First Report of Zebra Chip Disease and "Candidatus Liberibacter solanacearum" on Potatoes in Idaho.
In September 2011, potato (Solanum tuberosum L.) tubers graded in a packing facility in south-central Idaho were observed with internal discolorations suggestive of zebra chip disease (ZC). Symptoms were observed in 1 to 2% of tubers of cv. Russet Norkotah and included brown spots and streaks especially in and near the vascular tissue. Some tubers also showed a dark and sunken stolon attachment typical of ZC (1). Initially, tissue samples were taken from seven symptomatic tubers and tested by PCR for "Candidatus Liberibacter solanacearum", the bacterium associated with ZC. Primers specific for the 16S rDNA (primers CLipoF [4] and OI2c [3]) and the outer membrane protein (OMB 1482f and 2086r) (2) were used. Six of these samples were positive for the bacterium. The amplified 16S rDNA and OMB products from two symptomatic tubers of cv. Russet Norkotah were cloned and three clones of each were sequenced. The 16S sequences (1,071 bp; GenBank Accession Nos. JN848755 and JN848756) from the two tubers varied by one nucleotide and had 99 to 100% sequence identity to numerous "Ca. L. solanacearum" sequences in GenBank (e.g., Accession Nos. HM246509, FJ957897, and EU935004). Sequences of the two OMB clones (605 bp; GenBank Accession Nos. JN848757 and JN848758) had 97% sequence identity to the two "Ca. L. solanacearum" OMB sequences in GenBank (Accession Nos. CP002371 and FJ914617). Six of eight additional symptomatic field-collected cv. Russet Norkotah tubers corresponding to tubers collected in the packing facility were also positive for "Ca. L. solanacearum" by PCR. Additional severely symptomatic tubers of cvs. Russet Burbank, Yukon Gold, and raw cut French fries of Ranger Russet produced in south-central Idaho were subsequently tested by PCR and were found to be positive for "Ca. L. solanacearum" as well. On the basis of the symptoms, specific PCR amplification with two distinct primer pairs and DNA sequence analysis, zebra chip disease caused by "Ca. L. solanacearum" was determined to be present in Idaho. This disease has caused significant economic damage to potatoes in many regions, including Texas, Mexico, Central America, and New Zealand (1). Idaho is the largest potato-producing state in the United States, with over 150,000 ha planted this year, and therefore, ZC potentially poses a significant risk to agriculture in this state. References: (1) J. M. Crosslin et al. Online publication. doi:10.1094/PHP-2010-0317-01-RV, Plant Health Progress, 2010. (2) J. M. Crosslin et al. Southwest. Entomol. 36:125, 2011. (3) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (4) G. A. Secor. Plant Dis. 93:574, 2009.


Differentiation of human embryonic stem cells to hepatocytes using deleted variant of HGF and poly-amino-urethane-coated nonwoven polytetrafluoroethylene fabric.
Human embryonic stem (hES) cells have recently been studied as an attractive source for the development of a bioartificial liver (BAL). Here we evaluate the differentiation capacity of hES cells into hepatocytes. hES cells were subjected to suspension culture for 5 days, and then cultured onto poly-amino-urethane (PAU)-coated, nonwoven polytetrafluoroethylene (PTFE) fabric in the presence of fibroblast growth factor-2 (bFGF) (100 ng/ml) for 3 days, then with deleted variant of hepatocyte growth factor (dHGF) (100 ng/ml) and 1% dimethyl sulfoxide (DMSO) for 8 days, and finally with dexamethasone (10(-7) M) for 3 days. The hES cells showed gene expression of albumin in a time-dependent manner of the hepatic differentiation process. The resultant hES-derived hepatocytes metabolized the loaded ammonia and lidocaine at 7.8% and 23.6%, respectively. A million of such hepatocytes produced albumin and urea at 351.2 ng and urea at 7.0 microg. Scanning electron microscopy showed good attachment of the cells on the surface of the PTFE fabric and well-developed glycogen rosettes and Gap junction. In the present work we have demonstrated the efficient differentiation of hES cells to functional hepatocytes. The findings are useful to develop a BAL.


Molecular cloning and expression of mouse procalcitonin.
The nucleotide sequence for mouse calcitonin was determined from a cDNA obtained using a polymerase chain reaction (PCR) based method, the rapid amplification of cDNA ends (RACE)-PCR. Primers designed from highly conserved regions in the coding sequences of known rat and human calcitonin cDNAs were used to amplify calcitonin cDNA as 5'-end and 3'-end fragments from mouse thyroid RNA. The obtained cDNA is 850 bp in length most probably representing the entire mouse calcitonin mRNA. It contains an open reading frame coding for a 136 amino acid protein with a calculated M(r) of 15,143. Comparison of the deduced amino acid sequences of preprocalcitonin of mice with other species revealed highest homologies to the rat (93%) and human (77%) sequences. A recombinant form of mouse precalcitonin (rmPCT) of approximately 17 kDa was expressed as a fusion peptide in E.coli transformed with a PCR-cloned expression construct.


Actin-based features negatively regulated by protein kinase C-epsilon.
Cells exposed to phorbol 12-myristate 13-acetate (PMA) undergo a choreographed sequence of morphological changes. Some of these, including stimulation of membrane ruffles and the later appearance of stress fibers, rely on remodeling of the actin cytoskeleton. Although this process is poorly understood, it is important, because the same features are affected during oncogenic transformation. PMA also activates protein kinase C (PKC). Enzyme activation is followed by degradation. Either process might affect the remodeling of actin. The present studies determined whether any PKC isozymes were subject to degradation in tracheal epithelial cells by quantifying the amount of each isozyme present after PMA exposure. PKC-epsilon was the only isozyme to show declining content correlated with increased stress fiber accumulation. Stress fibers increased between 5 and 10 h, whereas PKC-epsilon declined to 38% of its starting value (95% confidence interval, 10-68%). The relationship could be fit by the function F(x) = 0.683 x exp[-0.841(x - 0.387)], where F is the frequency of fiber-containing cells and x is PKC-epsilon content. Fiber accumulation was further investigated after knockdown of PKC-epsilon with RNA interference and antisense oligodeoxynucleotide. Knockdown enhanced stress fibers in cells not yet exposed to PMA as well as the final frequency of fiber-containing cells after PMA exposure. With knockdown at both transcriptional and protein levels, approximately 15% of the original content was predicted and achieved, as judged from real-time PCR and PKC-epsilon content measurements. The results suggest that PKC-epsilon negatively regulates stress fibers, either by directly turning over one of their components or by regulating an upstream step affecting fiber organization.


Demonstration of reduced efficacy against cyathostomins without change in species composition after pyrantel embonate treatment in Swedish equine establishments.
Consisting of approximately 50 different species, the cyathostomin parasites are ubiquitous in grazing horses. Co-infection with several species is common, and large burdens can cause the fatal disease of larval cyathostominosis. Due to intense anthelmintic drug use, cyathostomin resistance has developed to all available anthelmintic drug groups. Resistance to the anthelmintic drug pyrantel (PYR) has been documented in over 90% of studies published over the past two decades. In Sweden, a study performed in the early 2000s only confirmed resistance in 4.5% of farms. Further, prescription-only administration of equine anthelmintic drugs was enforced in Sweden in 2007. However, it is unknown if this conservative drug use has maintained PYR efficacy in cyathostomins. The aim of the present study was to investigate the effect of PYR on cyathostomin infection in Sweden using fecal egg count reduction tests (FECRTs). Further, the effect of PYR treatment on cyathostomin species composition was studied using metabarcoding. Sixteen farms with at least six horses excreting a minimum of 100 eggs per gram feces were included. Using the current World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines, PYR resistance was demonstrated in nine of farms, with seven farms showing full susceptibility. Farms with low biosecurity measures had significantly lower efficacy of PYR treatment. The most common cyathostomin species were Cylicocyclus nassatus, Cyathostomum catinatum, Cylicostephanus longibursatus, Cys. calicatus, Cys. goldi, Cys. minutus, Coronocyclus coronatus and Cya. pateratum, accounting for 97% of all sequence reads prior to treatment. Of these, Cyc. nassatus and Cya. catinatum had the highest occurrence, accounting for 68% of all sequence reads prior to PYR treatment. Treatment did not significantly affect the species composition. The results highlight the importance of drug efficacy testing when using PYR to treat cyathostomin infection, even when selective anthelmintic treatment and thus low treatment intensity, is used on the farm.


Localization, quantification and interaction with host factors of endogenous HTLV-1 HBZ protein in infected cells and ATL.
Human T cell lymphotropic virus type 1 (HTLV-1) is the etiological agent of a severe form of neoplasia designated Adult T cell Leukaemia (ATL). It is widely accepted that the viral transactivator Tax-1 is the major viral product involved in the onset, but not in the maintenance, of neoplastic phenotype, as only 30-40% of ATL cells express Tax-1. It has been recently demonstrated that HBZ (HTLV-1 bZIP factor), a protein encoded by the minus strand of HTLV-1 genome, constantly expressed in infected cells and in ATL tumor cells, is also involved in the pathogenesis of leukaemia. The full role played by HBZ in oncogenesis is not clarified in detail also because of the limited availability of tools to assess quantitative expression, subcellular location and interaction of HBZ with host factors in ATL. By the use of the first reported monoclonal antibody against HBZ, 4D4-F3, generated in our laboratory it has been possible to carefully assess for the first time the above parameters in HTLV-1 chronically infected cells and, most importantly, in fresh leukemic cells from patients. Endogenous HBZ is expressed in speckle-like structures localized in the nucleus. The calculated number of endogenous HBZ molecules varies between 17.461 and 39.615 molecules per cell, 20- to 50-fold less than the amount expressed in HBZ transfected cells used by most investigators to assess the expression, function and subcellular localization of the viral protein. HBZ interacts in vivo with p300 and JunD and co-localizes only partially, and depending on the amount of expressed HBZ, not only with p300 and JunD but also with CBP and CREB2. The possibility to study endogenous HBZ in detail may significantly contribute to a better delineation of the role of HBZ during HTLV-1 infection and cellular transformation.


