In Vivo Interaction of the Hepatitis Delta Virus Small Antigen with the ELAV-Like Protein HuR.
The small and large delta antigens (S-HDAg and L-HDAg, respectively) represent two forms of the only protein encoded by the hepatitis delta virus (HDV) RNA genome. Consequently, HDV relies, at a large extent, on the host cell machinery for replication and transcription. Until now, only a limited number of cellular proteins were identified as S-HDAg or L-HDAg partners being involved in the modulation of the virus life cycle. In an attempt to identify cellular S-HDAg-binding proteins we made use of a yeast two-hybrid approach to screen a human liver cDNA library. We were able to identify HuR, a ubiquitously expressed protein involved in RNA stabilization, as an S-HDAg partner both in vitro and in vivo. HuR was found to be overexpressed and colocalize with HDAg in human hepatoma cells. siRNA knockdown of HuR mRNA resulted in inhibition of S-HDAg and L-HDAg expression.


Self antigens expressed by solid tumors Do not efficiently stimulate naive or activated T cells: implications for immunotherapy.
Induction and maintenance of cytotoxic T lymphocyte (CTL) activity specific for a primary endogenous tumor was investigated in vivo. The simian virus 40 T antigen (Tag) expressed under the control of the rat insulin promoter (RIP) induced pancreatic beta-cell tumors producing insulin, causing progressive hypoglycemia. As an endogenous tumor antigen, the lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) was introduced also under the control of the RIP. No significant spontaneous CTL activation against GP was observed. However, LCMV infection induced an antitumor CTL response which efficiently reduced the tumor mass, resulting in temporarily normalized blood glucose levels and prolonged survival of double transgenic RIP(GP x Tag2) mice (137 +/- 18 d) as opposed to control RIP-Tag2 mice (88 +/- 8 d). Surprisingly, the tumor-specific CTL response was not sustained despite the facts that the tumor cells continued to express MHC class I and LCMV-GP-specific CTLs were present and not tolerized. Subsequent adoptive transfer of virus activated spleen cells into RIP(GP x Tag2) mice further prolonged survival (168 +/- 11 d), demonstrating continued expression of the LCMV-GP tumor antigen and MHC class I. The data show that the tumor did not spontaneously induce or maintain an activated CTL response, revealing a profound lack of immunogenicity in vivo. Therefore, repetitive immunizations are necessary for prolonged antitumor immunotherapy. In addition, the data suggest that the risk for induction of chronic autoimmune diseases is limited, which may encourage immunotherapy against antigens selectively but not exclusively expressed by the tumor.


Automatic microfluidic platform for cell separation and nucleus collection.
This study reports a new biochip capable of cell separation and nucleus collection utilizing dielectrophoresis (DEP) forces in a microfluidic system comprising of micropumps and microvalves, operating in an automatic format. DEP forces operated at a low voltage (15 Vp-p) and at a specific frequency (16 MHz) can be used to separate cells in a continuous flow, which can be subsequently collected. In order to transport the cell samples continuously, a serpentine-shape (S-shape) pneumatic micropump device was constructed onto the chip device to drive the samples flow through the microchannel, which was activated by the pressurized air injection. The mixed cell samples were first injected into an inlet reservoir and driven through the DEP electrodes to separate specific samples. Finally, separated cell samples were collected individually in two outlet reservoirs controlled by microvalves. With the same operation principle, the nucleus of the specific cells can be collected after the cell lysis procedure. The pumping rate of the micropump was measured to be 39.8 microl/min at a pressure of 25 psi and a driving frequency of 28 Hz. For the cell separation process, the initial flow rate was 3 microl/min provided by the micropump. A throughput of 240 cells/min can be obtained by using the developed device. The DEP electrode array, microchannels, micropumps and microvalves are integrated on a microfluidic chip using micro-electro-mechanical-systems (MEMS) technology to perform several crucial procedures including cell transportation, separation and collection. The dimensions of the integrated chip device were measured to be 6x7 cm. By integrating an S-shape pump and pneumatic microvalves, different cells are automatically transported in the microchannel, separated by the DEP forces, and finally sorted to specific chambers. Experimental data show that viable and non-viable cells (human lung cancer cell, A549-luc-C8) can be successfully separated and collected using the developed microfluidic platform. The separation accuracy, depending on the DEP operating mode used, of the viable and non-viable cells are measured to be 84 and 81%, respectively. In addition, after cell lysis, the nucleus can be also collected using a similar scheme. The developed automatic microfluidic platform is useful for extracting nuclear proteins from living cells. The extracted nuclear proteins are ready for nuclear binding assays or the study of nuclear proteins.


Oral silymarin formulation efficacy in management of AC-T protocol induced hepatotoxicity in breast cancer patients: A randomized, triple blind, placebo-controlled clinical trial.
Chemotherapeutic agents, with or without other drugs and radiation, may cause indirect or direct hepatotoxicity. Doxorubicin-induced hepatotoxicity (DIH) is a major health concern in cancer patients receiving this cytotoxic drug that is mostly resulted from the production of reactive oxygen species leading to transient or permanent liver damages. Silymarin, a flavonoid extracted from the Silybum marianum, exhibits antioxidant and anti-inflammatory activities. This study aimed to investigate the clinical efficacy of systemic administration of silymarin in management of chemotherapy induced hepatotoxicity in patients with non-metastatic breast cancer who received doxorubicin/cyclophosphamide-paclitaxel (AC-T) regimen.Material: In this randomized, triple blind, placebo-controlled clinical trial, 30 patients who received AC-T who fulfilled the inclusion criteria were randomly allocated to silymarin (n = 15) or placebo (n = 15) groups to receive oral silymarin 140 mg three times a day or placebo tablets, respectively. Fatty liver severity was assessed by liver ultrasound imaging and FibroScan® and also measurement of liver function tests before and after the intervention. There was a non-significant trend toward more severe liver involvement in placebo group comparing to the silymarin group after intervention based on ultrasonography (p = 0.083). Besides, in silymarin group, hepatic involvement grade based on ultrasonography considerably reduced after intervention (p = 0.012). However, no difference was found between two groups based on FibroScan and liver function tests. Oral administration of silymarin could significantly reduce hepatotoxicity severity after 1 month of treatment in non-metastatic breast cancer patients treated with AC-T regimen.


Improving pathology and laboratory medicine in low-income and middle-income countries: roadmap to solutions.
Insufficient awareness of the centrality of pathology and laboratory medicine (PALM) to a functioning health-care system at policy and governmental level, with the resultant inadequate investment, has meant that efforts to enhance PALM in low-income and middle-income countries have been local, fragmented, and mostly unsustainable. Responding to the four major barriers in PALM service delivery that were identified in the first paper of this Series (workforce, infrastructure, education and training, and quality assurance), this second paper identifies potential solutions that can be applied in low-income and middle-income countries (LMICs). Increasing and retaining a quality PALM workforce requires access to mentorship and continuing professional development, task sharing, and the development of short-term visitor programmes. Opportunities to enhance the training of pathologists and allied PALM personnel by increasing and improving education provision must be explored and implemented. PALM infrastructure must be strengthened by addressing supply chain barriers, and ensuring laboratory information systems are in place. New technologies, including telepathology and point-of-care testing, can have a substantial role in PALM service delivery, if used appropriately. We emphasise the crucial importance of maintaining PALM quality and posit that all laboratories in LMICs should participate in quality assurance and accreditation programmes. A potential role for public-private partnerships in filling PALM services gaps should also be investigated. Finally, to deliver these solutions and ensure equitable access to essential services in LMICs, we propose a PALM package focused on these countries, integrated within a nationally tiered laboratory system, as part of an overarching national laboratory strategic plan.


Development and initial validation of a questionnaire to assess patients' perception of protective isolation following haematopoietic stem cell transplantation.
The aim of this study was to develop and psychometrically test a questionnaire assessing patients' perception of protective isolation following haematopoietic stem cell transplantation (HSCT). The conceptual framework for developing the questionnaire was a three-dimensional model that emerged from a metasynthesis: isolation-related suffering, relationship with oneself and relationship with others. Item selection was performed through a focus group, comparison with the findings of two phenomenological studies, and content validity with 22 experts. Cognitive interviews with five patients were used to verify face validity. A validation study was conducted in 10 Italian centres, all members of the Italian Group of stem cell transplant (GITMO). Patients completed the questionnaires between 7 and 9 days post-transplant. Dimensionality was tested through exploratory factor analysis (EFA). A total of 17 items yielded a content validity index (CVI) of 0.88. Participants included 186 adult patients receiving autologous (48%) or allogeneic (52%) HSCT in protective isolation. The EFA yielded a three-factor solution, explaining 55% of the variance. The scale showed adequate psychometric properties, with the exception of three items, which were eliminated. Future studies should test the psychometric properties of the questionnaire through confirmatory factor analysis and verify its transcultural validity.


Baicalin improves survival in a murine model of polymicrobial sepsis via suppressing inflammatory response and lymphocyte apoptosis.
An imbalance between overwhelming inflammation and lymphocyte apoptosis is the main cause of high mortality in patients with sepsis. Baicalin, the main active ingredient of the Scutellaria root, exerts anti-inflammatory, anti-apoptotic, and even antibacterial properties in inflammatory and infectious diseases. However, the therapeutic effect of baicalin on polymicrobial sepsis remains unknown. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in C57BL/6 mice. Mice were infused with baicalin intraperitoneally at 1 h, 6 h and 12 h after CLP. Survival rates were assessed over the subsequent 8 days. Bacterial burdens in blood and peritoneal cavity were calculated to assess the bacterial clearance. Neutrophil count in peritoneal lavage fluid was also calculated. Injuries to the lung and liver were detected by hematoxylin and eosin staining. Levels of cytokines, including tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-10 and IL-17, in blood and peritoneum were measured by enzyme-linked immunosorbent assay. Adaptive immune function was assessed by apoptosis of lymphocytes in the thymus and counts of different cell types in the spleen. Baicalin significantly enhanced bacterial clearance and improved survival of septic mice. The number of neutrophils in peritoneal lavage fluid was reduced by baicalin. Less neutrophil infiltration of the lung and liver in baicalin-treated mice was associated with attenuated injuries to these organs. Baicalin significantly reduced the levels of proinflammatory cytokines but increased the level of anti-inflammatory cytokine in blood and peritoneum. Apoptosis of CD3(+) T cell was inhibited in the thymus. The numbers of CD4(+), CD8(+) T lymphocytes and dendritic cells (DCs) were higher, while the number of CD4(+)CD25(+) regulatory T cells was lower in the baicalin group compared with the CLP group. Baicalin improves survival of mice with polymicrobial sepsis, and this may be attributed to its antibacterial property as well as its anti-inflammatory and anti-apoptotic effects.


