosage, renal disease, and female gender may be additional risk factors. Patients at higher risk of developing rhabdomyolysis should be closely monitored for signs and symptoms of the disease.


Functional analyses of variants reveal a significant role for dominant negative and common alleles in oligogenic Bardet-Biedl syndrome.
Technological advances hold the promise of rapidly catalyzing the discovery of pathogenic variants for genetic disease. However, this possibility is tempered by limitations in interpreting the functional consequences of genetic variation at candidate loci. Here, we present a systematic approach, grounded on physiologically relevant assays, to evaluate the mutational content (125 alleles) of the 14 genes associated with Bardet-Biedl syndrome (BBS). A combination of in vivo assays with subsequent in vitro validation suggests that a significant fraction of BBS-associated mutations have a dominant-negative mode of action. Moreover, we find that a subset of common alleles, previously considered to be benign, are, in fact, detrimental to protein function and can interact with strong rare alleles to modulate disease presentation. These data represent a comprehensive evaluation of genetic load in a multilocus disease. Importantly, superimposition of these results to human genetics data suggests a previously underappreciated complexity in disease architecture that might be shared among diverse clinical phenotypes.


Elevation of CpG frequencies in influenza A genome attenuates pathogenicity but enhances host response to infection.
Previously, we demonstrated that frequencies of CpG and UpA dinucleotides profoundly influence the replication ability of echovirus 7 (Tulloch et al., 2014). Here, we show that that influenza A virus (IAV) with maximised frequencies of these dinucleotides in segment 5 showed comparable attenuation in cell culture compared to unmodified virus and a permuted control (CDLR). Attenuation was also manifested in vivo, with 10-100 fold reduced viral loads in lungs of mice infected with 200PFU of CpG-high and UpA-high mutants. However, both induced powerful inflammatory cytokine and adaptive (T cell and neutralising antibody) responses disproportionate to their replication. CpG-high infected mice also showed markedly reduced clinical severity, minimal weight loss and reduced immmunopathology in lung, yet sterilising immunity to lethal dose WT challenge was achieved after low dose (20PFU) pre-immunisation with this mutant. Increasing CpG dinucleotide frequencies represents a generic and potentially highly effective method for generating safe, highly immunoreactive vaccines.


Heparin promotes the rapid fibrillization of a peptide with low intrinsic amyloidogenicity.
Amyloid deposits in vivo are complex mixtures composed of protein fibrils and nonfibrillar components, including polysaccharides of the glycosaminoglycan (GAG) class. It has been widely documented that GAGs influence the initiation and progress of self-assembly by several disease-associated amyloidogenic proteins and peptides in vitro. Here we investigated whether the GAG heparin can serve as a cofactor to induce amyloid-like fibril formation in a peptide predicted to have a weak propensity to aggregate and not associated with amyloid disorders. We selected the 23-residue peptide PLB(1-23), which corresponds to the acetylated cytoplasmic domain of the phospholamban transmembrane protein. PLB(1-23) remains unfolded in aqueous solution for >24 h and does not bind thioflavin T over this time period, in agreement with computer predictions that the peptide has a very low intrinsic amyloidogenicity. In the presence of low-molecular mass (5 kDa) heparin, which binds PLB(1-23) with micromolar affinity, the peptide undergoes spontaneous and rapid assembly into amyloid-like fibrils, the effect being more pronounced at pH 5.5 than at pH 7.4. At the lower pH, peptide aggregation is accompanied by a transition to a β-sheet rich structure. These results are consistent with the polyanionic heparin serving as a scaffold to enhance aggregation by aligning the peptide molecules in the correct orientation and with the appropriate periodicity. PLB(1-23) is toxic to cells when added in isolation, and promotion of fibril formation by heparin can reduce the toxicity of this peptide, consistent with the notion that amyloid-like fibrils represent a benign end stage of fibrillization. This work provides insight into the role that heparin and other glycosaminoglycans may play in amyloid formation and provides therapeutic avenues targeting the reduction of cytotoxicity of species along the amyloid formation pathway.


