Disruption of the Eng18B ENGase gene in the fungal biocontrol agent Trichoderma atroviride affects growth, conidiation and antagonistic ability.
The recently identified phylogenetic subgroup B5 of fungal glycoside hydrolase family 18 genes encodes enzymes with mannosyl glycoprotein endo-N-acetyl-β-D-glucosaminidase (ENGase)-type activity. Intracellular ENGase activity is associated with the endoplasmic reticulum associated protein degradation pathway (ERAD) of misfolded glycoproteins, although the biological relevance in filamentous fungi is not known. Trichoderma atroviride is a mycoparasitic fungus that is used for biological control of plant pathogenic fungi. The present work is a functional study of the T. atroviride B5-group gene Eng18B, with emphasis on its role in fungal growth and antagonism. A homology model of T. atroviride Eng18B structure predicts a typical glycoside hydrolase family 18 (αβ)(8) barrel architecture. Gene expression analysis shows that Eng18B is induced in dual cultures with the fungal plant pathogens Botrytis cinerea and Rhizoctonia solani, although a basal expression is observed in all growth conditions tested. Eng18B disruption strains had significantly reduced growth rates but higher conidiation rates compared to the wild-type strain. However, growth rates on abiotic stress media were significantly higher in Eng18B disruption strains compared to the wild-type strain. No difference in spore germination, germ-tube morphology or in hyphal branching was detected. Disruption strains produced less biomass in liquid cultures than the wild-type strain when grown with chitin as the sole carbon source. In addition, we determined that Eng18B is required for the antagonistic ability of T. atroviride against the grey mould fungus B. cinerea in dual cultures and that this reduction in antagonistic ability is partly connected to a secreted factor. The phenotypes were recovered by re-introduction of an intact Eng18B gene fragment in mutant strains. A putative role of Eng18B ENGase activity in the endoplasmic reticulum associated protein degradation pathway of endogenous glycoproteins in T. atroviride is discussed in relation to the observed phenotypes.


Macro-micro imaging of cardiac-neural circuits in co-cultures from normal and diseased hearts.
The autonomic nervous system plays an important role in the modulation of normal cardiac rhythm, but is also implicated in modulating the heart's susceptibility to re-entrant ventricular and atrial arrhythmias. The mechanisms by which the autonomic nervous system is pro-arrhythmic or anti-arrhythmic is multifaceted and varies for different types of arrhythmia and their cardiac substrates. Despite decades of research in this area, fundamental questions related to how neuron density and spatial organization modulate cardiac wave dynamics remain unanswered. These questions may be ill-posed in intact tissues where the activity of individual cells is often experimentally inaccessible. Development of simplified biological models that would allow us to better understand the influence of neural activation on cardiac activity can be beneficial. This Symposium Review summarizes the development of in vitro cardiomyocyte cell culture models of re-entrant activity, as well as challenges associated with extending these models to include the effects of neural activation.


Emotion assessment using the NIH Toolbox.
One of the goals of the NIH Toolbox for Assessment of Neurological and Behavioral Function was to identify or develop brief measures of emotion for use in prospective epidemiologic and clinical research. Emotional health has significant links to physical health and exerts a powerful effect on perceptions of life quality. Based on an extensive literature review and expert input, the Emotion team identified 4 central subdomains: Negative Affect, Psychological Well-Being, Stress and Self-Efficacy, and Social Relationships. A subsequent psychometric review identified several existing self-report and proxy measures of these subdomains with measurement characteristics that met the NIH Toolbox criteria. In cases where adequate measures did not exist, robust item banks were developed to assess concepts of interest. A population-weighted sample was recruited by an online survey panel to provide initial item calibration and measure validation data. Participants aged 8 to 85 years completed self-report measures whereas parents/guardians responded for children aged 3 to 12 years. Data were analyzed using a combination of classic test theory and item response theory methods, yielding efficient measures of emotional health concepts. An overview of the development of the NIH Toolbox Emotion battery is presented along with preliminary results. Norming activities led to further refinement of the battery, thus enhancing the robustness of emotional health measurement for researchers using the NIH Toolbox.


Statistical implications of using moving violations to determine crash responsibility in young driver crashes.
Traditional methods for determining crash responsibility - most commonly moving violation citations - may not accurately characterize at-fault status among crash-involved drivers given that: (1) issuance may vary by factors that are independent of fault (e.g., driver age, gender), and (2) these methods do not capture driver behaviors that are not illegal but still indicative of fault. We examined the statistical implications of using moving violations to determine crash responsibility in young driver crashes by comparing it with a method based on crash-contributing driver actions. We selected all drivers in police-reported passenger-vehicle crashes (2010-2011) that involved a New Jersey driver <21 years old (79,485 drivers<age 21, 61,355 drivers ≥age 21). For each driver, crash responsibility was determined from the crash report using two alternative methods: (1) issuance of a moving violation citation; and (2) presence of a driver action (e.g., failure to yield, inattention). Overall, 18% of crash-involved drivers were issued a moving violation while 50% had a driver action. Only 32.2% of drivers with a driver action were cited for a moving violation. Further, the likelihood of being cited given the presence of a driver action was higher among certain driver subgroups-younger drivers, male drivers, and drivers in single-vehicle and more severe crashes. Specifically among young drivers, those driving at night, carrying peer passengers, and having a suspended or no license were more often cited. Conversely, fatally-injured drivers were almost never cited. We also demonstrated that using citation data may lead to statistical bias in the characterization of at-fault drivers and of quasi-induced exposure measures. Studies seeking to accurately determine crash responsibility should thoughtfully consider the potential sources of bias that may result from using legal culpability methods. For many studies, determining driver responsibility via the identification of driver actions may yield more accurate characterizations of at-fault drivers.


Systematic review and consensus definitions for the Standardised Endpoints in Perioperative Medicine initiative: clinical indicators.
Clinical indicators are powerful tools to quantify the safety and quality of patient care. Their validity is often unclear and definitions extremely heterogeneous. As part of the International Standardised Endpoints in Perioperative Medicine (StEP) initiative, this study aimed to derive a set of standardised and valid clinical outcome indicators for use in perioperative clinical trials. We identified clinical indicators via a systematic review of the anaesthesia and perioperative medicine literature (PubMed/OVID, EMBASE, and Cochrane Library). We performed a three-stage Delphi consensus-gaining process that involved 54 clinician-researchers worldwide. Indicators were first shortlisted and the most suitable definitions for evaluation of quality and safety interventions determined. Indicators were then assessed for validity, reliability, feasibility, and clarity. We identified 167 clinical outcome indicators. Participation in the three Delphi rounds was 100% (n=13), 68% (n=54), and 85% (n= 6), respectively. A final list of eight outcome indicators was generated: surgical site infection at 30 days, stroke within 30 days of surgery, death within 30 days of coronary artery bypass grafting, death within 30 days of surgery, admission to the intensive care unit within 14 days of surgery, readmission to hospital within 30 days of surgery, and length of hospital stay (with or without in-hospital mortality). They were rated by the majority of experts as valid, reliable, easy to use, and clearly defined. These clinical indicators can be confidently used as endpoints in clinical trials measuring quality, safety, and improvement in perioperative care. PROSPERO 2016 CRD42016042102 (http://www.crd.york.ac.uk/PROSPERO/display_record.php? ID=CRD42016042102).


Promoting universal financial protection: evidence from seven low- and middle-income countries on factors facilitating or hindering progress.
Although universal health coverage (UHC) is a global health policy priority, there remains limited evidence on UHC reforms in low- and middle-income countries (LMICs). This paper provides an overview of key insights from case studies in this thematic series, undertaken in seven LMICs (Costa Rica, Georgia, India, Malawi, Nigeria, Tanzania, and Thailand) at very different stages in the transition to UHC.These studies highlight the importance of increasing pre-payment funding through tax funding and sometimes mandatory insurance contributions when trying to improve financial protection by reducing out-of-pocket payments. Increased tax funding is particularly important if efforts are being made to extend financial protection to those outside formal-sector employment, raising questions about the value of pursuing contributory insurance schemes for this group. The prioritisation of insurance scheme coverage for civil servants in the first instance in some LMICs also raises questions about the most appropriate use of limited government funds.The diverse reforms in these countries provide some insights into experiences with policies targeted at the poor compared with universalist reform approaches. Countries that have made the greatest progress to UHC, such as Costa Rica and Thailand, made an explicit commitment to ensuring financial protection and access to needed care for the entire population as soon as possible, while this was not necessarily the case in countries adopting targeted reforms. There also tends to be less fragmentation in funding pools in countries adopting a universalist rather than targeting approach. Apart from limiting cross-subsidies, fragmentation of pools has contributed to differential benefit packages, leading to inequities in access to needed care and financial protection across population groups; once such differentials are entrenched, they are difficult to overcome. Capacity constraints, particularly in purchasing organisations, are a pervasive problem in LMICs. The case studies also highlighted the critical role of high-level political leadership in pursuing UHC policies and citizen support in sustaining these policies.This series demonstrates the value of promoting greater sharing of experiences on UHC reforms across LMICs. It also identifies key areas of future research on health care financing in LMICs that would support progress towards UHC.


Functionally independent columns of rat somatosensory barrel cortex revealed with voltage-sensitive dye imaging.
Whisker movement is somatotopically represented in rodent neocortex by electrical activity in clearly defined barrels, which can be visualized in living brain slices. The functional architecture of this part of the cortex can thus be mapped in vitro with respect to its physiological input and compared with its anatomical architecture. The spatial extent of excitation was measured at high temporal resolution by imaging optical signals from voltage-sensitive dye evoked by stimulation of individual barrels in layer 4. The optical signals correlated closely with subthreshold EPSPs recorded simultaneously from excitatory neurons in layer 4 and layer 2/3, respectively. Excitation was initially (<2 msec) limited to the stimulated barrel and subsequently (>3 msec) spread in a columnar manner into layer 2/3 and then subsided in both layers after approximately 50 msec. The lateral extent of the response was limited to the cortical column defined structurally by the barrel in layer 4. Two experimental interventions increased the spread of excitation. First, blocking GABA(A) receptor-mediated synaptic inhibition caused excitation to spread laterally throughout wide regions of layer 2/3 and layer 5 but not into neighboring barrels, suggesting that the local excitatory connections within layer 4 are restricted to single barrels and that inhibitory neurons control spread in supragranular and infragranular layers. Second, NMDA receptor-dependent increase of the spread of excitation was induced by pairing repetitive stimulation of a barrel column with coincident stimulation of layer 2/3 in a neighboring column. Such plasticity in the spatial extent of excitation in a barrel column could underlie changes in cortical map structure induced by alterations of sensory experience.