Structure of Broadhaven virus by cryoelectron microscopy: correlation of structural and antigenic properties of Broadhaven virus and bluetongue virus outer capsid proteins.
The three-dimensional structure of Broadhaven virus (BRDV) has been determined to 23 A resolution by cryoelectron microscopy and image processing. As predicted from sequence homology, the BRDV structure resembles that of bluetongue virus (BTV) with the notable exception of one of the outer shell proteins. The cores of BRDV and BTV are identical at medium resolution; they have a diameter of 710 A and the VP7 trimers are arranged on a T = 13 icosahedral lattice. The outer shell proteins, VP5 of BRDV and BTV, have roughly the same molecular weight while VP4 of BRDV is only half the molecular weight of the corresponding VP2 of BTV. This size difference allows unambiguous determination of the identity of the triskelion shape as trimers of VP4 of BRDV (VP2 of BTV). The VP4 of BRDV sits on the VP7 trimers and projects outwards 40 A, giving the capsid an overall diameter of 790 A. This contrasts with VP2 of BTV, which projects outwards 95 A to give the capsid a diameter of 900 A. The difference in accessibility of the outer shell proteins of BRDV and BTV correlates with the difference in antigenic properties of these viral proteins. The shape of the BRDV VP5 indicates that it too is a trimer, thus implying that there are 360 copies of VP5 and 180 copies of VP4 per virion.


A Marfan syndrome-like phenotype caused by a neocentromeric supernumerary ring chromosome 15.
Small supernumerary marker chromosomes (sSMC) are abnormal chromosomes that cannot be characterized by standard banding cytogenetic techniques. A minority of sSMC contain a neocentromere, which is an ectopic centromere lacking the characteristic alpha-satellite DNA. The phenotypic manifestations of sSMC and neocentromeric sSMC are variable and range from severe intellectual disability and multiple congenital anomalies to a normal phenotype. Here we report a patient with a diagnosis of Marfan syndrome and infertility found to have an abnormal karyotype consisting of a chromosome 15 deletion and a ring-type sSMC likely stabilized by a neocentromere derived via a mechanism initially described by Barbara McClintock in 1938. Analysis of the sSMC identified that it contained the deleted chromosome 15 material and also one copy of FBN1, the gene responsible for Marfan syndrome. We propose that the patient's diagnosis arose from disruption of the FBN1 allele on the sSMC. To date, a total of 29 patients have been reported with an sSMC derived from a chromosomal deletion. We review these cases with a specific focus on the resultant phenotypes and note significant difference between this class of sSMC and other types of sSMC. Through this review we also identified a patient with a clinical diagnosis of neurofibromatosis type 1 who lacked a family history of the condition but was found to have a chromosome 17-derived sSMC that likely contained NF1 and caused the patient's disorder. We also review the genetic counseling implications and recommendations for a patient or family harboring an sSMC. © 2016 Wiley Periodicals, Inc.


Bryostatin 1: a potential anti-leukemic agent for chronic myelomonocytic leukemia.
Bryostatin 1 is a macrocyclic lactone which activates protein kinase C (PKC), and is able to induce maturation in cells from some cases of acute myelogenous leukemia. This paper reports that bryostatin inhibits the spontaneous in vitro proliferation of chronic myelomonocytic leukemia cells (CMMoL) in semi-solid medium at concentrations between 10(-8) and 10(-10) M. Growth inhibition was equivalent to or greater than that seen with phorbol-12-myristate-13-acetate. Bryostatin acted primarily as a cytotoxic agent, rather than as a cytostatic agent. The spontaneous in vitro proliferation of CMMoL cells is due to autocrine or paracrine secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF). Bryostatin 1 actually increased GM-CSF secretion by CMMoL cells while inhibiting their proliferation. Bryostatin 1 also increased tumor necrosis factor alpha (TNF alpha) secretion by CMMoL cells, and in 2/5 cases the cytotoxic effect of bryostatin 1 on fresh CMMoL cells could be substantially reversed by the addition of antibody to TNF alpha to the culture medium. Bryostatin 1 may produce a cytotoxic effect on CMMoL cells in part by increasing the secretion of, or sensitivity to, TNF alpha, and may have therapeutic potential in CMMoL.


FRAGILE ENVIRONMENT, SEASONALITY AND MATERNAL AND CHILDHOOD UNDERNUTRITION IN BANGLADESH.
This study assessed whether agricultural and household incomes were the same across different agro-ecological environments in Bangladesh. An in-depth analysis of the effect of unfavourable ecologies on maternal and child malnutrition was carried out. Data were from a longitudinal data set comprising a nationally representative data sample collected in 2014 and the Food Security Nutrition Surveillance Project (FSNSP) conducted in 2011 and 2012. Anthropometric indices were used to assess the nutritional status of mothers and under-five children. The key variables of interest were food seasonality and geographical location. Data were analysed using the General Linear Model and multinomial and binary logistic regression analysis. Panel data analysis showed that household income was not equal across agro-ecological zones, indicating that the fragility of the environment affects a household's ability to access food, and thus the nutritional status of mothers and children. Coastal areas of Bangladesh were found to be less dependent on agriculture, particularly cultivation, which had diminished during last few decades. Per capita income has been increasing in coastal areas of Bangladesh, led by remittance (money sent home by migrant workers) growing at 8% per year against 6% in other areas. Regression analysis showed that a household in a coastal zone earned 19% less than one in more favourable zones. Although the income from farm practices was found to be lower in unfavourable areas, the deficiency was compensated by increased non-farm incomes. The results from the FSNSP data showed that overall the rates of stunting and wasting among under-five children were 37% and 11.7%, respectively, and nearly 28% of mothers suffered from undernutrition. A highly significant regional heterogeneity in undernutrition was found, with alarmingly high levels in the Haor Basin and coastal belt areas. There were significantly higher rates of underweight and wasting in the monsoon season compared with the two harvest seasons among children under the age of five. The findings stress the importance of bringing geographical location and seasonality thinking into debates on hunger and nutrition in Bangladesh.


STR null alleles complicate parentage testing in South Africa.
Null alleles complicate parentage testing because they do not contribute positively to phenotypes.Objectives. To survey South African populations for null alleles at short tandem repeat (STR) loci used in parentage testing. Paternity case data were scanned for apparent contradictions compared with Mendelian inheritance that could be due to null alleles. Estimates of null allele frequencies were obtained from tallies of apparent carriers. Three of 15 loci appeared to have null alleles at appreciable frequencies and five showed no evidence of null alleles. A null allele at the vWA locus reached a frequency of ~0.3% in the black population and approximately three times this frequency in the coloured population. No apparent vWA null carriers were detected in whites suggesting that the Khoisan were the major contributors of the null alleles to coloureds. The apparent genotypes of a sample of TPOX null carriers changed from homozygous to heterozygous when they were retyped using different polymerase chain reaction primers. The revealed allele was allele 6 in every case. A D13S317 null allele was detected at relatively low frequencies in the black and coloured samples but 2/145 Indian (Asian) parents appear to be carriers, suggesting that it could be common in Indians. Three of the 15 forensically relevant STR loci investigated had null alleles at significant frequencies in South African populations. Failure to allow for the presence of null alleles can have a large impact on the outcome of parentage tests.


Invasive rats and recent colonist birds partially compensate for the loss of endemic New Zealand pollinators.
Reported declines of pollinator populations around the world have led to increasing concerns about the consequences for pollination as a critical ecosystem function and service. Pollination could be maintained through compensation if remaining pollinators increase their contribution or if novel species are recruited as pollinators, but empirical evidence of this compensation is so far lacking. Using a natural experiment in New Zealand where endemic vertebrate pollinators still occur on one offshore island reserve despite their local extinction on the adjacent North Island, we investigated whether compensation could maintain pollination in the face of pollinator extinctions. We show that two recently arrived species in New Zealand, the invasive ship rat (Rattus rattus) and the recent colonist silvereye (Zosterops lateralis; a passerine bird), at least partly maintain pollination for three forest plant species in northern New Zealand, and without this compensation, these plants would be significantly more pollen-limited. This study provides empirical evidence that widespread non-native species can play an important role in maintaining ecosystem functions, a role that needs to be assessed when planning invasive species control or eradication programmes.


An autosomal dominant, qualitative platelet disorder associated with multimerin deficiency, abnormalities in platelet factor V, thrombospondin, von Willebrand factor, and fibrinogen and an epinephrine aggregation defect.
Multimerin is a massive soluble, multimeric protein found in platelets and endothelial cells. Recent studies identified multimerin as a specific coagulation factor V binding protein, complexed with platelet, but not plasma, factor V. These findings led us to investigate individuals with inherited factor V deficiencies for possible multimerin abnormalities. Platelet proteins were evaluated using immunoassays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, immunoprecipitation, and direct binding studies. Patients with factor V Quebec, a disorder with abnormal platelet factor V, had a quantitative deficiency in multimerin (n = 11 tested; mean, 12.5%; range, 5% to 27% of the normal pool; normal range, 45% to 214%) with a normal multimer pattern. Quantitative and qualitative abnormalities were detected in their platelet factor V. An unrelated patient who was deficient in platelet and plasma factor V had normal platelet multimerin. The levels of platelet beta-thromboglobulin, von Willebrand factor, thrombospondin, and fibrinogen antigen were normal in the factor V Quebec patients. However, proteins with abnormal mobility were detected in their platelet lysate and releasate, and their platelet thrombospondin, von Willebrand factor, and fibrinogen showed evidence of proteolytic degradation. Platelet counts of the factor V Quebec patients ranged from mildly thrombocytopenic to low normal (mean, 159 x 10(9)/L; range, 104 to 198 x 10(9)/L). In addition, their platelets failed to aggregate in response to 6 to 10 micromol/L epinephrine despite normal numbers of platelet alpha 2-adrenergic receptors. These data indicate that patients with factor V Quebec have an inherited bleeding disorder distinct from other platelet disorders and associated with multiple abnormalities, including multimerin deficiency, abnormal platelet factor V, thrombospondin, von Willebrand factor, and fibrinogen, and an epinephrine aggregation defect.


