Frequency of respiratory virus infections and next-generation analysis of influenza A/H1N1pdm09 dynamics in the lower respiratory tract of patients admitted to the ICU.
Recent molecular diagnostic methods have significantly improved the diagnosis of viral pneumonia in intensive care units (ICUs). It has been observed that 222G/N changes in the HA gene of H1N1pdm09 are associated with increased lower respiratory tract (LRT) replication and worse clinical outcome. In the present study, the frequency of respiratory viruses was assessed in respiratory samples from 88 patients admitted to 16 ICUs during the 2014-2015 winter-spring season in Lombardy. Sixty-nine out of 88 (78.4%) patients were positive for a respiratory viral infection at admission. Of these, 57/69 (82.6%) were positive for influenza A (41 A/H1N1pdm09 and 15 A/H3N2), 8/69 (11.6%) for HRV, 2/69 (2.9%) for RSV and 2/69 (2.9%) for influenza B. Phylogenetic analysis of influenza A/H1N1pdm09 strains from 28/41 ICU-patients and 21 patients with mild respiratory syndrome not requiring hospitalization, showed the clear predominance of subgroup 6B strains. The median influenza A load in LRT samples of ICU patients was higher than that observed in the upper respiratory tract (URT) (p<0.05). Overall, a greater number of H1N1pdm09 virus variants were observed using next generation sequencing on partial HA sequences (codons 180-286) in clinical samples from the LRT as compared to URT. In addition, 222G/N/A mutations were observed in 30% of LRT samples from ICU patients. Finally, intra-host evolution analysis showed the presence of different dynamics of viral population in LRT of patients hospitalized in ICU with a severe influenza infection.


Enhanced performance of dye-sensitized solar cells based on TiO2 nanotube membranes using an optimized annealing profile.
We use free-standing TiO2 nanotube membranes that are transferred onto FTO slides in front-side illuminated dye-sensitized solar cells (DSSCs). We investigate the key parameters for solar cell arrangement of self-ordered anodic TiO2 nanotube layers on the FTO substrate, namely the influence of the annealing procedure on the DSSC light conversion efficiency. The results show that using an optimal temperature annealing profile can significantly enhance the DSSC efficiency (in our case η = 9.8%), as it leads to a markedly lower density of trapping states in the tube oxide, and thus to strongly improved electron transport properties.


Prevalence of burnout and its correlates among residents in a tertiary medical center in Kerala, India: A cross-sectional study.
Residents work in emotionally demanding environments with multiple stressors. The risk for burnout is high in them and it has significant negative consequences for their career. Burnout is also associated with consequences in terms of physical and mental health including insomnia, cardiovascular disease, depression and suicidal ideation. Thus, the study aimed to study the prevalence of burn out and its correlates among interns and residents at Government Medical College, Thiruvananthapuram, Kerala, India. Cross Sectional Study at Government Medical College, Thiruvananthapuram, Kerala, India. It was a cross Sectional study of 558 interns and residents of Government Medical College, Thiruvananthapuram, Kerala, India. Data was collected which included the Copenhagen Burnout Inventory [CBI]which assesses burnout in the dimensions of Personal burnout, Work burnout and Patient related burnout, with a cut off score of 50 for each dimension. Age, sex, year of study, department the resident belonged to, or an intern, junior resident or a super speciality senior resident (resident doing super speciality course after their post graduate masters degree) were the correlates assessed. Univariate analysis. More than one third of the participants were found to have burnout in one or another dimension of the CBI. Burnout was found to be the highest among the interns in the domains of personal burnout (64.05 %) and patient related burnout (68.62 %) and in junior residents for work related burnout (40%). Super specialty senior residents had the least prevalence of burnout in all three dimensions. Among the residents, Non Medical/Non Surgical residents had the least prevalence of burnout in all three dimensions, whereas surgical speciality residents had the highest of personal burnout (57.92 %) and Medical speciality residents had the highest patient related burnout (27.13%). Both medical and surgical specialty residents had equal prevalence of work burnout. The study also showed that as the number of years of residency increased, the burnout also increased in all three dimensions. A between gender difference in burnout was not noticed in our study. Burnout was found to be present in a large number of residents in our study. Nationwide studies and assessment of more correlates will be needed to understand this phenomenon and also for formulating measures for preventing and managing it.


The transcriptional landscape of hematopoietic stem cell ontogeny.
Transcriptome analysis of adult hematopoietic stem cells (HSCs) and their progeny has revealed mechanisms of blood differentiation and leukemogenesis, but a similar analysis of HSC development is lacking. Here, we acquired the transcriptomes of developing HSCs purified from >2,500 murine embryos and adult mice. We found that embryonic hematopoietic elements clustered into three distinct transcriptional states characteristic of the definitive yolk sac, HSCs undergoing specification, and definitive HSCs. We applied a network-biology-based analysis to reconstruct the gene regulatory networks of sequential stages of HSC development and functionally validated candidate transcriptional regulators of HSC ontogeny by morpholino-mediated knockdown in zebrafish embryos. Moreover, we found that HSCs from in vitro differentiated embryonic stem cells closely resemble definitive HSCs, yet lack a Notch-signaling signature, likely accounting for their defective lymphopoiesis. Our analysis and web resource will enhance efforts to identify regulators of HSC ontogeny and facilitate the engineering of hematopoietic specification.


Adherence to adjuvant hormonal therapy and its relationship to breast cancer recurrence and survival among low-income women.
Although clinical trials have demonstrated the benefit of adjuvant hormonal therapy for hormone receptor-positive breast cancer, it is not known whether poor medication adherence might impact outcomes, particularly in the context of a low-income population traditionally underrepresented in clinical trials. We explored the relationship between adherence to tamoxifen or selective aromatase inhibitors with cancer recurrence and death in a low-income, Medicaid-insured population. Using a Medicaid claims-tumor registry and National Death Index data, we evaluated adherence to adjuvant hormonal therapy [defined by the medication possession ratio (MPR)], cancer recurrence, and cancer-specific survival for female breast cancer diagnosed from 1998 to 2002, in North Carolina. Multivariate Cox proportional hazards models and logistic regression models were used to examine the role of adherence on cancer recurrence and survival. The sample consisted of 857 cases, mean age 67.7 years, 56.9% white, 60.9% local stage, with a mean follow-up of 4.4 years. Mean first-year MPR was 77%. MPR adherence was not significantly associated with cancer-related death [adjusted hazards ratio=1.18 (95% confidence interval, 0.54-2.59)], or recurrence [adjusted odds ratio=1.49 (95% confidence interval, 0.78-2.84)]. There was also no significant interaction between adherence and use of concurrent CYP2D6 enzyme inhibitors. Hormonal therapy adherence was not associated with breast cancer outcomes in this low-income population with relatively poor adherence. Although suboptimal adherence is considered to be an important clinical problem, its effects on breast cancer outcomes may be masked by patient genetic profiles, tumor characteristics, and behavioral factors.


Detection of epileptogenic focus using advanced dynamic statistical parametric mapping with magnetoencephalography in a patient with MRI-negative focal cortical dysplasia type IIB.
Advanced dynamic statistical parametric mapping (AdSPM) with magnetoencephalography (MEG) was used to identify MRI-negative epileptogenic lesions in this report. A 15-year-old girl had MRI-negative and pharmacology-resistant focal-onset epilepsy. She experienced two types of seizures. Type I consisted of her arousal from sleep, staring, and a forced head-turning movement to the left, followed by secondary generalization. Type II began with an aura of dizziness followed by staring and postictal headache with fatigue. Scalp video-electroencephalography (EEG) captured two type I seizures originating from the right frontocentral region. MEG showed scattered dipoles over the right frontal region. AdSPM identified the spike source at the bottom of the right inferior frontal sulcus. Intracranial video-EEG captured one type I seizure, which originated from the depth electrode at the bottom of the sulcus and correlated with the AdSPM spike source. Accordingly, the patient underwent resection of the middle and inferior frontal gyri, including the AdSPM-identified spike source. Histopathological examination revealed that the patient had focal cortical dysplasia type IIB. To date, the patient has been seizure free for 2 years while receiving topiramate treatment. This is the first preliminary report to identify MRI-negative epilepsy using AdSPM. Further investigation of AdSPM would be valuable for cases of MRI-negative focal epilepsy.


Energy Requirements of Odor Transduction in the Chemosensory Cilia of Olfactory Sensory Neurons Rely on Oxidative Phosphorylation and Glycolytic Processing of Extracellular Glucose.
The mechanisms that power the physiological events occurring in cilia, flagella, and microvilli are of fundamental importance for the functions of these important and ubicuous organelles. The olfactory epithelium is mostly populated by ciliated olfactory sensory neurons (OSNs) and surrounding sustentacular cells (SCs) with apical microvilli. The only OSN dendrite extends to the surface forming a knob projecting several chemosensory cilia of ∼50 × 0.2 μm, devoid of inner membranes embedded in a mucus layer. Upon odorant binding, odor receptors couple to G-protein activating adenylyl cyclase, producing cAMP. cAMP opens cyclic nucleotide-gated channels allowing a Ca2+ influx that opens Ca2+-activated Cl- channels, generating the receptor potential. Many enzymes are activated in chemotransduction to hydrolyze ATP. The knob contains approximately two mitochondria; assuming that the cilia ATP is 1 mm and diffuses along it at ∼10 μm in 500 ms, ATP from the knob mitochondria may not fulfill the demands of transduction over the full length of the cilium, which suggests an additional ATP source. We measured millimolar glucose in rat mucus; we detected glucose transporter GLUT3 in rat and toad (Caudiverbera caudiverbera) OSN cilia, SC microvilli, and glycolytic enzymes in rat cilia. We also found that the cilia and knob can incorporate and accumulate 2-deoxyglucose (glucose analog), but not when blocking GLUT. Glucose removal and the inhibition of glycolysis or oxidative phospholylation impaired the odor response. This evidence strongly suggests that glycolysis in the cilia and knob oxidative phosphorylation together fuel chemotransduction.SIGNIFICANCE STATEMENT How processes occurring in cilia and flagella are powered is a matter of general interest. Substantial progress has been made in unraveling the sensory transduction mechanisms, commonly occurring in such structures; however, the energy sources powering them have been scarcely explored. Accessibility to the specialized sensory organelles and their small dimensions have been limiting factors. Olfactory sensory neurons chemosensory cilia are elongated, mucus embedded, fully exposed structures particularly amenable for a multidisciplinary study of this problem, as done here. We demonstrate the occurrence and functionality of glucose uptake and glycolysis in the cilia. We support that odor transduction relies on ATP generated by oxidative phosphorylation in the dendrite and glycolytically in the cilia using glucose internalized from the mucus.