Imaging findings in dogs with caudal intervertebral disc herniation.
The radiographic and magnetic resonance imaging (MRI) findings for four dogs with herniation of the Cd1-Cd2 intervertebral disc are described. All dogs were 7 years of age at the time of presentation, with one neutered male and three neutered females. Breeds included one Beagle, one Bassett hound, and two large, mixed breed dogs. All dogs had tail pain on manipulation, two had pain during defecation, and two maintained an abnormal tail position. Three dogs had radiographs in which mineralization within the disc space was apparent. Two of these dogs also had mineralization within the vertebral canal. Three dogs underwent MRI, which was characterized by varying degrees of disc herniation and nerve root compression at Cd1-Cd2. Mobility may be a factor predisposing to disc herniation in the cranial aspect of the caudal spine. We documented that caudal disc herniation does occur occasionally in dogs and that radiography and MRI may be used to identify this disease. Caudal intervertebral disc herniation should be considered as a differential for dogs with caudal vertebral pain, pain with tail manipulation, pain during defecation, or abnormal tail carriage.


Shift of equilibrium density induced by 3,3'-diaminobenzidine cytochemistry: a new procedure for the analysis and purification of peroxidase-containing organelles.
Galactosylated BSA (galBSA) and its conjugate to horseradish peroxidase (galBSA-HRP) enter the galactose-specific pathway of hepatocytes. 10 min after intravenous injection, structures containing either ligand sediment mostly between 33,000 and 3 X 10(6) g X min (LP fraction) and have an equilibrium density of 1.11-1.13 g/ml in sucrose gradients (Quintart, J., P. J. Courtoy, J. N. Limet, and P. Baudhuin, 1983, Eur. J. Biochem., 131:105-112). Such low density fractions, prepared from rats given galBSA-HRP, were incubated for 30 min at 25 degrees C in 5.5 mM 3,3'-diaminobenzidine (DAB) and 11 mM H2O2 in buffered sucrose. Upon equilibration in a second sucrose gradient, the galBSA-HRP distribution shifted towards higher (approximately 1.19 g/ml) density, but the bulk of protein remained at low density. In the absence of H2O2, galBSA-HRP distribution was also found at low density. As observed by electron microscopy, particles equilibrating at higher density after DAB cytochemistry were largely made of vesicles or tubules filled with DAB reaction product. The density shift of galBSA-HRP-containing organelles after incubation with DAB and H2O2 is attributed to the trapping of HRP-oxidized DAB inside the host organelles. If the low density fractions isolated from a rat injected with [3H]galBSA-HRP were mixed in vitro with similar fractions from another rat given [14C]galBSA, the 3H distribution shifted after DAB cytochemistry, but the 14C distribution was essentially unaffected. By contrast, if both derivatives were injected simultaneously, a concomitant density shift was observed. In conclusion, the DAB-induced density shift was specific to ligand-HRP-containing organelles. The potentials of the method include the purification of HRP-containing particles and the study of their association to ligands, fluid-phase tracers, or marker enzymes.


Does Kisspeptin Belong to the Proposed RF-Amide Peptide Family?
Kisspeptin (KISS) plays a key role in regulating reproduction by binding to its receptor, GPR54. Because of the Arg-Phe (RF) sequence at its carboxyl terminus, KISS has been proposed to be a member of the RF-amide peptide family consisting of neuropeptide FF (NPFF), neuropeptide VF (NPVF), pyroglutamylated RF-amide peptide (QRFP), and prolactin-releasing hormone (PRLH). Evolutionary relationships of protein families can be determined through phylogenetic analysis. However, phylogenetic analysis among related peptide families often fails to provide sufficient information because only short mature peptide sequences from full preprohormone sequences are conserved. Considering the concept of the coevolution of peptide ligands and their cognate receptors, evolutionary relationships among related receptor families provide clues to explore relationships between their peptides. Although receptors for NPFF, NPVF, and QRFP are phylogenetically clustered together, receptors for PRLH and KISS are on different branches of the phylogenetic tree. In particular, KISS has been proposed to be a member of the KISS/galanin/spexin family based on synteny analysis and the phylogenetic relationship between their receptors. This article discusses the evolutionary history of the receptors for the proposed RF-amide peptide family and proposes that, from an evolutionary aspect, KISS has emerged from an ancestor, which is distinct from those of the other RF-amide peptides, and so should be classed separately.


Health assessment and seroepidemiologic survey of potential pathogens in wild Antillean manatees (Trichechus manatus manatus).
The Antillean manatee (Trichechus manatus manatus), a subspecies of the West Indian manatee, inhabits fresh, brackish, and warm coastal waters distributed along the eastern border of Central America, the northern coast of South America, and throughout the Wider Caribbean Region. Threatened primarily by human encroachment, poaching, and habitat degradation, Antillean manatees are listed as endangered by the International Union for the Conservation of Nature. The impact of disease on population viability remains unknown in spite of concerns surrounding the species' ability to rebound from a population crash should an epizootic occur. To gain insight on the baseline health of this subspecies, a total of 191 blood samples were collected opportunistically from wild Antillean manatees in Belize between 1997 and 2009. Hematologic and biochemical reference intervals were established, and antibody prevalence to eight pathogens with zoonotic potential was determined. Age was found to be a significant factor of variation in mean blood values, whereas sex, capture site, and season contributed less to overall differences in parameter values. Negative antibody titers were reported for all pathogens surveyed except for Leptospira bratislava, L. canicola, and L. icterohemorrhagiae, Toxoplasma gondii, and morbillivirus. As part of comprehensive health assessment in manatees from Belize, this study will serve as a benchmark aiding in early disease detection and in the discernment of important epidemiologic patterns in the manatees of this region. Additionally, it will provide some of the initial tools to explore the broader application of manatees as sentinel species of nearshore ecosystem health.


Hospice and palliative care development in India: a multimethod review of services and experiences.
Palliative care has been developing in India since the mid-1980s, but there is a dearth of evidence about service provision on which to base national policy and practice. The aim of this study was to assess the current state of palliative care in India, mapping the existence of services state by state, and documenting the perspectives and experiences of those involved. A multimethod review was used, which included synthesis of evidence from published and grey literature, ethnographic field visits, qualitative interviews with 87 individuals from 12 states, and collation of existing public health data. The review identified 138 hospice and palliative care services in 16 states and union territories. These are mostly concentrated in large cities, with the exception of Kerala, where they are much more widespread. Nongovernmental organizations, public and private hospitals, and hospices are the predominant sources of provision. We were unable to identify palliative care services in 19 states/union territories. Development of services is uneven, with greater provision evident in the south than the north, but for the majority of states, coverage is poor. Barriers to the development of palliative care include: poverty, population density, geography, opioid availability, workforce development, and limited national palliative care policy. Successful models exist for the development of affordable, sustainable community-based palliative care services. These have arisen from adapting Western models of hospice and palliative care for implementation in the Indian cultural context. Further work is required to ensure that the growing interest in hospice and palliative care in India is used to increase the momentum of progress.


Improved expression systems for regulated expression in Salmonella infecting eukaryotic cells.
In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the P(m) promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/P(sal) system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning site and different combinations of the following elements: (i) the nasF transcriptional attenuator, which reduces basal expression levels, (ii) a strong ribosome binding site, and (iii) the Type III Secretion System (TTSS) signal peptide from the effector protein SspH2 to deliver proteins directly to the eukaryotic cytosol following bacterial infection of animal cells. We show that different expression module versions can be used to direct a broad range of protein production levels. Furthermore, we demonstrate that the efficient reduction of basal expression by the nasF attenuator allows the cloning of genes encoding highly cytotoxic proteins such as colicin E3 even in the absence of its immunity protein. Additionally, we show that the Salmonella TTSS is able to translocate most of the protein produced by this regulatory cascade to the cytoplasm of infected HeLa cells. Our results indicate that these vectors represent useful tools for the regulated overproduction of heterologous proteins in bacterial culture or in animal cells, for the cloning and expression of genes encoding toxic proteins and for pathogenesis studies.


Regional fisheries management: Virtual decision making in a pandemic.
The global COVID-19 pandemic is impacting on the fisheries sector and posing significant challenges for the management of transboundary fisheries. Due to travel bans and border closures, regional organizations are not able to hold face-to-face meetings. This commentary provides a summary of the meeting procedures of Regional Fisheries Management Organizations and Regional Organizations during the global pandemic. Most organizations have transitioned to online platforms and are holding virtual meetings. These online meetings impose significant challenges concerning sustainable fisheries management, such as limited discussions and negotiations on important issues. Thus to continue their work effectively, these organizations need to develop new decision-making procedures that are more resilient in the upcoming future.


Modeling vaccination rollouts, SARS-CoV-2 variants and the requirement for non-pharmaceutical interventions in Italy.
Despite progress in clinical care for patients with coronavirus disease 2019 (COVID-19)1, population-wide interventions are still crucial to manage the pandemic, which has been aggravated by the emergence of new, highly transmissible variants. In this study, we combined the SIDARTHE model2, which predicts the spread of SARS-CoV-2 infections, with a new data-based model that projects new cases onto casualties and healthcare system costs. Based on the Italian case study, we outline several scenarios: mass vaccination campaigns with different paces, different transmission rates due to new variants and different enforced countermeasures, including the alternation of opening and closure phases. Our results demonstrate that non-pharmaceutical interventions (NPIs) have a higher effect on the epidemic evolution than vaccination alone, advocating for the need to keep NPIs in place during the first phase of the vaccination campaign. Our model predicts that, from April 2021 to January 2022, in a scenario with no vaccine rollout and weak NPIs ([Formula: see text] = 1.27), as many as 298,000 deaths associated with COVID-19 could occur. However, fast vaccination rollouts could reduce mortality to as few as 51,000 deaths. Implementation of restrictive NPIs ([Formula: see text] = 0.9) could reduce COVID-19 deaths to 30,000 without vaccinating the population and to 18,000 with a fast rollout of vaccines. We also show that, if intermittent open-close strategies are adopted, implementing a closing phase first could reduce deaths (from 47,000 to 27,000 with slow vaccine rollout) and healthcare system costs, without substantive aggravation of socioeconomic losses.


