Evaluation of adenosine deaminase activity and antibody to Mycobacterium tuberculosis antigen 5 in cerebrospinal fluid and the radioactive bromide partition test for the early diagnosis of tuberculosis meningitis.
A number of different biochemical and serological tests have been described recently for the early and accurate diagnosis of tuberculous meningitis. None of these tests has yet gained widespread acceptance in clinical medicine or in microbiology laboratories. To investigate this problem we evaluated adenosine deaminase activity (ADA), an enzyme linked immunosorbent assay (ELISA) that detects antibody to antigen 5 of Mycobacterium tuberculosis, and the radioactive bromide partition test (BPT) in the cerebrospinal fluid (CSF). Cerebrospinal fluid specimens from children with tuberculous, pyogenic, and viral meningitis as well as from patients with pulmonary tuberculosis without meningitis and from controls with normal CSFs were included inn the study. In addition, we estimated ADAs in serum samples from selected children in these groups. The sensitivity and specificity of the three tests evaluated in the CSF were: ADA assay 73% and 71%; BPT 92% and 92%; and ELISA for antibody to antigen 5, 53% and 90%, 40% and 94%, and 27% and 100%, respectively, at tires of more than or equal to 1:20, 1:40, and 1:80. The serum ADA was lower (11.0 +/- 6.15 IU/l) in children with tuberculous meningitis when compared with those with pulmonary tuberculosis alone (25.8 +/- 20.9 IU/l). The BPT was found to be the most reliable test in the early differentiation of tuberculous from other causes of meningitis and remained abnormal for a period of up to five months after the beginning of treatment. Accordingly, we believe that the BPT should be used in conjunction with bacterial and fungal antigen detection systems for the initial differentiation of clinically suspicious tuberculous meningitis from Gram or culture negative cases, or both, of bacterial and fungal meningitis.


Effects of amino acid changes in the extracellular domain of the human immunodeficiency virus type 1 gp41 envelope glycoprotein.
Changes were introduced into conserved amino acids within the ectodomain of the human immunodeficiency virus type 1 (HIV-1) gp41 transmembrane envelope glycoprotein. The effect of these changes on the structure and function of the HIV-1 envelope glycoproteins was examined. The gp41 glycoprotein contains an amino-terminal fusion peptide (residues 512 to 527) and a disulfide loop near the middle of the extracellular domain (residues 598 to 604). Mutations affecting the hydrophobic sequences between these two regions resulted in two phenotypes. Some changes in amino acids 528 to 562 resulted in a loss of the noncovalent association between gp41 and the gp120 exterior glycoprotein. Amino acid changes in other parts of the gp41 glycoprotein (residues 608 and 628) also resulted in subunit dissociation. Some changes affecting amino acids 568 to 596 resulted in envelope glycoproteins partially or completely defective in mediating membrane fusion. Syncytium formation was more sensitive than virus entry to these changes. Changes in several amino acids from 647 to 675 resulted in higher-than-wild-type syncytium-forming ability. One of these amino acid changes affecting tryptophan 666 resulted in escape from neutralization by an anti-gp41 human monoclonal antibody, 2F5. These results contribute to an understanding of the functional regions of the HIV-1 gp41 ectodomain.


Objective assessment of the compensatory effect of clinical hind limb lameness in horses: 37 cases (2011-2014).
OBJECTIVE To characterize and describe the compensatory load redistribution that results from unilateral hind limb lameness in horses. DESIGN Retrospective case series. ANIMALS 37 client-owned horses. PROCEDURES Medical records were reviewed to identify horses with unilateral hind limb lameness that responded positively (by objective assessment) to diagnostic local anesthesia during lameness evaluation and that were evaluated before and after diagnostic local anesthesia with an inertial sensor-based lameness diagnosis system. Horses were grouped as having hind limb lameness only, hind limb and ipsilateral forelimb lameness, or hind limb and contralateral forelimb lameness. Measures of head and pelvic movement asymmetry before (baseline) and after diagnostic local anesthesia were compared. The effect of group on baseline pelvic movement asymmetry variables was analyzed statistically. RESULTS Maximum pelvic height significantly decreased from the baseline value after diagnostic local anesthesia in each of the 3 lameness groups and in all horses combined. Minimum pelvic height significantly decreased after the procedure in all groups except the hind limb and contralateral forelimb lameness group. Head movement asymmetry was significantly decreased after diagnostic local anesthesia for horses with hind limb and ipsilateral forelimb lameness and for all horses combined, but not for those with hind limb lameness only or those with hind limb and contralateral forelimb lameness. CONCLUSIONS AND CLINICAL RELEVANCE Results supported that hind limb lameness can cause compensatory load redistribution evidenced as ipsilateral forelimb lameness. In this population of horses, contralateral forelimb lameness was not compensatory and likely reflected true lameness. Further studies are needed to investigate the source of the contralateral forelimb lameness in such horses.


Activated human astrocyte-derived extracellular vesicles modulate neuronal uptake, differentiation and firing.
Astrocytes in the central nervous system (CNS) provide supportive neural functions and mediate inflammatory responses from microglia. Increasing evidence supports their critical roles in regulating brain homoeostasis in response to pro-inflammatory factors such as cytokines and pathogen/damage-associated molecular pattern molecules in infectious and neurodegenerative diseases. However, the underlying mechanisms of the trans-cellular communication are still unclear. Extracellular vesicles (EVs) can transfer a large diversity of molecules such as lipids, nucleic acids and proteins for cellular communications. The purpose of this study is to characterize the EVs cargo proteins derived from human primary astrocytes (ADEVs) under both physiological and pathophysiological conditions. ADEVs were isolated from human primary astrocytes after vehicle (CTL) or interleukin-1β (IL-1β) pre-treatment. Label-free quantitative proteomic profiling revealed a notable up-regulation of proteins including actin-associated molecules, integrins and major histocompatibility complex in IL-1β-ADEVs compared to CTL-ADEVs, which were involved in cellular metabolism and organization, cellular communication and inflammatory response. When fluorescently labelled ADEVs were added into primary cultured mouse cortical neurons, we found a significantly increased neuronal uptake of IL-1β-ADEVs compared to CTL-ADEVs. We further confirmed it is likely due to the enrichment of surface proteins in IL-1β-ADEVs, as IL-1β-ADEVs uptake by neurons was partially suppressed by a specific integrin inhibitor. Additionally, treatment of neurons with IL-1β-ADEVs also reduced neurite outgrowth, branching and neuronal firing. These findings provide insight for the molecular mechanism of the ADEVs' effects on neural uptake, neural differentiation and maturation, and its alteration in inflammatory conditions.


What do we really know about food storage, surplus, and feasting in preagricultural communities?
In studying the origins of agriculture it is critical that we envision food production as a long-term human process that centers on the control and management of cycles of plant reproduction, including the harvesting, storage, and planting of seed stock. Drawing upon a growing body of literature illustrating multiple trajectories and pathways to agriculture, I see domestication as developing through coevolution between human beings and the resources they exploited. A more detailed understanding of the process and pathways of the origins of agriculture requires us to disentangle a complex knot of different yet interrelated factors, including food storage, food surplus, and feasting. I argue that archaeologists have yet to develop a detailed understanding of the scale and economic contributions of food storage in preagriculturalist communities. Evidence from the Near East indicates that use of storage practices increased dramatically after domestication. Analysis indicates that while there was a level of food storage in predomesticate and agricultural context, it was small scale. Finally, I argue that in some cases, discussions of food storage and feasting been reduced to claims of universal importance rather than a contextualized and detailed exploration within a specific cultural, temporal, and geographical case study.


Functionalization of 3D scaffolds with protein-releasing biomaterials for intracellular delivery.
Appropriate combinations of mechanical and biological stimuli are required to promote proper colonization of substrate materials in regenerative medicine. In this context, 3D scaffolds formed by compatible and biodegradable materials are under continuous development in an attempt to mimic the extracellular environment of mammalian cells. We have here explored how novel 3D porous scaffolds constructed by polylactic acid, polycaprolactone or chitosan can be decorated with bacterial inclusion bodies, submicron protein particles formed by releasable functional proteins. A simple dipping-based decoration method tested here specifically favors the penetration of the functional particles deeper than 300μm from the materials' surface. The functionalized surfaces support the intracellular delivery of biologically active proteins to up to more than 80% of the colonizing cells, a process that is slightly influenced by the chemical nature of the scaffold. The combination of 3D soft scaffolds and protein-based sustained release systems (Bioscaffolds) offers promise in the fabrication of bio-inspired hybrid matrices for multifactorial control of cell proliferation in tissue engineering under complex architectonic setting-ups.


Environmental Enteric Dysfunction Is Associated With Altered Bile Acid Metabolism.
Environmental enteric dysfunction (EED), a clinically asymptomatic condition characterized by inflammation of the small bowel mucosa, villous atrophy, and increased gut permeability, is common among children in developing countries. Because of abnormal gut mucosa and altered gut microbiome, EED could potentially affect the metabolism and enterohepatic circulation of bile acids. In 313 children, aged 12 to 59 months, EED was assessed by the dual sugar absorption test. Serum bile acids were measured using stable-isotope liquid chromatography-tandem mass spectrometry. In the overall study population, serum cholic acid and chenodeoxycholic acid were lower, whereas glycocholic acid, taurodeoxycholic acid, glycodeoxycholic acid, glycolithocholic acid, and glycoursodeoxycholic acid were significantly higher at older ages. Independent of age, serum taurochenodeoxycholic acid, tauromuricholic acid, and glycoursodeoxycholic acid were significantly different between 244 children with EED and 69 children without EED. Total serum bile acids (median, interquartile range) were 4.51 (2.45, 7.51) and 5.10 (3.32, 9.01) μmol/L in children with and without EED, respectively (age-adjusted, P = 0.0009). The proportion of bile acids conjugated with taurine instead of glycine was higher in children with EED (P < 0.0001). EED is associated with altered bile acid metabolism in young children in rural Malawi. Further work is needed to determine the generalizability of these findings in other study populations.