The role of tree nuts and peanuts in the prevention of coronary heart disease: multiple potential mechanisms.
Epidemiologic and clinical trial evidence has demonstrated consistent benefits of nut and peanut consumption on coronary heart disease (CHD) risk and associated risk factors. The epidemiologic studies have reported various endpoints, including fatal CHD, total CHD death, total CHD, and nonfatal myocardial infarct. A pooled analysis of 4 U.S. epidemiologic studies showed that subjects in the highest intake group for nut consumption had an approximately 35% reduced risk of CHD incidence. The reduction in total CHD death was due primarily to a decrease in sudden cardiac death. Clinical studies have evaluated the effects of many different nuts and peanuts on lipids, lipoproteins, and various CHD risk factors, including oxidation, inflammation, and vascular reactivity. Evidence from these studies consistently shows a beneficial effect on these CHD risk factors. The LDL cholesterol-lowering response of nut and peanut studies is greater than expected on the basis of blood cholesterol-lowering equations that are derived from changes in the fatty acid profile of the diet. Thus, in addition to a favorable fatty acid profile, nuts and peanuts contain other bioactive compounds that explain their multiple cardiovascular benefits. Other macronutrients include plant protein and fiber; micronutrients including potassium, calcium, magnesium, and tocopherols; and phytochemicals such as phytosterols, phenolic compounds, resveratrol, and arginine. Nuts and peanuts are food sources that are a composite of numerous cardioprotective nutrients and if routinely incorporated in a healthy diet, population risk of CHD would therefore be expected to decrease markedly.


Amphetamine derivatives induce locomotor hyperactivity by acting as indirect serotonin agonists.
Derivatives of amphetamine are potent releasers of both dopamine (DA) and serotonin (5-HT), but the relative contributions of DA and 5-HT release to the behavioral effects of these drugs have not been established. Previously, S-(+)3,4-methylenedioxymethamphetamine (S-(+)MDMA) was found to produce locomotor hyperactivity in rats which was dependent on 5-HT release. The present study found that MBDB (1.25, 2.5, 5.0 or 10.0 mg/kg), the alpha-ethyl derivative of MDMA that produces little or no direct DA release, also induced locomotor hyperactivity that lasted for greater than 60 min after the 5.0 and 10.0 mg/kg doses. MBDB produced spatial patterns of locomotor hyperactivity and suppression of exploratory activity (holepokes and rearings) very similar to the behavioral syndrome produced by MDMA. MBDB-induced hyperactivity was blocked by pretreatment with the selective 5-HT uptake inhibitor fluoxetine (2.5 or 10 mg/kg), suggesting that MBDB produced behavioral effects via uptake-carrier mediated release of 5-HT. Similarly, fluoxetine pretreatment blocked the locomotor hyperactivity produced by S-(+)3,4-methylenedioxyamphetamine (3.0 mg/kg) or p-chloroamphetamine (2.5 mg/kg), supporting a serotonergic basis for the action of these drugs. Tissue levels of 5-HT and its metabolite 5-HIAA were decreased 40 min after administration of S-(+)MDMA (3.0 mg/kg) or MBDB (5.0 mg/kg), and these decreases were prevented by fluoxetine pretreatment. S-(+)MDMA also produced a fluoxetine-sensitive increase of tissue DA levels, suggesting that 5-HT release may indirectly result in increased DA release, although MBDB did not significantly increase DA levels.(ABSTRACT TRUNCATED AT 250 WORDS)


Comparative Evaluation of Radioiodine and Technetium-Labeled DARPin 9_29 for Radionuclide Molecular Imaging of HER2 Expression in Malignant Tumors.
High expression of human epidermal growth factor receptor 2 (HER2) in breast and gastroesophageal carcinomas is a predictive biomarker for treatment using HER2-targeted therapeutics (antibodies trastuzumab and pertuzumab, antibody-drug conjugate trastuzumab DM1, and tyrosine kinase inhibitor lapatinib). Radionuclide molecular imaging of HER2 expression might permit stratification of patients for HER2-targeting therapies. In this study, we evaluated a new HER2-imaging probe based on the designed ankyrin repeat protein (DARPin) 9_29. DARPin 9_29 was labeled with iodine-125 by direct radioiodination and with [99mTc]Tc(CO)3 using the C-terminal hexahistidine tag. DARPin 9_29 preserved high specificity and affinity of binding to HER2-expressing cells after labeling. Uptake of [125I]I-DARPin 9_29 and [99mTc]Tc(CO)3-DARPin 9_29 in HER2-positive SKOV-3 xenografts in mice at 6 h after injection was 3.4 ± 0.7 %ID/g and 2.9 ± 0.7 %ID/g, respectively. This was significantly (p < 0.00005) higher than the uptake of the same probes in HER2-negative Ramos lymphoma xenografts, 0.22 ± 0.09 %ID/g and 0.30 ± 0.05 %ID/g, respectively. Retention of [125I]I-DARPin 9_29 in the lung, liver, spleen, and kidneys was appreciably lower compared with [99mTc]Tc(CO)3-DARPin 9_29, which resulted in significantly (p < 0.05) higher tumor-to-organ ratios. The biodistribution data were confirmed by SPECT/CT imaging. In conclusion, radioiodine is a preferable label for DARPin 9_29.


Dopamine gene therapy for Parkinson's disease in a nonhuman primate without associated dyskinesia.
In Parkinson's disease, degeneration of specific neurons in the midbrain can cause severe motor deficits, including tremors and the inability to initiate movement. The standard treatment is administration of pharmacological agents that transiently increase concentrations of brain dopamine and thereby discontinuously modulate neuronal activity in the striatum, the primary target of dopaminergic neurons. The resulting intermittent dopamine alleviates parkinsonian symptoms but is also thought to cause abnormal involuntary movements, called dyskinesias. To investigate gene therapy for Parkinson's disease, we simulated the disease in macaque monkeys by treating them with the complex I mitochondrial inhibitor 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, which induces selective degeneration of dopamine-producing neurons. In this model, we demonstrated that injection of a tricistronic lentiviral vector encoding the critical genes for dopamine synthesis (tyrosine hydroxylase, aromatic L-amino acid decarboxylase, and guanosine 5'-triphosphate cyclohydrolase 1) into the striatum safely restored extracellular concentrations of dopamine and corrected the motor deficits for 12 months without associated dyskinesias. Gene therapy-mediated dopamine replacement may be able to correct Parkinsonism in patients without the complications of dyskinesias.


Community engagement in health status instrument development: experience with the HIV disability questionnaire.
People with HIV face a variety of health-related challenges from HIV, concurrent health conditions, and side effects of treatment. These health-related challenges may be termed disability. Community involvement in conceptualizing and understanding disability is essential to developing appropriate measurement tools. To describe a community-academic partnership in the development of a new self-administered health status instrument, the HIV Disability Questionnaire (HDQ), using a community-engaged approach. With a Community Advisory Committee, we used categories from the Episodic Disability Framework, a conceptual framework developed in an earlier stage of research, as a foundation for item generation. The purpose of the 70-item HDQ is to characterize the presence, severity, and episodic nature of disability experienced by adults with HIV. The Community Advisory Committee suggested items beyond those in the Episodic Disability Framework, refined item phrasing, order, response options, and questionnaire instructions to enhance feasibility and relevance of the HDQ to adults with HIV, advised on how to structure the questionnaire to capture the episodic nature of disability, and suggested next steps for testing and implementation. The HDQ is the first HIV-specific questionnaire to describe disability experienced by adults with HIV. Engaging community research partners in the development of the HDQ enhanced the questionnaire's relevance, feasibility, and face and content validities. This approach may be considered by others interested in health status instrument development.


Investigating the impact of a research-based integrated curriculum on self-perceived research experiences of medical students in community placements: a pre- and post-test analysis of three student cohorts.
To build research capacity among graduating medical students, the teaching of research and critical analysis was integrated into the University of Wollongong (UoW) new, graduate-entry medical curriculum. This study examined whether the self-perceived research experiences of medical students, and consequent research capability, were influenced by exposure to this innovative research and critical analysis curriculum, which incorporated a 12-month community-based research project, and associated assessment tasks. The first three medical students cohorts (N = 221) completed a self-assessment of their research experiences in ten areas of research activity. Their responses were collected: before and after they undertook an individual community-based research project within a 12-month regional/rural clinical placement. The research areas investigated by the self-assessment tool were: (i) defining a research question/idea; (ii) writing a research protocol; (iii) finding relevant literature; (iv) critically reviewing the literature; (v) using quantitative research methods; (vi) using qualitative research methods; (vii) analysing and interpreting results; (viii) writing and presenting a research report; (ix) publishing results; and (x) applying for research funding. Participation rates of 94% (207/221) pre-placement and 99% (219/221) post-placement were achieved from the three student cohorts. Following the successful completion of the research projects and their assessment tasks, the median responses were significantly higher (p < 0.05) in nine of the ten research areas. The only area of research for which there was no increase recorded for any one of the three cohorts, or overall, was (x) applying for research funding. This activity was not a component of the UoW research and critical analysis curriculum and the item was included as a test of internal validity. Significant gains were also seen between cohorts in some key research areas. Improved research capability among medical students was evidenced by increased scores in various areas of research experience in the context of successful completion of relevant assessment tasks. The results suggest that research capability of medical students can be positively influenced by the provision of a research-based integrated medical curriculum and further consolidated by authentic learning experiences, gained through conducting 'hands-on' research projects, under the supervision and mentoring of research-qualified academics.


Cancer screening and genetics: a tale of two paradigms.
The long-standing medical tradition to "first do no harm" is reflected in population-wide evidence-based recommendations for cancer screening tests that focus primarily on reducing morbidity and mortality. The conventional cancer screening process is predicated on finding early-stage disease that can be treated effectively; yet emerging genetic and genomic testing technologies have moved the target earlier in the disease development process to identify a probabilistic predisposition to disease. Genetic risk information can have varying implications for the health and well-being of patients and their relatives, and has raised important questions about the evaluation and value of risk information. This article explores the paradigms that are being applied to the evaluation of conventional cancer screening tests and emerging genetic and genomic tests of cancer susceptibility, and how these perspectives are shifting and evolving in response to advances in our ability to detect cancer risks. We consider several challenges germane to the evaluation of both categories of tests, including defining benefits and harms in terms of personal and clinical utility, addressing healthcare consumers' information preferences, and managing scientific uncertainty. We encourage research and dialogue aimed at developing a better understanding of the value of all risk information, nongenetic and genetic, to people's lives. Cancer Epidemiol Biomarkers Prev; 23(6); 909-16. ©2014 AACR.