Visible-light-driven water oxidation with nanoscale Co(3)O(4) : new optimization strategies.
Water oxidation is the "bottleneck" of artificial photosynthesis on the way to clean and sustainable solar fuels production. Although spinel-type Co3 O4 keeps attracting considerable research interest as a robust and low-cost water oxidation catalyst (WOC), full control over its manifold performance parameters remains a preparative and analytical challenge. The present study screens a wide spectrum of influential factors (such as surface area and adsorption processes, morphology, crystallinity, and cobalt oxidation states) with respect to straightforward WOC optimization strategies. Tuning of crystallinity and cobalt valence states emerges as a major guideline for spinel catalyst synthesis, thus adding a key complementary factor to the main design paradigm of surface area maximization. In contrast to the prevailing amorphous characteristics of electrocatalytic WOCs, crystallinity was found to be crucial for photochemical WOC development. The interplay of synthetic history, crystallinity, and cobalt valence states in hydrothermally synthesized Co3 O4 WOCs was investigated with a variety of analytical techniques, including electron paramagnetic resonance (EPR), X-ray photoelectron spectroscopy (XPS), and various electron microscopy and spectroscopic techniques. Complementary "top down" and "bottom up" optimization strategies introduce new Co3 O4 WOC design parameters for facile catalyst production.


Improving the reliability of the U.S. vaccine supply: an evaluation of alternatives.
Since taking office, President Clinton has devoted considerable attention to childhood immunization and to the overall U.S. policy toward vaccine development, delivery, and pricing. But the reliability of U.S. vaccine supplies has received far less attention, despite several recent interruptions in vaccine production and supply. Moreover, the increasing producer concentration in the U.S. and global vaccine industries raises the possibility that more and more important vaccines will be produced by a single firm, often in a single production facility. Is a federal policy for vaccine supply assurance necessary, and what form should it take? Are sole-source suppliers a weak link in the U.S. vaccine supply chain? Are the recommendations of the Institute of Medicine's Committee on Emerging Infections for a publicly owned "standby production capacity" of critical vaccines feasible or cost-effective? We consider these and other issues in our discussion of the U.S. vaccine industry, the potential role of foreign vaccine suppliers, and the use of existing federal facilities and policies to address a possible interruption in critical vaccine supplies.


IgG subclass distribution in serum and rectal mucosa of monozygotic twins with or without inflammatory bowel disease.
Serum samples from 26 monozygotic twin pairs concordant or discordant with regard to inflammatory bowel disease, and rectal biopsies from 42 twins of the same subject group, were examined for IgG subclasses. They were all compared with normal controls. Almost all affected twins were in clinical remission. Paired immunofluorescence staining of the rectal mucosa showed that those with ulcerative colitis had a significantly higher (p < 0.01) proportion of IgG1 producing mucosal immunocytes than normal controls (78.1% v 55.9%). Conversely, the IgG2 cell fraction was significantly reduced (15.9% v 34.6%). Healthy twins from ulcerative colitis pairs tended to show a raised proportion of IgG1 cells and the IgG2 cell fraction was significantly reduced (p < 0.05). In discordant ulcerative colitis twin pairs, no difference appeared in the cellular IgG subclass pattern between healthy and affected twins. Furthermore, the proportion of IgG1 in these healthy and diseased twins showed good correlation (T = 0.867). The results in rectal mucosa of twins with Crohn's disease were widely scattered and affected twins did not differ significantly from normal controls. Healthy twins, however, showed a marginally raised IgG1 cell proportion, but no correlation was seen between the IgG subclass fractions in discordant Crohn's disease twin pairs. The serum concentrations of IgG1 and IgG2 did not differ from normal controls in twins of either category. These results suggested that in ulcerative colitis, the aberrant mucosal production of IgG1 and IgG2 does not depend on active disease, but is apparently at least partially explained by a genetic impact. Conversely, the mucosal IgG subclass pattern in Crohn's disease appears to be determined mainly by exogenous variables.