Plasma lipids predict myocardial infarction, but not stroke, in patients with established cerebrovascular disease.
To evaluate the role of plasma lipids in recurrent vascular events, including stroke, among individuals with established cerebrovascular disease. Plasma total cholesterol, HDL cholesterol, and triglycerides were measured at baseline among individuals participating in the Perindopril Protection Against Recurrent Stroke (PROGRESS) study, a randomized clinical trial of blood pressure lowering among patients with previous stroke or transient ischaemic attack. A series of nested case-control studies were used to investigate the association between each of these lipid variables and the risk of subsequent haemorrhagic stroke, ischaemic stroke, myocardial infarction (MI), and heart failure. A total of 895 patients were selected as cases (83 haemorrhagic stroke, 472 ischaemic stroke, 206 MI, and 258 heart failure) and each was matched with one to three controls. After adjustment for other major cardiovascular risk factors, none of the lipid variables was associated with the risk of either stroke subtype. There were significant positive and negative associations for total cholesterol and HDL, respectively, with the risk of MI; the odds ratio comparing the highest and lowest thirds of each of these lipid variables was 2.00 (95% CI: 1.30-3.09) for total cholesterol and 0.58 (95% CI: 0.37-0.90) for HDL. HDL was inversely associated with the risk of heart failure; however, this result was of borderline statistical significance (P=0.05). Lipid variables are associated with the risk of MI, but not recurrent stroke, in patients with established cerebrovascular disease.


Effective diffusion coefficients of K+ and Cl- ions in ion channel models.
We have used molecular dynamics simulations, corresponding to a total simulation time of 11 ns, to investigate the effective short-time local diffusion coefficient of potassium and chloride ions in a series of model ion channels. These models, which include channels formed by the fungal peptide alamethicin, by a synthetic leucine-serine peptide, and by the pore-lining M2 helix bundle of the nicotinic acetylcholine receptor, have a range of different secondary structures, diameters and hydrophobicities. We find that the diffusion coefficients of both ions are appreciably reduced in the narrower channels, the extent of the reduction being similar for both the anionic and cationic species. This suggests that a difference in mobility cannot be the source of the ion selectivity exhibited by some of the channels (for example, the leucine-serine peptide). We find no evidence for a reduction in mobility of either ion in the nAChR model. These results are broadly in line with a previous similar study of Na+ ions, and may be useful in Poisson-Nernst-Planck, Eyring rate theory or Brownian dynamics calculations of channel conductance.


The Influence of State-Specific Quitline Numbers on Call Volume During a National Tobacco Education Campaign Promoting 1-800-QUIT-NOW.
Previous research has shown that the first federally funded national tobacco education campaign (Tips) increased calls to the national quitline portal (1-800-QUIT-NOW). Quitlines in 13 states have alternate state-specific telephone numbers. This study examined quitline calls to 1-800-QUIT-NOW in states with and without alternate numbers during the Tips campaign. We used data on calls to 1-800-QUIT-NOW from all US states and the District of Columbia from 2 weeks before to 2 weeks after the 2012 Tips campaign. Similar data were obtained for California's alternate number, 1-800-NO-BUTTS. Multivariate linear models examined whether an interaction existed between Tips exposure, as measured by gross rating points, and presence of an alternate quitline number as well as the effect of Tips on calls to California's 1-800-NO-BUTTS. Having an alternate quitline number did not affect the rate of increase in calls to 1-800-QUIT-NOW, but it was associated with lower absolute numbers of calls to 1-800-QUIT-NOW. On average, states with alternate numbers had 98 fewer calls to 1-800-QUIT-NOW per week in a given area code than those without an alternate number (P < .001). In California, Tips gross rating points were positively correlated with calls to 1-800-QUIT-NOW (b = 38.5, P < .001) and to 1-800-NO-BUTTS (b = 14.1, P < .05). The Tips campaign had the same effect in increasing calls to 1-800-QUIT-NOW in states with and without alternate quitline numbers and had a modest spillover effect on calls to California's alternate number. States may consider the advantages and disadvantages of having alternate quitline numbers given continued national promotions of 1-800-QUIT-NOW. This is the first study that assesses whether the impact of a national tobacco education campaign promoting the national quitline portal number was influenced by the presence of state-specific quitline numbers and whether there was any spillover effect on calls to states' alternate quitline numbers. This study provides important information for states to consider the advantages and disadvantages of maintaining state-specific quitline numbers.


Canine CNGA3 Gene Mutations Provide Novel Insights into Human Achromatopsia-Associated Channelopathies and Treatment.
Cyclic nucleotide-gated (CNG) ion channels are key mediators underlying signal transduction in retinal and olfactory receptors. Genetic defects in CNGA3 and CNGB3, encoding two structurally related subunits of cone CNG channels, lead to achromatopsia (ACHM). ACHM is a congenital, autosomal recessive retinal disorder that manifests by cone photoreceptor dysfunction, severely reduced visual acuity, impaired or complete color blindness and photophobia. Here, we report the first canine models for CNGA3-associated channelopathy caused by R424W or V644del mutations in the canine CNGA3 ortholog that accurately mimic the clinical and molecular features of human CNGA3-associated ACHM. These two spontaneous mutations exposed CNGA3 residues essential for the preservation of channel function and biogenesis. The CNGA3-R424W results in complete loss of cone function in vivo and channel activity confirmed by in vitro electrophysiology. Structural modeling and molecular dynamics (MD) simulations revealed R424-E306 salt bridge formation and its disruption with the R424W mutant. Reversal of charges in a CNGA3-R424E-E306R double mutant channel rescued cGMP-activated currents uncovering new insights into channel gating. The CNGA3-V644del affects the C-terminal leucine zipper (CLZ) domain destabilizing intersubunit interactions of the coiled-coil complex in the MD simulations; the in vitro experiments showed incompetent trimeric CNGA3 subunit assembly consistent with abnormal biogenesis of in vivo channels. These newly characterized large animal models not only provide a valuable system for studying cone-specific CNG channel function in health and disease, but also represent prime candidates for proof-of-concept studies of CNGA3 gene replacement therapy for ACHM patients.


An intriguing case of locked jaw secondary to melioidosis.
A 56-year-old woman presented with fever, pain and restriction of movement of the right temporomandibular joint. She was premorbidly diagnosed to have type 2 diabetes mellitus and rheumatoid arthritis. Local examination revealed a poorly demarcated severely tender, erythematous swelling in the right preauricular region. All haematological and biochemical investigations were within normal limits. MRI of the neck revealed the presence of a masticator space infection with intramuscular abscess involving the masseter and the temporalis muscles along with intracranial extension. Osteomyelitic changes were detected in the right mandibular condyle, temporal bone and in the temporomandibular joint. Melioidosis was suspected due to this unique clinical presentation of an abscess at an unusual and atypical site. Blood cultures identified the Gram-negative bacilli Burkholderia pseudomallei, which established the diagnosis of Melioidosis. Remarkable improvement was attained with antibiotics meropenem and cotrimoxazole, deferring the need for any surgical intervention.


A Practical Neighbor Discovery Framework for Wireless Sensor Networks.
Neighbor discovery is a crucial operation frequently executed throughout the life cycle of a Wireless Sensor Network (WSN). Various protocols have been proposed to minimize the discovery latency or to prolong the lifetime of sensors. However, none of them have addressed that all the critical concerns stemming from real WSNs, including communication collisions, latency constraints and energy consumption limitations. In this paper, we propose Spear, the first practical neighbor discovery framework to meet all these requirements. Spear offers two new methods to reduce communication collisions, thus boosting the discovery rate of existing neighbor discovery protocols. Spear also takes into consideration latency constraints and facilitates timely adjustments in order to reduce the discovery latency. Spear offers two practical energy management methods that evidently prolong the lifetime of sensor nodes. Most importantly, Spear automatically improves the discovery results of existing discovery protocols, on which no modification is required. Beyond reporting details of different Spear modules, we also present experiment evaluations on several notable neighbor discovery protocols. Results show that Spear greatly improves the discovery rate from 33.0% to 99.2%, and prolongs the sensor nodes lifetime up to 6.47 times.


Predictors of response to radioiodine therapy in hyperthyroid cats.
The objective of this retrospective study was to evaluate hyperthyroid cats for pretreatment factors that would predict response to radioiodine therapy. Hyperthyroidism was diagnosed in 193 cats based on elevated serum thyroxine levels and/or elevated thyroid to salivary gland ratios on thyroid scintigraphy. All cats were treated with an intravenous bolus of 4 mCi of radioiodine and follow-up serum thyroxine levels were evaluated at 1 week and 1, 3, 6, and 12 months post-therapy. There was a significant relationship between pretreatment thyroxine values and post-treatment thyroxine values at all of the follow-up time points (p < 0.001). There was also a relationship between thyroid to salivary gland technetium scan ratio results and serum thyroxine values at pretreatment and at 1 week post-treatment (p = 0.02, 0.005, respectively). A greater scan ratio was associated with higher thyroxine levels at these time points, but not at 1, 3, 6 or 12 months post-therapy. Ninety-eight cats pretreated with methimazole were analyzed for the effect of this drug on response to therapy. Methimazole was discontinued > or = 5 days before radioiodine therapy in 58 cats and < 5 days in 31 cats, in 9 cats the number of days off methimazole was unknown. There was no difference in response to radioiodine based upon when methimazole was discontinued (p = 0.70).


Integration of 2D CMUT arrays with front-end electronics for volumetric ultrasound imaging.
For three-dimensional (3D) ultrasound imaging, connecting elements of a two-dimensional (2D) transducer array to the imaging system's front-end electronics is a challenge because of the large number of array elements and the small element size. To compactly connect the transducer array with electronics, we flip-chip bond a 2D 16 x 16-element capacitive micromachined ultrasonic transducer (CMUT) array to a custom-designed integrated circuit (IC). Through-wafer interconnects are used to connect the CMUT elements on the top side of the array with flip-chip bond pads on the back side. The IC provides a 25-V pulser and a transimpedance preamplifier to each element of the array. For each of three characterized devices, the element yield is excellent (99 to 100% of the elements are functional). Center frequencies range from 2.6 MHz to 5.1 MHz. For pulse echo operation, the average - 6-dB fractional bandwidth is as high as 125%. Transmit pressures normalized to the face of the transducer are as high as 339 kPa and input-referred receiver noise is typically 1.2 to 2.1 mPa/pHz. The flip-chip bonded devices were used to acquire 3D synthetic aperture images of a wire-target phantom. Combining the transducer array and IC, as shown in this paper, allows for better utilization of large arrays, improves receive sensitivity, and may lead to new imaging techniques that depend on transducer arrays that are closely coupled to IC electronics.


