Productive infection of CD4+ and CD8+ mature human T cell populations and clones by human herpesvirus 6. Transcriptional down-regulation of CD3.
The susceptibility to infection by human herpes-virus 6 (HHV-6) of mature human T lymphocytes belonging to the two major subpopulations (i.e., CD3+ CD4+ CD8- and CD3+ CD4- CD8+) was investigated by using CD4+ or CD8+ T cell populations and clones derived from normal adult peripheral blood. Productive HHV-6 infection was observed in both CD4+ and CD8+ T cells. By days 2 to 6 after infection, increasing numbers of cells exhibited characteristic morphologic alterations, becoming enlarged, uniformly rounded and refractile as a consequence of the virus-induced cytopathic effect. During the course of HHV-6 infection, analysis of the surface membrane phenotype of the T cell populations and clones revealed a progressive decline in the expression of the CD3/TCR complex, whereas other T cell-associated markers (e.g., CD2) were unaffected. Northern blot analysis of mRNA extracted from HHV-6-infected T cells demonstrated a dramatic loss of the specific messages for the gamma-, delta-, and epsilon-chains of CD3. Infection by HHV-6, but not by HSV-1 or human CMV, elicited CD3/TCR down-regulation also in the neoplastic T cell line Jurkat. The down-regulation of CD3/TCR was dependent upon live virus infection, because previous inactivation of HHV-6 by heat (56 degrees C for 1 h) or UV light (16 J/m2) totally abrogated the effect. Expression of the immediate early or early genes of HHV-6 was not sufficient to induce CD3/TCR modulation, as indicated by studies with the viral DNA polymerase inhibitor phosphonoformic acid. The observation that both major subsets of mature TCR-alpha beta+ T lymphocytes are susceptible to HHV-6 infection indicates that this virus may have a broad spectrum of activity on the immune system. The transcriptional down-regulation of the CD3/TCR complex, by affecting a critical T cell recognition function, could be relevant to HHV-6 pathogenesis.


Characterization of the chromosomal binding sites and dimerization partners of the viral oncoprotein Meq in Marek's disease virus-transformed T cells.
Marek's disease virus (MDV) is an acute transforming alphaherpesvirus that causes T-cell lymphomas in chickens. We previously reported the identification of a putative oncogene, meq, that is encoded only by the oncogenic serotype of MDV. The gene product, Meq, is a latent protein that is consistently expressed in MDV-transformed lymphoblastoid cells and tumor cells. Meq has a bZIP (basic leucine zipper) structure resembling the family of Jun/Fos. The mechanism whereby Meq transforms T cells remains poorly understood. In this study, we explored the properties of Meq as a transcriptional factor. We analyzed Meq's dimerization partners and its target genes in MSB-1, an MDV-transformed T-cell line. By using in vitro assays, we first demonstrated Meq's potential to dimerize with a variety of bZIP proteins. We then identified c-Jun as the primary dimerization partner of Meq. Both are found to be colocalized in the nucleus and corecruited to promoters with AP-1 sequences. By using chromatin immunoprecipitation (ChIP), we scanned the entire MDV genome for Meq binding sites and found three regions that were enriched with Meq binding: the MDV lytic replication origin, the promoter for Meq, and the promoter for ICP4. Transactivation assays using the above promoters showed that Meq/Meq homodimers exhibited repression activity, whereas Meq/Jun heterodimers showed activation. Finally, we were able to show by ChIP that Meq is recruited to the interleukin-2 promoter in a region encompassing an AP-1 site. Thus, in addition to providing general knowledge about the transcriptional properties of Meq, our studies revealed for the first time the ability of Meq to interact with the latent MDV and host genomes. Our data suggest, therefore, a role for Meq in viral genome regulation during latency, in addition to its putative causal role in T-cell transformation.


Separation of Nickel-Titanium Rotary and Reciprocating Instruments: A Mini-Review of Clinical Studies.
This review aimed to evaluate clinical studies that assessed separated NiTi rotary and reciprocating instruments. This review assessed clinical studies involving treatments performed by undergraduate students, graduate students, and endodontic specialists. This review evaluated studies using rotary instruments, reciprocating instruments, and hybrid techniques. The number of uses of the different NiTi rotary and reciprocating systems was also assessed. The incidence of separation for rotary instruments ranged from 0% to 23%. Rotary instruments were used from 1 to 50 times depending on the instrument and tooth type. The lowest rate of incidence separation for rotary instruments was obtained by undergraduate students, using a hybrid technique. The separation incidence for reciprocating instruments ranged from 0% to 1.71%. Reciprocating instruments were mostly single-used; one study reported their use up to 3 times. Separation rate in reciprocating instruments was similar in single-use or in multiple uses 0.2%. Separation of instruments has dropped recently and seems to be a minor problem in current Endodontics. Multiple uses of NiTi rotary instruments are a possibility without significantly increasing the risk of instrument separation. Single and multiple uses of NiTi reciprocating instruments are also associated with low incidence of separation. Attempting to remove separated instruments should be carefully evaluated.


Endoscopic retrograde computed tomography of the pancreas.
Endoscopic retrograde cholangiopancreatography (ERCP) and computed tomography (CT) are well established diagnostic studies for pancreatic evaluation. We conducted a study to determine whether performing these examinations in tandem increased diagnostic accuracy. Patients undergoing ERCP with successful cannulation of the pancreatic duct and who could be placed in the gantry of our CT scanner within 30 min were included in the study. The combined examination, or endoscopic retrograde computed tomography of the pancreas (ERCTP), provided additional useful information in several types of patients including those who had intrapancreatic air without clinical abscess, those with apparent total obstruction of the pancreatic duct on ERCP, and those who were post partial pancreatectomy and were being considered for additional surgery. No additional useful information was provided in patients with a normal ERCP or in routine cases of pancreatitis.


A new basal sauropodomorph (Dinosauria: Saurischia) from Quebrada del Barro Formation (Marayes-El Carrizal Basin), northwestern Argentina.
Argentinean basal sauropodomorphs are known by several specimens from different basins; Ischigualasto, El Tranquilo, and Mogna. The Argentinean record is diverse and includes some of the most primitive known sauropodomorphs such as Panphagia and Chromogisaurus, as well as more derived forms, including several massospondylids. Until now, the Massospondylidae were the group of basal sauropodomorphs most widely spread around Pangea with a record in almost all continents, mostly from the southern hemisphere, including the only record from Antarctica. We describe here a new basal sauropodomorph, Leyesaurus marayensis gen. et sp. nov., from the Quebrada del Barro Formation, an Upper Triassic-Lower Jurassic unit that crops out in northwestern Argentina. The new taxon is represented by a partial articulated skeleton that includes the skull, vertebral column, scapular and pelvic girdles, and hindlimb. Leyesaurus is diagnosed by a set of unique features, such as a sharply acute angle (50 degrees) formed by the ascending process of the maxilla and the alveolar margin, a straight ascending process of the maxilla with a longitudinal ridge on its lateral surface, noticeably bulging labial side of the maxillary teeth, greatly elongated cervical vertebrae, and proximal articular surface of metatarsal III that is shelf-like and medially deflected. Phylogenetic analysis recovers Leyesaurus as a basal sauropodomorph, sister taxon of Adeopapposaurus within the Massospondylidae. Moreover, the results suggest that massospondylids achieved a higher diversity than previously thought. Our phylogenetic results differ with respect to previous analyses by rejecting the massospondylid affinities of some taxa from the northern hemisphere (e.g., Seitaad, Sarahsaurus). As a result, the new taxon Leyesaurus, coupled with other recent discoveries, suggests that the diversity of massospondylids in the southern hemisphere was higher than in other regions of Pangea. Finally, the close affinities of Leyesaurus with the Lower Jurassic Massospondylus suggest a younger age for the Quebrada del Barro Formation than previously postulated.


Rapid online assessment of reading ability.
An accurate model of the factors that contribute to individual differences in reading ability depends on data collection in large, diverse and representative samples of research participants. However, that is rarely feasible due to the constraints imposed by standardized measures of reading ability which require test administration by trained clinicians or researchers. Here we explore whether a simple, two-alternative forced choice, time limited lexical decision task (LDT), self-delivered through the web-browser, can serve as an accurate and reliable measure of reading ability. We found that performance on the LDT is highly correlated with scores on standardized measures of reading ability such as the Woodcock-Johnson Letter Word Identification test (r = 0.91, disattenuated r = 0.94). Importantly, the LDT reading ability measure is highly reliable (r = 0.97). After optimizing the list of words and pseudowords based on item response theory, we found that a short experiment with 76 trials (2-3 min) provides a reliable (r = 0.95) measure of reading ability. Thus, the self-administered, Rapid Online Assessment of Reading ability (ROAR) developed here overcomes the constraints of resource-intensive, in-person reading assessment, and provides an efficient and automated tool for effective online research into the mechanisms of reading (dis)ability.


Effects of a Dynamic Warm-Up, Static Stretching or Static Stretching with Tendon Vibration on Vertical Jump Performance and EMG Responses.
The purpose of this study was to investigate the short-term effects of static stretching, with vibration given directly over Achilles tendon, on electro-myographic (EMG) responses and vertical jump (VJ) performances. Fifteen male, college athletes voluntarily participated in this study (n=15; age: 22±4 years old; body height: 181±10 cm; body mass: 74±11 kg). All stages were completed within 90 minutes for each participant. Tendon vibration bouts lasted 30 seconds at 50 Hz for each volunteer. EMG analysis for peripheral silent period, H-reflex, H-reflex threshold, T-reflex and H/M ratio were completed for each experimental phases. EMG data were obtained from the soleus muscle in response to electro stimulation on the popliteal post tibial nerve. As expected, the dynamic warm-up (DW) increased VJ performances (p=0.004). Increased VJ performances after the DW were not statistically substantiated by the EMG findings. In addition, EMG results did not indicate that either static stretching (SS) or tendon vibration combined with static stretching (TVSS) had any detrimental or facilitation effect on vertical jump performances. In conclusion, using TVSS does not seem to facilitate warm-up effects before explosive performance.