Coercion and compulsion in mental healthcare-an international perspective.
Coercion has always existed in psychiatry and is increasingly debated. The 'move into the community' in many countries over recent decades and the evolution of community services have substantially altered the locus of coercion. In many countries psychiatric services remain poorly funded and patchy. Substantial differences between regions and countries in the provision of services, the role of the family, and the wider economic and political climate are likely to lead to different sources and experiences of coercion. This paper explores a number of factors that may affect the prevalence and type of coercion in psychiatric services and in society and their impact upon those with severe mental illnesses. Differences in service provision are explored and wider societal issues that may impact are considered along with relevant evidence. Coercion is commonly experienced by those with severe mental illnesses but is poorly understood. The vast majority of research relates to High Income Group countries with developed community services and formal mental health legislation that adopt the so-called 'medical model'. Further research and collaboration is urgently required to increase our understanding of these issues, which are difficult to define and measure. An evidence base that is relevant worldwide, not just to a small group of countries, is needed to inform training and the care of all patients. A particular focus must be expanding our knowledge and understanding of coercion in cultures outside those where such research has traditionally taken place to date.


Preliminary experiences with intraoperative radiation therapy (IORT) for the treatment of brain tumors.
Ten brain tumor patients underwent wide resection of the tumor followed by Intraoperative Radiation Therapy (IORT) at the first surgery or at the second salvage surgery after failure of conventional external beam irradiation. Two patients(1 meningioma, 1 glioblastoma multiforme) were treated at the first surgery and 8 patients(3 anaplastic astrocytoma, 3 glioblastoma multiforme, 1 meningioma, 1 gliosarcoma) were treated after salvage surgery. The IORT doses were ranged from 15-25 Gy depending on the tumor volume and previous radiation therapy. The neurological status(Karnofsky performance status) was improved in 4 cases, not changed in 6 cases after IORT. There were several complications after IORT; radiation necrosis, communicating hydrocephalus, wound infection, and abnormal CT findings such as diffuse low density area in an around operation site. The radiation necrosis was confirmed by operation in a recurrent meningioma patient 12 months after IORT. At follow-up, ranging from 1 to 16 months, there was no deaths. Based on our limited experiences, the IORT might be one of the adjuvant therapeutic modalities especially for the malignant brain tumors and unresectable huge meningioma.


Percutaneous venovenous CO2 removal with regional anticoagulation in an ovine model.
Extracorporeal CO2 removal may reduce minute ventilation requirements and allow for better tolerance of low tidal volume ventilating strategies in patients with severe respiratory insufficiency. Conventional extracorporeal gas exchange is labor-intensive, expensive, and usually requires systemic anticoagulation. In this study, a simplified venovenous circuit was developed by using regional citrate anticoagulation to avoid potential complications associated with systemic heparin. Five healthy adult sheep underwent percutaneous placement of a double-lumen 18F catheter into the internal jugular vein. The extracorporeal circuit consisted of a hollow fiber oxygenator and a variable speed roller pump. Regional anticoagulation consisted of a continuous citrate infusion to the inflow limb of the circuit. Systemic calcium levels were maintained by calcium chloride infusion through a central line. CO2 transfer was measured at varying levels of blood and gas flow. CO2 transfer ranged from 31 ml/min (500 ml/min blood flow; 2 l/min gas flow) to 150 ml/min (1000 ml/min blood flow; 15 l/min gas flow) and was directly proportional to blood flow and gas flow (p < 0.05). Normocapnia was maintained despite a 75% reduction in minute ventilation. At 24 hours, there was no significant clot formation in the circuit.


Hunter-Gatherers and the Origins of Religion.
Recent studies of the evolution of religion have revealed the cognitive underpinnings of belief in supernatural agents, the role of ritual in promoting cooperation, and the contribution of morally punishing high gods to the growth and stabilization of human society. The universality of religion across human society points to a deep evolutionary past. However, specific traits of nascent religiosity, and the sequence in which they emerged, have remained unknown. Here we reconstruct the evolution of religious beliefs and behaviors in early modern humans using a global sample of hunter-gatherers and seven traits describing hunter-gatherer religiosity: animism, belief in an afterlife, shamanism, ancestor worship, high gods, and worship of ancestors or high gods who are active in human affairs. We reconstruct ancestral character states using a time-calibrated supertree based on published phylogenetic trees and linguistic classification and then test for correlated evolution between the characters and for the direction of cultural change. Results indicate that the oldest trait of religion, present in the most recent common ancestor of present-day hunter-gatherers, was animism, in agreement with long-standing beliefs about the fundamental role of this trait. Belief in an afterlife emerged, followed by shamanism and ancestor worship. Ancestor spirits or high gods who are active in human affairs were absent in early humans, suggesting a deep history for the egalitarian nature of hunter-gatherer societies. There is a significant positive relationship between most characters investigated, but the trait "high gods" stands apart, suggesting that belief in a single creator deity can emerge in a society regardless of other aspects of its religion.


Differential activation of human core, non-core and auditory-related cortex during speech categorization tasks as revealed by intracranial recordings.
Speech perception requires that sounds be transformed into speech-related objects with lexical and semantic meaning. It is unclear at what level in the auditory pathways this transformation emerges. Primary auditory cortex has been implicated in both representation of acoustic sound attributes and sound objects. While non-primary auditory cortex located on the posterolateral superior temporal gyrus (PLST) is clearly involved in acoustic-to-phonetic pre-lexical representations, it is unclear what role this region plays in auditory object formation. Additional data support the importance of prefrontal cortex in the formation of auditory objects, while other data would implicate this region in auditory object selection. To help clarify the respective roles of auditory and auditory-related cortex in the formation and selection of auditory objects, we examined high gamma activity simultaneously recorded directly from Heschl's gyrus (HG), PLST and prefrontal cortex, while subjects performed auditory semantic detection tasks. Subjects were patients undergoing evaluation for treatment of medically intractable epilepsy. We found that activity in posteromedial HG and early activity on PLST was robust to sound stimuli regardless of their context, and minimally modulated by tasks. Later activity on PLST could be strongly modulated by semantic context, but not by behavioral performance. Activity within prefrontal cortex also was related to semantic context, and did co-vary with behavior. We propose that activity in posteromedial HG and early activity on PLST primarily reflect the representation of spectrotemporal sound attributes. Later activity on PLST represents a pre-lexical processing stage and is an intermediate step in the formation of word objects. Activity in prefrontal cortex appears directly involved in word object selection. The roles of other auditory and auditory-related cortical areas in the formation of word objects remain to be explored.


Corrosion of titanium under simulated inflammation conditions: clinical context and in vitro investigations.
Titanium and alloys thereof are widely utilized for biomedical applications in the fields of orthopedics and dentistry. The corrosion resistance and perceived biocompatibility of such materials are essentially related to the presence of a thin passive oxide layer on the surface. However, during inflammation phases, the immune system and its leukocytic cells generate highly aggressive molecules, such as hydrogen peroxide and radicals, that can significantly alter the passive film resulting in the degradation of the titanium implants. In combination with mechanical factors, this can lead to the release of metal ions, nanoparticles or microscaled debris in the surrounding tissues (which may sustain chronic inflammation), bring about relevant health issues and contribute to implant loss or failure. After briefly presenting the context of inflammation, this review article analyses the state-of-the-art knowledge of the in vitro corrosion of titanium, titanium alloys and coated titanium by reactive oxygen species and by living cells with an emphasis on electrochemical and microstructural aspects. STATEMENT OF SIGNIFICANCE: Inflammation involves the production of reactive oxygen species that are known to alter the passive layer protecting titanium implants against the aggressive environment of the human body. Inflammatory processes therefore contribute to the deterioration of biomedical devices. Although review articles on biomaterials for implant applications are regularly published in the literature, none has ever focused specifically on the topic of inflammation. After briefly recalling the clinical context, this review analyses the in vitro studies on titanium corrosion under simulated inflammation conditions from the pioneer works of the 80s and the 90s till the most recent investigations. It reports about the status of this research area for a multidisciplinary readership covering the fields of materials science, corrosion and implantology.


Aged-care nurses' knowledge of nursing documentation: an Australian perspective.
This study investigated registered nurses' knowledge of documentation used in aged-care nursing home facilities in Queensland, Australia. The purpose of nursing documentation is to communicate health information, facilitate quality assurance and research, demonstrate nurses' accountability and, within Australia, to support funding of residents' care. Little is known about the relationship between RNs' knowledge of nursing documentation, the documentation process within residential aged care and the outcomes of the documentation. Cross-sectional, retrospective design. The study was conducted with a large sample of RNs (n = 360) located in 162 Queensland aged-care facilities. Participants completed a postage-return questionnaire in which they identified factors that influence their knowledge and understanding of documentation. Participants reported that they have considerable knowledge of nursing documentation. They also indicated that they were most knowledgeable about policies on documentation and writing discharge instructions. However, their knowledge of nursing assessments ranked fifth and they were least knowledgeable about reading reports each shift. The modified version of Edelstein's questionnaire provided a valid and reliable instrument for measuring RNs' knowledge of nursing documentation. A factor analysis of the 16 items in the Knowledge scale showed excellent reliability. The data indicated that RNs in aged-care facilities have high levels of knowledge about documentation. Specific recommendations relate to the implementation of comprehensive documentation education programs that reflect the needs of organisations and the level of RNs' skills and knowledge concerning documentation. Accurate nursing documentation is relevant to residents' care outcomes and to government funding allocations. Measuring RNs' knowledge of nursing documentation can identify factors that impede and facilitate their documentation of care.


