From Drug Screening to Target Deconvolution: a Target-Based Drug Discovery Pipeline Using Leishmania Casein Kinase 1 Isoform 2 To Identify Compounds with Antileishmanial Activity.
Existing therapies for leishmaniases present significant limitations, such as toxic side effects, and are rendered inefficient by parasite resistance. It is of utmost importance to develop novel drugs targeting Leishmania that take these two limitations into consideration. We thus chose a target-based approach using an exoprotein kinase, Leishmania casein kinase 1.2 (LmCK1.2) that was recently shown to be essential for intracellular parasite survival and infectivity. We developed a four-step pipeline to identify novel selective antileishmanial compounds. In step 1, we screened 5,018 compounds from kinase-biased libraries with Leishmania and mammalian CK1 in order to identify hit compounds and assess their specificity. For step 2, we selected 88 compounds among those with the lowest 50% inhibitory concentration to test their biological activity on host-free parasites using a resazurin reduction assay and on intramacrophagic amastigotes using a high content phenotypic assay. Only 75 compounds showed antileishmanial activity and were retained for step 3 to evaluate their toxicity against mouse macrophages and human cell lines. The four compounds that displayed a selectivity index above 10 were then assessed for their affinity to LmCK1.2 using a target deconvolution strategy in step 4. Finally, we retained two compounds, PP2 and compound 42, for which LmCK1.2 seems to be the primary target. Using this four-step pipeline, we identify from several thousand molecules, two lead compounds with a selective antileishmanial activity.


Generalized quantum cumulant dynamics.
A means of unifying some semiclassical models of computational chemistry is presented; these include quantized Hamiltonian dynamics, quantal cumulant dynamics, and semiclassical Moyal dynamics (SMD). A general method for creating the infinite hierarchy of operator dynamics in the Heisenberg picture is derived together with a general method for truncation (or closure) of that series, and in addition, we provide a simple link to the phase space methods of SMD. Operator equations of arbitrary order may be created readily, avoiding the tedious algebra identified previously. Truncation is based on a simple recurrence formula which is related to, but avoids the more complex contractions of, Wick's theorem. This generalized method is validated against a number of trial problems considered using the previous methods. We also touch on some of the limitations involved using such methods, noting, in particular, that any truncation will lead to a state which is in some sense unphysical. Finally, we briefly introduce our quantum algebra package QuantAL which provides an automated method for the generation of the required equation set, the initial conditions for all variables from any start, and all the higher order approximations necessary for truncation of the series, at essentially arbitrary order.


Cold Atmospheric Plasma Promotes Killing of Staphylococcus aureus by Macrophages.
Macrophages are important immune cells that are involved in the elimination of microbial pathogens. Following host invasion, macrophages are recruited to the site of infection, where they launch antimicrobial defense mechanisms. Effective microbial clearance by macrophages depends on phagocytosis and phagolysosomal killing mediated by oxidative burst, acidification, and degradative enzymes. However, some pathogenic microorganisms, including some drug-resistant bacteria, have evolved sophisticated mechanisms to prevent phagocytosis or escape intracellular degradation. Cold atmospheric plasma (CAP) is an emerging technology with promising bactericidal effects. Here, we investigated the effect of CAP on Staphylococcus aureus phagocytosis by RAW 264.7 macrophage-like cells. We demonstrate that CAP treatment increases intracellular concentrations of reactive oxygen species (ROS) and nitric oxide and promotes the elimination of both antibiotic-sensitive and antibiotic-resistant S. aureus by RAW 264.7 cells. This effect was inhibited by antioxidants indicating that the bactericidal effect of CAP was mediated by oxidative killing of intracellular bacteria. Furthermore, we show that CAP promotes the association of S. aureus to lysosomal-associated membrane protein 1 (LAMP-1)-positive phagosomes, in which bacteria are exposed to low pH and cathepsin D hydrolase. Taken together, our results provide the first evidence that CAP activates defense mechanisms of macrophages, ultimately leading to bacterial elimination. IMPORTANCE Staphylococcus aureus is the most frequent cause of skin and soft tissue infections. Treatment failures are increasingly common due to antibiotic resistance and the emergence of resistant strains. Macrophages participate in the first line of immune defense and are critical for coordinated defense against pathogenic bacteria. However, S. aureus has evolved sophisticated mechanisms to escape macrophage killing. In the quest to identify novel antimicrobial therapeutic approaches, we investigated the activity of cold atmospheric plasma (CAP) on macrophages infected with S. aureus. Here, we show that CAP treatment promotes macrophage ability to eliminate internalized bacteria. Importantly, CAP could trigger killing of both antibiotic-sensitive and antibiotic-resistant strains of S. aureus. While CAP did not affect the internalization capacity of macrophages, it increased oxidative-dependent bactericidal activity and promoted the formation of degradative phagosomes. Our study shows that CAP has beneficial effects on macrophage defense mechanisms and may potentially be useful in adjuvant antimicrobial therapies.


DeepPVP: phenotype-based prioritization of causative variants using deep learning.
Prioritization of variants in personal genomic data is a major challenge. Recently, computational methods that rely on comparing phenotype similarity have shown to be useful to identify causative variants. In these methods, pathogenicity prediction is combined with a semantic similarity measure to prioritize not only variants that are likely to be dysfunctional but those that are likely involved in the pathogenesis of a patient's phenotype. We have developed DeepPVP, a variant prioritization method that combined automated inference with deep neural networks to identify the likely causative variants in whole exome or whole genome sequence data. We demonstrate that DeepPVP performs significantly better than existing methods, including phenotype-based methods that use similar features. DeepPVP is freely available at https://github.com/bio-ontology-research-group/phenomenet-vp . DeepPVP further improves on existing variant prioritization methods both in terms of speed as well as accuracy.


Intracellular tortuosity underlies slow cAMP diffusion in adult ventricular myocytes.
3',5'-Cyclic adenosine monophosphate (cAMP) signals in the heart are often confined to concentration microdomains shaped by cAMP diffusion and enzymatic degradation. While the importance of phosphodiesterases (degradative enzymes) in sculpting cAMP microdomains is well established in cardiomyocytes, less is known about cAMP diffusivity (DcAMP) and factors affecting it. Many earlier studies have reported fast diffusivity, which argues against sharply defined microdomains. [cAMP] dynamics in the cytoplasm of adult rat ventricular myocytes were imaged using a fourth generation genetically encoded FRET-based sensor. The [cAMP]-response to the addition and removal of isoproterenol (β-adrenoceptor agonist) quantified the rates of cAMP synthesis and degradation. To obtain a read out of DcAMP, a stable [cAMP] gradient was generated using a microfluidic device which delivered agonist to one half of the myocyte only. After accounting for phosphodiesterase activity, DcAMP was calculated to be 32 µm(2)/s; an order of magnitude lower than in water. Diffusivity was independent of the amount of cAMP produced. Saturating cAMP-binding sites with the analogue 6-Bnz-cAMP did not accelerate DcAMP, arguing against a role of buffering in restricting cAMP mobility. cAMP diffused at a comparable rate to chemically unrelated but similar sized molecules, arguing for a common physical cause of restricted diffusivity. Lower mitochondrial density and order in neonatal cardiac myocytes allowed for faster diffusion, demonstrating the importance of mitochondria as physical barriers to cAMP mobility. In adult cardiac myocytes, tortuosity due to physical barriers, notably mitochondria, restricts cAMP diffusion to levels that are more compatible with microdomain signalling.


Research Resource: The Dexamethasone Transcriptome in Hypothalamic Embryonic Neural Stem Cells.
Exposure to excess glucocorticoids during fetal development has long-lasting physiological and behavioral consequences, although the mechanisms are poorly understood. The impact of prenatal glucocorticoids exposure on stress responses in juvenile and adult offspring implicates the developing hypothalamus as a target of adverse prenatal glucocorticoid action. Therefore, primary cultures of hypothalamic neural-progenitor/stem cells (NPSCs) derived from mouse embryos (embryonic day 14.5) were used to identify the glucocorticoid transcriptome in both males and females. NPSCs were treated with vehicle or the synthetic glucocorticoid dexamethasone (dex; 100nM) for 4 hours and total RNA analyzed using RNA-Sequencing. Bioinformatic analysis demonstrated that primary hypothalamic NPSC cultures expressed relatively high levels of a number of genes regulating stem cell proliferation and hypothalamic progenitor function. Interesting, although these cells express glucocorticoid receptors (GRs), only low levels of sex-steroid receptors are expressed, which suggested that sex-specific differentially regulated genes identified are mediated by genetic and not hormonal influences. We also identified known or novel GR-target coding and noncoding genes that are either regulated equivalently in male and female NPSCs or differential responsiveness in one sex. Using gene ontology analysis, the top functional network identified was cell proliferation and using bromodeoxyuridine (BrdU) incorporation observed a reduction in proliferation of hypothalamic NPSCs after dexamethasone treatment. Our studies provide the first characterization and description of glucocorticoid-regulated pathways in male and female embryonically derived hypothalamic NPSCs and identified GR-target genes during hypothalamic development. These findings may provide insight into potential mechanisms responsible for the long-term consequences of fetal glucocorticoid exposure in adulthood.


Arginine vasopressin attenuates phenylephrine-induced forearm vasoconstriction in men.
1. The aim of this study was to examine whether arginine vasopressin modulates the vasoconstricting action of phenylephrine in human forearms. 2. In seven healthy subjects, we determined the percentage increases in forearm vascular resistance evoked by intra-arterial infusion of phenylephrine at graded doses during simultaneous intra-arterial infusion of saline or arginine vasopressin at two doses. Similarly, in another seven subjects, we examined the effects of intra-arterial infusions of saline or angiotensin II on the vasoconstricting action of intra-arterial phenylephrine. 3. Arginine vasopressin and angiotensin II caused small, but insignificant, increases in baseline forearm vascular resistance. Arginine vasopressin at the two doses significantly attenuated the percentage increases in forearm vascular resistance evoked with phenylephrine at graded doses. Angiotensin II did not alter the forearm vascular responses to phenylephrine. 4. Intra-arterial infusion of arginine vasopressin at doses of 0.6 and 1.8 ng/min raised the plasma arginine vasopressin concentration in the venous effluents from 1.6 +/- 0.3 (control) to 4.0 +/- 0.9 and to 16.4 +/- 6.9 pg/ml, respectively. 5. These results suggest that arginine vasopressin at physiological concentrations with a minimal direct effect on resistance arteries attenuates the vasoconstricting action of phenylephrine in human forearms.