Tobacco control approaches and inequity--how far have we come and where are we going?
Despite Australia's success in reducing smoking rates, substantial inequities persist--with high smoking prevalence among disadvantaged groups. This article uses Fair Foundations: The VicHealth framework for health equity to identify promising strategies for promoting equity within tobacco control policies and programmes. A rapid review of the Australian and international literature was conducted in March 2014 using Pubmed, ISI web of Science and Scopus, Cochrane library and Google Scholar. A search of the grey literature was conducted to identify promising policy interventions. Population health surveys suggest that tobacco-related inequities in Victoria are beginning to decline. Data from the Victorian Smoking Survey shows that the inequity gap is narrowing, and in recent years, the prevalence of regular smoking declined fastest among disadvantaged smokers. Future approaches to accelerate reductions in tobacco-related inequities include: (i) continue proven population-based tobacco control policies--especially increasing the price of tobacco (while remaining cognisant of the increased economic burden for those smokers who do not quit), and continuing mass media campaigns; (ii) strengthening social policies to promote equity in early child development; educational experiences; quality of local environments; employment and working conditions; (iii) identifying and investing in targeted approaches to influence social norms and more effectively identify and support disadvantaged smokers to quit; (iv) within tobacco control programmes, give greatest priority to interventions focused on adult smokers (including pregnant women and their partners).


Biological Role of Nutrients, Food and Dietary Patterns in the Prevention and Clinical Management of Major Depressive Disorder.
Major Depressive Disorder (MDD) is a growing disabling condition affecting around 280 million people worldwide. This complex entity is the result of the interplay between biological, psychological, and sociocultural factors, and compelling evidence suggests that MDD can be considered a disease that occurs as a consequence of an evolutionary mismatch and unhealthy lifestyle habits. In this context, diet is one of the core pillars of health, influencing multiple biological processes in the brain and the entire body. It seems that there is a bidirectional relationship between MDD and malnutrition, and depressed individuals often lack certain critical nutrients along with an aberrant dietary pattern. Thus, dietary interventions are one of the most promising tools to explore in the field of MDD, as there are a specific group of nutrients (i.e., omega 3, vitamins, polyphenols, and caffeine), foods (fish, nuts, seeds fruits, vegetables, coffee/tea, and fermented products) or dietary supplements (such as S-adenosylmethionine, acetyl carnitine, creatine, amino acids, etc.), which are being currently studied. Likewise, the entire nutritional context and the dietary pattern seem to be another potential area of study, and some strategies such as the Mediterranean diet have demonstrated some relevant benefits in patients with MDD; although, further efforts are still needed. In the present work, we will explore the state-of-the-art diet in the prevention and clinical support of MDD, focusing on the biological properties of its main nutrients, foods, and dietary patterns and their possible implications for these patients.


Location of Two Seams in the Proximity of the C2v ππ* Minimum Energy Path of Formaldehyde.
Photochemical reactions rationalization is a key aspect for the understanding and setup of novel experiment and novel photoinitiated pathways. In this respect, the relationship between minimum energy paths over an excited-state and the intersection to lower potential energy surfaces is fundamental. In order to help the understanding of this relationship, in this study we present a novel kind of constraint for geometry optimizations, namely, an "orthogonality" constraint. Its possible applications are described. A complete example on how to retrieve the direct relationship between a minimum energy path over an excited-state potential energy surface and a conical intersection seam is given for C2v symmetry constrained formaldehyde. The advantages of using the novel constraint when rationalizing a (photo)chemical reaction are presented.


The ecology of religious beliefs.
Although ecological forces are known to shape the expression of sociality across a broad range of biological taxa, their role in shaping human behavior is currently disputed. Both comparative and experimental evidence indicate that beliefs in moralizing high gods promote cooperation among humans, a behavioral attribute known to correlate with environmental harshness in nonhuman animals. Here we combine fine-grained bioclimatic data with the latest statistical tools from ecology and the social sciences to evaluate the potential effects of environmental forces, language history, and culture on the global distribution of belief in moralizing high gods (n = 583 societies). After simultaneously accounting for potential nonindependence among societies because of shared ancestry and cultural diffusion, we find that these beliefs are more prevalent among societies that inhabit poorer environments and are more prone to ecological duress. In addition, we find that these beliefs are more likely in politically complex societies that recognize rights to movable property. Overall, our multimodel inference approach predicts the global distribution of beliefs in moralizing high gods with an accuracy of 91%, and estimates the relative importance of different potential mechanisms by which this spatial pattern may have arisen. The emerging picture is neither one of pure cultural transmission nor of simple ecological determinism, but rather a complex mixture of social, cultural, and environmental influences. Our methods and findings provide a blueprint for how the increasing wealth of ecological, linguistic, and historical data can be leveraged to understand the forces that have shaped the behavior of our own species.


Management of Patients With Venous Leg Ulcers: Challenges and Current Best Practice.
Introduction It is well documented that the prevalence of venous leg ulcers (VLUs) is increasing, coinciding with an ageing population. Accurate global prevalence of VLUs is difficult to estimate due to the range of methodologies used in studies and accuracy of reporting. (1) Venous ulceration is the most common type of leg ulceration and a significant clinical problem, affecting approximately 1% of the population and 3% of people over 80 years of age (2) in westernised countries. Moreover, the global prevalence of VLUs is predicted to escalate dramatically, as people are living longer, often with multiple comorbidities. Recent figures on the prevalence of VLUs are based on a small number of studies, conducted in Western countries, and the evidence is weak. However, it is estimated that 93% of VLUs will heal in 12 months, and 7% remain unhealed after five years. (3) Furthermore, the recurrence rate within 3 months after wound closure is as high as 70%. (4) (-6) Thus, cost-effective adjunct evidence-based treatment strategies and services are needed to help prevent these ulcers, facilitate healing when they occur and prevent recurrence. The impact of a VLU represents social, personal, financial and psychological costs on the individual and further economic drain on the health-care system. This brings the challenge of providing a standardised leg ulcer service which delivers evidence-based treatment for the patient and their ulcer. It is recognised there are variations in practice and barriers preventing the implementation of best practice. There are patients not receiving appropriate and timely treatment in the initial development of VLUs, effective management of their VLU and preventing recurrence once the VLU has healed. Health-care professionals (HCPs) and organisations must have confidence in the development process of clinical practice guidelines and have ownership of these guidelines to ensure those of the highest quality guide their practice. These systematic judgments can assist in policy development, and decision making, improve communication, reduce errors and improve patient outcomes. There is an abundance of studies and guidelines that are available and regularly updated, however, there is still variation in the quality of the services offered to patients with a VLU. There are also variations in the evidence and some recommendations contradict each other, which can cause confusion and be a barrier to implementation. (7) The difference in health-care organisational structures, management support and the responsibility of VLU management can vary in different countries, often causing confusion and a barrier to seeking treatment. These factors further complicate the guideline implementation process, which is generally known to be a challenge with many diseases. (8).


Role of the spike glycoprotein of human Middle East respiratory syndrome coronavirus (MERS-CoV) in virus entry and syncytia formation.
Little is known about the biology of the emerging human group c betacoronavirus, Middle East Respiratory Syndrome coronavirus (MERS-CoV). Because coronavirus spike glycoproteins (S) mediate virus entry, affect viral host range, and elicit neutralizing antibodies, analyzing the functions of MERS-CoV S protein is a high research priority. MERS-CoV S on lentivirus pseudovirions mediated entry into a variety of cell types including embryo cells from New World Eptesicus fuscus bats. Surprisingly, a polyclonal antibody to the S protein of MHV, a group a murine betacoronavirus, cross-reacted in immunoblots with the S2 domain of group c MERS-CoV spike protein. MERS pseudovirions released from 293T cells contained only uncleaved S, and pseudovirus entry was blocked by lysosomotropic reagents NH4Cl and bafilomycin and inhibitors of cathepsin L. However, when MERS pseudovirions with uncleaved S protein were adsorbed at 4°C to Vero E6 cells, brief trypsin treatment at neutral pH triggered virus entry at the plasma membrane and syncytia formation. When 293T cells producing MERS pseudotypes co-expressed serine proteases TMPRSS-2 or -4, large syncytia formed at neutral pH, and the pseudovirions produced were non-infectious and deficient in S protein. These experiments show that if S protein on MERS pseudovirions is uncleaved, then viruses enter by endocytosis in a cathepsin L-dependent manner, but if MERS-CoV S is cleaved, either during virus maturation by serine proteases or on pseudovirions by trypsin in extracellular fluids, then viruses enter at the plasma membrane at neutral pH and cause massive syncytia formation even in cells that express little or no MERS-CoV receptor. Thus, whether MERS-CoV enters cells within endosomes or at the plasma membrane depends upon the host cell type and tissue, and is determined by the location of host proteases that cleave the viral spike glycoprotein and activate membrane fusion.


Prevalence and Predictors of Uncircumcised, Kenyan Men's Desire for Circumcision for Their Infant Sons.
Men are key decision makers for their son's circumcision, so understanding their beliefs is important for the uptake of early infant male circumcision in countries in sub-Saharan Africa that have high HIV prevalence. We analyzed men's preferences for circumcising their sons using data from a population-representative survey of 1501 uncircumcised men aged 25-49 years in western Kenya. Most men (59%) reported they would "definitely" want their son circumcised if a son was born to them within the next year. However, only 25% intended to become circumcised themselves. In multivariable Poisson regression models to estimate prevalence ratios, key predictors of the desire to circumcise their sons included knowledge that circumcision reduces HIV acquisition, having a supportive partner, discussing circumcision with the partner, altruism, and intention to be circumcised himself. Focusing on partner dynamics may have the greatest capacity to increase demand since 55% had not talked to their partner about circumcision.