Combination of FASP and StageTip-based fractionation allows in-depth analysis of the hippocampal membrane proteome.
Membrane proteomics is challenging because the desirable strong detergents are incompatible with downstream analysis. Recently, we demonstrated efficient removal of SDS by the filter aided sample preparation method (FASP). Here we combine FASP with our previously described small-scale membrane enrichment protocol. Analysis of a single mouse hippocampus enables identification of more than 1000 membrane proteins in a single LC-MS/MS run without protein or peptide prefractionation. To extend proteome coverage, we developed a simple anion exchange fractionation method in a StageTip format. When separating peptides into six fractions, a duplicate analysis resulted in identification of 4206 proteins of which 64% were membrane proteins. This data set covers 83% of glutamate and GABA receptor subunits identified in hippocampus in the Allen Brain Atlas and adds further isoforms. The combined method provides a streamlined protocol for rapid and sensitive membrane proteome mapping. We also provide a generic protocol for combining FASP with StageTip-based ion exchange fractionation, which is generally applicable to proteome analysis.


Pulmonary angiography using 16 slice multidetector computed tomography in normal dogs.
We report a canine computed tomography (CT) pulmonary angiography technique using multidetector CT (MDCT). CT pulmonary angiography using a 16 slice MDCT was performed on five healthy, anesthetized beagles. A helical acquisition with pitch of 1.4 was used. The time delay for the angiographic study was determined using a bolus-tracking program. A dose of 400 mg I/kg of nonionic contrast medium (Iohexol 300 mg I/ml) was administered to each dog via a cephalic catheter using an angiographic power injector at a rate of 5 ml/s. In two dogs a second study, using a contrast medium dose of 200 and 600 mg I/kg was performed. Arterial enhancement of transverse and reformatted images was classified subjectively as excellent, good, or poor, and assessed objectively by measuring Hounsfield units at the right main pulmonary artery. Angiographic studies were evaluated by two radiologists to determine the number of subsegmental arterial branches visualized. The median number of subsegmental arterial branches identified was five (range: 2-7). Based on the time attenuation curve obtained by the bolus-tracking program, there was consistent enhancement of the right main pulmonary artery beginning at 6 s and peaking at 8 s in 4/5 dogs. The contrast medium dose of 400 mg I/kg produced good to excellent vascular enhancement in the same 4/5 dogs. A dose of 200 mg I/kg resulted in poor enhancement. CT pulmonary angiography using MDCT and an automated bolus-tracking program allows rapid, consistent evaluation of the pulmonary vasculature using a single dose of 400 mg I/kg of contrast medium.


A distinct three-helix centipede toxin SSD609 inhibits I(ks) channels by interacting with the KCNE1 auxiliary subunit.
KCNE1 is a single-span transmembrane auxiliary protein that modulates the voltage-gated potassium channel KCNQ1. The KCNQ1/KCNE1 complex in cardiomyocytes exhibited slow activated potassium (I(ks)) currents. Recently, a novel 47-residue polypeptide toxin SSD609 was purified from Scolopendra subspinipes dehaani venom and showed I(ks) current inhibition. Here, chemically synthesized SSD609 was shown to exert I(ks) inhibition in extracted guinea pig cardiomyocytes and KCNQ1/KCNE1 current attenuation in CHO cells. The K(+) current attenuation of SSD609 showed decent selectivity among different auxiliary subunits. Solution nuclear magnetic resonance analysis of SSD609 revealed a distinctive three-helix conformation that was stabilized by a new disulfide bonding pattern as well as segregated surface charge distribution. Structure-activity studies demonstrated that negatively charged Glu19 in the amphipathic extracellular helix of KCNE1 was the key residue that interacted with SSD609. The distinctive three-helix centipede toxin SSD609 is known to be the first polypeptide toxin acting on channel auxiliary subunit KCNE1, which suggests a new type of pharmacological regulation for ion channels in cardiomyocytes.


Human casein alpha s1 induces proinflammatory cytokine expression in monocytic cells by TLR4 signaling.
Human casein alpha S1 (CSN1S1) is a milk-derived protein, which gives rise to sustained antibody formation after breast feeding in infancy and induces the expression of proinflammatory cytokines. So far, the cellular CSN1S1 receptor is unrecognized. Our objective was to identify the receptor employed by CSN1S1 to induce proinflammatory effects. CSN1S1 concentration and time dependently induced expression of IL-1β, IL-8, and IL-6 in monocytic cells despite addition of polymyxin B, which completely abrogated LPS effects. Coincubation of monocytic cells with a neutralizing antibody to Toll-like receptor 4 (TLR4) but not to TLR2 inhibited CSN1S1-induced effects. In TLR4/MD2/CD14-cotransfected HEK293 cells CSN1S1 increased IL-8 expression, while untransfected cells were completely unresponsive. Furthermore, CSN1S1-induced IL1-β secretion was impeded by inhibition of MyD88 and caspase-1. Flow cytometric in vitro assays confirmed binding of CSN1S1 to TLR4. Phosphorylation of CSN1S1 by casein-kinase 2 completely abolished proinflammatory cytokine induction and binding to TLR4. CSN1S1 is a multifunctional milk protein that exerts proinflammatory properties via TLR4 and the inflammasome pathway in a phosphorylation-dependent manner. The contribution of CSN1S1 in mother milk to the development of a potent immune system in breastfed individuals should be further assessed.


Species-specific impact of microplastics on coral physiology.
There is evidence that microplastic (MP) pollution can negatively influence coral health; however, mechanisms are unknown and most studies have used MP exposure concentrations that are considerably higher than current environmental conditions. Furthermore, whether MP exposure influences coral susceptibility to other stressors such as ocean warming is unknown. Our objective was to determine the physiology response of corals exposed to MP concentrations that have been observed in-situ at ambient and elevated temperature that replicates ocean warming. Here, two sets of short-term experiments were conducted at ambient and elevated temperature, exposing the corals Acroporasp. and Seriatopora hystrix to microspheres and microfibres. Throughout the experiments, gross photosynthesis and net respiration was quantified using a 4-chamber coral respirometer, and photosynthetic yields of photosystem II were measured using Pulse-Amplitude Modulated (PAM) fluorometry. Results indicate the effect of MP exposure is dependent on MP type, coral species, and temperature. MP fibres (but not spheres) reduced photosynthetic capability of Acropora sp., with a 41% decrease in photochemical efficiency at ambient temperature over 12 days. No additional stress response was observed at elevated temperature; photosynthetic performance significantly increased in Seriatopora hystrix exposed to MP spheres. These findings show that a disruption to coral photosynthetic ability can occur at MP concentrations that have been observed in the marine environment and that MP pollution impact on corals remains an important aspect for further research.


Sticky Pi is a high-frequency smart trap that enables the study of insect circadian activity under natural conditions.
In the face of severe environmental crises that threaten insect biodiversity, new technologies are imperative to monitor both the identity and ecology of insect species. Traditionally, insect surveys rely on manual collection of traps, which provide abundance data but mask the large intra- and interday variations in insect activity, an important facet of their ecology. Although laboratory studies have shown that circadian processes are central to insects' biological functions, from feeding to reproduction, we lack the high-frequency monitoring tools to study insect circadian biology in the field. To address these issues, we developed the Sticky Pi, a novel, autonomous, open-source, insect trap that acquires images of sticky cards every 20 minutes. Using custom deep learning algorithms, we automatically and accurately scored where, when, and which insects were captured. First, we validated our device in controlled laboratory conditions with a classic chronobiological model organism, Drosophila melanogaster. Then, we deployed an array of Sticky Pis to the field to characterise the daily activity of an agricultural pest, Drosophila suzukii, and its parasitoid wasps. Finally, we demonstrate the wide scope of our smart trap by describing the sympatric arrangement of insect temporal niches in a community, without targeting particular taxa a priori. Together, the automatic identification and high sampling rate of our tool provide biologists with unique data that impacts research far beyond chronobiology, with applications to biodiversity monitoring and pest control as well as fundamental implications for phenology, behavioural ecology, and ecophysiology. We released the Sticky Pi project as an open community resource on https://doc.sticky-pi.com.


Power evolution along phase-sensitive parametric amplifiers: an experimental survey.
We propose and experimentally demonstrate a method based on Brillouin optical time-domain analysis to measure the longitudinal signal power distribution along phase-sensitive fiber-optical parametric amplifiers (PS-FOPAs). Experimental results show that the amplification of a PS-FOPA could go through different longitudinal profiles and yet finish with the same overall gain. This behavior is in sheer contrast with theoretical expectations, according to which longitudinal gain distribution should follow certain profiles determined by the initial relative phase difference but can never end up in the same overall gain. The gap between theory and experiment only becomes evident when the pump wavelength is within the fluctuation range of the zero dispersion wavelength (ZDW) of the PS-FOPA.


Effects of nanofiber/stem cell composite on wound healing in acute full-thickness skin wounds.
Acute full-thickness skin wounds (FTSW) caused by extensive burns or high-energy trauma are not adequately addressed by current clinical treatments. This study hypothesized that biomimetic nanofiber scaffolds (NFSs) functionalized with rich attachment of bone-marrow-derived mesenchymal stem cells (BM-MSCs) can promote wound healing in acute FTSW. Results in a rat model showed that both NFS and BM-MSCs contributed to the wound healing. Wounds in NFS group with a higher density of BM-MSCs achieved complete closure 8 days earlier than the control group. Implanted BM-MSCs were found to promote epithelial edge ingrowth and collagen synthesis. The colocation of BM-MSCs (tagged with quantum-dots) with the expression of keratin 10 and filaggrin indicated the participation of BM-MSCs in epidermal differentiation at early and intermediate stages under the local wounding environment. Overall, this study suggests a great potential of using NFS/BM-MSC composites for the treatment of acute FTSW.


Expression, purification and preliminary X-ray analysis of a fibrillarin homolog from Methanococcus jannaschii, a hyperthermophile.
Fibrillarin plays a central role in ribosome biogenesis as a ribosomal RNA-processing protein. A Methanococcus jannaschii homolog of fibrillarin has been overexpressed, purified and crystallized. Crystals belong to the C2 space group with unit-cell parameters a = 121.4, b = 43.2, c = 55.3 A, beta = 96.9 degrees. Under flash-frozen conditions and using synchrotron radiation, the crystals diffract to 1.8 A resolution. For structural determination, a selenomethionine derivative of the protein has also been crystallized.