Comparative trypanotolerance of the small east African breed of goats from different localities to Trypanosoma congolense infection.
Differences in susceptibility of the Small East African breed of goats to an experimental Trypanosoma congolense infection were investigated. The goats were obtained from different areas of East Africa, Morogoro and Arusha (Tanzania), Imbo and Lambwe Valley (Kenya). Morogoro goats were found to be more tolerant, followed by Arusha, Lambwe Valley and Imbo goats, in that order. The Imbo goats had highest parasitaemia, more severe anaemia, marked weight losses and highest mortality rate. The Morogoro goats showed a milder infection with lower parasitaemia, less severe anaemia, weight losses and lowest mortality rate. The Lambwe Valley and Arusha goats were intermediate to the Morogoro (resistant) and Imbo (susceptible) goats. The significance of these findings in relation to existence of heterogeneity in the Small East African goats is discussed.


Moraxella catarrhalis uses a twin-arginine translocation system to secrete the β-lactamase BRO-2.
Moraxella catarrhalis is a human-specific gram-negative bacterium readily isolated from the respiratory tract of healthy individuals. The organism also causes significant health problems, including 15-20% of otitis media cases in children and ~10% of respiratory infections in adults with chronic obstructive pulmonary disease. The lack of an efficacious vaccine, the rapid emergence of antibiotic resistance in clinical isolates, and high carriage rates reported in children are cause for concern. Virtually all Moraxella catarrhalis isolates are resistant to β-lactam antibiotics, which are generally the first antibiotics prescribed to treat otitis media in children. The enzymes responsible for this resistance, BRO-1 and BRO-2, are lipoproteins and the mechanism by which they are secreted to the periplasm of M. catarrhalis cells has not been described. Comparative genomic analyses identified M. catarrhalis gene products resembling the TatA, TatB, and TatC proteins of the well-characterized Twin Arginine Translocation (TAT) secretory apparatus. Mutations in the M. catarrhalis tatA, tatB and tatC genes revealed that the proteins are necessary for optimal growth and resistance to β-lactams. Site-directed mutagenesis was used to replace highly-conserved twin arginine residues in the predicted signal sequence of M. catarrhalis strain O35E BRO-2, which abolished resistance to the β-lactam antibiotic carbanecillin. Moraxella catarrhalis possesses a TAT secretory apparatus, which plays a key role in growth of the organism and is necessary for secretion of BRO-2 into the periplasm where the enzyme can protect the peptidoglycan cell wall from the antimicrobial activity of β-lactam antibiotics.


Illness, medical expenditure and household consumption: observations from Taiwan.
Illness conditions lead to medical expenditure. Even with various types of medical insurance, there can still be considerable out-of-pocket costs. Medical expenditure can affect other categories of household consumptions. The goal of this study is to provide an updated empirical description of the distributions of illness conditions and medical expenditure and their associations with other categories of household consumptions. A phone-call survey was conducted in June and July of 2012. The study was approved by ethics review committees at Xiamen University and FuJen Catholic University. Data was collected using a Computer-Assisted Telephone Survey System (CATSS). "Household" was the unit for data collection and analysis. Univariate and multivariate analyses were conducted, examining the distributions of illness conditions and the associations of illness and medical expenditure with other household consumptions. The presence of chronic disease and inpatient treatment was not significantly associated with household characteristics. The level of per capita medical expenditure was significantly associated with household size, income, and household head occupation. The presence of chronic disease was significantly associated with levels of education, insurance and durable goods consumption. After adjusting for confounders, the associations with education and durable goods consumption remained significant. The presence of inpatient treatment was not associated with consumption levels. In the univariate analysis, medical expenditure was significantly associated with all other consumption categories. After adjusting for confounding effects, the associations between medical expenditure and the actual amount of entertainment expenses and percentages of basic consumption, savings, and insurance (as of total consumption) remained significant. This study provided an updated description of the distributions of illness conditions and medical expenditure in Taiwan. The findings were mostly positive in that illness and medical expenditure were not observed to be significantly associated with other consumption categories. This observation differed from those made in some other Asian countries and could be explained by the higher economic status and universal basic health insurance coverage of Taiwan.


Utilization of computers in clinical practice--role of physician networks: preliminary communication.
This study examines the influence of physician networks on the utilization of computers in clinical practice. Data on patient referrals, consultations, professional discussions, and on-call coverage were collected from 24 physicians who comprise a private group practice. Their utilization of a computerized hospital medical information system (HIS) in caring for patients admitted to a 1160-bed private, university affiliated, teaching hospital was determined. A matrix representing the professional relations among these physicians was subjected to smallest space analysis, a form of multidimensional scaling. Also, a number of indices that describe structural and interactional properties of the network and individual physicians were computed. The three-dimensional representation of the network that resulted from the analysis suggests a two-step process of adoption and utilization of medical technology. Physicians who were engaged in outside professional activities and in the training of medical students and house staff were more likely to utilize the system in caring for patients. These physicians, who were more centrally located and dominant in the referral and consultation process, were more likely to influence the practice patterns of their colleagues. The use of network analysis to identify influential physicians whose involvement is essential to the success of efforts aimed at introducing computers into clinical practice is discussed.


Effects of viscosity and fermentability of dietary fibre on nutrient digestibility and digesta characteristics in ileal-cannulated grower pigs.
Relative contributions of two functional properties, viscosity and fermentability of dietary fibre, on apparent ileal digestibility (AID), apparent total tract digestibility (ATTD), digesta passage rate, N retention and SCFA concentration have not been established. Thus, eight ileal-cannulated pigs randomised in a double 4 × 4 Latin square were fed four diets based on maize starch and casein supplemented with 5 % of actual fibre in a 2 × 2 factorial arrangement: low-fermentable, low-viscous cellulose (CEL); low-fermentable, high-viscous carboxymethylcellulose (CMC); high-fermentable, low-viscous oat β-glucan (LBG); high-fermentable, high-viscous oat β-glucan (HBG). Viscosity and fermentability interacted to affect (P < 0·001) digesta viscosity and AID and ATTD of nutrients. These properties tended to interact to affect (P < 0·10) digesta passage rate and butyrate. Pigs fed the CMC diet had the lowest (P < 0·05) digesta passage rate and the highest (P < 0·001) AID of energy, crude protein and DM, and ATTD of energy and DM. Post-ileal DM digestibility was highest (P < 0·001) for pigs fed the CEL and HBG diets. Post-ileal DM digestibility had a negative, curvilinear relationship with the AID of energy and crude protein (R2 0·85 and 0·72, respectively; P < 0·001). Digesta viscosity had a less strong relationship with the AID of energy and crude protein (R2 0·45 and 0·36, respectively; P < 0·001). In conclusion, high-viscous, low-fermentable dietary fibre increases the proportion of a diet that is digested in the small intestine by reducing digesta passage rate.


HIV and identity: the experience of AIDS support group members who unexpectedly tested HIV negative in Uganda.
Living with HIV, for many of those infected, has meant adjusting to life with a stigmatised condition and, until recently, the threat of looming death. We explore the adjustment of a group of long-term former clients of The AIDS Support Organisation (TASO) in Uganda who, when tested for HIV during the rollout of antiretroviral therapy in 2004, were found to be HIV negative. In-depth semi-structured interviews with 34 former TASO clients were conducted between 2005 and 2007. Their narratives reveal a great deal about the biographical disruption they have faced, and the biographical work that they have undertaken in both the personal and the social dimensions of their lives in order to manage their new-found HIV-uninfected status. After the negative test result, as they were no longer HIV-infected, they had to leave TASO and that support was sorely missed, as was the friendship of TASO members to whom they often felt reluctant to disclose their new status. The identity 'reversal' or change was often handled privately. Compared with their transition to an HIV-positive identity, they now lacked a social dimension to their identity transformation as they managed their new identity in the face of self- and public doubt.


A geometrical approach to control and controllability of nonlinear dynamical networks.
In spite of the recent interest and advances in linear controllability of complex networks, controlling nonlinear network dynamics remains an outstanding problem. Here we develop an experimentally feasible control framework for nonlinear dynamical networks that exhibit multistability. The control objective is to apply parameter perturbation to drive the system from one attractor to another, assuming that the former is undesired and the latter is desired. To make our framework practically meaningful, we consider restricted parameter perturbation by imposing two constraints: it must be experimentally realizable and applied only temporarily. We introduce the concept of attractor network, which allows us to formulate a quantifiable controllability framework for nonlinear dynamical networks: a network is more controllable if the attractor network is more strongly connected. We test our control framework using examples from various models of experimental gene regulatory networks and demonstrate the beneficial role of noise in facilitating control.


Seroreversion of positive anti-hepatitis C virus antibodies in left ventricular assist device recipients: Now you see them, now you don't.
The clinical significance of positive anti-hepatitis C virus (anti-HCV) antibody tests in recipients of left ventricular assist devices remains unclear. In light of emerging evidence suggesting the possibility of persistent low-level HCV infection in patients with positive anti-HCV antibody test but negative HCV ribonucleic acid, it is very important to distinguish the truly false positive HCV antibodies, in recipients of continuous flow left ventricular assist devices, from those suggestive of a prior clinically resolved infection or one where a low-level viremia may have persisted. We conducted a retrospective analysis of left ventricular assist device recipients at our institution. While the total incidence of positive HCV antibody with concomitantly negative HCV ribonucleic acid test (19.2%) was in keeping with the incidences reported in prior cross-sectional studies, we longitudinally followed our patients and observed a 100% seroreversion. Seroreversion, which has not been reported in other studies, occurred either during continued left ventricular assist device support (10 out of 26) or after heart transplant (7 out of 26). Hundred percent seroreversion strongly suggested that the anti-HCV antibodies were truly false positive.


Exposure to an environmental estrogen breaks down sexual isolation between native and invasive species.
Environmental change can increase the likelihood of interspecific hybridization by altering properties of mate recognition and discrimination between sympatric congeners. We examined how exposure to an environmentally widespread endocrine-disrupting chemical (EDC), bisphenol A (BPA), affected visual communication signals and behavioral isolation between an introduced freshwater fish and a native congener (genus: Cyprinella). Exposure to BPA induced changes in the expression of male secondary traits as well as male and female mate choice, leading to an overall reduction in prezygotic isolation between congeners. Changes in female mate discrimination were not tightly linked to changes in male phenotypic traits, suggesting that EDC exposure may alter female choice thresholds independently of the effects of exposure on males. These findings indicate that environmental exposure to EDCs can lead to population declines via the erosion of species boundaries and by promoting the establishment and spread of non-native species via hybridization.