Synthesis of Conductive Carbon Aerogels Decorated with β-Tricalcium Phosphate Nanocrystallites.
There has been substantial interest in research aimed at conductive carbon-based supports since the discovery that the electrical stimulus can have dramatic effect on cell behavior. Among these carbon-aerogels decorated with biocompatible polymers were suggested as future materials for tissue engineering. However, high reaction temperatures required for the synthesis of the aerogels tend to impair the stability of the polymeric networks. Herein, we report a synthetic route towards carbon-aerogel scaffolds decorated with biocompatible ceramic nanoparticles of tricalcium phosphate. The composites can be prepared at temperature as high as 1100 °C without significant effect on the morphology of the composite which is comparable with the original aerogel framework. Although the conductivity of the composites tends to decrease with the increasing ceramic content the measured conductivity values are similar to those previously reported on polymer-functionalized carbon-aerogels. The cell culture study revealed that the developed constructs support cell proliferation and provide good cell attachment suggesting them as potentially good candidates for tissue-engineering applications.


Augmented insulin action on rates of protein synthesis after resistance exercise in rats.
This study investigated whether insulin has a modulatory effect on protein synthesis rates in skeletal muscle after four sessions of resistance exercise. Male rats engaged in resistance exercise (Acute) that required full extension of the hindlimbs with weights over the scapula or performed the standing movement with no additional weight (Nonex). Two separate studies were conducted. Rates of protein synthesis for study 1 (Acute, n = 6; Nonex, n = 6) were assessed 16 h postexercise by incorporation of [3H]phenylalanine ([3H]F) into muscle protein by use of an in vivo flooding dose protocol. Rates of protein synthesis in soleus of Acute (100 +/- 9 nmol F.g-1.h-1) were significantly higher than in Nonex (72 +/- 9 nmol F.g-1.h-1, P < 0.05). Rates of protein synthesis were significantly higher in gastrocnemius of Acute vs. Nonex (48 +/- 7 vs. 25 +/- 2 nmol F.g-1.h-1) but not in extensor digitorum longus (EDL). Assessment of protein synthesis rates for study 2 was conducted 16 h after resistance exercise with use of [3H]F incorporation into muscle protein during in situ bilateral hindlimb perfusion, with each leg perfused simultaneously but separately. Perfusion medium for one leg, but not the other, contained insulin (6.25 ng/ml). Soleus and gastrocnemius of Acute had higher protein synthesis rates than Nonex only in the leg that received insulin. For gastrocnemius, rates of protein synthesis in Acute without insulin were significantly lower than in Nonex with or without insulin. Insulin had no effect on protein synthesis rates for any muscle in Nonex rats. Neither exercise nor insulin affected protein synthesis rates in EDL. We conclude that insulin is a necessary component in elevated protein synthesis rates after resistance exercise in muscles composed of primarily slow-or fast-twitch fibers, and that a physiological perturbation (resistance exercise in this study) is required to observe such modulation, because rates of protein synthesis in Nonex muscles were not influenced by insulin.


Germline allele-specific expression of TGFBR1 confers an increased risk of colorectal cancer.
Much of the genetic predisposition to colorectal cancer (CRC) in humans is unexplained. Studying a Caucasian-dominated population in the United States, we showed that germline allele-specific expression (ASE) of the gene encoding transforming growth factor-beta (TGF-beta) type I receptor, TGFBR1, is a quantitative trait that occurs in 10 to 20% of CRC patients and 1 to 3% of controls. ASE results in reduced expression of the gene, is dominantly inherited, segregates in families, and occurs in sporadic CRC cases. Although subtle, the reduction in constitutive TGFBR1 expression alters SMAD-mediated TGF-beta signaling. Two major TGFBR1 haplotypes are predominant among ASE cases, which suggests ancestral mutations, but causative germline changes have not been identified. Conservative estimates suggest that ASE confers a substantially increased risk of CRC (odds ratio, 8.7; 95% confidence interval, 2.6 to 29.1), but these estimates require confirmation and will probably show ethnic differences.


Sparse deep predictive coding captures contour integration capabilities of the early visual system.
Both neurophysiological and psychophysical experiments have pointed out the crucial role of recurrent and feedback connections to process context-dependent information in the early visual cortex. While numerous models have accounted for feedback effects at either neural or representational level, none of them were able to bind those two levels of analysis. Is it possible to describe feedback effects at both levels using the same model? We answer this question by combining Predictive Coding (PC) and Sparse Coding (SC) into a hierarchical and convolutional framework applied to realistic problems. In the Sparse Deep Predictive Coding (SDPC) model, the SC component models the internal recurrent processing within each layer, and the PC component describes the interactions between layers using feedforward and feedback connections. Here, we train a 2-layered SDPC on two different databases of images, and we interpret it as a model of the early visual system (V1 & V2). We first demonstrate that once the training has converged, SDPC exhibits oriented and localized receptive fields in V1 and more complex features in V2. Second, we analyze the effects of feedback on the neural organization beyond the classical receptive field of V1 neurons using interaction maps. These maps are similar to association fields and reflect the Gestalt principle of good continuation. We demonstrate that feedback signals reorganize interaction maps and modulate neural activity to promote contour integration. Third, we demonstrate at the representational level that the SDPC feedback connections are able to overcome noise in input images. Therefore, the SDPC captures the association field principle at the neural level which results in a better reconstruction of blurred images at the representational level.


Concept for estimating mitochondrial DNA haplogroups using a maximum likelihood approach (EMMA).
The assignment of haplogroups to mitochondrial DNA haplotypes contributes substantial value for quality control, not only in forensic genetics but also in population and medical genetics. The availability of Phylotree, a widely accepted phylogenetic tree of human mitochondrial DNA lineages, led to the development of several (semi-)automated software solutions for haplogrouping. However, currently existing haplogrouping tools only make use of haplogroup-defining mutations, whereas private mutations (beyond the haplogroup level) can be additionally informative allowing for enhanced haplogroup assignment. This is especially relevant in the case of (partial) control region sequences, which are mainly used in forensics. The present study makes three major contributions toward a more reliable, semi-automated estimation of mitochondrial haplogroups. First, a quality-controlled database consisting of 14,990 full mtGenomes downloaded from GenBank was compiled. Together with Phylotree, these mtGenomes serve as a reference database for haplogroup estimates. Second, the concept of fluctuation rates, i.e. a maximum likelihood estimation of the stability of mutations based on 19,171 full control region haplotypes for which raw lane data is available, is presented. Finally, an algorithm for estimating the haplogroup of an mtDNA sequence based on the combined database of full mtGenomes and Phylotree, which also incorporates the empirically determined fluctuation rates, is brought forward. On the basis of examples from the literature and EMPOP, the algorithm is not only validated, but both the strength of this approach and its utility for quality control of mitochondrial haplotypes is also demonstrated.


Chromosomics: Bridging the Gap between Genomes and Chromosomes.
The recent advances in DNA sequencing technology are enabling a rapid increase in the number of genomes being sequenced. However, many fundamental questions in genome biology remain unanswered, because sequence data alone is unable to provide insight into how the genome is organised into chromosomes, the position and interaction of those chromosomes in the cell, and how chromosomes and their interactions with each other change in response to environmental stimuli or over time. The intimate relationship between DNA sequence and chromosome structure and function highlights the need to integrate genomic and cytogenetic data to more comprehensively understand the role genome architecture plays in genome plasticity. We propose adoption of the term 'chromosomics' as an approach encompassing genome sequencing, cytogenetics and cell biology, and present examples of where chromosomics has already led to novel discoveries, such as the sex-determining gene in eutherian mammals. More importantly, we look to the future and the questions that could be answered as we enter into the chromosomics revolution, such as the role of chromosome rearrangements in speciation and the role more rapidly evolving regions of the genome, like centromeres, play in genome plasticity. However, for chromosomics to reach its full potential, we need to address several challenges, particularly the training of a new generation of cytogeneticists, and the commitment to a closer union among the research areas of genomics, cytogenetics, cell biology and bioinformatics. Overcoming these challenges will lead to ground-breaking discoveries in understanding genome evolution and function.


Bivalirudin versus heparin in non-ST and ST-segment elevation myocardial infarction-a registry-based randomized clinical trial in the SWEDEHEART registry (the VALIDATE-SWEDEHEART trial).
The optimal anticoagulant for patients with acute coronary syndrome treated with percutaneous coronary intervention (PCI) has not been validated in current practice of radial approach and pretreatment with potent P2Y12 inhibitors. Several studies have indicated increased bleeding rate and, in some instances, even increased mortality by the routine use of heparin and glycoprotein IIb/IIIa inhibitors compared to bivalirudin. Direct comparison of bivalirudin versus heparin alone has yielded contradictory results depending on study designs. The VALIDATE-SWEDEHEART trial is a multicenter, prospective, randomized, registry-based, controlled, and open-label clinical trial in patients with ST-segment elevation myocardial infarction (STEMI) or non-STEMI undergoing PCI pretreated with ticagrelor, prasugrel, or cangrelor. We hypothesize that bivalirudin is superior to heparin alone in reducing death, myocardial infarction, and major bleeding events at 180 days (primary end point). The trial will enroll 3,000 patients with STEMI and 3,000 patients with non-STEMI undergoing PCI. The trial will use a hybrid registry-based randomized clinical trial design where inclusion, randomization, and baseline data collection are performed using The Swedish Web-system for Enhancement and Development of Evidence-based care in Heart disease Evaluated According to Recommended Therapies registry. The primary composite end point (death, myocardial infarction, or major bleeding events at 180 days) will be identified through active screening after 7 and 180 days and adjudicated by a blinded central end point committee. Secondary end points and long-term outcomes will be recorded from national registries. The VALIDATE-SWEDEHEART trial is founded on a nationwide clinical registry and uses a hybrid registry-based randomized clinical trial (RRCT) design methodology to evaluate efficacy and safety of bivalirudin as compared to heparin alone for acute coronary syndrome, in a large population receiving contemporary recommended therapies including predominantly radial invasive approach and pretreatment with potent P2Y12 inhibitors.