Synthesis of α-tribromomethylamines via Mg-mediated addition of bromoform to imines.
Mg-mediated addition of bromoform to electron deficient imines such as N-sulfonylimines affords α-tribromomethylated N-sulfonylamines in good to excellent yields. The procedure could be further simplified by transforming the imine precursors, α-sulfonyl-N-tosyl- and Boc-amines, in one pot to the corresponding α-tribromomethyl derivatives. Facile removal of the Boc protecting group in nearly quantitative yield and a silver carbonate mediated monodebromination have also been demonstrated.


Internal fixation compared with total hip replacement for displaced femoral neck fractures in the elderly. A randomised, controlled trial.
The treatment algorithms for displaced fractures of the femoral neck need to be improved if we are to reduce the need for secondary surgery. We have studied 102 patients of mean age 80 years, with an acute displaced fracture of the femoral neck. They were randomly placed into two groups, treated either by internal fixation (IF) with two cannulated screws or total hip replacement (THR). None showed severe cognitive dysfunction, all were able to walk independently, and all lived in their own home. They were reviewed at four, 12 and 24 months after surgery. Outcome measurements included hip complications, revision surgery, hip function according to Charnley and the health-related quality of life (HRQoL) according to EuroQol (EQ-5D). The failure rate after 24 months was higher in the IF group than in the THR group with regard to hip complications (36% and 4%, respectively; p < 0.001), and the number of revision procedures (42% and 4%, p < 0.001). Hip function was significantly better in the THR group at all follow-up reviews regarding pain (p < 0.005), movement (p < 0.05 except at 4 months) and walking (p < 0.05). The reduction in HRQoL (EQ-5D index score) was also significantly lower in the THR group than in the IF group, comparing the pre-fracture situation with that at all follow-up reviews (p < 0.05). The results of our study strongly suggest that THR provides a better outcome than IF for elderly, relatively healthy, lucid patients with a displaced fracture of the femoral neck.


Mortality in patients with pituitary disease.
Pituitary disease is associated with increased mortality predominantly due to vascular disease. Control of cortisol secretion and GH hypersecretion (and cardiovascular risk factor reduction) is key in the reduction of mortality in patients with Cushing's disease and acromegaly, retrospectively. For patients with acromegaly, the role of IGF-I is less clear-cut. Confounding pituitary hormone deficiencies such as gonadotropins and particularly ACTH deficiency (with higher doses of hydrocortisone replacement) may have a detrimental effect on outcome in patients with pituitary disease. Pituitary radiotherapy is a further factor that has been associated with increased mortality (particularly cerebrovascular). Although standardized mortality ratios in pituitary disease are falling due to improved treatment, mortality for many conditions are still elevated above that of the general population, and therefore further measures are needed. Craniopharyngioma patients have a particularly increased risk of mortality as a result of the tumor itself and treatment to control tumor growth; this is a key area for future research in order to optimize the outcome for these patients.


Fast population coding.
Uncertainty coming from the noise in its neurons and the ill-posed nature of many tasks plagues neural computations. Maybe surprisingly, many studies show that the brain manipulates these forms of uncertainty in a probabilistically consistent and normative manner, and there is now a rich theoretical literature on the capabilities of populations of neurons to implement computations in the face of uncertainty. However, one major facet of uncertainty has received comparatively little attention: time. In a dynamic, rapidly changing world, data are only temporarily relevant. Here, we analyze the computational consequences of encoding stimulus trajectories in populations of neurons. For the most obvious, simple, instantaneous encoder, the correlations induced by natural, smooth stimuli engender a decoder that requires access to information that is nonlocal both in time and across neurons. This formally amounts to a ruinous representation. We show that there is an alternative encoder that is computationally and representationally powerful in which each spike contributes independent information; it is independently decodable, in other words. We suggest this as an appropriate foundation for understanding time-varying population codes. Furthermore, we show how adaptation to temporal stimulus statistics emerges directly from the demands of simple decoding.


Neural Integration Underlying a Time-Compensated Sun Compass in the Migratory Monarch Butterfly.
Migrating eastern North American monarch butterflies use a time-compensated sun compass to adjust their flight to the southwest direction. Although the antennal genetic circadian clock and the azimuth of the sun are instrumental for proper function of the compass, it is unclear how these signals are represented on a neuronal level and how they are integrated to produce flight control. To address these questions, we constructed a receptive field model of the compound eye that encodes the solar azimuth. We then derived a neural circuit model that integrates azimuthal and circadian signals to correct flight direction. The model demonstrates an integration mechanism, which produces robust trajectories reaching the southwest regardless of the time of day and includes a configuration for remigration. Comparison of model simulations with flight trajectories of butterflies in a flight simulator shows analogous behaviors and affirms the prediction that midday is the optimal time for migratory flight.


Factors influencing the collagenase digestion phase of human islet isolation.
Substantial advances in human islet isolation technology have occurred during the past decade. However, it is still difficult to recover the entire quantity of islets contained in a pancreas. A major obstacle to successful human islet isolation has been the variability of the collagenase digestion phase of islet isolation. Future advances in enzyme technology will make it possible to optimally liberate islets with enzyme blends "tailor-made" for each individual donor pancreas. Such innovative strategies will be advantageous in improving islet isolation efficiency, recovery, viability, and ultimately posttransplant function.


Visualizing request-flow comparison to aid performance diagnosis in distributed systems.
Distributed systems are complex to develop and administer, and performance problem diagnosis is particularly challenging. When performance degrades, the problem might be in any of the system's many components or could be a result of poor interactions among them. Recent research efforts have created tools that automatically localize the problem to a small number of potential culprits, but research is needed to understand what visualization techniques work best for helping distributed systems developers understand and explore their results. This paper compares the relative merits of three well-known visualization approaches (side-by-side, diff, and animation) in the context of presenting the results of one proven automated localization technique called request-flow comparison. Via a 26-person user study, which included real distributed systems developers, we identify the unique benefits that each approach provides for different problem types and usage modes.


PEDOT doped with algal, mammalian and synthetic dopants: polymer properties, protein and cell interactions, and influence of electrical stimulation on neuronal cell differentiation.
Poly(3,4-ethylenedioxythiophene) (PEDOT) films were electrochemically polymerised with several synthetic (dodecylbenzosulfonic acid (DBSA)) and biological (dextran sulphate (DS), chondroitin sulphate (CS), alginic acid (ALG) and ulvan (ULV)) dopant anions, and their physical, mechanical and electrochemical properties characterised. PEDOT films incorporating the biological dopants ALG and ULV produced films of the greatest surface roughness (46 ± 5.1 and 31 ± 1.9 nm, respectively), and demonstrated significantly lower shear modulus values relative to all other PEDOT films (2.1 ± 0.1 and 1.2 ± 0.2 MPa, respectively). Quartz crystal microgravimetry was used to study the adsorption of the important extracellular matrix protein fibronectin, revealing protein adsorption to be greatest on PEDOT doped with DS, followed by DBSA, ULV, CS and ALG. Electrical stimulation experiments applying a pulsed current using a biphasic waveform (250 Hz) were undertaken using PEDOT doped with either DBSA or ULV. Electrical stimulation had a significant influence on cell morphology and cell differentiation for PEDOT films with either dopant incorporated, with the degree of branching per cell increased by 10.5× on PEDOT-DBSA and 6.5× on PEDOT-ULV relative to unstimulated cells, and mean neurite length per cell increasing 2.6× and 2.2× on stimulated vs. unstimulated PEDOT-DBSA and PEDOT-ULV, respectively. We demonstrate the cytocompatibility of synthetic and biologically doped PEDOT biomaterials, including the new algal derived polysaccharide dopant ulvan, which, along with DBSA doped PEDOT, is shown to significantly enhance the differentiation of PC12 neuronal cells under electrical stimulation.


Percutaneous laser disc decompression: clinical experience at SCTIMST and long term follow up.
Low backache (LBA) is now increasing in younger population due to misdirected spinal kinetics secondary to improper posture, heavy load lifting and motorbike driving. Hence minimally invasive procedures are increasingly sought after. Among these, PLDD is currently popular and in use. We present our long term follow-up in the use of Nd:YAG laser for PLDD. To evaluate the efficacy of PLDD in treatment of contained herniation of lumbar discs & long term follow up results. Forty patients with contained lumbar disc herniation on MRI and who did not respond to 6 weeks conservative treatment were subjected to PLDD. L4-5 disc was treated in 31, L5-S1 in 12 and L1-2 and L3-4 in one each. Nd:YAG laser at 1064 nm was used for the procedure. Total laser energy of 1500-2000 Joules was delivered at the disc space depending upon the size. There was immediate pain relief in 32/40 (80%). According to MacNab criteria good to fair response was seen in 37/40 (92%) and 3 patients (7.5%) responded poorly to this treatment. On follow up which ranged from 1 to 7 years, 34/40 (85%) had pain relief with no need for further treatment. Significant pain at local puncture site was experienced by 8 (20%), pain during lasing was experienced by one. One patient developed muscular spasm. Percutaneous laser disc decompression is a safe, relatively noninvasive and effective treatment modality for contained, nonsequestered, herniated lumbar disc disease in carefully selected patients.


Why have sex? The population genetics of sex and recombination.
One of the greatest puzzles in evolutionary biology is the high frequency of sexual reproduction and recombination. Given that individuals surviving to reproductive age have genomes that function in their current environment, why should they risk shuffling their genes with those of another individual? Mathematical models are especially important in developing predictions about when sex and recombination can evolve, because it is difficult to intuit the outcome of evolution with several interacting genes. Interestingly, theoretical analyses have shown that it is often quite difficult to identify conditions that favour the evolution of high rates of sex and recombination. For example, fitness interactions among genes (epistasis) can favour sex and recombination but only if such interactions are negative, relatively weak and not highly variable. One reason why an answer to the paradox of sex has been so elusive is that our models have focused unduly on populations that are infinite in size, unstructured and isolated from other species. Yet most verbal theories for sex and recombination consider a finite number of genotypes evolving in a biologically and/or physically complex world. Here, we review various hypotheses for why sex and recombination are so prevalent and discuss theoretical results indicating which of these hypotheses is most promising.