Erythropoietin withdrawal alters interactions between young red blood cells, splenic endothelial cells, and macrophages: an in vitro model of neocytolysis.
We have described the rapid destruction of young red blood cells (neocytolysis) in astronauts adapting to microgravity, in polycythemic high altitude dwellers who descend to sea level, and in patients with kidney disorders. This destruction results from a decrease in erythropoietin (EPO) production. We hypothesized that such EPO withdrawal could trigger physiological changes in cells other than red cell precursors and possibly lead to the uptake and destruction of young red cells by altering endothelial cell-macrophage interactions, most likely occurring in the spleen. We identified EPO receptors on human splenic endothelial cells (HSEC) and investigated the responses of these cells to EPO withdrawal. A monolayer of HSEC, unlike human endothelial cells from aorta, glomerulus, or umbilical vein, demonstrated an increase in permeability upon EPO withdrawal that was accompanied by unique morphological changes. When HSEC were cultured with monocyte-derived macrophages (but not when either cell type was cultured alone), EPO withdrawal induced an increased ingestion of young red cells by macrophages when compared with the constant presence or absence of EPO. HSEC may represent a unique cell type that is able to respond to EPO withdrawal by increasing permeability and interacting with phagocytic macrophages, which leads to neocytolysis.


Inter-laboratory validation of bioaccessibility testing for metals.
Bioelution assays are fast, simple alternatives to in vivo testing. In this study, the intra- and inter-laboratory variability in bioaccessibility data generated by bioelution tests were evaluated in synthetic fluids relevant to oral, inhalation, and dermal exposure. Using one defined protocol, five laboratories measured metal release from cobalt oxide, cobalt powder, copper concentrate, Inconel alloy, leaded brass alloy, and nickel sulfate hexahydrate. Standard deviations of repeatability (sr) and reproducibility (sR) were used to evaluate the intra- and inter-laboratory variability, respectively. Examination of the sR:sr ratios demonstrated that, while gastric and lysosomal fluids had reasonably good reproducibility, other fluids did not show as good concordance between laboratories. Relative standard deviation (RSD) analysis showed more favorable reproducibility outcomes for some data sets; overall results varied more between- than within-laboratories. RSD analysis of sr showed good within-laboratory variability for all conditions except some metals in interstitial fluid. In general, these findings indicate that absolute bioaccessibility results in some biological fluids may vary between different laboratories. However, for most applications, measures of relative bioaccessibility are needed, diminishing the requirement for high inter-laboratory reproducibility in absolute metal releases. The inter-laboratory exercise suggests that the degrees of freedom within the protocol need to be addressed.


RapGEF2 is essential for embryonic hematopoiesis but dispensable for adult hematopoiesis.
RapGEF2 is one of many guanine nucleotide exchange factors (GEFs) that specifically activate Rap1. Here, we generated RapGEF2 conditional knockout mice and studied its role in embryogenesis and fetal as well as adult hematopoietic stem cell (HSC) regulation. RapGEF2 deficiency led to embryonic lethality at ~ E11.5 due to severe yolk sac vascular defects. However, a similar number of Flk1(+) cells were present in RapGEF2(+/+) and RapGEF2(-/-) yolk sacs indicating that the bipotential early progenitors were in fact generated in the absence of RapGEF2. Further analysis of yolk sacs and embryos revealed a significant reduction of CD41 expressing cells in RapGEF2(-/-) genotype, suggesting a defect in the maintenance of definitive hematopoiesis. RapGEF2(-/-) cells displayed defects in proliferation and migration, and the in vitro colony formation ability of hematopoietic progenitors was also impaired. At the molecular level, Rap1 activation was impaired in RapGEF2(-/-) cells that in turn lead to defective B-raf/ERK signaling. Scl/Gata transcription factor expression was significantly reduced, indicating that the defects observed in RapGEF2(-/-) cells could be mediated through Scl/Gata deregulation. Inducible deletion of RapGEF2 during late embryogenesis in RapGEF2(cko/cko)ER(cre) mice leads to defective fetal liver erythropoiesis. Conversely, inducible deletion in the adult bone marrow, or specific deletion in B cells, T cells, HSCs, and endothelial cells has no impact on hematopoiesis.


Leaf gas exchange, carbon isotope discrimination, and grain yield in contrasting rice genotypes subjected to water deficits during the reproductive stage.
Genotypic variations in leaf gas exchange and yield were analysed in five upland-adapted and three lowland rice cultivars subjected to a differential soil moisture gradient, varying from well-watered to severely water-stressed conditions. A reduction in the amount of water applied resulted in a significant decrease in leaf gas exchange and, subsequently, in above-ground dry mass and grain yield, that varied among genotypes and distance from the line source. The comparison between the variable J and the Delta values in recently synthesized sugars methods, yielded congruent estimations of mesophyll conductance (g(m)), confirming the reliability of these two techniques. Our data demonstrate that g(m) is a major determinant of photosynthesis (A), because rice genotypes with inherently higher g(m) were capable of keeping higher A in stressed conditions. Furthermore, A, g(s), and g(m) of water-stressed genotypes rapidly recovered to the well-watered values upon the relief of water stress, indicating that drought did not cause any lasting metabolic limitation to photosynthesis. The comparisons between the A/C(i) and corresponding A/C(c) curves, measured in the genotypes that showed intrinsically higher and lower instantaneous A, confirmed this finding. Moreover, the effect of drought stress on grain yield was correlated with the effects on both A and total diffusional limitations to photosynthesis. Overall, these data indicate that genotypes which showed higher photosynthesis and conductances were also generally more productive across the entire soil moisture gradient. The analysis of Delta revealed a substantial variation of water use efficiency among the genotypes, both on the long-term (leaf pellet analysis) and short-term scale (leaf soluble sugars analysis).


CD23-mediated transport of IgE/immune complexes across human intestinal epithelium: role of p38 MAPK.
We previously reported that CD23/FcepsilonRII (low-affinity IgE receptor) is expressed on human intestinal epithelial cells and is responsible for transepithelial transport of IgE. In this study, we compared the transport of IgE with that of immune complexes in both the apical-to-serosal and the serosal-to-apical directions across HT29 epithelial cell layers and examined the effects of two p38 MAPK inhibitors, SKF86002 and SB203580, on the expression and function of CD23. Our study showed that both p38 MAPK inhibitors at 10 microM significantly inhibited constitutive and IL-4-upregulated CD23 protein expression in epithelial cells. Both inhibitors, in a concentration-dependent manner, also significantly reduced IgE binding and uptake into cells. Transepithelial transport of IgE and immune complexes across the epithelial barrier were similarly inhibited. IL-4 upregulated the phosphorylation and activity of p38 MAPK and the phosphorylation of the downstream substrate MAPKAPK-2 (MK-2). The inhibitors exerted effects in the pathway post the p38 MAPK; SB203580 significantly inhibited the phosphorylation of MK-2. Our results indicate that CD23 expression in these human intestinal epithelial cells is mediated through the p38 MAPK pathway and that inhibition of p38 MAPK consequently interferes with the transport of IgE and immune complexes across the intestinal epithelial barrier.


About females and males: continuity and discontinuity in flies.
Through the decades of relentless and dedicated studies in Drosophila melanogaster, the pathway that governs sexual development has been elucidated in great detail and has become a paradigm in understanding fundamental cell-fate decisions. However, recent phylogenetic studies show that the molecular strategy used in Drosophila deviates in some important aspects from those found in other dipteran flies and suggest that the Drosophila pathway is likely to be a derivative of a simpler and more common principle. In this essay, I will discuss the evolutionary plasticity of the sex-determining pathway based on studies in the common housefly, Musca domestica. Diversification appears to primarily arise from subtle differences in the regulation of the key switch gene transformer at the top of the pathway. On the basis of these findings I propose a new idea on how the Drosophila pathway may have evolved from a more archetypal system such as in M. domestica. In essence, the arrival of an X counting mechanism mediated by Sex-lethal to compensate for X linked gene dose differences set the stage for an intimate coupling of the two pathways. Its precedent recruitment to the dosage compensation pathway allowed for an intervention in the regulation of transformer where it gradually and eventually' completely substituted for a need of transformer autoregulation.


Occurrence of QoI Resistance and Detection of the G143A Mutation in Michigan Populations of Venturia inaequalis.
Control strategies for Venturia inaequalis rely heavily on chemical fungicides. Single-site fungicides such as the quinone-outside inhibitors (QoI) have been used in Michigan apple orchards for more than 11 years. In 2008, we sampled eight commercial orchards in the Fruit Ridge growing region of Michigan in which apple scab control failures were observed on 'McIntosh' apple following applications of kresoxim-methyl or trifloxystrobin. QoI resistance was assessed in 210 total isolates (a total of 17 orchards) using a spore germination assay and in 319 isolates using a polymerase chain reaction (PCR) assay to detect the G143A mutation located within the V. inaequalis cytochrome b gene (CYTB). The G143A mutation is known to confer high-level QoI resistance in plant-pathogenic fungi. QoI resistance was confirmed in 50 and 64% of the isolates tested with the spore germination and PCR assays, respectively, and there was a 97% concordance observed between the assays. In 2009, we sampled and examined an additional 1,201 V. inaequalis isolates from 64 orchards in Michigan and 86 isolates from four baseline sites in Ohio. All of these isolates were assayed for the G143A mutation and it was detected within 67 and 0% of the Michigan and Ohio isolates, respectively. Our results indicate the widespread occurrence of QoI resistance in Michigan commercial orchard populations of V. inaequalis. Loss of QoI fungicides further limits the arsenal of fungicides available to commercial apple growers for successful scab management.


Tuning of laser pulse shapes in grating-based compressors for optimal electron acceleration in plasmas.
The temporal shape (rise time, fall time, skewness) of 50-200-fs Ti:sapphire laser pulses has been controlled by appropriate adjustment of a grating-pair compressor. It was found that the skewness of the laser pulse envelope is particularly sensitive to the third-order component of the spectral phase. Introducing such a third-order phase offset by detuning the grating pair relative to the optimum pulse compression settings allowed the generation of skewed pulses. As an example of an application, these skewed pulses were used to optimize a laser-plasma electron accelerator.