Differential induction of dyskinesia and neuroinflammation by pulsatile versus continuous l-DOPA delivery in the 6-OHDA model of Parkinson's disease.
Neuroinflammation is associated with l-DOPA treatment in Parkinson's disease (PD), suggesting a role in l-DOPA-induced dyskinesia (LID), however it is unclear whether increased inflammation is specifically related to the dyskinetic outcome of l-DOPA treatment. Diversely from oral l-DOPA, continuous intrajejunal l-DOPA infusion is associated with very low dyskinetic outcome in PD patients. We reproduced these regimens of administration in 6-OHDA-lesioned hemiparkinsonian rats, where dyskinetic responses and striatal neuroinflammation induced by chronic pulsatile (DOPAp) or continuous (DOPAc) l-DOPA were compared. Moreover, we investigated the contribution of a peripheral inflammatory challenge with lipopolysaccharide (LPS), to DOPAp-induced dyskinetic and neuroinflammatory responses. Rats 6-OHDA-infused in the medial forebrain bundle received two weeks treatment with DOPAp, DOPAc via subcutaneous osmotic minipumps, or DOPAp followed by DOPAc. l-DOPA plasma levels were measured in all experimental groups. An independent group of rats received one peripheral dose of LPS 24h before DOPAp treatment. Abnormal involuntary movements (AIMs) were evaluated as a rat model of LID. Immunoreactivity (IR) for OX-42, microglial and neuronal TNF-α, iNOS and GFAP was quantified in denervated and contralateral striatum. In addition, serum TNF-α was measured. The 6-OHDA denervation induced a mild microgliosis in the striatum two weeks after neurotoxin infusion, and increased TNF-α IR in microglia. Rats receiving the DOPAp treatment developed AIMs and displayed increased striatal OX-42, microglial TNF-α, iNOS and GFAP. Moreover, TNF-α IR was also increased in a subpopulation of striatal neurons. Conversely, DOPAc did not induce AIMs or inflammatory responses in either drug-naïve animals or rats that were previously dyskinetic when exposed to DOPAp. Serum TNF-α was not altered by any l-DOPA treatment. LPS pre-treatment increased the degree of DOPAp-induced AIMs and striatal IR for OX-42, TNF-α, iNOS and GFAP. Altogether the present findings indicate that in the 6-OHDA model, chronic l-DOPA induces striatal inflammatory responses, which however depend upon the administration regimen and the dyskinetic outcome of drug treatment. The potentiation of dyskinetic responses by LPS suggests a reciprocal causal link between neuroinflammation and LID.


Volition and eye movements.
Although the conceptual distinction between voluntary and automatic acts seems intuitively obvious, its neural basis remains opaque. Assigning volition--or some paraphrase such as action selection--to discrete parts of the brain arguably tells us nothing about what volition actually is in neural terms. Equally, exploring the relative sensitivity of discrete brain areas to manipulations of action choice, including its asymptote--free choice--would only be informative if voluntary processes could thereby be reliably isolated. Unfortunately, such manipulations are subject to ineliminable confounds, such as the complexity of the underlying condition-action associations. Here we propose an adaptation of a classic oculomotor task--saccadic choice with asynchronous targets--where the processes engaged in free choice manifest as interference in the performance of an automatic task, thereby circumventing the difficulties in parameterising volition. We suggest that this task may be useful in probing deficits in voluntary action in pathological states.


Oncology Nurses' Knowledge of Pharmacogenomics Before and After Implementation of an Education Module.
To assess the efficacy of an interactive continuing education module in improving knowledge of pharmacogenomics in oncology nursing practice. 434 inpatient and outpatient oncology nurses from a large teaching hospital in Florida and oncology nurses who practice in North Carolina. An interactive continuing education module was created based on key information elicited from a focus group of inpatient and outpatient oncology nurses regarding their lack of knowledge on pharmacogenomics. A pre-/post-test design was implemented. Purposive sampling of oncology nurses was used. The mean pretest score was 72.7 and the post-test score was 85.9. A statistically significant difference was found between these scores. No difference in scores were found between the oncology nurses employed at urban hospitals compared to nurses at community hospitals or outpatient settings. Educational opportunities for pharmacogenomics should be threaded throughout nursing competencies. The continuing education module in the current article has been shown to significantly improve oncology nurses' knowledge of genomic and pharmacogenomic information.


Use of capillary electrophoresis to quantitate carbamylated hemoglobin concentrations in dogs with renal failure.
To evaluate quantification of the amount of carbamylated hemoglobin (CarbHb), using capillary electrophoresis (CE) and a new dynamic capillary coating system to separate hemoglobin derivatives, and to assess the use of CarbHb amounts to evaluate long-term urea exposure and differential diagnoses of azotemia in dogs. 8 dogs with renal failure, 2 dogs with diabetes mellitus, and 7 control dogs. Optimal analytic conditions for separation of CarbHb and other hemoglobin derivatives in blood samples obtained from dogs were determined, using a commercial analysis system developed for the detection of glycohemoglobin Hb A1c (GlycHb) in human blood samples. Relative content of hemoglobin derivatives in blood from 10 dogs with renal failure or endocrine diseases were compared with values for 7 dogs without renal or endocrine diseases. Satisfactory resolution of hemoglobin derivatives was obtained, which permitted identification and quantitation of the amount of CarbHb as a percentage of the total amount of hemoglobin. Normal or increased amounts of GlycHb did not interfere with CarbHb analysis. Dogs with chronic renal failure had considerably higher peak amounts of CarbHb than dogs with acute renal failure, a dog with chronic renal failure that was treated by use of hemodialysis, or dogs without renal disease. Amounts of CarbHb in blood samples obtained from dogs can be readily quantified by use of capillary electrophoresis. Assessment of the amount of CarbHb can be used to facilitate evaluation of the cause of azotemia in dogs.


Fear-avoidance and endurance-related responses to pain: new models of behavior and their consequences for clinical practice.
Patients' beliefs and expectations about their pain have been identified as important disabling factors in chronic musculoskeletal pain. Besides fear-avoidance beliefs and pain-related fear, cognitions such as thought suppression as well as pain/task persistence behavior have been shown to be associated with pain and disability. The aim of this report is to present a critical evaluation of research, based on the avoidance-endurance model of pain. A qualitative review of the literature concerning the occurrence of fear-avoidance- versus endurance-related pain responses as well as concerning the association of these pain responses and different self-reported and overtly measured outcomes. While consistent evidence has confirmed the role of fear-avoidance responses to pain and pain-related disability measured via self-report, the influence of fear-avoidance responses on objectively assessed physical activity is less clear. There is preliminary evidence that avoidance-endurance model based subgroups showing a pattern of cognitions of thought suppression, anxious/depressive mood and task/pain persistence behavior (distress endurance responses) or a pattern of cognitions of ignoring/minimizing pain, positive mood despite pain and task/pain persistence behavior (eustress endurance responses) will develop more pain prospectively and show higher levels of specific strain postures, measured by accelerometer, than patients showing adaptive pain responses. Although both, fear-avoidance and endurance responses have been identified in patients with chronic musculoskeletal pain, currently evidence to confirm their hypothesized consequences for daily functioning is incomplete. Finally, thoughts on the development of differentially targeted and individually scheduled behavioral interventions are reported, including suggestions for further research.


Do premorbid predictors of alcohol dependence also predict the failure to recover from alcoholism?
In a search for viable endophenotypes of alcoholism, this longitudinal study attempted to identify premorbid predictors of alcohol dependence that also predicted the course of alcoholism. The 202 male subjects who completed a 40-year follow-up were originally selected from a Danish birth cohort (N = 9,182). Two thirds of the subjects were high-risk biological sons of treated alcoholics. A large number of measures (361) were obtained at different periods before any subject had developed an alcohol-use disorder. At age 40, a psychiatrist provided mutually exclusive lifetime diagnoses of alcohol abuse or alcohol dependence that were characterized as currently active or currently in remission according to Diagnostic and Statistical Manual of Mental Disorders, Third Edition, Revised, course specifiers. The majority of subjects with a diagnosis of alcohol abuse were in remission at age 40 compared with those with a diagnosis of alcohol dependence (88% vs. 58%). Treatment did not predict remission. Fourteen of the 18 predictors of remission that also predicted dependence were submitted to an exploratory factor analysis (varimax). Two premorbid dimensions were identified: cognitive efficiency and early behavioral dyscontrol in childhood. Both factors predicted the failure to remit (low cognitive efficiency and high behavioral dyscontrol) even when lifetime alcoholism severity was controlled. This 4-decade study found a striking disconnect between measures that predicted alcohol dependence and measures that predicted remission from alcohol dependence. Reduced cognitive efficiency and increased behavioral dyscontrol may be basic to gaining a fuller understanding of the etiology of alcoholism.


Bioactive hydrogel-filament scaffolds for nerve repair and regeneration.
The design of novel biomaterials is crucial for the advancement of tissue engineering in nerve regeneration. In this study we developed and evaluated novel biosynthetic scaffolds comprising collagen crosslinked with a terpolymer of poly(N-isopropylacrylamide) (PNiPAAm) as conduits for nerve growth. These collagen-terpolymer (collagen-TERP) scaffolds grafted with the laminin pentapeptide YIGSR were previously used as corneal substitutes in pigs and demonstrated enhanced nerve regeneration compared to allografts. The purpose of this project was to enhance neuronal growth on the collagen-TERP scaffolds through the incorporation of supporting fibers. Neuronal growth on these matrices was assessed in vitro using isolated dorsal root ganglia as a nerve source. Statistical significance was assessed using a one-way ANOVA. The incorporation of fibers into the collagen-TERP scaffolds produced a significant increase in neurite extension (p<0.05). The growth habit of the nerves varied with the type of fiber and included directional growth of the neurites along the surface of certain fiber types. Furthermore, the presence of fibers in the collagen-TERP scaffolds appeared to influence neurite morphology and function; neurites grown on fibers-incorporated collagen-TERP scaffolds expressed higher levels of Na channels compared to the scaffolds without fiber. Overall, our results suggest that incorporation of supporting fibers enhanced neurite outgrowth and that surface properties of the scaffold play an important role in promoting and guiding nerve regeneration. More importantly, this study demonstrates the potential value of tissue engineered collagen-TERP hybrid scaffolds as conduits in peripheral nerve repair.