Paradoxical reduction of synaptic inhibition by vigabatrin.
GABAergic inhibition, a primary target for pharmacological modulation of excitability in the CNS, can be altered by multiple mechanisms including alteration of GABA metabolism. Gamma-vinyl GABA (vigabatrin, GVG) is an irreversible inhibitor of the GABA catabolic enzyme GABA transaminase, thus its anticonvulsant properties are thought to result from an elevation of brain GABA levels. We examined the effects of GVG on GABAergic synaptic transmission in hippocampal slices. GVG unexpectedly reduced miniature and evoked inhibitory postsynaptic currents (IPSCs) in dentate granule cells. The reduction in synaptic events was accompanied by an increase in tonic GABA(A) receptor-mediated current. These effects developed slowly and persisted following wash out of GVG. The GVG pretreatment reduced sucrose-evoked GABA release as well as postsynaptic sensitivity to exogenous GABA, indicating that both pre- and postsynaptic mechanisms contributed to the reduction in synaptic currents. These results suggest that tonic rather than phasic increases in GABA underlie the anticonvulsant properties of GVG, and that mechanisms that elevate brain neurotransmitter levels do not necessarily correlate with enhanced synaptic release.


Inhibition of adiposity and related metabolic disturbances by polyphenol-rich extract of Boswellia serrata gum through alteration of adipo/cytokine profiles.
The role of proinflammatory cytokines in adiposity is well established. The anti-inflammatory and antihyperglycemia effects of Boswellia serrata (B. Serrata) gum have been demonstrated by many investigators. The present study aimed to investigate the anti-obesity potential of B. serrata extract. The effects of B. serrata extract on lipase activity and acute food intake were investigated. The present study aimed to investigate the anti-obesity potential of B. serrata extract. The effects on lipase activity and acute food intake were investigated. Body weight changes, biochemical and histopathological markers were demonstrated in rats fed a high-fat diet. Boswellia serrata extract inhibited alterations in pancreatic lipase activity, but orlistat was more efficacious. B. serrata and ephidrene, but not orlistat, significantly suppressed cumulative food intake in mice. In obese rats, B. serrata or orlistat significantly decreased weight gain and weight of visceral white adipose tissue. B. serrata-treated animals exhibited a significant reduction in serum glucose, TC, TG, LDL-C, FFA, IL-1β, TNF-α, insulin and leptin levels of obese rat groups while HDL-C and adiponectin levels were significantly increased by orlistat or B. serrata extract. Histopathological examination of the liver revealed that B. serrata was more effective than orlistat in alleviating steatosis and adipocyte hypertrophy shown in obese control rats. Boswellia serrata is as effective as orlistat in preventing obesity, hyperlipidemia, steatosis and insulin resistance. These actions may be mediated by suppression of food intake and decrease levels of TNF-α, IL-1β and leptin resistance along with increasing adiponectin.


No Differences in Achieving Hepatitis C Virus Care Milestones Between Patients Identified by Birth Cohort or Risk-Based Screening.
National hepatitis C virus (HCV) screening guidelines recommended 1-time testing of persons born between 1945 and 1965. We performed a retrospective study to compare care milestones achieved by HCV-infected patients identified by birth cohort versus risk-based screens. We determined the proportions of patients newly identified with HCV infection who met care milestones (viral load, referral to and evaluation by a specialist, offer of treatment, initiation of treatment, and sustained viral response) and the time it took to reach them. We found no differences in HCV care milestones for patients identified via birth cohort testing versus risk-based screening. Overall, only 43% of HCV antibody-positive patients were referred to care, and less than 4% started treatment. The time to each care milestone was lengthy and varied greatly; treatment was initiated in a median of 308 days. Although birth cohort testing will likely increase identification of patients with HCV infection, it does not seem to increase the number of patients that meet management milestones. New methods are needed to increase access to care and establish efficient models of health care delivery.


Building an evidence base for occupational health interventions.
This article summarizes arguments for building an evidence base for occupational health. Evidence is needed on the most effective ways of eliminating health hazards in the workplace and at work, enhancing healthy behavior or the empowerment of workers, and preventing and treating occupational diseases and occupational disability. An evidence base for occupational health must include systematic reviews. The Cochrane Collaboration has brought together some of the evidence; however, a search for systematic reviews on the top priorities in occupational health research showed that systematic reviews are lacking in many areas. Current reviewing methods can be adapted to the special features of occupational health. It is concluded that more effort should be invested in the preparation, maintenance, and dissemination of systematic reviews in order to create a necessary evidence base for occupational health interventions. Occupational health could benefit considerably from greater awareness of the evidence for and against various types of intervention.


Structural Changes In Cells Imaged by Soft X-ray Cryo-Tomography During Hepatitis C Virus Infection.
Chronic hepatitis C virus (HCV) infection causes severe liver disease in millions of humans worldwide. Pathogenesis of HCV infection is strongly driven by a deficient immune response of the host, although intersection of different aspects of the virus life cycle with cellular homeostasis is emerging as an important player in the pathogenesis and progression of the disease. Cryo soft X-ray tomography (cryo-SXT) was performed to investigate the ultrastructural alterations induced by the interference of HCV replication with cellular homeostasis. Native, whole cell, three-dimensional (3D) maps were obtained in HCV replicon-harboring cells and in a surrogate model of HCV infection. Tomograms from HCV-replicating cells show blind-ended endoplasmic reticulum tubules with pseudospherical extrusions and marked alterations of mitochondrial morphology that correlated spatially with the presence of endoplasmic reticulum alterations, suggesting a short-range influence of the viral machinery on mitochondrial homeostasis. Both mitochondrial and endoplasmic reticulum alterations could be reverted by a combination of sofosbuvir/daclatasvir, which are clinically approved direct-acting antivirals for the treatment of chronic HCV infection. In addition to providing structural insight into cellular aspects of HCV pathogenesis, our study illustrates how cryo-SXT is a powerful 3D wide-field imaging tool for the assessment and understanding of complex cellular processes in a setting of near-native whole hydrated cells. Our results also constitute a proof of concept for the use of cryo-SXT as a platform that enables determining the potential impact of candidate compounds on the ultrastructure of the cell that may assist drug development at a preclinical level.


Looking back on 10 years of global road safety.
Every year more than 1.35 million people lose their lives on the road and tens of millions more are injured, some permanently. Since the early 2000s there has been renewed focus on the issue, with the United Nations, World Health Organization and the World Bank placing the issue higher on their agendas. Guided by the United Nations General Assembly, World Health Assembly resolutions and ministerial-level conferences on the global road safety crisis, multisectoral partnerships have synthesised the evidence, advocated for action (there are two Sustainable Development Goal targets with an ambitious goal of reducing deaths and injuries from road traffic crashes by 50%), raised public awareness, generated funding, piloted interventions and monitored progress. And yet the total number of deaths has plateaued despite some sporadic country-level successes. More needs to be done-more people need to be trained in countries to deliver, monitor and evaluate a systems approach to road safety, more solid evidence of what works in low-resource settings is needed (including sustainable transportation options) and there needs to be a greater focus on optimising care and support for those injured in crashes-if we are to begin to see numbers come down in the next decade.


Droplet formation in microfluidic T-junction generators operating in the transitional regime. II. Modeling.
This is the second part of a two-part study on the generation of droplets at a microfluidic T-junction operating in the transition regime. In the preceding paper [Phys. Rev. E 85, 016322 (2012)], we presented our experimental observations of droplet formation and decomposed the process into three sequential stages defined as the lag, filling, and necking stages. Here we develop a model that describes the performance of microfluidic T-junction generators working in the squeezing to transition regimes where confinement of the droplet dominates the formation process. The model incorporates a detailed geometric description of the drop shape during the formation process combined with a force balance and necking criteria to define the droplet size, production rate, and spacing. The model inherently captures the influence of the intersection geometry, including the channel width ratio and height-to-width ratio, capillary number, and flow ratio, on the performance of the generator. The model is validated by comparing it to speed videos of the formation process for several T-junction geometries across a range of capillary numbers and viscosity ratios.


Distinct neurobehavioural effects of cannabidiol in transmembrane domain neuregulin 1 mutant mice.
The cannabis constituent cannabidiol (CBD) possesses anxiolytic and antipsychotic properties. We have previously shown that transmembrane domain neuregulin 1 mutant (Nrg1 TM HET) mice display altered neurobehavioural responses to the main psychoactive constituent of cannabis, Δ(9)-tetrahydrocannabinol. Here we investigated whether Nrg1 TM HET mice respond differently to CBD and whether CBD reverses schizophrenia-related phenotypes expressed by these mice. Adult male Nrg1 TM HET and wild type-like littermates (WT) received vehicle or CBD (1, 50 or 100 mg/kg i.p.) for 21 days. During treatment and 48 h after withdrawal we measured behaviour, whole blood CBD concentrations and autoradiographic receptor binding. Nrg1 HET mice displayed locomotor hyperactivity, PPI deficits and reduced 5-HT(2A) receptor binding density in the substantia nigra, but these phenotypes were not reversed by CBD. However, long-term CBD (50 and 100 mg/kg) selectively enhanced social interaction in Nrg1 TM HET mice. Furthermore, acute CBD (100 mg/kg) selectively increased PPI in Nrg1 TM HET mice, although tolerance to this effect was manifest upon repeated CBD administration. Long-term CBD (50 mg/kg) also selectively increased GABA(A) receptor binding in the granular retrosplenial cortex in Nrg1 TM HET mice and reduced 5-HT(2A) binding in the substantia nigra in WT mice. Nrg1 appears necessary for CBD-induced anxiolysis since only WT mice developed decreased anxiety-related behaviour with repeated CBD treatment. Altered pharmacokinetics in mutant mice could not explain our findings since no genotype differences existed in CBD blood concentrations. Here we demonstrate that Nrg1 modulates acute and long-term neurobehavioural effects of CBD, which does not reverse the schizophrenia-relevant phenotypes.


Surface structure of sodium dodecyl sulfate surfactant and oil at the oil-in-water droplet liquid/liquid interface: a manifestation of a nonequilibrium surface state.
We present sum frequency scattering spectra on kinetically stabilized emulsions consisting of nanoscopic oil droplets in water, stabilized with sodium dodecyl sulfate (SDS). We have measured the interfacial structure of the alkyl chains of the surfactant molecules, the alkyl chain of the oil molecules, the weakly dispersive D(2)O response, and the interference between SDS and the oil. We find a big difference in chain conformation: SDS has many chain defects, whereas the oil has very few. Our spectra are interpreted to originate from a surface structure with oil molecules predominantly oriented parallel with respect to the plane of the interface. The SDS headgroup is surrounded by water molecules. The SDS alkyl tail is in a disordered state and partially in contact with water. Such a conformation of surfactant occupies a surface area of several hundreds of squared angstroms.