"MRI Stealth" robot for prostate interventions.
The paper reports an important achievement in MRI instrumentation, a pneumatic, fully actuated robot located within the scanner alongside the patient and operating under remote control based on the images. Previous MRI robots commonly used piezoelectric actuation limiting their compatibility. Pneumatics is an ideal choice for MRI compatibility because it is decoupled from electromagnetism, but pneumatic actuators were hardly controllable. This achievement was possible due to a recent technology breakthrough, the invention of a new type of pneumatic motor, PneuStep 1, designed for the robot reported here with uncompromised MRI compatibility, high-precision, and medical safety. MrBot is one of the "MRI stealth" robots today (the second is described in this issue by Zangos et al.). Both of these systems are also multi-imager compatible, being able to operate with the imager of choice or cross-imaging modalities. For MRI compatibility the robot is exclusively constructed of nonmagnetic and dielectric materials such as plastics, ceramics, crystals, rubbers and is electricity free. Light-based encoding is used for feedback, so that all electric components are distally located outside the imager's room. MRI robots are modern, digital medical instruments in line with advanced imaging equipment and methods. These allow for accessing patients within closed bore scanners and performing interventions under direct (in scanner) imaging feedback. MRI robots could allow e.g. to biopsy small lesions imaged with cutting edge cancer imaging methods, or precisely deploy localized therapy at cancer foci. Our robot is the first to show the feasibility of fully automated in-scanner interventions. It is customized for the prostate and operates transperineally for needle interventions. It can accommodate various needle drivers for different percutaneous procedures such as biopsy, thermal ablations, or brachytherapy. The first needle driver is customized for fully automated low-dose radiation seed brachytherapy. This paper gives an introduction to the challenges of MRI robot compatibility and presents the solutions adopted in making the MrBot. Its multi-imager compatibility and other preclinical tests are included. The robot shows the technical feasibility of MRI-guided prostate interventions, yet its clinical utility is still to be determined.


Mercury vapor and female reproductive toxicity.
Epidemiological studies finding menstrual cycle abnormalities among women occupationally exposed to Hg degrees prompted us to investigate the mechanisms of reproductive toxicity of Hg degrees in the female rat. Nose-only Hg degrees vapor inhalation exposures were conducted on regularly cycling rats 80-90 days of age in dose-response and acute time-course studies, which have previously proven useful as a model to identify ovarian toxicants. Vaginal smears were evaluated daily and serum hormone levels were correlated with cycle and with ovarian morphology at necropsy. Exposure concentration-related effects of Hg degrees were evaluated by exposing rats to 0, 1, 2, or 4 mg/m3 Hg degrees vapor 2 h/day for 11 consecutive days. Tissue Hg levels correlated with exposure concentration and duration. Exposure of rats to 4 mg/m3 (but not 1 or 2 mg/m3) Hg vapor for 11 days resulted in significant decreases in body weights relative to controls. Estrous cycles were slightly prolonged in the 2 and 4 mg/m3 dose groups, and serum estradiol and progesterone levels were significantly different in the 4 mg/m3 group compared to controls. The alterations in cycle and hormones at the 4 mg/m3 exposure concentration were attributed to body weight loss and generalized toxicity. In the time-course study, rats were exposed to 2 mg/m3 Hg degrees or air beginning in metestrus and evaluated daily for 8 days. A lengthening of the cycle was detected and morphological changes were observed in the corpora lutea (CL) after exposure for 6 days. To determine if changes in the CL and cyclicity correlated with a functional defect, rats were exposed to Hg degrees vapor and evaluated for pregnancy outcome. There were no significant effects on pregnancy rate or numbers of implantation sites when rats were exposed to 1 or 2 mg/m3 Hg degrees for 8 days prior to breeding, or when exposed for 8 days after breeding. These studies indicate that exposure to Hg degrees vapor altered estrous cyclicity, but had no significant effect on ovulation, implantation, or maintenance of first pregnancy during exposure of short duration in female rats.


Perturbation of cytochrome c maturation reveals adaptability of the respiratory chain in Mycobacterium tuberculosis.
Mycobacterium tuberculosis depends on aerobic respiration for growth and utilizes an aa3-type cytochrome c oxidase for terminal electron transfer. Cytochrome c maturation in bacteria requires covalent attachment of heme to apocytochrome c, which occurs outside the cytoplasmic membrane. We demonstrate that in M. tuberculosis the thioredoxin-like protein Rv3673c, which we named CcsX, is required for heme insertion in cytochrome c. Inactivation of CcsX resulted in loss of c-type heme absorbance, impaired growth and virulence of M. tuberculosis, and induced cytochrome bd oxidase. This suggests that the bioenergetically less efficient bd oxidase can compensate for deficient cytochrome c oxidase activity, highlighting the flexibility of the M. tuberculosis respiratory chain. A spontaneous mutation in the active site of vitamin K epoxide reductase (VKOR) suppressed phenotypes of the CcsX mutant and abrogated the activity of the disulfide bond-dependent alkaline phosphatase, which shows that VKOR is the major disulfide bond catalyzing protein in the periplasm of M. tuberculosis. Mycobacterium tuberculosis requires oxygen for growth; however, the biogenesis of respiratory chain components in mycobacteria has not been explored. Here, we identified a periplasmic thioredoxin, CcsX, necessary for heme insertion into cytochrome c. We investigated the consequences of disrupting cytochrome c maturation (CCM) for growth and survival of M. tuberculosis in vitro and for its pathogenesis. Appearance of a second-site suppressor mutation in the periplasmic disulfide bond catalyzing protein VKOR indicates the strong selective pressure for a functional cytochrome c oxidase. The observation that M. tuberculosis is able to partially compensate for defective CCM by upregulation of the cytochrome bd oxidase exposes a functional role of this alternative terminal oxidase under normal aerobic conditions and during pathogenesis. This suggests that targeting both oxidases simultaneously might be required to effectively disrupt respiration in M. tuberculosis.


Orthobunyavirus spike architecture and recognition by neutralizing antibodies.
Orthobunyaviruses (OBVs) form a distinct genus of arthropod-borne bunyaviruses that can cause severe disease upon zoonotic transmission to humans. Antigenic drift or genome segment re-assortment have in the past resulted in new pathogenic OBVs, making them potential candidates for causing emerging zoonoses in the future. Low-resolution electron cryo-tomography studies have shown that OBV particles feature prominent trimeric spikes, but their molecular organization remained unknown. Here we report X-ray crystallography studies of four different OBVs showing that the spikes are formed by an N-terminal extension of the fusion glycoprotein Gc. Using Schmallenberg virus, a recently emerged OBV, we also show that the projecting spike is the major target of the neutralizing antibody response, and provide X-ray structures in complex with two protecting antibodies. We further show that immunization of mice with the spike domains elicits virtually sterilizing immunity, providing fundamental knowledge essential in the preparation for potential newly emerging OBV zoonoses.


Improving the sensing performance of double gold gratings by oblique incident light.
Here we demonstrate a simple method to improve the plasmonic sensing performance of gold gratings. The gratings consist of periodic polymer gratings covered with a gold layer, created by nanoimprint lithography and metal deposition. We investigated the effect of gold thickness and the incident angles on the plasmonic sensing performance. With the optimized gold layer, the full-width at half maximum of this grating was reduced by 60% by using the oblique incident light instead of the normal incident light. A maximum value of the figure of merit at oblique incidence is 12, which is double the one at normal incidence.


Characterization of hTERT-immortalized osteoblast cell lines generated from wild-type and connexin43-null mouse calvaria.
The gap junction protein connexin43 (Cx43) has been proposed to play key roles in bone differentiation and mineralization, but underlying cellular mechanisms are not totally understood. To further explore roles of Cx43 in these processes, we immortalized calvarial osteoblasts from wild-type and Cx43-null mice using human telomerase reverse transcriptase (hTERT). Osteoblastic (MOB) cell lines were generated from three individual wild-type and three individual Cx43-null mouse calvaria. Average population doubling times of the cell lines were higher than of the primary osteoblasts but did not greatly differ with regard to genotype. Modest to high level of Cx45 expression was detected in MOBs of both genotypes. Most of the cell lines expressed osteoblastic markers [Type I collagen, osteopontin, osteocalcin, parathyroid hormone/parathyroid hormone-related peptide receptor (PTH/PTHrP), periostin (OSF-2), osterix (Osx), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP)], and mineralization was comparable to that of primary osteoblasts. Two MOB cell lines from each genotype with most robust maintenance of osteoblast lineage markers were analyzed in greater detail, revealing that the Cx43-null cell lines showed a significant delay in early differentiation (up to 9 days in culture). Matrix mineralization was markedly delayed in one of the Cx43-null lines and slightly delayed in the other. These findings comparing new and very stable wild-type and Cx43-null osteoblastic cell lines define a role for Cx43 in early differentiation and mineralization stages of osteoblasts and further support the concept that Cx43 plays important role in the cellular processes associated with skeleton function.


A field-based approach to support improved diabetes care in rural states.
Diabetes care is a challenge in rural areas where primary care practices are faced with limited resources, few clinical information systems, and relative isolation from education programs and diabetes centers with multispecialty teams. This report describes an effective field-based approach to support improved care for patients with diabetes in primary care practices in rural states. A collaborative effort between diabetes prevention and control programs in Montana and Wyoming and the University of North Dakota was established to provide support to rural primary care practices for improvement in diabetes care. Field teams from each state diabetes program approached primary care practices. After assessment and orientation of office staff, a computer-based registry was established in each practice. Baseline data were collected in 1997 in Montana and in 1998 in Wyoming; follow-up occurred on July 31, 2004. Health department staff provided ongoing technical support for implementing and evaluating quality-improvement interventions. Forty primary care practices, providing care to more than 7000 patients with diabetes, participated in this quality-improvement effort at follow-up. Of the 37 primary care practices participating in the quality-improvement program for 6 or more months at follow-up, there were significant improvements in Montana in rates of hemoglobin A1c testing, blood glucose control, low-density lipoprotein cholesterol testing, foot and dilated retinal examinations, and pneumococcal vaccinations, and there were significant improvements in pneumococcal vaccinations in Wyoming. A field-based approach in which individual practices maintain and use their own registries for both clinical care and quality improvement with ongoing support is a sustainable and an effective strategy for improving diabetes care for rural populations.