Glycodelin is a potential novel follow-up biomarker for malignant pleural mesothelioma.
Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor with a short survival time arising from the mesothelial cells of the pleura. Soluble mesothelin-related peptide (SMRP), osteopontin or EFEMP1 (Fibulin-3) are well described biomarkers for malignant mesothelioma with moderate sensitivity and specificity. In this study, we characterized the expression of glycodelin, a marker for risk pregnancy, in MPM by RNA and protein analyses and investigated its potential as a MPM biomarker. We were able to detect glycodelin in the serum of MPM patients. Compared to benign lung diseases, the serum levels were significant increased. Patients with high glycodelin serum levels revealed a worse overall survival. The glycodelin serum levels correlated with the tumor response to treatment. A comparison of SMRP and glycodelin serum measurement in a large patient cohort demonstrated that the detection of both soluble factors can increase the reliable diagnostic of MPM. Glycodelin was highly expressed in MPM tumors. Analyses of a tissue micro array indicated that the immunomodulatory form glycodelin A was expressed in MPM and correlated with the survival of the patients. Altogether, glycodelin seems to be a new potential biomarker for the aggressive malignant pleural mesothelioma.


Effect of age on hypertensive stimuli and the development of hypertension in Brattleboro rats.
1. In rats with inherited diabetes insipidus, unilateral nephrectomy plus drinking of 0.6% NaCl solution (saline) did not influence blood pressure in adult rats. However, when these factors applied before puberty, they produced hypertension. 2. We therefore analysed whether saline administration before puberty or unilateral nephrectomy before puberty was more important for this hypertensive response. 3. Saline drinking was found to be necessary for the response because hypertension was elicited by unilateral nephrectomy in adult rats only if saline consumption began before puberty.


Spatio-temporal patterns of perkinsosis in the Manila clam Ruditapes philippinarum from Arcachon Bay (SW France).
Pathogens belonging to the genus Perkinsus infect many bivalve molluscan species around the world, including the Manila clam Ruditapes philippinarum. We investigated the spatial distribution of this parasite at 34 stations throughout Arcachon Bay (SW France). Prevalence of perkinsosis was 93% and mean infection abundance was 96 x 10(3) cells g(-1) wet gill. Lowest mean abundances were found close to the Leyre River mouth and a significant negative correlation was observed between mean abundance and salinity. Perkinsosis was rare at the oceanic site where salinities and other environmental parameters were stable. A second aim of this study was to survey perkinsosis during annual cycles at 4 sites within Arcachon Bay. Prevalence and intensities (+/- SE) of the disease were high, on average between 70 and 100%, and 130 x 10(3) +/- 6.7 x 10(3) cells g(-1) wet gill. No seasonal cycle was evident. Clams were infected at 9 mm shell length and infection increased with clam size. The third objective was to determine the disinfection and infection kinetics through a 21 mo reciprocal transplantation between a nearly Perkinsus sp.-free area and a highly affected site. Disinfection appeared to be a very slow process and was similar at the site with favorable conditions for Perkinsus sp. as at the site with unfavorable conditions. Conversely, infection acquisition appeared to be episodic with spatially defined areas. Consequently, the overall lack of a clear seasonal infection pattern is interpreted as the combination of episodic infection events and slow disinfection kinetics.


Cross contamination of Escherichia coli O157:H7 between lettuce and wash water during home-scale washing.
Lettuce and leafy greens have been implicated in multiple foodborne disease outbreaks. This study quantifies cross contamination between lettuce pieces in a small-scale home environment. A five-strain cocktail of relevant Escherichia coli O157:H7 strains was used. Bacterial transfer between single inoculated lettuce leaf pieces to 10 non-inoculated lettuce leaf pieces that were washed in a stainless steel bowl of water for 30 s, 1 min, 2 min, and 5 min was quantified. Regardless of washing time, the wash water became contaminated with 90-99% of bacteria originally present on the inoculated lettuce leaf piece. The E. coli O157:H7 concentration on initially inoculated leaf pieces was reduced ∼ 2 log CFU. Each initially uncontaminated lettuce leaf piece had ∼ 1% of the E. coli O157:H7 from the inoculated lettuce piece transferred to it after washing, with more transfer occurring during the shortest (30 s) and longest (5 min) wash times. In all cases the log percent transfer rates were essentially normally distributed. In all scenarios, most of the E. coli O157:H7 (90-99%) transferred from the inoculated lettuce pieces to the wash water. Washing with plain tap water reduces levels of E. coli O157:H7 on the inoculated lettuce leaf pieces, but also spreads contamination to previously uncontaminated leaf pieces.


Thermal control of virulence factors in bacteria: a hot topic.
Pathogenic bacteria sense environmental cues, including the local temperature, to control the production of key virulence factors. Thermal regulation can be achieved at the level of DNA, RNA or protein and although many virulence factors are subject to thermal regulation, the exact mechanisms of control are yet to be elucidated in many instances. Understanding how virulence factors are regulated by temperature presents a significant challenge, as gene expression and protein production are often influenced by complex regulatory networks involving multiple transcription factors in bacteria. Here we highlight some recent insights into thermal regulation of virulence in pathogenic bacteria. We focus on bacteria which cause disease in mammalian hosts, which are at a significantly higher temperature than the outside environment. We outline the mechanisms of thermal regulation and how understanding this fundamental aspect of the biology of bacteria has implications for pathogenesis and human health.


Trauma exposure and post-traumatic stress disorder in the general population.
To examine the lifetime prevalence of trauma experiences and post-traumatic stress disorder (PTSD). Questionnaire-assessed PTSD, the type of traumatic event experienced, perceived trauma impact, and trauma frequency in 1824 randomly selected men and women. PTSD lifetime prevalence was estimated at 5.6% with a 1 : 2 male-to-female ratio, in spite of men reporting greater trauma exposure. The highest PTSD risk was associated with sexual and physical assault, robbery and multiple trauma experiences. Controlling for trauma type did not account for gender differences, while controlling for experienced distress did. The conditional probability for PTSD varied as a function of trauma type, frequency and impact of the event, with increased rates associated with prevalent trauma exposure and higher perceived distress. The latter accounted for the gender effect, suggesting that gender differences in PTSD in part represent a generally greater vulnerability to stress in women.


Adult stem cell response to doped bioactive borate glass.
Bioactive glasses have transformed healthcare due to their versatility. Bioactive borate glass, in particular, has shown remarkable healing properties for both hard and soft tissues. Incorporating dopants into the composition of bioactive glass helps to control mechanical properties, and it increases their usefulness for clinical applications. Using a bioactive borate glass, 13-93B3 (B3), we investigated eleven dopants on the viability and migration potential of adipose stem cells (ASCs), a therapeutic source of cells used in tissue engineering and cell therapy. Our results show that under standard cell culture conditions, only Cu-doped B3 decreased cell viability, while only Y-doped B3 attracted ASCs as it dissolved in cell culture media. Using a transwell invasion assay, priming ASCs with Co, Fe, Ga, I, Sr, or Zn-doped B3 increased their homing capacity. Because there is widespread interest in optimizing and enhancing the homing efficiency of ASCs and other therapeutic cells, we then tested if priming bone marrow mesenchymal stem cells (BMSCs) with dopants also increased their homing capacity. In the case of BMSCs, there was a significant increase in invasion when cells were primed with any of the doped-B3 glasses. This work shows that incorporating dopants into borate glasses can provide a platform for a safe and efficient method that stimulates endogenous cells and healing mechanisms.


Development of the corticothalamic projections.
In this review we discuss recent advances in the understanding of corticothalamic axon guidance; patterning of the early telencephalon, the sequence and choreography of the development of projections from subplate, layers 5 and 6. These cortical subpopulations display different axonal outgrowth kinetics and innervate distinct thalamic nuclei in a temporal pattern determined by cortical layer identity and subclass specificity. Guidance by molecular cues, structural cues, and activity-dependent mechanisms contribute to this development. There is a substantial rearrangement of the corticofugal connectivity outside the thalamus at the border of and within the reticular thalamic nucleus, a region that shares some of the characteristics of the cortical subplate during development. The early transient circuits are not well understood, nor the extent to which this developmental pattern may be driven by peripheral sensory activity. We hypothesize that transient circuits during embryonic and early postnatal development are critical in the matching of the cortical and thalamic representations and forming the cortical circuits in the mature brain.


A microfluidic traps system supporting prolonged culture of human embryonic stem cells aggregates.
The unlimited proliferative and differentiative capacities of embryonic stem cells (ESCs) are tightly regulated by their microenvironment. Local concentrations of soluble factors, cell-cell interactions and extracellular matrix signaling are just a few variables that influence ESC fate. A common method employed to induce ESC differentiation involves the formation of cell aggregates called embryoid bodies (EBs), which recapitulate early stages of embryonic development. EBs are normally formed in suspension cultures, producing heterogeneously shaped and sized aggregates. The present study demonstrates the usage of a microfluidic traps system which supports prolonged EB culturing. The traps are uniquely designed to facilitate cell capture and aggregation while offering efficient gas/nutrients exchange. A finite element simulation is presented with emphasis on several aspects critical to appropriate design of such bioreactors for ESC culture. Finally, human ESC, mouse Nestin-GFP ESC and OCT4-EGFP ESCs were cultured using this technique and demonstrated extended viability for more than 5 days. In addition, EBs developed and maintained a polarized differentiation pattern, possibly as a result of the nutrient gradients imposed by the traps bioreactor. The novel microbioreactor presented here can enhance future embryogenesis research by offering tight control of culturing conditions.


Prevalence of the sick building syndrome symptoms in office workers before and after being exposed to a building with an improved ventilation system.
To find if the prevalence of symptoms associated with sick building syndrome decreased among office workers after moving to a building with improved ventilation (after controlling for potential confounders). Workers in five buildings in 1991 all moved in 1992 into a single building with improved design, operation, and maintenance of the ventilation system. All buildings had sealed windows with mechanical ventilation, air conditioning, and humidification. Workers completed a self administered questionnaire during normal working hours in February 1991 and February 1992. The questionnaire encompassed symptoms of the eyes, nose and throat, respiratory system, skin, fatigue, headache, and difficulty concentrating, personal, psychosocial, and work related factors. During normal office hours of the same week environmental variables were measured. The study population comprised 1390 workers in 1991 and 1371 workers in 1992 who represented more than 80% of the eligible population. The prevalence of most symptoms decreased when workers moved to the new building: skin (54%), respiratory system (53%), nose and throat (46%), fatigue (44%), headache (37%), eyes (23%). These findings were all significant and remained generally similar after controlling for personal, psychosocial, and work related factors. Furthermore, more than 60% of workers symptomatic in 1991 were asymptomatic in 1992 for all types of symptoms. In contrast, less than 15% of workers were asymptomatic in 1991 but symptomatic in 1992 for all types of symptoms. In this study, the prevalence of most symptoms usually associated with the sick building syndrome decreased by 40% to 50% after workers were transferred to a building with an improved ventilation system. The results show that it is possible to diminish the prevalence of symptoms associated with the sick building syndrome among office workers occupying a building with mechanical ventilation, air conditioning, and sealed windows.