Folliculocentric cutaneous presentation of disseminated Candida krusei infection in a patient with acute myeloid leukemia.
Candida krusei (C. krusei) is a multidrug-resistant opportunistic fungal pathogen that may cause disseminated infections in immunocompromised hosts. However, its clinical and histologic features are not well-characterized. We present a unique case to contribute to the growing knowledge base associated with this organism. During hospitalization for neutropenic fever, a 19-year-old man with acute myeloid leukemia, who underwent hematopoietic stem cell transplantation, developed a generalized folliculocentric eruption following initiation of antifungal therapy for newly diagnosed C. krusei fungemia. Despite adequate antifungal coverage and negative blood cultures, the follicular-based erythematous papules persisted. Biopsies demonstrated yeast within ruptured follicles, without angiotropism or involvement of the interfollicular dermis, subcutaneous tissue, or stratum corneum. Concurrent skin tissue cultures confirmed C. krusei. The patient remained febrile despite aggressive antifungal therapy, with relapse of leukemia and subsequent death. Our case is unusual given the development of cutaneous lesions following clearance of fungemia, with yeast limited to ruptured follicular lumina, possibly indicating a primary cutaneous source or early transfollicular/transepidermal elimination. Given the limited available descriptions of cutaneous histopathology for C. krusei, we seek to add to the understanding of its pathophysiology and aid in the diagnosis and treatment of this often fatal infection.


Osteopathic Manipulation in the Management of Chronic Pain: Current Perspectives.
Chronic pain is a common condition that often interferes with work or other activities. Guidelines support the use of non-pharmacological treatments, such as spinal manipulation, in patients with chronic pain. Osteopathic physicians in the United States are uniquely positioned to manage chronic pain because their professional philosophy embraces the biopsychosocial model and they are trained in the use of osteopathic manipulative treatment (OMT) to complement conventional medical care. This narrative review provides current perspectives on the osteopathic approach to chronic pain management, including evidence for the efficacy of OMT based on systematic searches of the biomedical literature and the ClinicalTrials.gov database. Men, persons with low levels of education, and non-White and Hispanic patients are significantly less likely to have received OMT during their lifetime. Patients with low back and neck pain are most likely to be treated with OMT, and osteopathic manipulative medicine specialty physicians and family medicine physicians most often use OMT. However, many osteopathic physicians report using OMT infrequently. Although OMT is considered safe, based on millions of patient encounters over more than a century, there is limited evidence on its efficacy in treating chronic pain. The lone exception involves chronic low back pain, wherein there is evidence from systematic reviews, a large clinical trial, and observational studies. There is lesser evidence to support cost effectiveness and patient satisfaction associated with OMT for chronic pain. The only clinical practice guideline established by the American Osteopathic Association recommends that OMT should be used to treat chronic low back pain in patients with somatic dysfunction. Given the philosophy of osteopathic medicine, universal training of osteopathic physicians to use OMT, and national guidelines supporting non-pharmacological treatments for chronic pain, it is unclear why OMT use is reported to be remarkably low in physician surveys.


Short report: isolation and partial characterization of a Puumala virus from a human case of nephropathia epidemica in France.
Puumala (PUU) virus (Bunyaviridae: Hantavirus), the etiologic agent of nephropathia epidemica (NE), the mid form of hemorrhagic fever with renal syndrome, is enzootic in Europe and has been known to occur in France since 1983. We report the first isolation of PUU virus in France and western Europe from a case of NE acquired in France. The virus was isolated from a serum collected in the acute phase of the clinical course by successive blind passages in Vero E6 cells. Serologic typing using monoclonal antibodies confirmed the identity of the virus as PUU. The sequence of an 832-nucleotide fragment of the virus medium RNA segment obtained by the polymerase chain reaction (PCR) also classified it as a PUU virus. The sequence of this isolate from a human case in France is closely related to the sequence of a PUU virus obtained by the PCR from a German patient.


Muscle mechanics: adaptations with exercise-training.
Based on the MHC isoform pattern, adult mammalian limb skeletal muscles contain two and, in some species, three types of fast fibers (Type IIa, IIx, and IIb), and one slow fiber (Type I). Slow muscles, such as the soleus, contain primarily the slow Type I fiber, whereas fast-twitch muscles are composed primarily of a mixture of the fast myosin isozymes. Force generation involves cross-bridge interaction and transition from a weakly bound, low-force state (AM-ADP-P(i)) to the strongly bound, high-force state (AM-ADP). This transition is thought to be rate limiting in terms of dP/dt, and the high-force state is the dominant cross-bridge form during a peak isometric contraction. Intact fast and slow skeletal muscles generate approximately the same amount of peak force (Po) of between 200 and 250 kN.m-2. However, the rate of transition from the low- to high-force state shows Ca2+ sensitivity and is 7-fold higher in fast-twitch, as compared to slow-twitch, skeletal muscle fibers. Fiber Vo or the maximal cross-bridge cycle rate is highly correlated with and thought to be dependent on the specific activity of the myosin or myofibrillar ATPase. The hierarchy for Vo is the Type IIb > IIx > IIa > I. This functional difference for the fast fiber types explains the higher Vo observed in the predominantly Type IIb SVL vs. the mixed fast Type IIa and IIb EDL muscle. A plot of Vo vs. species size demonstrates that an inverse relationship exists between Vo and body mass. From the standpoint of work capacity, the important property is power output. An analysis of individual muscles indicates that peak power is obtained at loads considerably below 50% of Po. Individuals with a high percentage of fast-twitch fibers generate a greater torque and higher power at a given velocity than those with predominantly slow-twitch fibers. In humans, mean peak power occurred in a ratio of 10:5:1 for the Type IIb, IIa, and I fibers. The in vivo measurement of the torque-velocity relationship and Vmax in human muscle is difficult because of limitations inherent in the equipment used and the inability to study the large limb muscles independently. Nevertheless, the in vivo torque-velocity relationships are similar to those measured in vitro in animals. This observation suggests that little central nervous system inhibition exists and that healthy subjects are able to achieve maximal activation of their muscles. Although peak isometric tension is not dependent on fiber type distribution, a positive correlation exists between the percentage of fast fibers and peak torque output at moderate-to-high angular isokinetic velocities. Consequently, peak power output is substantially greater in subjects possessing a predominance of fast fibers. The mechanical properties of slow and fast muscles do adapt to programs of regular exercise. Endurance exercise training has been shown to increase the Vo of the slow soleus by 20%. This increase could have been caused by either a small increase in all, or most, of the fibers, or to a conversion of a few fibers from slow to fast. Recently, the increase was shown to be caused by the former, as the individual slow Type I fibers of the soleus showed a 20% increase in Vo, but there was little or no change in the percentage of fast fibers. The increased Vo was correlated with, and likely caused by, an increased fiber ATPase. We hypothesize that the increased ATPase and cross-bridge cycling speed might be attributable to an increased expression of fast MLCs in the slow Type I fibers (Fig. 14.10). This hypothesis is based on the fact that light chains have been shown to be involved in the power stroke, and removal of light chains depresses force and velocity. Regular endurance exercise training had no effect on fiber size, but with prolonged durations of daily training it depressed Po and peak power. When the training is maintained over prolonged periods, it may even induce atrophy of the slow Type I and fast Type IIa fibers. (ABSTRACT TRUNCATED)


SFCHECK: a unified set of procedures for evaluating the quality of macromolecular structure-factor data and their agreement with the atomic model.
In this paper we present SFCHECK, a stand-alone software package that features a unified set of procedures for evaluating the structure-factor data obtained from X-ray diffraction experiments and for assessing the agreement of the atomic coordinates with these data. The evaluation is performed completely automatically, and produces a concise PostScript pictorial output similar to that of PROCHECK [Laskowski, MacArthur, Moss & Thornton (1993). J. Appl. Cryst. 26, 283-291], greatly facilitating visual inspection of the results. The required inputs are the structure-factor amplitudes and the atomic coordinates. Having those, the program summarizes relevant information on the deposited structure factors and evaluates their quality using criteria such as data completeness, structure-factor uncertainty and the optical resolution computed from the Patterson origin peak. The dependence of various parameters on the nominal resolution (d spacing) is also given. To evaluate the global agreement of the atomic model with the experimental data, the program recomputes the R factor, the correlation coefficient between observed and calculated structure-factor amplitudes and Rfree (when appropriate). In addition, it gives several estimates of the average error in the atomic coordinates. The local agreement between the model and the electron-density map is evaluated on a per-residue basis, considering separately the macromolecule backbone and side-chain atoms, as well as solvent atoms and heterogroups. Among the criteria are the normalized average atomic displacement, the local density correlation coefficient and the polymer chain connectivity. The possibility of computing these criteria using the omit-map procedure is also provided. The described software should be a valuable tool in monitoring the refinement procedure and in assessing structures deposited in databases.


In vitro evaluation of the efficacy of a veterinary dry heat fluid warmer.
To evaluate the efficacy of a veterinary dry heat fluid warmer on ambient and prewarmed crystalloid fluids and refrigerated packed red blood cells (pRBC). Prospective in vitro study. University teaching hospital. None. Ambient and prewarmed crystalloid fluids and refrigerated pRBC were delivered via a standard fluid administration set at various rates. A thermistor continuously monitored fluid outflow temperature with and without a dry heat veterinary fluid warmer (study device). The outflow temperature was significantly higher with the study device as compared to control conditions for all fluids and rates tested. The maximum outflow temperature of approximately 35°C (95°F) occurred when the study device was applied to either ambient or prewarmed crystalloid fluids at 50 mL/h. In the study device trials, the outflow temperature of ambient crystalloid fluids ranged from 35.1° to 27.3°C (95.2° to 81.1°F) as the fluid rate increased from 50 to 999 mL/h. Control trials of prewarmed crystalloids produced outflow temperatures that rapidly approached ambient temperature. Addition of the study device to prewarmed crystalloids resulted in outflow temperatures that were similar to that of the corresponding ambient crystalloid trials. Control trials of refrigerated pRBC achieved ambient temperature at rates from 10 to 500 mL/h. With the study device, pRBC were maximally warmed to an outflow temperature of 35.8°C (96.4°F) at 100 mL/h. Although the study device generated statistically significant increases in outflow temperature of crystalloid fluids and pRBC, the ability of the device to decrease the metabolic cost of fluid administration is limited to specific clinical scenarios. The use of prewarmed crystalloid fluids with or without the study device offers minimal benefit over ambient temperature crystalloids. Substantial warming of pRBC occurs during administration, even without use of the study device.


