Raman Imaging of Plant Cell Walls in Sections of Cucumis sativus.
Raman microspectra combine information on chemical composition of plant tissues with spatial information. The contributions from the building blocks of the cell walls in the Raman spectra of plant tissues can vary in the microscopic sub-structures of the tissue. Here, we discuss the analysis of 55 Raman maps of root, stem, and leaf tissues of Cucumis sativus, using different spectral contributions from cellulose and lignin in both univariate and multivariate imaging methods. Imaging based on hierarchical cluster analysis (HCA) and principal component analysis (PCA) indicates different substructures in the xylem cell walls of the different tissues. Using specific signals from the cell wall spectra, analysis of the whole set of different tissue sections based on the Raman images reveals differences in xylem tissue morphology. Due to the specifics of excitation of the Raman spectra in the visible wavelength range (532 nm), which is, e.g., in resonance with carotenoid species, effects of photobleaching and the possibility of exploiting depletion difference spectra for molecular characterization in Raman imaging of plants are discussed. The reported results provide both, specific information on the molecular composition of cucumber tissue Raman spectra, and general directions for future imaging studies in plant tissues.


Effects of leukotriene D4 on glomerular dynamics in the rat.
Glomerular micropuncture studies were performed in euvolemic, anesthetized, adult, male Munich-Wistar rats to evaluate the glomerular microcirculatory actions of leukotriene D4 (LTD4). In preliminary experiments, intrarenal arterial administration of LTD4 (1 microgram X kg-1 X min-1) was found to cause a rise in mean systemic arterial pressure, a loss of plasma volume, and a fall in renal blood flow, effects identical to those previously reported for LTC4. To assess the local glomerular actions of LTD4 independently of these systemic effects, glomerular microcirculatory dynamics were assessed during LTD4 infusion while constancy of renal perfusion pressure and plasma volume were maintained by partial aortic constriction and isoncotic plasma infusion, respectively. To ascertain the LTD4 actions that are independent of angiotensin II, saralasin (5 micrograms X kg-1 X min-1 iv) was also given throughout the study. LTD4 caused a significant increase in efferent arteriolar resistance in association with a fall in glomerular plasma flow rate (QA) and a rise in glomerular capillary hydraulic pressure. Despite the latter, single-nephron filtration rate fell due to combined reductions in QA and the glomerular ultrafiltration coefficient. These local glomerular constrictor actions of LTD4 support the possibility that this eicosanoid might play an important intermediary role in the functional impairment accompanying some forms of inflammatory injury.


BosR (BB0647) controls the RpoN-RpoS regulatory pathway and virulence expression in Borrelia burgdorferi by a novel DNA-binding mechanism.
In Borrelia burgdorferi (Bb), the Lyme disease spirochete, the alternative σ factor σ⁵⁴ (RpoN) directly activates transcription of another alternative σ factor, σ(S) (RpoS) which, in turn, controls the expression of virulence-associated membrane lipoproteins. As is customary in σ⁵⁴-dependent gene control, a putative NtrC-like enhancer-binding protein, Rrp2, is required to activate the RpoN-RpoS pathway. However, recently it was found that rpoS transcription in Bb also requires another regulator, BosR, which was previously designated as a Fur or PerR homolog. Given this unexpected requirement for a second activator to promote σ⁵⁴-dependent gene transcription, and the fact that regulatory mechanisms among similar species of pathogenic bacteria can be strain-specific, we sought to confirm the regulatory role of BosR in a second virulent strain (strain 297) of Bb. Indeed, BosR displayed the same influence over lipoprotein expression and mammalian infectivity for strain Bb 297 that were previously noted for Bb strain B31. We subsequently found that recombinant BosR (rBosR) bound to the rpoS gene at three distinct sites, and that binding occurred despite the absence of consensus Fur or Per boxes. This led to the identification of a novel direct repeat sequence (TAAATTAAAT) critical for rBosR binding in vitro. Mutations in the repeat sequence markedly inhibited or abolished rBosR binding. Taken together, our studies provide new mechanistic insights into how BosR likely acts directly on rpoS as a positive transcriptional activator. Additional novelty is engendered by the facts that, although BosR is a Fur or PerR homolog and it contains zinc (like Fur and PerR), it has other unique features that clearly set it apart from these other regulators. Our findings also have broader implications regarding a previously unappreciated layer of control that can be involved in σ⁵⁴-dependent gene regulation in bacteria.