Local Integration Accounts for Weak Selectivity of Mouse Neocortical Parvalbumin Interneurons.
Dissecting the functional roles of excitatory and inhibitory neurons in cortical circuits is a fundamental goal in neuroscience. Of particular interest are their roles in emergent cortical computations such as binocular integration in primary visual cortex (V1). We measured the binocular response selectivity of genetically defined subpopulations of excitatory and inhibitory neurons. Parvalbumin (PV+) interneurons received strong inputs from both eyes but lacked selectivity for binocular disparity. Because broad selectivity could result from heterogeneous synaptic input from neighboring neurons, we examined how individual PV+ interneuron selectivity compared to that of the local neuronal network, which is primarily composed of excitatory neurons. PV+ neurons showed functional similarity to neighboring neuronal populations over spatial distances resembling measurements of synaptic connectivity. On the other hand, excitatory neurons expressing CaMKIIα displayed no such functional similarity with the neighboring population. Our findings suggest that broad selectivity of PV+ interneurons results from nonspecific integration within local networks. VIDEO ABSTRACT.


Epithelial integrin alpha 6 beta 4: complete primary structure of alpha 6 and variant forms of beta 4.
The integrin alpha 6 beta 4 is a heterodimer predominantly expressed by epithelia. While no definite receptor function has yet been assigned to it, this integrin may mediate adhesive and/or migratory functions of epithelial cells. We have determined the complete primary structure of both the alpha 6 and beta 4 subunits from cDNA clones isolated from pancreatic carcinoma cell line libraries. The deduced amino acid sequence of alpha 6 is homologous to other integrin alpha chains (18-26% identity). Antibodies to an alpha 6 carboxy terminus peptide immunoprecipitated alpha 6 beta 4 complexes from carcinoma cells and alpha 6 beta 1 complexes from platelets, providing further evidence for the association of alpha 6 with more than one beta subunit. The deduced amino acid sequence of beta 4 predicts an extracellular portion homologous to other integrin beta chains, and a unique cytoplasmic domain comprised of greater than 1,000 residues. This agrees with the structures of the beta 4 cDNAs from normal epithelial cells (Suzuki, S., and Y. Naitoh. 1990. EMBO [Eur. Mol. Biol. Organ.] J. 9:757-763; Hogervost, F., I. Kuikman, A. E. G. Kr. von dem Borne, and A. Sonnenberg. 1990. EMBO [Eur. Mol. Biol. Organ.] J. 9:765-770). Compared to these structures, however, the beta 4 cDNAs that we have cloned from carcinoma cells contain extra sequences. One of these is located in the 5'-untranslated region, and may encode regulatory sequences. Another specifies a segment of 70 amino acids in the cytoplasmic tail. Amplification by reverse transcription-polymerase chain reaction of mRNA indicated that multiple forms of beta 4 may exist, possibly due to cell-type specific alternative splicing. The unique structure of beta 4 suggests its involvement in novel cytoskeletal interactions. Consistent with this possibility, alpha 6 beta 4 is mostly concentrated on the basal surface of epithelial cells, but does not colocalize with components of adhesion plaques.


Management of Recurrent Rectourethral Fistula by York Mason Posterior Transrectal Transsphincteric Approach.
Rectourethral fistula (RUF) may develop after ureterovesical and rectal intervention or radiation therapy (RT) rarely, but it is associated with significant morbidity and mortality. The patient will typically present with pneumaturia, faecaluria, and urinary drainage from the rectum. Diagnosis can be easily done with digital rectal examination, cystography, and urethrocystoscopy. Conservative supportive management of RUF does not appear to be successful in most patients, and management with surgical intervention remains the best treatment option. Several surgical techniques have been described including transabdominal, transanal, transperineal, combined abdominoperineal, anterior and posterior transsphincteric, transsacral, laparoscopic, robotic, and endoscopic minimally invasive approaches. There have been very few data about treatment of recurrent RUF. We would like to report the management of recurrent RUF following transurethral resection of prostate and RT for prostate carcinoma in an immunosuppressed, 75-year-old patient by York Mason posterior transrectal transsphincteric approach.


Knowledge-driven genomic interactions: an application in ovarian cancer.
Effective cancer clinical outcome prediction for understanding of the mechanism of various types of cancer has been pursued using molecular-based data such as gene expression profiles, an approach that has promise for providing better diagnostics and supporting further therapies. However, clinical outcome prediction based on gene expression profiles varies between independent data sets. Further, single-gene expression outcome prediction is limited for cancer evaluation since genes do not act in isolation, but rather interact with other genes in complex signaling or regulatory networks. In addition, since pathways are more likely to co-operate together, it would be desirable to incorporate expert knowledge to combine pathways in a useful and informative manner. Thus, we propose a novel approach for identifying knowledge-driven genomic interactions and applying it to discover models associated with cancer clinical phenotypes using grammatical evolution neural networks (GENN). In order to demonstrate the utility of the proposed approach, an ovarian cancer data from the Cancer Genome Atlas (TCGA) was used for predicting clinical stage as a pilot project. We identified knowledge-driven genomic interactions associated with cancer stage from single knowledge bases such as sources of pathway-pathway interaction, but also knowledge-driven genomic interactions across different sets of knowledge bases such as pathway-protein family interactions by integrating different types of information. Notably, an integration model from different sources of biological knowledge achieved 78.82% balanced accuracy and outperformed the top models with gene expression or single knowledge-based data types alone. Furthermore, the results from the models are more interpretable because they are framed in the context of specific biological pathways or other expert knowledge. The success of the pilot study we have presented herein will allow us to pursue further identification of models predictive of clinical cancer survival and recurrence. Understanding the underlying tumorigenesis and progression in ovarian cancer through the global view of interactions within/between different biological knowledge sources has the potential for providing more effective screening strategies and therapeutic targets for many types of cancer.


Microbial responses to microgravity and other low-shear environments.
Microbial adaptation to environmental stimuli is essential for survival. While several of these stimuli have been studied in detail, recent studies have demonstrated an important role for a novel environmental parameter in which microgravity and the low fluid shear dynamics associated with microgravity globally regulate microbial gene expression, physiology, and pathogenesis. In addition to analyzing fundamental questions about microbial responses to spaceflight, these studies have demonstrated important applications for microbial responses to a ground-based, low-shear stress environment similar to that encountered during spaceflight. Moreover, the low-shear growth environment sensed by microbes during microgravity of spaceflight and during ground-based microgravity analogue culture is relevant to those encountered during their natural life cycles on Earth. While no mechanism has been clearly defined to explain how the mechanical force of fluid shear transmits intracellular signals to microbial cells at the molecular level, the fact that cross talk exists between microbial signal transduction systems holds intriguing possibilities that future studies might reveal common mechanotransduction themes between these systems and those used to sense and respond to low-shear stress and changes in gravitation forces. The study of microbial mechanotransduction may identify common conserved mechanisms used by cells to perceive changes in mechanical and/or physical forces, and it has the potential to provide valuable insight for understanding mechanosensing mechanisms in higher organisms. This review summarizes recent and future research trends aimed at understanding the dynamic effects of changes in the mechanical forces that occur in microgravity and other low-shear environments on a wide variety of important microbial parameters.


Predictors of 30-day mortality and hospital costs in patients with ventilator-associated pneumonia attributed to potentially antibiotic-resistant gram-negative bacteria.
To identify predictors of 30-day mortality and hospital costs in patients with ventilator-associated pneumonia (VAP) attributed to potentially antibiotic-resistant Gram-negative bacteria (PARGNB) [Pseudomonas aeruginosa, Acinetobacter species, and Stenotrophomonas maltophilia]. A retrospective, single-center, observational cohort study. Barnes-Jewish Hospital, a 1,200-bed urban teaching hospital. Adult patients requiring hospitalization with microbiologically confirmed VAP attributed to PARGNB. Retrospective data collection from automated hospital, microbiology, and pharmacy databases. Seventy-six patients with VAP attributed to PARGNB were identified over a 5-year period. Nineteen patients (25.0%) died during hospitalization. Patients receiving their first dose of appropriate antibiotic therapy within 24 h of BAL sampling had a statistically lower 30-day mortality rate compared to patients receiving the first dose of appropriate therapy >24 h after BAL (17.2% vs 50.0%; p = 0.005). VAP due to Acinetobacter species was most often initially treated with an inappropriate antibiotic regimen, followed by S maltophilia and P aeruginosa (66.7% vs 33.3% vs 17.2%; p = 0.017). Overall, total hospitalization costs were statistically similar in patients initially treated with an inappropriate antibiotic regimen compared to an appropriate regimen ($68,597 +/- $55,466 vs $86,644 +/- $64,433; p = 0.390). These data suggest that inappropriate initial antibiotic therapy of microbiologically confirmed VAP attributed to PARGNB is associated with greater 30-day mortality. High rates of VAP attributed to antibiotic-resistant bacteria (eg, Acinetobacter species) may require changes in the local empiric antibiotic treatment of VAP in order to optimize the prescription of appropriate initial therapy.


Adapting to changing memory retrieval demands: evidence from event-related potentials.
This study investigated preparatory processes involved in adapting to changing episodic memory retrieval demands. Event-related potentials (ERPs) were recorded while participants performed a general old/new recognition task and a specific task that also required retrieval of perceptual details. The relevant task remained either constant or changed (predictably or randomly) across trials. Responses were slowed when participants switched from the specific to the general task but not vice versa. Hence, asymmetrical switch costs were observed, suggesting that retrieval preparation is dependent not only on the current retrieval goal but also influenced by recent retrieval attempts. Consistently, over posterior scalp regions ERPs associated with advance preparation were modulated by the preceding task, reflecting increased attentional selection requirements for the general task, and by the foreknowledge about the task sequence. When retrieval demands remained constant, frontal slow-waves elicited by retrieval-cues were more positive going for the specific task, indicating full implementation of a retrieval orientation that allows more efficient retrieval of perceptual details.