Fontan-Associated Liver Disease: Proceedings from the American College of Cardiology Stakeholders Meeting, October 1 to 2, 2015, Washington DC.
Over the past decade, as the majority of patients with single ventricle anatomy who have undergone the Fontan operation reach adulthood, a newly recognized disease process, Fontan-associated liver disease (FALD), has emerged. FALD is an extracardiac complication that may lead to substantial comorbid disease and premature mortality. The risk factors, pathophysiology, longitudinal consequences, and therapeutic options related to FALD remain poorly defined. Although we recognize that Fontan circulatory properties are associated with extracardiac organ dysfunction, numerous gaps in our understanding of the nature of this relationship exist. Such extracardiac manifestations, in addition to other late complications of the circulation, can significantly affect quality of life and healthcare use. Therefore, to initiate a formal evaluation of FALD, the American College of Cardiology (ACC) sponsored a stakeholders meeting on October 1 to 2, 2015, in Washington, DC. The goal of the meeting was to bring together subspecialty experts in the fields of adult and pediatric hepatology, congenital cardiology (adult congenital and pediatric cardiology), heart failure/transplant, epidemiology, and cardiothoracic surgery, as well as patient advocates, patients, parents of children and young adults who have had the Fontan procedure, and research organizations and societies to discuss the current state of FALD. Topics included gaps in knowledge, optimal care, research opportunities and barriers, and sound practices to guide providers, patients, and families. This report summarizes findings from the stakeholders meeting and seeks to establish a platform for understanding and addressing FALD.


Cell-Specific Loss of SNAP25 from Cortical Projection Neurons Allows Normal Development but Causes Subsequent Neurodegeneration.
Synaptosomal associated protein 25 kDa (SNAP25) is an essential component of the SNARE complex regulating synaptic vesicle fusion. SNAP25 deficiency has been implicated in a variety of cognitive disorders. We ablated SNAP25 from selected neuronal populations by generating a transgenic mouse (B6-Snap25tm3mcw (Snap25-flox)) with LoxP sites flanking exon5a/5b. In the presence of Cre-recombinase, Snap25-flox is recombined to a truncated transcript. Evoked synaptic vesicle release is severely reduced in Snap25 conditional knockout (cKO) neurons as shown by live cell imaging of synaptic vesicle fusion and whole cell patch clamp recordings in cultured hippocampal neurons. We studied Snap25 cKO in subsets of cortical projection neurons in vivo (L5-Rbp4-Cre; L6-Ntsr1-Cre; L6b-Drd1a-Cre). cKO neurons develop normal axonal projections, but axons are not maintained appropriately, showing signs of swelling, fragmentation and eventually complete absence. Onset and progression of degeneration are dependent on the neuron type, with L5 cells showing the earliest and most severe axonal loss. Ultrastructural examination revealed that cKO neurites contain autophagosome/lysosome-like structures. Markers of inflammation such as Iba1 and lipofuscin are increased only in adult cKO cortex. Snap25 cKO can provide a model to study genetic interactions with environmental influences in several disorders.


Roots Structure and Development of Austrobaileya scandens (Austrobaileyaceae) and Implications for Their Evolution in Angiosperms.
Abstract: Since the resolution of the ANA grade [Amborellales, Nymphaeales, Austrobaileyales] as sister to all other flowering plants, a few comparative studies of root structure have suggested that some of their anatomical traits could be of importance to understanding root evolutionary development and angiosperm phylogeny. However, there is still a paucity of information on root structure and apical meristems (RAMs) in these lineages and especially the sister to all other Austrobaileyales, Austrobaileya scandens. We used microtome sections and bright field, epifluorescence, laser confocal, and scanning electron microscopy to study adventitious root RAMs and tissues of A. scandens. Our results indicate that root structure is relatively simple in A. scandens. The epidermis has a thick cuticle and lacks root hairs. The stele is typically diarch, or some modification thereof, and surrounded by a cortex differentiated into a uniseriate endodermis, a middle region sometimes packed with starch, some oil cells, and colonized by arbuscular mycorrhizal fungi, and a multiseriate exodermis. Secondary growth produced many vessel elements in the secondary xylem and scattered sclerenchymatous fibers in secondary phloem. The absence of distinct patterning within the RAM and between the RAM and derivative differentiating tissues shows that the RAM is open and characterized by common initials. Roots structure and anatomy of A. scandens are thus essentially similar to some previously described in Amborella or Illicium in the ANA grade and many magnoliids, and suggest that the first woody flowering plants likely had an open RAM with common initials. Their functional and evolutionary significance in woody early-diverging and basal lineages of flowering plants and gymnosperms remains unclear, but they are clearly ancestral traits.


Fluorescent multicolor multiplex homogeneous assay for the simultaneous analysis of the two most common hemochromatosis mutations.
We report the development of a qualitative fluorescent multiplex homogeneous assay designed for the detection of the two most common hemochromatosis mutations using dual-labeled fluorescent probes. The assay is able to detect four allelic variants in a single closed tube using a single thermocycling protocol. The procedure combines the great sensitivity of the polymerase chain reaction, the specificity provided by allele-specific oligonucleotide hybridization using the 5(') nuclease assay format, and the higher throughput of a multicolor fluorescence detection procedure. Genomic DNA was prepared from whole blood specimens using standard procedures. Following DNA sample preparation, two regions of the hemochromatosis gene (HFE) including the H63D and C282Y mutations were coamplified and detected in real-time by four different fluorescently labeled allele-specific oligonucleotide probes. Assay specificity was demonstrated by a blind methods comparison study that included 37 DNA samples from individuals with a known HFE genotype. Results from the study showed that the multicolor multiplex HFE assay unambiguously classified all possible genotypes for the HFE gene C282Y and H63D mutations(1). This technique will be useful for research and molecular diagnostic laboratories and can be easily adapted for the detection of other single nucleotide polymorphisms.


Clinical guidelines for the diagnosis and treatment of lower urinary tract dysfunction in patients with spinal cord injury.
The present article is an abridged English translation of the Japanese clinical guidelines for the diagnosis and treatment of lower urinary tract dysfunction in patients with spinal cord injury updated as of July 2019. The patients are adult spinal cord injured patients with lower urinary tract dysfunction; special consideration of pediatric and elderly populations is presented separately. The target audience is healthcare providers who are engaged in the medical care of patients with spinal cord injury. The mandatory assessment includes medical history, physical examination, frequency-volume chart, urinalysis, blood chemistry, transabdominal ultrasonography, measurement of post-void residual urine, uroflowmetry and video-urodynamic study. Optional assessments include questionnaires on the quality of life, renal scintigraphy and cystourethroscopy. The presence or absence of risk factors for renal damage and symptomatic urinary tract infection affects urinary management, as well as pharmacological treatments. Further treatment is recommended if the maximum conservative treatment fails to improve or prevent renal damage and symptomatic urinary tract infection. In addition, management of urinary incontinence should be considered individually in patients with risk factors for urinary incontinence and decreased quality of life.


Surgical myocardial revascularization: off-pump use of bilateral internal thoracic artery grafting.
Advances in surgical myocardial revascularization (MR) have introduced additional benefits with the off-pump (OP) technique and the use of bilateral internal thoracic artery (ITA) grafting. Off-pump surgical MR has been associated with improved immediate outcomes and reduced incidence of perioperative complications, and the use of bilateral ITA grafts provides increased survival and decreased cardiovascular events in the long term. To present the initial experience with the combined use of these advances in surgical MR. A total of 35 consecutive patients undergoing off-pump MR with bilateral ITA grafting were studied; the left ITA was directed toward the anterior descending artery and the right ITA was anastomosed to the circumflex artery branches. The predominant comorbidities were previous myocardial infarction in 71.4% of the patients, diabetes mellitus in 34.2%, and renal failure in 14.2%. RESULTS No patients presented electrocardiographic changes or enzyme elevation in the postoperative period. The number of bypasses per patient ranged from two to four (median of three bypasses/patient). Postoperative hospital stay ranged from three to 12 days (mean of 4.7 + 1.7 days). No cases of sternal dehiscence or infection were observed, but one patient suffered a stroke on the fourth postoperative day and died. Late follow-up lasted from four to 48 months. No late deaths occurred, 31 patients are asymptomatic, and three have residual angina. The combination of these technical advances in surgical myocardial revascularization proved efficient and able to contribute to improved benefits in the long term.


Characterizing time series via complexity-entropy curves.
The search for patterns in time series is a very common task when dealing with complex systems. This is usually accomplished by employing a complexity measure such as entropies and fractal dimensions. However, such measures usually only capture a single aspect of the system dynamics. Here, we propose a family of complexity measures for time series based on a generalization of the complexity-entropy causality plane. By replacing the Shannon entropy by a monoparametric entropy (Tsallis q entropy) and after considering the proper generalization of the statistical complexity (q complexity), we build up a parametric curve (the q-complexity-entropy curve) that is used for characterizing and classifying time series. Based on simple exact results and numerical simulations of stochastic processes, we show that these curves can distinguish among different long-range, short-range, and oscillating correlated behaviors. Also, we verify that simulated chaotic and stochastic time series can be distinguished based on whether these curves are open or closed. We further test this technique in experimental scenarios related to chaotic laser intensity, stock price, sunspot, and geomagnetic dynamics, confirming its usefulness. Finally, we prove that these curves enhance the automatic classification of time series with long-range correlations and interbeat intervals of healthy subjects and patients with heart disease.


UTX regulates mesoderm differentiation of embryonic stem cells independent of H3K27 demethylase activity.
To investigate the role of histone H3K27 demethylase UTX in embryonic stem (ES) cell differentiation, we have generated UTX knockout (KO) and enzyme-dead knock-in male ES cells. Deletion of the X-chromosome-encoded UTX gene in male ES cells markedly decreases expression of the paralogous UTY gene encoded by Y chromosome, but has no effect on global H3K27me3 level, Hox gene expression, or ES cell self-renewal. However, UTX KO cells show severe defects in mesoderm differentiation and induction of Brachyury, a transcription factor essential for mesoderm development. Surprisingly, UTX regulates mesoderm differentiation and Brachyury expression independent of its enzymatic activity. UTY, which lacks detectable demethylase activity, compensates for the loss of UTX in regulating Brachyury expression. UTX and UTY bind directly to Brachyury promoter and are required for Wnt/β-catenin signaling-induced Brachyury expression in ES cells. Interestingly, male UTX KO embryos express normal levels of UTY and survive until birth. In contrast, female UTX KO mice, which lack the UTY gene, show embryonic lethality before embryonic day 11.5. Female UTX KO embryos show severe defects in both Brachyury expression and embryonic development of mesoderm-derived posterior notochord, cardiac, and hematopoietic tissues. These results indicate that UTX controls mesoderm differentiation and Brachyury expression independent of H3K27 demethylase activity, and suggest that UTX and UTY are functionally redundant in ES cell differentiation and early embryonic development.