[Promoter methylation of DAPK1, RAR-β and MGMT in exfoliated cervical cytology and its clinical application].
To assess the correlation of promoter methylation of DAPK1, RAR-β and MGMT with cervical lesions from cytology to histology, and to reveal the clinical value of DNA methylation in diagnosis of cervical intraepithelial neoplasia (CIN). A total of 103 random-selected cervical samples were collected from residual liquid-based cytology specimens after clinical use in cytopathological diagnosis in outpatient clinic of obstetrics and gynecology, Peking Union Medical Collage Hospital from March 2010 to October 2010. Informed consent was obtained from each woman before the initiation of the study. The methylation sensitive-high resolution melt (MS-HRM) assay was used to evaluate promoter methylation of three genes (DAPK1, RAR-β and MGMT) in 103 biopsy-confirmed liquid-based cervical cytology samples. Methylation levels and high-risk HPV DNA loading (HCII values) were analyzed in relation to both cytological and histological diagnosis. The methylation level of all three genes showed significant difference among the different cytological groups (P = 0.000, 0.011 and 0.002, respectively). The methylation level of DAPK1 and RAR-β showed significant difference among the different histological groups (P = 0.000 and 0.021), while there was no significant difference for MGMT. DAPK1 methylation levels was 1.47% in the CINII/high-grade precancerous lesions group, and 20.98% in the normal/CINI groups (P = 0.000), but there was no significant difference between CINII/high-grade precancerous lesions and normal/CINI groups for RAR-β and MGMT. The combination of DAPK1/HR-HPV loading showed a sensitivity of 0.825 and an area under the receiver operating characteristic curve (ROC) curve (AUC) of 0.695 as diagnostic methods for detecting CINII/high-grade precancerous lesions. DNA methylation such as DAPK1 and RAR-β, in combination with HR-HPV detection, may serve as biomarkers to detect CINII/high-grade precancerous lesions. Detection of methylated DNA from liquid-based cervical cytology specimens is technically feasible with the MS-HRM assay.


Complex patterns in fossilized stromatolites revealed by hyperspectral imaging (400-2496 nm).
Hyperspectral imaging (400-2496 nm) was used to quantitatively map surface textures and compositional variations in stromatolites to determine whether complexity of textures could be used as evidence to support biogenicity in the absence of preserved biomarkers. Four samples of 2.72-2.4 Ga stromatolites from a variety of settings, encompassing marine and lacustrine environments, were selected for hyperspectral imaging. Images of the sawn surfaces of samples were processed to identify reflectance and mineral absorption features and quantify their intensity (as an index of mineral abundance) using automated feature extraction. Amounts of ferrous iron were quantified using a ratio of reflectance at 1650 and 1299 nm. Visible near infrared imagery (400-970 nm) did not reveal additional textural patterns to those obtained from visual inspection. Shortwave infrared imagery (1000-2496 nm), however, revealed complex laminar and convoluted patterns, including a distinctive texture of sharp peaks and broad, low troughs in one sample, similar to living tufted microbial mats. Spectral analysis revealed another sample to be composed of dolomite. Two other samples were dominated by calcite or chlorite ± illite. Large variations in amounts of ferrous iron were found, but ferric iron was exclusively located in the oxidation crust. Hyperspectral imaging revealed large differences between parts of a sample of biogenic and non-biogenic origin. The former was characterized by calcite with varying amounts of ferrous iron, distributed in lenticular, convoluted patterns; the latter by Mg-Fe chlorite with large amounts of aluminium silicate, distributed as fine laminar layers. All minerals identified by hyperspectral imaging were confirmed by thin section petrography and XRD analyses. Spatial statistics generated from quantitative minerals maps showed different patterns between these different parts of the sample. Thus, hyperspectral imaging was shown to be a powerful tool for detecting structures in stromatolites that could be used, together with other lines of evidence, to support biogenicity.


Duvoglustat HCl Increases Systemic and Tissue Exposure of Active Acid α-Glucosidase in Pompe Patients Co-administered with Alglucosidase α.
Duvoglustat HCl (AT2220, 1-deoxynojirimycin) is an investigational pharmacological chaperone for the treatment of acid α-glucosidase (GAA) deficiency, which leads to the lysosomal storage disorder Pompe disease, which is characterized by progressive accumulation of lysosomal glycogen primarily in heart and skeletal muscles. The current standard of care is enzyme replacement therapy with recombinant human GAA (alglucosidase alfa [AA], Genzyme). Based on preclinical data, oral co-administration of duvoglustat HCl with AA increases exposure of active levels in plasma and skeletal muscles, leading to greater substrate reduction in muscle. This phase 2a study consisted of an open-label, fixed-treatment sequence that evaluated the effect of single oral doses of 50 mg, 100 mg, 250 mg, or 600 mg duvoglustat HCl on the pharmacokinetics and tissue levels of intravenously infused AA (20 mg/kg) in Pompe patients. AA alone resulted in increases in total GAA activity and protein in plasma compared to baseline. Following co-administration with duvoglustat HCl, total GAA activity and protein in plasma were further increased 1.2- to 2.8-fold compared to AA alone in all 25 Pompe patients; importantly, muscle GAA activity was increased for all co-administration treatments from day 3 biopsy specimens. No duvoglustat-related adverse events or drug-related tolerability issues were identified.


Atrial Fibrillation as a Prognostic Indicator in Medium to Large-Sized Dogs with Myxomatous Mitral Valvular Degeneration and Congestive Heart Failure.
The prevalence and prognostic importance of atrial fibrillation (AF) on survival in nonsmall breed dogs with myxomatous mitral valvular disease (MMVD) and congestive heart failure (CHF) remain unknown. To identify the prevalence of AF in nonsmall breed dogs with CHF because of MMVD and to characterize the impact of AF on survival outcome. Sixty-four client-owned dogs (>15 kg) with MMVD and CHF. Retrospective review of medical records for dogs weighing >15 kg with MMVD treated for CHF. Thirty-three dogs presented with AF or developed AF during follow-up examinations, and 31 dogs were free of AF until cardiac-related death. For dogs with AF, median survival time (MST) was 142 days (range: 9-478) while dogs without AF lived 234 days (range: 13-879 days). AF increased risk of cardiac-related death (HR = 2.544; 95% CI = 1.41-4.59; P = .0019) when compared to dogs without AF. MST was significantly prolonged for dogs with AF whose rates were adequately controlled (<160 bpm; 171 days; n = 13) when compared to dogs that failed to respond to negative chronotropic agents (61 days; n = 20; P = .032). The administration of combination treatment (diltiazem and digoxin) significantly decreased median HR to 144 bpm (range: 84-218 bpm) in dogs with AF and significantly prolonged MST (diltiazem+digoxin: 130 days versus diltiazem: 35 days, P = .0241) when compared to diltiazem alone. Inadequately controlled AF is associated with a higher rate of mortality. Optimization of therapeutic strategies for the rate control of AF remains determined.


Size-dependent optical properties of BaTiO3-SrTiO3 superlattices.
Artificial BaTiO(3)-SrTiO(3) superlattices with stacking periodicity varying between 27 and 1670 A in separate films were grown on MgO substrates by pulsed laser deposition. Both the static and active optical properties were found to be sensitive on the stacking periodicity. Birefringence decreased with increasing individual layer thickness due to relaxation of the interface originated stress. The electro-optic response also showed a layer thickness dependence, reaching a maximum at an individual layer thickness of 13 unit cells.


Intravenous N-acetylcysteine in pediatric patients with nonacetaminophen acute liver failure: a placebo-controlled clinical trial.
N-acetylcysteine (NAC) was found to improve transplantation-free survival in only those adults with nonacetaminophen (non-APAP) acute liver failure (ALF) and grade 1-2 hepatic encephalopathy (HE). Because non-APAP ALF differs significantly between children and adults, the Pediatric Acute Liver Failure (PALF) Study Group evaluated NAC in non-APAP PALF. Children from birth through age 17 years with non-APAP ALF enrolled in the PALF registry were eligible to enter an adaptively allocated, doubly masked, placebo-controlled trial using a continuous intravenous infusion of NAC (150 mg/kg/day in 5% dextrose in water [D5W]) or placebo (D5W) for up to 7 days. The primary outcome was 1-year survival. Secondary outcomes included liver transplantation-free survival, liver transplantation (LTx), length of intensive care unit (ICU) and hospital stays, organ system failure, and maximum HE score. A total of 184 participants were enrolled in the trial with 92 in each arm. The 1-year survival did not differ significantly (P = 0.19) between the NAC (73%) and placebo (82%) treatment groups. The 1-year LTx-free survival was significantly lower (P = 0.03) in those who received NAC (35%) than those who received placebo (53%), particularly, but not significantly so, among those less than 2 years old with HE grade 0-1 (NAC 25%; placebo 60%; P = 0.0493). There were no significant differences between treatment arms for hospital or ICU length of stay, organ systems failing, or highest recorded grade of HE. NAC did not improve 1-year survival in non-APAP PALF. One-year LTx-free survival was significantly lower with NAC, particularly among those <2 years old. These results do not support broad use of NAC in non-APAP PALF and emphasizes the importance of conducting controlled pediatric drug trials, regardless of results in adults.


Nutritional and fluid requirements: high-output stomas.
Based on the current available evidence, this article explores the nutritional management of those with a high-output stoma. The main alterations required to the intake of patients with a high-output stoma include the use of an oral rehydration solution to ensure optimum absorption of fluid and sodium, and a high-calorie, high-protein diet, with the aim of optimizing nutritional status. Diet advice should be delivered by a dietitian with experience in managing these complex patients. Monitoring of electrolytes and micronutrients is essential, and long-term follow up from a multidisciplinary nutrition support team is invaluable in coordinating this. Patients with high-output stomas can enjoy good quality of life and long-term health if their condition is managed effectively by a well-organized multidisciplinary team.