Sympathy for the devil: a conservation strategy for devil and manta rays.
International trade for luxury products, medicines, and tonics poses a threat to both terrestrial and marine wildlife. The demand for and consumption of gill plates (known as Peng Yu Sai, "Fish Gill of Mobulid Ray") from devil and manta rays (subfamily Mobulinae, collectively referred to as mobulids) poses a significant threat to these marine fishes because of their extremely low productivity. The demand for these gill plates has driven an international trade supplied by largely unmonitored and unregulated catches from target and incidental fisheries around the world. Scientific research, conservation campaigns, and legal protections for devil rays have lagged behind those for manta rays despite similar threats across all mobulids. To investigate the difference in attention given to devil rays and manta rays, we examined trends in the scientific literature and updated species distribution maps for all mobulids. Using available information on target and incidental fisheries, and gathering information on fishing and trade regulations (at international, national, and territorial levels), we examined how threats and protective measures overlap with species distribution. We then used a species conservation planning approach to develop the Global Devil and Manta Ray Conservation Strategy, specifying a vision, goals, objectives, and actions to advance the knowledge and protection of both devil and manta rays. Our literature review revealed that there had been nearly 2.5-times more "manta"-titled publications, than "mobula" or "devil ray"-titled publications over the past 4.5 years (January 2012-June 2016). The majority of these recent publications were reports on occurrence of mobulid species. These publications contributed to updated Area of Occupancy and Extent of Occurrence maps which showed expanded distributions for most mobulid species and overlap between the two genera. While several international protections have recently expanded to include all mobulids, there remains a greater number of national, state, and territory-level protections for manta rays compared to devil rays. We hypothesize that there are fewer scientific publications and regulatory protections for devil rays due primarily to perceptions of charisma that favour manta rays. We suggest that the well-established species conservation framework used here offers an objective solution to close this gap. To advance the goals of the conservation strategy we highlight opportunities for parity in protection and suggest solutions to help reduce target and bycatch fisheries.


3D angle-independent Doppler and speckle tracking for the myocardium and blood flow
A technology based on velocity ratio indices is described for application in the myocardium. Angle-independent Doppler indices, such as the pulsatility index, which employ velocity ratios, can be measured even if the ultrasound beam vector at the moving target and the motion vector are not in a known plane. The unknown plane situation is often encountered when an ultrasound beam interrogates sites in the myocardium. The velocities employed in an index calculation must be close to the same or opposite directions. The Doppler velocity ratio indices are independent of angle in 3D space as are ratio indices based on 1D strain and 1D speckle tracking. Angle-independent results with spectral Doppler methods are discussed. Possible future imaging techniques based on velocity ratios are presented. By using indices that involve ratios, several other sources of error cancel in addition to that of angular dependence for example errors due to less than optimum gain settings and beam distortion. This makes the indices reliable as research or clinical tools. Ratio techniques can be readily implemented with current commercial blood flow pulsed wave duplex Doppler equipment or with pulsed wave tissue Doppler equipment. In 70 patients where the quality of the real-time B-mode looked suitable for the Doppler velocity ratio technique, there was only one case where clear spectra could not be obtained for both the LV wall and the septum. A reproducibility study of spectra from the septum of the heart shows a 12% difference in velocity ratios in the repeat measurements.


Follow-up care in general practice of patients with myocardial infarction or angina pectoris: initial results of the SHIP trial. Southampton Heart Integrated Care Project.
We aimed to assess the effectiveness of a nurse-led programme to ensure that follow-up care is provided in general practice after hospital diagnosis of myocardial infarction (MI) or angina pectoris. We conducted a randomized controlled trial with stratified random allocation of practices to intervention and control groups within all 67 practices in Southampton and South-West Hampshire, England. The subjects were 422 adult patients with a MI and 175 patients with a new diagnosis of angina recruited during hospital admission or chest pain clinic attendance between April 1995 and September 1996. Intervention involved a programme of secondary preventive care led by specialist liaison nurses in which we sought to improve communication between hospital and general practice and to encourage general practice nurses to provide structured follow-up. The main outcome measures were: extent of general practice follow-up; attendance for cardiac rehabilitation; medication prescribed at hospital discharge; self-reported smoking, diet and exercise; and symptoms of chest pain and shortness of breath. Follow-ups of 90.1 % of subjects at 1 month and 80.6% at 4 months were carried out. Median attendance for nurse follow-up in the 4 months following diagnosis was 3 (IQR 2-5) in intervention practices and 0 (IQR 0-1) in control practices; the median number of visits to a doctor was the same in both groups. At hospital discharge, levels of prescribing of preventive medication were low in both intervention and control groups: aspirin 77 versus 74% (P = 0.32), cholesterol lowering agents 9 versus 10% (P = 0.8). Conversely, 1 month after diagnosis, the vast majority of patients in both groups reported healthy lifestyles: 90 versus 84% reported eating healthy food (P = 0.53); 73 versus 67% taking regular exercise (P = 0.13); 89 versus 92% not smoking (P = 0.77). Take up of cardiac rehabilitation was 37% in the intervention group and 22% in the control group (P = 0.001); the median number of sessions attended was also higher (5 versus 3 out of 6). The intervention of a liaison nurse is effective in ensuring that general practice nurses follow-up patients after hospital discharge. It does not alter the number of follow-up visits made by the patient to the doctor. Levels of prescribing and reported changes in behaviour at hospital discharge indicate that the main tasks facing practice nurses during follow-up are to help patients to sustain changes in behaviour, to encourage doctors to prescribe appropriate medication and to encourage patients to adhere to medication while returning to an active life. These are very different tasks to those traditionally undertaken by practice nurses in relation to primary prevention, where the emphasis has been on identifying risk and motivating change. Assessment of the effectiveness of practice nurses in undertaking these new tasks requires a longer follow-up.


MO-F-213AB-01: Improving Dose Uniformity in Patch-Field Proton Therapy Using Beam Current Modulation.
To improve the dose homogeneity in patch-field proton therapy using beam current modulation. MCNPX simulations were performed for various configurations of the Mevion S250 beamline. Spread-out Bragg peak (SOBP) dose distributions were calculated, and lateral and distal dose profiles were extracted for through- and patch-field targets at depths ranging from 5.0 to 27.5 cm. We define 'dose non-uniformity' (DNU) as the difference between the maximum and minimum dose relative to the prescription observed in a patched dose distribution. DNU was first calculated for dose distributions combining through-field lateral dose profiles and with patch-field distal dose profiles, assuming a 'standard' delivery with constant beam current. Distal dose profiles were then optimized using beam current modulation to better complement the lateral dose profiles when combined into a patched dose distribution. The DNU for optimized dose distributions was then calculated and compared to the DNU found for standard delivery. Using standard deliveries, DNU was 10% or less when patching lateral profiles 12.5-17.5 cm deep. Significantly greater DNU was observed for patches outside of this range, sometimes exceeding 35%. Using optimized distal profiles, DNU was reduced to 10% or less for all lateral profiles deeper than 12.5 cm. Patches with lateral profiles shallower than 12.5 cm were not improved, as distal dose dropoff could not be improved to better match the sharp lateral dose dropoff. Optimizing beam current modulation can create distal profiles with more gradual dose falloff than found in a standard delivery, allowing optimized distal dose distributions to sum more homogeneously with lateral dose distributions. Thus, the hot or cold spots that often appear in patched dose distributions from standard deliveries can be mitigated by optimizing beam current. This method may also be applied to feathering methods or scanned beam deliveries to further improve patch-field dose homogeneity. Partially supported by MEVION Medical Systems, Inc.


Anatabine lowers Alzheimer's Aβ production in vitro and in vivo.
Brain Aβ accumulation represents a key pathological hallmark in Alzheimer's disease. In this study, we investigated the impact of anatabine, a minor alkaloid present in plants of the Solanacea family on Aβ production in vitro using a cell line overexpressing the human amyloid precursor protein (APP) and in vivo using a transgenic mouse model of Alzheimer's disease. In vitro, anatabine lowers Aβ₁₋₄₀ and Aβ₁₋₄₂ levels in a dose dependent manner and reduces sAPPβ production without impacting sAPPα levels suggesting that anatabine lowers Aβ production by mainly impacting the β-cleavage of APP. Additionally, we show that anatabine lowers NFκB activation at doses that inhibit Aβ production in vitro. Since NFκB is known to regulate BACE-1 expression (the rate limiting enzyme responsible for Aβ production), we determined the impact of anatabine on BACE-1 transcription. We show that anatabine inhibits BACE-1 transcription and reduces BACE-1 protein levels in human neuronal like SHSY-5Y cells suggesting that the Aβ lowering properties of anatabine are mediated via a regulation of BACE-1 expression. In vivo, we show that an acute treatment with anatabine for four days significantly lowers brain soluble Aβ₁₋₄₀ and Aβ₁₋₄₂ levels in a transgenic mouse model of Alzheimer's disease. Altogether our data suggest that anatabine may represent an interesting compound for regulating brain Aβ accumulation.


Synergistic drug combinations from electronic health records and gene expression.
Using electronic health records (EHRs) and biomolecular data, we sought to discover drug pairs with synergistic repurposing potential. EHRs provide real-world treatment and outcome patterns, while complementary biomolecular data, including disease-specific gene expression and drug-protein interactions, provide mechanistic understanding. We applied Group Lasso INTERaction NETwork (glinternet), an overlap group lasso penalty on a logistic regression model, with pairwise interactions to identify variables and interacting drug pairs associated with reduced 5-year mortality using EHRs of 9945 breast cancer patients. We identified differentially expressed genes from 14 case-control human breast cancer gene expression datasets and integrated them with drug-protein networks. Drugs in the network were scored according to their association with breast cancer individually or in pairs. Lastly, we determined whether synergistic drug pairs found in the EHRs were enriched among synergistic drug pairs from gene-expression data using a method similar to gene set enrichment analysis. From EHRs, we discovered 3 drug-class pairs associated with lower mortality: anti-inflammatories and hormone antagonists, anti-inflammatories and lipid modifiers, and lipid modifiers and obstructive airway drugs. The first 2 pairs were also enriched among pairs discovered using gene expression data and are supported by molecular interactions in drug-protein networks and preclinical and epidemiologic evidence. This is a proof-of-concept study demonstrating that a combination of complementary data sources, such as EHRs and gene expression, can corroborate discoveries and provide mechanistic insight into drug synergism for repurposing.