Comparison the prognostic value of galectin-3 and serum markers of cardiac extracellular matrix turnover in patients with chronic systolic heart failure.
Galectin-3 (Gal-3) shows the ability of survival prediction in heart failure (HF) patients. However, Gal-3 is strongly associated with serum markers of cardiac extracellular matrix (ECM) turnover. The aim of this study is to compare the impact of Gal-3 and serum markers of cardiac ECM turnover on prognostic prediction of chronic systolic HF patients. Serum Gal-3, brain natriuretic peptide (BNP), extracellular matrix including type I and III aminoterminal propeptide of procollagen (PINP and PIIINP), matrix metalloproteinase-2, 9 (MMP-2, 9), and tissue inhibitor of metalloproteinase-1 (TIMP-1) were analyzed. Cox regression analysis was used for survival analysis. A total of 105 (81 male) patients were enrolled. During 980±346 days follow-up, 17 patients died and 36 episodes of HF admission happened. Mortality of these patients was significantly associated with the log PIIINP (β= 15.380; P=0.042), log TIMP-1(β= 44.530; P=0.003), log MMP-2 (β= 554.336; P<0.001), log BNP (β= 28.273; P=0.034). Log Gal-3 (β= 7.484; P=0.066) is borderline associated with mortality. Mortality or first HF admission of these patients was significantly associated with the log TIMP-1(β= 16.496; P=0.006), log MMP-2 (β= 221.864; P<0.001), log BNP (β= 5.999; P=0.034). Log Gal-3 (β= 4.486; P=0.095) only showed borderline significance. In several models adjusting clinical parameters, log MMP-2 was significantly associated with clinical outcome. In contrast, log Gal-3 was not. The prognostic strength of MMP-2 to clinical outcome prediction in HF patients is stronger than Gal-3.


Development of a PBPK model for monoclonal antibodies and simulation of human and mice PBPK of a radiolabelled monoclonal antibody.
Physiology based pharmacokinetic (PBPK) modeling and simulation is a useful method for prediction of biodistribution of both macromolecules and small molecules. It can enhance our understanding of the underlying mechanisms of biodistribution and hence may help in rational design of macromolecules used as diagnostic and therapeutic agents. In this review we discuss PBPK modeling and simulation of a radiolabelled Monoclonal Antibody ((111)In-DOTA-hAFP31 IgG) ("MAB") in mice without tumor and in a human with tumor. This study is part of Xemet Co.'s effort to develop a more accurate and reliable PBPK model and simulation platform, which is applicable both for small molecules and macromolecules. The simulated results were fitted to experimental time series data by varying parameters which were not fixed a priori. It was demonstrated that the PBPK model describes the main features of the pharmacokinetics of the studied systems. It was also shown that simulation can be used for evaluating the parameters of the system and scaling up the pharmacokinetics of MAB from mice to man. We identified several areas of improvement and further development needed to improve the accuracy of PBPK simulation for MAB and other macromolecules. It was concluded that the transvascular permeabilities are the most important parameters and more research is needed to enable prediction of permeabilities from molecular characteristics of macromolecules. It would also be necessary to understand better and describe with a more detailed model the microstructure of the tumor and to measure or predict the antigen concentration in tumor. Non-specific, non-saturable binding in other organs/tissues should be understood better and the kinetic constants of the binding should be measured experimentally. Although the metabolism and clearance were neglected in this study they need to be included in more detailed studies. Also the intracellular trafficking of macromolecules, which was not included in this study, shall be included in the more accurate models.