Extraction of the EPP component from the surface EMG.
A surface electromyogram (EMG), especially when recorded near the neuromuscular junction, is expected to contain the endplate potential (EPP) component which can be extracted with an appropriate signal filter. Two factors are important: the EMG must be recorded in monopolar fashion, and the recording must be done so the low frequency signal corresponding the EPP is not eliminated. This report explains how to extract the EPP component from the EMG of the masseter muscle in a human subject. The surface EMG is recorded from eight sites using traditional disc electrodes aligned along over the muscle, with equal inter-electrode distance from the zygomatic arch to the angle of mandible in response to quick gum clenching. A reference electrode is placed on the tip of the nose. The EPP component is extracted from the raw EMGs by applying a high-cut digital filter (2nd dimension Butterworth filter) with a range of 10-35 Hz. When the filter is set to 10 Hz, the extracted EPP wave deflects either negative or positive depending on the recording site. The difference in the polarity reflects the sink-source relation of the end plate current, with the site showing the most negative deflection corresponding to the neuromuscular junction. In the case of the masseter muscle, the neuromuscular junction is estimated to be located in the inferior portion close to the angle of mandible. The EPP component exhibits an interesting oscillation when the cut-off frequency of the high-cut digital filter is set to 30 Hz. The EPP oscillation indicates that muscle contraction is adjusted in an intermittent manner. Abnormal tremors accompanying various sorts of diseases may be substantially due to this EPP oscillation, which becomes slower and is difficult to cease.


Protein structure prediction with in-cell photo-crosslinking mass spectrometry and deep learning.
While AlphaFold2 can predict accurate protein structures from the primary sequence, challenges remain for proteins that undergo conformational changes or for which few homologous sequences are known. Here we introduce AlphaLink, a modified version of the AlphaFold2 algorithm that incorporates experimental distance restraint information into its network architecture. By employing sparse experimental contacts as anchor points, AlphaLink improves on the performance of AlphaFold2 in predicting challenging targets. We confirm this experimentally by using the noncanonical amino acid photo-leucine to obtain information on residue-residue contacts inside cells by crosslinking mass spectrometry. The program can predict distinct conformations of proteins on the basis of the distance restraints provided, demonstrating the value of experimental data in driving protein structure prediction. The noise-tolerant framework for integrating data in protein structure prediction presented here opens a path to accurate characterization of protein structures from in-cell data.


Purification, pharmacological modulation, and biochemical characterization of interactors of endogenous human gamma-secretase.
Gamma-secretase is a unique intramembrane-cleaving protease complex, which cleaves the Alzheimer's disease-associated beta-amyloid precursor protein (APP) and a number of other type I membrane proteins. Human gamma-secretase consists of the catalytic subunit presenilin (PS) (PS1 or PS2), the substrate receptor nicastrin, APH-1 (APH-1a or APH-1b), and PEN-2. To facilitate in-depth biochemical analysis of gamma-secretase, we developed a fast and convenient multistep purification procedure for the endogenous enzyme. The enzyme was purified from HEK293 cells in an active form and had a molecular mass of approximately 500 kDa. Purified gamma-secretase was capable of producing the major amyloid-beta peptide (Abeta) species, such as Abeta40 and Abeta42, from a recombinant APP substrate in physiological ratios. Abeta generation could be modulated by pharmacological gamma-secretase modulators. Moreover, the Abeta42/Abeta40 ratio was strongly increased by purified PS1 L166P, an aggressive familial Alzheimer's disease mutant. Tandem mass spectrometry analysis revealed the consistent coisolation of several proteins with the known gamma-secretase core subunits. Among these were the previously described gamma-secretase interactors CD147 and TMP21 as well as other known interactors of these. Interestingly, the Niemann-Pick type C1 protein, a cholesterol transporter previously implicated in gamma-secretase-mediated processing of APP, was identified as a major copurifying protein. Affinity capture experiments using a biotinylated transition-state analogue inhibitor of gamma-secretase showed that these proteins are absent from active gamma-secretase complexes. Taken together, we provide an effective procedure for isolating endogenous gamma-secretase in considerably high grade, thus aiding further characterization of this pivotal enzyme. In addition, we provide evidence that the copurifying proteins identified are unlikely to be part of the active gamma-secretase enzyme.


One-shot genitalia are not an evolutionary dead end - regained male polygamy in a sperm limited spider species.
Monogynous mating systems with extremely low male mating rates have several independent evolutionary origins and are associated with drastic adaptations involving self-sacrifice, one-shot genitalia, genital damage, and termination of spermatogenesis immediately after maturation. The combination of such extreme traits likely restricts evolutionary potential perhaps up to the point of making low male mating rates irreversible and hence may constitute an evolutionary dead end. Here, we explore the case of a reversion to multiple mating from monogynous ancestry in golden orb-web spiders, Nephila senegalensis. Male multiple mating is regained by the loss of genital damage and sexual cannibalism but spermatogenesis is terminated with maturation, restricting males to a single loading of their secondary mating organs and a fixed supply of sperm. However, males re-use their mating organs and by experimentally mating males to many females, we show that the sperm supply is divided between copulations without reloading the pedipalps. By portioning their precious sperm supply, males achieve an average mating rate of four females which effectively doubles the maximal mating rate of their ancestors. A heritage of one-shot genitalia does not completely restrict the potential to increase mating rates in Nephila although an upper limit is defined by the available sperm load. Future studies should now investigate how males use this potential in the field and identify selection pressures responsible for a reversal from monogynous to polygynous mating strategies.


Functional shifts in insect microRNA evolution.
MicroRNAs (miRNAs) are short endogenous RNA molecules that regulate gene expression at the posttranscriptional level and have been shown to play critical roles during animal development. The identification and comparison of miRNAs in metazoan species are therefore paramount for our understanding of the evolution of body plans. We have characterized 203 miRNAs from the red flour beetle Tribolium castaneum by deep sequencing of small RNA libraries. We can conclude, from a single study, that the Tribolium miRNA set is at least 15% larger than that in the model insect Drosophila melanogaster (despite tens of high-throughput sequencing experiments in the latter). The rate of birth and death of miRNAs is high in insects. Only one-third of the Tribolium miRNA sequences are conserved in D. melanogaster, and at least 18 Tribolium miRNAs are conserved in vertebrates but lost in Drosophila. More than one-fifth of miRNAs that are conserved between Tribolium and Drosophila exhibit changes in the transcription, genomic organization, and processing patterns that lead to predicted functional shifts. For example, 13% of conserved miRNAs exhibit seed shifting, and we describe arm-switching events in 11% of orthologous pairs. These shifts fundamentally change the predicted targets and therefore function of orthologous miRNAs. In general, Tribolium miRNAs are more representative of the insect ancestor than Drosophila miRNAs and are more conserved in vertebrates.


Gamma-Glutamyltransferase 1 Promotes Clear Cell Renal Cell Carcinoma Initiation and Progression.
Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. While the localized form of this disease can be treated surgically, advanced and metastatic stages are resistant to chemotherapies. Although more innovative treatments, such as targeted or immune-based therapies, exist, the need for new therapeutic options remains. ccRCC presents unique metabolic signatures and multiple studies have reported a significant increase in levels of reduced glutathione (GSH) and its precursors in ccRCC tumor samples compared with normal kidney tissues. These observations led us to investigate the effects of blocking the GSH pathway, particularly the gamma-glutamyltransferase 1 (GGT1) enzyme, in multiple ccRCC cell lines. In this study, we provide in vitro and in vivo evidence that GGT1/GSH pathway inhibition impacts ccRCC cell growth, through increased cell-cycle arrest. Of note, GGT1 inhibition also impairs ccRCC cell migration. Finally, pharmacologic GSH pathway inhibition decreases ccRCC cell proliferation and increases sensitivity to standard chemotherapy. Our results suggest that GGT1/GSH pathway inhibition represents a new strategy to overcome ccRCC chemoresistance. IMPLICATIONS: GGT1/GSH pathway inhibition represents a promising therapeutic strategy to overcome chemoresistance and inhibit progression of ccRCC tumors.


Region-specific alterations in brain development in one- to three-year-old boys with fragile X syndrome.
Longitudinal neuroimaging investigation of fragile X syndrome (FXS), the most common cause of inherited intellectual disability and autism, provides an opportunity to study the influence of a specific genetic factor on neurodevelopment in the living human brain. We examined voxel-wise gray and white matter volumes (GMV, WMV) over a 2-year period in 1- to 3-year-old boys with FXS (n = 41) and compared these findings to age- and developmentally matched controls (n = 28). We found enlarged GMV in the caudate, thalamus, and fusiform gyri and reduced GMV in the cerebellar vermis in FXS at both timepoints, suggesting early, possibly prenatal, genetically mediated alterations in neurodevelopment. In contrast, regions in which initial GMV was similar, followed by an altered growth trajectory leading to increased size in FXS, such as the orbital gyri, basal forebrain, and thalamus, suggests delayed or otherwise disrupted synaptic pruning occurring postnatally. WMV of striatal-prefrontal regions was greater in FXS compared with controls, and group differences became more exaggerated over time, indicating the possibility that such WM abnormalities are the result of primary FMRP-deficiency-related axonal pathology, as opposed to secondary connectional dysregulation between morphologically atypical brain structures. Our results indicate that structural abnormalities of different brain regions in FXS evolve differently over time reflecting time-dependent effects of FMRP deficiency and provide insight into their neuropathologic underpinnings. The creation of an early and accurate human brain phenotype for FXS in humans will significantly improve our capability to detect whether new disease-specific treatments can "rescue" the FXS phenotype in affected individuals.


A role for the mitochondrial deacetylase Sirt3 in regulating energy homeostasis.
Here, we demonstrate a role for the mitochondrial NAD-dependent deacetylase Sirt3 in the maintenance of basal ATP levels and as a regulator of mitochondrial electron transport. We note that Sirt3(-/-) mouse embryonic fibroblasts have a reduction in basal ATP levels. Reconstitution with wild-type but not a deacetylase-deficient form of Sirt3 restored ATP levels in these cells. Furthermore in wild-type mice, the resting level of ATP correlates with organ-specific Sirt3 protein expression. Remarkably, in mice lacking Sirt3, basal levels of ATP in the heart, kidney, and liver were reduced >50%. We further demonstrate that mitochondrial protein acetylation is markedly elevated in Sirt3(-/-) tissues. In addition, in the absence of Sirt3, multiple components of Complex I of the electron transport chain demonstrate increased acetylation. Sirt3 can also physically interact with at least one of the known subunits of Complex I, the 39-kDa protein NDUFA9. Functional studies demonstrate that mitochondria from Sirt3(-/-) animals display a selective inhibition of Complex I activity. Furthermore, incubation of exogenous Sirt3 with mitochondria can augment Complex I activity. These results implicate protein acetylation as an important regulator of Complex I activity and demonstrate that Sirt3 functions in vivo to regulate and maintain basal ATP levels.