Schedule-selective biochemical modulation of 5-fluorouracil: a phase II study in advanced colorectal cancer.
Based on experimental findings suggesting that 5-fluorouracil (FUra) may have different mechanisms of action depending on the schedule of administration, we generated the hypothesis that biochemical modulation of this fluoropyrimidine should be schedule specific. We thus tested the activity of a hybrid regimen consisting of two biweekly cycles of FUra bolus (600 mg/m2) modulated by pretreatment (24-h interval) with methotrexate (200 mg/m2), alternating with a 3-week continuous infusion of FUra (200 mg/m2/day) modulated by low-dose (6S)leucovorin (20 mg/m2 bolus weekly). Thirty-three consecutive patients with advanced measurable colorectal cancer and no prior therapy for metastatic disease entered the study from February 1992 to August 1993. They were treated with two biweekly cycles of FUra bolus (600 mg/m2) preceded by (24-h interval) methotrexate (200 mg/m2), alternating with a 3-week continuous infusion of FUra (200 mg/m2/day) modulated by low-dose (6S)leucovorin (20 mg/m2 bolus weekly). The median Eastern Cooperative Oncology Group performance status was 1; the liver was the only metastatic site in 17 patients. Treatment outcome was evaluated by computed tomographic scan in all patients, except for two. Three complete and 13 partial responses were obtained among these 33 patients (response rate, 48%; 95% confidence limits, 31-66%). Performance status (Eastern Cooperative Oncology Group) influenced clinical response. The combined complete response and partial response rate was 69%, 33%, and 25% in patients with an Eastern Cooperative Oncology Group performance status of 0, 1, and 2, respectively (chi2, 4.6, P = 0.032, two-tailed Mantel test for trend). After a median follow-up time of 26 months, 10 patients are still alive. The median progression-free survival and overall survival were 9.5 and 20.2 months, respectively. No toxic deaths or grade 4 toxicity occurred. The incidence of grade 3 toxicity per patient in any cycle was: mucositis 6%, diarrhea 3%, and vomiting 3% for the bolus part and 21%, 3%, and 6%, respectively, for the continuous infusion part of the regimen. Hand-foot syndrome occurred in 27% of the patients treated with the continuous infusion regimen.


Implementing healthy lifestyle promotion in primary care: a quasi-experimental cross-sectional study evaluating a team initiative.
Non-communicable diseases are a leading cause of death and can largely be prevented by healthy lifestyles. Health care organizations are encouraged to integrate healthy lifestyle promotion in routine care. This study evaluates the impact of a team initiative on healthy lifestyle promotion in primary care. A quasi-experimental, cross-sectional design compared three intervention centres that had implemented lifestyle teams with three control centres that used a traditional model of care. Outcomes were defined using the RE-AIM framework: reach, the proportion of patients receiving lifestyle promotion; effectiveness, self-reported attitudes and competency among staff; adoption, proportion of staff reporting regular practice of lifestyle promotion; implementation, fidelity to the original lifestyle team protocol. Data collection methods included a patient questionnaire (n = 888), a staff questionnaire (n = 120) and structured interviews with all practice managers and, where applicable, team managers (n = 8). The chi square test and problem-driven content analysis was used to analyse the questionnaire and interview data, respectively. Reach: patients at control centres (48%, n = 211) received lifestyle promotion significantly more often compared with patients at intervention centres (41%, n = 169). Effectiveness: intervention staff was significantly more positive towards the effectiveness of lifestyle promotion, shared competency and how lifestyle promotion was prioritized at their centre. Adoption: 47% of staff at intervention centres and 58% at control centres reported that they asked patients about their lifestyle on a daily basis. all intervention centres had implemented multi-professional teams and team managers and held regular meetings but struggled to implement in-house referral structures for lifestyle promotion, which was used consistently among staff. Intervention centres did not show higher rates than control centres on reach of patients or adoption among staff at this stage. All intervention centres struggled to implement working referral structures for lifestyle promotion. Intervention centres were more positive on effectiveness outcomes, attitudes and competency among staff, however. Thus, lifestyle teams may facilitate lifestyle promotion practice in terms of increased responsiveness among staff, illustrated by positive attitudes and perceptions of shared competency. More research is needed on lifestyle promotion referral structures in primary care regarding their configuration and implementation.


Determinants within the C-terminus of the human norepinephrine transporter dictate transporter trafficking, stability, and activity.
The function of the human norepinephrine transporter (hNET) depends on its presence at the cell surface. A role for the hNET C-terminus in trafficking the transporter to the surface has been suggested by the report of a bovine NET C-terminal splice variant that accumulates within heterologous host cells, and a human variant homolog has also been reported. We examined the relevance of the C-terminus of hNET to trafficking and function using transfected LLC-PK1 cells. The intracellular and surface expression of NET proteins was evaluated by Western blots, and their functional capacities were assessed using transport assays. We found that the C-terminal residues encoded by hNET 1a enable the efficient maturation and surface expression of hNET and therefore critically impact transporter activity. Alternative splicing causes the retention of immature hNETs within the cell, whereas introduced C-terminal deletions result in significant degradation. The loss of the terminal isoleucine alone (Delta617-hNET) is sufficient to cause the degradation of hNET, an effect that can be mimicked by nonconservative point mutations at the terminal position. The phenotype of Delta617-hNET is recapitulated in neuronal SK-N-MC cells, but is significantly less severe in HEK-293 cells, suggesting a role for host cell factors in enabling the biosynthetic progression of wild-type hNET. Additional proximal residues may act at other steps to affect the expression of the fully mature protein on the cell surface (Q608A) and to more directly affect transporter activity (F609A). Together our studies document a critical contribution of the hNET C-terminus to transporter trafficking, stability, and function.


A definition and ethical evaluation of overdiagnosis.
Overdiagnosis is an emerging problem in health policy and practice: we address its definition and ethical implications. We argue that the definition of overdiagnosis should be expressed at the level of populations. Consider a condition prevalent in a population, customarily labelled with diagnosis A. We propose that overdiagnosis is occurring in respect of that condition in that population when (1) the condition is being identified and labelled with diagnosis A in that population (consequent interventions may also be offered); (2) this identification and labelling would be accepted as correct in a relevant professional community; but (3) the resulting label and/or intervention carries an unfavourable balance between benefits and harms. We identify challenges in determining and weighting relevant harms, then propose three central ethical considerations in overdiagnosis: the extent of harm done, whether harm is avoidable and whether the primary goal of the actor/s concerned is to benefit themselves or the patient, citizen or society. This distinguishes predatory (avoidable, self-benefiting), misdirected (avoidable, other-benefiting) and tragic (unavoidable, other-benefiting) overdiagnosis; the degree of harm moderates the justifiability of each type. We end with four normative challenges: (1) methods for adjudicating between professional standards and identifying relevant harms and benefits should be procedurally just; (2) individuals, organisations and states are differently responsible for addressing overdiagnosis; (3) overdiagnosis is a matter for distributive justice: the burdens of both overdiagnosis and its prevention could fall on the least-well-off; and (4) communicating about overdiagnosis risks harming those unaware that they may have been overdiagnosed. These challenges will need to be addressed as the field develops.


Oral absorption of the somatostatin analogue SMS 201-995: theoretical and practical implications.
Oral administration of SMS 201-995 (SMS), a subcutaneously injectable somatostatin analogue, was investigated in five healthy volunteers, who drank 2 mg of SMS with 75 g of glucose. Mean maximal plasma SMS concentrations after the 2 mg oral dose were comparable with those after subcutaneous injection of 50 micrograms, although the peak was delayed (90 vs 15 min). Biological activity of absorbed SMS was shown by significant and lasting suppression of plasma insulin concentrations, resulting in significant hyperglycaemia at 90 and 120 min compared with the control study. The feasibility of oral administration of SMS may extend its use in the treatment of acromegaly and gut endocrine tumours. Other peptide hormone analogues, structurally 'protected' against enzymatic degradation, may also be active orally and thus be useful therapeutically.


Low-volume muscle endurance training prevents decrease in muscle oxidative and endurance function during 21-day forearm immobilization.
To examine the effects of low-volume muscle endurance training on muscle oxidative capacity, endurance and strength of the forearm muscle during 21-day forearm immobilization (IMM-21d). The non-dominant arm (n = 15) was immobilized for 21 days with a cast and assigned to an immobilization-only group (Imm-group; n = 7) or an immobilization with training group (Imm+Tr-group; n = 8). Training comprised dynamic handgrip exercise at 30% of pre-intervention maximal voluntary contraction (MVC) at 1 Hz until exhaustion, twice a week during the immobilization period. The duration of each exercise session was 51.7 +/- 3.4 s (mean +/- SE). Muscle oxidative capacity was evaluated by the time constant for phosphocreatine recovery (tau(off)PCr) after a submaximal handgrip exercise using (31)phosphorus-magnetic resonance spectroscopy. An endurance test was performed at 30% of pre-intervention MVC, at 1 Hz, until exhaustion. tau(off)PCr was significantly prolonged in the Imm-group after 21 days (42.0 +/- 2.8 and 64.2 +/- 5.1 s, pre- and post-intervention respectively; P < 0.01) but did not change for the Imm+Tr-group (50.3 +/- 3.0 and 48.8 +/- 5.0 s, ns). Endurance decreased significantly for the Imm-group (55.1 +/- 5.1 and 44.7 +/- 4.6 s, P < 0.05) but did not change for the Imm+Tr-group (47.9 +/- 3.0 and 51.7 +/- 4.0 s, ns). MVC decreased similarly in both groups (P < 0.01). Twice-weekly muscle endurance training sessions, each lasting approx. 50 s, effectively prevented a decrease in muscle oxidative capacity and endurance; however, there was no effect on MVC decline with IMM-21d.


