Isolation of crt mutants constitutive for transcription of the DNA damage inducible gene RNR3 in Saccharomyces cerevisiae.
Ribonucleotide reductase is an essential enzyme that catalyzes the rate limiting step for production of the deoxyribonucleotides required for DNA synthesis. It is encoded by three genes, RNR1, RNR2 and RNR3, each of which is inducible by agents that damage DNA or block DNA replication. To probe the signaling pathway mediating this DNA damage response, we have designed a general selection system for isolating spontaneous trans-acting mutations that alter RNR3 expression using a chromosomal RNR3-URA3 transcriptional fusion and an RNR3-lacZ reporter plasmid. Using this system, we have isolated 202 independent trans-acting crt (constitutive RNR3 transcription) mutants that express high levels of RNR3 in the absence of DNA damaging agents. Of these, 200 are recessive and fall into 9 complementation groups. In some crt groups, the expression of RNR1 and RNR2 are also elevated, suggesting that all three RNR genes share a common regulatory pathway. Mutations in most CRT genes confer additional phenotypes, among these are clumpiness, hydroxyurea sensitivity, temperature sensitivity and slow growth. Five of the CRT genes have been identified as previously cloned genes; CRT4 is TUP1, CRT5 is POL1/CDC17, CRT6 is RNR2, CRT7 is RNR1, and CRT8 is SSN6. crt6-68 and crt7-240 are the first ts alleles of RNR2 and RNR1, respectively, and arrest with a large budded, cdc terminal phenotype at the nonpermissive temperature. The isolation of crt5-262, an additional cdc allele of POL1/CDC17, suggests for the first time that directly blocking DNA replication can provide a signal to induce the DNA damage response. crt2 mutants show a defect in basal level expression of RNR1-lacZ reporter constructs. These are the first mutants isolated in yeast that alter the regulation of DNA damage inducible genes and the identification of their functions sheds light on the DNA damage sensory network.


Impact of Genetic Testing on Risk-Management Behavior of Black Breast Cancer Survivors: A Longitudinal, Observational Study.
Black women are overrepresented among premenopausal breast cancer (BC) survivors. These patients warrant genetic testing (GT) followed by risk-reducing behaviors. This study documented patterns and predictors of cancer risk-management behaviors among young black BC survivors after GT. Black women (n = 143) with a diagnosis of BC at the age of 50 years or younger received GT. At 1 year after GT, participants reported receipt of risk-reducing mastectomy, risk-reducing salpingo-oophorectomy, mammogram, breast magnetic resonance imaging (MRI), CA125 test, and transvaginal/pelvic ultrasound. Logistic regression was used to examine predictors of BC risk management (risk-reducing mastectomy or breast MRI) and ovarian cancer risk management (risk-reducing salpingo-oophorectomy, CA125 test, or transvaginal/pelvic ultrasound). Of the study participants, 16 (11%) were BRCA1/2-positive, 43 (30%) had a variant of uncertain significance, and 84 (59%) were negative. During the 12 months after GT, no women received risk-reducing mastectomy. The majority (93%) received a mammogram, and a smaller proportion received breast MRI (33%), risk-reducing salpingo-oophorectomy (10%), CA125 test (11%), or transvaginal/pelvic ultrasound (34%). Longer time since the BC diagnosis predicted lower likelihood of BC risk management (odds ratio [OR] 0.54). BRCA1/2 carrier status (OR 4.57), greater perceived risk of recurrence (OR 8.03), and more hereditary breast and ovarian cancer knowledge (OR 1.37) predicted greater likelihood of ovarian cancer risk management. Young black BC survivors appropriately received mammograms and ovarian cancer risk management based on their BRCA1/2 test result. However, the low usage of MRI among BRCA1/2 carriers contrasts with national guidelines. Future research should examine barriers to MRI among black BC survivors. Finally, modifiable variables predicting risk management after GT were identified, providing implications for future interventions.


IMGT/HLA and IMGT/MHC: sequence databases for the study of the major histocompatibility complex.
The IMGT/HLA database (http://www.ebi.ac.uk/imgt/hla) has provided a centralized repository for the sequences of the alleles named by the WHO Nomenclature Committee for Factors of the HLA System for the past four years. Since its initial release the database has grown and is the primary source of information for the study of sequences of the human major histocompatibilty complex. The initial release of the database contained a limited number of tools. As a result of feedback from our users and developments in HLA we have been able to provide new tools and facilities. The HLA sequences have also been extended to include intron sequences and the 3' and 5' untranslated regions in the alignments and also the inclusion of new genes such as MICA. The IMGT/MHC database (http://www.ebi.ac.uk/imgt/mhc) was released in March 2002 to provide a similar resource for other species. The first release of IMGT/MHC contains the sequences of non-human primates (apes, new and old world monkeys), canines and feline sequences. Further species will be added shortly and the database aims to become the primary source of MHC data for non-human sequences.


Psychopathology as a predictor of treatment outcome in alcoholics.
We performed a one-year follow-up study of 266 alcoholics who had received extensive psychiatric assessment, including diagnosis with the National Institute of Mental Health Diagnostic Interview Schedule and DSM-III criteria, during their index treatment episode. The aims were to evaluate the relationship between additional DSM-III diagnoses in alcoholics and outcome at follow-up, assess the relative prognostic power of different ways of measuring psychopathology by comparing categorical DSM-III diagnoses and a global symptom severity measure, and assess whether ratings of psychopathology add to the prognostic power of an alcohol-dependence measure. While coexistent psychiatric diagnoses generally predicted poorer treatment outcome, there were significant interactions in the relationship between diagnoses and treatment outcome for men and women. For men, having an additional diagnosis of major depression, antisocial personality, or drug abuse was associated with poorer outcome. For women, having major depression was associated with a better outcome in drinking-related measures, while antisocial personality and drug abuse were associated with poorer prognosis. The value of determining psychiatric diagnosis was supported by covariance analyses that suggested that prognostic significance of specific disorders was not accounted for by general psychopathology or general dependence dimensions.


Antibacterial effect and cytocompatibility of nano-structured TiO2 film containing Cl.
The aim of this study was to investigate the antibacterial effect and cytocompatibility of a nano-structured TiO2 film that contained Cl and had been coated onto commercially pure titanium. First, we prepared nano-structured TiO2 by anodization with hydrofluoric acid. Then, to confer an antibacterial effect, we performed a second anodization with NaCl solutions of different concentrations (0.5 M, 1 M, 2 M). The morphology, composition, and wettability of the surface were investigated by SEM, EDS, and a video contact angle measuring system. The antibacterial effect was evaluated by film adhesion method. And cytotoxicity was determined by the viability of MG-63 cells in a MTT assay. The SEM and EDS results showed that the TiO2 nano-structure containing Cl had successfully formed after the second anodization. The contact angle analysis showed that the anodized titanium had a hydrophilic character. The results of this in vitro investigation demonstrated that the TiO2 nano-structure film anodized in 1 M NaCl had an antibacterial effect and good cell compatibility.


Julius--a template based supplementary electronic health record system.
EHR systems are widely used in hospitals and primary care centres but it is usually difficult to share information and to collect patient data for clinical research. This is partly due to the different proprietary information models and inconsistent data quality. Our objective was to provide a more flexible solution enabling the clinicians to define which data to be recorded and shared for both routine documentation and clinical studies. The data should be possible to reuse through a common set of variable definitions providing a consistent nomenclature and validation of data. Another objective was that the templates used for the data entry and presentation should be possible to use in combination with the existing EHR systems. We have designed and developed a template based system (called Julius) that was integrated with existing EHR systems. The system is driven by the medical domain knowledge defined by clinicians in the form of templates and variable definitions stored in a common data repository. The system architecture consists of three layers. The presentation layer is purely web-based, which facilitates integration with existing EHR products. The domain layer consists of the template design system, a variable/clinical concept definition system, the transformation and validation logic all implemented in Java. The data source layer utilizes an object relational mapping tool and a relational database. The Julius system has been implemented, tested and deployed to three health care units in Stockholm, Sweden. The initial responses from the pilot users were positive. The template system facilitates patient data collection in many ways. The experience of using the template system suggests that enabling the clinicians to be in control of the system, is a good way to add supplementary functionality to the present EHR systems. The approach of the template system in combination with various local EHR systems can facilitate the sharing and reuse of validated clinical information from different health care units. However, future system developments for these purposes should consider using the openEHR/CEN models with shareable archetypes.


Tumor-Associated CSF MicroRNAs for the Prediction and Evaluation of CNS Malignancies.
Cerebrospinal fluid (CSF) is a readily reachable body fluid that is reflective of the underlying pathological state of the central nervous system (CNS). Hence it has been targeted for biomarker discovery for a variety of neurological disorders. CSF is also the major route for seeding metastases of CNS malignancies and its analysis could be informative for diagnosis and risk stratification of brain cancers. Recently, modern high-throughput, microRNAs (miRNAs) measuring technology has enabled sensitive detection of distinct miRNAs that are bio-chemicallystable in the CSF and can distinguish between different types of CNS cancers. Owing to the fact that a CSF specimen can be obtained with relative ease, analysis of CSF miRNAs could be a promising contribution to clinical practice. In this review, we examine the current scientific knowledge on tumor associated CSF miRNAs that could guide diagnosis of different brain cancer types, or could be helpful in predicting disease progression and therapy response. Finally, we highlight their potential applications clinically as biomarkers and discuss limitations.


Adjuvant bisphosphonates in early breast cancer: consensus guidance for clinical practice from a European Panel.
Bisphosphonates have been studied in randomised trials in early breast cancer to investigate their ability to prevent cancer treatment-induced bone loss (CTIBL) and reduce the risk of disease recurrence and metastasis. Treatment benefits have been reported but bisphosphonates do not currently have regulatory approval for either of these potential indications. This consensus paper provides a review of the evidence and offers guidance to breast cancer clinicians on the use of bisphosphonates in early breast cancer. Using the nominal group methodology for consensus, a systematic review of the literature was augmented by a workshop held in October 2014 for breast cancer and bone specialists to present and debate the available pre-clinical and clinical evidence for the use of adjuvant bisphosphonates. This was followed by a questionnaire to all members of the writing committee to identify areas of consensus. The panel recommended that bisphosphonates should be considered as part of routine clinical practice for the prevention of CTIBL in all patients with a T score of <-2.0 or ≥2 clinical risk factors for fracture. Compelling evidence from a meta-analysis of trial data of >18,000 patients supports clinically significant benefits of bisphosphonates on the development of bone metastases and breast cancer mortality in post-menopausal women or those receiving ovarian suppression therapy. Therefore, the panel recommends that bisphosphonates (either intravenous zoledronic acid or oral clodronate) are considered as part of the adjuvant breast cancer treatment in this population and the potential benefits and risks discussed with relevant patients.