Simultaneous measurement of retardance and fast axis angle of a quarter-wave plate using one photoelastic modulator.
A method for simultaneous measurement of the retardance and the fast axis angle of quarter-wave plate using one photoelastic modulator is presented. A laser beam passes through a polarizer, a photoelastic modulator, the quarter-wave plate to be measured, and an analyzer to be detected. Before and after the quarter-wave plate is rotated 45° at any initial fast axis direction, two detection signals are obtained to resolve simultaneously the retardance and the fast axis angle. In experiments, a quarter-wave plate was measured with fast axis angles from -89° to 90°. The average and the standard deviation of the retardances at different fast axis directions are respectively 89.50° and 0.17°. The maximum measurement deviation of the fast axis angle is 0.5°. The usefulness of the method is verified.


Selective GPER activation decreases proliferation and activates apoptosis in tumor Leydig cells.
We have previously shown that estrogens binding to estrogen receptor (ER) α increase proliferation of Leydig tumor cells. Estrogens can also bind to G protein-coupled ER (GPER) and activation of this receptor can either increase or decrease cell proliferation of several tumor types. The aim of this study was to investigate GPER expression in R2C rat tumor Leydig cells, evaluate effects of its activation on Leydig tumor cell proliferation and define the molecular mechanisms triggered in response to its activation. R2C cells express GPER and its activation, using the specific ligand G-1, is associated with decreased cell proliferation and initiation of apoptosis. Apoptosis after G-1 treatment was asserted by appearance of DNA condensation and fragmentation, decrease in Bcl-2 and increase in Bax expression, cytochrome c release, caspase and poly (ADP-ribose) polymerase-1 (PARP-1) activation. These effects were dependent on GPER activation because after silencing of the gene, using a specific small interfering RNA, cyt c release, PARP-1 activation and decrease in cell proliferation were abrogated. These events required a rapid, however, sustained extracellular regulated kinase 1/2 activation. G-1 was able to decrease the growth of R2C xenograft tumors in CD1 nude mice while increasing the number of apoptotic cells. In addition, in vivo administration of G-1 to male CD1 mice did not cause any alteration in testicular morphology, while cisplatin, the cytotoxic drug currently used for the therapy of Leydig tumors, severely damaged testicular structure, an event associated with infertility in cisplatin-treated patients. These observations indicate that GPER targeting for the therapy of Leydig cell tumor may represent a good alternative to cisplatin to preserve fertility in Leydig tumor patients.


The burden of sickness absence from musculoskeletal causes in Great Britain.
National initiatives to prevent and/or manage sickness absence require a database from which trends can be monitored. To evaluate the information provided by surveillance schemes and publicly available data sets on sickness absence nationally from musculoskeletal disorders (MSDs). A grey literature search was undertaken using the search engine Google, supplemented by leads from consultees from academia, industry, employers, lay interest groups and government. We abstracted data on the outcomes and populations covered and made quantitative estimates of MSD-related sickness absence, overall and, where distinguishable, by subdiagnosis. The coverage and limitations of each source were evaluated. Sources included the Labour Force Survey (LFS) and its Self-reported Work-related Illness survey module, the THOR-GP surveillance scheme, surveys by national and local government, surveys by employers' organizations and a database of benefit statistics. Each highlighted MSDs as a leading cause of sickness absence. Data limitations varied by source, but typically included lack of diagnostic detail and restriction of focus to selected subgroups (e.g. work-ascribed or benefit-awarded cases, specific employment sectors). Additionally, some surveys had very low response rates, were completed only by proxy respondents or ranked only the perceived importance of MSD-related sickness absence, rather than measuring it. National statistics on MSD-related sickness absence are piecemeal and incomplete. This limits capacity to plan and monitor national policies in an important area of public health. Simple low-cost additions to the LFS would improve the situation.