Cell size and growth rate are major determinants of replicative lifespan.
Yeast cells, like mammalian cells, enlarge steadily as they age. Unabated cell growth can promote cellular senescence; however, the significance of the relationship between size and cellular lifespan is not well understood. Herein, we report a genetic link between cell size, growth rate and lifespan. Mutations that increase cell size concomitantly increase growth rate and decrease lifespan. As a result, large cells grow, divide and age dramatically faster than small cells. Conversely, small cell mutants age slowly and are long-lived. Investigation of the mechanisms involved suggests that attainment of a maximal size modulates lifespan. Indeed, cumulative results revealed that life expectancy is size-dependent, and that the rate at which cells age is determined in large part by the amount of cell growth per generation.


Exposure to cats and dogs, and symptoms of asthma, rhinoconjunctivitis, and eczema.
Associations between exposure to cats and dogs and respiratory and allergic outcomes in children have been reported in affluent countries, but little is known about such associations in less-affluent countries. The International Study of Asthma and Allergies in Childhood, phase 3 was carried out in children aged 6-7 years and adolescents aged 13-14 years across the world. Questions about cats and dogs in the home were included in an additional questionnaire. Using logistic regression, we investigated the association between such exposures and symptoms of asthma, rhinoconjunctivitis, and eczema. Adjustments were made for sex, region of the world, language, gross national income per capita, and 10 other covariates. Among children (6-7 years of age), cat exposure in the first year of life was associated with current symptoms of asthma, wheeze, rhinoconjunctivitis, and eczema, especially in less-affluent countries. Among adolescents (13-14 years of age), we found a positive association between exposure to cats or dogs and symptom prevalence in more-affluent and less-affluent countries. The global multivariate odds ratios for children with complete covariate data were 1.17 (95% confidence interval = 1.08-1.29) for current symptoms of asthma, 1.13 (1.05-1.23) for rhinoconjunctivitis, and 1.38 (1.26-1.52) for eczema. Smaller odds ratios were found for exposure to only dogs. Exposure to only cats was associated with eczema. Early-life exposure to cats is a risk factor for symptoms of asthma, rhinoconjunctivitis, and eczema in 6- to 7-year-old children, especially in less-affluent countries. Current exposure to cats and dogs combined, and only to dogs, is a risk factor for symptom reporting by 13- to 14-year-old adolescents worldwide.


A Systematic Review of Interventions and Programs Targeting Appropriate Prescribing of Opioids.
Canada and the United States have the highest levels of prescription opioid consumption in the world. In an attempt to curb the opioid epidemic, a variety of interventions have been implemented. Thus far, evidence regarding their effectiveness has not been consolidated. The objectives of this study were to: 1) identify interventions that target opioid prescribing; 2) assess and compare the effectiveness of interventions on opioid prescription and related harms; 3) determine the methodological quality of evaluation studies. The study involved a systematic review of the literature including bibliographical databases and gray literature sources. Systematic review including bibliographical databases and gray literature sources. We searched MEDLINE, Embase, and LILACS databases from January 1, 2005 to September 23, 2016 for any intervention that targeted the prescription of opioids. We also examined websites of relevant organizations and scanned bibliographies of included articles and reviews for additional references. The target population was that of all health care providers (HCPs) or users of opioids with no restriction on indication. Endpoints were those related to process (implementation), outcomes (effectiveness), or impact. Sources were screened independently by 2 reviewers using pre-defined eligibility criteria. Synthesis of findings was qualitative; no pooling of results was conducted. Literature search yielded 12,278 unique sources. Of these, 142 were retained. During full-text review, 75 were further excluded. Searches of the gray literature and bibliographies yielded 49 additional sources. Thus, a total of 95 distinct interventions were identified. Over half consisted of prescription monitoring programs (PMPs) and mainly targeted HCPs. Evaluation studies addressed mainly opioid prescription rate (30.6%), opioid use (19.4%), or doctor shopping or diversion (9.7%). Fewer studies considered overdose death (9.7%), abuse (9.7%), misuse (4.2%), or diversion (5.6%). Study designs consisted of cross-sectional surveys (23.3%), pre-post intervention (26.7%), or time series without a comparison group (13.3%), which limit the robustness of the evidence. Although PMPs and policies have been associated with a reduction in opioid prescription, their impact on appropriateness of use according to clinical guidelines and restriction of access to patients in need is inconsistent. Continuing medical education (CME) and pain management programs were found effective in improving chronic pain management, but studies were conducted in specific settings. The impact of interventions on abuse and overdose-death is conflicting. Due to the very large number of publications and programs found, it was difficult to compare interventions owing to the heterogeneity of the programs and to the methodologies of evaluation studies. No assessment of publication bias was done in the review. Evidence of effectiveness of interventions targeting the prescription of opioids is scarce in the literature. Although PMPs have been associated with a reduction in the overall prescription rates of Schedule II opioids, their impact on the appropriateness of use taking into consideration benefits, misuse, legal and illegal use remains elusive. Our review suggests that existing interventions have not addressed all determinants of inappropriate opioid prescribing and usage. A well-described theoretical framework would be the backdrop against which targeted interventions or policies may be developed. Opioid, prescription, abuse, misuse, diversion, interventions, prescription monitoring programs.


Tuberculosis knowledge, attitudes and health-seeking behaviour in rural Uganda.
To assess tuberculosis (TB) knowledge, attitudes and health-seeking behaviour to inform the design of communication and social mobilisation interventions. Iganga/Mayuge Demographic Surveillance Site, Uganda. Between June and July 2008, 18 focus group discussions and 12 key informant interviews were conducted, including parents of infants and adolescents and key informant interviews with community leaders, traditional healers and patients with TB. People viewed TB as contagious, but not necessarily an airborne pathogen. Popular TB aetiologies included sharing utensils, heavy labour, smoking, bewitchment and hereditary transmission. TB patients were perceived to seek care late or to avoid care. Combining care from traditional healers and the biomedical system was common. Poverty, drug stock-outs, fear of human immunodeficiency virus (HIV) testing and length of TB treatment negatively affect health-seeking behaviour. Stigma and avoidance of persons with TB often reflects an assumption of HIV co-infection. The community's concerns about pill burden, quality of care, financial barriers, TB aetiology, stigma and preference for pluralistic care need to be addressed to improve early detection. Health education messages should emphasise the curability of TB, the feasibility of treatment and the engagement of traditional healers as partners in identifying cases and facilitating adherence to treatment.


Quadrivalent vaccine against human papillomavirus to prevent high-grade cervical lesions.
Human papillomavirus types 16 (HPV-16) and 18 (HPV-18) cause approximately 70% of cervical cancers worldwide. A phase 3 trial was conducted to evaluate a quadrivalent vaccine against HPV types 6, 11, 16, and 18 (HPV-6/11/16/18) for the prevention of high-grade cervical lesions associated with HPV-16 and HPV-18. In this randomized, double-blind trial, we assigned 12,167 women between the ages of 15 and 26 years to receive three doses of either HPV-6/11/16/18 vaccine or placebo, administered at day 1, month 2, and month 6. The primary analysis was performed for a per-protocol susceptible population that included 5305 women in the vaccine group and 5260 in the placebo group who had no virologic evidence of infection with HPV-16 or HPV-18 through 1 month after the third dose (month 7). The primary composite end point was cervical intraepithelial neoplasia grade 2 or 3, adenocarcinoma in situ, or cervical cancer related to HPV-16 or HPV-18. Subjects were followed for an average of 3 years after receiving the first dose of vaccine or placebo. Vaccine efficacy for the prevention of the primary composite end point was 98% (95.89% confidence interval [CI], 86 to 100) in the per-protocol susceptible population and 44% (95% CI, 26 to 58) in an intention-to-treat population of all women who had undergone randomization (those with or without previous infection). The estimated vaccine efficacy against all high-grade cervical lesions, regardless of causal HPV type, in this intention-to-treat population was 17% (95% CI, 1 to 31). In young women who had not been previously infected with HPV-16 or HPV-18, those in the vaccine group had a significantly lower occurrence of high-grade cervical intraepithelial neoplasia related to HPV-16 or HPV-18 than did those in the placebo group. (ClinicalTrials.gov number, NCT00092534 [ClinicalTrials.gov].).


Survival of hendra virus in the environment: modelling the effect of temperature.
Hendra virus (HeV), a highly pathogenic zoonotic paramyxovirus recently emerged from bats, is a major concern to the horse industry in Australia. Previous research has shown that higher temperatures led to lower virus survival rates in the laboratory. We develop a model of survival of HeV in the environment as influenced by temperature. We used 20 years of daily temperature at six locations spanning the geographic range of reported HeV incidents to simulate the temporal and spatial impacts of temperature on HeV survival. At any location, simulated virus survival was greater in winter than in summer, and in any month of the year, survival was higher in higher latitudes. At any location, year-to-year variation in virus survival 24 h post-excretion was substantial and was as large as the difference between locations. Survival was higher in microhabitats with lower than ambient temperature, and when environmental exposure was shorter. The within-year pattern of virus survival mirrored the cumulative within-year occurrence of reported HeV cases, although there were no overall differences in survival in HeV case years and non-case years. The model examines the effect of temperature in isolation; actual virus survivability will reflect the effect of additional environmental factors.