Skin permeability prediction with MD simulation sampling spatial and alchemical reaction coordinates.
A molecular-level understanding of skin permeation may rationalize and streamline product development, and improve quality and control, of transdermal and topical drug delivery systems. It may also facilitate toxicity and safety assessment of cosmetics and skin care products. Here, we present new molecular dynamics simulation approaches that make it possible to efficiently sample the free energy and local diffusion coefficient across the skin's barrier structure to predict skin permeability and the effects of chemical penetration enhancers. In particular, we introduce a new approach to use two-dimensional reaction coordinates in the accelerated weight histogram method, where we combine sampling along spatial coordinates with an alchemical perturbation virtual coordinate. We present predicted properties for 20 permeants, and demonstrate how our approach improves correlation with ex vivo/in vitro skin permeation data. For the compounds included in this study, the obtained log KPexp-calc mean square difference was 0.9 cm2 h-2.


Role of SRP RNA in the GTPase cycles of Ffh and FtsY.
The bacterial homologues of the signal recognition particle (SRP) and its receptor, the Ffh*4.5S RNA ribonucleoprotein complex and the FtsY protein, respectively, form a unique complex in which both Ffh and FtsY act as GTPase activating proteins for one another, resulting in the mutual stimulation of GTP hydrolysis by both proteins. Previous work showed that 4.5S RNA enhances the GTPase activity in the presence of both Ffh and FtsY, but it was not clear how this was accomplished. In this work, kinetic and thermodynamic analyses of the GTPase reactions of Ffh and FtsY have provided insights into the role of 4.5S RNA in the GTPase cycles of Ffh and FtsY. We found that 4.5S RNA accelerates the association between Ffh and FtsY 400-fold in their GTP-bound form, analogous to its 200-fold catalytic effect on Ffh*FtsY association previously observed with the GppNHp-bound form [Peluso, P., et al. (2000) Science 288, 1640-1643]. Further, Ffh-FtsY association is rate-limiting for the observed GTPase reaction with subsaturating Ffh and FtsY, thereby accounting for the apparent stimulatory effect of 4.5S RNA on the GTPase activity observed previously. An additional step, GTP hydrolysis from the Ffh*FtsY complex, is also moderately facilitated by 4.5S RNA. These results suggest that 4.5S RNA modulates the conformation of the Ffh*FtsY complex and may, in turn, regulate its GTPase activity during the SRP functional cycle.


Principal component analysis of blood microRNA datasets facilitates diagnosis of diverse diseases.
Early, ideally pre-symptomatic, recognition of common diseases (e.g., heart disease, cancer, diabetes, Alzheimer's disease) facilitates early treatment or lifestyle modifications, such as diet and exercise. Sensitive, specific identification of diseases using blood samples would facilitate early recognition. We explored the potential of disease identification in high dimensional blood microRNA (miRNA) datasets using a powerful data reduction method: principal component analysis (PCA). Using Qlucore Omics Explorer (QOE), a dynamic, interactive visualization-guided bioinformatics program with a built-in statistical platform, we analyzed publicly available blood miRNA datasets from the Gene Expression Omnibus (GEO) maintained at the National Center for Biotechnology Information at the National Institutes of Health (NIH). The miRNA expression profiles were generated from real time PCR arrays, microarrays or next generation sequencing of biologic materials (e.g., blood, serum or blood components such as platelets). PCA identified the top three principal components that distinguished cohorts of patients with specific diseases (e.g., heart disease, stroke, hypertension, sepsis, diabetes, specific types of cancer, HIV, hemophilia, subtypes of meningitis, multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, mild cognitive impairment, aging, and autism), from healthy subjects. Literature searches verified the functional relevance of the discriminating miRNAs. Our goal is to assemble PCA and heatmap analyses of existing and future blood miRNA datasets into a clinical reference database to facilitate the diagnosis of diseases using routine blood draws.


Using phylogenomic patterns and gene ontology to identify proteins of importance in plant evolution.
We use measures of congruence on a combined expressed sequenced tag genome phylogeny to identify proteins that have potential significance in the evolution of seed plants. Relevant proteins are identified based on the direction of partitioned branch and hidden support on the hypothesis obtained on a 16-species tree, constructed from 2,557 concatenated orthologous genes. We provide a general method for detecting genes or groups of genes that may be under selection in directions that are in agreement with the phylogenetic pattern. Gene partitioning methods and estimates of the degree and direction of support of individual gene partitions to the overall data set are used. Using this approach, we correlate positive branch support of specific genes for key branches in the seed plant phylogeny. In addition to basic metabolic functions, such as photosynthesis or hormones, genes involved in posttranscriptional regulation by small RNAs were significantly overrepresented in key nodes of the phylogeny of seed plants. Two genes in our matrix are of critical importance as they are involved in RNA-dependent regulation, essential during embryo and leaf development. These are Argonaute and the RNA-dependent RNA polymerase 6 found to be overrepresented in the angiosperm clade. We use these genes as examples of our phylogenomics approach and show that identifying partitions or genes in this way provides a platform to explain some of the more interesting organismal differences among species, and in particular, in the evolution of plants.


Sensitivity and specificity of procedures for the differential diagnosis of epileptic and non-epileptic seizures: a systematic review.
Non-epileptic seizures (NES) present a considerable challenge in clinical practice. This paper reviews published evidence for the reliability of a number of procedures for the differential diagnosis of NES and epilepsy. Papers identified from MEDLINE and PsychInfo Databases (1980-2001) and additional hand searches were independently reviewed using methods for evaluating evidence in systematic reviews [Liddle, J., Williamson, M. Irwig, L. Method for evaluating research guideline evidence. New South Wales Department of Health; 1996 [State Health publication no. (CEB) 96-204]; SIGN. An introduction to SIGN methodology for the development of evidence based clinical guidelines. Scottish Intercollegiate Network; 1999]. Included studies had to have an NES group and a control group of people with epilepsy (each n> or =10), allocated using EEG linked video-recording of concurrent behaviour, and sensitivity and specificity values had to be stated or be calculable. Thirty-three papers were identified, of which 13 satisfied criteria. Excluded studies are briefly described. Those retained comprised a range of procedures [seizure induction, MMPI assessment, physiological assessment (prolactin, SPECT), pre-ictal pseudosleep, and ictal/post-ictal characteristics]. No procedure emerged with both high sensitivity and specificity and adequately replicated findings, although high levels of specificity were more commonly reported than high levels of sensitivity. This suggests that procedures were generally better at excluding a possible diagnosis. No procedure attains reliability equivalent to EEG video-telemetry. Further rigorous evaluation, using standardised and replicable methodologies, is required. The range of symptoms presented in NES suggests that a multi-method approach may be required. This too would require evaluation.


Mouse cardiac acyl coenzyme a synthetase 1 deficiency impairs Fatty Acid oxidation and induces cardiac hypertrophy.
Long-chain acyl coenzyme A (acyl-CoA) synthetase isoform 1 (ACSL1) catalyzes the synthesis of acyl-CoA from long-chain fatty acids and contributes the majority of cardiac long-chain acyl-CoA synthetase activity. To understand its functional role in the heart, we studied mice lacking ACSL1 globally (Acsl1(T-/-)) and mice lacking ACSL1 in heart ventricles (Acsl1(H-/-)) at different times. Compared to littermate controls, heart ventricular ACSL activity in Acsl1(T-/-) mice was reduced more than 90%, acyl-CoA content was 65% lower, and long-chain acyl-carnitine content was 80 to 90% lower. The rate of [(14)C]palmitate oxidation in both heart homogenate and mitochondria was 90% lower than in the controls, and the maximal rates of [(14)C]pyruvate and [(14)C]glucose oxidation were each 20% higher. The mitochondrial area was 54% greater than in the controls with twice as much mitochondrial DNA, and the mRNA abundance of Pgc1α and Errα increased by 100% and 41%, respectively. Compared to the controls, Acsl1(T-/-) and Acsl1(H-/-) hearts were hypertrophied, and the phosphorylation of S6 kinase, a target of mammalian target of rapamycin (mTOR) kinase, increased 5-fold. Our data suggest that ACSL1 is required to synthesize the acyl-CoAs that are oxidized by the heart, and that without ACSL1, diminished fatty acid (FA) oxidation and compensatory catabolism of glucose and amino acids lead to mTOR activation and cardiac hypertrophy without lipid accumulation or immediate cardiac dysfunction.


Describing and predicting developmental profiles of externalizing problems from childhood to adulthood.
This longitudinal study considers externalizing behavior problems from ages 5 to 27 (N = 585). Externalizing problem ratings by mothers, fathers, teachers, peers, and self-report were modeled with growth curves. Risk and protective factors across many different domains and time frames were included as predictors of the trajectories. A major contribution of the study is in demonstrating how heterotypic continuity and changing measures can be handled in modeling changes in externalizing behavior over long developmental periods. On average, externalizing problems decreased from early childhood to preadolescence, increased during adolescence, and decreased from late adolescence to adulthood. There was strong nonlinear continuity in externalizing problems over time. Family process, peer process, stress, and individual characteristics predicted externalizing problems beyond the strong continuity of externalizing problems. The model accounted for 70% of the variability in the development of externalizing problems. The model's predicted values showed moderate sensitivity and specificity in prediction of arrests, illegal drug use, and drunk driving. Overall, the study showed that by using changing, developmentally relevant measures and simultaneously taking into account numerous characteristics of children and their living situations, research can model lengthy spans of development and improve predictions of the development of later, severe externalizing problems.


Ultrafast all-optical logic gates based on soliton trapping in fibers.
Ultrafast all-optical soliton-trapping logic gates, including an inverter, exclusive OR, and AND, are experimentally demonstrated in birefringent fibers. The soliton-trapping logic gates are three terminal devices with orthogonally polarized inputs, phase-insensitive nonlinear operation, and switching energies of ~42 pJ. Using a 0.2-THz bandpass filter, the contrast ratio for the exclusive-OR gate is ~8:1, but the output pulses are ~10 times broader than the input pulse width. By widening the filter bandpass to 0.58 THz, an inverter is demonstrated with an ~4:1 contrast ratio and output pulses that can propagate as solitons in a fiber. Numerical simulations show that the output from the inverter can be cascaded to other trapping gates.