MO-F-BRCD-01: Stereotactic Body Radiation Therapy: Updates on Clinical, Biological, and Physics/QA:SBRT (Part 1): Biological and Clinical Updates.
Stereotactic Body Radiation Therapy (SBRT) is an important form of cancer therapy with increasingly broad application across a spectrum of tumor types in primary and metastatic settings. In this presentation the radiation biology, clinical experience from various trials, and cautionary updates on normal tissue tolerances will be presented. The effective radiobiology of SBRT and hypofractionated courses of therapy has become more evident with the increasing reports of retrospective clinical outcomes and prospective clinical trial results. Current open multi-institutional national cooperative trials will be reviewed. Accumulating clinical experiences are yielding new insights into practical aspects of tumor and normal tissue responses to high dose per fraction treatment. Indeed, SBRT has produced profound tumoricidal and ablative effects, however there is potential for grave toxicity and this demands that clinicians be knowledgeable regarding normal tissue tolerances for various hypofractionated courses. As a final note, the technology associated with SBRT has evolved remarkably in the last decade, and procedures that originally required hours to plan, with cumbersome quality assurance methods, arduous set-up times, and long protracted deliveries can now be performed in ever shorter time periods. Given these technology improvements and recognizing the great palliative potential of hypofractionated radiation therapy to relieve cancer symptoms quickly and efficiently, a new strategy to deliver SBRT in a single session called STAT RAD is presented for discussion.


Prolonged muscle vibration increases stretch reflex amplitude, motor unit discharge rate, and force fluctuations in a hand muscle.
The purpose of this study was to compare the influence of prolonged vibration of a hand muscle on the amplitude of the stretch reflex, motor unit discharge rate, and force fluctuations during steady, submaximal contractions. Thirty-two young adults performed 10 isometric contractions at a constant force (5.0 +/- 2.3% of maximal force) with the first dorsal interosseus muscle. Each contraction was held steady for 10 s, and then stretch reflexes were evoked. Subsequently, 20 subjects had vibration applied to the relaxed muscle for 30 min, and 12 subjects received no vibration. The muscle vibration induced a tonic vibration reflex. The intervention (vibration or no vibration) was followed by 2 sets of 10 constant-force contractions with applied stretches (After and Recovery trials). The mean electromyogram amplitude of the short-latency component of the stretch reflex increased by 33% during the After trials (P < 0.01) and by 38% during the Recovery trials (P < 0.01). The standard deviation of force during the steady contractions increased by 21% during the After trials (P < 0.05) and by 28% during the Recovery trials (P < 0.01). The discharge rate of motor units increased from 10.3 +/- 2.7 pulses/s (pps) before vibration to 12.2 +/- 3.1 pps (P < 0.01) during the After trials and to 11.9 +/- 2.6 pps during the Recovery trials (P < 0.01). There was no change in force fluctuations or stretch reflex magnitude for the subjects in the Control group. The results indicate that prolonged vibration increased the short-latency component of the stretch reflex, the discharge rate of motor units, and the fluctuations in force during contractions by a hand muscle. These adjustments were necessary to achieve the target force due to the vibration-induced decrease in the force capacity of the muscle.


Selective BET bromodomain inhibition as an antifungal therapeutic strategy.
Invasive fungal infections cause significant morbidity and mortality among immunocompromised individuals, posing an urgent need for new antifungal therapeutic strategies. Here we investigate a chromatin-interacting module, the bromodomain (BD) from the BET family of proteins, as a potential antifungal target in Candida albicans, a major human fungal pathogen. We show that the BET protein Bdf1 is essential in C. albicans and that mutations inactivating its two BDs result in a loss of viability in vitro and decreased virulence in mice. We report small-molecule compounds that inhibit C. albicans Bdf1 with high selectivity over human BDs. Crystal structures of the Bdf1 BDs reveal binding modes for these inhibitors that are sterically incompatible with the human BET-binding pockets. Furthermore, we report a dibenzothiazepinone compound that phenocopies the effects of a Bdf1 BD-inactivating mutation on C. albicans viability. These findings establish BET inhibition as a promising antifungal therapeutic strategy and identify Bdf1 as an antifungal drug target that can be selectively inhibited without antagonizing human BET function.