Physicians' perceptions of capacity building for managing chronic disease in seniors using integrated interprofessional care models.
To explore the barriers to and facilitators of adapting and expanding a primary care memory clinic model to integrate care of additional complex chronic geriatric conditions (heart failure, falls, chronic obstructive pulmonary disease, and frailty) into care processes with the goal of improving outcomes for seniors. Mixed-methods study using quantitative (questionnaires) and qualitative (interviews) methods. Ontario. Family physicians currently working in primary care memory clinic teams and supporting geriatric specialists. Family physicians currently working in memory clinic teams (n = 29) and supporting geriatric specialists(n = 9) were recruited as survey participants. Interviews were conducted with memory clinic lead physicians (n = 16).Statistical analysis was done to assess differences between family physician ratings and geriatric specialist ratings related to the capacity for managing complex chronic geriatric conditions, the role of interprofessional collaboration within primary care, and funding and staffing to support geriatric care. Results from both study methods were compared to identify common findings. Results indicate overall support for expanding the memory clinic model to integrate care for other complex conditions. However, the current primary care structure is challenged to support optimal management of patients with multiple comorbidities, particularly as related to limited funding and staffing resources. Structured training, interprofessional teams, and an active role of geriatric specialists within primary care were identified as important facilitators. The memory clinic model, as applied to other complex chronic geriatric conditions, has the potential to build capacity for high-quality primary care, improve health outcomes,promote efficient use of health care resources, and reduce healthcare costs.


Cracking the molecular origin of intrinsic tyrosine kinase activity through analysis of pathogenic gain-of-function mutations.
The basal (ligand-independent) kinase activity of receptor tyrosine kinases (RTKs) promotes trans-phosphorylation on activation loop tyrosines upon ligand-induced receptor dimerization, thus upregulating intrinsic kinase activity and triggering intracellular signaling. To understand the molecular determinants of intrinsic kinase activity, we used X-ray crystallography and NMR spectroscopy to analyze pathogenic FGF receptor mutants with gradations in gain-of-function activity. These structural analyses revealed a "two-state" dynamic equilibrium model whereby the kinase toggles between an "inhibited," structurally rigid ground state and a more dynamic and heterogeneous active state. The pathogenic mutations have different abilities to shift this equilibrium toward the active state. The increase in the fractional population of FGF receptors in the active state correlates with the degree of gain-of-function activity and clinical severity. Our data demonstrate that the fractional population of RTKs in the active state determines intrinsic kinase activity and underscore how a slight increase in the active population of kinases can have grave consequences for human health.


Multilevel correlates of behavioral resilience among children in child welfare.
Resilience, defined as positive adaptation and functioning following exposure to significant adversity, is an important topic of investigation in child welfare. The current study used data from the Ontario Looking After Children (OnLAC) project to estimate the prevalence of behavioral resilience (i.e., lower frequency of conduct and emotional problems, higher frequency of prosocial behavior) in 531 5-9 year olds living in out-of-home care, and to determine how behaviorally-resilient children are functioning in other domains (i.e., peer relationships and academic performance). Furthermore, hierarchical linear modeling was used to examine the contribution of four levels of analysis (i.e., child, family, child welfare worker, and child welfare agency) on behaviors and to identify the contribution of predictor variables within each of these levels. Findings indicated that 50-70% of children exhibited resilience on one behavioral outcome while approximately 30% showed resilience on at least two of the outcomes. Also, 8.4-9.6% exhibited resilience on one of the behavioral outcomes in addition to peer relationships and academic performance. The child level accounted for the highest proportion of total explained variance in behavioral outcomes, followed by the family-, child welfare worker-, and child welfare agency-levels. A number of child and foster family variables predicted behavioral functioning. Findings indicate that it is important to inquire about children's functioning across multiple domains to obtain a comprehensive developmental assessment. Also, child and foster family characteristics appear to play considerable roles in the promotion of behavioral resilience.


Clinical prediction rule for identifying children with cerebrospinal fluid pleocytosis at very low risk of bacterial meningitis.
Children with cerebrospinal fluid (CSF) pleocytosis are routinely admitted to the hospital and treated with parenteral antibiotics, although few have bacterial meningitis. We previously developed a clinical prediction rule, the Bacterial Meningitis Score, that classifies patients at very low risk of bacterial meningitis if they lack all of the following criteria: positive CSF Gram stain, CSF absolute neutrophil count (ANC) of at least 1000 cells/microL, CSF protein of at least 80 mg/dL, peripheral blood ANC of at least 10,000 cells/microL, and a history of seizure before or at the time of presentation. To validate the Bacterial Meningitis Score in the era of widespread pneumococcal conjugate vaccination. A multicenter, retrospective cohort study conducted in emergency departments of 20 US academic medical centers through the Pediatric Emergency Medicine Collaborative Research Committee of the American Academy of Pediatrics. All children aged 29 days to 19 years who presented at participating emergency departments between January 1, 2001, and June 30, 2004, with CSF pleocytosis (CSF white blood cells > or =10 cells/microL) and who had not received antibiotic treatment before lumbar puncture. The sensitivity and negative predictive value of the Bacterial Meningitis Score. Among 3295 patients with CSF pleocytosis, 121 (3.7%; 95% confidence interval [CI], 3.1%-4.4%) had bacterial meningitis and 3174 (96.3%; 95% CI, 95.5%-96.9%) had aseptic meningitis. Of the 1714 patients categorized as very low risk for bacterial meningitis by the Bacterial Meningitis Score, only 2 had bacterial meningitis (sensitivity, 98.3%; 95% CI, 94.2%-99.8%; negative predictive value, 99.9%; 95% CI, 99.6%-100%), and both were younger than 2 months old. A total of 2518 patients (80%) with aseptic meningitis were hospitalized. This large multicenter study validates the Bacterial Meningitis Score prediction rule in the era of conjugate pneumococcal vaccine as an accurate decision support tool. The risk of bacterial meningitis is very low (0.1%) in patients with none of the criteria. The Bacterial Meningitis Score may be helpful to guide clinical decision making for the management of children presenting to emergency departments with CSF pleocytosis.


Differential role of mitogen-activated protein kinase in three distinct phases of memory for sensitization in Aplysia.
The mitogen-activated protein kinase (MAPK) pathway has been implicated recently in synaptic plasticity and memory. Here we used tail shock-induced sensitization of the tail-elicited siphon withdrawal reflex in Aplysia to examine the role of MAPK in three different phases of memory. We show that a specific pattern of serotonin (5-HT) application that produces intermediate-term and long-term synaptic facilitation (ITF and LTF, respectively) of the sensory-motor (SN-MN) synapses in Aplysia leads to sustained activation of extracellular signal-regulated kinase in the ventrocaudal cluster sensory neurons (SNs), which include the tail SNs. Furthermore, repeated tail shocks that induce intermediate-term and long-term memory (ITM and LTM, respectively) for sensitization also lead to sustained MAPK activation in the SNs. Given these results, we next examined the requirement of MAPK activity in (1) SN-MN synaptic facilitation and (2) memory for sensitization in Aplysia, by inhibiting MEK, the upstream kinase that phosphorylates and activates MAPK. In cellular experiments, we show that MAPK activity is required for ITF of tail SN-tail MN synapses, and, in parallel behavioral experiments, we show that ITM requires MAPK activity for its induction but not its expression. In contrast, short-term memory for sensitization does not require MAPK activity. Finally, 5-HT-induced LTF has been shown previously to require MAPK activity. Here we show that LTM for sensitization also requires MAPK activity. These results provide evidence that MAPK plays important roles specifically in long-lasting phases of synaptic plasticity and memory.


The integrated landscape of driver genomic alterations in glioblastoma.
Glioblastoma is one of the most challenging forms of cancer to treat. Here we describe a computational platform that integrates the analysis of copy number variations and somatic mutations and unravels the landscape of in-frame gene fusions in glioblastoma. We found mutations with loss of heterozygosity in LZTR1, encoding an adaptor of CUL3-containing E3 ligase complexes. Mutations and deletions disrupt LZTR1 function, which restrains the self renewal and growth of glioma spheres that retain stem cell features. Loss-of-function mutations in CTNND2 target a neural-specific gene and are associated with the transformation of glioma cells along the very aggressive mesenchymal phenotype. We also report recurrent translocations that fuse the coding sequence of EGFR to several partners, with EGFR-SEPT14 being the most frequent functional gene fusion in human glioblastoma. EGFR-SEPT14 fusions activate STAT3 signaling and confer mitogen independence and sensitivity to EGFR inhibition. These results provide insights into the pathogenesis of glioblastoma and highlight new targets for therapeutic intervention.


Hydrodynamic trapping measures the interaction between membrane-associated molecules.
How membrane proteins distribute and behave on the surface of cells depends on the molecules' chemical potential. However, measuring this potential, and how it varies with protein-to-protein distance, has been challenging. Here, we present a method we call hydrodynamic trapping that can achieve this. Our method uses the focused liquid flow from a micropipette to locally accumulate molecules protruding above a lipid membrane. The chemical potential, as well as information about the dimensions of the studied molecule, are obtained by relating the degree of accumulation to the strength of the trap. We have used this method to study four representative proteins, with different height-to-width ratios and molecular properties; from globular streptavidin, to the rod-like immune cell proteins CD2, CD4 and CD45. The data we obtain illustrates how protein shape, glycosylation and flexibility influence the behaviour of membrane proteins, as well as underlining the general applicability of the method.


A person-centred intervention for providing information to parents of children with cancer. Experiences and effects.
The aim of this paper is to describe the experiences of participating in a person-centred information intervention aimed at parents of children with cancer. Eight parents participated in the intervention, beginning two months after their child's diagnosis. The intervention was based upon the representational approach to patient education and a mixed method approach was employed in the study. The experiences of parents and intervention nurses were captured via qualitative interviews and the effects of the intervention on parental psychosocial measures, primarily perceived stress, were evaluated using a single-case design with web-based questionnaires. Parents expressed high satisfaction with the intervention, as reported in the follow-up interviews and on the scale measuring satisfaction. However, no changes were seen in the quantitative measures of psychosocial distress. The nurses performing the intervention felt it was useful and feasible. A representational approach to providing person-centred information to parents of children with cancer was appreciated and considered feasible by both the parents and the intervention nurses. However, further research is needed considering the lack of effect on the parents' perceived stress.


Polarization correlations in backscattering from media with different optical densities.
Random electromagnetic fields have a number of distinctive statistical properties that may depend on their origin. We show that, when two mutually coherent fields overlap, their individual characteristics are not completely lost. If assumptions can be made regarding the coherence properties of one of the fields, then the correlation length of the second one can be retrieved using the higher-order polarization properties of the combined field. We demonstrate experimentally that colloidal particles of different sizes can be identified based on polarization correlations measured even in situations of strong multiple scattering.