Hemodynamics and Metabolic Parameters in Normothermic Kidney Preservation Are Linked With Donor Factors, Perfusate Cells, and Cytokines.
Kidney transplantation is the best renal-replacement option for most patients with end-stage renal disease. Normothermic machine preservation (NMP) of the kidney has been studied extensively during the last two decades and implemented in clinical trials. Biomarker research led to success in identifying molecules with diagnostic, predictive and therapeutic properties in chronic kidney disease. However, perfusate biomarkers and potential predictive mechanisms in NMP have not been identified yet. Twelve discarded human kidneys (n = 7 DBD, n = 5 DCD) underwent NMP for up to 24 h. Eight were perfused applying urine recirculation (URC), four with replacement of urine (UR) using Ringer's lactate. The aim of our study was to investigate biomarkers (NGAL, KIM-1, and L-FABP), cells and cytokines in the perfusate in context with donor characteristics, perfusate hemodynamics and metabolic parameters. Cold ischemia time did not correlate with any of the markers. Perfusates of DBD kidneys had a significantly lower number of leukocytes after 6 h of NMP compared to DCD. Arterial flow, pH, NGAL and L-FABP correlated with donor creatinine and eGFR. Arterial flow was higher in kidneys with lower perfusate lactate. Perfusate TNF-α was higher in kidneys with lower arterial flow. The cytokines IL-1β and GM-CSF decreased during 6 h of NMP. Kidneys with more urine output had lower perfusate KIM-1 levels. Median and 6-h values of lactate, arterial flow, pH, NGAL, KIM-1, and L-FABP correlated with each other indicating a 6-h period being applicable for kidney viability assessment. The study results demonstrate a comparable cytokine and cell profile in perfusates with URC and UR. In conclusion, clinically available perfusate and hemodynamic parameters correlate well with donor characteristics and measured biomarkers in a discarded human NMP model.


RH5.1-CyRPA-Ripr antigen combination vaccine shows little improvement over RH5.1 in a preclinical setting.
RH5 is the leading vaccine candidate for the Plasmodium falciparum blood stage and has shown impact on parasite growth in the blood in a human clinical trial. RH5 binds to Ripr and CyRPA at the apical end of the invasive merozoite form, and this complex, designated RCR, is essential for entry into human erythrocytes. RH5 has advanced to human clinical trials, and the impact on parasite growth in the blood was encouraging but modest. This study assessed the potential of a protein-in-adjuvant blood stage malaria vaccine based on a combination of RH5, Ripr and CyRPA to provide improved neutralizing activity against P. falciparum in vitro. Mice were immunized with the individual RCR antigens to down select the best performing adjuvant formulation and rats were immunized with the individual RCR antigens to select the correct antigen dose. A second cohort of rats were immunized with single, double and triple antigen combinations to assess immunogenicity and parasite neutralizing activity in growth inhibition assays. The DPX® platform was identified as the best performing formulation in potentiating P. falciparum inhibitory antibody responses to these antigens. The three antigens derived from RH5, Ripr and CyRPA proteins formulated with DPX induced highly inhibitory parasite neutralising antibodies. Notably, RH5 either as a single antigen or in combination with Ripr and/or CyRPA, induced inhibitory antibodies that outperformed CyRPA, Ripr. An RCR combination vaccine may not induce substantially improved protective immunity as compared with RH5 as a single immunogen in a clinical setting and leaves the development pathway open for other antigens to be combined with RH5 as a next generation malaria vaccine.


Tackling the 'dyslexia paradox': reading brain and behavior for early markers of developmental dyslexia.
Developmental dyslexia is an unexplained inability to acquire accurate or fluent reading that affects approximately 5-17% of children. Dyslexia is associated with structural and functional alterations in various brain regions that support reading. Neuroimaging studies in infants and pre-reading children suggest that these alterations predate reading instruction and reading failure, supporting the hypothesis that variant function in dyslexia susceptibility genes lead to atypical neural migration and/or axonal growth during early, most likely in utero, brain development. Yet, dyslexia is typically not diagnosed until a child has failed to learn to read as expected (usually in second grade or later). There is emerging evidence that neuroimaging measures, when combined with key behavioral measures, can enhance the accuracy of identification of dyslexia risk in pre-reading children but its sensitivity, specificity, and cost-efficiency is still unclear. Early identification of dyslexia risk carries important implications for dyslexia remediation and the amelioration of the psychosocial consequences commonly associated with reading failure.


Palindromic GOLGA8 core duplicons promote chromosome 15q13.3 microdeletion and evolutionary instability.
Recurrent deletions of chromosome 15q13.3 associate with intellectual disability, schizophrenia, autism and epilepsy. To gain insight into the instability of this region, we sequenced it in affected individuals, normal individuals and nonhuman primates. We discovered five structural configurations of the human chromosome 15q13.3 region ranging in size from 2 to 3 Mb. These configurations arose recently (∼0.5-0.9 million years ago) as a result of human-specific expansions of segmental duplications and two independent inversion events. All inversion breakpoints map near GOLGA8 core duplicons-a ∼14-kb primate-specific chromosome 15 repeat that became organized into larger palindromic structures. GOLGA8-flanked palindromes also demarcate the breakpoints of recurrent 15q13.3 microdeletions, the expansion of chromosome 15 segmental duplications in the human lineage and independent structural changes in apes. The significant clustering (P = 0.002) of breakpoints provides mechanistic evidence for the role of this core duplicon and its palindromic architecture in promoting the evolutionary and disease-related instability of chromosome 15.


Caseworker judgments and substantiation.
Substantiation can have an important effect on what interventions are pursued for children investigated for maltreatment, but researchers lack knowledge about how the decision to substantiate is made. Using information from 4,515 children from a national probability study of children investigated for maltreatment, this study examined how caseworker judgments of harm, risk, and evidence predicted substantiation. The substantiation rate was 29.9%, but the majority of cases were substantiated when caseworkers reported at least moderate harm, at least moderate risk, and/or probably to clearly sufficient evidence. Each judgment variable significantly predicted substantiation in a multivariable model, with evidence the strongest predictor. Child gender and age were significant predictors beyond harm, risk, and evidence, suggesting that other judgments also influence substantiation. In 9 of 100 cases, reports were not substantiated despite moderate to severe harm. Thus, substantiation is generally based on judgments of harm, risk, and evidence but not exclusively. The findings underline previous researchers' conclusions that substantiation is a flawed measure of child maltreatment and suggest that policy and practice related to substantiation are due for a fresh appraisal by state child welfare service agencies.


Tobacco use among southwestern Alaska Native people.
We examined the characteristics, attitudes, beliefs, and exposure to tobacco products in a cohort of rural dwelling Alaska Native (AN) people. We conducted a study of 400 of AN adult tobacco users and nonusers living in Southwestern Alaska. Questionnaires covered variables such as demographics, tobacco-use history, current tobacco use and dependence scales, general health status, attitudes and beliefs about tobacco, and quitting history. The study population smoked 7.8 cigarettes per day compared with 16.8 on average for the U.S. population: a significant proportion of the population engaged in dual use of cigarettes and smokeless tobacco products. Over one third (40.9%), first tried tobacco at age 11 or younger. The mean measures of tobacco addiction (e.g., Fagerstrom Test for Nicotine Dependence, Severson Scale of Smokeless Tobacco Dependence) scores were lower compared with other U.S. populations. Very high tobacco-use prevalence, dual product use, and early tobacco use are observed in Southwestern AN people. Unexpectedly these did not appear to be correlated with heavier individual tobacco use or higher levels of addiction in this population.


Do financial incentives in a globally budgeted healthcare payment system produce changes in the way patients are categorized? A five-year study.
To assess the responses to financial incentives after a change in the payment system in a capitation-style healthcare payment system over a 5-year period. Cross-sectional and longitudinal examination of cost, utilization, and diagnostic data. Using Veterans Health Administration (VHA) administrative data on healthcare users between fiscal years 1998 and 2002, we calculated the proportion of new patients entering each of the payment classes, the illness burden of patients entering the payment classes, and the profitability index (a ratio of payment to costs) for each class suspected of gaming and each control class. Our main dependent variables of interest were the differences in the measures between each utilization-based class and each diagnosis-based class. We used 2 different analytic approaches to assess whether these differences increased or decreased over time. No clear evidence of gaming behavior was present in our results. A few comparisons were significant, but they did not show a consistent pattern of responses to incentives. For example, 6 of 16 comparisons of profitability index were significant, but (contrary to the hypothesis) 4 of these had a negative value for the time parameter, indicating decreasing profitability in the utilization-based classes versus the diagnosis-based classes. Although the payment system could be manipulated to increase payment to VHA networks, no such consistent gaming behavior was observed. More research is needed to better understand the effects of financial incentives in other healthcare payment systems.


Association between angiographic complications and clinical outcomes among patients with acute coronary syndrome undergoing percutaneous coronary intervention: an EARLY ACS (Early Glycoprotein IIb/IIIa Inhibition in Non-ST-Segment Elevation Acute Coronary Syndrome) angiographic substudy.
The goal of this analysis was to determine the association between intraprocedural complications and clinical outcomes among patients with high-risk non-ST-segment elevation acute coronary syndrome (NSTEACS) undergoing percutaneous coronary intervention (PCI). Among patients undergoing PCI for NSTEACS, the relationship between intraprocedural complications and clinical outcomes, independent of epicardial and myocardial perfusion, has not been well characterized. The EARLY ACS (Early Glycoprotein IIb/IIIa Inhibition in Non-ST-Segment Elevation Acute Coronary Syndrome) trial enrolled 9,406 patients with high-risk NSTEACS undergoing an early invasive strategy. Of these, 1,452 underwent angiographic assessment in an independent core laboratory and did not have a myocardial infarction (MI) between enrollment and angiography. We assessed the relationship between abrupt closure, loss of side branch(es), distal embolization, and no-reflow phenomenon and 30-day clinical outcomes in these patients. Of the patients, 166 (11.4%) experienced an intraprocedural complication. Baseline clinical characteristics were similar between patients who did and did not have complications. The 30-day composite of death or MI was significantly higher among patients with an intraprocedural complication (28.3% vs. 7.8%, odds ratio [OR]: 4.68, 95% confidence interval [CI]: 3.2 to 7.0, p < 0.001). Individually, both mortality (3.0% vs. 0.9%, OR: 3.60, 95% CI: 1.2 to 10.5, p = 0.019) and MI (27.1% vs. 7.4%, OR: 4.66, 95% CI: 3.1 to 7.0, p < 0.001) were significantly increased. After adjusting for differences in post-PCI epicardial and myocardial perfusion, the association with 30-day death or MI remained significant. Among high-risk NSTEACS patients undergoing an invasive strategy, the incidence of intraprocedural complications is high, and the occurrence of these complications is associated with worse clinical outcomes independent of epicardial and myocardial perfusion. (Early Glycoprotein IIb/IIIa Inhibition in Patients With Non-ST-segment Elevation Acute Coronary Syndrome [EARLY ACS]; NCT00089895).