In-silico insights on the prognostic potential of immune cell infiltration patterns in the breast lobular epithelium.
Scattered inflammatory cells are commonly observed in mammary gland tissue, most likely in response to normal cell turnover by proliferation and apoptosis, or as part of immunosurveillance. In contrast, lymphocytic lobulitis (LLO) is a recurrent inflammation pattern, characterized by lymphoid cells infiltrating lobular structures, that has been associated with increased familial breast cancer risk and immune responses to clinically manifest cancer. The mechanisms and pathogenic implications related to the inflammatory microenvironment in breast tissue are still poorly understood. Currently, the definition of inflammation is mainly descriptive, not allowing a clear distinction of LLO from physiological immunological responses and its role in oncogenesis remains unclear. To gain insights into the prognostic potential of inflammation, we developed an agent-based model of immune and epithelial cell interactions in breast lobular epithelium. Physiological parameters were calibrated from breast tissue samples of women who underwent reduction mammoplasty due to orthopedic or cosmetic reasons. The model allowed to investigate the impact of menstrual cycle length and hormone status on inflammatory responses to cell turnover in the breast tissue. Our findings suggested that the immunological context, defined by the immune cell density, functional orientation and spatial distribution, contains prognostic information previously not captured by conventional diagnostic approaches.


Exogenous expression of N-cadherin in breast cancer cells induces cell migration, invasion, and metastasis.
E- and N-cadherin are calcium-dependent cell adhesion molecules that mediate cell-cell adhesion and also modulate cell migration and tumor invasiveness. The loss of E-cadherin-mediated adhesion has been shown to play an important role in the transition of epithelial tumors from a benign to an invasive state. However, recent evidence indicates that another member of the cadherin family, N-cadherin, is expressed in highly invasive tumor cell lines that lacked E-cadherin expression. These findings have raised the possibility that N-cadherin contributes to the invasive phenotype. To determine whether N-cadherin promotes invasion and metastasis, we transfected a weakly metastatic and E-cadherin-expressing breast cancer cell line, MCF-7, with N-cadherin and analyzed the effects on cell migration, invasion, and metastasis. Transfected cells expressed both E- and N-cadherin and exhibited homotypic cell adhesion from both molecules. In vitro, N-cadherin-expressing cells migrated more efficiently, showed an increased invasion of Matrigel, and adhered more efficiently to monolayers of endothelial cells. All cells produced low levels of the matrix metalloproteinase MMP-9, which was dramatically upregulated by treatment with FGF-2 only in N-cadherin-expressing cells. Migration and invasion of Matrigel were also greatly enhanced by this treatment. When injected into the mammary fat pad of nude mice, N-cadherin-expressing cells, but not control MCF-7 cells, metastasized widely to the liver, pancreas, salivary gland, omentum, lung, lymph nodes, and lumbar spinal muscle. The expression of both E- and N-cadherin was maintained both in the primary tumors and metastatic lesions. These results demonstrate that N-cadherin promotes motility, invasion, and metastasis even in the presence of the normally suppressive E-cadherin. The increase in MMP-9 production by N-cadherin-expressing cells in response to a growth factor may endow them with a greater ability to penetrate matrix protein barriers, while the increase in their adherence to endothelium may improve their ability to enter and exit the vasculature, two properties that may be responsible for metastasis of N-cadherin-expressing cells.