Drivers of temporal changes in temperate forest plant diversity vary across spatial scales.
Global biodiversity is affected by numerous environmental drivers. Yet, the extent to which global environmental changes contribute to changes in local diversity is poorly understood. We investigated biodiversity changes in a meta-analysis of 39 resurvey studies in European temperate forests (3988 vegetation records in total, 17-75 years between the two surveys) by assessing the importance of (i) coarse-resolution (i.e., among sites) vs. fine-resolution (i.e., within sites) environmental differences and (ii) changing environmental conditions between surveys. Our results clarify the mechanisms underlying the direction and magnitude of local-scale biodiversity changes. While not detecting any net local diversity loss, we observed considerable among-site variation, partly explained by temporal changes in light availability (a local driver) and density of large herbivores (a regional driver). Furthermore, strong evidence was found that presurvey levels of nitrogen deposition determined subsequent diversity changes. We conclude that models forecasting future biodiversity changes should consider coarse-resolution environmental changes, account for differences in baseline environmental conditions and for local changes in fine-resolution environmental conditions.


Identification of stiffness-induced signalling mechanisms in cells from patent and fused sutures associated with craniosynostosis.
Craniosynostosis is a bone developmental disease where premature ossification of the cranial sutures occurs leading to fused sutures. While biomechanical forces have been implicated in craniosynostosis, evidence of the effect of microenvironmental stiffness changes in the osteogenic commitment of cells from the sutures is lacking. Our aim was to identify the differential genetic expression and osteogenic capability between cells from patent and fused sutures of children with craniosynostosis and whether these differences are driven by changes in the stiffness of the microenvironment. Cells from both sutures demonstrated enhanced mineralisation with increasing substrate stiffness showing that stiffness is a stimulus capable of triggering the accelerated osteogenic commitment of the cells from patent to fused stages. The differences in the mechanoresponse of these cells were further investigated with a PCR array showing stiffness-dependent upregulation of genes mediating growth and bone development (TSHZ2, IGF1), involved in the breakdown of extracellular matrix (MMP9), mediating the activation of inflammation (IL1β) and controlling osteogenic differentiation (WIF1, BMP6, NOX1) in cells from fused sutures. In summary, this study indicates that stiffer substrates lead to greater osteogenic commitment and accelerated bone formation, suggesting that stiffening of the extracellular environment may trigger the premature ossification of the sutures.


Eph signaling controls mitotic spindle orientation and cell proliferation in neuroepithelial cells.
Mitotic spindle orientation must be tightly regulated during development and adult tissue homeostasis. It determines cell-fate specification and tissue architecture during asymmetric and symmetric cell division, respectively. Here, we uncover a novel role for Ephrin-Eph intercellular signaling in controlling mitotic spindle alignment in Drosophila optic lobe neuroepithelial cells through aPKC activity-dependent myosin II regulation. We show that conserved core components of the mitotic spindle orientation machinery, including Discs Large1, Mud/NuMA, and Canoe/Afadin, mislocalize in dividing Eph mutant neuroepithelial cells and produce spindle alignment defects in these cells when they are down-regulated. In addition, the loss of Eph leads to a Rho signaling-dependent activation of the PI3K-Akt1 pathway, enhancing cell proliferation within this neuroepithelium. Hence, Eph signaling is a novel extrinsic mechanism that regulates both spindle orientation and cell proliferation in the Drosophila optic lobe neuroepithelium. Similar mechanisms could operate in other Drosophila and vertebrate epithelia.


Microfluidic synthesis of advanced microparticles for encapsulation and controlled release.
We describe droplet microfluidic strategies used to fabricate advanced microparticles that are useful structures for the encapsulation and release of actives; these strategies can be further developed to produce microparticles for advanced drug delivery applications. Microfluidics enables exquisite control in the fabrication of polymer vesicles and thermosensitive microgels from single and higher-order multiple emulsion templates. The strategies used to create the diversity of microparticle structures described in this review, coupled with the scalability of microfluidics, will enable fabrication of large quantities of novel microparticle structures that have potential uses in controlled drug release applications.


A randomized controlled trial of group cognitive-behavioral therapy vs. enhanced supportive therapy for auditory hallucinations.
There has been little research examining group cognitive-behavioral therapy (CBT) for schizophrenia, especially compared to an active control treatment. The purpose of this study was to investigate the effectiveness of group CBT for auditory hallucinations compared to an enhanced supportive therapy (ST). Sixty five participants with schizophrenia spectrum disorders and persistent hallucinations were randomly assigned to group CBT or enhanced group ST. Primary outcomes focused on beliefs about voices and global auditory hallucinations severity. Secondary outcomes included psychotic symptoms, self-esteem, social functioning, insight, depression, and hospitalization. Controlling for baseline levels, these outcomes were evaluated across post-treatment, 3 month and 12 month follow-ups. Participants who received enhanced ST were less likely to both resist voices and to rate them as less malevolent through 12-month follow-up relative to participants who received CBT. Group CBT was associated with lower general and total symptom scores on the PANSS through 12-month-followup relative to participants who received enhanced ST. Outcomes improved through 12-month follow-up in both therapy groups, with enhanced ST having more specific impact on auditory hallucinations, and CBT impacting general psychotic symptoms.


Acute renal failure in critically ill patients: a multinational, multicenter study.
Although acute renal failure (ARF) is believed to be common in the setting of critical illness and is associated with a high risk of death, little is known about its epidemiology and outcome or how these vary in different regions of the world. To determine the period prevalence of ARF in intensive care unit (ICU) patients in multiple countries; to characterize differences in etiology, illness severity, and clinical practice; and to determine the impact of these differences on patient outcomes. Prospective observational study of ICU patients who either were treated with renal replacement therapy (RRT) or fulfilled at least 1 of the predefined criteria for ARF from September 2000 to December 2001 at 54 hospitals in 23 countries. Occurrence of ARF, factors contributing to etiology, illness severity, treatment, need for renal support after hospital discharge, and hospital mortality. Of 29 269 critically ill patients admitted during the study period, 1738 (5.7%; 95% confidence interval [CI], 5.5%-6.0%) had ARF during their ICU stay, including 1260 who were treated with RRT. The most common contributing factor to ARF was septic shock (47.5%; 95% CI, 45.2%-49.5%). Approximately 30% of patients had preadmission renal dysfunction. Overall hospital mortality was 60.3% (95% CI, 58.0%-62.6%). Dialysis dependence at hospital discharge was 13.8% (95% CI, 11.2%-16.3%) for survivors. Independent risk factors for hospital mortality included use of vasopressors (odds ratio [OR], 1.95; 95% CI, 1.50-2.55; P<.001), mechanical ventilation (OR, 2.11; 95% CI, 1.58-2.82; P<.001), septic shock (OR, 1.36; 95% CI, 1.03-1.79; P = .03), cardiogenic shock (OR, 1.41; 95% CI, 1.05-1.90; P = .02), and hepatorenal syndrome (OR, 1.87; 95% CI, 1.07-3.28; P = .03). In this multinational study, the period prevalence of ARF requiring RRT in the ICU was between 5% and 6% and was associated with a high hospital mortality rate.


Development of methods for avian oil toxicity studies using the double crested cormorant (Phalacrocorax auritus).
Oral and external dosing methods replicating field exposure were developed using the double crested cormorant (DCCO) to test the toxicity of artificially weathered Deepwater Horizon Mississippi Canyon 252 oil. The majority of previous oil dosing studies conducted on wild-caught birds used gavage methods to dose birds with oil and determine toxicity. However, rapid gut transit time of gavaged oil likely reduces oil absorption. In the present studies, dosing relied on injection of oil into live feeder fish for oral dosing of these piscivorous birds, or applying oil to body contour feathers resulting in transdermal oil exposure and oral exposure through preening. Both oral and external oil dosing studies identified oil-related toxicity endpoints associated with oxidative stress such as hemolytic anemia, liver and kidney damage, and immuno-modulation or compromise. External oil application allowed for controlled study of thermoregulatory stress as well. Infrared thermal images indicated significantly greater surface temperatures and heat loss in treated birds following external oil applications; however, measurements collected by coelomically implanted temperature transmitters showed that internal body temperatures were stable over the course of the study period. Birds exposed to oil externally consumed more fish than control birds, indicating metabolic compensation for thermal stress. Conversely, birds orally dosed with oil experienced hypothermia and consumed less fish compared to control birds.


An Outward-Facing Aromatic Amino Acid Is Crucial for Signaling between the Membrane-Spanning and Nucleotide-Binding Domains of Multidrug Resistance Protein 1 (MRP1; ABCC1).
The 190-kDa human MRP1 is an ATP-binding cassette multidrug and multiorganic anion efflux transporter. The 17 transmembrane helices of its three membrane-spanning domains, together with its two nucleotide binding domains (NBDs), form a stabilizing network of domain-domain interactions that ensure substrate binding in the cytoplasm is efficiently coupled to ATP binding and hydrolysis to effect solute efflux into the extracellular milieu. Here we show that Ala substitution of Phe583 in an outward-facing loop between the two halves of the transporter essentially eliminates the binding of multiple organic anions by MRP1. Conservative substitutions with Trp and Tyr had little or no effect. The F583A mutation also caused a substantial increase in orthovanadate-induced trapping of azidoADP by the cytoplasmic NBDs of MRP1, although the binding of ATP was unaffected. These observations indicate that the loss of the aromatic side chain at position 583 impairs the release of ADP and thus effectively locks the transporter in a low-affinity solute binding state. Phe583 is the first outward-facing amino acid in MRP1 found to be critical for its transport function. Our data provide evidence for long-range coupling, presumably via allosteric interaction, between this outward-facing region of MRP1 and both the solute binding and nucleotide binding regions of the transporter. Cryoelectron microscopy structural and homology models of MRP1 indicate that the orientation of the Phe583 side chain is altered by ATP binding but are currently unable to provide insights into the molecular mechanism by which this long-range signaling is propagated.