Apparent Lack of BRAFV600E Derived HLA Class I Presented Neoantigens Hampers Neoplastic Cell Targeting by CD8+ T Cells in Langerhans Cell Histiocytosis.
Langerhans Cell Histiocytosis (LCH) is a neoplastic disorder of hematopoietic origin characterized by inflammatory lesions containing clonal histiocytes (LCH-cells) intermixed with various immune cells, including T cells. In 50-60% of LCH-patients, the somatic BRAFV600E driver mutation, which is common in many cancers, is detected in these LCH-cells in an otherwise quiet genomic landscape. Non-synonymous mutations like BRAFV600E can be a source of neoantigens capable of eliciting effective antitumor CD8+ T cell responses. This requires neopeptides to be stably presented by Human Leukocyte Antigen (HLA) class I molecules and sufficient numbers of CD8+ T cells at tumor sites. Here, we demonstrate substantial heterogeneity in CD8+ T cell density in n = 101 LCH-lesions, with BRAFV600E mutated lesions displaying significantly lower CD8+ T cell:CD1a+ LCH-cell ratios (p = 0.01) than BRAF wildtype lesions. Because LCH-lesional CD8+ T cell density had no significant impact on event-free survival, we investigated whether the intracellularly expressed BRAFV600E protein is degraded into neopeptides that are naturally processed and presented by cell surface HLA class I molecules. Epitope prediction tools revealed a single HLA class I binding BRAFV600E derived neopeptide (KIGDFGLATEK), which indeed displayed strong to intermediate binding capacity to HLA-A*03:01 and HLA-A*11:01 in an in vitro peptide-HLA binding assay. Mass spectrometry-based targeted peptidomics was used to investigate the presence of this neopeptide in HLA class I presented peptides isolated from several BRAFV600E expressing cell lines with various HLA genotypes. While the HLA-A*02:01 binding BRAF wildtype peptide KIGDFGLATV was traced in peptides isolated from all five cell lines expressing this HLA subtype, KIGDFGLATEK was not detected in the HLA class I peptidomes of two distinct BRAFV600E transduced cell lines with confirmed expression of HLA-A*03:01 or HLA-A*11:01. These data indicate that the in silico predicted HLA class I binding and proteasome-generated neopeptides derived from the BRAFV600E protein are not presented by HLA class I molecules. Given that the BRAFV600E mutation is highly prevalent in chemotherapy refractory LCH-patients who may qualify for immunotherapy, this study therefore questions the efficacy of immune checkpoint inhibitor therapy in LCH.


Involvement of D₁/D₂ dopamine antagonists upon open-arms exploratory behaviours induced by intra-nucleus accumbens shell administration of N-methyl-D-aspartate.
Glutamatergic system stimulation in some parts of the brain may affect anxiety-related behaviours, aversive learning and memory. This system retains many interactions with dopaminergic neurotransmission. We have studied the effect of nucleus accumbens (NAc) shell glutamatergic system activation on anxiety-related behaviours as well as aversive learning and memory in adult male Wistar rats using the N-methyl-D-aspartate (NMDA) receptor agonist, NMDA. Furthermore, the possible involvement of the NAc shell dopamine D1 and D2 receptors upon NMDA-induced effects was evaluated. The elevated plus-maze task was used to assess the drugs' concomitant effects on anxiety, learning and memory in rats. All drugs were delivered into the NAc shell via bilaterally implanted indwelling cannulae. The NMDA-induced anxiolytic-like behaviours upon retest could possibly be attributed to the further avoidance acquisition impairments. Moreover, the inhibition of dopaminergic system using SCH 23390 and sulpiride induced an anxiolytic-like response and impaired the aversive memory acquisition during retest. However, the concurrent intra-NAc shell microinjection of the subthreshold dose of SCH 23390 and sulpiride (0.125 µg/rat) reversed the anxiolytic-like effect and blocked the aversive memory impairment induced by intra-NAc shell NMDA. Our results suggest a modulatory role of the NAc shell dopaminergic system on NMDA-induced effects in the aversive memory.


Assessment of therapeutic response of Plasmodium falciparum to chloroquine and sulfadoxine-pyrimethamine in an area of low malaria transmission in Colombia.
Although chloroquine (CQ) resistance was first reported in Colombia in 1961 and sulfadoxine-pyrimethamine (SP) resistance in 1981, the frequency of treatment failures to these drugs in Colombia is unclear. A modified World Health Organization 14-day in vivo drug efficacy test for uncomplicated Plasmodium falciparum malaria in areas with intense malaria transmission was adapted to reflect the clinical and epidemiologic features of a low-intensity malaria transmission area in the Pacific Coast Region of Colombia. Patients > or =1 year of age with a parasite density > or =1,000 asexual parasites per microliter were enrolled in this study. Forty-four percent (24 of 54) of the CQ-treated patients were therapeutic failures, including 7 early treatment failures (ETFs) and 17 late treatment failures (LTFs). Four (6%) of 67 SP-treated patients were therapeutic failures (2 ETFs and 2 LTFs). Therapeutic failure in the CQ-treated group was associated with an age <15 years old (P < 0.01), but was not associated with initial parasite density, the presence of CQ or sulfa-containing drugs in urine, or a history of malaria. The high level of therapeutic failures to CQ detected in this study underscores the need and importance of drug efficacy evaluation in the development of a rational national antimalarial drug policy. The relatively low level of therapeutic failures to SP compared with other South American countries raises further questions regarding factors that might have prevented the rapid development of in vivo resistance to this drug combination.


[Why rational drug use must be a priority?].
According to the World Health Organization, rational drug use means that patients receive medicines appropriate to their clinical needs, in doses that meet their individual requirements, for an adequate period of time, and at the lowest cost to them and their community. However, as far as one can see, the reality is very different. In Brazil, at least, 35% of medicines are purchased for self-medication. Medicaments are responsible for 27% of intoxications in Brazil and for 16% of deaths due to poisoning are caused by medicaments. Moreover, 50% of medicaments are prescribed, dispensed or used inappropriately and hospitals spend about 15 to 20% of their budgets to deal with the troubles caused by misuse of medicaments. The promise of immediate relief of suffering is a very attractive appeal, but it has its price. Sometimes the price we pay is our health. Rational drug use requires a very complex logical chain involving a number of variables and participation of different social actors including patients, health professionals, legislators, policy makers, industry, commerce and the government.


Comparison of the biological activity of recombinant human thyroid-stimulating hormone with bovine thyroid-stimulating hormone and evaluation of recombinant human thyroid-stimulating hormone in healthy dogs of different breeds.
To evaluate whether use of recombinant human (rh) thyroid-stimulating hormone (TSH) induces equivalent stimulation, compared with bovine TSH (bTSH), and to evaluate activity of rhTSH in dogs of various large breeds. 18 healthy research Beagles and 20 healthy client-owned dogs of various breeds with body weight > 20 kg. The 18 Beagles were randomly assigned to 3 groups, and each dog received either 75 microg of rhTSH, IM or IV, or 1 unit of bTSH, IM, respectively, in a crossover design. The 20 client-owned dogs received 75 microg of rhTSH, IV. Blood samples were taken before and 6 hours after TSH administration for determination of total serum thyroxine (T(4)) concentration. Additional blood samples were taken after 2 and 4 hours in Beagles that received rhTSH, IM. There was a significant increase in T(4) concentration in all dogs, but there were no differences between values obtained after administration of bTSH versus rhTSH or IV versus IM administration of rhTSH. Although there was a significant difference in age and body weight between Beagles and non-Beagles, there was no difference in post-TSH simulation T(4) concentration between the 2 groups. Results indicated an equivalent biological activity of rhTSH, compared with bTSH. Use of 75 microg of rhTSH, IV, did not induce a different magnitude of stimulation in large-breed dogs, compared with Beagles. Euthyroidism was confirmed if post-TSH simulation T(4) concentration was > or = 2.5 microg/dL and at least 1.5 times basal T(4) concentration.


Preliminary safety and acceptability of a carrageenan gel for possible use as a vaginal microbicide.
We sought to determine the safety and acceptability of vaginal gel formulation PC-503 among low risk, abstinent women. The active ingredient was 2% pharmaceutical grade lambda carrageenan, a sulphated polymer that is generally recognised as safe by the US Food and Drug Administration. 35 women in five sites applied 5 ml of the PC-503 gel vaginally once a day for 7 days while abstaining from sexual intercourse. Visual vaginal examinations were performed on days 1, 4, and 8. STI testing and vaginal pool Gram stain preparations were done on days 1 and 8. Participants were asked about product acceptability. 34 of the 35 women enrolled completed 7 days' use. Following product use, five reported mild symptoms including "bladder fullness," "genital warmth," or discomfort, and lower abdominal pain, and one had moderate pale yellow cervical discharge. Using the Nugent criteria, three women had bacterial vaginosis (BV) before and after use; three had BV before but not after, and two had BV after but not before. Most of the women found PC-503 to be pleasant or neutral in feel and smell and considered extra lubrication to be an advantage; however, one third found it to be messy. Vaginal use of PC-503 gel did not cause significant adverse effects in a small number of low risk, sexually abstinent women. Further testing in larger numbers of sexually active women is planned. A smaller volume of gel may be more acceptable to some women.