Coupling of diversification and pH adaptation during the evolution of terrestrial Thaumarchaeota.
The Thaumarchaeota is an abundant and ubiquitous phylum of archaea that plays a major role in the global nitrogen cycle. Previous analyses of the ammonia monooxygenase gene amoA suggest that pH is an important driver of niche specialization in these organisms. Although the ecological distribution and ecophysiology of extant Thaumarchaeota have been studied extensively, the evolutionary rise of these prokaryotes to ecological dominance in many habitats remains poorly understood. To characterize processes leading to their diversification, we investigated coevolutionary relationships between amoA, a conserved marker gene for Thaumarchaeota, and soil characteristics, by using deep sequencing and comprehensive environmental data in Bayesian comparative phylogenetics. These analyses reveal a large and rapid increase in diversification rates during early thaumarchaeotal evolution; this finding was verified by independent analyses of 16S rRNA. Our findings suggest that the entire Thaumarchaeota diversification regime was strikingly coupled to pH adaptation but less clearly correlated with several other tested environmental factors. Interestingly, the early radiation event coincided with a period of pH adaptation that enabled the terrestrial Thaumarchaeota ancestor to initially move from neutral to more acidic and alkaline conditions. In contrast to classic evolutionary models, whereby niches become rapidly filled after adaptive radiation, global diversification rates have remained stably high in Thaumarchaeota during the past 400-700 million years, suggesting an ongoing high rate of niche formation or switching for these microbes. Our study highlights the enduring importance of environmental adaptation during thaumarchaeotal evolution and, to our knowledge, is the first to link evolutionary diversification to environmental adaptation in a prokaryotic phylum.


A novel approach to analyzing concentration polarization of polysaccharide solutions.
This paper describes a novel approach to analyzing the concentration polarization (CP) in the short-time constant flux filtration of polysaccharide solutions. With application of the pearl-necklace model based polymer solution permeability correlation, a transmembrane pressure (TMP) increase rate model was developed to relate the linear TMP increase rate measured in the short-time constant flux filtration to the solid concentration at the membrane surface, the polymer critical overlapping concentration, and the radius of gyration of the polymer molecules. Establishment of such a theoretical model allows analysis of the gelation propensity of the CP layer formed by polysaccharides based on the experimentally determined linear TMP increase rate.


Augmenting clinical interpretability of thiopurine methyltransferase laboratory evaluation.
Individuals with decreased thiopurine methyltransferase (TPMT) activity are at risk of adverse effects of thiopurine administration whereas its increased activity may inactivate drugs faster. We evaluated genotype-phenotype correlations in patients with suspected hematological malignancies and inflammatory bowel disease from our region based on findings of nonlinear TPMT enzyme kinetics previously unreported. The study group comprised 267 individuals. They were screened for the most common variants of low TPMT activity. TPMT activity was measured in erythrocytes using the HPLC rate-blanked method. Thirty-three patients (12.4%) were heterozygous (26 were TPMT*1/*3A, 5 TPMT*1/*2, 2 TPMT *1/*3C) and 1 was a compound heterozygote (*2/*3A). Normal and low normal TPMT activities substantially overlapped in wild-type and heterozygous individuals, whereas high activities were found in 29 wild-type genotyped patients. Extreme and life-threatening toxicity was observed in the compound heterozygote patient. Activity measurement performed at diagnosis provides clinicians with information on immediate pharmacokinetic-related adverse events and/or hypermetabolism, and genotyping may indicate the rate of pharmacodynamic thioguanine nucleotide accumulation due to slower overall thiopurine metabolism.


Surgical reimplantation of penile glans amputation in children during circumcision.
Circumcision is one of the oldest and most commonly performed surgical procedures. Unfortunately, various complications may occur during circumcision, ranging from trivial to tragic such as penile amputation which is a serious complication and a challenging injury to treat. We describe two cases of non-microsurgical successful reattachment of a distal penile glans which were amputated during circumcision. In the first case, a 5-year-old child underwent circumcision by an urologist under local anesthesia. In the second one, a 3-year-old child underwent circumcision by a general practitioner who used to make circumcision. In this article, the literature is reviewed; results and potential complications of this surgery are also discussed. Glans sensation was present, early morning erection was maintained, and there was an erectile response during penile manipulation in both cases. Although circumcision is not technically difficult, it should be taken seriously. The use of microsurgical reattachment is not always possible, especially in pediatric cases; it also requires special equipment and training.