Noonan syndrome and systemic lupus erythematosus: presentation in childhood.
Noonan syndrome is a relatively common disorder which is usually diagnosed in childhood because of the presence of distinctive facial features, short stature and cardiac anomalies. Previous clinical studies of Noonan syndrome have suggested that autoimmune disorders may be another possible manifestation and there have been two recent reports of Noonan syndrome patients with systemic lupus erythematosus. In this report we present an 11-year-old boy with Noonan syndrome and systemic lupus erythematosus, supporting the previous observations of an association between these two conditions.


How to Link SNOMED CT Procedure and WHO International Classification of Health Interventions (ICHI).
Among different used healthcare terminology resources there is a need to link them to ease their interoperability. SNOMED CT is the most detailed clinical reference terminology for procedure used in the Electronic Health Record (EHR). WHO developed since 2006 an aggregated classification named ICHI required for statistics and resource allocation. It is based on an ontology framework defined in ISO 1828 named Categorial Structure (CAST) for surgical procedures. We present ICHI coding structure, ISO 1828 CAST standard for surgical procedures and SNOMED CT procedures hierarchy concept model and their relations. We demonstrate the obstacles to align ICHI coding structure with ISO 1828 CAST which can be linked with SNOMED CT procedures hierarchy concept model. We recommend to update ICHI coding structure to decrease the gap with ISO 1828 CAST and SNOMED CT concept model for procedures hierarchy to allow users to share the different terminology resources.


The ZtvelB Gene Is Required for Vegetative Growth and Sporulation in the Wheat Pathogen Zymoseptoria tritici.
The ascomycete fungus Zymoseptoria tritici is the causal agent of Septoria Tritici Blotch (STB), a major disease of wheat across Europe. Current understanding of the genetic components and the environmental cues which influence development and pathogenicity of this fungus is limited. The velvet B gene, velB, has conserved roles in development, secondary metabolism, and pathogenicity across fungi. The function of this gene is best characterised in the model ascomycete fungus Aspergillus nidulans, where it is involved in co-ordinating the light response with downstream processes. There is limited knowledge of the role of light in Z. tritici, and of the molecular mechanisms underpinning the light response. We show that Z. tritici is able to detect light, and that the vegetative morphology of this fungus is influenced by light conditions. We also identify and characterise the Z. tritici velB gene, ZtvelB, by gene disruption. The ΔztvelB deletion mutants were fixed in a filamentous growth pattern and are unable to form yeast-like vegetative cells. Their morphology was similar under light and dark conditions, showing an impairment in light-responsive growth. In addition, the ΔztvelB mutants produced abnormal pycnidia that were impaired in macropycnidiospore production but could still produce viable infectious micropycnidiospores. Our results show that ZtvelB is required for yeast-like growth and asexual sporulation in Z. tritici, and we provide evidence for a role of ZtvelB in integrating light perception and developmental regulation in this important plant pathogenic fungus.


Lipid raft association and cholesterol sensitivity of P2X1-4 receptors for ATP: chimeras and point mutants identify intracellular amino-terminal residues involved in lipid regulation of P2X1 receptors.
Cholesterol-rich lipid rafts act as signaling microdomains and can regulate receptor function. We have shown in HEK293 cells recombinant P2X1-4 receptors (ATP-gated ion channels) are expressed in lipid rafts. Localization to flotillin-rich lipid rafts was reduced by the detergent Triton X-100. This sensitivity to Triton X-100 was concentration- and subunit-dependent, demonstrating differential association of P2X1-4 receptors with lipid rafts. The importance of raft association to ATP-evoked P2X receptor responses was determined in patch clamp studies. The cholesterol-depleting agents methyl-β-cyclodextrin or filipin disrupt lipid rafts and reduced P2X1 receptor currents by >90%. In contrast, ATP-evoked P2X2-4 receptor currents were unaffected by lipid raft disruption. To determine the molecular basis of cholesterol sensitivity, we generated chimeric receptors replacing portions of the cholesterol-sensitive P2X1 receptor with the corresponding region from the insensitive P2X2 receptor. These chimeras identified the importance of the intracellular amino-terminal region between the conserved protein kinase C site and the first transmembrane segment for the sensitivity to cholesterol depletion. Mutation of any of the variant residues between P2X1 and P2X2 receptors in this region in the P2X1 receptor (residues 20-23 and 27-29) to cysteine removed cholesterol sensitivity. Cholesterol depletion did not change the ATP sensitivity or cell surface expression of P2X1 receptors. This suggests that cholesterol is normally needed to facilitate the opening/gating of ATP-bound P2X1 receptor channels, and mutations in the pre-first transmembrane segment region remove this requirement.


Computer-Aided Diagnosis with Deep Learning Architecture: Applications to Breast Lesions in US Images and Pulmonary Nodules in CT Scans.
This paper performs a comprehensive study on the deep-learning-based computer-aided diagnosis (CADx) for the differential diagnosis of benign and malignant nodules/lesions by avoiding the potential errors caused by inaccurate image processing results (e.g., boundary segmentation), as well as the classification bias resulting from a less robust feature set, as involved in most conventional CADx algorithms. Specifically, the stacked denoising auto-encoder (SDAE) is exploited on the two CADx applications for the differentiation of breast ultrasound lesions and lung CT nodules. The SDAE architecture is well equipped with the automatic feature exploration mechanism and noise tolerance advantage, and hence may be suitable to deal with the intrinsically noisy property of medical image data from various imaging modalities. To show the outperformance of SDAE-based CADx over the conventional scheme, two latest conventional CADx algorithms are implemented for comparison. 10 times of 10-fold cross-validations are conducted to illustrate the efficacy of the SDAE-based CADx algorithm. The experimental results show the significant performance boost by the SDAE-based CADx algorithm over the two conventional methods, suggesting that deep learning techniques can potentially change the design paradigm of the CADx systems without the need of explicit design and selection of problem-oriented features.


Magnetophoretic Transport Line System for Rapid On-Chip Attomole Protein Detection.
A lab-on-a-chip traveling wave magnetophoresis approach for sensitive and rapid protein detection is reported. In this method, a chip-based magnetic microarray comprising lines of micrometer-sized thin film magnetic elements was used to control the movement of magnetic beads (MBs). The MBs and the chip were functionalized, forming a sandwich-type assay. The MBs were transported across a detection area, and the presence of target molecules resulted in the immobilization of MBs within this area. Target quantification was accomplished by MB counting in the detection area using an optical microscope. In order to demonstrate the versatility of the microarray, biotinylated antiavidin was selected as the target protein. In this case, avidin-functionalized MBs and an avidin-functionalized detection area were used. With a total assay time of 1 to 1.5 h (depending on the labeling approach used), a limit of detection in the attomole range was achieved. Compared to on-chip surface plasmon resonance biodetection systems, our method has a larger dynamic range and is about a factor of 500 times more sensitive. Furthermore, our MB transportation system can operate in any chip-based biosensor platform, thereby significantly improving traditional biosensors.


Association of Breastfeeding and the Federal Poverty Level: National Survey of Family Growth, 2011-2013.
Breastfeeding is strongly endorsed in the Healthy People 2020 goals; however, there remain many disparities in breastfeeding prevalence. The purpose of this study was to examine the association between breastfeeding and the Federal Poverty Level in the United States. Data from 5,397 women in the National Survey of Family Growth 2011-2013 survey were included in this study. The data were analyzed for descriptive features and logistic regressions of the Federal Poverty Level on breastfeeding. There were 64.1% of women who reported breastfeeding. Over one-third (35.2%) of women reported having a household income of 0-99% of the Federal Poverty Level. There were 15.2% of women who reported an income of 400% and above the Federal Poverty Level. With statistical adjustment for maternal age, race/ethnicity, education, marital status, parity, preterm birth, birth weight, insurance, and dwelling, the Federal Poverty Level was not significantly associated with breastfeeding. In this recent survey of mothers, Federal Poverty Level was not shown to be a significant factor in breastfeeding.


Perinatal outcomes according to the mode of delivery in women with a triplet pregnancy in The Netherlands.
Objective: In women with a triplet pregnancy, there is debate on the preferred mode of delivery. We performed a nationwide cohort study to assess the impact of mode of delivery on perinatal outcome in women with a triplet pregnancy. Methods: Nationwide cohort study on women with a triplet pregnancy who delivered between 26 + 0 and 40 + 0 weeks of gestation in the years 1999-2008. We compared perinatal outcomes according to the intended mode of delivery and the actual mode of delivery. Outcome measures were perinatal mortality and neonatal morbidity. Perinatal outcomes were analyzed taking into account the dependency between the children of the same triplet pregnancy ("any mortality" and "any morbidity") and were also analyzed separately per child. Results: We identified 386 women with a triplet pregnancy in the study period. Mean gestational age at delivery was 33.1 weeks (SD 2.5 weeks; range 26.0-40.0 weeks). Perinatal mortality was 2.3% for women with a planned caesarean section and 2.4% in women with a planned vaginal delivery (aOR 0.37; 95% confidence interval (CI) 0.09-1.5) and neonatal morbidity was 26.0% versus 36.0%, (aOR 0.88; 95% CI 0.51-1.4) respectively. In the subgroup analyses according to gestational age and in the analysis of perinatal outcomes per child separately, there were also no large differences in perinatal outcomes. The same applied for perinatal outcomes according to the actual mode of delivery. Conclusion: In this large cohort study among women with a triplet pregnancy, caesarean delivery is not associated with reduced perinatal mortality and morbidity.