Improved detection of acute myocardial infarction using a diagnostic algorithm based on calculated epicardial potentials.
New methods for detecting myocardial infarction in patients with suspected acute coronary syndromes are needed particularly in an era where the majority of patients with myocardial infarction present with non-diagnostic 12-lead electrocardiograms (ECG). We compared a novel epicardial diagnostic algorithm using epicardial potentials from the 80-lead body surface map with other electrocardiographic techniques in detection of myocardial infarction. Between February 1999 and February 2001, consecutive patients (n=427) with ischemic type chest pain had an initial 12-lead ECG and body surface map recorded. Detecting myocardial infarction using an epicardial algorithm was first performed in a training set (n=213) and tested in a validation set of patients (n=214). The results from this epicardial algorithm in myocardial infarction detection were compared with the physician's interpretation of the 12-lead ECG, the body surface map algorithm (PRIME) and physician's interpretation of the body surface map. Myocardial infarction occurred in 205 patients (creatine kinase >or=2x upper limit of normal with creatine kinase-MB >or=7% CK). The physician's interpretation of the 12-lead ECG identified 122 with myocardial infarction (sensitivity 60%, specificity 99%), the body surface map algorithm 137 (sensitivity 67%, specificity 89%), the physician's interpretation of the body surface map 153 (sensitivity 75%, specificity 91%) and the epicardial algorithm 158 (sensitivity 77% specificity 99%). Combining the physician's interpretation of the 12-lead ECG with the epicardial algorithm increased significantly the detection of myocardial infarction (sensitivity 85%, specificity 98%, p<0.001) compared with the 12-lead ECG. An epicardial algorithm based on epicardial potentials increases significantly the detection of myocardial infarction particularly among those with non-diagnostic 12-lead ECG's.


Multicentre outcomes of robot-assisted partial nephrectomy after major open abdominal surgery.
To evaluate the outcomes of robot-assisted partial nephrectomy RAPN after major prior abdominal surgery (PAS) using a large multicentre database. We identified 1 686 RAPN from five academic centres between 2006 and 2014. In all, 216 patients had previously undergone major PAS, defined as having an open upper midline/ipsilateral incision. Perioperative outcomes were compared with those 1 470 patients who had had no major PAS. The chi-squared test and Mann-Whitney U-test were used for categorical and continuous variables, respectively. There was no statistically significant difference in Charlson comorbidity index, tumour size, R.E.N.A.L. nephrometry score or preoperative estimated glomerular filtration rate (eGFR) between the groups. Age and body mass index were higher in patients with PAS. The PAS group had a higher estimated blood loss (EBL) but this did not lead to a higher transfusion rate. A retroperitoneal approach was used more often in patients with major PAS (11.2 vs 5.4%), although this group did not have a higher percentage of posterior tumours (38.8 vs 43.3%, P = 0.286). Operative time, warm ischaemia time, length of stay, positive surgical margin, percentage change in eGFR, and perioperative complications were not significantly different between the groups. RAPN in patients with major PAS is safe and feasible, with increased EBL but no increased rate of transfusion. Patients with major PAS had almost twice the likelihood of having a retroperitoneal approach.


Incidence and Outcomes of Cardiopulmonary Resuscitation in PICUs.
To determine the incidence of cardiopulmonary resuscitation in PICUs and subsequent outcomes. Multicenter prospective observational study of children younger than 18 years old randomly selected and intensively followed from PICU admission to hospital discharge in the Collaborative Pediatric Critical Care Research Network December 2011 to April 2013. Among 10,078 children enrolled, 139 (1.4%) received cardiopulmonary resuscitation for more than or equal to 1 minute and/or defibrillation. Of these children, 78% attained return of circulation, 45% survived to hospital discharge, and 89% of survivors had favorable neurologic outcomes. The relative incidence of cardiopulmonary resuscitation events was higher for cardiac patients compared with non-cardiac patients (3.4% vs 0.8%, p <0.001), but survival rate to hospital discharge with favorable neurologic outcome was not statistically different (41% vs 39%, respectively). Shorter duration of cardiopulmonary resuscitation was associated with higher survival rates: 66% (29/44) survived to hospital discharge after 1-3 minutes of cardiopulmonary resuscitation versus 28% (9/32) after more than 30 minutes (p < 0.001). Among survivors, 90% (26/29) had a favorable neurologic outcome after 1-3 minutes versus 89% (8/9) after more than 30 minutes of cardiopulmonary resuscitation. These data establish that contemporary PICU cardiopulmonary resuscitation, including long durations of cardiopulmonary resuscitation, results in high rates of survival-to-hospital discharge (45%) and favorable neurologic outcomes among survivors (89%). Rates of survival with favorable neurologic outcomes were similar among cardiac and noncardiac patients. The rigorous prospective, observational study design avoided the limitations of missing data and potential selection biases inherent in registry and administrative data.


Maxillomandibular advancement surgery in patients with obstructive sleep apnea: mandibular morphology and stability.
Causal associations between various craniofacial morphologic variables and obstructive sleep apnea have been inferred and serve to justify many treatments. The purposes of this study were to examine the presurgical and postsurgical mandibular morphology of patients with obstructive sleep apnea who were undergoing maxillomandibular advancement and to assess the stability of the observed changes. Various mandibular morphologic variables of 32 male subjects were measured on presurgical, immediately postsurgical, and short-term and long-term postsurgical radiographs. The results demonstrated that presurgical mandibular morphology was not significantly different from that of control samples derived from the literature. The presurgical mandibular plane-hyoid measurement was an average of 11.4 mm greater than that in matched controls. On average, surgery resulted in a significantly longer mandible, a greater gonial angle, and a reduced mandibular plane-hyoid distance, although the response of the hyoid was quite variable. The surgical changes in mandibular length were relatively stable over the long-term. Obstructive sleep apnea did not appear to be related to abnormal presurgical mandibular morphology in this sample.


Biomolecular bases of the senescence process and cancer. A new approach to oncological treatment linked to ageing.
Human ageing is associated with a gradual decline in the physiological functions of the body at multiple levels and it is a key risk factor for many diseases, including cancer. Ageing process is intimately related to widespread cellular senescence, characterised by an irreversible loss of proliferative capacity and altered functioning associated with telomere attrition, accumulation of DNA damage and compromised mitochondrial and metabolic function. Tumour and senescent cells may be generated in response to the same stimuli, where either cellular senescence or transformation would constitute two opposite outcomes of the same degenerative process. This paper aims to review the state of knowledge on the biomolecular relationship between cellular senescence, ageing and cancer. Importantly, many of the cell signalling pathways that are found to be altered during both cellular senescence and tumourigenesis are regulated through shared epigenetic mechanisms and, therefore, they are potentially reversible. MicroRNAs are emerging as pivotal players linking ageing and cancer. These small RNA molecules have generated great interest from the point of view of future clinical therapy for cancer because successful experimental results have been obtained in animal models. Micro-RNA therapies for cancer are already being tested in clinical phase trials. These findings have potential importance in cancer treatment in aged people although further research-based knowledge is needed to convert them into an effective molecular therapies for cancer linked to ageing.


Correcting biases in psychiatric diagnostic practice in Northwest Russia: comparing the impact of a general educational program and a specific diagnostic training program.
A general education in psychiatry does not necessary lead to good diagnostic skills. Specific training programs in diagnostic coding are established to facilitate implementation of ICD-10 coding practices. However, studies comparing the impact of these two different educational approaches on diagnostic skills are lacking. The aim of the current study was to find out if a specific training program in diagnostic coding improves the diagnostic skills better than a general education program, and if a national bias in diagnostic patterns can be minimised by a specific training in diagnostic coding. A pre post design study with two groups was carried in the county of Archangelsk, Russia. The control group (39 psychiatrists) took the required course (general educational program), while the intervention group (45 psychiatrists) were given a specific training in diagnostic coding. Their diagnostic skills before and after education were assessed using 12 written case-vignettes selected from the entire spectrum of psychiatric disorders. There was a significant improvement in diagnostic skills in both the intervention group and the control group. However, the intervention group improved significantly more than did the control group. The national bias was partly corrected in the intervention group but not to the same degree in the control group. When analyzing both groups together, among the background factors only the current working place impacted the outcome of the intervention. Establishing an internationally accepted diagnosis seems to be a special skill that requires specific training and needs to be an explicit part of the professional educational activities of psychiatrists. It does not appear that that skill is honed without specific training. The issue of national diagnostic biases should be taken into account in comparative cross-cultural studies of almost any character. The mechanisms of such biases are complex and need further consideration in future research. Future research should also address the question as to whether the observed improvement in diagnostic skills after specific training actually leads to changes in routine diagnostic practice.


Development of high-yield autofluorescent protein microarrays using hybrid cell-free expression with combined Escherichia coli S30 and wheat germ extracts.
Protein-based microarray platforms offer considerable promise as high-throughput technologies in proteomics. Particular advantages are provided by self-assembling protein microarrays and much interest centers around analysis of eukaryotic proteins and their molecular interactions. Efficient cell-free protein synthesis is paramount for the production of self-assembling protein microarrays, requiring optimal transcription, translation, and protein folding. The Escherichia coli S30 extract demonstrates high translation rates but lacks the protein-folding efficiency of its eukaryotic counterparts derived from rabbit reticulocyte and wheat germ extract. In comparison to E. coli, eukaryotic extracts, on the other hand, exhibit slower translation rates and poor overall protein yields. A cell-free expression system that synthesizes folded eukaryotic proteins in considerable yields would optimize in vitro translation for protein microarray assembly. Self-assembling autofluorescent protein microarrays were produced by in situ transcription and translation of chimeric proteins containing a C-terminal Green Fluorescent Protein tag. Proteins were immobilized as array elements using an anti-GFP monoclonal antibody. The amounts of correctly-folded chimeric proteins were quantified by measuring the fluorescence intensity from each array element. During cell-free expression, very little or no fluorescence was observed from GFP-tagged multidomain eukaryotic plant proteins when in vitro translation was performed with E. coli S30 extract. Improvement was seen using wheat germ extract, but fluorescence intensities were still low because of poor protein yields. A hybrid in vitro translation system, combining S30 and wheat germ extracts, produced high levels of correctly-folded proteins for most of the constructs that were tested. The results are consistent with the hypothesis that the wheat germ extract enhances the protein folding capabilities of the in vitro system by providing eukaryotic ribosomes and chaperones and, at the same time, the E. coli S30 extract, which includes an ATP regeneration system, translates the polypeptides at high rates. This hybrid cell-free expression system allows the facile production of high-yield protein arrays suitable for downstream assays.