Flexible ureterorenoscopy versus semirigid ureteroscopy for the treatment of proximal ureteral stones: a retrospective comparative analysis of 124 patients.
To investigate and compare the stone clearence and complication rates of flexible ureteroscopy (URS) with semirigid URS in patients having proximal ureteral stones. The data of 124 patients with proximal ureteral stones who underwent semirigid or flexible ureterorenoscopic lithotripsy between March 2008 and December 2012 were retrospectively investigated. The patients were divided into 2 groups according to the operation types. Group 1 included 63 patients who were treated with semirigid URS and group 2 was consisted from 61 patients who underwent flexible URS. Each group was compared in terms of stone diameter, successful access to the stone, operation time, reoperation rates, stone free status at postoperative 1st and 3rd month and complications. Successful access was achieved in 48/63 (76%) of the cases in group 1 and 57/61 (93%) of the patients in group 2 (P < .05). Initial stone free status was 63.4% (40/63) and 86.8% (53/61) in groups 1 and 2, respectively (P < .05). Third month radiologic investigations revelaed a stone free rate of 77.7% (49/57) in group 1 and 93.4% (57/61) in group 2 (P < .05). Reoperation was required in 20.6% (13/63) of cases in group 1 and this value was only 6% (4/61) in group 2 (P < .05). There was not any statistically significant difference between 2 groups in terms of complication rates (P > .05). Flexible URS is a favorable option for patients having proximal ureteral stones with higher stone free rate; on the other hand semirigid URS seems a less successful alternative for treatment of proximal ureteral stones.


Aberrant neuromagnetic activation in the motor cortex in children with acute migraine: a magnetoencephalography study.
Migraine attacks have been shown to interfere with normal function in the brain such as motor or sensory function. However, to date, there has been no clinical neurophysiology study focusing on the motor function in children with migraine during headache attacks. To investigate the motor function in children with migraine, twenty-six children with acute migraine, meeting International Classification of Headache Disorders criteria and age- and gender-matched healthy children were studied using a 275-channel magnetoencephalography system. A finger-tapping paradigm was designed to elicit neuromagnetic activation in the motor cortex. Children with migraine showed significantly prolonged latency of movement-evoked magnetic fields (MEF) during finger movement compared with the controls. The correlation coefficient of MEF latency and age in children with migraine was significantly different from that in healthy controls. The spectral power of high gamma (65-150 Hz) oscillations during finger movement in the primary motor cortex is also significantly higher in children with migraine than in controls. The alteration of responding latency and aberrant high gamma oscillations suggest that the developmental trajectory of motor function in children with migraine is impaired during migraine attacks and/or developmentally delayed. This finding indicates that childhood migraine may affect the development of brain function and result in long-term problems.


The use of portable ultrasound devices in low- and middle-income countries: a systematic review of the literature.
To review the scientific literature pertaining to the use of hand-carried and hand-held ultrasound devices in low- and middle-income countries (LMIC), with a focus on clinical applications, geographical areas of use, the impact on patient management and technical features of the devices used. The electronic databases PubMed and Google Scholar were searched. No language or date restrictions were applied. Case reports and original research describing the use of hand-carried ultrasound devices in LMIC were included if agreed upon as relevant by two-reviewer consensus based on our predefined research questions. A total of 644 articles were found and screened, and 36 manuscripts were included for final review. Twenty-seven studies were original research articles, and nine were case reports. Several reports describe the successful diagnosis and management of difficult, often life-threatening conditions, using hand-carried and hand-held ultrasound. These portable ultrasound devices have also been studied for cardiac screening exams, as well as a rapid triage tool in rural areas and after natural disaster. Most applications focus on obstetrical and abdominal complaints. Portable ultrasound may have an impact on clinical management in up to 70% of all cases. However, no randomised controlled trials have evaluated the impact of ultrasound-guided diagnosis and treatment in resource-constrained settings. The exclusion of articles published in journals not listed in the large databases may have biased our results. Our findings are limited by the lack of higher quality evidence (e.g. controlled trials). Hand-carried and hand-held ultrasound is successfully being used to triage, diagnose and treat patients with a variety of complaints in LMIC. However, the quality of the current evidence is low. There is an urgent need to perform larger clinical trials assessing the impact of hand-carried ultrasound in LMIC.


Temporal Association between Abdominal Weight Status and Healthy Aging: Findings from the 2011-2018 National Health and Aging Trends Study.
The longitudinal effect of abdominal weight status (AWS) defined by waist circumference (WC) on healthy aging has not yet been comprehensively examined. Therefore, the purpose of the present study was to examine the temporal association between WC-defined AWS and a comprehensive assessment for healthy aging. This study utilized data from 5211 respondents aged 65+ who participated in the National Health and Aging Trends Study from 2011 to 2018. Mixed effects regression models were used to examine the association between baseline AWS and the annual change rate in healthy aging score (HAS) via interaction terms (AWS*round) adjusting for confounding effects. Further multiple mixed models examined the relationship of AWS and HAS over an 8-year period. There were no annual change rate differences in HAS by baseline AWS, regardless of sex. However, males with abdominal obesity were more likely to have a lower HAS than males with normal AWS (β = -0.20, 95% CI: -0.30, -0.10, p < 0.001) but no difference in HAS was observed between males with overweight and normal AWS. A similar pattern was observed among females. Study results indicate that AWS was associated with HAS but it did not modify annual HAS change rate over time.


A qualitative study of anticipated barriers and facilitators to the implementation of nurse-delivered alcohol screening, brief intervention, and referral to treatment for hospitalized patients in a Veterans Affairs medical center.
Unhealthy alcohol use includes the spectrum of alcohol consumption from risky drinking to alcohol use disorders. Routine alcohol screening, brief intervention (BI) and referral to treatment (RT) are commonly endorsed for improving the identification and management of unhealthy alcohol use in outpatient settings. However, factors which might impact screening, BI, and RT implementation in inpatient settings, particularly if delivered by nurses, are unknown, and must be identified to effectively plan randomized controlled trials (RCTs) of nurse-delivered BI. The purpose of this study was to identify the potential barriers and facilitators associated with nurse-delivered alcohol screening, BI and RT for hospitalized patients. We conducted audio-recorded focus groups with nurses from three medical-surgical units at a large urban Veterans Affairs Medical Center. Transcripts were analyzed using modified grounded theory techniques to identify key themes regarding anticipated barriers and facilitators to nurse-delivered screening, BI and RT in the inpatient setting. A total of 33 medical-surgical nurses (97% female, 83% white) participated in one of seven focus groups. Nurses consistently anticipated the following barriers to nurse-delivered screening, BI, and RT for hospitalized patients: (1) lack of alcohol-related knowledge and skills; (2) limited interdisciplinary collaboration and communication around alcohol-related care; (3) inadequate alcohol assessment protocols and poor integration with the electronic medical record; (4) concerns about negative patient reaction and limited patient motivation to address alcohol use; (5) questionable compatibility of screening, BI and RT with the acute care paradigm and nursing role; and (6) logistical issues (e.g., lack of time/privacy). Suggested facilitators of nurse-delivered screening, BI, and RT focused on provider- and system-level factors related to: (1) improved provider knowledge, skills, communication, and collaboration; (2) expanded processes of care and nursing roles; and (3) enhanced electronic medical record features. RCTs of nurse-delivered alcohol BI for hospitalized patients should include consideration of the following elements: comprehensive provider education on alcohol screening, BI and RT; record-keeping systems which efficiently document and plan alcohol-related care; a hybrid model of implementation featuring active roles for interdisciplinary generalists and specialists; and ongoing partnerships to facilitate generation of additional evidence for BI efficacy in hospitalized patients.


Re-evaluating the use of Voronoi Tessellations in the assessment of oxygen supply from capillaries in muscle.
The ability to characterise capillary supply plays a key role in developing effective therapeutic interventions for numerous pathological conditions, such as capillary loss in skeletal or cardiac muscle. However, quantifying capillary supply is fraught with difficulties. Averaged measures such as capillary density or mean inter-capillary distance cannot account for the local geometry of the underlying capillary distribution, and thus can only highlight a tissue wide, global hypoxia. Detailed tissue geometry, such as muscle fibre size, has been incorporated into indices of capillary supply by considering the distribution of Voronoi tessellations generated from capillary locations in a plane perpendicular to muscle fibre orientation, implicitly assuming that each Voronoi polygon represents the area of supply of its enclosed capillary. Using a modelling framework to assess the capillary supply capacity under maximal sustainable conditions in muscle, we theoretically demonstrate that Voronoi tessellations often provide an accurate representation of the regions supplied by each capillary. However, we highlight that this use of Voronoi tessellations is inappropriate and inaccurate in the presence of extensive capillary rarefaction and pathological variations in oxygen tension of different capillaries. In such cases, oxygen flux trapping regions are developed to provide a more general representation of the capillary supply regions, in particular incorporating the additional influences of heterogeneity that are absent in the consideration of Voronoi tessellations.


Targeting Splicing in the Treatment of Human Disease.
The tightly regulated process of precursor messenger RNA (pre-mRNA) alternative splicing (AS) is a key mechanism in the regulation of gene expression. Defects in this regulatory process affect cellular functions and are the cause of many human diseases. Recent advances in our understanding of splicing regulation have led to the development of new tools for manipulating splicing for therapeutic purposes. Several tools, including antisense oligonucleotides and trans-splicing, have been developed to target and alter splicing to correct misregulated gene expression or to modulate transcript isoform levels. At present, deregulated AS is recognized as an important area for therapeutic intervention. Here, we summarize the major hallmarks of the splicing process, the clinical implications that arise from alterations in this process, and the current tools that can be used to deliver, target, and correct deficiencies of this key pre-mRNA processing event.