Genome-wide association analysis identifies PDE4D as an asthma-susceptibility gene.
Asthma, a chronic airway disease with known heritability, affects more than 300 million people around the world. A genome-wide association (GWA) study of asthma with 359 cases from the Childhood Asthma Management Program (CAMP) and 846 genetically matched controls from the Illumina ICONdb public resource was performed. The strongest region of association seen was on chromosome 5q12 in PDE4D. The phosphodiesterase 4D, cAMP-specific (phosphodiesterase E3 dunce homolog, Drosophila) gene (PDE4D) is a regulator of airway smooth-muscle contractility, and PDE4 inhibitors have been developed as medications for asthma. Allelic p values for top SNPs in this region were 4.3 x 10(-07) for rs1588265 and 9.7 x 10(-07) for rs1544791. Replications were investigated in ten independent populations with different ethnicities, study designs, and definitions of asthma. In seven white and Hispanic replication populations, two PDE4D SNPs had significant results with p values less than 0.05, and five had results in the same direction as the original population but had p values greater than 0.05. Combined p values for 18,891 white and Hispanic individuals (4,342 cases) in our replication populations were 4.1 x 10(-04) for rs1588265 and 9.2 x 10(-04) for rs1544791. In three black replication populations, which had different linkage disequilibrium patterns than the other populations, original findings were not replicated. Further study of PDE4D variants might lead to improved understanding of the role of PDE4D in asthma pathophysiology and the efficacy of PDE4 inhibitor medications.


Catechol-O-methyltransferase Val158Met polymorphism is associated with somatosensory amplification and nocebo responses.
A large number of unwanted adverse events and symptoms reported by patients in clinical trials are not caused by the drug provided, since most of adverse events also occur in corresponding placebo groups. These nocebo effects also play a major role in drug discontinuation in clinical practice, negatively affecting treatment efficacy as well as patient adherence and compliance. Experimental and clinical data document a large interindividual variability in nocebo responses, however, data on psychological, biological or genetic predictors of nocebo responses are lacking. Thus, with an established paradigm of behaviorally conditioned immunosuppressive effects we analyzed possible genetic predictors for nocebo responses. We focused on the genetic polymorphisms in the catechol-O-methyltransferase (COMT) gene (Val158Met) and analyzed drug specific and general side effects before and after immunosuppressive medication and subsequent placebo intake in 62 healthy male subjects. Significantly more drug-specific as well as general side effects were reported from homozygous carriers of the Val158 variant during medication as well as placebo treatment compared to the other genotype groups. Val158/Val158 carriers also had significantly higher scores in the somatosensory amplification scale (SSAS) and the BMQ (beliefs about medicine questionnaire). Together these data demonstrate potential genetic and psychological variables predicting nocebo responses after drug and placebo intake, which might be utilized to minimize nocebo effects in clinical trials and medical practice.


Epigenetic inactivation of retinoid X receptor genes in non-small cell lung cancer and the relationship with clinicopathologic features.
Retinoid X receptors (RXRs) are nuclear receptors for retinoids that play a critical role in the regulation of growth and differentiation in normal and tumor cells. Deregulation of RXR expression has been reported in non-small cell lung cancer (NSCLC); however, the mechanism underlying the impaired expression of RXRs in lung cancer is not known. Aberrant methylation of promoter CpG islands is known to be a major mechanism for inactivation of tumor suppressor genes. We investigated the methylation status of the RXR genes in 139 surgically resected NSCLCs and correlated the results with the clinicopathologic characteristics of the patients. Methylation in the tumors was detected in all three genes: RXRA, 5.7%; RXRB, 4.3%; RXRG, 23.7%. Reverse transcriptase-polymerase chain reaction analysis showed that RXRG methylation correlates with mRNA expression. Methylation of the RXRG gene was not significantly associated with the prognosis of patients. When the patients were categorized by smoking status, however, the effect of RXRG methylation on prognosis was significantly different between never- and ever-smokers (P=0.003, test for homogeneity). Specifically, RXRG methylation was associated with a significantly worse survival in never-smokers; a trend to better survival outcome was observed for ever-smokers, although not statistically significant. This finding suggests that methylation-associated downregulation of the RXRG gene may play a differential role in the carcinogenesis of NSCLCs according to smoking status, but further studies are needed to confirm this.


Risk factors for osteoporosis related to their outcome: fractures.
The aim of the study was to determine to what extent easy obtainable bone mineral density (BMD)-related risk factors are associated with the occurrence of fractures and to what extent changes in these determinants during a patient's lifetime are relevant. A cross-sectional population-based study was carried out on 4725 postmenopausal women, 50-80 years of age, registered with 23 general practitioners (GPs). The women were questioned and examined. BMD of the lumbar spine was measured using dual-energy X-ray absorptiometry (QDR-1000, Hologic). We analyzed the total population as well as a random sample of 1155 women for whom additional data were collected on recalled weight at age 20-30 years and on self-reported height. Body mass index (BMI) was estimated in two ways: (2) objective BMI [= measured weight/(measured height)2]; (2) recalled BMI [= recalled body weight at age 20-30/(self-reported height)2]. Fractures (dependent variable) were categorized as: (1) fractures sustained during the patient's lifetime; (2) fractures after the age of 50 years; (3) fractures that had occurred during the 5 years before BMD measurement took place. Multivariate stepwise backward and forward logistic regression analyses, using fractures as the dependent variable, were performed with all discrete and non-discrete variables (divided into quartiles). The relationship between the presence of osteoporosis and the presence of fractures was related to the changes in BMI (recalled BMI versus objective BMI). More advanced age, positive family history of fractures and BMD had a positive association with the presence of fractures. Low recalled BMI was a statistically significant predictor of 'fractures during the patient's lifetime' and of 'fractures after the age of 50'. Hysterectomy was associated with a higher prevalence of 'fractures during the patient's lifetime'. Perimenopausal complaints in the history seemed to be associated with a lower prevalence of 'fractures after the age of 50'. Moderate (and heavy) occupational exercise in the past were associated with the presence of fractures 'after the age of 50' and 'fractures during the past 5 years'. Sporting activities in the past showed a slightly positive relationship with the presence of 'fractures during the patient's lifetime' and 'fractures after the age of 50'. Bivariate analysis revealed that current smokers had not sustained significantly more fractures than current nonsmokers, but within the subgroup of current smokers, the prevalence of fractures was significantly higher among those women who had smoked for more than 35 years. Smoking was statistically significantly associated with early menopause. Early menopause was not statistically significantly related to the presence of osteoporosis but appeared to be statistically significantly associated with the prevalence of fractures in the age categories over 65 years. The absolute risks of sustaining one or more fractures ranged from 3% to 44%. Women in the lowest quartile of recalled and objective BMI were often osteoporotic (40%). In this category, women with normal BMD had a statistically significant lower fracture risk than osteoporotic women. Women with a possibly decreased BMI were most often osteoporotic and had sustained more 'fractures during the past 5 years' than expected. Women who had (probably) always been obese were less often osteoporotic and had a much lower fracture risk. It is concluded that decreased BMI is associated with a higher risk of developing fractures at an older age. Prevention of fractures should include fall prevention. In addition, in lean women treatment of low BMD is important.


Assessment of neonatal, cord, and adult platelet granule trafficking and secretion.
Despite the transient hyporeactivity of neonatal platelets, full-term neonates do not display a bleeding tendency, suggesting potential compensatory mechanisms which allow for balanced and efficient neonatal hemostasis. This study aimed to utilize small-volume, whole blood platelet functional assays to assess the neonatal platelet response downstream of the hemostatic platelet agonists thrombin and adenosine diphosphate (ADP). Thrombin activates platelets via the protease-activated receptors (PARs) 1 and 4, whereas ADP signals via the receptors P2Y1 and P2Y12 as a positive feedback mediator of platelet activation. We observed that neonatal and cord blood-derived platelets exhibited diminished PAR1-mediated granule secretion and integrin activation relative to adult platelets, correlating to reduced PAR1 expression by neonatal platelets. PAR4-mediated granule secretion was blunted in neonatal platelets, correlating to lower PAR4 expression as compared to adult platelets, while PAR4 mediated GPIIb/IIIa activation was similar between neonatal and adult platelets. Under high shear stress, cord blood-derived platelets yielded similar thrombin generation rates but reduced phosphatidylserine expression as compared to adult platelets. Interestingly, we observed enhanced P2Y1/P2Y12-mediated dense granule trafficking in neonatal platelets relative to adults, although P2Y1/P2Y12 expression in neonatal, cord, and adult platelets were similar, suggesting that neonatal platelets may employ an ADP-mediated positive feedback loop as a potential compensatory mechanism for neonatal platelet hyporeactivity.


Respiratory effects in humans of a 5-day elevation of end-tidal PCO2 by 8 Torr.
The aims of this study were to determine 1) whether ventilatory adaptation occurred over a 5-day exposure to a constant elevation in end-tidal PCO2 and 2) whether such an exposure altered the sensitivity of the chemoreflexes to acute hypoxia and hypercapnia. Ten healthy human subjects were studied over a period of 13 days. Their ventilation, chemoreflex sensitivities, and acid-base status were measured daily before, during, and after 5 days of elevated end-tidal PCO2 at 8 Torr above normal. There was no major adaptation of ventilation during the 5 days of hypercapnic exposure. There was an increase in ventilatory chemosensitivity to acute hypoxia (from 1.35 +/- 0.08 to 1.70 +/- 0.07 l/min/%; P < 0.01) but no change in ventilatory chemosensitivity to acute hypercapnia. There was a degree of compensatory metabolic alkalosis. The results do not support the hypothesis that the ventilatory adaptation to chronic hypercapnia would be much greater with constant elevation of alveolar PCO2 than with constant elevation of inspired PCO2, as has been used in previous studies and in which the feedback loop between ventilation and alveolar PCO2 is left intact.