A new analysis of Mars "Special Regions": findings of the second MEPAG Special Regions Science Analysis Group (SR-SAG2).
A committee of the Mars Exploration Program Analysis Group (MEPAG) has reviewed and updated the description of Special Regions on Mars as places where terrestrial organisms might replicate (per the COSPAR Planetary Protection Policy). This review and update was conducted by an international team (SR-SAG2) drawn from both the biological science and Mars exploration communities, focused on understanding when and where Special Regions could occur. The study applied recently available data about martian environments and about terrestrial organisms, building on a previous analysis of Mars Special Regions (2006) undertaken by a similar team. Since then, a new body of highly relevant information has been generated from the Mars Reconnaissance Orbiter (launched in 2005) and Phoenix (2007) and data from Mars Express and the twin Mars Exploration Rovers (all 2003). Results have also been gleaned from the Mars Science Laboratory (launched in 2011). In addition to Mars data, there is a considerable body of new data regarding the known environmental limits to life on Earth-including the potential for terrestrial microbial life to survive and replicate under martian environmental conditions. The SR-SAG2 analysis has included an examination of new Mars models relevant to natural environmental variation in water activity and temperature; a review and reconsideration of the current parameters used to define Special Regions; and updated maps and descriptions of the martian environments recommended for treatment as "Uncertain" or "Special" as natural features or those potentially formed by the influence of future landed spacecraft. Significant changes in our knowledge of the capabilities of terrestrial organisms and the existence of possibly habitable martian environments have led to a new appreciation of where Mars Special Regions may be identified and protected. The SR-SAG also considered the impact of Special Regions on potential future human missions to Mars, both as locations of potential resources and as places that should not be inadvertently contaminated by human activity.


What Are the Long-term Results of MUTARS® Modular Endoprostheses for Reconstruction of Tumor Resection of the Distal Femur and Proximal Tibia?
Modular endoprostheses are commonly used to reconstruct defects of the distal femur and proximal tibia after bone tumor resection. Because limb salvage surgery for bone sarcomas is relatively new, becoming more frequently used since the 1980s, studies focusing on the long-term results of such prostheses in treatment of primary tumors are scarce. (1) What proportion of patients experience a mechanical complication with the MUTARS® modular endoprosthesis when used for tumor reconstruction around the knee, and what factors may be associated with mechanical failure? (2) What are the nonmechanical complications? (3) What are the implant failure rates at 5, 10, and 15 years? (4) How often is limb salvage achieved using this prosthesis? Between 1995 and 2010, endoprostheses were the preferred method of reconstruction after resection of the knee in adolescents and adults in our centers. During that period, we performed 114 MUTARS® knee replacements in 105 patients; no other endoprosthetic systems were used. Four patients (four of 105 [4%]) were lost to followup, leaving 110 reconstructions in 101 patients for review. The reverse Kaplan-Meier method was used to calculate median followup, which was equal to 8.9 years (95% confidence interval [CI], 8.0-9.7). Mean age at surgery was 36 years (range, 13-82 years). Predominant diagnoses were osteosarcoma (n = 56 [55%]), leiomyosarcoma of bone (n = 10 [10%]), and chondrosarcoma (n = 9 [9%]). In the early period of our study, we routinely used uncemented uncoated implants for primary reconstructions. Later, hydroxyapatite (HA)-coated implants were the standard. Eighty-nine reconstructions (89 of 110 [81%]) were distal femoral replacements (78 uncemented [78 of 89 {88%}, 42 of which were HA-coated [42 of 78 {54%}]) and 21 (21 of 110 [19%]) were proximal tibial replacements. In 26 reconstructions (26 of 110 [24%]), the reconstruction was performed for a failed previous reconstruction. We used a competing risk model to estimate the cumulative incidence of implant failure. Complications of soft tissue or instability occurred in seven reconstructions (seven of 110 [6%]). With the numbers we had, for uncemented distal femoral replacements, we could not detect a difference in loosening between revision (five of 17 [29%]) and primary reconstructions (eight of 61 [13%]) (hazard ratio [HR], 1.72; 95% CI, 0.55-5.38; p = 0.354). Hydroxyapatite-coated uncemented implants had a lower risk of loosening (two of 42 [5%]) than uncoated uncemented implants (11 of 36 [31%]) (HR, 0.23; 95% CI, 0.05-1.06; p = 0.060). Structural complications occurred in 15 reconstructions (15 of 110 [14%]). Infections occurred in 14 reconstructions (14 of 110 [13%]). Ten patients had a local recurrence (10 of 101 [10%]). With failure for mechanical reasons as the endpoint, the cumulative incidences of implant failure at 5, 10, and 15 years were 16.9% (95% CI, 9.6-24.2), 20.7% (95% CI, 12.5-28.8%), and 37.9% (95% CI, 16.1-59.7), respectively. We were able to salvage some of the failures so that at followup, 90 patients (90 of 101 [89%]) had a MUTARS® in situ. Although no system has yet proved ideal to restore normal function and demonstrate long-term retention of the implant, MUTARS® modular endoprostheses represent a reliable long-term option for knee replacement after tumor resection, which seems to be comparable to other modular implants available to surgeons. Although the number of patients is relatively small, we could demonstrate that with this prosthesis, an uncemented HA-coated implant is useful in achieving durable fixation. Level IV, therapeutic study.


Heterogeneity and clinical significance of ETV1 translocations in human prostate cancer.
A fluorescence in situ hybridisation (FISH) assay has been used to screen for ETV1 gene rearrangements in a cohort of 429 prostate cancers from patients who had been diagnosed by trans-urethral resection of the prostate. The presence of ETV1 gene alterations (found in 23 cases, 5.4%) was correlated with higher Gleason Score (P=0.001), PSA level at diagnosis (P=<0.0001) and clinical stage (P=0.017) but was not linked to poorer survival. We found that the six previously characterised translocation partners of ETV1 only accounted for 34% of ETV1 re-arrangements (eight out of 23) in this series, with fusion to the androgen-repressed gene C15orf21 representing the commonest event (four out of 23). In 5'-RACE experiments on RNA extracted from formalin-fixed tissue we identified the androgen-upregulated gene ACSL3 as a new 5'-translocation partner of ETV1. These studies report a novel fusion partner for ETV1 and highlight the considerable heterogeneity of ETV1 gene rearrangements in human prostate cancer.


2014 MERS-CoV outbreak in Jeddah--a link to health care facilities.
A marked increase in the number of cases of Middle East respiratory syndrome coronavirus (MERS-CoV) infection occurred in Jeddah, Saudi Arabia, in early 2014. We evaluated patients with MERS-CoV infection in Jeddah to explore reasons for this increase and to assess the epidemiologic and clinical features of this disease. We identified all cases of laboratory-confirmed MERS-CoV infection in Jeddah that were reported to the Saudi Arabian Ministry of Health from January 1 through May 16, 2014. We conducted telephone interviews with symptomatic patients who were not health care personnel, and we reviewed hospital records. We identified patients who were reported as being asymptomatic and interviewed them regarding a history of symptoms in the month before testing. Descriptive analyses were performed. Of 255 patients with laboratory-confirmed MERS-CoV infection, 93 died (case fatality rate, 36.5%). The median age of all patients was 45 years (interquartile range, 30 to 59), and 174 patients (68.2%) were male. A total of 64 patients (25.1%) were reported to be asymptomatic. Of the 191 symptomatic patients, 40 (20.9%) were health care personnel. Among the 151 symptomatic patients who were not health care personnel, 112 (74.2%) had data that could be assessed, and 109 (97.3%) of these patients had had contact with a health care facility, a person with a confirmed case of MERS-CoV infection, or someone with severe respiratory illness in the 14 days before the onset of illness. The remaining 3 patients (2.7%) reported no such contacts. Of the 64 patients who had been reported as asymptomatic, 33 (52%) were interviewed, and 26 of these 33 (79%) reported at least one symptom that was consistent with a viral respiratory illness. The majority of patients in the Jeddah MERS-CoV outbreak had contact with a health care facility, other patients, or both. This highlights the role of health care-associated transmission. (Supported by the Ministry of Health, Saudi Arabia, and by the U.S. Centers for Disease Control and Prevention.).


Class III PI3K regulates organismal glucose homeostasis by providing negative feedback on hepatic insulin signalling.
Defective hepatic insulin receptor (IR) signalling is a pathogenic manifestation of metabolic disorders including obesity and diabetes. The endo/lysosomal trafficking system may coordinate insulin action and nutrient homeostasis by endocytosis of IR and the autophagic control of intracellular nutrient levels. Here we show that class III PI3K--a master regulator of endocytosis, endosomal sorting and autophagy--provides negative feedback on hepatic insulin signalling. The ultraviolet radiation resistance-associated gene protein (UVRAG)-associated class III PI3K complex interacts with IR and is stimulated by insulin treatment. Acute and chronic depletion of hepatic Vps15, the regulatory subunit of class III PI3K, increases insulin sensitivity and Akt signalling, an effect that requires functional IR. This is reflected by FoxO1-dependent transcriptional defects and blunted gluconeogenesis in Vps15 mutant cells. On depletion of Vps15, the metabolic syndrome in genetic and diet-induced models of insulin resistance and diabetes is alleviated. Thus, feedback regulation of IR trafficking and function by class III PI3K may be a therapeutic target in metabolic conditions of insulin resistance.


Experimental airborne transmission of porcine postweaning multisystemic wasting syndrome.
The objective of these studies was to investigate if porcine postweaning multisystemic wasting syndrome (PMWS) could be induced in healthy pigs following contact with air from pigs with clinical signs of PMWS. The pigs were housed in different units. Either 31 (study I) or 25 (study II) pigs with clinical symptoms of PMWS from a PMWS-affected herd and 25 healthy pigs from a PMWS-free, but PCV2-positive, herd were housed in unit A. Fifty pigs from a PMWS-free herd were housed in unit B, which were connected by pipes to unit A. In unit C, 30 pigs from a PMWS-free herd were housed as controls. In study II, the pigs in units A and B from the PMWS-free herd developed clinical signs of PMWS 2-3 weeks after arrival. PMWS was confirmed at necropsy and the diseased pigs had increased PCV2 load and increased antibody titers against PCV2 in serum that coincided with the development of clinical signs typical of PMWS. Sequence analysis revealed that the PCV2 isolate belonged to genotype 2b. In conclusion, the present study showed that PMWS can be induced in pigs from a PMWS-free herd by airborne contact with pigs from a PMWS-affected herd.


The affirmation of self: a new perspective on the immune system.
The fundamental concepts of autopoiesis, which emphasize the circular organization underlying both living organisms and cognition, have been criticized on the grounds that since they are conceived as a tight logical chain of definitions and implications, it is often not clear whether they are indeed a scientific theory or rather just a potential scientific vocabulary of doubtful utility to working scientists. This article presents the deployment of the concepts of autopoiesis in the field of immunology, a discipline where working biologists themselves spontaneously have long had recourse to "cognitive" metaphors: "recognition"; a "repertoire" of recognized molecular shapes; "learning" and "memory"; and, most striking of all, a "self versus non-self" distinction. It is shown that in immunology, the concepts of autopoiesis can be employed to generate clear novel hypotheses, models demonstrating these ideas, testable predictions, and novel therapeutic procedures. Epistemologically, it is shown that the self-non-self distinction, while quite real, is misleadingly named. When a real mechanism for generating this distinction is identified, it appears that the actual operational distinction is between (a) a sufficiently numerous set of initial antigens, present from the start of ontogeny, in conditions that allow for their participation in the construction of the system's organization and operation, and (b) single antigens that are first presented to the system after two successive phases of maturation. To call this a self-non-self distinction obscures the issue by presupposing what it ought to be the job of scientific investigation to explain.