Magnetic resonance imaging evaluation of hepatic cysts in early autosomal-dominant polycystic kidney disease: the Consortium for Radiologic Imaging Studies of Polycystic Kidney Disease cohort.
The objective of this study was to investigate the prevalence of hepatic cysts by age and gender in patients with early autosomal-dominant polycystic kidney disease (ADPKD) and to determine whether hepatic cyst volume is related to renal and renal cyst volumes by using magnetic resonance imaging (MRI). A total of 230 patients with ADPKD (94 men and 136 women) who were aged 15 to 46 yr and had relatively preserved renal function were studied. MRI images of the kidney and liver were obtained to measure renal, renal cyst, and hepatic cyst volumes. These volume measurements and hepatic cyst prevalence were compared in all patients and in subgroups on the basis of gender and age (15 to 24, 25 to 34, and 35 to 46 yr). The overall prevalence of hepatic cysts was 83%; the prevalence was 58, 85, and 94% in the sequential age groups and 85% in women and 79% in men. The prevalence was related directly to renal volume (chi2 = 4.30, P = 0.04) and to renal cyst volume (chi2 = 5.59, P = 0.02). The total hepatic cyst volume was significantly greater in women than in men (a logarithmic transformation mean of 5.27 versus 1.94 ml; P = 0.003). The average hepatic cyst volume was 0.25, 5.75, and 22.78 ml in sequential age groups. Hepatic cysts are evident in 94% of patients who are older than 35 yr and in 55% of individuals who are younger than 25 yr. Hepatic cysts are more prevalent and larger in total cyst volume in women than in men. Hepatic cyst prevalence and aggregate total hepatic cyst volume increased with age.


Second-dose ChAdOx1 and BNT162b2 COVID-19 vaccines and thrombocytopenic, thromboembolic and hemorrhagic events in Scotland.
We investigated thrombocytopenic, thromboembolic and hemorrhagic events following a second dose of ChAdOx1 and BNT162b2 using a self-controlled case series analysis. We used a national prospective cohort with 2.0 million(m) adults vaccinated with two doses of ChAdOx or 1.6 m with BNT162b2. The incidence rate ratio (IRR) for idiopathic thrombocytopenic purpura (ITP) 14-20 days post-ChAdOx1 second dose was 2.14, 95% confidence interval (CI) 0.90-5.08. The incidence of ITP post-second dose ChAdOx1 was 0.59 (0.37-0.89) per 100,000 doses. No evidence of an increased risk of CVST was found for the 0-27 day risk period (IRR 0.83, 95% CI 0.16 to 4.26). However, few (≤5) events arose within this risk period. It is perhaps noteworthy that these events all clustered in the 7-13 day period (IRR 4.06, 95% CI 0.94 to 17.51). No other associations were found for second dose ChAdOx1, or any association for second dose BNT162b2 vaccination. Second dose ChAdOx1 vaccination was associated with increased borderline risks of ITP and CVST events. However, these events were rare thus providing reassurance about the safety of these vaccines. Further analyses including more cases are required to determine more precisely the risk profile for ITP and CVST after a second dose of ChAdOx1 vaccine.


Strategies for manipulating the p53 pathway in the treatment of human cancer.
Human cancer progression is driven in part by the mutation of oncogenes and tumour-suppressor genes which, under selective environmental pressures, give rise to evolving populations of biochemically altered cells with enhanced tumorigenic and metastatic potential. Given that human cancers are biologically and pathologically quite distinct, it has been quite surprising that a common event, perturbation of the p53 pathway, occurs in most if not all types of human cancers. The central role of p53 as a tumour-suppressor protein has fuelled interest in defining its mechanism of function and regulation, determining how its inactivation facilitates cancer progression, and exploring the possibility of restoring p53 function for therapeutic benefit. This review will highlight the key biochemical properties of p53 protein that affect its tumour-suppressor function and the experimental strategies that have been developed for the re-activation of the p53 pathway in cancers.


Angiotensin II regulates adrenal vascular tone through zona glomerulosa cell-derived EETs and DHETs.
Elevated concentrations of aldosterone are associated with several cardiovascular diseases. Angiotensin II (Ang II) increases aldosterone secretion and adrenal blood flow. This concurrent increase in steroidogenesis and adrenal blood flow is not understood. We investigated the role of zona glomerulosa (ZG) cells in the regulation of vascular tone of bovine adrenal cortical arteries by Ang II. ZG cells enhanced endothelium-dependent relaxations to Ang II. The ZG cell-dependent relaxations to Ang II were unchanged by removing the endothelium-dependent response to Ang II. These ZG cell-mediated relaxations were ablated by cytochrome P450 inhibition, epoxyeicosatrienoic acid (EET) antagonism, and potassium channel blockade. Analysis of ZG cell EET production by liquid chromatography/mass spectrometry demonstrated an increase in EETs and dihydroxyeicosatrienoic acids with Ang II stimulation. These EETs and dihydroxyeicosatrienoic acids produced similar concentration-dependent relaxations of adrenal arteries, which were attenuated by EET antagonism. Whole-cell potassium currents of adrenal artery smooth muscle cells were increased by Ang II stimulation in the presence of ZG cells but decreased in the absence of ZG cells. This increase in potassium current was abolished by iberiotoxin. Similarly, 14,15-EET induced concentration-dependent increases in potassium current, which was abolished by iberiotoxin. ZG cell aldosterone release was not directly altered by EETs. These data suggest that Ang II stimulates ZG cells to release EETs and dihydroxyeicosatrienoic acids, resulting in potassium channel activation and relaxation of adrenal arteries. This provides a mechanism by which Ang II concurrently increases adrenal blood flow and steroidogenesis.


No evidence for circulating mesenchymal stem cells in patients with organ injury.
Mesenchymal stem cells (MSC) are present in the bone marrow, from where they are thought to migrate through the blood stream to the sites of injury. However, virtually all tissues contain resident MSC that may contribute to local regenerative and immunomodulatory processes, thereby hypothetically preempting the need for recruiting MSC through the bloodstream. Although there is some indication for circulating MSC in animal models, there is little solid evidence for the mobilization and migration of MSC in the human circulation. In the present study, we were unable to detect MSC in the blood of healthy individuals. We then searched for MSC in the blood of ten patients with end-stage renal disease, ten patients with end-stage liver disease, and in eight heart transplant patients with biopsy-proven rejection by culturing of mononuclear cells under MSC-supporting culture conditions. In none of these patient categories, MSC were identified in the blood. MSC were, however, found in the blood of a severe trauma patient with multiple fractures, suggesting that disruption of bone marrow leads to the release of MSC into the blood stream. The conclusion of this study is that MSC are not recruited into the circulation in patients with injured solid organs and during aggressive immune responses after transplantation.


Signs and symptoms associated with primary tooth eruption: a clinical trial of nonpharmacological remedies.
To evaluate disturbances in primary tooth eruption and their management with nonpharmacological remedies. In this nonrandomized clinical trial, 270 children aged between 8 and 36 months were selected and divided into 5 groups with 54 children initially enrolled in each group. The children were seen during an 8-day period during tooth eruption. At each appointment data were recorded from oral examination, tympanic temperature measurement and a questionnaire. The five methods used as remedies to reduce teething symptoms were: 1) cuddle therapy, 2) ice, 3) rubbing the gums, 4) teething rings and 5) food for chewing. Teething symptoms, the type of erupted tooth, symptoms of recovery and the mother's satisfaction with treatment were evaluated. Two hundred and fifty four children (mean age 16 ± 7.2 months) completed the study. The most frequent teething symptoms were drooling (92%), sleep disturbances (82.3%) and irritability (75.6%). These symptoms were more pronounced in low birth weight children (p > 0.05). Canine eruption led to more loss of appetite than incisor (p = 0.033) or molars eruption (p = 0.014). Low grade increases in body temperature were observed only on the day of eruption (36.70 ± 0.39 °C), when body temperature was significantly different compared to the day before and the day after eruption (both p < 0.001). There was no significant correlation between fever as reported by mothers and temperature readings obtained by the investigators. The most favorable results for time to recovery and the mother's satisfaction were seen when teething rings were used, followed by cuddle therapy and rubbing the gums. There was no association between teething and symptoms such as fever or diarrhea. Low birth weight children may have more teething symptoms. Teething rings, cuddle therapy and rubbing the gums were the most effective methods to reduce symptoms. Iranian Registry of Clinical Trials: code IRCT201211127402N3.


Serum Prolidase Enzyme Activity Level: Not a Predictive Biomarker for Epilepsy.
Oxidative stress (OS) and inflammation are considered responsible for the pathogenesis of epilepsy. Prolidase has an extremely important role in proline recycling for collagen synthesis. Higher than normal proline levels have been shown to increase OS. Furthermore, prolidase activity is associated with inflammation during fibrotic process. No study has yet investigated the relationship between epilepsy and prolidase enzyme activity (PEA). In this study, we aimed to contribute to the existing literature by assessing postictal PEA levels, which are correlated with inflammation and OS, to determine whether PEA levels may be used as a biomarker for epilepsy. This study included patients with epilepsy who presented to the emergency department within first 6 h of a seizure. The epileptic group included 27 patients (16 males, 11 females) and the control group included 31 healthy individuals (11 males, 20 females). The mean age of the epilepsy (n=27) and healthy control group (n=31) was 43.1±20.2 and 51.9±21 years, respectively. Serum PEA levels were 1171.90±343.3 in the epileptic group and 1137.1±295.6 in the control group. There were no significant differences between two groups (p>0.05). Our study results suggest that although PEA is an enzyme associated with OS and inflammation, it is still not an ideal biomarker for epileptic patients. This study is important because it investigated PEA in patients with idiopathic epilepsy for the first time.


Dipeptidyl peptidase-4 inhibitor improves neovascularization by increasing circulating endothelial progenitor cells.
Current methods used to treat critical limb ischaemia (CLI) are hampered by a lack of effective strategies, therefore, therapeutic vasculogenesis may open up a new field for the treatment of CLI. In this study we investigated the ability of the DPP-4 inhibitor, sitagliptin, originally used as a hypoglycaemic agent, to induce vasculogenesis in vivo. Sitagliptin were administered daily to C57CL/B6 mice and eGFP transgenic mouse bone marrow-transplanted ICR mice that had undergone hindlimb ischaemic surgery. Laser Doppler imaging and flow cytometry were used to evaluate the degree of neovasculogenesis and circulating levels of endothelial progenitor cells (EPCs) respectively. Cell surface markers of EPCs and endothelial NOS (eNOS) in vessels were studied. Sitagliptin elevated plasma glucagon-like peptide-1 (GLP-1) levels in mice subjected to ischaemia, decreased plasma dipeptidyl peptidase-4 (DPP-4) concentration, and augmented ischaemia-induced increases in stromal cell-derived factor-1 (SDF-1) in a dose-dependent manner. Blood flow in the ischaemic limb was significantly improved in mice treated with sitagliptin. Circulating levels of EPCs were also increased after sitagliptin treatment. Sitagliptin also enhanced the expression of CD 34 and eNOS in ischaemic muscle. In addition, sitagliptin promoted EPC mobilization and homing to ischaemic tissue in eGFP transgenic mouse bone marrow-transplanted ICR mice. Circulating EPC levels and neovasculogenesis were augmented by the DPP-4 inhibitor, sitagliptin and this effect was dependent on an eNOS-related pathway in a mouse model of hindlimb ischaemia. The results indicate that oral administration of sitagliptin has therapeutic potential as an inducer of vasculogenesis.