Serendipity in the diagnosis of pheochromocytoma.
Pheochromocytomas are increasingly being discovered incidentally on imaging studies performed without clinical suspicion of the existence of an adrenal lesion. We aimed to determine the rate of diagnosis of adrenal pheochromocytoma as an incidental finding during a recent 7-year period. We obtained the Department of Pathology database to study all the patients at our institution with newly diagnosed pheochromocytomas in the 7-year period from 2005 to 2011 to determine the clinical presentation and the means of diagnosis. In 40 (70.2%) of the 57 patients, an adrenal pheochromocytoma was detected in an imaging study performed without suspicion of an adrenal lesion. There were 13 chest computed tomography studies-8 to evaluate for possible pulmonary emboli. Other indications included abdominal pain or discomfort (n = 8), trauma (n = 3), abnormal liver function tests (n = 3), suspect renal artery stenosis (n = 3), hematuria (n = 2), colitis (n = 2), and 4 miscellaneous indications. Our study documents that the commonest current means of initial detection of pheochromocytoma is by serendipitous discovery. In 16 of our 40 patients with serendipitously discovered pheochromocytomas, there were no clinical symptoms of pheochromocytoma; these were true incidentalomas. More than two thirds of the new cases of pheochromocytoma were detected by serendipity (found during studies not performed to evaluate for pheochromocytoma), approximately one third were true incidentalomas (pheochromocytomas in patients without symptoms). In a 7-year period at a single institution, 40 patients, 70% of new cases of surgically proven pheochromocytoma, were initially detected by serendipity. Sixteen patients, 40% of those incidentally discovered represented true examples of "incidentalomas."


Mind shift, mode shift: a lifestyle approach to reducing car ownership and use based on behavioural economics and social marketing.
This paper sets out a new approach to the Wicked Problems of obesity and climate change, and the linked and causative Wicked Problem of increasing car ownership and use. Policies to bring about modal shift from car dependence to forms of transport that cause lower emissions, and are less obesogenic, are conventionally based on framing car use as an externality to be addressed by policies such as taxation or regulation. These policies have been hampered by the reluctance of politicians and policymakers to countenance electoral risk by impinging on the individual's perceived right to personal mobility. This paper's approach combines insights and methods from behavioural economics and social marketing: it shifts the focus by considering car ownership and use not as an externality to be addressed in the aggregate, but as the product of individual behaviours and lifestyle choices. Behavioural economics can help to uncover the motivations, heuristics and cognitive biases behind such behaviours. Social marketing builds on the premise that people will only change their behaviour if they are sufficiently motivated to do so. It can be used to design interventions which help sectors of the population make transport choices that are more optimal--both for the individuals concerned and for all of us who are affected by these Wicked Problems.


A study on the genotoxic effects of 8-Cl-cAMP on human lymphocytes in vitro.
8-chloro-cyclic adenosine 3',5'-monophosphate (8-Cl-cAMP) is the most potent cAMP analogue that selectively inhibits a variety of cancer cell lines in vitro and tumors in vivo. Its action toward a variety of tumors, especially when coupled with other antitumor agents, have lead to phase I clinical investigations and recently phase II clinical investigations. Until today very little was done to evaluate its genotoxic potential. In order to evaluate its genotoxic potential we used the cytogenetic and cytokinesis block micronucleus assay in vitro on peripheral blood lymphocytes of healthy individuals. Using three concentrations (1 microM, 5 microM and 15 microM), 8-Cl-cAMP in normal human peripheral blood lymphocytes did not induce any cytogenetic aberrations of the structural type [chromatid breakage, isochromatid breakage and gaps], but did induce premature centromere separation (PCS) in all respective doses and increased the frequency of micronuclei (p <0.05) only in the highest dose (15 microM). Antiproliferative action of 8-Cl-cAMP was estimated by using the cytokinesis block nuclear division index (NDI). The results showed a decrease in the NDI of cells exposed to all doses of 8-Cl-cAMP when compared to control. Therefore, the overall results show a genotoxic potential of 8-Cl-cAMP in peripheral blood lymphocytes in vitro.


Zika Virus Disease in Colombia - Preliminary Report.
Colombia began official surveillance for Zika virus disease (ZVD) in August 2015. In October 2015, an outbreak of ZVD was declared after laboratory-confirmed disease was identified in nine patients. Using the national population-based surveillance system, we assessed patients with clinical symptoms of ZVD from August 9, 2015, to April 2, 2016. Laboratory test results and pregnancy outcomes were evaluated for a subgroup of pregnant women. Concurrently, we investigated reports of microcephaly for evidence of congenital ZVD. By April 2, 2016, there were 65,726 cases of ZVD reported in Colombia, of which 2485 (4%) were confirmed by means of reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assay. The overall reported incidence of ZVD among female patients was twice that in male patients. A total of 11,944 pregnant women with ZVD were reported in Colombia, with 1484 (12%) of these cases confirmed on RT-PCR assay. In a subgroup of 1850 pregnant women, more than 90% of women who were reportedly infected during the third trimester had given birth, and no infants with apparent abnormalities, including microcephaly, have been identified. A majority of the women who contracted ZVD in the first or second trimester were still pregnant at the time of this report. Among the cases of microcephaly investigated from January 2016 through April 2016, four patients had laboratory evidence of congenital ZVD; all were born to asymptomatic mothers who were not included in the ZVD surveillance system. Preliminary surveillance data in Colombia suggest that maternal infection with the Zika virus during the third trimester of pregnancy is not linked to structural abnormalities in the fetus. However, the monitoring of the effect of ZVD on pregnant women in Colombia is ongoing. (Funded by Colombian Instituto Nacional de Salud and the Centers for Disease Control and Prevention.).


The basis of Stroop interference involving the multimodal correlates of auditory pitch.
A pure auditory tone has a range of multimodal qualities that are determined by its pitch. A reaction-time task was used to demonstrate that subjects respond immediately and automatically to these qualities. Subjects were required to press one of two keys depending on which word, from a limited set, appeared on a microcomputer screen. The words were antonyms that represented multimodal stimulus qualities, and they were assigned to alternative responses so that the two words that shared the same response were correlated in the same way with pitch. As an incidental stimulus, either a 50 Hz tone or a 5500 Hz tone accompanied the presentation of each word. Subjects were found to respond more slowly when the multimodal qualities of the tone were incongruent with the qualities represented by the test word. When the stimulus-response mapping rules were changed, however, the Stroop effect did not occur; suggesting that a polarised semantic code of the incidental tone, that embraces its multimodal features, accesses the same semantic register as the equivalent code for the test word itself.


Metachromatic leukodystrophy (sulfatide lipidoses) cultured in vitro.
Cerebral white matter from a patient with metachromatic leukodystrophly was successfully cultured in vitro. A luxuriant growth in Rose chambers and in roller tubes was apparent by the 14th day. The outgrown cells contained large numbers of granules which stained dark brown with cresyl violetacetic acid. The growth and metachromasia were maintained up to 80 days in vitro.


Management of acquired coagulopathy in acute paediatrics.
Acquired coagulopathy is a relatively uncommon occurrence in acute paediatrics but when it occurs is usually associated with significant underlying pathology and often with critical illness. It can be caused by a number of disease processes but infection, blood loss, iatrogenic causes and liver dysfunction are among the commonest. The blood coagulation cascade is complex and intersects with many other physiological pathways. It is also subject to developmental changes, and 'normal' coagulation and haemostasis change considerably during early life. The diagnosis of abnormal coagulation and when treatment should be initiated is influenced both by age and developmental status and limited by the range of tests routinely available to clinicians. Treatment has predominantly involved transfusion of plasma products (usually fresh frozen plasma and cryoprecipitate) but a number of pharmaceutical and human-derived options are now available. Although plasma products are less frequently transfused than red cells or platelets, their use continues to increase and has not followed the reducing usage of other blood components. This article discusses the aetiology of coagulopathy, describes the commonly available diagnostic tests and outlines the evidence available to guide paediatricians when treating acutely ill children with acquired coagulopathy.


Fracture toughness design in horse hoof keratin.
An engineering fracture mechanics approach was applied to the analysis of the fracture resistance of equine hoof-wall. The relationship between fracture toughness and the morphological organization of the keratin hoof tissue was investigated. Fracture toughness was evaluated using the J-integral analysis method which employs the compact tension test geometry. Tensile tests were also conducted to evaluate the effect of the morphological organization on the stress-strain behaviour. Hoof-wall has greatest fracture resistance for cracks running proximally, parallel to the tubular component of the wall keratin. For fully hydrated material tested in this direction the mean critical J-integral value at failure was 1.19 X 10(4)J m-2. This was nearly three times greater than the value determined for the weakest orientation, in which the crack ran parallel to the material between the tubules. The lower fracture toughness of the intertubular material dominates the fracture behaviour of this tissue. The tubular components of the wall appear to reinforce against fracture along the weak plane and the entire wall organization provides the mechanical capability for limiting and controlling fracture in this tissue.


An SNP in an ultraconserved regulatory element affects Dlx5/Dlx6 regulation in the forebrain.
Dlx homeobox genes play a crucial role in the migration and differentiation of the subpallial precursor cells that give rise to various subtypes of gamma-aminobutyric acid (GABA)-expressing neurons of the forebrain, including local-circuit cortical interneurons. Aberrant development of GABAergic interneurons has been linked to several neurodevelopmental disorders, including epilepsy, schizophrenia, Rett syndrome and autism. Here, we report in mice that a single-nucleotide polymorphism (SNP) found in an autistic proband falls within a functional protein binding site in an ultraconserved cis-regulatory element. This element, I56i, is involved in regulating Dlx5/Dlx6 homeobox gene expression in the developing forebrain. We show that the SNP results in reduced I56i activity, predominantly in the medial and caudal ganglionic eminences and in streams of neurons tangentially migrating to the cortex. Reduced activity is also observed in GABAergic interneurons of the adult somatosensory cortex. The SNP affects the affinity of Dlx proteins for their binding site in vitro and reduces the transcriptional activation of the enhancer by Dlx proteins. Affinity purification using I56i sequences led to the identification of a novel regulator of Dlx gene expression, general transcription factor 2 I (Gtf2i), which is among the genes most often deleted in Williams-Beuren syndrome, a neurodevelopmental disorder. This study illustrates the clear functional consequences of a single nucleotide variation in an ultraconserved non-coding sequence in the context of developmental abnormalities associated with disease.