Guidelines for the emergency management of asthma in adults. CAEP/CTS Asthma Advisory Committee. Canadian Association of Emergency Physicians and the Canadian Thoracic Society.
To develop a set of comprehensive, standardized evidence-based guidelines for the assessment and treatment of acute asthma in adults in the emergency setting. The use of medications was evaluated by class, dose, route, onset of action and optimal mode of delivery. The use of objective measurements and clinical features to assess response to therapy were evaluated in relation to the decision to admit or discharge the patient or arrange for follow-up care. Control of symptoms and disease reflected in hospital admission rates, frequency of treatment failures following discharge, resolution of symptoms and improvement of spirometric test results. Previous guidelines, articles retrieved through a search of MEDLINE, emergency medical abstracts and information from members of the expert panel were reviewed by members of the Canadian Association of Emergency Physicians (CAEP) and the Canadian Thoracic Society. Where evidence was not available, consensus was reached by the expert panel. The resulting guidelines were reviewed by members of the parent organizations. The evidence-based methods and values of the Canadian Task Force on the Periodic Health Examination were used. As many as 80% of the approximate 400 deaths from asthma each year in Canada are felt to be preventable. The use of guidelines, aggressive emergency management and consistent use of available options at discharge are expected to decrease the rates of unnecessary hospital admissions and return visits to emergency departments because of treatment failures. Substantial decreases in costs are expected from the use of less expensive drugs, or drug delivery systems, fewer hospital admissions and earlier return to full activity after discharge. Beta2-agonists are the first-line therapy for the management of acute asthma in the emergency department (grade A recommendation). Bronchodilators should be administered by the inhaled route and titrated using objective and clinical measures of airflow limitation (grade A). Metered-dose inhalers are preferred to wet nebulizers, and a chamber (spacer device) is recommended for severe asthma (grade A). Anticholinergic therapy should be added to beta 2 agonist therapy in severe and life-threatening cases and may be considered in cases of mild to moderate asthma (grade A). Aminophylline is not recommended for use in the first 4 hours of therapy (grade A). Ketamine and succinylcholine are recommended for rapid sequence intubation in life-threatening cases (grade B). Adrenaline (administered subcutaneously or intravenously), salbutamol (administered intravenously) and anesthetics (inhaled) are recommended as alternatives to conventional therapy in unresponsive life-threatening cases (grade B). Severity of airflow limitation should be determined according to the forced expiratory volume at 1 second or the peak expiratory flow rate, or both, before and after treatment and at discharge (grade A). Consideration for discharge should be based on both spirometric test results and assessment of clinical risk factors for relapse (grade A). All patients should be considered candidates for systemic corticosteroid therapy at discharge (grade A). Those requiring corticosteroid therapy should be given 30 to 60 mg of prednisone orally (or equivalent) per day for 7 to 14 days; no tapering is required (grade A). Inhaled corticosteroids are an integral component of therapy and should be prescribed for all patients receiving oral corticosteroid therapy at discharge (grade A). Patients should be given a discharge treatment plan and clear instructions for follow-up care (grade C). The guidelines share the same principles of those from the British Thoracic Society and the National Institutes of Health. Two specific validation initiatives have been undertaken: (a) several Canadian centres have been involved in the collection of comprehensive administrative data to assess compliance and outcome measures and (b) a survey of Canadian emergency physicians conducted to gather baseline informaton of treatment patterns, was conducted before development of the guidelines and will be repeated to re-evaluate emergency management of asthma.


The Effects of GABAergic Polarity Changes on Episodic Neural Network Activity in Developing Neural Systems.
Early in development, neural systems have primarily excitatory coupling, where even GABAergic synapses are excitatory. Many of these systems exhibit spontaneous episodes of activity that have been characterized through both experimental and computational studies. As development progress the neural system goes through many changes, including synaptic remodeling, intrinsic plasticity in the ion channel expression, and a transformation of GABAergic synapses from excitatory to inhibitory. What effect each of these, and other, changes have on the network behavior is hard to know from experimental studies since they all happen in parallel. One advantage of a computational approach is that one has the ability to study developmental changes in isolation. Here, we examine the effects of GABAergic synapse polarity change on the spontaneous activity of both a mean field and a neural network model that has both glutamatergic and GABAergic coupling, representative of a developing neural network. We find some intuitive behavioral changes as the GABAergic neurons go from excitatory to inhibitory, shared by both models, such as a decrease in the duration of episodes. We also find some paradoxical changes in the activity that are only present in the neural network model. In particular, we find that during early development the inter-episode durations become longer on average, while later in development they become shorter. In addressing this unexpected finding, we uncover a priming effect that is particularly important for a small subset of neurons, called the "intermediate neurons." We characterize these neurons and demonstrate why they are crucial to episode initiation, and why the paradoxical behavioral change result from priming of these neurons. The study illustrates how even arguably the simplest of developmental changes that occurs in neural systems can present non-intuitive behaviors. It also makes predictions about neural network behavioral changes that occur during development that may be observable even in actual neural systems where these changes are convoluted with changes in synaptic connectivity and intrinsic neural plasticity.


An atlas of gene expression from seed to seed through barley development.
Assaying relative and absolute levels of gene expression in a diverse series of tissues is a central step in the process of characterizing gene function and a necessary component of almost all publications describing individual genes or gene family members. However, throughout the literature, such studies lack consistency in genotype, tissues analyzed, and growth conditions applied, and, as a result, the body of information that is currently assembled is fragmented and difficult to compare between different studies. The development of a comprehensive platform for assaying gene expression that is available to the entire research community provides a major opportunity to assess whole biological systems in a single experiment. It also integrates detailed knowledge and information on individual genes into a unified framework that provides both context and resource to explore their contributions in a broader biological system. We have established a data set that describes the expression of 21,439 barley genes in 15 tissues sampled throughout the development of the barley cv. Morex grown under highly controlled conditions. Rather than attempting to address a specific biological question, our experiment was designed to provide a reference gene expression data set for barley researchers; a gene expression atlas and a comparative data set for those investigating genes or regulatory networks in other plant species. In this paper we describe the tissues sampled and their transcriptomes, and provide summary information on genes that are either specifically expressed in certain tissues or show correlated expression patterns across all 15 tissue samples. Using specific examples and an online tutorial, we describe how the data set can be interrogated for patterns and levels of barley gene expression and how the resulting information can be used to generate and/or test specific biological hypotheses.


'Let the computer choose?': the experience of participants in a randomised preference trial of medical versus surgical termination of pregnancy.
The termination of pregnancy trial (Newcastle upon Tyne, UK), is the only randomised trial on termination of pregnancy methods incorporating a qualitative element that aimed to understand the experiences of women participating in the trial. Based on the results of this qualitative work, this article aims to provide insights into two strands of understanding; firstly, women's experience of participating in research about abortion and secondly, their experience of participating in a randomised preference trial. Semi-structured interviews were conducted of up to 90 minutes with 30 participants recruited at a single hospital site. A total of 20 women from the preference arm and 10 from the random arm were interviewed. The analysis and discussion of our findings use reflexive modernisation as a framework for understanding and interpreting some of the actions of social agents, that is, the participants and trial recruiters in the course of a clinical trial as an expert system. We found that the factors that shape women's experiences and decisions include trust in the expert system and reflexivity and agency on the part of both participants and trial recruiters.


Possible sources of discrepancies in the use of the Semmes-Weinstein monofilament. Impact on prevalence of insensate foot and workload requirements.
The purpose of this study was to evaluate the effects of different testing sites and buckling strengths on the sensitivity and specificity of using the Semmes-Weinstein monofilament to detect patients with insensate foot. The impact on workload required to educate and follow up these high-risk individuals was estimated by modeling in our patient population with a documented status of neuropathy. Using the 5.07/10-g monofilament, one observer tested 132 randomly selected subjects with diabetes at five sites on the right foot. The sensitivity and specificity of each site and combinations of sites in detecting vibration perception threshold > 40 was calculated. In addition, two monofilaments, one with a buckling force of 5 g and the other with a force of 15 g, were compared by testing 200 randomly selected patients. An estimate of the prevalence of insensate foot and workload was made by modeling the findings to the 5,270 patients with neuropathy status registered on our computerized database. Specificity of the 5.07/10-g monofilament to detect insensate foot at each of the five sites is high, at approximately 90%, but there is considerably more variation and lower sensitivity, ranging from 44-71%. Data derived from the use of different combinations of sites showed that more stringent criteria are associated with lower sensitivity but higher specificity. If the foot is considered insensate when either of sites 3 and 4 (plantar aspect of the first and fifth metatarsal heads, respectively) cannot feel the monofilament, there is reasonable sensitivity and specificity (80-86%, respectively). By modeling on our diabetes center population, it can be demonstrated that the choice of different methodologies leads to different conclusions about the prevalence of severe neuropathy, ranging from 3.4 to 29.3%. Using a combination of sites 3 and 4 for monofilament testing gives a reasonable compromise for time, sensitivity, and specificity. Minor changes in sensitivity and specificity can lead to major changes in the prevalence of neuropathy, with implications for workload.


Imaging of Bronchial Pathology in Antibody Deficiency: Data from the European Chest CT Group.
Studies of chest computed tomography (CT) in patients with primary antibody deficiency syndromes (ADS) suggest a broad range of bronchial pathology. However, there are as yet no multicentre studies to assess the variety of bronchial pathology in this patient group. One of the underlying reasons is the lack of a consensus methodology, a prerequisite to jointly document chest CT findings. We aimed to establish an international platform for the evaluation of bronchial pathology as assessed by chest CT and to describe the range of bronchial pathologies in patients with antibody deficiency. Ffteen immunodeficiency centres from 9 countries evaluated chest CT scans of patients with ADS using a predefined list of potential findings including an extent score for bronchiectasis. Data of 282 patients with ADS were collected. Patients with common variable immunodeficiency disorders (CVID) comprised the largest subgroup (232 patients, 82.3%). Eighty percent of CVID patients had radiological evidence of bronchial pathology including bronchiectasis in 61%, bronchial wall thickening in 44% and mucus plugging in 29%. Bronchiectasis was detected in 44% of CVID patients aged less than 20 years. Cough was a better predictor for bronchiectasis than spirometry values. Delay of diagnosis as well as duration of disease correlated positively with presence of bronchiectasis. The use of consensus diagnostic criteria and a pre-defined list of bronchial pathologies allows for comparison of chest CT data in multicentre studies. Our data suggest a high prevalence of bronchial pathology in CVID due to late diagnosis or duration of disease.