Disruption of an M. tuberculosis membrane protein causes a magnesium-dependent cell division defect and failure to persist in mice.
The identification of Mycobacterium tuberculosis genes necessary for persistence in vivo provides insight into bacterial biology as well as host defense strategies. We show that disruption of M. tuberculosis membrane protein PerM (Rv0955) resulted in an IFN-γ-dependent persistence defect in chronic mouse infection despite the mutant's near normal growth during acute infection. The perM mutant required increased magnesium for replication and survival; incubation in low magnesium media resulted in cell elongation and lysis. Transcriptome analysis of the perM mutant grown in reduced magnesium revealed upregulation of cell division and cell wall biosynthesis genes, and live cell imaging showed PerM accumulation at the division septa in M. smegmatis. The mutant was acutely sensitive to β-lactam antibiotics, including specific inhibitors of cell division-associated peptidoglycan transpeptidase FtsI. Together, these data implicate PerM as a novel player in mycobacterial cell division and pathogenesis, and are consistent with the hypothesis that immune activation deprives M. tuberculosis of magnesium.


Efficacy of three neuroprotective drugs in secondary progressive multiple sclerosis (MS-SMART): a phase 2b, multiarm, double-blind, randomised placebo-controlled trial.
Neurodegeneration is the pathological substrate that causes major disability in secondary progressive multiple sclerosis. A synthesis of preclinical and clinical research identified three neuroprotective drugs acting on different axonal pathobiologies. We aimed to test the efficacy of these drugs in an efficient manner with respect to time, cost, and patient resource. We did a phase 2b, multiarm, parallel group, double-blind, randomised placebo-controlled trial at 13 clinical neuroscience centres in the UK. We recruited patients (aged 25-65 years) with secondary progressive multiple sclerosis who were not on disease-modifying treatment and who had an Expanded Disability Status Scale (EDSS) score of 4·0-6·5. Participants were randomly assigned (1:1:1:1) at baseline, by a research nurse using a centralised web-based service, to receive twice-daily oral treatment of either amiloride 5 mg, fluoxetine 20 mg, riluzole 50 mg, or placebo for 96 weeks. The randomisation procedure included minimisation based on sex, age, EDSS score at randomisation, and trial site. Capsules were identical in appearance to achieve masking. Patients, investigators, and MRI readers were unaware of treatment allocation. The primary outcome measure was volumetric MRI percentage brain volume change (PBVC) from baseline to 96 weeks, analysed using multiple regression, adjusting for baseline normalised brain volume and minimisation criteria. The primary analysis was a complete-case analysis based on the intention-to-treat population (all patients with data at week 96). This trial is registered with ClinicalTrials.gov, NCT01910259. Between Jan 29, 2015, and June 22, 2016, 445 patients were randomly allocated amiloride (n=111), fluoxetine (n=111), riluzole (n=111), or placebo (n=112). The primary analysis included 393 patients who were allocated amiloride (n=99), fluoxetine (n=96), riluzole (n=99), and placebo (n=99). No difference was noted between any active treatment and placebo in PBVC (amiloride vs placebo, 0·0% [95% CI -0·4 to 0·5; p=0·99]; fluoxetine vs placebo -0·1% [-0·5 to 0·3; p=0·86]; riluzole vs placebo -0·1% [-0·6 to 0·3; p=0·77]). No emergent safety issues were reported. The incidence of serious adverse events was low and similar across study groups (ten [9%] patients in the amiloride group, seven [6%] in the fluoxetine group, 12 [11%] in the riluzole group, and 13 [12%] in the placebo group). The most common serious adverse events were infections and infestations. Three patients died during the study, from causes judged unrelated to active treatment; one patient assigned amiloride died from metastatic lung cancer, one patient assigned riluzole died from ischaemic heart disease and coronary artery thrombosis, and one patient assigned fluoxetine had a sudden death (primary cause) with multiple sclerosis and obesity listed as secondary causes. The absence of evidence for neuroprotection in this adequately powered trial indicates that exclusively targeting these aspects of axonal pathobiology in patients with secondary progressive multiple sclerosis is insufficient to mitigate neuroaxonal loss. These findings argue for investigation of different mechanistic targets and future consideration of combination treatment trials. This trial provides a template for future simultaneous testing of multiple disease-modifying medicines in neurological medicine. Efficacy and Mechanism Evaluation (EME) Programme, an MRC and NIHR partnership, UK Multiple Sclerosis Society, and US National Multiple Sclerosis Society.


TdT Expression Is a Marker of Better Survival in Merkel Cell Carcinoma, and Expression of B-Cell Markers Is Associated With Merkel Cell Polyomavirus.
Merkel cell carcinoma is a rare but very aggressive cutaneous tumor. We evaluated the prognostic potential of B-cell markers (terminal deoxynucleotidyl transferase [TdT], PAX5, CD117), follicular stem cell markers (CK15, CK19), p63, p53, RB, and Merkel cell polyomavirus (MCPyV; CM2B4) in 136 primary cutaneous Merkel cell carcinomas. Clinical, histopathologic, and immunohistochemical analyses were performed. The results were correlated with patient outcomes by Fisher exact test, log-rank tests, and Cox multivariate models. By Fisher exact test, although TdT significantly correlated with both lack of progression (P = .0087) and alive status (P = .0056), MCPyV status correlated only with alive status (P = .031). In univariate analyses, TdT, MCPyV, and RB significantly correlated with improved overall survival, whereas p63 and CK15 correlated with worse overall survival. However, in multivariate analyses, only TdT expression remained as an independent predictor of improved overall survival, Merkel cell carcinoma-specific survival, and progression-free survival. By linear regression analyses, significant correlations between MCPyV vs TdT, PAX5, and CD117 were observed. TdT expression is a potential marker of better survival in Merkel cell carcinoma. Expression of B-cell markers is associated with MCPyV, suggesting that clonal viral integration might play a role in the expression of these markers.


Overexpression of the c-MET/HGF receptor gene in human thyroid carcinomas.
The receptor for Hepatocyte Growth Factor is a transmembrane tyrosine kinase encoded by the c-MET oncogene. We have previously shown that the Met protein is expressed in several human epithelial tissues. The receptor is barely detectable, however, in normal thyroids and in specimens from patients affected by non-neoplastic thyroid diseases. Now we report that the expression of the Met/HGF receptor is increased a hundred fold in 22 out of 41 human carcinomas derived from the thyroid follicular epithelium. A comprehensive analysis of 15 cases showed that the overexpressing carcinomas belong to histotype variants correlated with negative prognosis and in all but one case there were evidences of locally advanced disease and/or distant metastases. The 11 benign adenomas and the 5 medullary carcinomas tested were negative. Western blot analysis with monoclonal antibodies directed against either the intracellular or the extracellular receptor domains failed to reveal major structural alterations. Southern blot analysis also demonstrated that the c-MET gene was not amplified nor rearranged. These data suggest a role for the overexpression of c-MET oncogene in the pathogenesis and progression of thyroid tumors derived from the follicular epithelium.


Targeting of adenovirus E1A and E4-ORF3 proteins to nuclear matrix-associated PML bodies.
The PML protein was first identified as part of a fusion product with the retinoic acid receptor alpha (RAR alpha), resulting from the t(15;17) chromosomal translocation associated with acute promyelocytic leukemia (APL). It has been previously demonstrated that PML, which is tightly bound to the nuclear matrix, concentrates in discrete subnuclear compartments that are disorganized in APL cells due to the expression of the PML-RAR alpha hybrid. Here we report that adenovirus infection causes a drastic redistribution of PML from spherical nuclear bodies into fibrous structures. The product encoded by adenovirus E4-ORF3 is shown to be responsible for this reorganization and to colocalize with PML into these fibers. In addition, we demonstrate that E1A oncoproteins concentrate in the PML domains, both in infected and transiently transfected cells, and that this association requires the conserved amino acid motif (D)LXCXE, common to all viral oncoproteins that bind pRB or the related p107 and p130 proteins. The SV-40 large T antigen, another member of this oncoprotein family is also found in close association with the PML nuclear bodies. Taken together, the present data indicate that the subnuclear domains containing PML represent a preferential target for DNA tumor viruses, and therefore suggest a more general involvement of the PML nuclear bodies in oncogenic processes.


A dual-labeled knottin peptide for PET and near-infrared fluorescence imaging of integrin expression in living subjects.
Previously, we used directed evolution to engineer mutants of the Ecballium elaterium trypsin inhibitor (EETI-II) knottin that bind to αvβ3 and αvβ5 integrin receptors with low nanomolar affinity, and showed that Cy5.5- or (64)Cu-DOTA-labeled knottin peptides could be used to image integrin expression in mouse tumor models using near-infrared fluorescence (NIRF) imaging or positron emission tomography (PET). Here, we report the development of a dual-labeled knottin peptide conjugated to both NIRF and PET imaging agents for multimodality imaging in living subjects. We created an orthogonally protected peptide-based linker for stoichiometric coupling of (64)Cu-DOTA and Cy5.5 onto the knottin N-terminus and confirmed that conjugation did not affect binding to αvβ3 and αvβ5 integrins. NIRF and PET imaging studies in tumor xenograft models showed that Cy5.5 conjugation significantly increased kidney uptake and retention compared to the knottin peptide labeled with (64)Cu-DOTA alone. In the tumor, the dual-labeled (64)Cu-DOTA/Cy5.5 knottin peptide showed decreased wash-out leading to significantly better retention (p < 0.05) compared to the (64)Cu-DOTA-labeled knottin peptide. Tumor uptake was significantly reduced (p < 0.05) when the dual-labeled knottin peptide was coinjected with an excess of unlabeled competitor and when tested in a tumor model with lower levels of integrin expression. Finally, plots of tumor-to-background tissue ratios for Cy5.5 versus (64)Cu uptake were well-correlated over several time points post injection, demonstrating pharmacokinetic cross validation of imaging labels. This dual-modality NIRF/PET imaging agent is promising for further development in clinical applications where high sensitivity and high resolution are desired, such as detection of tumors located deep within the body and image-guided surgical resection.