Targeted overexpression of CKI-insensitive cyclin-dependent kinase 4 increases functional β-cell number through enhanced self-replication in zebrafish.
β-Cells of the islet of Langerhans produce insulin to maintain glucose homeostasis. Self-replication of β-cells is the predominant mode of postnatal β-cell production in mammals, with about 20% of rodent β cells dividing in a 24-hour period. However, replicating β-cells are rare in adults. Induction of self-replication of existing β-cells is a potential treatment for diabetes. In zebrafish larvae, β-cells rarely self-replicate, even under conditions that favor β-cell genesis such overnutrition and β-cell ablation. It is not clear why larval β-cells are refractory to replication. In this study, we tested the hypothesis that insufficient activity of cyclin-dependent kinase 4 may be responsible for the low replication rate by ectopically expressing in β-cells a mutant CDK4 (CDK4(R24C)) that is insensitive to inhibition by cyclin-dependent kinase inhibitors. Our data show that expression of CDK4(R24C) in β-cells enhanced β-cell replication. CDK4(R24C) also dampened compensatory β-cell neogenesis in larvae and improved glucose tolerance in adult zebrafish. Our data indicate that CDK4 inhibition contributes to the limited β-cell replication in larval zebrafish. To our knowledge, this is the first example of genetically induced β-cell replication in zebrafish.


National Patterns of Medication Treatment for Depression, 1987 to 2001.
BACKGROUND: We investigated trends in antidepressant use, as well as broader changes in depression treatment, following the availability of selective serotonin reuptake inhibitors (SSRIs). METHOD: Using data from the National Disease and Therapeutic Index, a nationally representative survey of U.S. office-based physicians conducted by IMS HEALTH, we analyzed trends in antidepressant prescribing patterns from 1987 through the third quarter of 2001. Annual sample sizes of physician visits by patients reported to have depression ranged from 3901 visits in 1987 to 6639 in 1998. Outcomes examined included the frequency of depression visits, the likelihood of antidepressant therapy, and the use of specific medications. RESULTS: The estimated national number of physician visits by patients with depression increased from 14.4 million visits in 1987 to 24.5 million in 2001 (annualized). The rate of antidepressant medication treatment in these patients also increased from 70% in 1987 to 89% in 2001. In 1987, tricyclic antidepressants were prescribed to 47% of patients with depression. The most common individual antidepressants were amitriptyline (14%), trazodone (12%), doxepin (8%), and desipramine (6%). In 1989, a year after its introduction, fluoxetine was prescribed to 21% of patients with depression. The introduction of other SSRIs led aggregate SSRI use to grow to 38% in 1992, 60% in 1996, and 69% in 2000. In 2001, sertraline (18%), paroxetine (16%), fluoxetine (14%), citalopram (13%), and bupropion (9%) were the leading antidepressants, while tricyclics were used in only 2% of patients. The use of benzodiazepines in depression declined from 21% of patients in 1987 to 8% in 2001. CONCLUSION: The increasing therapeutic dominance of SSRIs may have contributed to other changes in depression treatment, including declining benzodiazepine use, increased aggregate antidepressant treatment rates, and increased reporting of depression.


Concerted transcriptional regulation by BRCA1 and COBRA1 in breast cancer cells.
Cofactor of BRCA1 (COBRA1) was first identified as a protein that binds to the breast cancer susceptibility gene product BRCA1. COBRA1 modulates estrogen-dependent and independent transcription and suppresses the growth of breast cancer cells. Its expression is significantly reduced in metastatic and recurrent breast cancer, pointing to a tumor suppressor function in breast cancer development. In light of these initial implications of COBRA1 in human breast cancer, the current investigation sought to obtain more direct functional evidence that links COBRA1 with BRCA1 in transcriptional regulation in breast cancer cells. Small hairpin RNA (shRNA)-mediated gene knockdown and gene expression microarray were used to study the impact of COBRA1 and BRCA1 on global transcription in the same breast cancer cell background. The gene expression profiling study in tissue culture cells uncovers a significant overlap of COBRA1- and BRCA1-regulated genes, many of which have been previously implicated in breast cancer progression. The data shown herein support the notion that COBRA1 and BRCA1 may engage in common gene regulatory pathways to suppress breast cancer progression.


A Multi-Omics Characterization of the Natural Product Potential of Tropical Filamentous Marine Cyanobacteria.
Microbial natural products are important for the understanding of microbial interactions, chemical defense and communication, and have also served as an inspirational source for numerous pharmaceutical drugs. Tropical marine cyanobacteria have been highlighted as a great source of new natural products, however, few reports have appeared wherein a multi-omics approach has been used to study their natural products potential (i.e., reports are often focused on an individual natural product and its biosynthesis). This study focuses on describing the natural product genetic potential as well as the expressed natural product molecules in benthic tropical cyanobacteria. We collected from several sites around the world and sequenced the genomes of 24 tropical filamentous marine cyanobacteria. The informatics program antiSMASH was used to annotate the major classes of gene clusters. BiG-SCAPE phylum-wide analysis revealed the most promising strains for natural product discovery among these cyanobacteria. LCMS/MS-based metabolomics highlighted the most abundant molecules and molecular classes among 10 of these marine cyanobacterial samples. We observed that despite many genes encoding for peptidic natural products, peptides were not as abundant as lipids and lipopeptides in the chemical extracts. Our results highlight a number of highly interesting biosynthetic gene clusters for genome mining among these cyanobacterial samples.


Belatacept-based immunosuppression in de novo liver transplant recipients: 1-year experience from a phase II randomized study.
This exploratory phase II study evaluated the safety and efficacy of belatacept in de novo adult liver transplant recipients. Patients were randomized (N = 260) to one of the following immunosuppressive regimens: (i) basiliximab + belatacept high dose [HD] + mycophenolate mofetil (MMF), (ii) belatacept HD + MMF, (iii) belatacept low dose [LD] + MMF, (iv) tacrolimus + MMF, or (v) tacrolimus alone. All received corticosteroids. Demographic characteristics were similar among groups. The proportion of patients who met the primary end point (composite of acute rejection, graft loss, death by month 6) was higher in the belatacept groups (42–48%) versus tacrolimus groups (15–38%), with the highest number of deaths and grafts losses in the belatacept LD group. By month 12, the proportion surviving with a functioning graft was higher with tacrolimus + MMF (93%) and lower with belatacept LD (67%) versus other groups (90%: basiliximab + belatacept HD; 83%: belatacept HD; 88%: tacrolimus). Mean calculated GFR was 15–34 mL/min higher in belatacept-treated patients at 1 year. Two cases of posttransplant lymphoproliferative disease and one case of progressive multifocal leukoencephalopathy occurred in belatacept-treated patients. Follow-up beyond month 12 revealed an increase in death and graft loss in another belatacept group (belatacept HD), after which the study was terminated.


Synthetic biology platform of CoryneBrick vectors for gene expression in Corynebacterium glutamicum and its application to xylose utilization.
Currently, the majority of tools in synthetic biology have been designed and constructed for model organisms such as Escherichia coli and Saccharomyces cerevisiae. In order to broaden the spectrum of organisms accessible to such tools, we established a synthetic biological platform, called CoryneBrick, for gene expression in Corynebacterium glutamicum as a set of E. coli-C. glutamicum shuttle vectors whose elements are interchangeable with BglBrick standard parts. C. glutamicum is an established industrial microorganism for the production of amino acids, proteins, and commercially promising chemicals. Using the CoryneBrick vectors, we showed various time-dependent expression profiles of a red fluorescent protein. This CoryneBrick platform was also applicable for two-plasmid expression systems with a conventional C. glutamicum expression vector. In order to demonstrate the practical application of the CoryneBrick vectors, we successfully reconstructed the xylose utilization pathway in the xylose-negative C. glutamicum wild type by fast BglBrick cloning methods using multiple genes encoding for xylose isomerase and xylulose kinase, resulting in a growth rate of 0.11 ± 0.004 h(-1) and a xylose uptake rate of 3.35 mmol/gDW/h when 1 % xylose was used as sole carbon source. Thus, CoryneBrick vectors were shown to be useful engineering tools in order to exploit Corynebacterium as a synthetic platform for the production of chemicals by controllable expression of the genes of interest.


Clinical gestalt and the prediction of massive transfusion after trauma.
Early recognition and treatment of trauma patients requiring massive transfusion (MT) has been shown to reduce mortality. While many risk factors predicting MT have been demonstrated, there is no universally accepted method or algorithm to identify these patients. We hypothesised that even among experienced trauma surgeons, the clinical gestalt of identifying patients who will require MT is unreliable. Transfusion and mortality outcomes after trauma were observed at 10 U.S. Level-1 trauma centres in patients who survived ≥ 30 min after admission and received ≥ 1 unit of RBC within 6h of arrival. Subjects who received ≥ 10 units within 24h of admission were classified as MT patients. Trauma surgeons were asked the clinical gestalt question "Is the patient likely to be massively transfused?" 10 min after the patients arrival. The performance of clinical gestalt to predict MT was assessed using chi-square tests and ROC analysis to compare gestalt to previously described scoring systems. Of the 1245 patients enrolled, 966 met inclusion criteria and 221 (23%) patients received MT. 415 (43%) were predicted to have a MT and 551(57%) were predicted to not have MT. Patients predicted to have MT were younger, more often sustained penetrating trauma, had higher ISS scores, higher heart rates, and lower systolic blood pressures (all p<0.05). Gestalt sensitivity was 65.6% and specificity was 63.8%. PPV and NPV were 34.9% and 86.2% respectively. Data from this large multicenter trial demonstrates that predicting the need for MT continues to be a challenge. Because of the increased mortality associated with delayed therapy, a more reliable algorithm is needed to identify and treat these severely injured patients earlier.