A Web-Based Application for Personalized Ecological Momentary Assessment in Psychiatric Care: User-Centered Development of the PETRA Application.
Smartphone self-monitoring of mood, symptoms, and contextual factors through ecological momentary assessment (EMA) provides insights into the daily lives of people undergoing psychiatric treatment. Therefore, EMA has the potential to improve their care. To integrate EMA into treatment, a clinical tool that helps clients and clinicians create personalized EMA diaries and interpret the gathered data is needed. This study aimed to develop a web-based application for personalized EMA in specialized psychiatric care in close collaboration with all stakeholders (ie, clients, clinicians, researchers, and software developers). The participants were 52 clients with mood, anxiety, and psychotic disorders and 45 clinicians (psychiatrists, psychologists, and psychiatric nurses). We engaged them in interviews, focus groups, and usability sessions to determine the requirements for an EMA web application and repeatedly obtained feedback on iteratively improved high-fidelity EMA web application prototypes. We used human-centered design principles to determine important requirements for the web application and designed high-fidelity prototypes that were continuously re-evaluated and adapted. The iterative development process resulted in Personalized Treatment by Real-time Assessment (PETRA), which is a scientifically grounded web application for the integration of personalized EMA in Dutch clinical care. PETRA includes a decision aid to support clients and clinicians with constructing personalized EMA diaries, an EMA diary item repository, an SMS text message-based diary delivery system, and a feedback module for visualizing the gathered EMA data. PETRA is integrated into electronic health record systems to ensure ease of use and sustainable integration in clinical care and adheres to privacy regulations. PETRA was built to fulfill the needs of clients and clinicians for a user-friendly and personalized EMA tool embedded in routine psychiatric care. PETRA is unique in this codevelopment process, its extensive but user-friendly personalization options, its integration into electronic health record systems, its transdiagnostic focus, and its strong scientific foundation in the design of EMA diaries and feedback. The clinical effectiveness of integrating personalized diaries via PETRA into care requires further research. As such, PETRA paves the way for a systematic investigation of the utility of personalized EMA for routine mental health care.


Pomegranate Extract Improves Maximal Performance of Trained Cyclists after an Exhausting Endurance Trial: A Randomised Controlled Trial.
The efficacy of pomegranate (Punica granatum) extract (PE) for improving performance and post-exercise recovery in an active population was equivocal in previous studies. In this study, a randomised, double-blinded, placebo-controlled, balanced, cross-over trial with two arms was conducted. Eligibility criteria for participants were as follows: male, amateur cyclist, with a training routine of 2 to 4 sessions per week (at least one hour per session). The cyclists (n = 26) were divided into treatment (PE) and placebo (PLA) groups for a period of 15 days. After physical tests, the groups were exchanged after a 14-day washout period. Exercise tests consisted of endurance bouts (square-wave endurance exercise test followed by an incremental exercise test to exhaustion) and an eccentric exercise drill. The objective was to assess the efficacy of PE in performance outcomes and post-exercise muscular recovery and force restoration after a prolonged submaximal effort. Twenty-six participants were included for statistical analysis. There was a statistically significant difference in total time to exhaustion (TTE)(17.66⁻170.94 s, p < 0.02) and the time to reach ventilatory threshold 2 (VT2)(26.98⁻82.55 s, p < 0.001), with greater values for the PE compared to the PLA group. No significant results were obtained for force restoration in the isokinetic unilateral low limb test. PE, after a prolonged submaximal effort, may be effective in improving performance outcomes at maximal effort and might help to restore force in the damaged muscles.


How are "teaching the teachers" courses in evidence based medicine evaluated? A systematic review.
Teaching of evidence-based medicine (EBM) has become widespread in medical education. Teaching the teachers (TTT) courses address the increased teaching demand and the need to improve effectiveness of EBM teaching. We conducted a systematic review of assessment tools for EBM TTT courses. To summarise and appraise existing assessment methods for teaching the teachers courses in EBM by a systematic review. We searched PubMed, BioMed, EmBase, Cochrane and Eric databases without language restrictions and included articles that assessed its participants. Study selection and data extraction were conducted independently by two reviewers. Of 1230 potentially relevant studies, five papers met the selection criteria. There were no specific assessment tools for evaluating effectiveness of EBM TTT courses. Some of the material available might be useful in initiating the development of such an assessment tool. There is a need for the development of educationally sound assessment tools for teaching the teachers courses in EBM, without which it would be impossible to ascertain if such courses have the desired effect.


Inflammation-mediating cytokine response to acute handcycling exercise with/without functional electrical stimulation-evoked lower-limb cycling.
This feasibility study compared the plasma inflammation-mediating cytokine response to an acute bout of handcycling (HC) with and without the addition of functional electrical stimulation (FES)-evoked lower-limb cycling. On two separate occasions, five recreationally active, community-based participants with motor complete paraplegia (thoracic 5- 7) performed 30 min HC and hybrid exercise (HYB) at a fixed power output. Venous blood samples were collected at rest, immediately postexercise, 1 h postexercise (post+1) and 2 h postexercise (post+2). Plasma interleukin (IL)-6, IL-10, IL-1 receptor antagonist (IL-1ra), adrenaline, and cortisol concentrations were determined via enzyme-linked immunoassay. Plasma IL-6 concentrations were significantly (p < 0.04) elevated (~2.5-fold) at post+1 and post+2 in HYB only. A small (0.5-fold), nonsignificant (p > 0.05) increase in IL-6 was observed at post+1 in HC, with concentrations significantly higher in HYB at post+2 (p < 0.02). Plasma IL-1ra was unaffected in both trials. Although not reaching statistical significance (p = 0.15), a ~1-fold increase in IL-10 concentration was seen in HYB at post+2. In contrast, increases in adrenaline (p < 0.04) and cortisol (p = 0.08) were observed immediately postexercise in HC and HYB. Initial findings suggest paralyzed skeletal muscle releases IL-6 in response to FES-evoked contractions. HYB may provide a greater anti-inflammatory potential in individuals with a thoracic spinal cord injury compared with HC alone.


[Soil soluble organic nitrogen content in different forest stands in Wanmulin Nature Reserve].
An investigation was made on the soil soluble organic nitrogen (SON) in two natural forests Altingia gracilies (ALG) and Castanopsis carlesii (CAC) and an adjacent 35-year-old Cunninghamia lanceolata (CUL) plantation in Wanmulin Nature Reserve in Jianou, Fujian Province. Among the three forest stands, ALG had a significantly higher content of soil SON, being 95.3, 78.3, and 72.5 mg x kg(-1) in 0-5 cm, 5-10 cm, and 10-20 cm soil layers, respectively (P<0.05), while CAC and CUL had lesser differences in their soil SON content, which was 74.5, 70.1, and 65.6 mg x kg(-1) in the three soil layers for CAC, and 78.6, 68.9, and 69.1 mg x kg(-1) in the three soil layers for CUL, respectively. The proportion of SON to total soluble nitrogen (TSN) in 0-20 cm soil layer was 79.17-80.78% for CAC, 68.64%-74.51% for CUL, and 59.97%-69.66% for ALG. With increasing soil depth, the proportion of soil SON to soil TSN and total nitrogen (TN) for the three forest stands all increased. Soil SON content had a significant positive correlation with soil organic carbon (r=0.982, P<0.01), and also, had definite correlations with soil TSN, TN, NH4(+)-N, and NO3(-)-N. It also correlated with the factors such as forest type, topography, altitude, and tree age.


Is cohabitation as a form of union formation a licence to intimate partner physical violence in Uganda?
The study examined the argument that cohabitation as a form of union increases physical violence victimization among women. The study's aim was to assess the association between physical violence and other socio-demographic factors that influence physical violence among women. Self-reported data were extracted from the 2016 Uganda Demographic and Health Survey (UDHS), with a sample of 2479 couples, from the couple file. Chi-squared tests and multivariate Firth-logit regression models were used to examine the relationship between intimate partner violence (IPV) victimization and marital status controlling for other social-demographic factors. There was no significant evidence that women in cohabiting union have a higher risk of exposure to physical violence in the Ugandan context. The risk of experiencing physical violence perpetration varied by birth cohort, with the most recent cohorts exhibiting a slightly higher risk of experiencing partner violence than previous cohorts. Significant factors found to be associated with an increased risk of experiencing IPV included being in the poorer, middle and richer compared with the poorest wealth tertile of income, residing in Eastern or Northern regions compared with the Central region, being affiliated to the Catholic faith compared with Anglican and having five or more children compared with 4 or fewer children. In conclusion, there is no evidence that physical violence is more pronounced among women in cohabiting unions compared with married women in Uganda.


Modified Blalock Taussig shunt: a not-so-simple palliative procedure.
Thirty-two consecutive isolated modified Blalock Taussig (BT) shunts performed in infancy since 2004 were reviewed and analysed to identify the risk factors for shunt intervention and mortality. Sternotomy was the only approach used. Median age and weight were 10.5 (range 1-74) days and 2.9 (1.9-4.4) kg, respectively. Shunt palliation was performed for biventricular hearts (Tetralogy of Fallot/double outlet right ventricle/transposition of great arteries_ventricular septal defect_pulmonary stenosis/pulmonary atresia_ventricular septal defect/others) in 21, and univentricular hearts in 11, patients. Hypoplastic left heart syndrome patients were excluded. Two procedures required cardiopulmonary bypass. Median shunt size was 3.5 (3-4) mm and median shunt size/kg body weight was 1.2 (0.9-1.7) mm/kg. Reduction in shunt size was necessary in 5 of 32 (16%) patients. Three of 32 (9%) patients died after 3 (1-15) days due to cardiorespiratory decompensation. Lower body weight (P = 0.04) and bigger shunt size/kg of body weight (P = 0.004) were significant risk factors for mortality. Acute shunt thrombosis was observed in 3 of 32 (9%), none leading to death. Need for cardiac decongestive therapy was associated with univentricular hearts (P < 0.001), bigger shunt size (P = 0.054) and longer hospital stay (P = 0.005). Twenty-eight patients have undergone a successful shunt takedown at a median age of 5.5 (0.5-11.9) months, without late mortality. Palliation with a modified BT shunt continues to be indicated despite increased thrust on primary corrective surgery. Though seemingly simple, it is associated with significant morbidity and mortality. Effective over-shunting and acute shunt thrombosis are the lingering problems of shunt therapy.