The cardiocardiac sympathetic reflex during coronary occlusion in anesthetized dogs.
Cardiac sympathetic afferent fibers activated during coronary occlusion exert an excitatory influence on sympathetic discharge to the heart in cats after spinal cord section. The significance of this cardiocardiac sympathetic reflex response during myocardial ischemia in animals with an intact neuraxis is unknown. We studied the responses of efferent cardiac sympathetic nerve activity (CSNA), arterial pressure, and heart rate to coronary occlusion in two groups of dogs with cardiac sympathetic reflexes intact and with other reflex inputs affecting CSNA sectioned or controlled. In group I (n = 10), the vagi were sectioned, the spinal cord remained intact, and the carotid sinuses were isolated and perfused to maintain baroreceptor input constant. Coronary occlusion was performed at moderate and low basal levels of CSNA by setting carotid sinus pressure at 125-150 and 200 mm Hg, respectively. Under these conditions, CSNA was not altered by occlusion of either the anterior descending or the circumflex coronary artery. In group II (n = 4), the vagi were sectioned and the spinal cord was interrupted. In these dogs, CSNA increased significantly (61 +/- 19%) during coronary occlusion. These results show that an excitatory cardiocardiac sympathetic reflex can be demonstrated in dogs with spinal cords sectioned but not with spinal cords intact. This finding is consistent with the view that inhibitory bulbospinal pathways minimize the influence of the spinal cardiocardiac sympathetic reflex during myocardial ischemia in anesthetized dogs.


Endogenous siRNAs derived from transposons and mRNAs in Drosophila somatic cells.
Small interfering RNAs (siRNAs) direct RNA interference (RNAi) in eukaryotes. In flies, somatic cells produce siRNAs from exogenous double-stranded RNA (dsRNA) as a defense against viral infection. We identified endogenous siRNAs (endo-siRNAs), 21 nucleotides in length, that correspond to transposons and heterochromatic sequences in the somatic cells of Drosophila melanogaster. We also detected endo-siRNAs complementary to messenger RNAs (mRNAs); these siRNAs disproportionately mapped to the complementary regions of overlapping mRNAs predicted to form double-stranded RNA in vivo. Normal accumulation of somatic endo-siRNAs requires the siRNA-generating ribonuclease Dicer-2 and the RNAi effector protein Argonaute2 (Ago2). We propose that endo-siRNAs generated by the fly RNAi pathway silence selfish genetic elements in the soma, much as Piwi-interacting RNAs do in the germ line.


MAPK phosphatase-3 promotes hepatic gluconeogenesis through dephosphorylation of forkhead box O1 in mice.
Insulin resistance results in dysregulated hepatic gluconeogenesis that contributes to obesity-related hyperglycemia and progression of type 2 diabetes mellitus (T2DM). Recent studies show that MAPK phosphatase-3 (MKP-3) promotes gluconeogenic gene transcription in hepatoma cells, but little is known about the physiological role of MKP-3 in vivo. Here, we have shown that expression of MKP-3 is markedly increased in the liver of diet-induced obese mice. Consistent with this, adenovirus-mediated MKP-3 overexpression in lean mice promoted gluconeogenesis and increased fasting blood glucose levels. Conversely, shRNA knockdown of MKP-3 in both lean and obese mice resulted in decreased fasting blood glucose levels. In vitro experiments identified forkhead box O1 (FOXO1) as a substrate for MKP-3. MKP-3-mediated dephosphorylation of FOXO1 at Ser256 promoted its nuclear translocation and subsequent recruitment to the promoters of key gluconeogenic genes. In addition, we showed that PPARγ coactivator-1α (PGC-1α) acted downstream of FOXO1 to mediate MKP-3-induced gluconeogenesis. These data indicate that MKP-3 is an important regulator of hepatic gluconeogenesis in vivo and suggest that inhibition of MKP-3 activity may provide new therapies for T2DM.


Presynaptic NMDARs in the hippocampus facilitate transmitter release at theta frequency.
A rise in [Ca(2+)](i) provides the trigger for neurotransmitter release at neuronal boutons. We have used confocal microscopy and Ca(2+) sensitive dyes to directly measure the action potential-evoked [Ca(2+)](i) in the boutons of Schaffer collaterals. This reveals that the trial-by-trial amplitude of the evoked Ca(2+) transient is bimodally distributed. We demonstrate that "large" Ca(2+) transients occur when presynaptic NMDA receptors are activated following transmitter release. Presynaptic NMDA receptor activation proves critical in producing facilitation of transmission at theta frequencies. Because large Ca(2+) transients "report" transmitter release, their frequency on a trial-by-trial basis can be used to estimate the probability of release, p(r). We use this novel estimator to show that p(r) increases following the induction of long-term potentiation.


Response Styles to Positive Affect and Depression: Concurrent and Prospective Associations in a Community Sample.
We examined the concurrent and prospective relations between response styles to positive affect and depression in a community sample. Participants (n = 345) completed self-report measures of current and past depressive episodes, depressive symptoms, anhedonia, and responses to positive affect (including dampening and positive rumination) at two time points, with a 5-month interval. Higher levels of dampening responses to positive affect were related to higher concurrent levels of depressive symptoms. The tendency to positively ruminate on positive affect was negatively related to concurrent anhedonic symptoms. When controlling for current depressive symptomatology, formerly depressed individuals had a higher tendency to dampen positive affect than never-depressed controls, and did not differ from a currently depressed group. Dampening responses did not predict depressive symptoms prospectively, but lower levels of (self-focused) positive rumination did predict higher levels of future anhedonic symptoms. Results indicate that not only currently but also formerly depressed individuals engage in dysfunctional (dampening) strategies in response to positive affect. It is possible that currently as well as formerly depressed individuals might benefit from interventions that are directed at the remediation of disturbed regulation of positive affect. However, our prospective results make clear that more research is needed to examine the precise conditions under which dampening would be a detrimental (and positive rumination a beneficial) response style in the course of depression.


Results of a multicenter randomized phase II trial of thalidomide and prednisone maintenance therapy for multiple myeloma after autologous stem cell transplant.
We report a multicenter, randomized phase II trial conducted to assess the tolerability of combined thalidomide and prednisone maintenance in multiple myeloma. Eligibility required administration of melphalan (200 mg/m2) with blood stem cell support within 1 year of treatment onset and initiation of maintenance within 60 to 100 days after stem cell infusion. All patients received 50 mg of prednisone by mouth on alternate days and thalidomide at a starting dose of either 200 or 400 mg daily by mouth. The primary end point was the incidence of dropout or dose reduction due to treatment toxicity within 6 months. Sixty-seven patients were enrolled. Median follow-up is 36.8 months. The primary end point was reached by 31% of patients on the 200 mg of thalidomide arm and 64% of patients on the 400 mg of thalidomide arm. Allowing for dose reduction, 76% of patients assigned to the 200 mg of thalidomide arm and 41% of patients assigned to the 400 mg of thalidomide arm remained on any maintenance therapy 18 months after registration. Eighty-eight percent of all patients dose-reduced thalidomide and 72% of all patients dose-reduced prednisone within 2 years of beginning maintenance. The median progression-free survival post-transplant is 32.3 months, or 42.2 months from diagnosis. Only the 200 mg of thalidomide arm of this trial met our definition of a tolerable maintenance therapy, defined as no dose reductions or discontinuation due to toxicity in at least 65% of patients for a minimum of 6 months, thus establishing a dosing schedule for phase III trials.


In vitro reconstitution of eukaryotic ion channels using droplet interface bilayers.
The ability to routinely study eukaryotic ion channels in a synthetic lipid environment would have a major impact on our understanding of how different lipids influence ion channel function. Here, we describe a straightforward, detergent-free method for the in vitro reconstitution of eukaryotic ion channels and ionotropic receptors into droplet interface bilayers and measure their electrical activity at both the macroscopic and single-channel level. We explore the general applicability of this method by reconstitution of channels from a wide range of sources including recombinant cell lines and native tissues, as well as preparations that are difficult to study by conventional methods including erythrocytes and mitochondria.


Evidence that HLA class I and II associations with type 1 diabetes, autoantibodies to GAD and autoantibodies to IA-2, are distinct.
A major feature of type 1 diabetes is the appearance of islet autoantibodies before diagnosis. However, although the genetics of type 1 diabetes is advanced, the genetics of islet autoantibodies needs further investigation. The primary susceptibility loci in type 1 diabetes, the HLA class I and II genes, are believed to determine the specificity and magnitude of the autoimmune response to islet antigens. We investigated the association of glutamic acid decarboxylase autoantibodies (GADA) and insulinoma-associated antigen-2 autoantibodies (IA-2A) with the HLA region. Associations of GADA and IA-2A with HLA-DRB1, HLA-DQB1, HLA-B, HLA-C, HLA-A, MICA, and 3,779 single nucleotide polymorphisms (SNPs) were analyzed in 2,531 childhood-onset case subjects (median time since diagnosis 5 years). All analyses were adjusted for age-at-diagnosis and duration of diabetes. GADA and IA-2A were associated with an older age-at-diagnosis (P < 10(-19)). For GADA, the primary association was with HLA-DQB1 (P = 9.00 × 10(-18)), with evidence of a second independent effect in the HLA class I region with SNP, rs9266722 (P = 2.84 × 10(-6)). HLA-DRB1 had the strongest association with IA-2A (P = 1.94 × 10(-41)), with HLA-A*24 adding to the association, albeit negatively (P = 1.21 × 10(-10)). There was no evidence of association of either IA-2A or GADA with the highly type 1 diabetes predisposing genotype, HLA-DRB1*03/04. Despite genetic association of type 1 diabetes and the islet autoantibodies localizing to the same HLA class II genes, HLA-DRB1 and HLA-DQB1, the effects of the class II alleles and genotypes involved are quite different. Therefore, the presence of autoantibodies is unlikely to be causal, and their role in pathogenesis remains to be established.


Differential antimicrobial susceptibility profiles between symptomatic and asymptomatic non-typhoidal Salmonella infections in Vietnamese children.
Non-typhoidal Salmonella (NTS) serovars, sequences types and antimicrobial susceptibility profiles have specific associations with animal and human infections in Vietnam. Antimicrobial resistance may have an effect on the manifestation of human NTS infections, with isolates from asymptomatic individuals being more susceptible to antimicrobials than those associated with animals and human diarrhoea.