Maternal separation in childhood and diurnal cortisol patterns in mid-life: findings from the Whitehall II study.
Animal studies suggest that maternal separation is associated with alterations in the hypothalamic-pituitary-adrenal (HPA) axis through effects that occur in a critical period following birth. Evidence for an association of the diurnal cortisol rhythm with maternal separation in humans is equivocal. We examined whether maternal separation in childhood is associated with diurnal cortisol pattern in 3712 middle-aged men and women. Two aspects of cortisol release were examined: the cortisol awakening response (CAR) and the diurnal slope in cortisol throughout the day. Maternal separation in childhood was reported by 12% of participants. Those participants who reported maternal separation had a larger CAR and flatter slopes in cortisol levels compared to those who did not report maternal separation [adjusted mean CAR in those reporting no separation versus separation: 7.1, 95% confidence interval (CI) 6.7-7.5 v. 8.4, 95% CI 7.3-9.5, p = 0.02, corresponding to adjusted mean diurnal slope: -0.129 (95% CI -0.130 to -0.128) v. -0.126 (95% CI -0.128 to -0.124), p = 0.01]. In participants who reported maternal separation, the age of separation was not associated with either cortisol measure (p = 0.11). The association between maternal separation and slope in cortisol secretion was largely explained by smoking behaviour and marital status at the time of sample collection whereas that of the CAR was explained by childhood psychosocial, material factors and adult health behaviours. Our findings suggest that maternal separation in childhood is associated with alterations in the diurnal cortisol pattern in middle age. These associations are predominantly accounted for by adult circumstances and behaviours.


A gel-based PCR method to differentiate sheeppox virus field isolates from vaccine strains.
Sheeppox (SPP) and goatpox (GTP) caused by sheeppox virus (SPPV) and goatpox virus (GTPV), respectively of the genus Capripoxvirus in the family Poxviridae, are severely afflicting small ruminants' production systems in Africa and Asia. In endemic areas, SPP and GTP are controlled using vaccination with live attenuated vaccines derived from SPPV, GTPV or Lumpy skin disease virus (LSDV). Sometimes outbreaks occur following vaccination. In order to successfully control the spread of the virus, it is essential to identify whether the animals were infected by the field strain and the vaccine did not provide sufficient protection. Alternatively, in some cases the vaccine strain may cause adverse reactions in vaccinated animals or in rare occasions, re-gain virulence. Thus, diagnostic tools for differentiation of virulent strains from attenuated vaccine strains of the virus are needed. The aim of this study was to identify an appropriate diagnostic target region in the capripoxvirus genome by comparing the genomic sequences of SPPV field isolates with those of the most widely used SPP vaccine strains. A unique 84 base pair nucleotide deletion located between the DNA ligase gene and the VARV B22R homologue gene was found only in SPPV vaccines derived from the Romanian and Yugoslavian RM/65 strains and absent in SPPV field isolates originated from various geographical locations of Asia and Africa. In addition, we developed and evaluated a conventional PCR assay, exploiting the targeted intergenic region to differentiate SPPV vaccine virus from field isolates. The assay produced an amplicon size of 218 bp for the vaccine strains, while the SPPV field isolates resulted in a 302 bp PCR fragment. The assay showed good sensitivity and specificity, and the results were in full agreement with the sequencing data of the PCR amplicons. The developed assay is an improvement of currently existing diagnostic tools and, when combined with a capripox virus species-specific assay, will enhance SPP and GTP diagnosis and surveillance and facilitate epidemiological investigations in countries using live attenuated SPP vaccines. In addition, for laboratories with limited resources, the assay provides a simple and cost-effective alternative for sequencing.


Cellular and humoral immunity to hepatitis-B surface antigen in active chronic hepatitis.
The hepatitis-B surface antigen (HBsAG) may be persistently present in the serum in a few cases of active chronic hepatitis but the cause of the disease in most patients is unknown. In a study of 39 HBsAg-negative cases cell-mediated immunity to HBsAg was observed in 24 (62%), suggesting a high frequency of previous infection with the hepatitis-B virus. Hepatitis-B surface antibody was detectable by radioimmunoassay in six patients, in all of whom complexes of HBsAg were present in the serum on electron microscopy. Out of 12 patients with HBsAg-positive active chronic hepatitis who were also studied eight, including all those untreated at the time, showed a cellular response to the antigen. Evidence of sensitization to a liver-specific cell surface lipoprotein was found with similar frequency in the two groups. These results are consistent with the hypothesis that hepatitis-B virus infection is important in initiating the disease in many cases of active chronic hepatitis and that sensitization to the liver cell membrane antigen is the autoimmune process responsible for the perpetuation of the liver injury.


A Randomized Controlled Pilot Trial of Interventions to Improve Functional Recovery After Hospitalization in Older Adults: Feasibility and Adherence.
Hospitalization is a major risk factor for functional decline, disability, loss of independence, and mortality in older adults. Evidence-based interventions to improve functional recovery from hospitalization are difficult to evaluate and implement in geriatric patients. The goal of this pilot study was to test the feasibility of recruiting geriatric inpatients and implementing pragmatic interventions to improve physical function following hospitalization. Enrolled subjects were randomized to one of five 30-day posthospitalization interventions: isocaloric placebo (P), whey protein supplement (W), in-home rehabilitation+placebo (R+P), rehabilitation+whey protein (R+W), or testosterone (T). Data were collected from a single-site university hospital to determine: (i) institution-based feasibility (nonmodifiable factors including number of patients screened, eligible, contacted) and (ii) patient-based feasibility (modifiable factors including number of patients refusing, enrolled, randomized, intervention adherence, and withdrawal). From January 2014 to July 2016, 4,533 patients were chart screened; 594 (13.1%) were eligible to participate; 384 eligible subjects were contacted; 113 were enrolled; and 100 were randomized. Supplement adherence was 75% and was not different by age, education, level of independence, depression, supplement type, or dual intervention, but was significantly higher in subjects who completed the intervention (p < .01). Rehabilitation session adherence was 77% and did not vary significantly by age, education, level of independence, depression, or supplement type, but was significantly higher for sessions directly supervised (p < .01). Adherence was 100% in the testosterone arm with 94.7% of injections given within 24 hours of discharge. Findings from this clinical trial indicate that posthospitalization interventions in geriatric patients are feasible at both the institution and patient level.


Gene expression profiling to identify eggshell proteins involved in physical defense of the chicken egg.
As uricoletic animals, chickens produce cleidoic eggs, which are self-contained bacteria-resistant biological packages for extra-uterine development of the chick embryo. The eggshell constitutes a natural physical barrier against bacterial penetration if it forms correctly and remains intact. The eggshell's remarkable mechanical properties are due to interactions among mineral components and the organic matrix proteins. The purpose of our study was to identify novel eggshell proteins by examining the transcriptome of the uterus during calcification of the eggshell. An extensive bioinformatic analysis on genes over-expressed in the uterus allowed us to identify novel eggshell proteins that contribute to the egg's natural defenses. Our 14 K Del-Mar Chicken Integrated Systems microarray was used for transcriptional profiling in the hen's uterus during eggshell deposition. A total of 605 transcripts were over-expressed in the uterus compared with the magnum or white isthmus across a wide range of abundance (1.1- to 79.4-fold difference). The 605 highly-expressed uterine transcripts correspond to 469 unique genes, which encode 437 different proteins. Gene Ontology (GO) analysis was used for interpretation of protein function. The most over-represented GO terms are related to genes encoding ion transport proteins, which provide eggshell mineral precursors. Signal peptide sequence was found for 54 putative proteins secreted by the uterus during eggshell formation. Many functional proteins are involved in calcium binding or biomineralization--prerequisites for interacting with the mineral phase during eggshell fabrication. While another large group of proteins could be involved in proper folding of the eggshell matrix. Many secreted uterine proteins possess antibacterial properties, which would protect the egg against microbial invasion. A final group includes proteases and protease inhibitors that regulate protein activity in the acellular uterine fluid where eggshell formation takes place. Our original study provides the first detailed description of the chicken uterus transcriptome during formation of the eggshell. We have discovered a cache of about 600 functional genes and identified a large number of encoded proteins secreted into uterine fluid for fabrication of the eggshell and chemical protection of the egg. Some of these uterine genes could prove useful as biological markers for genetic improvement of phenotypic traits (i.e., egg and eggshell quality).


Selection against Robertsonian fusions involving housekeeping genes in the house mouse: integrating data from gene expression arrays and chromosome evolution.
Monobrachial homology resulting from Robertsonian (Rb) fusions is thought to contribute to chromosomal speciation through underdominance. Given the karyotypic diversity characterizing wild house mouse populations [Mus musculus domesticus, (MMU)], variation that results almost exclusively from Rb fusions (diploid numbers range from 22 to 40) and possibly whole arm reciprocal translocations (WARTs), this organism represents an excellent model for testing hypotheses of chromosomal evolution. Previous studies of chromosome size and recombination rates have failed to explain the bias for certain chromosomes to be involved more frequently than others in these rearrangements. Here, we show that the pericentromeric region of one such chromosome, MMU19, which is infrequently encountered as a fusion partner in wild populations, is significantly enriched for housekeeping genes when compared to other chromosomes in the genome. These data suggest that there is selection against breakpoints in the pericentromeric region and provide new insights into factors that constrain chromosomal reorganizations in house mice. Given the anticipated increase in vertebrate whole genome sequences, the examination of gene content and expression profiles of the pericentromeric regions of other mammalian lineages characterized by Rb fusions (i.e., other rodents, bats, and bovids, among others) is both achievable and crucial to developing broadly applicable models of chromosome evolution.


Breathing through a particular nostril can alter metabolism and autonomic activities.
There is increasing interest in the fact that breathing exclusively through one nostril may alter the autonomic functions. The present study aimed at checking whether such changes actually do occur, and whether breathing is consciously regulated. 48 male subjects, with ages ranging from 25 to 48 years were randomly assigned to different groups. Each group was asked to practice one out of three pranayamas (viz. right nostril breathing, left nostril breathing or alternate nostril breathing). These practices were carried out as 27 respiratory cycles, repeated 4 times a day for one month. Parameters were assessed at the beginning and end of the month, but not during the practice. The 'right nostril pranayama' group showed a significant increase, of 37% in baseline oxygen consumption. The 'alternate nostril' pranayama group showed an 18% increase, and the left nostril pranayama group also showed an increase, of 24%. This increase in metabolism could be due to increased sympathetic discharge to the adrenal medulla. The 'left nostril Pranayama' group showed an increase in volar galvanic skin resistance, interpreted as a reduction in sympathetic nervous system activity supplying the sweat glands. These results suggest that breathing selectively through either nostril could have a marked activating effect or a relaxing effect on the sympathetic nervous system. The therapeutic implications of being able to alter metabolism by changing the breathing pattern have been mentioned.