A test of climate, sun, and culture relationships from an 1810-year Chinese cave record.
A record from Wanxiang Cave, China, characterizes Asian Monsoon (AM) history over the past 1810 years. The summer monsoon correlates with solar variability, Northern Hemisphere and Chinese temperature, Alpine glacial retreat, and Chinese cultural changes. It was generally strong during Europe's Medieval Warm Period and weak during Europe's Little Ice Age, as well as during the final decades of the Tang, Yuan, and Ming Dynasties, all times that were characterized by popular unrest. It was strong during the first several decades of the Northern Song Dynasty, a period of increased rice cultivation and dramatic population increase. The sign of the correlation between the AM and temperature switches around 1960, suggesting that anthropogenic forcing superseded natural forcing as the major driver of AM changes in the late 20th century.


Both JNK and P38 MAPK pathways participate in the protection by dexmedetomidine against isoflurane-induced neuroapoptosis in the hippocampus of neonatal rats.
Dexmedetomidine, a highly selective α2-adrenergic agonist, has been reported to attenuate isoflurane-induced cognitive impairment and neuroapoptosis. However, the underlying molecular mechanisms remain poorly understood. The aim of this study was to investigate whether mitogen-activated protein kinase (MAPK) pathway was involved in dexmedetomidine-induced neuroprotection against isoflurane effects. Seven-day-old (P7) neonatal Sprague-Dawley rats were pretreated with various concentrations of dexmedetomidine, and then exposed to 0.75% isoflurane or air for 6h. Terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL) was used to detect neuronal apoptosis in their hippocampus. Activated caspase-3, extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH2-terminal kinases (JNK), p38, phospho-ERK1/2, phospho-JNK and phospho-p38 proteins were detected by Western blotting in the hippocampus at the end of exposure. Also, P7 rats were pretreated with 75 μg/kg dexmedetomidine alone, or given the ERK inhibitor U0126 before dexmedetomidine pretreatment, or pretreated with the p38 MAPK inhibitor SB203580 or JNK inhibitor SP600125 alone, and then exposed to 0.75% isoflurane for 6h. Isoflurane induced significant neuroapoptosis, increased the protein expression of phospho-JNK, phospho-c-Jun, phospho-p38 and phospho-nuclear factor-κB (NF-κB), decreased the level of phospho-ERK1/2 protein and reduced the ratio of Bcl-2/Bax in the hippocampus. Dexmedetomidine pretreatment inhibited isoflurane-induced neuroapoptosis and restored proteins expression of MAPK pathways and the Bcl-2/Bax ratio after isoflurane exposure. Moreover, SB203580 and SP600125 also partly attenuated the isoflurane-induced protein changes. However, U0126 did not reverse dexmedetomidine-induced neuroprotection. Our results indicate that the JNK and p38 pathways, not the ERK pathway are involved in dexmedetomidine-induced neuroprotection against isoflurane effects.


An East-West approach to the management of central post-stroke pain.
The development of neuropathic pain following stroke is not uncommon. The consequences include significant disabilities and depression. Treatment can often be difficult and responses unsatisfactory. We report a patient with severe central post-stroke pain (CPSP) of the right leg benefiting from a combination of Western multidisciplinary therapies AND acupuncture. A literature search has revealed that relatively few studies have been done on the management of CPSP, compared with other types of neuropathic pain. Amitriptyline and carbamazepine were found to produce positive effects on post-stroke pain in one small study; lamotrigine and gabapentin are two newer drugs which appear promising. To the best of our knowledge, the use of acupuncture for the treatment of CPSP has not been previously reported.


[The role of the personality in the feeding behavior disorders].
Personality disorders are common in eating disorders and preliminary reports indicate that this type of disorders implicate a poor prognosis in anorexia and bulimia nervosa. Cluster C personality disorders, particularly avoidant and dependent personality disorder are the most frequent in anorexia nervosa. In bulimia nervosa, however, cluster B personality disorders, including borderline and histrionic personality disorders, are more frequent. Furthermore, temperament could differentiate anorectic patients from bulimic patients. Eating disorders seem to be associated with high scores in neuroticism and harm avoidance. However, while anorexia nervosa patients might have higher persistence, bulimia nervosa patients seem to have an impulsive temperament. According to the character model of Cloninger, both anorexia and bulimia present lower scores in the dimension self-directedness.


Visualization of bronchial circulation at bronchial anastomotic site using bronchial fluorescein angiography technique.
Successful bronchial healing after a bronchoplastic procedure mainly depends on bronchial circulation at the anastomostic site. We developed a bronchial fluorescein angiography (B-FAG) technique for visualizing circulation on the bronchial surface. The technique was evaluated in animals. Fluorescein was used as a contrast agent and an autofluorescence imaging (AFI) bronchoscope as a detector. The left main pulmonary artery (PA) and main bronchus of 10 pigs were isolated. After transection of the left main bronchus and bronchial arteries and re-anastomosis of the bronchus, the pigs were randomly divided into two groups: the PA- group (n = 5), in which the pulmonary artery was transected; and the PA+ group (n = 5), in which the pulmonary artery was preserved. Following intravenous injection of fluorescein, the distal anastomotic site was observed for 30 min with autofluorescence imaging bronchoscopy. Bronchial specimens sampled 2 days after the surgical intervention were histologically evaluated. In the PA- group, there was no fluorescein enhancement in the distal bronchus throughout the observation time. However, enhancement, which turned the bronchial surface from magenta to bright green, was clearly observed in less than 207 ± 102.5 s in the PA+ group. The enhancement status detected by bronchial fluorescein angiography was related to the extent of tissue damage, as was proven histologically in the acute healing stage. Bronchial fluorescein angiography clearly visualized the circulatory status promptly after the anastomosis procedure at the central bronchus. This technique is a potentially practical approach to predict ischaemic airway complications following bronchial anastomosis.


Sim4cc: a cross-species spliced alignment program.
Advances in sequencing technologies have accelerated the sequencing of new genomes, far outpacing the generation of gene and protein resources needed to annotate them. Direct comparison and alignment of existing cDNA sequences from a related species is an effective and readily available means to determine genes in the new genomes. Current spliced alignment programs are inadequate for comparing sequences between different species, owing to their low sensitivity and splice junction accuracy. A new spliced alignment tool, sim4cc, overcomes problems in the earlier tools by incorporating three new features: universal spaced seeds, to increase sensitivity and allow comparisons between species at various evolutionary distances, and powerful splice signal models and evolutionarily-aware alignment techniques, to improve the accuracy of gene models. When tested on vertebrate comparisons at diverse evolutionary distances, sim4cc had significantly higher sensitivity compared to existing alignment programs, more than 10% higher than the closest competitor for some comparisons, while being comparable in speed to its predecessor, sim4. Sim4cc can be used in one-to-one or one-to-many comparisons of genomic and cDNA sequences, and can also be effectively incorporated into a high-throughput annotation engine, as demonstrated by the mapping of 64,000 Fagus grandifolia 454 ESTs and unigenes to the poplar genome.


Micro-shear bond strength of adhesive resins to enamel at different relative humidity conditions.
This study tested whether exhaled humid conditions would affect the adhesion of etch-and-rinse, two-step and one-step self-etch adhesive resins to enamel. Enamel surfaces of human maxillary anterior teeth (N=240, n=20) were exposed to four humid conditions (H1: 63-68%, H2: 73-78%, H3: 93-98%, H4: 36-45% RH) during bonding with Adper Single Bond 2 (SB2), Clearfil SE Bond (CSE) and Adper Easy Bond (AEB). Specimens were stored in distilled water at 37 ºC for 24 h and tested to failure using micro-shear bond strength (μSBS) test. Data were analyzed using two-way ANOVA and Tukey's test (p<0.05). The μSBS to enamel with SB2, CSE and AEB was not significantly affected by humidity parameters. AEB resulted in significantly lower μSBS in all conditions. The frequency of adhesive failures was the highest at H2, H3 for SB2, H3 for CSE and H1-4 for AEB indicating that humidity conditions may decrease adhesion quality to enamel.


The functions of nonsuicidal self-injury: converging evidence for a two-factor structure.
Research has identified more than a dozen functions of non-suicidal self-injury (NSI), but the conceptual and empirical overlap among these functions remains unclear. The present study examined the structure of NSI functions in two large samples of patients receiving acute-care treatment for NSI. Two different measures of NSI functions were utilized to maximize generalizability of findings: one sample (n = 946) was administered the Inventory of Statements About Self-injury (ISAS; Klonsky and Glenn in J Psychopathol Behav Assess 31:215-219, 2009), and a second sample (n = 211) was administered the Functional Assessment of Self-Mutilation (FASM; Lloyd et al. in Self-mutilation in a community sample of adolescents: descriptive characteristics and provisional prevalence rates. Poster session at the annual meeting of the Society for Behavioral Medicine, New Orleans, LA, 1997). Exploratory factor analyses revealed that both measures exhibited a robust two-factor structure: one factor represented Intrapersonal functions, such as affect regulation and anti-dissociation, and a second factor represented Social functions, such as interpersonal influence and peer bonding. In support of the two-factor structure's construct validity, the factors exhibited a pattern of correlations with indicators of NSI severity that was consistent with past research and theory. Findings have important implications for theory, research, and treatment. In particular, the two-factor framework should guide clinical assessment, as well as future research on the implications of NSI functions for course, prognosis, treatment, and suicide risk.