Circularly polarized guided modes in dielectrically chiral photonic crystal fiber.
The effect of dielectric chirality on the polarization states and mode indices of guided modes in photonic crystal fiber (PCF) is investigated by a modified plane-wave expansion (PWE) method. Using a solid-core chiral PCF as a numerical example, we show that circular polarization is the eigenstate of the fundamental mode. Mode index divergence between right-handed circularly polarized (RCP) and left-handed circularly polarized (LCP) states is demonstrated. Chirality's effect on mode index and circular birefringence (CB) in such a PCF is found to be similar to that in bulk chiral media.


Novel molecular determinants in the pore region of sodium channels regulate local anesthetic binding.
The pore of the Na+ channel is lined by asymmetric loops formed by the linkers between the fifth and sixth transmembrane segments (S5-S6). We investigated the role of the N-terminal portion (SS1) of the S5-S6 linkers in channel gating and local anesthetic (LA) block using site-directed cysteine mutagenesis of the rat skeletal muscle (Na(V)1.4) channel. The mutants examined have variable effects on voltage dependence and kinetics of fast inactivation. Of the cysteine mutants immediately N-terminal to the putative DEKA selectivity filter in four domains, only Q399C in domain I and F1236C in domain III exhibit reduced use-dependent block. These two mutations also markedly accelerated the recovery from use-dependent block. Moreover, F1236C and Q399C significantly decreased the affinity of QX-314 for binding to its channel receptor by 8.5- and 3.3-fold, respectively. Oddly enough, F1236C enhanced stabilization of slow inactivation by both hastening entry into and delaying recovery from slow inactivation states. It is noteworthy that symmetric applications of QX-314 on both external and internal sides of F1236C mutant channels reduced recovery from use-dependent block, indicating an allosteric effect of external QX-314 binding on the recovery of availability of F1236C. These observations suggest that cysteine mutation in the SS1 region, particularly immediate adjacent to the DEKA ring, may lead to a structural rearrangement that alters binding of permanently charged QX-314 to its receptor. The results lend further support for a role for the selectivity filter region as a structural determinant for local anesthetic block.


Quantitative analysis of the self-assembly strategies of intermediate filaments from tetrameric vimentin.
In vitro assembly of intermediate filaments from tetrameric vimentin consists of a very rapid phase of tetramers laterally associating into unit-length filaments and a slow phase of filament elongation. We focus in this paper on a systematic quantitative investigation of two molecular models for filament assembly, recently proposed in (Kirmse et al. J. Biol. Chem. 282, 52 (2007), 18563-18572), through mathematical modeling, model fitting, and model validation. We analyze the quantitative contribution of each filament elongation strategy: with tetramers, with unit-length filaments, with longer filaments, or combinations thereof. In each case, we discuss the numerical fitting of the model with respect to one set of data, and its separate validation with respect to a second, different set of data. We introduce a high-resolution model for vimentin filament self-assembly, able to capture the detailed dynamics of filaments of arbitrary length. This provides much more predictive power for the model, in comparison to previous models where only the mean length of all filaments in the solution could be analyzed. We show how kinetic observations on low-resolution models can be extrapolated to the high-resolution model and used for lowering its complexity.


Tactical Circuit Training Improves Blood Pressure and Vascular Health More Than Resistance Training.
Exercise training is known to reduce CVD risk factors; however, in tactical populations, like veterans and firefighters, the effects of different forms of exercise such as tactical circuit training (CT) or conventional resistance training (RT) is unclear. Thus, the purpose of this study was to compare changes in various CVD risk measures after 4-week tactical CT or RT programs. Thirty-seven firefighters (20 CT, 17 RT), 35% of whom were veterans, participated. Pre- and post-intervention measures included body fat (BF%), carotid artery intima media thickness (IMT), central and brachial BP, and indices of arterial stiffness (augmentation index, Aix@75), myocardial oxygenation (subendocardial viability ratio, SEVR), and endothelial function (flow-mediated dilation, FMD). Estimation of maximum oxygen consumption (VO2peak) for aerobic fitness, balance, muscular endurance, and strength were also compared. For the clinical laboratory values, there were no between group differences and the only within group change was found in triglyceride levels. Tactical CT lowered triglyceride levels by 24.2% (P < 0.05). Only tactical CT exercise lowered BP. Both brachial (4.6% reduction) and central (4.4 % reduction) systolic and diastolic SBP and DBP decreased with CT (all P ≤ 0.01). After training we found improvements in FMD and SEVR with tactical CT only. Percent FMD increased by 28.7% (P < 0.01) while SEVR increased by 4.4% (P < 0.05) in the tactical CT group. Fitness improved in both cohorts (P < 0.05). These data suggest that 4 weeks of a CT program improves several CVD-risk factors and may be more beneficial.


Influence of indomethacin on the systemic and pulmonary vascular resistance in man.
1. Indomethacin, an inhibitor of the cyclo-oxygenase system that converts arachidonic acid into prostaglandins and related substances, was infused intravenously in 12 healthy volunteer subjects. 2. Systemic systolic and diastolic blood pressures and heart rate were recorded in all subjects, and in most of them also the systemic arteriovenous oxygen difference, the total oxygen uptake and the pulmonary arterial and wedge pressures. 3. The infusion of indomethacin was followed by a decreased cardiac output (from 7.3 +/- 0.3 to 6.3 +/- 0.3 litres/min) and an increased mean systemic blood pressure (from 92 +/- 1 to 102 +/- 1 mmHg), indicating an elevation of the total systemic vascular resistance (from 98 +/- 4 to 124 +/- 5 kPa 1(-1) s) by indomethacin. The ventilation and the pulmonary vascular resistance did not change after the infusion of indomethacin. 4. The results suggest that products formed by the cyclo-oxygenase system at rest exert a relaxing effect in certain parts of the systemic vascular bed, thereby lowering the systemic vascular resistance.


Use of flash glucose-sensing technology (FreeStyle Libre) in youth with type 1 diabetes: AWeSoMe study group real-life observational experience.
Frequent glucose testing is required for optimal management of type 1 diabetes (T1D). Limited data are available regarding real-world experience of the novel technology for monitoring by continuous interstitial fluid glucose (IFG), using flash glucose-sensing technology (FSL-CGM). We aimed to assess the effect of FSL-CGM in a real-life clinical setting on glycemic control parameters, compliance, and adverse events among pediatric and young adult T1D patients. This observational multi-center study assessed FSL-CGM use (6-12 months) in T1D patients (mean ± SD age 13.4 ± 4.9 years) who purchased the device out-of-pocket. Outcome measures included HbA1c, mean IFG levels, CGM metrics [time in hypoglycemia (< 54 mg/dL; < 3 mmol/L), in target range (70-180 mg/dL; 3.9-10 mmol/L), and in hyperglycemia > 240 mg/dL; > 13.3 mmol/L)], frequency of self-monitoring of blood glucose, acute complications, skin reactions, and reasons for initiation/discontinuation. Among patients with regular use of the FSL-CGM (n = 59), mean HbA1c decreased from 8.86 ± 0.23 to 8.05 ± 0.2% (73.3-64.5 mmol/mol) in 3 months (p = 0.0001) and plateaued thereafter. A clinically significant reduction in HbA1c (defined as a decrease of ≥ 0.5%) was associated with shorter diabetes duration. Of 71 patients who initiated use of the FSL-CGM, 12 (16.9%) discontinued during the study period. No statistically significant changes were found after FSL-CGM use, in mean and standard deviation IFG levels, and in time of glucose levels in target, hypoglycemia, and hyperglycemia ranges. One patient with hypoglycemia unawareness was found dead-in-bed while using FSL-CGM. Real-life observational data in a self-selected young T1D population demonstrated a significant and sustained reduction in HbA1c with FSL-CGM in one-third of the participants. Surveillance of glucose monitoring should be individualized, especially for patients with hypoglycemia unawareness.


Cytochrome P450 oxidoreductase gene mutations and Antley-Bixler syndrome with abnormal genitalia and/or impaired steroidogenesis: molecular and clinical studies in 10 patients.
We report on molecular and clinical findings in 10 Japanese patients (four males and six females) from eight families (two pairs of siblings and six isolated cases) with Antley-Bixler syndrome accompanied by abnormal genitalia and/or impaired steroidogenesis. Direct sequencing was performed for all the 15 exons of cytochrome P450 oxidoreductase gene (POR), showing two missense mutations (R457H and Y578C), a 24-bp deletion mutation resulting in loss of nine amino acids and creation of one amino acid (L612_W620delinsR), a single bp insertion mutation leading to frameshift (I444fsX449), and a silent mutation (G5G). R457H has previously been shown to be a pathologic mutation, and computerized modeling analyses indicated that the 15A>G for G5G could disturb an exonic splicing enhancer motif, and the remaining three mutations should affect protein conformations. Six patients were compound heterozygotes, and three patients were R457H homozygotes; no mutation was identified on one allele of the remaining one patient. Clinical findings included various degrees of skeletal features, such as brachycephaly, radiohumeral synostosis, and digital joint contractures in patients of both sexes, normal-to-poor masculinization during fetal and pubertal periods in male patients, virilization during fetal life and poor pubertal development without worsening of virilization in female patients, and relatively large height gain and delayed bone age from the pubertal period in patients of both sexes, together with maternal virilization during pregnancy. Blood cholesterol was grossly normal, and endocrine studies revealed defective CYP17A1 and CYP21A2 activities. The results suggest that Antley-Bixler syndrome with abnormal genitalia and/or impaired steroidogenesis is caused by POR mutations, and that clinical features are variable and primarily explained by impaired activities of POR-dependent CYP51A1, CYP17A1, CYP21A2, and CYP19A1.


Prediction of relapse to frequent heroin use and the role of methadone prescription: an analysis of the Amsterdam Cohort Study among drug users.
The risk of relapse into frequent heroin use was studied among 732 participants of the Amsterdam Cohort Study (ACS) on HIV/AIDS among drug users, who experienced an episode of abstinence from or occasional use of heroin. Participants of the ACS were recruited primarily from easy access ("low-threshold") methadone programs. The duration of abstinence/occasional use and relative risks (RR) of relapse were estimated by analyzing 1577 episodes by means of survival analysis using characteristics of patients and methadone treatment as covariates. The majority of episodes (85.8%) were followed by relapse within 5 years. Less education, intense use of heroin prior to the episode of abstinence or well-controlled use, occasional use of heroin and intense use of cocaine during the episode, and having a drug-using partner or having no partner were significantly associated with a higher risk of relapse. Among frequent attendees of a "low-threshold" methadone program, relapse was associated with the daily dose of methadone: RR for dosages <40 and 40-60 mg, compared with doses of >100mg, were 1.45 (P<0.01) and 1.59 (P<0.01), respectively. No beneficial influence was revealed of methadone dosage or program attendance in itself on the risk of relapse into cocaine. High doses of methadone in a harm-reduction setting extend the duration of an episode of no or occasional use of heroin. Other factors, such as no occasional use of heroin during the episode, no use of cocaine, and having a non-using partner, seem to be equally important.