Cholesterol modulates the organization of the gammaM4 transmembrane domain of the muscle nicotinic acetylcholine receptor.
A 28-mer gammaM4 peptide, obtained by solid-state synthesis and corresponding to the fourth transmembrane segment of the nicotinic acetylcholine receptor gamma-subunit, possesses a single tryptophan residue (Trp453), making it an excellent model for studying peptide-lipid interactions in membranes by fluorescence spectroscopy. The gammaM4 peptide was reconstituted with synthetic lipids (vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, i.e., POPC) rich and poor in cholesterol and analyzed using steady-state and time-resolved fluorescence techniques. The decrease in gammaM4 intrinsic fluorescence lifetime observed upon incorporation into a cholesterol-rich lo phase could be rationalized on the basis of a dynamic self-quenching owing to the formation of peptide-rich patches in the membrane. This agrees with the low Förster type resonance energy transfer efficiency from the Trp453 residue to the fluorescent cholesterol analog, dehydroergosterol, in the lo phase. In the absence of cholesterol the gammaM4 nicotinic acetylcholine receptor peptide is randomly distributed in the POPC bilayer with its hydrophobic moiety matching the membrane thickness, whereas in the presence of cholesterol the increase in the membrane thickness and variation of the material properties favor the formation of peptide-enriched patches, i.e., interhelix interaction energy is essential for obtaining a stabilized structure. Thus, the presence of a cholesterol-rich, ordered POPC phase drives the organization of peptide-enriched patches, in which the gammaM4 peptide occupies approximately 30% of the patch area.


Genetic architecture of early pre-inflammatory stage transcription signatures of autoimmune diabetes in the pancreatic lymph nodes of the NOD mouse reveals significant gene enrichment on chromosomes 6 and 7.
Autoimmune diseases are characterized by the stimulation of an excessive immune response to self-tissues by inner and/or outer organism factors. Common characteristics in their etiology include a complex genetic predisposition and environmental triggers as well as the implication of the major histocompatibility (MHC) locus on human chromosome 6p21. A restraint number of non-MHC susceptibility genes, part of the genetic component of type 1 diabetes have been identified in human and in animal models, while the complete spectrum of genes involved remains unknown. We elaborate herein patterns of chromosomal organization of 162 genes differentially expressed in the pancreatic lymph nodes of Non-Obese Diabetic mice, carefully selected by early sub-phenotypic evaluation (presence or absence of insulin autoantibodies). Chromosomal assignment of these genes revealed a non-random distribution on five chromosomes (47%). Significant gene enrichment was observed in particular for two chromosomes, 6 and 7. While a subset of these genes coding for secreted proteins showed significant enrichment on both chromosomes, the overall pool of genes was significantly enriched on chromosome 7. The significance of this unexpected gene distribution on the mouse genome is discussed in the light of novel findings indicating that genes affecting common diseases map to recombination "hotspot" regions of mammalian genomes. The genetic architecture of transcripts differentially expressed in specific stages of autoimmune diabetes offers novel venues towards our understanding of patterns of inheritance potentially affecting the pathological disease mechanisms.


Vitamin D supplementation modulates the immune system and improves atopic dermatitis in children.
Vitamin D seems to influence the evolution of atopic dermatitis (AD) in children. We tested the vitamin D serum levels of 39 children with AD (AD group t0) and of 20 nonallergic healthy controls (C group). AD severity was evaluated using the AD scoring system (SCORAD index). Cytokine serum levels (IL-2, IL-4, IL-6, IFN-γ, TNF-α) and atopy biomarkers were also measured. The patients were then treated with vitamin D oral supplementation of 1,000 IU/day (25 mg/day) for 3 months. We then reevaluated the vitamin D serum levels, AD severity and cytokine serum levels in all of the treated children (AD group t1). The cross-sectional analysis on patients affected by AD (AD group t0) showed that the initial levels of all the tested cytokines except for TNF-α were higher than those of the healthy control group (C group), falling outside the normal range. After 3 months of supplementation the patients had significantly increased vitamin D levels (from 22.97 ± 8.03 to 29.41 ± 10.73 ng/ml; p = 0.01). A concomitant significant reduction of both the SCORAD index (46.13 ± 15.68 at the first visit vs. 22.57 ± 15.28 at the second visit; p < 0.001) and of all the altered cytokines (IL-2, IL-4, IL-6, IFN-γ) was also found. This study showed vitamin D supplementation to be an effective treatment in reducing AD severity in children through normalization of the Th1 and Th2 interleukin serum pattern.


Effects of parenteral infusion with medium-chain triglycerides and safflower oil emulsions on hepatic lipids, plasma amino acids and inflammatory mediators in septic rats.
This study was designed to investigate the effects of preinfusion with total parenteral nutrition (TPN) using medium-chain triglycerides (MCT) versus safflower oil (SO) emulsion as fat sources on hepatic lipids, plasma amino acid profiles, and inflammatory-related mediators in septic rats. Normal rats, with internal jugular catheters, were divided into two groups and received TPN. TPN provided 300kcal/kg/day with 40% of the non-protein energy provided as fat. All TPN solutions were isonitrogenous and identical in nutrient composition except for the fat emulsion, which was made of SO or a mixture of MCT and soybean oil (9:1) (MO). After receiving TPN for 6 days, each group of rats was further divided into control and sepsis subgroups. Sepsis was induced by cecal ligation and puncture, whereas control rats received sham operation. All rats were classified into four groups as follows: MCT control group (MOC, n= 8), MCT sepsis group (MOS, n= 8), safflower oil control group (SOC, n= 8), and safflower oil sepsis group (SOS, n= 11). The results of the study demonstrated that the MOS group had lower hepatic lipids than did the SOS group. Plasma leucine and isoleucine levels were significantly lower in the SOS than in the SOC group, but no differences in these two amino acids were observed between the MOC and MOS groups. Plasma arginine levels were significantly lower in septic groups than in those without sepsis despite whether MCT or safflower oil was infused. Plasma glutamine and alanine levels, however, did not differ between septic and non-septic groups either in the SO or MO groups. No differences in interleukin-1b, interleukin-6, tumor necrosis factor-alpha, and leukotriene B(4)concentrations in peritoneal lavage fluid were observed between the two septic groups. These results suggest that catabolic reaction is septic rats preinfused MCT is not as obvious as those preinfused safflower oil. Compared with safflower oil, TPN with MCT administration has better effects on reducing sepsis-induced liver fat deposition. Preinfusion with MCT before sepsis, however, had no effect on inflammatory-related cytokines or leukotriene in peritoneal lavage fluid. In addition, plasma arginine appears to be a more sensitive indicator than glutamine for septic insult.


Skeletal muscle degeneration and regeneration after injection of bothropstoxin-II, a phospholipase A2 isolated from the venom of the snake Bothrops jararacussu.
A myotoxic phospholipase A2, named bothropstoxin II (BthTX-II), was isolated from the venom of the South American snake Bothrops jararacussu and the pathogenesis of myonecrosis induced by this toxin was studied in mice. BthTX-II induced a rapid increase in plasma creatine kinase levels. Histological and ultrastructural observations demonstrate that this toxin affects muscle fibers by first disrupting the integrity of plasma membrane, as "delta lesions" were the earliest morphological alteration and since the plasma membrane was interrupted or absent in many portions. In agreement with this hypothesis, BthTX-II released peroxidase entrapped in negatively charged multilamellar liposomes and behaved as an amphiphilic protein in charge shift electrophoresis, an indication that its mechanism of action might be based on the interaction and disorganization of plasma membrane phospholipids. Membrane damage was followed by a complex series of morphological alterations in intracellular structures, most of which are probably related to an increase in cytosolic calcium levels. Myofilaments became hypercontracted into dense clumps which alternated with cellular spaces devoid of myofibrillar material. Later on, myofilaments changed to a hyaline appearance with a more uniform distribution. Mitochondria were drastically affected, showing high amplitude swelling, vesiculation of cristae, formation of flocculent densities, and membrane disruption. By 24 hr, abundant polymorphonuclear leucocytes and macrophages were observed in the interstitial space as well as inside necrotic fibers. Muscle regeneration proceeded normally, as abundant myotubes and regenerating myofibers were observed 7 days after BthTX-II injection. By 28 days regenerating fibers had a diameter similar to that of adult muscle fibers, although they presented two distinctive features: central location of nuclei and some fiber splitting. This good regenerative response may be explained by the observation that BthTX-II does not affect blood vessels, nerves, or basal laminae.


Effectively responding to tactile stimulation: do homologous cue and effector locations really matter?
We report a study designed to investigate the extent to which speeded behavioral responses following tactile stimulation are influenced by differences in neural conduction latencies at different body sites and/or by the characteristics of the compatibility between the cue and effector. The results showed that it may not be particularly desirable to present tactile cues (e.g., warning signals) to an interface operator's feet if a speeded foot response is required, for even though such an arrangement maximizes the set-level compatibility between the stimulus and the response, it turns out that response latencies are primarily determined by conduction latencies through the peripheral nervous system.


Beta-glucans in advanced CKD: role in endotoxaemia and inflammation.
(1-3)-β-D glucans (BG) are cellular components of yeasts and fungi. Elevated blood levels may be an adjunct in diagnosing invasive fungal infection, though can be high in dialysis patients without fungaemia. BG can also induce false positive signals in endotoxin detection assays (Limulus Amoebocyte Lysate [LAL] assay). We explored the relationship between BG levels, renal impairment, endotoxaemia and inflammation. We measured serum BG levels, markers of inflammation and blood endotoxin levels in 20 controls, 20 with stages 1-3 chronic kidney disease (CKD), 20 with stages 4-5 CKD, 15 on peritoneal dialysis (PD) and 60 on haemodialysis (HD). Another 30 patients were studied before and after HD initiation. BG levels increased with advancing CKD, being highest in HD patients, 22% of whom had elevated levels (> 80 pg/ml). Levels increased significantly following HD initiation. Levels also correlated positively with CRP, TNFα, IL-6 levels, independently of CKD stage. Blood endotoxin was detectable by LAL assays in 10-53% of the CKD cohort, being most prevalent in the HD group, and correlating positively with BG levels. Adding BG blocking agent to the assay reduced endotoxin detection confining it to only 5% of HD patients. Levels of inflammatory markers were higher in those with detectable endotoxin - whether false- or true positives. BG levels increased with decreasing renal function, being highest in dialysis patients. High BG levels were associated with false positive blood endotoxin signals, and with markers of inflammation, independently of CKD stage. The cause for high BG levels is unknown but could reflect increased gut permeability and altered mononuclear phagocytic system function.