The Development of Irritable Bowel Syndrome: A Prospective Community-Based Cohort Study.
It remains controversial whether psychosocial burden is an independent predictor of irritable bowel syndrome (IBS) or occurs concurrently as an epiphenomenon. Here we prospectively examine the individual contribution of psychosocial risk factors, demographic factors, somatic symptoms, and gastrointestinal infection within a non-clinical, IBS-free population before infection occurred. A prospective community-based cohort study including a consecutive sample of healthy participants with an elevated risk of developing gastrointestinal infection during long-distance travel was conducted. Potential predictive factors were investigated using validated self-report scales pre-travel, 1 week after return, and 7 months post-travel. IBS was assessed using the ROME-III Diagnostic Questionnaire. Of the 1,964 eligible long-distance travelers, 1,464 responded at follow-up directly after their journey, and 1,190 participants completed the study 7 months post-journey. Fifty-three percent of study completers were female, mean age was 39.9 (s.d.=15.7) years. The mean travel duration was 40.8 (s.d.=52.8) days, and 43.3% (95% confidence interval (CI)=40.4-46.1%) of participants experienced at least moderate infectious travelers' diarrhea. The incidence of newly developed IBS 7 months post-travel was 7.2% (95%CI=5.8-8.6%). In multivariate analyses, female gender, vulnerability to diarrhea under stress, baseline somatic symptom burden, baseline illness anxiety, diarrhea within the 4 months pre-travel, and travelers' diarrhea during the journey significantly predicted IBS post-travel. This study indicates that gastrointestinal infection as well as predisposing factors such as female gender, vulnerability to diarrhea under stress, illness anxiety, and somatic symptom burden predict the development of IBS. The results indicate the necessity of simultaneously addressing both somatic and psychological needs in patients with IBS as early as possible.


The utility of pitch elevation in the evaluation of oropharyngeal Dysphagia: preliminary findings.
To evaluate the utility of a pitch elevation task in the assessment of oropharyngeal dysphagia. This study was a pilot prospective cohort study including 40 consecutive patients (16 male and 24 female) who were referred by their physician for a swallowing evaluation. Patients were evaluated with a noninstrumental clinical examination and a videofluoroscopic swallow study, and participated in a pitch elevation task during videofluoroscopic image acquisition. Relationships between pitch elevation measurements (acoustic and perceptual) and swallow parameters (penetration/aspiration and residue) were investigated. Results of this pilot study revealed that both maximum fundamental frequency (F(0)) and perceptual evaluation of pitch elevation independently significantly predicted Penetration-Aspiration Scale scores for thin liquid swallows (p = .01 and .03, respectively). Vocal range (average pitch to falsetto) was not sensitive in predicting likelihood of oropharyngeal dysphagia. Findings indicate that reduced pitch elevation can be indicative of reduced airway protection and swallowing impairment in some dysphagia patients and may be a useful supplement to dysphagia screening and diagnosis. Further investigation is warranted to determine the optimal utility of this procedure for different diagnostic categories of patients.


Profiling oil sands mixtures from industrial developments and natural groundwaters for source identification.
The objective of this study was to identify chemical components that could distinguish chemical mixtures in oil sands process-affected water (OSPW) that had potentially migrated to groundwater in the oil sands development area of northern Alberta, Canada. In the first part of the study, OSPW samples from two different tailings ponds and a broad range of natural groundwater samples were assessed with historically employed techniques as Level-1 analyses, including geochemistry, total concentrations of naphthenic acids (NAs) and synchronous fluorescence spectroscopy (SFS). While these analyses did not allow for reliable source differentiation, they did identify samples containing significant concentrations of oil sands acid-extractable organics (AEOs). In applying Level-2 profiling analyses using electrospray ionization high resolution mass spectrometry (ESI-HRMS) and comprehensive multidimensional gas chromatography time-of-flight mass spectrometry (GC × GC-TOF/MS) to samples containing appreciable AEO concentrations, differentiation of natural from OSPW sources was apparent through measurements of O2:O4 ion class ratios (ESI-HRMS) and diagnostic ions for two families of suspected monoaromatic acids (GC × GC-TOF/MS). The resemblance between the AEO profiles from OSPW and from 6 groundwater samples adjacent to two tailings ponds implies a common source, supporting the use of these complimentary analyses for source identification. These samples included two of upward flowing groundwater collected <1 m beneath the Athabasca River, suggesting OSPW-affected groundwater is reaching the river system.


Genetic dissection of the α-globin super-enhancer in vivo.
Many genes determining cell identity are regulated by clusters of Mediator-bound enhancer elements collectively referred to as super-enhancers. These super-enhancers have been proposed to manifest higher-order properties important in development and disease. Here we report a comprehensive functional dissection of one of the strongest putative super-enhancers in erythroid cells. By generating a series of mouse models, deleting each of the five regulatory elements of the α-globin super-enhancer individually and in informative combinations, we demonstrate that each constituent enhancer seems to act independently and in an additive fashion with respect to hematological phenotype, gene expression, chromatin structure and chromosome conformation, without clear evidence of synergistic or higher-order effects. Our study highlights the importance of functional genetic analyses for the identification of new concepts in transcriptional regulation.


Trihalomethanes in public drinking water and stillbirth and low birth weight rates: an intervention study.
During 2003-2004, United Utilities water company in North West England introduced enhanced coagulation (EC) to four treatment works to mitigate disinfection by-product (DBP) formation. This enabled examination of the relation between DBPs and birth outcomes whilst reducing socioeconomic confounding. We compared stillbirth, and low and very low birth weight rates three years before (2000-2002) with three years after (2005-2007) the intervention, and in relation to categories of THM change. We created exposure metrics for EC and trihalomethane (THM) concentration change (n=258 water zones). We linked 429,599 live births and 2279 stillbirths from national birth registers to the water zone at birth. We used Poisson regression to model the differences in birth outcome rates with an interaction between before/after the intervention and EC or THM change. EC treatment reduced chloroform concentrations more than non-treatment (mean -29.7 µg/l vs. -14.5 µg/l), but not brominated THM concentrations. Only 6% of EC water zones received 100% EC water, creating exposure misclassification concerns. EC intervention was not associated with a statistically significant reduction in birth outcome rates. Areas with the highest chloroform decrease (30 - 65 μg/l) had the greatest percentage decrease in low -9 % (-12, -5) and very low birth weight -16% (-24, -8) rates. The interaction between before/after intervention and chloroform change was statistically significant only for very low birth weight, p=0.02. There were no significant decreases in stillbirth rates. In a novel approach for studying DBPs and adverse reproductive outcomes, the EC intervention to reduce DBPs did not affect birth outcome rates. However, a measured large decrease in chloroform concentrations was associated with statistically significant reductions in very low birth weight rates.


Distribution of sympathetic fiber areas in the sensory nerves of forearm: an immunohistochemical study in cadavers.
Secondary to peripheral nerve injuries, involvement of sympathetic fibers complications such as Complex Regional Pain Syndrome (CRPS) have been reported. There are no reports available in the distribution of the sympathetic fibers/areas of sensory nerves in the forearm. The present study aim is an attempt to find the distribution of sympathetic fibers in the anterior branch of medial antebrachial cutaneous nerve of forearm (AMACN), lateral antebrachial cutaneous nerve of forearm (LACN) and superficial branch of radial nerve (SBRN) at cubital fossae. We have studied on 17 fresh human cadaveric AMACN, LACN and SRBN samples. Frozen sections of these nerves were processed by immunohistochemical (tyrosine hydroxylase) method for sympathetic fibers. Sympathetic fibers area (Asym) was found to be more in SBRN when compared to AMACN and LACN. The comparison of the sympathetic index (SI = sympathetic fibers area / total fascicular area of the nerve) between AMACN and LACN (p-value < 0.001), AMACN and SBRN (p-value <0.001), LACN and SBRN (p-value < 0.001) were statistically significant. Sympathetic index (SI) for SBRN was more when compared to AMACN and LACN. SBRN had maximum percentage (5.16%) of Asym when compared with LACN and AMACN. Sympathetic fibers area (Asym), sympathetic index (SI) and percentage of sympathetic fibers area (Asym %) were found to be more in SBRN when compared with AMACN and LACN. These results of the study might help to explain sympathetic system-related diseases in the area of distribution of AMACN, LACN and SBRN.


Molecular recognition of macrocyclic peptidomimetic inhibitors by HIV-1 protease.
High-resolution crystal structures are described for seven macrocycles complexed with HIV-1 protease (HIVPR). The macrocycles possess two amides and an aromatic group within 15-17 membered rings designed to replace N- or C-terminal tripeptides from peptidic inhibitors of HIVPR. Appended to each macrocycle is a transition state isostere and either an acyclic peptide, nonpeptide, or another macrocycle. These cyclic analogues are potent inhibitors of HIVPR, and the crystal structures show them to be structural mimics of acyclic peptides, binding in the active site of HIVPR via the same interactions. Each macrocycle is restrained to adopt a beta-strand conformation which is preorganized for protease binding. An unusual feature of the binding of C-terminal macrocyclic inhibitors is the interaction between a positively charged secondary amine and a catalytic aspartate of HIVPR. A bicyclic inhibitor binds similarly through its secondary amine that lies between its component N-terminal and C-terminal macrocycles. In contrast, the corresponding tertiary amine of the N-terminal macrocycles does not interact with the catalytic aspartates. The amine-aspartate interaction induces a 1.5 A N-terminal translation of the inhibitors in the active site and is accompanied by weakened interactions with a water molecule that bridges the ligand to the enzyme, as well as static disorder in enzyme flap residues. This flexibility may facilitate peptide cleavage and product dissociation during catalysis. Proteases [Aba67,95]HIVPR and [Lys7,Ile33,Aba67,95]HIVPR used in this work were shown to have very similar crystal structures.


The putative flippase Apt1 is required for intracellular membrane architecture and biosynthesis of polysaccharide and lipids in Cryptococcus neoformans.
Flippases are responsible for the asymmetric distribution of phospholipids in biological membranes. In the encapsulated fungal pathogen Cryptococcus neoformans, the putative flippase Apt1 is an important regulator of polysaccharide secretion and pathogenesis in mice by unknown mechanisms. In this study, we analyzed the role of C. neoformans Apt1 in intracellular membrane architecture and synthesis of polysaccharide and lipids. Analysis of wild type (WT), apt1Δ (mutant) and apt1Δ::APT1 (complemented) strains by transmission electron microscopy revealed that deletion of APT1 resulted in the formation of irregular vacuoles. Disorganization of vacuolar membranes in apt1Δ cells was accompanied by a significant increase in the amounts of intra-vacuolar and pigment-containing vesicles. Quantitative immunogold labeling of C. neoformans cells with a monoclonal antibody raised to a major capsular component suggested impaired polysaccharide synthesis. APT1 deletion also affected synthesis of phosphatidylserine, phosphatidylethanolamine, inositolphosphoryl ceramide, glucosylceramide and ergosterylglycoside. These results reveal novel functions of Apt1 and are in agreement with the notion that this putative flippase plays an important role in the physiology of C. neoformans.