A comparative study on the effects of morphine on central, peripheral and ganglionic stores of catecholamines.
The turnover and metabolism of catecholamines after i.p. administration of 10 (low dose) or 40 (high dose) mg/kg of morphine sulfate were measured in the brain, lumbar spinal cord, heart, celiac and superior cervical ganglia (SCG) of demedullated rats with unilaterally decentralized SCG. Demedullation significantly increased norepinephrine concentration and turnover in both sham operated and decentralized SCG, but demedullation failed to change the turnover of dopamine (DA) in both ganglia. Low doses of morphine only increased DA metabolism in the caudate nucleus. High doses of morphine failed to change the turnover of norepinephrine stored in heart and sympathetic ganglia but produced variable changes in norepinephrine turnover in the spinal cord and brain. High doses of morphine significantly increased DA turnover and metabolism in the celiac ganglion, SCG, caudate nucleus and the spinal cord of demedullated rats. These effects of morphine were antagonized by naloxone. DA metabolism in decentralized SCG of demedullated and SCG of intact rats remained unchanged after high doses of morphine. The action of morphine on DA metabolism was facilitated by demedullation, suggesting that the adrenal medulla secretes a modulator that can counteract the effects of opiate ligands on ganglionic small intensely fluorescent cells. Atropine and hexamethonium failed to antagonize the increase in the metabolism of ganglionic DA produced by morphine; and the increase in ganglionic DA metabolism caused by oxotremorine was not antagonized by naloxone, thus confirming a disassociation between the action on ganglionic DA mediated by cholinergic and opiate receptors. Since decentralization of the SCG abolishes the increase in DA metabolism induced by morphine, stimulation of extraganglionic opiate receptors reflects the metabolism of DA located in small intensely fluorescent cells.


Zinc as chaperone-mimicking agent for retardation of amyloid β peptide fibril formation.
Metal ions have emerged to play a key role in the aggregation process of amyloid β (Aβ) peptide that is closely related to the pathogenesis of Alzheimer's disease. A detailed understanding of the underlying mechanistic process of peptide-metal interactions, however, has been challenging to obtain. By applying a combination of NMR relaxation dispersion and fluorescence kinetics methods we have investigated quantitatively the thermodynamic Aβ-Zn(2+) binding features as well as how Zn(2+) modulates the nucleation mechanism of the aggregation process. Our results show that, under near-physiological conditions, substoichiometric amounts of Zn(2+) effectively retard the generation of amyloid fibrils. A global kinetic profile analysis reveals that in the absence of zinc Aβ40 aggregation is driven by a monomer-dependent secondary nucleation process in addition to fibril-end elongation. In the presence of Zn(2+), the elongation rate is reduced, resulting in reduction of the aggregation rate, but not a complete inhibition of amyloid formation. We show that Zn(2+) transiently binds to residues in the N terminus of the monomeric peptide. A thermodynamic analysis supports a model where the N terminus is folded around the Zn(2+) ion, forming a marginally stable, short-lived folded Aβ40 species. This conformation is highly dynamic and only a few percent of the peptide molecules adopt this structure at any given time point. Our findings suggest that the folded Aβ40-Zn(2+) complex modulates the fibril ends, where elongation takes place, which efficiently retards fibril formation. In this conceptual framework we propose that zinc adopts the role of a minimal antiaggregation chaperone for Aβ40.


Development of a multiplex-PCR for direct detection of the genes for enterotoxin B and C, and toxic shock syndrome toxin-1 in Staphylococcus aureus isolates.
As well as conventional methods such as immunodiffusion, ELISA, or agglutination for the detection of toxin production in Staphylococcus aureus, amplification techniques like PCR allow a very sensitive and specific identification of the genes responsible for enterotoxin B and C, and TSST-1 production. These toxins might be a cause of the toxic shock syndrome (TSS). For that reason an easy and quick test system for determining the toxin production pattern of S. aureus isolates is desirable so that strains suspected to be toxin producers may be identified much faster and easier. In the present investigation, a new multiplex-PCR method was used that allowed single bacterial colonies grown on agar plates to be used directly in the PCR assay without preceding preparation. This procedure generated information concerning the presence of seb, sec-1 and tst genes within 4 h in a single test. To analyse the sensitivity and the specificity of this procedure, 100 methicillin-resistant S. aureus (MRSA), 50 coagulase-negative staphylococci and 50 other eubacterial isolates were tested initially with sets of single primer pairs followed by a combined multiplex-PCR. Results of this amplification technique were compared to a conventional and widely used method for toxin detection, reversed passive latex agglutination (RPLA). With the RPLA assay results as the basis, sensitivity and specificity of the seb and tst primer sets were 100%, whereas sensitivity and specificity of the sec-1 primer set were 100% and 82%, respectively. With the sec-1 primer set, two isolates were identified as carrying the corresponding toxin gene although the RPLA test did not show any detectable toxin. The multiplex-PCR rapidly generated reliable information concerning the toxin-producing capacity of staphylococcal strains and could be easily integrated into a multiplex procedure described previously. The latter enabled the identification of specific PCR products for eubacteria and staphylococci as well as the detection of the coa and mecA genes.


Recombination produces coherent bacterial species clusters in both core and accessory genomes.
Population samples show bacterial genomes can be divided into a core of ubiquitous genes and accessory genes that are present in a fraction of isolates. The ecological significance of this variation in gene content remains unclear. However, microbiologists agree that a bacterial species should be 'genomically coherent', even though there is no consensus on how this should be determined. We use a parsimonious model combining diversification in both the core and accessory genome, including mutation, homologous recombination (HR) and horizontal gene transfer (HGT) introducing new loci, to produce a population of interacting clusters of strains with varying genome content. New loci introduced by HGT may then be transferred on by HR. The model fits well to a systematic population sample of 616 pneumococcal genomes, capturing the major features of the population structure with parameter values that agree well with empirical estimates. The model does not include explicit selection on individual genes, suggesting that crude comparisons of gene content may be a poor predictor of ecological function. We identify a clearly divergent subpopulation of pneumococci that are inconsistent with the model and may be considered genomically incoherent with the rest of the population. These strains have a distinct disease tropism and may be rationally defined as a separate species. We also find deviations from the model that may be explained by recent population bottlenecks or spatial structure.


A p130Cas tyrosine phosphorylated substrate domain decoy disrupts v-crk signaling.
The adaptor protein p130Cas (Cas) has been shown to be involved in different cellular processes including cell adhesion, migration and transformation. This protein has a substrate domain with up to 15 tyrosines that are potential kinase substrates, able to serve as docking sites for proteins with SH2 or PTB domains. Cas interacts with focal adhesion plaques and is phosphorylated by the tyrosine kinases FAK and Src. A number of effector molecules have been shown to interact with Cas and play a role in its function, including c-crk and v-crk, two adaptor proteins involved in intracellular signaling. Cas function is dependent on tyrosine phosphorylation of its substrate domain, suggesting that tyrosine phosphorylation of Cas in part regulates its control of adhesion and migration. To determine whether the substrate domain alone when tyrosine phosphorylated could signal, we have constructed a chimeric Cas molecule that is phosphorylated independently of upstream signals. We found that a tyrosine phosphorylated Cas substrate domain acts as a dominant negative mutant by blocking Cas-mediated signaling events, including JNK activation by the oncogene v-crk in transient and stable lines and v-crk transformation. This block was the result of competition for binding partners as the chimera competed for binding to endogenous c-crk and exogenously expressed v-crk. Our approach suggests a novel method to study adaptor proteins that require phosphorylation, and indicates that mere tyrosine phosphorylation of the substrate domain of Cas is not sufficient for its function.


Recent burning of boreal forests exceeds fire regime limits of the past 10,000 years.
Wildfire activity in boreal forests is anticipated to increase dramatically, with far-reaching ecological and socioeconomic consequences. Paleorecords are indispensible for elucidating boreal fire regime dynamics under changing climate, because fire return intervals and successional cycles in these ecosystems occur over decadal to centennial timescales. We present charcoal records from 14 lakes in the Yukon Flats of interior Alaska, one of the most flammable ecoregions of the boreal forest biome, to infer causes and consequences of fire regime change over the past 10,000 y. Strong correspondence between charcoal-inferred and observational fire records shows the fidelity of sedimentary charcoal records as archives of past fire regimes. Fire frequency and area burned increased ∼6,000-3,000 y ago, probably as a result of elevated landscape flammability associated with increased Picea mariana in the regional vegetation. During the Medieval Climate Anomaly (MCA; ∼1,000-500 cal B.P.), the period most similar to recent decades, warm and dry climatic conditions resulted in peak biomass burning, but severe fires favored less-flammable deciduous vegetation, such that fire frequency remained relatively stationary. These results suggest that boreal forests can sustain high-severity fire regimes for centuries under warm and dry conditions, with vegetation feedbacks modulating climate-fire linkages. The apparent limit to MCA burning has been surpassed by the regional fire regime of recent decades, which is characterized by exceptionally high fire frequency and biomass burning. This extreme combination suggests a transition to a unique regime of unprecedented fire activity. However, vegetation dynamics similar to feedbacks that occurred during the MCA may stabilize the fire regime, despite additional warming.


Influence of internal viscoelastic modes on the Brownian motion of a λ-DNA coated colloid.
We study the influence of grafted polymers on the diffusive behaviour of a colloidal particle. Our work demonstrates how such additional degrees of freedom influence the Brownian motion of the particle, focusing on internal viscoelastic coupling between the polymer and colloid. Specifically, we study the mean-squared displacements (MSDs) of λ-DNA grafted colloids using Brownian dynamics simulation. Our simulations reveal the non-trivial effect of internal modes, which gives rise to a crossover from the short-time viscoelastic to long-time diffusional behaviour. We also show that basic features can be captured by a simple theoretical model considering the relative motion of a colloid to a part of the polymer corona. This model describes well a MSD calculated from an extremely long trajectory of a single λ-DNA coated colloid from experiment and allows characterisation of the λ-DNA hairs. Our study suggests that the access to the internal relaxation modes via the colloid trajectory offers a novel method for the characterisation of soft attachments to a colloid.