Interparticle distance as a key factor for controlling the dual-emission properties of carbon dots.
Due to their distinct and useful features, multi-color-emissive carbon dots (CDs) have gained much attention, however, their lack of red emission has so far limited their practical use for various applications, thus understanding of their emission origins is required. Concentration-dependent photoluminescence (PL) is a unique feature of CDs and is regarded as one way to realize tunable PL, providing information on their PL mechanism. In this work, we synthesized concentration-dependent blue/red dual-color-emissive CDs under single excitation at 365 nm. With the increase in concentration, the blue emission band showed a large red-shift from 431 to 500 nm, while the red emission band at around 650 nm showed only a relative intensity change, without a significant spectral shift. We performed spectroscopic analysis by changing the solution environment including the solvent type and pH to investigate the origins of the two emission bands. These results, combined with the zeta potential and fluorescence microscopy results, confirmed that the interparticle distance of CDs is a key factor for their dual-emissive PL behavior. With the advantage of unique PL features, we showed the potential of the CDs for white light-emitting diode (WLED) and ratiometric pH sensing applications.


The use and effectiveness of cadaveric workshops in higher surgical training: a systematic review.
The aim of this systematic review is to describe the use of cadavers in postgraduate surgical training, to determine the effect of cadaveric training sessions on surgical trainees' technical skills performance and to determine how trainees perceive the use of cadaveric workshops as a training tool. An electronic literature search was performed, restricted to the English language, of MEDLINE(®), Embase™, the Cumulative Index to Nursing and Allied Health Literature (CINAHL(®)), Centre for Agricultural Bioscience (CAB) Abstracts, the Educational Resources Information Center (ERIC™) database, the British Education Index, the Australian Education Index, the Cochrane Library and the Best Evidence in Medical Education website. Studies that were eligible for review included primary studies evaluating the use of human cadaveric surgical workshops for surgical skills training in postgraduate surgical trainees and those that included a formal assessment of skills performance or trainee satisfaction after the training session. Eight studies were identified as satisfying the eligibility criteria. One study showed a benefit from cadaveric workshop training with regard to the ability of trainees to perform relatively simple emergency procedures and one showed weak evidence of a benefit in performing more complex surgical procedures. Three studies showed that trainees valued the experience of cadaveric training. Evidence for the effectiveness of cadaveric workshops in surgical training is currently limited. In particular, there is little research into how these workshops improve the performance of surgical trainees during subsequent live surgery. However, both trainees and assessors hold them in high regard and feel they help to improve operative skills. Further research into the role of cadaveric workshops is required.


Genetic and physical mapping of a type 1 diabetes susceptibility gene (IDDM12) to a 100-kb phagemid artificial chromosome clone containing D2S72-CTLA4-D2S105 on chromosome 2q33.
Polymorphic markers within the CTLA4 gene on chromosome 2q33 have been shown to be associated with type 1 diabetes. Therefore, a gene responsible for the disease (IDDM12) most likely lies within a region of <1-2 cM of CTLA4. To define more precisely the IDDM12 interval, we genotyped a multiethnic (U.S. Caucasian, Mexican-American, French, Spanish, Korean, and Chinese) collection of 178 simplex and 350 multiplex families for 10 polymorphic markers within a genomic interval of approximately 300 kb, which contains the candidate genes CTLA4 and CD28. The order of these markers (D2S346, CD28, GGAA19E07, D2S307, D2S72, CTLA4, D2S105, and GATA52A04) was determined by sequence tagged site content mapping of bacterial artificial chromosome (BAC) and yeast artificial chromosome (YAC) clones. The transmission disequilibrium test (TDT) analyses of our data revealed significant association/linkage with three markers within CTLA4 and two immediate flanking markers (D2S72 and D2S105) on each side of CTLA4 but not with more distant markers including the candidate gene CD28. Tsp analyses revealed significant association only with the three polymorphic markers within the CTLA4 gene. The markers linked and associated with type 1 diabetes are contained within a phagemid artificial chromosome clone of 100 kb, suggesting that the IDDM12 locus is either CTLA4 or an unknown gene in very close proximity.


A search for quantitative trait loci for ovulation rate in cattle.
Seventy-seven polymorphic microsatellites were analysed in offspring of three elite sires that were part of the foundation of an experimental population selected for twinning rate at the US Meat Animal Research Center, Clay Center, Nebraska. All females were assessed for ovulation rate by rectal palpation of corpora lutea over 8-10 consecutive oestrous cycles from approximately 12 to 18 months of age, and associations between ovulation rate and sire allele were examined in each of the three sire groups. A preliminary analysis was performed using selectively genotyped daughters of each sire. Markers found significant or approaching significance were also genotyped in all daughters, sons and granddaughters of these sires. A test of marker associations limited to the granddaughter data provided an independent confirmation of marker effect and significance relative to the initial test with daughter data. Putative ovulation rate quantitative trait loci were detected on chromosomes 7 and 23. Marker UWCA20 on chromosome 7 was associated with an effect in excess of one phenotypic standard deviation and accounted for approximately 10% of phenotypic variation ovulation rate. Marker CYP21 (steroid 21-hydroxylase) on chromosome 23 was associated with an effect of slightly less than half a phenotypic standard deviation and accounted for approximately 4% of phenotypic variation.


Association of CaV1.3 L-type calcium channels with Shank.
Neurons express multiple types of voltage-gated calcium (Ca2+) channels. Two subtypes of neuronal L-type Ca2+ channels are encoded by CaV1.2 and CaV1.3 pore-forming subunits. Both CaV1.2 and CaV1.3 subunits contain class I PDZ (postsynaptic density-95/Discs large/zona occludens-1) domain-binding consensus at their C termini. In yeast two-hybrid screen of rat brain cDNA library with the C-terminal bait of CaV1.3a (long C-terminal splice variant) L-type Ca2+ channel subunit, we isolated multiple clones of postsynaptic adaptor protein Shank. We demonstrated a specific association of CaV1.3a C termini, but not of CaV1.2 C termini, with Shank PDZ domain in vitro. We further demonstrated that the proline-rich region present in C termini of CaV1.3a subunit binds to Shank Src homology 3 domain. We established that CaV1.3a and Shank localized to postsynaptic locations in cultured rat hippocampal neurons. By expressing epitope-tagged recombinant CaV1.3 subunits in rat hippocampal neuronal cultures, we demonstrated that the presence of Shank-binding motifs in CaV1.3a sequence is both necessary and sufficient for synaptic clustering of CaV1.3 L-type Ca2+ channels. In experiments with dominant-negative peptides and dihydropyridine-resistant CaV1.3a mutants, we demonstrated an importance of Shank-binding motif in CaV1.3a sequence for phosphorylated cAMP response element-binding protein (pCREB) signaling in cultured hippocampal neurons. Our results directly link CaV1.3 neuronal L-type Ca2+ channels to macromolecular signaling complex formed by Shank and other modular adaptor proteins at postsynaptic density and provide novel information about the role played by CaV1.3 L-type Ca2+ channels in pCREB signaling.


Navigating Barriers to Patient Access and Reimbursement in Mohs Micrographic Surgery.
Insurance companies have implemented new policies including excessive prior authorization (PA) requirements, high-deductible plans, and complicated billing structures in an effort to curb rising health care costs. Studies investigating the real-time impact on providers and patients are emerging, but few within the field of dermatology have been published. To assess the impact of cost-cutting policies on patients and physicians. A survey was electronically distributed to members of the American College of Mohs Surgery (ACMS). The majority of respondents (78.2%) practiced in a private setting, with no other demographic differences. The majority of respondents (70%) dedicated 1 to 2 employees to obtaining PAs. Fifty percent reported an average time of 30 minutes spent per PA. Fifty-six percent of respondents obtained PA from private insurance before Mohs surgery, whereas only 24.5% obtained PA from Medicare. Forty-nine percent of practitioners provided patients with a financial disclosure prior to Mohs surgery. Moreover, many practitioners reported screening patients for high-deductible policies and request an advanced deposit against the deductible. Sixty percent reported difficulty obtaining payment for service in the absence of an advanced deposit. The burden of restrictive health care policies will have long-term consequences for the patient-provider interaction and patient outcomes.


Cellular and molecular mechanisms underlying NOD2 risk-associated polymorphisms in Crohn's disease.
The discovery that polymorphisms in the NOD2 (nucleotide-binding oligomerization domain containing 2) gene are associated with a greatly increased risk for the development of Crohn's disease has provided a means to achieve a deeper understanding of the dysregulation of mucosal immune responses to the commensal intestinal organisms that is thought to underlie this disease. NOD2 is a NOD-like receptor (NLR) family member that senses and responds to bacterial wall peptides; thus, the most widely held view of the relation of the NOD2 polymorphisms with Crohn's disease is that these polymorphisms lead to deficient immune responses to gut bacteria, and these, in turn, lead to quantitative or qualitative changes in the bacterial population in the gut lumen or lamina propria that cause inflammation at this site. Initially, this view was based mainly on the observation that defective NOD2 function can result in reduced α-defensin production by intestinal Paneth cells and that such impairment leads to loss of host defense against gut bacteria. In this review, we reconsider this possibility and marshal evidence that it is not in fact likely to be a prime element of Crohn's disease causation. More recently, evidence has been accumulating that the NOD2 dysfunction leads to Crohn's inflammation by inducing changes in the gut microbiome that influence immune effector or regulatory function. We review the strengths and weaknesses of this emerging hypothesis. Finally, we consider the possibility that NOD2 dysfunction can lead to inflammation because of a second and somewhat overlooked aspect of its function, that as an immunoregulator of innate immune responses. In particular, we review the body of evidence that NOD2 stimulation activates a cross-tolerance response that downregulates and thus prevents excessive TLR responses that cause Crohn's inflammation.