Lung function and microbiota diversity in cystic fibrosis.
Chronic infection and concomitant airway inflammation is the leading cause of morbidity and mortality for people living with cystic fibrosis (CF). Although chronic infection in CF is undeniably polymicrobial, involving a lung microbiota, infection surveillance and control approaches remain underpinned by classical aerobic culture-based microbiology. How to use microbiomics to direct clinical management of CF airway infections remains a crucial challenge. A pivotal step towards leveraging microbiome approaches in CF clinical care is to understand the ecology of the CF lung microbiome and identify ecological patterns of CF microbiota across a wide spectrum of lung disease. Assessing sputum samples from 299 patients attending 13 CF centres in Europe and the USA, we determined whether the emerging relationship of decreasing microbiota diversity with worsening lung function could be considered a generalised pattern of CF lung microbiota and explored its potential as an informative indicator of lung disease state in CF. We tested and found decreasing microbiota diversity with a reduction in lung function to be a significant ecological pattern. Moreover, the loss of diversity was accompanied by an increase in microbiota dominance. Subsequently, we stratified patients into lung disease categories of increasing disease severity to further investigate relationships between microbiota characteristics and lung function, and the factors contributing to microbiota variance. Core taxa group composition became highly conserved within the severe disease category, while the rarer satellite taxa underpinned the high variability observed in the microbiota diversity. Further, the lung microbiota of individual patient were increasingly dominated by recognised CF pathogens as lung function decreased. Conversely, other bacteria, especially obligate anaerobes, increasingly dominated in those with better lung function. Ordination analyses revealed lung function and antibiotics to be main explanators of compositional variance in the microbiota and the core and satellite taxa. Biogeography was found to influence acquisition of the rarer satellite taxa. Our findings demonstrate that microbiota diversity and dominance, as well as the identity of the dominant bacterial species, in combination with measures of lung function, can be used as informative indicators of disease state in CF. Video Abstract.


A novel fibroblast growth factor-1 (FGF1) mutant that acts as an FGF antagonist.
Crosstalk between integrins and FGF receptors has been implicated in FGF signaling, but the specifics of the crosstalk are unclear. We recently discovered that 1) FGF1 directly binds to integrin alphavbeta3, 2) the integrin-binding site and FGF receptor (FGFR) binding site are distinct, and 3) the integrin-binding-defective FGF1 mutant (R50E) is defective in inducing FGF signaling although R50E still binds to FGFR and heparin and induces transient ERK1/2 activation. We tested if excess R50E affect DNA synthesis and cell survival induced by WT FGF1 in BaF3 mouse pro-B cells expressing human FGFR1. R50E suppressed DNA synthesis and cell proliferation induced by WT FGF1. We tested if WT FGF1 and R50E generate integrin-FGF1-FGFR ternary complex. WT FGF1 induced ternary complex formation (integrin-FGF-FGFR1) and recruitment of SHP-2 to the complex in NIH 3T3 cells and human umbilical endothelial cells, but R50E was defective in these functions. It has been reported that sustained ERK1/2 activation is integrin-dependent and crucial to cell cycle entry upon FGF stimulation. We thus determined the time-course of ERK1/2 activation induced by WT FGF1 and R50E. We found that WT FGF1 induced sustained activation of ERK1/2, but R50E was defective in this function. Our results suggest that 1) R50E is a dominant-negative mutant, 2) Ternary complex formation is involved in FGF signaling, 3) The defect of R50E to bind to integrin may be directly related to the antagonistic action of R50E. Taken together, these results suggest that R50E has potential as a therapeutic in cancer.


Expression and silencing of the microtubule-associated protein Tau in breast cancer cells.
The microtubule-associated protein Tau has been reported to be a predictive factor for clinical response to taxanes in metastatic breast cancer. We generated a panel of eight taxane-resistant variants from four human breast cancer cell lines (MCF-7, T-47D, MDA-MB-231, and BT-549). Four variants had higher levels of Tau compared with their T-47D and MDA-MB-231 parental cells. Using isoform-specific primers, we found that Tau 0N, 1N, 2N, 3R, and 4R isoforms are overexpressed in the resistant variants, as is Tau exon 6 but not exons 4A or 8. To determine whether Tau overexpression produces resistance to taxanes, we derived three independent T-47D clones stably overexpressing Tau 3R and 4R isoforms. Tau overexpression did not result in taxane resistance compared with parental cells transfected with vector alone. We then knocked down Tau expression in three cell lines that expressed Tau constitutively (MCF-7 and ZR-75-1 breast cancer cells, and OVCAR-3 ovarian cancer cells). Lentivirus-mediated silencing of Tau expression in MCF-7 and OVCAR-3 cells did not result in increased taxane sensitivity compared with luciferase short hairpin RNA-infected cells and uninfected parental cells. Transient silencing using Tau-specific small interfering RNAs also did not alter taxane sensitivity relative to nontargeting controls in both MCF-7 and ZR-75-1 cells. These results show that neither overexpression nor depletion of Tau modulates cellular sensitivity to taxanes. Although Tau overexpression has been reported to be a predictive marker of taxane resistance, it is not likely to be a direct mechanism of taxane resistance in breast cancer.


Protein and fat utilization by humans as affected by calcium phosphate, calcium carbonate, and manganese gluconate supplements.
The object of this study was to determine the effect of calcium carbonate or calcium phosphate supplements on dietary protein and fat utilization from low and high manganese diets. During the 63-day study, the 14 human adult subjects ate a constant laboratory controlled diet. In separate periods, subjects consumed the basal diet alone or with supplements of calcium carbonate, calcium carbonate plus manganese gluconate, calcium phosphate, or calcium phosphate plus manganese gluconate. Contrast analyses of data indicated that manganese gluconate supplementation of diets, when combined with either calcium phosphate or calcium carbonate, increased fecal losses of fat. When used as single supplements, both calcium phosphate and calcium carbonate depressed fecal fat loss in comparison with values when no supplements were used. Manganese gluconate supplements depressed fecal nitrogen losses calculated as a percentage of dry fecal weight.


Thrombocytopenia model with minimal manipulation of blood cells allowing whole blood assessment of platelet function.
In vitro models of thrombocytopenia are useful research tools. Previously published models have shortcomings altering properties of platelets and other blood components. The aim of the present study was to develop a whole blood method to induce thrombocytopenia with minimal manipulation, and to describe platelet function in induced thrombocytopenia in individuals with healthy platelets. Hirudin anticoagulated blood was obtained from 20 healthy volunteers. One part of the blood was gently centrifuged at 130g for 15 minutes. The platelet-rich plasma was replaced with phosphate-buffered saline to establish thrombocytopenia. Various levels of thrombocytopenia were achieved by combining different volumes of baseline whole blood and thrombocytopenic blood. Platelet counts were measured by flow cytometry (Navios, Beckman Coulter) and routine haematological analyser (Sysmex XE-5000). Platelet function was analysed by impedance aggregometry (Multiplate® Analyzer, Roche) and by flow cytometry (Navios, Beckman Coulter) using collagen, adenosine diphosphate, thrombin receptor activating peptide-6 and ristocetin as agonists. Median baseline platelet count was 227×10(9)/l. The in vitro model yielded median platelet counts at 51×10(9)/l (range 26-93×10(9)/l). We observed minor, yet significant, changes in platelet size and maturity from baseline to modelled thrombocytopenia. In the thrombocytopenic samples, significant and positive linear associations were found between platelet count and platelet aggregation across all agonists (all p-values<0.001). Platelet function assessed by flow cytometry showed minimal alterations in the thrombocytopenic samples. A new whole blood-based model of thrombocytopenia was established and validated. This new model serves as a useful future tool, particularly to explore platelet function in patients with thrombocytopenia.


Screening for Auditory Impairment-Which Hearing Assessment Test (SAI-WHAT): RCT design and baseline characteristics.
Effective screening programs should not merely detect presence of disease, but also lead to long-term benefit. We describe the rationale and design of the first randomized clinical trial to study the long-term effects of routine screening for hearing loss. We also describe the baseline characteristics of the randomized cohort. We randomized 2305 veterans age 50 years or older to a control arm without screening, or to screening with: physiologic testing (AudioScope), a self-administered questionnaire (Hearing Handicap Inventory for the Elderly-Screening version [HHIE-S]), or both tests. The primary outcome measure will be hearing aid use one year after screening. We will also study a number of secondary outcomes, including appointments made with and visits to an audiologist, cases of aidable hearing loss, hearing aids dispensed, self-rated communication ability, and hearing-related quality of life. Baseline demographic and health status measures were evenly distributed across the screening arms. The percentage of patients who screened positive for hearing loss was 18.6%, 59.2%, and 63.6% for the AudioScope, HHIE-S, and combined screening arms, respectively. Long-term results are needed to gain insight into whether the AudioScope is associated with high rates of false negative screening, the HHIE-S is associated with high rates of false positive screening, or a combination of both. Identifying the best screening program will depend on determining which strategy leads to successful hearing aid use.


Innate immunity triggers IL-32 expression by fibroblast-like synoviocytes in rheumatoid arthritis.
Interleukin-32 (IL-32) is a recently described cytokine that is a strong inducer of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, IL-1β, IL-6, and IL-8. The expression of this cytokine is highly increased in the rheumatoid synovium and correlated with the severity of joint inflammation. Little is known regarding the innate immune-related regulation of IL-32 by fibroblast-like synoviocytes (FLSs). We therefore investigated the effect of innate immune stimulation by ligands of Toll-like receptor (TLR)2, TLR3, and TLR4, and cytokines such as TNF-α and interferon (IFN)-γ, on IL-32 expression by FLSs. FLSs were isolated from patients with rheumatoid arthritis (RA) according to the ACR criteria. Quantitative RT-PCR, confocal analysis, and ELISA were performed to evaluate IL-32 mRNA induction and IL-32 release by FLSs stimulated with TLR2 (BLP), TLR3 (poly I:C), and TLR4 (lipopolysaccharide) ligands, TNF-α and IFN-γ. TLR2, -3, and -4 ligands as well as IFN-γ and TNF-α induced IL-32 β, γ and δ mRNA expression by RA FLSs. Mature IL-32 was expressed intracellularly and released by cells stimulated with the various activators. The IL-32α isoform was expressed intracellularly in response to TNF-α and poly I:C and not released in culture supernatants. Stimulation of FLS with TNF-α, BLP, lipopolysaccharide, or poly I:C concomitant with IFN-γ increased IL-32 expression compared with stimulation with IFN-γ alone. IL-32 synthesis by FLSs is tightly regulated by innate immunity in rheumatoid arthritis. Thus TNF-α, IFN-γ, double-strand RNA, hyaluronic acid, or other damage-associated molecular patterns (DAMPs), highly secreted in synovial tissues of RA patients, might trigger IL-32 secretion by FLSs. IL-32 might therefore represent a relevant therapeutic target in RA.