Identification of hub genes related to the recovery phase of irradiation injury by microarray and integrated gene network analysis.
Irradiation commonly causes long-term bone marrow injury charactertized by defective HSC self-renewal and a decrease in HSC reserve. However, the effect of high-dose IR on global gene expression during bone marrow recovery remains unknown. Microarray analysis was used to identify differentially expressed genes that are likely to be critical for bone marrow recovery. Multiple bioinformatics analyses were conducted to identify key hub genes, pathways and biological processes. 1) We identified 1302 differentially expressed genes in murine bone marrow at 3, 7, 11 and 21 days after irradiation. Eleven of these genes are known to be HSC self-renewal associated genes, including Adipoq, Ccl3, Ccnd1, Ccnd2, Cdkn1a, Cxcl12, Junb, Pten, Tal1, Thy1 and Tnf; 2) These 1302 differentially expressed genes function in multiple biological processes of immunity, including hematopoiesis and response to stimuli, and cellular processes including cell proliferation, differentiation, adhesion and signaling; 3) Dynamic Gene Network analysis identified a subgroup of 25 core genes that participate in immune response, regulation of transcription and nucleosome assembly; 4) A comparison of our data with known irradiation-related genes extracted from literature showed 42 genes that matched the results of our microarray analysis, thus demonstrated consistency between studies; 5) Protein-protein interaction network and pathway analyses indicated several essential protein-protein interactions and signaling pathways, including focal adhesion and several immune-related signaling pathways. Comparisons to other gene array datasets indicate that global gene expression profiles of irradiation damaged bone marrow show significant differences between injury and recovery phases. Our data suggest that immune response (including hematopoiesis) can be considered as a critical biological process in bone marrow recovery. Several critical hub genes that are key members of significant pathways or gene networks were identified by our comprehensive analysis.


Chronological age, biological age, and individual variation in the stress response in the European starling: a follow-up study.
The strength of the avian stress response declines with age. A recently published study of European starlings (Sturnus vulgaris) found that a marker of biological age predicted the strength of the stress response even in individuals of the same chronological age. Specifically, birds that had experienced greater developmental telomere attrition (DTA) showed a lower peak corticosterone (CORT) response to an acute stressor, and more rapid recovery of CORT levels towards baseline. Here, we performed a follow-up study using the same capture-handling-restraint stressor in a separate cohort of starlings that had been subjected to a developmental manipulation of food availability and begging effort. We measured the CORT response at two different age points (4 and 18 months). Our data suggest a decline in the strength of the CORT response with chronological age: peak CORT was lower at the second age point, and there was relatively more reduction in CORT between 15 and 30 min. Individual consistency between the two age points was low, but there were modest familial effects on baseline and peak CORT. The manipulation of begging effort affected the stress response (specifically, the reduction in CORT between 15 and 30 min) in an age-dependent manner. However, we did not replicate the associations with DTA observed in the earlier study. We meta-analysed the data from the present and the earlier study combined, and found some support for the conclusions of the earlier paper.


Quantitative proteome comparison of human hearts with those of model organisms.
Delineating human cardiac pathologies and their basic molecular mechanisms relies on research conducted in model organisms. Yet translating findings from preclinical models to humans present a significant challenge, in part due to differences in cardiac protein expression between humans and model organisms. Proteins immediately determine cellular function, yet their large-scale investigation in hearts has lagged behind those of genes and transcripts. Here, we set out to bridge this knowledge gap: By analyzing protein profiles in humans and commonly used model organisms across cardiac chambers, we determine their commonalities and regional differences. We analyzed cardiac tissue from each chamber of human, pig, horse, rat, mouse, and zebrafish in biological replicates. Using mass spectrometry-based proteomics workflows, we measured and evaluated the abundance of approximately 7,000 proteins in each species. The resulting knowledgebase of cardiac protein signatures is accessible through an online database: atlas.cardiacproteomics.com. Our combined analysis allows for quantitative evaluation of protein abundances across cardiac chambers, as well as comparisons of cardiac protein profiles across model organisms. Up to a quarter of proteins with differential abundances between atria and ventricles showed opposite chamber-specific enrichment between species; these included numerous proteins implicated in cardiac disease. The generated proteomics resource facilitates translational prospects of cardiac studies from model organisms to humans by comparisons of disease-linked protein networks across species.


Quantifying the risk from ovine BSE and the impact of control strategies.
Although no naturally infected sheep with bovine spongiform encephalopathy (BSE) has ever been discovered, it remains possible that BSE once infected the UK sheep population, has been transmitted between sheep, and is still present today. We constructed a mathematical model to assess the current maximum theoretical exposure to consumers from BSE-infected ovine material and to estimate the risk reduction that could be achieved by abattoir-based control options if BSE-infected sheep were ever found in the national flock. We predict that, if present, the exposure to consumers from a single BSE-infected sheep would be high: one sheep, close to the end of its incubation period, is likely to contribute 10-1000 times more infectious material than a fully infectious cow. Furthermore, 30% of this exposure comes from infectivity residing in lymphatic and peripheral tissue that cannot be completely removed from a carcass. We are 95% confident that throughout Great Britain, no more than four sheep flocks currently harbour an ongoing BSE epidemic. However, since the exposure from a single infected sheep is high, the annual human exposure from four 'typical' BSE-infected flocks could be considerable. Small reductions in exposure could be achieved by strategies based on tissue testing, a 12-month age restriction or expanded definitions of high-risk tissues. A six-month age restriction is likely to be more effective and genotype-based strategies the most effective.


Brain responses to chronic social defeat stress: effects on regional oxidative metabolism as a function of a hedonic trait, and gene expression in susceptible and resilient rats.
Chronic social defeat stress, a depression model in rats, reduced struggling in the forced swimming test dependent on a hedonic trait-stressed rats with high sucrose intake struggled less. Social defeat reduced brain regional energy metabolism, and this effect was also more pronounced in rats with high sucrose intake. A number of changes in gene expression were identified after social defeat stress, most notably the down-regulation of Gsk3b and Map1b. The majority of differences were between stress-susceptible and resilient rats. Conclusively, correlates of inter-individual differences in stress resilience can be identified both at gene expression and oxidative metabolism levels.


Characterization of 16 microsatellite markers for the Oreinotinus clade of Viburnum (Adoxaceae).
Microsatellite loci were isolated from four species of Viburnum (Adoxaceae) to study population structure and assess species boundaries among morphologically similar South American Viburnum species of the Oreinotinus clade. Using a microsatellite-enriched library and mining next-generation sequence data, 16 microsatellites were developed. Each locus was tested on two populations of V. triphyllum and one population of V. pichinchense. For nuclear loci, one to 13 alleles were recovered, expected heterozygosity ranged from 0 to 0.8975, Simpson diversity index ranged from 0.0167 to 1.000, and Shannon diversity index ranged from 0 to 2.3670 in a given population. For the mitochondrial locus, three to six alleles were recovered and unbiased haploid diversity values ranged from 0.756 to 0.853 in a given population. The 16 microsatellite loci developed for the Oreinotinus clade (Viburnum, Adoxaceae) will inform investigations of population structure and species boundaries within this group.


Managing demographic risk.
In developed nations, the workforce is aging rapidly. That trend has serious implications. Companies could face severe labor shortages in a few years as workers retire, taking critical knowledge with them. Businesses may also see productivity decline among older employees, especially in physically demanding jobs. The authors, partners at Boston Consulting Group, offer managers a systematic way to assess these dual threats--capacity risk and productivity risk--at their companies. It involves studying the age distribution of their employees to see if large percentages fall within high age brackets and then projecting--by location, unit, and job category--how the distribution will change over the next 15 years. Managers must also factor in both the impact of strategic moves on personnel needs and the future supply of workers in the market. When RWE Power analyzed its trends, the company learned that in 2018 almost 80% of its workers would be over 50. What's more, in certain critical areas its labor surplus was about to become a sizable shortfall. For instance, a shortage of specialized engineers would develop in the company just as their ranks in the job market thinned and competition to hire them intensified. Reversing its downsizing course, RWE Power took steps to increase its supply of workers in those key positions. The authors show how companies that face talent gaps, as RWE Power did, can close them through training, transfers, recruitment, retention, productivity improvements, and outsourcing. They also describe measures that companies can take to keep older workers productive, including workplace accommodations, revised compensation structures, performance incentives, and targeted health care management. The key is to identify and address potential problems early. Firms that do so will gain an edge on rivals that are still relentlessly focused on reducing head count.


Blast shockwaves propagate Ca(2+) activity via purinergic astrocyte networks in human central nervous system cells.
In a recent study of the pathophysiology of mild, blast-induced traumatic brain injury (bTBI) the exposure of dissociated, central nervous system (CNS) cells to simulated blast resulted in propagating waves of elevated intracellular Ca(2+). Here we show, in dissociated human CNS cultures, that these calcium waves primarily propagate through astrocyte-dependent, purinergic signaling pathways that are blocked by P2 antagonists. Human, compared to rat, astrocytes had an increased calcium response and prolonged calcium wave propagation kinetics, suggesting that in our model system rat CNS cells are less responsive to simulated blast. Furthermore, in response to simulated blast, human CNS cells have increased expressions of a reactive astrocyte marker, glial fibrillary acidic protein (GFAP) and a protease, matrix metallopeptidase 9 (MMP-9). The conjoint increased expression of GFAP and MMP-9 and a purinergic ATP (P2) receptor antagonist reduction in calcium response identifies both potential mechanisms for sustained changes in brain function following primary bTBI and therapeutic strategies targeting abnormal astrocyte activity.