Molecular dynamics study of zinc binding to cysteines in a peptide mimic of the alcohol dehydrogenase structural zinc site.
The binding of zinc (Zn) ions to proteins is important for many cellular events. The theoretical and computational description of this binding (as well as that of other transition metals) is a challenging task. In this paper the binding of the Zn ion to four cysteine residues in the structural site of horse liver alcohol dehydrogenase (HLADH) is studied using a synthetic peptide mimic of this site. The study includes experimental measurements of binding constants, classical free energy calculations from molecular dynamics (MD) simulations and quantum mechanical (QM) electron structure calculations. The classical MD results account for interactions at the molecular level and reproduce the absolute binding energy and the hydration free energy of the Zn ion with an accuracy of about 10%. This is insufficient to obtain correct free energy differences. QM correction terms were calculated from density functional theory (DFT) on small clusters of atoms to include electronic polarisation of the closest waters and covalent contributions to the Zn-S coordination bond. This results in reasonably good agreement with the experimentally measured binding constants and Zn ion hydration free energies in agreement with published experimental values. The study also includes the replacement of one cysteine residue to an alanine. Simulations as well as experiments showed only a small effect of this upon the binding free energy. A detailed analysis indicate that the sulfur is replaced by three water molecules, thereby changing the coordination number of Zn from four (as in the original peptide) to six (as in water).


The European Association of Urology Robotic Urology Section (ERUS) survey of robot-assisted radical prostatectomy (RARP).
To evaluate surgeons adherence to current clinical practice, with the available evidence, for robot-assisted radical prostatectomy (RARP) and offer a baseline assessment to measure the impact of the Pasadena recommendations. Recently, the European Association of Urology Robotic Urology Section (ERUS) supported the Pasadena Consensus Conference on best practices in RARP. This survey was performed in January 2012. A specific questionnaire was sent, by e-mail, to 145 robotic surgeons who were included in the mailing-list of ERUS members and working in different urological institutions. Participating surgeons were invited to answer a multiple-choice questionnaire including 24-items evaluating the main RARP surgical steps. In all, 116 (79.4%) invited surgeons answered the questionnaire and accepted to participate to the ERUS survey. In all, 47 (40.5%) surgeons performed >100 RARPs; 41 (35.3%) between 50 and 100, and 28 (24.1%) <50 yearly. The transperitoneal, antegrade technique was the preferred approach. Minimising bladder neck dissection and the use of athermal dissection of the neurovascular bundles (NVBs) were also popular. There was more heterogeneity in the use of energy for seminal vesicle dissection, the preservation of the tips of the seminal vesicle and the choice between intra- and interfascial planes during the antero-lateral dissection of the NVBs. There was also large variability in the posterior and/or anterior reconstruction steps. The present study is the first international survey evaluating surgeon preferences during RARP. Considering that the results were collected before the publication of the Pasadena recommendations, the data might be considered an important baseline evaluation to test the dissemination and effects of the Pasadena recommendations in subsequent years.


Association of an increase in CD8+ T cells with the presence of Trypanosoma cruzi antigens in chronic, human, chagasic myocarditis.
The role of Trypanosoma cruzi in the pathogenesis of myocarditis in the chronic phase of Chagas' disease is still controversial, with autoimmune mechanisms frequently being proposed. In the present work, we demonstrate that higher numbers of CD8+ T cells are correlated with the presence of parasite antigens, suggesting an important role for the parasite in the development of myocardial inflammation. Quantification of the mean numbers of CD8+ and CD4+ T cells per 400x microscopic field was performed in myocardial specimens from 33 chronic chagasic patients with heart failures (nine biopsies and 24 necropsies), using an immunoperoxidase technique. The cases were grouped according to a semiquantitative score of the relative amounts of T. cruzi antigens: group 1 = absent (14 cases); group 2 = scarce extracellular or intramacrophagic antigens (12 cases); group 3 = many extracellular or intramacrophagic antigens plus T. cruzi intramyocytic pseudocysts (seven cases). The mean numbers of CD8+ and CD4+ T cells in groups 1,2, and 3 were 6.94 and 3.79, 13.89 and 6.24, and 17.91 and 5.97, respectively. The numbers of CD8+ T cells in groups 2 and 3 were significantly higher compared with group 1 (no T. cruzi antigens), but were not different from each other. Scarce, extramyocytic T. cruzi antigens were associated with an intense inflammatory infiltrate, suggesting that delayed-type hypersensitivity immune mechanism is induced by the parasite; intact myocardiocytes containing parasites did not show an inflammatory reaction around them. A poor inflammatory response was frequently associated with many extramyocytic antigens and myocardial parasite pseudocysts, suggesting that active proliferation and dissemination of the parasites occur when the immunologic response is diminished. The number of CD4+ T cells did not vary significantly among the three groups. We conclude that the CD8+ T cell is the main cell type responsible for immune activation in chronic, human, chagasic myocarditis and is probably activated by the presence of T. cruzi antigens associated with internal myocytic host antigens. The absence of a significant member of CD4+ T cells in the presence of T. cruzi antigens suggests inhibition of CD4+ T cell activation or the lack of a class II major histocompatibility complex molecule presentation mechanism.


Seroepidemiologic Survey of Potential Pathogens in Obligate and Facultative Scavenging Avian Species in California.
Throughout the world, populations of scavenger birds are declining rapidly with some populations already on the brink of extinction. Much of the current research into the factors contributing to these declines has focused on exposure to drug residues, lead, and other toxins. Despite increased monitoring of these declining populations, little is known about infectious diseases affecting scavenger bird species. To assess potential infectious disease risks to both obligate and facultative scavenger bird species, we performed a serosurvey for eleven potential pathogens in three species of scavenging birds in California: the California condor (Gymnogyps californianus), turkey vulture (Cathartes aura) and golden eagle (Aquila chrysaetos). California condors were seropositive for avian adenovirus, infectious bronchitis virus, Mycoplasma gallisepticum, avian paramyxovirus-2, West Nile virus (WNV) and Toxoplasma gondii. Golden eagles were seropositive for avian adenovirus, Chlamydophila psittaci and Toxoplasma gondii, and turkey vultures were seropositive for avian adenovirus, Chlamydophila psittaci, avian paramyxovirus-1, Toxoplasma gondii and WNV. Risk factor analyses indicated that rearing site and original release location were significantly associated with a positive serologic titer to WNV among free-flying condors. This study provides preliminary baseline data on infectious disease exposure in these populations for aiding in early disease detection and provides potentially critical information for conservation of the endangered California condor as it continues to expand its range and encounter new infectious disease threats.


Angiotensin II mediates epithelial-to-mesenchymal transformation in tubular cells by ANG 1-7/MAS-1-dependent pathways.
Epithelial-to-mesenchymal transformation (EMT) of tubular cells into a myofibroblastic phenotype is an important mediator of renal scarring in chronic nephropathy. This study examines the role of the renin-angiotensin system (RAS) in this process. NRK-52E cells were exposed to angiotensin (ANG) II and ANG 1-7 in the presence or absence of inhibitors and agonists of RAS signaling. EMT was assessed at 3 days by expression of alpha-smooth muscle actin (alpha-SMA) and E-cadherin and the induction of a myofibroblastic phenotype. Expression of fibrogenic growth factors and matrix proteins was assessed by RT-PCR and immunofluorescence microscopy. To confirm findings in vivo, rats were also infused with ANG 1-7 (24 microg*kg(-1)*h(-1)) or saline via an osmotic minipump for 10 days, and renal fibrogenesis was then assessed. Treatment of NRK-52E cells with ANG II induced characteristic changes of EMT. Selective blockade of the AT(1) receptor or the AT(2) receptor failed to inhibit ANG II-induced EMT. However, blockade of the ANG 1-7 receptor, Mas-1, was able to prevent ANG II-dependent EMT. To confirm these findings, both ANG 1-7 and the selective Mas receptor agonist, AVE-0991, were able to induce NRK-52E cells in a dose-dependent manner. Exposing cells to recombinant ACE2 was also able to induce EMT. In addition, an infusion of ANG 1-7 induced the tubular expression of alpha-SMA and the expression of matrix proteins in the kidney. ANG II is a potent stimulus for EMT, but not through conventional pathways. This study points to the possible limitations of conventional RAS blockade, which not only fails to antagonize this pathway, but also may enhance it via augmenting the synthesis of ANG 1-7.


Evolution of a carbohydrate binding module into a protein-specific binder.
A carbohydrate binding module, CBM4-2, derived from the xylanase (Xyn 10A) of Rhodothermus marinus has been used as a scaffold for molecular diversification. Its binding specificity has been evolved to recognise a quite different target, a human monoclonal IgG4. In order to understand the basis for this drastic change in specificity we have further investigated the target recognition of the IgG4-specific CBMs. Firstly, we defined that the structure target recognised by the selected CBM-variants was the protein and not the carbohydrates attached to the glycoprotein. We also identified key residues involved in the new specificity and/or responsible for the swap in specificity, from xylan to human IgG4. Specific changes present in all these CBMs included mutations not introduced in the design of the library from which the specific clones were selected. Reversion of such mutations led to a complete loss of binding to the target molecule, suggesting that they are critical for the recognition of human IgG4. Together with the mutations introduced at will, they had transformed the CBM scaffold into a protein binder. We have thus shown that the scaffold of CBM4-2 is able to harbour molecular recognition for either carbohydrate or protein structures.


The written summary as a group psychotherapy technique.
During the past 18 months, we have been preparing detailed written summaries of the events of group therapy meetings, which have then been mailed to the group members. This technique was introduced initially as a device to provide structure in a very anxious group. We soon realized that the summary had a great potential for enhancing therapist effectiveness and it came to assume a number of other functions. In this report, we describe our experience with this technique, emphasizing its importance as a tool to improve the cognitive integration of the group therapy experience for both patient and therapist.