Treatment with C1-esterase inhibitor concentrate in type I or II hereditary angioedema: a systematic literature review.
Hereditary angioedema (HAE) due to C1 esterase inhibitor (HAE-C1-INH) deficiency is a rare genetic disorder presenting with recurrent episodes of skin swellings, abdominal pain attacks, and potentially fatal laryngeal edema. This study was designed to review the efficacy and safety of pasteurized, human, plasma-derived C1-INH concentrate for the treatment of patients with HAE-C1-INH. A systematic search of electronic databases up to December 2011 was performed without language or date restrictions. Two reviewers completed the study selection using predefined inclusion criteria, tabulated, and analyzed the data. The data were inappropriate for meta-analysis; thus, a qualitative synthesis was performed. We identified 89 studies (≍2000 patients) that investigated C1-INH. Replacement therapy with C1-INH significantly shortened time to onset of symptom relief in HAE attacks compared with placebo in a randomized controlled trial, and similar improvements were consistently reported in observational and descriptive studies, accompanied by improvements in patients' quality of life. C1-INH has been shown to be effective for patients receiving home therapy and short- and long-term prophylaxis. Treatment with C1-INH was generally well tolerated. Administration of C1-INH was not associated with transmission of viruses or development of autoantibodies irrespective of treatment duration. This research provides additional confirmation of the efficacy of C1-INH in the treatment and prevention of HAE attacks. C1-INH is generally safe and well tolerated and has an excellent safety record for over 25 years of clinical use.


Efficient image classification via multiple rank regression.
The problem of image classification has aroused considerable research interest in the field of image processing. Traditional methods often convert an image to a vector and then use a vector-based classifier. In this paper, a novel multiple rank regression model (MRR) for matrix data classification is proposed. Unlike traditional vector-based methods, we employ multiple-rank left projecting vectors and right projecting vectors to regress each matrix data set to its label for each category. The convergence behavior, initialization, computational complexity, and parameter determination are also analyzed. Compared with vector-based regression methods, MRR achieves higher accuracy and has lower computational complexity. Compared with traditional supervised tensor-based methods, MRR performs better for matrix data classification. Promising experimental results on face, object, and hand-written digit image classification tasks are provided to show the effectiveness of our method.


The relationship between symptom severity and symptom interference, education, age, marital status, and type of chemotherapy treatment in Israeli women with early-stage breast cancer.
To examine symptom severity's relationship to symptom interference, education, age, marital status, and type of chemotherapy treatment in Israeli women with stage I or II breast cancer. Cross-sectional, descriptive, correlational design. Hadassah University Hospital's oncology daytime care unit in Israel. 51 women with stage I or II breast cancer who were receiving an adjuvant chemotherapy protocol that included doxorubicin. Women receiving adjuvant chemotherapy were given the M.D. Anderson Symptom Inventory (MDASI), a modified version of the Breast Cancer Prevention Trial Hot Flashes Subscale (BCPT-HFS), and a demographic and treatment questionnaire to assess their symptoms toward the end of their chemotherapy treatment. Symptom severity, symptom interference, education, age, marital status, and type of chemotherapy treatment. The most frequent and severe symptoms were fatigue, sleep disturbance, and drowsiness. The MDASI symptom severity total scores were positively correlated with total scores of interference with activities of daily life, with most individual symptoms being significantly related to the total interference scores. The strongest relationships were found with fatigue, distress, and sadness. Education was inversely related to the MDASI general symptom severity total scores; age was inversely related to the BCPT-HFS total scores. Patients who received treatment with doxorubicin plus cyclophosphamide or doxorubicin, cyclophosphamide, plus fluorouracil had greater symptom severity than those who received doxorubicin plus cyclophosphamide followed by paclitaxel and had their symptoms evaluated after receiving paclitaxel. Increased symptom severity disrupts daily function and life in women with breast cancer. Evidence-based symptom profiles for different chemotherapy protocols are needed.


Functional GATA-3 binding sites within murine CD8 alpha upstream regulatory sequences.
Genes encoding the accessory molecules CD8 and CD4 are activated early in thymocyte development, generating CD4+8+ double positive intermediates, which give rise to two functionally distinct mature T cell subsets that express either CD4 or CD8. The mechanisms that govern the activation or suppression of the CD8 gene are likely to be central to the T cell development program. To identify the key regulatory factors, we have initiated an analysis of the transcriptional regulation of the murine CD8 alpha gene. We have identified three CD8+ cell-specific DNAase I hypersensitive sites (HSS) located upstream of the murine CD8 alpha gene. In vitro mobility shift analysis of the -4.0-kb HSS region has revealed multiple binding sites for the T cell-restricted transcription factor GATA-3. In vitro translated murine GATA-3 binds specifically to both CD8 GATA sites, and coexpression of this factor in transient transfection assays transactivates a reporter construct containing these sequences. These results provide the first evidence for the role of a T cell-restricted factor in the regulation of either CD8 or CD4 genes.


Experiences of alien control in schizophrenia reflect a disorder in the central monitoring of action.
Twenty-three acute psychotic patients who were drug free at the time of testing performed a motor task designed to elicit many errors. Normal subjects and many of the psychotic patients were able to correct these errors in the absence of visual feedback. The ability to make such corrections depends on the subject knowing what response he has just made. Patients with experiences of alien control of their thoughts and actions who formed a subgroup of those classified as schizophrenic, were significantly less likely to make error corrections in the absence of visual feedback. This result is consistent with our previous suggestion (Frith, 1987) that these symptoms are a consequence of problems with the central monitoring of responses.


Methotrexate Restores Regulatory T Cell Function Through Demethylation of the FoxP3 Upstream Enhancer in Patients With Rheumatoid Arthritis.
We have previously shown, in a cohort of untreated rheumatoid arthritis (RA) patients, that the suppressive function of Treg cells is defective. However, other studies in cohorts of patients with established RA have shown that Treg cell function is normal. We hypothesized that treatment may restore Treg cell function and lead to reduced disease activity. The aim of this study was to investigate whether treatment with methotrexate (MTX) can result in epigenetic changes that lead to restoration of the Treg cell suppressive function in RA. Peripheral blood samples from RA patients were assessed using (3) H-thymidine incorporation to measure Treg cell suppression of T cell proliferation, and by enzyme-linked immunosorbent assay to determine Treg cell suppression of interferon-γ production. CTLA-4 and FoxP3 expression was measured by flow cytometry and quantitative polymerase chain reaction (qPCR) in Treg cells from healthy individuals and RA patients. CD4+ T cells isolated from healthy individuals were cultured with interleukin-2 (IL-2), IL-6, and tumor necrosis factor α in the presence or absence of MTX, and FoxP3 expression was determined using qPCR and flow cytometry. Methylation of the FOXP3 upstream enhancer was analyzed by bisulfite sequencing PCR. Defective Treg cell function was observed only in RA patients who had not been treated with MTX, whereas Treg cells from MTX-exposed RA patients had restored suppressive function. This restored suppression was associated with increased expression of FoxP3 and CTLA-4 in Treg cells. Bisulfite sequencing PCR of Treg cells cultured in MTX revealed a significant reduction in methylation of the FOXP3 upstream enhancer. This study identifies a novel mechanism of action of MTX, in which treatment of RA patients with MTX restores defective Treg cell function through demethylation of the FOXP3 locus, leading to a subsequent increase in FoxP3 and CTLA-4 expression.


The therapeutic role of endothelial progenitor cells in Type 1 diabetes mellitus.
Pancreatic β-cells sense and adjust the blood glucose level by secretion of insulin. In Type 1 diabetes mellitus, these insulin-producing cells are destroyed, leaving the patients incapable of regulating blood glucose homeostasis. At the time of diagnosis, most patients still have 20-30% of their original β-cell mass remaining. These residual β-cells are targets for intervention therapies aimed at preventing further autoimmune destruction, in addition to increasing the number of existing β-cells. Such a therapeutic option is highly desirable since it may lead to a full recovery of newly diagnosed patients, with no need for further treatment with immunosuppressant drugs or exogenous insulin administration. In this article, we propose that endothelial progenitor cells, a cell type known to promote and support neovascularization following endothelial injury, may be used as part of a combinational stem cell therapy aimed to improve the vascularization, survival and proliferation of β-cells.


Normal inter-limb differences during the straight leg raise neurodynamic test: a cross sectional study.
The straight leg raise (SLR) neurodynamic test is commonly used to examine the sensitivity of the lower quarter nervous system to movement. Range of motion during the SLR varies considerably, due to factors such as age, sex and activity level. Knowing intra-individual, inter-limb differences may provide a normative measure that is not influenced by such demographic characteristics. This study aimed to determine normal asymmetries between limbs in healthy, asymptomatic individuals during SLR testing and the relationship of various demographic characteristics. The limb elevation angle was measured using an inclinometer during SLR neurodynamic testing that involved pre-positioning the ankle in plantar flexion (PF/SLR) and neutral dorsiflexion (DF/SLR). Phase 1 of the study included 20 participants where the ankle was positioned using an ankle brace replicating research testing conditions. Phase 2 included 20 additional participants where the ankle was manually positioned to replicate clinical testing conditions. The group average range of motion during PF/SLR was 57.1 degrees (SD: 16.8 degrees) on the left and 56.7 degrees (SD: 17.2 degrees) on the right while during DF/SLR the group average was 48.5 degrees (SD: 16.1 degrees) on the left and 48.9 degrees (SD: 16.4 degrees) on the right. The range of motion during SLR was moderately correlated to weight (-0.40 to -0.52), body mass index (-0.41 to -0.52), sex (0.40 to 0.42) and self-reported activity level (0.50 to 0.57). Intra-individual differences between limbs for range of motion during PF/SLR averaged 5.0 degrees (SD: 3.5 degrees) (95% CI: 3.8 degrees, 6.1 degrees) and during DF/SLR averaged 4.1 degrees (SD: 3.2 degrees) (95% CI: 3.1 degrees, 5.1 degrees) but were not correlated with any demographic characteristic. There were no significant differences between Phase 1 and Phase 2. Overall range of motion during SLR was related to sex, weight, BMI and activity level, which is likely reflected in the high variability documented. We can be 95% confident that inter-limb differences during SLR neurodynamic testing fall below 11 degrees in 90% of the general population of healthy individuals. In addition, inter-limb differences were not affected by demographic factors and thus may be a more valuable comparison for test interpretation.