Mediation of Developmental Risk Factors for Psychosis by White Matter Microstructure in Young Adults With Psychotic Experiences.
White matter (WM) abnormalities have been identified in schizophrenia at the earliest stages of the disorder. Individuals in the general population with psychotic experiences (PEs) may show similar changes, suggesting dysfunction due to aberrant neurodevelopment. Studying such people is a powerful means of understanding the nature of neurodevelopmental problems without the confound of clinical management and allows other potential risk factors associated with the schizophrenia spectrum to be taken into account. To compare WM microstructure and myelination in young adults with and without PEs identified from a population-based cohort using diffusion and relaxometry magnetic resonance imaging and to quantify potential mediating effects of WM on several known risk factors for psychosis. In this case-control study, participants were drawn from the UK Avon Longitudinal Study of Parents and Children. Psychotic experiences were assessed using a semistructured interview. Magnetic resonance imaging was carried out at age 20 years in 123 participants who had PEs and 124 individuals serving as controls. Participants with PEs were subdivided into those with operationally defined suspected PEs, definite PEs, and psychotic disorder. Diffusion tensor magnetic resonance imaging and relaxometry-derived myelin water fractions were used to measure WM microstructure and myelination, respectively. Differences in quantitative WM indices were assessed using tract-based spatial statistics. A binary model and a continuum-like ordinal model of PEs were tested. Among the 123 participants who had PEs (mean [SE] age, 20.01 [0.004] years), 37 were male and 86 were female. Among the 124 controls (mean [SE] age, 20.11 [0.004] years), 49 were male and 76 were female. Fractional anisotropy in left frontomedial WM was significantly reduced in individuals with PEs (Montreal Neurological Institute [MNI] coordinates, -18, 37, -2; P = .0046). The ordinal model identified a similar but more widespread effect, with a corresponding increase in radial diffusivity (MNI coordinates, -15, 29, 21; P = .0042). Low birth weight (ρ = -0.155; P = .015) and childhood IQ (ρ = -0.188; P = .003) were associated with the presence of PEs. Results of mediation analysis were consistent with the association between birth weight (21.1% mediation effect; P = 6.20 × 10-3) and childhood IQ (7.9% mediation effect; P = .041) and by PEs being mediated by fractional anisotropy changes in these regions. The results of the study imply the presence of abnormal WM microstructure in young adults with PEs. The results are consistent with the hypothesis that neurodevelopmental factors cause alterations in the cellular composition of WM circuits critical to higher cognitive function. Such alterations may first manifest in childhood as reduced IQ and later contribute to PEs in early adulthood.


Sights of touching activates the somatosensory cortex in humans.
We report our observations of cross-modal interactions between sight and touch using functional magnetic resonance imaging (fMRI). Experiments were devised to show that sight and touch are linked in a cross-modal arrangement, and two separate experiments were done in an MRI scanner. In the first, the subject's right hand was stimulated with a brush; in the second, a video sequence was presented to the subject inside the scanner through video goggles in visual three-dimensional stereo, showing one brushstroke every second on a hand in the same manner as the subject had just previously experienced. The result was that both the primary and the secondary somatosensory cortexes were activated in the participants when the hands were touched, and when the subjects saw only a hand being touched in the same manner. The results indicated cross-modal links between sight and touch of the hand in humans.


In vivo characterization of distinct modality-specific subsets of somatosensory neurons using GCaMP.
Mechanistic insights into pain pathways are essential for a rational approach to treating this vast and increasing clinical problem. Sensory neurons that respond to tissue damage (nociceptors) may evoke pain sensations and are typically classified on the basis of action potential velocity. Electrophysiological studies have suggested that most of the C-fiber nociceptors are polymodal, responding to a variety of insults. In contrast, gene deletion studies in the sensory neurons of transgenic mice have frequently resulted in modality-specific deficits. We have used an in vivo imaging approach using the genetically encoded fluorescent calcium indicator GCaMP to study the activity of dorsal root ganglion sensory neurons in live animals challenged with painful stimuli. Using this approach, we can visualize spatially distinct neuronal responses and find that >85% of responsive dorsal root ganglion neurons are modality-specific, responding to either noxious mechanical, cold, or heat stimuli. These observations are mirrored in behavioral studies of transgenic mice. For example, deleting sodium channel Nav1.8 silences mechanical- but not heat-sensing sensory neurons, consistent with behavioral deficits. In contrast, primary cultures of axotomized sensory neurons show high levels of polymodality. After intraplantar treatment with prostaglandin E2, neurons in vivo respond more intensely to noxious thermal and mechanical stimuli, and additional neurons (silent nociceptors) are unmasked. Together, these studies define polymodality as an infrequent feature of nociceptive neurons in normal animals.


Anthropometric measurements and vertebral deformities. European Vertebral Osteoporosis Study (EVOS) Group.
To investigate the association between anthropometric indices and morphometrically determined vertebral deformity, the authors carried out a cross-sectional study using data from the European Vertebral Osteoporosis Study (EVOS), a population-based study of vertebral osteoporosis in 36 European centers from 19 countries. A total of 16,047 EVOS subjects were included in this analysis, of whom 1,973 subjects (915 males, 1,058 females) (12.3%) aged 50 years or over had one or more vertebral deformities ("cases"). The cases were compared with the 14,074 subjects (6,539 males, 7,535 females) with morphometrically normal spines ("controls"). Data were collected on self-reported height at age 25 years and minimum weight after age 25 years, as well as on current measured height and weight. Body mass index (BMI) and height and weight change were calculated from these data. The relations between these variables and vertebral deformity were examined separately by sex with logistic regression adjusting for age, smoking, and physical activity. In females, there was a significant trend of decreasing risk with increasing quintile of current weight, current BMI, and weight gain since age 25 years. In males, subjects in the lightest quintile for these measures were at increased risk but there was no evidence of a trend. An ecologic analysis by country revealed a negative correlation between mean BMI and the prevalence of deformity in females but not in males. The authors conclude that low body weight is associated with presence of vertebral deformity.


Mast cells augment lesion size and persistence during experimental Leishmania major infection in the mouse.
Mast cells are a source of a variety of cytokines that may influence the host response to Leishmania major. To investigate the role of mast cells during L. major infection, we performed a morphometric analysis of mast cells at cutaneous sites in resistant C57BL/6 mice and susceptible BALB/c mice injected with L. major. Extensive dermal mast cell degranulation was found at sites of L. major infection in both strains of mice. We also examined the course of L. major infection in genetically mast cell-deficient WBB6F1-W/Wv or WCB6F1-Sl/Sld mice, their respective congenic normal (WBB6F1-(+/+) or WCB6F1-(+/+)) littermates, and WBB6F1-W/Wv mice that had been selectively and locally repaired of their cutaneous mast cell deficiency. We found that mast cells significantly augmented the intensity and maximal size of the cutaneous lesions at sites of L. major infection, and in some cases substantially prolonged the persistence of the reactions. However, the lesions ultimately resolved in both the mast cell-deficient and the congenic normal mice. In addition, the presence or absence of mast cells had little or no effect on the numbers of viable parasites recovered from the cutaneous lesions. Moreover, mast cell-deficient W/Wv mice and the congenic normal (+/+) mice produced similar levels of IFN-gamma mRNA in lymph nodes draining the cutaneous lesions whereas no IL-4 mRNA was detectable. Taken together, these data suggest that mast cells significantly augment the size of cutaneous lesions during L. major infection in mice. However, mast cells do not appear to influence significantly either the parasite burden or the ultimate resolution of the infection.


Care of diabetic patients in hospital clinics and general practice clinics: a study in Dudley.
A five year retrospective casenote review was undertaken of 452 patients attending 11 different general practice diabetic clinics, and 506 patients attending a diabetic clinic at hospital A and 210 patients attending a diabetic clinic at hospital B. The populations attending the clinics, the degree of glycaemic control achieved and the monitoring for the development of diabetic complications were investigated. Insulin dependent patients comprised 57.9% of patients attending the diabetic clinic at hospital A, 35.7% at hospital B and 25.0% of patients attending the diabetic clinics at general practices. Of these 55.6%, 37.3% and 30.1% respectively received multiple daily insulin injections. Hospital A had a higher proportion of patients under 40 years old than hospital B or the general practice clinics. The ages of diabetic patients attending the general practice diabetic clinics were broadly similar to those attending hospital B. Significantly more general practice patients, both insulin and non-insulin dependent, had a mean blood glucose level of less than 11 mmol l-1 compared with patients attending clinics at hospitals A and B (P < 0.001). Glycosylated haemoglobin levels did not differ between patients attending hospital A and the general practice clinics. More non-insulin dependent and insulin dependent diabetic patients attending the general practice clinics and hospital A had been monitored satisfactorily for diabetic retinopathy (general practice clinic 68.8% and 39.7% respectively, hospital A 61.7% and 43.5%) than at hospital B (43.0% and 19.4%). Referral rates among all groups for ophthalmological assessment were similar.(ABSTRACT TRUNCATED AT 250 WORDS)


Neural mapping of guilt: a quantitative meta-analysis of functional imaging studies.
Guilt is a self-conscious emotion associated with the negative appraisal of one's behavior. In recent years, several neuroimaging studies have investigated the neural correlates of guilt, but no meta-analyses have yet identified the most robust activation patterns. A systematic review of literature found 16 functional magnetic resonance imaging studies with whole-brain analyses meeting the inclusion criteria, for a total of 325 participants and 135 foci of activation. A meta-analysis was then conducted using activation likelihood estimation. Additionally, Meta-Analytic Connectivity Modeling (MACM) analysis was conducted to investigate the functional connectivity of significant clusters. The analysis revealed 12 significant clusters of brain activation (voxel-based FDR-corrected p < 0.05) located in the prefrontal, temporal and parietal regions, mainly in the left hemisphere. Only the left dorsal cingulate cluster survived stringent FWE correction (voxel-based p < 0.05). Secondary analyses (voxel-based FDR-corrected p < 0.05) on the 7 studies contrasting guilt with another emotional condition showed an association with clusters in the left precuneus, the anterior cingulate, the left medial frontal gyrus, the right superior frontal gyrus and the left superior temporal gyrus. MACM demonstrated that regions associated with guilt are highly interconnected. Our analysis identified a distributed neural network of left-lateralized regions associated with guilt. While voxel-based FDR-corrected results should be considered exploratory, the dorsal cingulate was robustly associated with guilt. We speculate that this network integrates cognitive and emotional processes involved in the experience of guilt, including self-representation, theory of mind, conflict monitoring and moral values. Limitations of our meta-analyses comprise the small sample size and the heterogeneity of included studies, and concerns about naturalistic validity.


Altered p16(INK4) and RB1 Expressions Are Associated with Poor Prognosis in Patients with Nonsmall Cell Lung Cancer.
p16(INK4) and RB1 are two potent cell cycle regulators to control the G1/S transition by interacting with CDK4/6, E2F, and D-type cyclins, respectively. Depending on the tumour type, genetic alterations resulting in the functional inactivation have frequently been reported in both genes. By contrast, much less is known regarding the overexpression of these proteins in the tumor cells. In this study, expressions of p16(INK4) RB1, and CDKN2A copy number variances (CNV) in the tumor cells were assessed by immunohistochemistry and fluorescence in situ hybridization (FISH), respectively, in 73 nonsmall cell lung cancer (NSCLC) with known 5-year survivals. The histologic type (P = 0.01), p16(INK4) (P = 0.004), and RB1 (P < 0.001) were predictive of survivals. The CDKN2A CNV (P < 0.05) was also significant when compared to those cases without CNV. Therefore, among the molecular genetic prognostic factors, expressions of RB1 and p16(INK4) in the tumor cells were the most strongly predictive of adverse outcomes in stage I and II nonsquamous NSCLC.