Stress-induced hyperglycemia is associated with higher mortality in severe traumatic brain injury.
An association between stress-induced hyperglycemia (SIH) and increased mortality has been demonstrated following trauma. Experimental animal model data regarding the association between hyperglycemia and outcomes following traumatic brain injury (TBI) are inconsistent, suggesting that hyperglycemia may be harmful, neutral, or beneficial. The purpose of this study was to examine the effects of SIH versus diabetic hyperglycemia (DH) on severe TBI. Admission glycosylated hemoglobin (HbA1c), glucose levels, and comorbidity data were collected during a 4-year period from September 2009 to December 2013 for patients with severe TBI (i.e., admission Glasgow Coma Scale [GCS] score of 3-8 and head Abbreviated Injury Scale [AIS] score ≥ 3). Diabetes mellitus was determined by patient history or admission HbA1c of 6.5% or greater. SIH was determined by the absence of diabetes mellitus and admission glucose of 200 mg/dL or greater. A Cox proportional hazards model adjusted for age, sex, injury mechanism, and Injury Severity Score (ISS) was used to calculate hazard ratios (HRs) and associated 95% confidence intervals (CIs) for the association between SIH and the outcomes of interest. During the study period, a total of 626 patients were included in the study group, having severe TBI defined by both GCS score of 3 to 8 and head AIS score being 3 or greater and also had available HbA1c and admission glucose levels. A total of 184 patients were admitted with hyperglycemia; 152 patients (82.6%) were diagnosed with SIH, and 32 patients (17.4%) were diagnosed with DH. When comparing patients with severe TBI adjusted for age, sex, injury mechanism, ISS, Revised Trauma Score (RTS), and lactic acid greater than 2.5 mmol/L, patients with SIH had a 50% increased mortality (HR, 1.49; 95% CI, 1.13-1.95) compared with the nondiabetic normoglycemia patients. DH patients did not have a significant increase in mortality (HR, 0.94; 95% CI, 0.56-1.58). SIH is associated with higher mortality after severe TBI. This association was not observed among patients with DH, which suggests that hyperglycemia related to diabetes is of less importance compared with SIH in terms of mortality in the acute trauma and TBI patient. Further research is warranted to identify mechanisms causing SIH and subsequent worse outcomes after TBI. Prognostic/epidemiologic study, leve III.


Prospective audit of emergency department transit times associated with entonox analgesia for reduction of the acute, traumatic dislocated shoulder.
To establish current UK practice for the management of the acute traumatic shoulder dislocation with respect to analgesia and reduction manoeuvres. To compare the transit times of patients through an emergency department (ED) after the use of intravenous analgesia and/ or sedation compared to entonox +/- simple oral analgesia. A postal questionnaire was sent to 100 UK ED consultants to establish current practice. The treating clinicians were allowed to choose the method of analgesia provided to reduce the patient's dislocated shoulder, provided the patient was happy with it. They administered either (1) traditional intravenous morphine and/or midazolam or (2) entonox +/- simple oral analgesia to facilitate reduction. A prospective audit was conducted to compare the transit times of the two groups of patients. The postal questionnaire revealed that intravenous morphine and midazolam are widely used during reduction of the acute shoulder dislocation in the UK. The audit showed that this was associated with a significantly prolonged transit time through the ED, compared to entonox alone, (mean 77 min versus 177 min, respectively, p<0.001) without compromise in reduction success. Entonox +/- simple oral analgesia significantly decreases ED transit times as compared to IV morphine and/or midazolam for the reduction of the acute traumatic dislocated shoulder. Further studies should be done into patient pain scores and into the best combination of oral analgesia and entonox.


Survival analyses of social support and trauma among homeless male and female veterans who abuse substances.
This appears to be the 1st study of gender differences in how well various forms of trauma and social support predict homeless substance abusers' tenure in the community without rehospitalization. Sexual and physical abuses at different stages of the life span, combat exposure, and recent traumatic events were analyzed with social support factors via Cox's proportional hazard model of survival in a 2-year follow-up. The survival models showed similarities and dissimilarities in predictors of tenure in the community for women compared to men among homeless veterans. Traumata and related factors (e.g., depression and suicidal thoughts) were more potent (negative) predictors of tenure, and family and friends were more important social supports, for women than for men. Men's tenure was more positively associated with job satisfaction and more negatively related to substance abuse, combat exposure, cognitive impairments, aggression, and physical health problems. The intervention implications of these findings are discussed.


Visual Control for Multirobot Organized Rendezvous.
This paper addresses the problem of visual control of a set of mobile robots. In our framework, the perception system consists of an uncalibrated flying camera performing an unknown general motion. The robots are assumed to undergo planar motion considering nonholonomic constraints. The goal of the control task is to drive the multirobot system to a desired rendezvous configuration relying solely on visual information given by the flying camera. The desired multirobot configuration is defined with an image of the set of robots in that configuration without any additional information. We propose a homography-based framework relying on the homography induced by the multirobot system that gives a desired homography to be used to define the reference target, and a new image-based control law that drives the robots to the desired configuration by imposing a rigidity constraint. This paper extends our previous work, and the main contributions are that the motion constraints on the flying camera are removed, the control law is improved by reducing the number of required steps, the stability of the new control law is proved, and real experiments are provided to validate the proposal.


Stigmergic construction and topochemical information shape ant nest architecture.
The nests of social insects are not only impressive because of their sheer complexity but also because they are built from individuals whose work is not centrally coordinated. A key question is how groups of insects coordinate their building actions. Here, we use a combination of experimental and modeling approaches to investigate nest construction in the ant Lasius niger. We quantify the construction dynamics and the 3D structures built by ants. Then, we characterize individual behaviors and the interactions of ants with the structures they build. We show that two main interactions are involved in the coordination of building actions: (i) a stigmergic-based interaction that controls the amplification of depositions at some locations and is attributable to a pheromone added by ants to the building material; and (ii) a template-based interaction in which ants use their body size as a cue to control the height at which they start to build a roof from existing pillars. We then develop a 3D stochastic model based on these individual behaviors to analyze the effect of pheromone presence and strength on construction dynamics. We show that the model can quantitatively reproduce key features of construction dynamics, including a large-scale pattern of regularly spaced pillars, the formation and merging of caps over the pillars, and the remodeling of built structures. Finally, our model suggests that the lifetime of the pheromone is a highly influential parameter that controls the growth and form of nest architecture.


Tumor-suppressive effect of LRIG1, a negative regulator of ErbB, in non-small cell lung cancer harboring mutant EGFR.
Epidermal growth factor receptor (EGFR) is a member of the ErbB (HER) family that is known to play important roles in the pathogenesis of various human cancers. Mutations of the EGFR gene are commonly found as oncogenic driver mutations and have been targeted for treatment of non-small cell lung cancer (NSCLC). Leucine-rich repeat and immunoglobulin-like domain protein-1 (LRIG1) is a cell-surface protein that is known as a negative regulator of the ErbB (HER) family. In this study, we first confirmed that the expression levels of LRIG1 were much lower in NSCLC than in non-malignant cells or tissues. Next, we focused on the effect of LRIG1 in NSCLC. For this purpose, we established clones stably overexpressing LRIG1, using EGFR-mutant (HCC827, HCC4011 and NCI-H1975) and wild-type (A549) cells. Transfection of LRIG1 was associated with a decrease in the expression and phosphorylation levels of EGFR in the HCC827, HCC4011 and NCI-H1975 cells. It was also associated with strong suppression of the cell proliferative, invasive, migratory and tumorigenic potential of the HCC827 cells. On the other hand, no such effects were observed in the A549 cells. In addition, LRIG1 also downregulated the expression and phosphorylation levels of other tyrosine kinase receptors, such as HER2, HER3, MET and IGF-1R, and prevented the epithelial-to-mesenchymal transition induced by TGF-β in the HCC827 cells. These findings suggest that LRIG1 exerts important tumor-suppressive effects in EGFR-mutant NSCLC and has the potential to become a novel therapeutic target for EGFR-mutant NSCLC.


Organometallic Rhenium Complexes Divert Doxorubicin to the Mitochondria.
Doxorubicin, a well-established chemotherapeutic agent, is known to accumulate in the cell nucleus. By using ICP-MS, we show that the conjugation of two small organometallic rhenium complexes to this structural motif results in a significant redirection of the conjugates from the nucleus to the mitochondria. Despite this relocation, the two bioconjugates display excellent toxicity toward HeLa cells. In addition, we carried out a preliminarily investigation of aspects of cytotoxicity and present evidence that the conjugates disrupt the mitochondrial membrane potential, are strong inhibitors of human Topoisomerase II, and induce apoptosis. Such derivatives may enhance the therapeutic index of the aggressive parent drug and overcome drug resistance by influencing nuclear and mitochondrial homeostasis.


Evaluation of high throughput gene expression platforms using a genomic biomarker signature for prediction of skin sensitization.
Allergic contact dermatitis (ACD) develops upon exposure to certain chemical compounds termed skin sensitizers. To reduce the occurrence of skin sensitizers, chemicals are regularly screened for their capacity to induce sensitization. The recently developed Genomic Allergen Rapid Detection (GARD) assay is an in vitro alternative to animal testing for identification of skin sensitizers, classifying chemicals by evaluating transcriptional levels of a genomic biomarker signature. During assay development and biomarker identification, genome-wide expression analysis was applied using microarrays covering approximately 30,000 transcripts. However, the microarray platform suffers from drawbacks in terms of low sample throughput, high cost per sample and time consuming protocols and is a limiting factor for adaption of GARD into a routine assay for screening of potential sensitizers. With the purpose to simplify assay procedures, improve technical parameters and increase sample throughput, we assessed the performance of three high throughput gene expression platforms--nCounter®, BioMark HD™ and OpenArray®--and correlated their performance metrics against our previously generated microarray data. We measured the levels of 30 transcripts from the GARD biomarker signature across 48 samples. Detection sensitivity, reproducibility, correlations and overall structure of gene expression measurements were compared across platforms. Gene expression data from all of the evaluated platforms could be used to classify most of the sensitizers from non-sensitizers in the GARD assay. Results also showed high data quality and acceptable reproducibility for all platforms but only medium to poor correlations of expression measurements across platforms. In addition, evaluated platforms were superior to the microarray platform in terms of cost efficiency, simplicity of protocols and sample throughput. We evaluated the performance of three non-array based platforms using a limited set of transcripts from the GARD biomarker signature. We demonstrated that it was possible to achieve acceptable discriminatory power in terms of separation between sensitizers and non-sensitizers in the GARD assay while reducing assay costs, simplify assay procedures and increase sample throughput by using an alternative platform, providing a first step towards the goal to prepare GARD for formal validation and adaption of the assay for industrial screening of potential sensitizers.