A species-specific nematocide that results in terminal embryogenesis.
Nematode-insect interactions are ubiquitous, complex and constantly changing as the host and nematode coevolve. The entomophilic nematode Pristionchus pacificus is found on a myriad beetle species worldwide, although the molecular dynamics of this relationship are largely unknown. To better understand how host cues affect P. pacificus embryogenesis, we characterized the threshold of sensitivity to the pheromone (Z)-7-tetradecen-2-one (ZTDO) by determining the minimum exposure duration and developmental window that results in P. pacificus embryonic lethality. We found early-stage embryos exposed to volatile ZTDO for as few as 4 h all display terminal embryogenesis, characterized by punctuated development up to 48 h later, with abnormal morphology and limited cavity formation. To determine if the pheromone arrests pre-hatching development by suffocating or permeabilizing the eggshells, we raised embryos under anoxic conditions and also examined eggshell permeability using the lipophilic dye FM4-64. We found that asphyxiating the embryos arrested embryogenesis in a reversible manner but did not phenocopy the effects of ZTDO exposure, whereas the ZTDO-induced disruption of embryogenesis did correlate with increased eggshell permeability. The effects of ZTDO are also highly specific, as other lipid insect compounds do not produce any detectable embryocidal effect. The high specificity and unusual teratogenic effect of ZTDO may be important in mediating the host-nematode relationship by regulating P. pacificus development.


Microlenses with defined contour shapes.
Ink-jet printing of optical ink over SU-8 pillars is here proposed as a technology for obtaining microlenses with shape control. To demonstrate the flexibility of this method, microlenses with five different contour shapes (ranging from circular and elliptical to toric or more advanced geometries) have been fabricated. Furthermore, the optical properties of the different fabricated lenses have been experimentally investigated. Focal distance, numerical aperture (NA) and full-width at half maximum (FWHM) of the microlenses have been determined. Arrays of microlenses showed an identical behavior with a standard deviation in the total intensity of only 7%. Additionally, the focal plane of the fabricated symmetric microlenses and the Sturm interval of the non-symmetric ones have been obtained. The experimental results demonstrate the validity and flexibility of the proposed technology.


Prevention of Craniofacial Pain Secondary to Harvesting of Temporalis Fascia - A Novel Technique.
A lesser known after effect of harvesting temporalis fascia is the post-surgical craniofacial pain. The aim of the study was to evaluate this pain after tympanomastoid surgeries and the effectiveness of silastic sheet interpositioning to prevent this pain. This pilot study that spanned one year, included patients who underwent tympanoplasty with or without mastoidectomy involving the harvesting of temporalis fascia. At the end of surgery, the wound was closed after silastic sheet was secured over the donor site in cases and without silastic sheet in controls. In the post-operative period, patients scored their temporal pain, tenderness and pain during opening of mouth and mastication on a visual analogue scale (VAS) on day 7, 15, 30 and 90. Visual analogue scale (VAS) scores of the silastic group were lower than the control group on day 7 and 15 after surgery and the difference was statistically significant. In the control group, temporal pain and tenderness were 74% and 81% respectively on day 7. VAS scores of both groups decreased over time and were negligible after 3 months. There were no significant postoperative complications in either group and no reaction or rejection of silastic sheet in the cases. Post-surgical craniofacial pain secondary to the harvesting of temporalis fascia is observed in a majority of the patients. This novel technique involving silastic sheet interposition can decimate early post-operative temporal pain, tenderness and masticatory pain.


The cholesteryl ester transfer protein (CETP) gene and the risk of Alzheimer's disease.
Like the apolipoprotein E (APOE) gene, the most common genetic determinant for Alzheimer's disease (AD), the cholesteryl ester transfer protein (CETP) is involved in lipid metabolism. We studied the I405V polymorphism of the CETP gene in relation to AD. We genotyped 544 AD cases and 5,404 controls from the Rotterdam study, using a TaqMan allelic discrimination assay. Odds ratios (ORs) for AD were estimated using logistic regression analysis. CETP VV carriers showed significantly increased high-density lipoprotein levels compared to the IV and II carriers. In the overall analysis of AD, the risk of disease for the VV carriers of the CETP polymorphism was non-significantly increased compared to II carriers OR(VV) = 1.33, 95% confidence interval (CI) 0.96-1.90 p = 0.08). In those without the APOE*4 allele, the risk of AD for VV carriers was increased 1.67-fold (95% CI 1.11-2.52, p = 0.01). The difference in the relationship between CETP and AD between APOE*4 carriers and APOE*4 non-carriers was statistically significant (p for interaction = 0.04). Our results suggest that the VV genotype of the I405V polymorphism of the CETP gene increases the risk of AD in the absence of the APOE*4 allele, probably through a cholesterol metabolism pathway in the brain.


Trends and interaction of polypharmacy and potentially inappropriate prescribing in primary care over 15 years in Ireland: a repeated cross-sectional study.
To examine: (1) changes in polypharmacy in 1997, 2002, 2007 and 2012 and; (2) changes in potentially inappropriate prescribing (PIP) prevalence and the relationship between PIP and polypharmacy in individuals aged ≥65 years over this period in Ireland. This repeated cross-sectional study using pharmacy claims data included all individuals eligible for the General Medical Services scheme in the former Eastern Health Board region of Ireland in 1997, 2002, 2007 and 2012 (range 338,025-539,752 individuals). Outcomes evaluated were prevalence of polypharmacy (being prescribed ≥5 regular medicines) and excessive polypharmacy (≥10 regular medicines) in all individuals and PIP prevalence in those aged ≥65 years determined by 30 criteria from the Screening Tool for Older Persons' Prescriptions. The prevalence of polypharmacy increased from 1997 to 2012, particularly among older individuals (from 17.8% to 60.4% in those aged ≥65 years). The adjusted incident rate ratio for polypharmacy in 2012 compared to 1997 was 4.16 (95% CI 3.23 to 5.36), and for excessive polypharmacy it was 10.53 (8.58 to 12.91). Prevalence of PIP rose from 32.6% in 1997 to 37.3% in 2012. High-dose aspirin and digoxin prescribing decreased over time, but long-term proton pump inhibitors at maximal dose increased substantially (from 0.8% to 23.8%). The odds of having any PIP in 2012 were lower compared to 1997 after controlling for gender and level of polypharmacy, OR 0.39 (95% CI 0.39 to 0.4). Accounting for the marked increase in polypharmacy, prescribing quality appears to have improved with a reduction in the odds of having PIP from 1997 to 2012. With growing numbers of people taking multiple regular medicines, strategies to address the related challenges of polypharmacy and PIP are needed.


Periodic solutions and refractory periods in the soliton theory for nerves and the locust femoral nerve.
Close to melting transitions it is possible to propagate solitary electromechanical pulses which reflect many of the experimental features of the nerve pulse including mechanical dislocations and reversible heat production. Here we show that one also obtains the possibility of periodic pulse generation when the constraint for the nerve is the conservation of the overall length of the nerve. This condition generates an undershoot beneath the baseline ('hyperpolarization') and a 'refractory period', i.e., a minimum distance between pulses. In this paper, we outline the theory for periodic solutions to the wave equation and compare these results to action potentials from the femoral nerve of the locust (Locusta migratoria). In particular, we describe the frequently occurring minimum-distance doublet pulses seen in these neurons and compare them to the periodic pulse solutions.


Effects of hyperandrogenism on tear function and tear drainage in patients with polycystic ovary syndrome.
Polycystic ovary syndrome (PCOS) is an endocrinopathy characterized by chronic anovulation and hyperandrogenism. Hormonal changes can affect ocular surface and tear functions. We evaluated dry eye symptoms, clinical tear film alterations, and tear clearance in patients with PCOS. Thirty-five women with PCOS and 22 healthy individuals were enrolled in the study. An ocular surface disease index (OSDI) questionnaire was administered, and after the routine ophthalmologic examination tear film breakup time (TBUT) and Schirmer tests were performed. Tear clearance was assessed via fluorescein dye disappearance test (FDDT). No difference was observed in Schirmer test between the groups, however OSDI scores were significantly higher (p = 0.01) and TBUT was significantly lower (p = 0.01) in PCOS patients than in the control women. FDDTs were not different between the PCOS and control subjects. Our findings suggest that patients diagnosed with PCOS should be advised to obtain an ophthalmic examination, especially for dry eye.


Mild obesity is protective after severe burn injury.
To assess the impact of obesity on morbidity and mortality in severely burned patients. Despite the increasing number of people with obesity, little is known about the impact of obesity on postburn outcomes. A total of 405 patients were prospectively enrolled as part of the multicenter trial Inflammation and the Host Response to Injury Glue Grant with the following inclusion criteria: 0 to 89 years of age, admitted within 96 hours after injury, and more than 20% total body surface area burn requiring at least 1 surgical intervention. Body mass index was used in adult patients to stratify according to World Health Organization definitions: less than 18.5 (underweight), 18.5 to 29.9 (normal weight), 30 to 34.9 (obese I), 35 to 39.9 (obese II), and body mass index more than 40 (obese III). Pediatric patients (2 to ≤18 years of age) were stratified by using the Centers for Disease Control and Prevention and World Health Organization body mass index-for-age growth charts to obtain a percentile ranking and then grouped as underweight (<5th percentile), normal weight (5th percentile to <95th percentile), and obese (≥95th percentile). The primary outcome was mortality and secondary outcomes were clinical markers of patient recovery, for example, multiorgan function, infections, sepsis, and length of stay. A total of 273 patients had normal weight, 116 were obese, and 16 were underweight; underweight patients were excluded from the analyses because of insufficient patient numbers. There were no differences in primary and secondary outcomes when normal weight patients were compared with obese patients. Further stratification in pediatric and adult patients showed similar results. However, when adult patients were stratified in obesity categories, log-rank analysis showed improved survival in the obese I group and higher mortality in the obese III group compared with obese I group (P < 0.05). Overall, obesity was not associated with increased morbidity and mortality. Subgroup analysis revealed that patients with mild obesity have the best survival, whereas morbidly obese patients have the highest mortality. (NCT00257244).