Adult Lymphoma-Associated Hemophagocytic Lymphohistiocytosis: A Clinical Case Series in a Predominantly Hispanic Cohort.
Hemophagocytic lymphohistiocytosis (HLH) is a systemic inflammation disorder secondary to immune dysregulation. Patients may present with fevers, splenomegaly, bone marrow failure and hemophagocytosis, among other clinical and laboratory findings. Lymphoma-associated HLH (LA-HLH) is a puzzling diagnosis given both conditions overlapping presentation. There are currently no established treatment guidelines for LA-HLH. We conducted a retrospective search of the tumor registry and pathology database at the University of Miami/Jackson Memorial Hospital using Pathology Laboratory Information System (LIS) and natural language search. We identified adult patients with a combined diagnosis of lymphoma and HLH between January 2008 and July 2018. Data from nine LA-HLH patients were identified and reviewed. The median age was 53 years (range 19 - 73), with 78% of cases of Hispanic origin. Lymphoma subtypes consisted of six T-cell/NK-cell neoplasms: two peripheral T-cell lymphoma (PTCL), not otherwise specified (NOS); two Epstein-Barr virus (EBV)+ extranodal NK-/T-cell lymphomas; one EBV+, CD8+, PTCL, NOS; one EBV+, post-transplant lymphoproliferative disorder-anaplastic large cell lymphoma, anaplastic lymphoma kinase negative (PTLD ALCL ALK-); and three B-cell neoplasms: one EBV+ diffuse large B-cell lymphoma (DLBCL); two DLBCL, NOS. HLH and lymphoma were diagnosed simultaneously in six out of nine cases. Hemophagocytosis phenomena were demonstrated in seven out of nine cases. Treatment consisted of combined HLH and lymphoma therapies in four cases, while lymphoma-directed therapy was applied to four patients; another case was treated with a modified version of the HLH-1994 protocol. Overall, a total of five cases were exposed to HLH-directed regimens (HLH-1994/2004). Three patients had refractory LA-HLH and entered hospice care, whereas another three cases succumbed to treatment-related complications. Of the seven cases that were evaluable for lymphoma response, four cases (57%) achieved complete response (CR), and three of them (43%) were alive with no evidence of recurrence at 10, 16 and 52 months as of the last contact. Herein, we describe our unique experience of an LA-HLH case series in a predominantly Hispanic population in South Florida. The diagnosis is challenging, often delayed, and the prognosis is dismal in refractory cases despite currently available rescue therapies. Furthermore, we describe for the first time the association between HLH and PTLD ALCL.


Limitations and possibilities of low cell number ChIP-seq.
Chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq) offers high resolution, genome-wide analysis of DNA-protein interactions. However, current standard methods require abundant starting material in the range of 1-20 million cells per immunoprecipitation, and remain a bottleneck to the acquisition of biologically relevant epigenetic data. Using a ChIP-seq protocol optimised for low cell numbers (down to 100,000 cells/IP), we examined the performance of the ChIP-seq technique on a series of decreasing cell numbers. We present an enhanced native ChIP-seq method tailored to low cell numbers that represents a 200-fold reduction in input requirements over existing protocols. The protocol was tested over a range of starting cell numbers covering three orders of magnitude, enabling determination of the lower limit of the technique. At low input cell numbers, increased levels of unmapped and duplicate reads reduce the number of unique reads generated, and can drive up sequencing costs and affect sensitivity if ChIP is attempted from too few cells. The optimised method presented here considerably reduces the input requirements for performing native ChIP-seq. It extends the applicability of the technique to isolated primary cells and rare cell populations (e.g. biobank samples, stem cells), and in many cases will alleviate the need for cell culture and any associated alteration of epigenetic marks. However, this study highlights a challenge inherent to ChIP-seq from low cell numbers: as cell input numbers fall, levels of unmapped sequence reads and PCR-generated duplicate reads rise. We discuss a number of solutions to overcome the effects of reducing cell number that may aid further improvements to ChIP performance.


Model of three-dimensional structure of VDR bound with Vitamin D3 analogs substituted at carbon-2.
All Vitamin D analogs possessing the A ring modified at C-2 and showing calcemic activities nest themselves in the VDR binding pocket, oriented towards Tyr 143. Such topology resembles the position of the Vitamin D hormone in hVDRmt [Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 5491]. Conversely, inactive 2beta-methyl-19-nor-analogs anchor the receptor cavity in a distinguishably different manner, namely by their side chain. Moreover, these inactive vitamins have a different conformation around C(6)-C(7) bond. Topology of modeled complexes suggests that a Vitamin D analog will be biologically active if its intercyclic 5,7-diene moiety assumes parallel position to tryptophan aromatic rings; such orientation allows for creating pi-pi interactions. The broad comparison of calcemic activities of the analogs, and their interactions with VDR, revealed that specific hydrophobic contacts are involved in bone calcium mobilization (BCM). These contacts occur between 21-methyl group and a few amino acids (V296, L305 and L309), conserved in the nuclear receptor superfamily. In the inactive 2beta-methyl-19-nor analogs such contacts do not exist. We speculate that two hydrophobic receptor patches, being in close contact with ligand methyl groups, might influence interaction with co-modulators involved in calcium homeostasis.


Arginase 1 promotes retinal neurovascular protection from ischemia through suppression of macrophage inflammatory responses.
The lack of effective therapies to limit neurovascular injury in ischemic retinopathy is a major clinical problem. This study aimed to examine the role of ureohydrolase enzyme, arginase 1 (A1), in retinal ischemia-reperfusion (IR) injury. A1 competes with nitric oxide synthase (NOS) for their common substrate L-arginine. A1-mediated L-arginine depletion reduces nitric oxide (NO) formation by NOS leading to vascular dysfunction when endothelial NOS is involved but prevents inflammatory injury when inducible NOS is involved. Studies were performed using wild-type (WT) mice, global A1+/- knockout (KO), endothelial-specific A1 KO, and myeloid-specific A1 KO mice subjected to retinal IR injury. Global as well as myeloid-specific A1 KO mice showed worsened IR-induced neuronal loss and retinal thinning. Deletion of A1 in endothelial cells had no effect, while treatment with PEGylated (PEG) A1 improved neuronal survival in WT mice. In addition, A1+/- KO mice showed worsened vascular injury manifested by increased acellular capillaries. Western blotting analysis of retinal tissue showed increased inflammatory and necroptotic markers with A1 deletion. In vitro experiments showed that macrophages lacking A1 exhibit increased inflammatory response upon LPS stimulation. PEG-A1 treatment dampened this inflammatory response and decreased the LPS-induced metabolic reprogramming. Moreover, intravitreal injection of A1 KO macrophages or systemic macrophage depletion with clodronate liposomes increased neuronal loss after IR injury. These results demonstrate that A1 reduces IR injury-induced retinal neurovascular degeneration via dampening macrophage inflammatory responses. Increasing A1 offers a novel strategy for limiting neurovascular injury and promoting macrophage-mediated repair.


Selective expression of CD45 isoforms on functional subpopulations of CD34+ hemopoietic cells from human bone marrow.
We have found that the small population of cells in human marrow that are characterized by their expression of CD34 can be readily subdivided into two apparently nonoverlapping subpopulations of approximate equal size, one expressing CD45RO and one CD45R. Functional studies of these subpopulations revealed that all of the primitive erythroid colony-forming cells (BFU-E) are CD34+ CD45RO+. Similarly, more primitive cells that give rise to both erythroid and granulopoietic colony-forming cells after being maintained for 5 wk on confluent irradiated long-term marrow culture feeder layers, also show this phenotype. In contrast, most granulopoietic colony-forming cells are CD34+ CD45RO- cells. The differential expression of CD45 isoforms on distinct functional subpopulations of hemopoietic cells is consistent with the concept that these molecules play an important role in the differentiation or activation of primitive, normally quiescent, hemopoietic cells. The presence of CD45RO and the lack of CD45R on human cells capable of initiating hemopoiesis in the long-term marrow culture system correspond to the reported lack of CD45R on transplantable hemopoietic stem cells in rodents and may be a useful addition to strategies for human stem cell purification, or for purging CD45R+ leukemic cells.


The role of cefiderocol in clinical practice.
Cefiderocol is a new antimicrobial with a chemical structure similar to ceftazidime and cefepime. In this review we will focus on the role of cefiderocol in different clinical scenarios produced by resistant Gram-negative microorganisms, especially to carbapenems. In infections caused by Gram-negative microorganisms, inappropriate antibiotic treatment increased the risk of mortality almost fourfold. In patients with hospital-acquired infection and septic shock; with sepsis and poor functional reserve due to fragility; in immunocompromised patients; and in those with local ecology, individual history of colonization or previous infection and risk factors for carbapenem-resistant Enterobacteriaceae (CRE) such as the presence of chronic multi-morbidities, the best option would be to start an active empirical treatment against gram-negative bacteria resistant to carbapenems and later in 24-36 h with the information obtained from the cultures we could decide on a definitive empirical or directed treatment and avoid unnecessary overuse of these antibiotics. Cefiderocol would be in these cases a good candidate due to its excellent in vitro activity against all classes of beta-lactamase-producing Gram-negatives (including carbapenemase class A, B and D producers), as well as against non-fermenting Gram-negatives such as P. aeruginosa, Acinetobacter spp. and S. maltophilia. It is necessary to optimize the use of new antibiotics such as cefiderocol, guaranteeing the best available treatment to patients while delaying the emergence and spread of resistance.


Inverted Internal Limiting Membrane Flap versus Internal Limiting Membrane Peeling for Macular Hole Retinal Detachment in High Myopia.
To compare surgical outcomes between the inverted internal limiting membrane (ILM) flap technique and ILM peeling for macular hole retinal detachment (MHRD) in eyes with high myopia. Multicenter cohort study. We retrospectively reviewed medical records of consecutive patients treated between June 2008 and September 2018 at 7 hospitals and included 100 eyes with MHRD associated with high myopia in our study. All eyes underwent vitrectomy with the inverted ILM flap technique (57 eyes) or ILM peeling (43 eyes) and were followed up for more than 6 months. We estimated odds ratios and their 95% confidence intervals (CIs) for macular hole (MH) closure using multivariate logistic regression analysis. We also examined factors associated with the postoperative best-corrected visual acuity (BCVA) at the final visit using multiple linear regression analysis. Macular hole closure and postoperative BCVA at the final visit. The MH closure rate was significantly higher in the inverted ILM flap group (80.7%) than in the ILM peeling group (37.2%; P < 0.001). Moreover, postoperative BCVA at the final visit was significantly better in the former group (0.88 ± 0.48 vs. 0.99 ± 0.48; P = 0.03). The retinal attachment rate (ILM flap, 91.2%; ILM peeling, 79.5%; P = 0.229) and recovery rates for the external limiting membrane and ellipsoid zone line (ILM flap, 10.9%; ILM peeling, 0%; P = 0.12) showed no significant intergroup differences. After adjustment for age, axis, tamponade substance, and dye for ILM staining, the inverted ILM flap technique was associated strongly and positively with MH closure (odds ratio, 7.14; 95% CI, 2.72-18.7; P = 0.001). Moreover, the inverted ILM flap technique and preoperative BCVA were associated significantly and positively with the postoperative BCVA at the final visit. Our findings suggest that the MH closure rate and postoperative visual outcome for eyes with high myopia-associated MHRD are better with the inverted ILM flap technique than with ILM peeling. Thus, vitrectomy with the inverted ILM flap technique should be considered as the initial surgery for MHRD associated with high myopia.


Why we don't come: patient perceptions on no-shows.
Patients who schedule clinic appointments and fail to keep them have a negative impact on the workflow of a clinic in many ways. This study was conducted to identify the reasons patients in an urban family practice setting give for not keeping scheduled appointments. Semistructured interviews were conducted with 34 adult patients coming to the clinic for outpatient care. Interviews were audiotaped and transcribed verbatim. A multidisciplinary team used an immersion-crystallization organizing style to analyze the content of the qualitative interviews individually and in team meetings. Participants identified 3 types of issues related to missing appointments without notifying the clinic staff: emotions, perceived disrespect, and not understanding the scheduling system. Although they discussed logistical issues of appointment keeping, participants did not identify these issues as key reasons for nonattendance. Appointment making among these participants was driven by immediate symptoms and a desire for self-care. At the same time, many of these participants experienced anticipatory fear and anxiety about both procedures and bad news. Participants did not feel obligated to keep a scheduled appointment in part because they felt disrespected by the health care system. The effect of this feeling was compounded by participants' lack of understanding of the scheduling system. The results of this study suggest that reducing no-show rates among patients who sometimes attend might be addressed by reviewing waiting times and participants' perspectives of personal respect.


High Dose of Metformin Decreases Susceptibility to Occlusive Arterial Thrombosis in Diabetic Mice.
Metformin is the most prescribed medication in Diabetes Mellitus(DM). Metformin has shown to decrease mean platelet volume, with promising antiplatelet effects. High doses of Metformin have also been associated with hypercoagulation. We hypothesize that Metformin will protect DM mice from occlusive arterial thrombus formation by altering platelet activation and mitochondrial bioenergetics. DM was developed by low dose of Streptozotocin, non-DM (healthy) mice are controls. Either vehicle or Metformin was administered twice daily via oral gavage for 7-days. Ferric chloride (FeCl3) arterial thrombosis and tail bleeding time were performed. Whole blood aggregometry, platelet activation/adhesion and mitochondrial bioenergetics were evaluated. Metformin decreased susceptibility of DM mice to arterial thrombosis. Platelet bioenergetics show DM mice have increased platelet mitochondrial respiration, but no differences were observed with Metformin treatment. In non-DM (healthy) mice, Metformin modulated ADP-dependent increase in platelet adhesion. Non-DM (healthy) mice, Metformin shortens bleeding time with faster thrombotic occlusion. Metformin also increased platelet mitochondrial maximal respiration and spare respiratory capacity uniquely in non-DM (healthy) mice. Metformin regulates platelet bioenergetics and ADP-mediated platelet function in DM mice which attenuates susceptibility to arterial thrombosis. Future studies will evaluate clinically relevant doses of Metformin that regulates thrombotic function in diabetic platelets.


Characterization of Inflammasome Components in Pig Intestine and Analysis of the Influence of Probiotic Enterococcus Faecium during an Escherichia Coli Challenge.
The aim of the present study was to investigate systematically the expression of inflammasome components in pig intestine and to analyze the influence of age and long-term supplementation with the probiotic Enterococcus faecium NCIMB 10415 (E. faecium). In order to examine probiotic effects on the inflammasomes during a challenge with pathogens, enterotoxigenic Escherichia coli (ETEC) and E. faecium were directly added to pig jejunum in Ussing chambers. The mRNA expression of inflammasome components generally decreased in an oral-aboral direction in intestinal tissues. In 29-day-old piglets, the expression levels of NLRP3 were significantly higher and ASC (apoptotic speck-like protein containing a caspase recruitment domain) expression were lower compared with those in the ileum of 70-day-old pigs (p ≤ 0.05). Long-term supplementation with E. faecium significantly increased ASC expression levels in the jejunum and ileum of 29-day-old piglets compared to control animals (p ≤ 0.05). Ex vivo addition of ETEC or E. faecium did not affect mRNA expression of inflammasome components significantly, whereas IL-1β protein release was significantly elevated in ETEC-incubated jejunum (p ≤ 0.05), providing evidence for the functional activation of the inflammasome, which was prevented by pre-incubation with E. faecium. We conclude that pre-incubation with E. faecium has a protective effect during ETEC challenge; this effect is probably not located at the inflammasome transcription level. The results of this study of the expression and regulation of inflammasome components in pigs are similar to those obtained in humans, reinforcing the use of pigs as a suitable model for translational inflammasome research.


Visuomotor adaptation to a visual rotation is gravity dependent.
Humans perform vertical and horizontal arm motions with different temporal patterns. The specific velocity profiles are chosen by the central nervous system by integrating the gravitational force field to minimize energy expenditure. However, what happens when a visuomotor rotation is applied, so that a motion performed in the horizontal plane is perceived as vertical? We investigated the dynamic of the adaptation of the spatial and temporal properties of a pointing motion during prolonged exposure to a 90° visuomotor rotation, where a horizontal movement was associated with a vertical visual feedback. We found that participants immediately adapted the spatial parameters of motion to the conflicting visual scene in order to keep their arm trajectory straight. In contrast, the initial symmetric velocity profiles specific for a horizontal motion were progressively modified during the conflict exposure, becoming more asymmetric and similar to those appropriate for a vertical motion. Importantly, this visual effect that increased with repetitions was not followed by a consistent aftereffect when the conflicting visual feedback was absent (catch and washout trials). In a control experiment we demonstrated that an intrinsic representation of the temporal structure of perceived vertical motions could provide the error signal allowing for this progressive adaptation of motion timing. These findings suggest that gravity strongly constrains motor learning and the reweighting process between visual and proprioceptive sensory inputs, leading to the selection of a motor plan that is suboptimal in terms of energy expenditure.


Adolescent Marijuana Use, Marijuana-Related Perceptions, and Use of Other Substances Before and After Initiation of Retail Marijuana Sales in Colorado (2013-2015).
Due to the recentness of changes to marijuana policies in a number of states, the effect on adolescent use and perceptions is not yet well understood. This study examines change in adolescent marijuana use and related perceptions in Colorado, before and after the implementation of legal commercial sale of recreational marijuana for adults starting on January 1, 2014. The data are from a repeated cross-sectional survey of a representative sample of Colorado high school students, with separately drawn samples surveyed in fall 2013 (prior to implementation) and fall 2015 (18 months after implementation). We examined change in the prevalence of adolescent marijuana use, measured by lifetime use, past 30-day use, frequent use, and use on school property. To consider the possibility of heterogeneity in the change in marijuana use, we examined change in past 30-day marijuana use by demographic characteristics (sex, grade, race/ethnicity), school characteristics (poverty, percent minority), urbanicity of the school district, and whether the city or county permitted retail marijuana stores. There was an absence of significant effects for change in lifetime or past 30-day marijuana use. Among those reporting past 30-day use, frequent use and use on school property declined. There was a significant decline in the perceived harm associated with marijuana use, but we did not find a significant effect for perceived wrongfulness, perceived ease of access, or perceived parental disapproval. We did not find significant variability in past 30-day use by demographic characteristics or by school and community factors from 2013 to 2015. We did not find a significant effect associated with the introduction of legal sales of recreational marijuana to adults in Colorado on adolescent (illegal) use, but ongoing monitoring is warranted, including consideration of heterogeneity in the effects of marijuana policies.


First molecular detection of Apis mellifera filamentous virus in honey bees (Apis mellifera) in Hungary.
Western honey bees (Apis mellifera) are important pollinators in the ecosystem and also play a crucial economic role in the honey industry. During the last decades, a continuous decay was registered in honey bee populations worldwide, including Hungary. In our study, we used metagenomic approaches and conventional PCR screening on healthy and winter mortality affected colonies from multiple sites in Hungary. The major goal was to discover presumed bee pathogens with viral metagenomic experiments and gain prevalence and distribution data by targeted PCR screening. We examined 664 honey bee samples that had been collected during winter mortality from three seemingly healthy colonies and from one colony infested heavily by the parasitic mite Varroa destructor in 2016 and 2017. The subsequent PCR screening of honey bee samples revealed the abundant presence of Apis mellifera filamentous virus (AmFV) for the first time in Central Europe. Based on phylogeny reconstruction, the newly-detected virus was found to be most closely related to a Chinese AmFV strain. More sequence data from multiple countries would be needed for studying the detailed phylogeographical patterns and worldwide spreading process of AmFV. Here we report the prevalent presence of this virus in Hungarian honey bee colonies.


Enabling freehand lateral scanning of optical coherence tomography needle probes with a magnetic tracking system.
We present a high-resolution three-dimensional position tracking method that allows an optical coherence tomography (OCT) needle probe to be scanned laterally by hand, providing the high degree of flexibility and freedom required in clinical usage. The method is based on a magnetic tracking system, which is augmented by cross-correlation-based resampling and a two-stage moving window average algorithm to improve upon the tracker's limited intrinsic spatial resolution, achieving 18 µm RMS position accuracy. A proof-of-principle system was developed, with successful image reconstruction demonstrated on phantoms and on ex vivo human breast tissue validated against histology. This freehand scanning method could contribute toward clinical implementation of OCT needle imaging.