Autosomal ring chromosomes in human genetic disorders.
Ring chromosomes arise following breakage and rejoining in both chromosome arms. They are heterogeneous with variable size and genetic content and can originate from any chromosome. Phenotypes associated with ring chromosomes are highly variable as apart from any deletion caused by ring formation, imbalances from ring instability can also occur. Of interest is ring chromosome 20 which has a significant association with epilepsy with seizure onset in early childhood. Severe growth deficiency without major malformations is a common finding in the ring chromosome carrier. This phenotype associated with ring behaviour and mitotic instability and independent of the chromosome involved has been termed the "ring syndrome". Precise genotype-phenotype correlations for ring chromosomes may not be possible as influencing factors vary depending on the extent of deletion in ring formation, ring instability and the level of mosaicism. Although ring chromosomes usually arise as de novo events, familial transmission of rings from carrier to offspring has been described and prenatal diagnosis for any pregnancies should always be considered.


Direct interaction of focal adhesion kinase with p190RhoGEF.
Focal adhesion kinase (FAK) is a protein-tyrosine kinase that associates with multiple cell surface receptors and signaling proteins through which it can modulate the activity of several intracellular signaling pathways. FAK activity can influence the formation of distinct actin cytoskeletal structures such as lamellipodia and stress fibers in part through effects on small Rho GTPases, although the molecular interconnections of these events are not well defined. Here, we report that FAK interacts with p190RhoGEF, a RhoA-specific GDP/GTP exchange factor, in neuronal cells and in brain tissue extracts by co-immunoprecipitation and co-localization analyses. Using a two-hybrid assay and deletion mutagenesis, the binding site of the FAK C-terminal focal adhesion targeting (FAT) domain was identified within the C-terminal coiled-coil domain of p190RhoGEF. Binding was independent of a LD-like binding motif within p190RhoGEF, yet FAK association was disrupted by a mutation (Leu-1034 to Ser) that weakens the helical bundle structure of the FAK FAT domain. Neuro-2a cell binding to laminin increased endogenous FAK and p190RhoGEF tyrosine phosphorylation, and co-transfection of a dominant-negative inhibitor of FAK activity, termed FRNK, inhibited lamininstimulated p190RhoGEF tyrosine phosphorylation and p21 RhoA GTP binding. Overexpression of FAK in Neuro-2a cells increased both endogenous p190RhoGEF tyrosine phosphorylation and RhoA activity, whereas these events were inhibited by FRNK co-expression. Because insulin-like growth factor 1 treatment of Neuro-2a cells increased FAK tyrosine phosphorylation and enhanced p190RhoGEF-mediated activation of RhoA, our results support the conclusion that FAK association with p190RhoGEF functions as a signaling pathway downstream of integrins and growth factor receptors to stimulate Rho activity.


O-Glycosylation of the N-terminal region of the serine-rich adhesin Srr1 of Streptococcus agalactiae explored by mass spectrometry.
Serine-rich (Srr) proteins exposed at the surface of Gram-positive bacteria are a family of adhesins that contribute to the virulence of pathogenic staphylococci and streptococci. Lectin-binding experiments have previously shown that Srr proteins are heavily glycosylated. We report here the first mass-spectrometry analysis of the glycosylation of Streptococcus agalactiae Srr1. After Srr1 enrichment and trypsin digestion, potential glycopeptides were identified in collision induced dissociation spectra using X! Tandem. The approach was then refined using higher energy collisional dissociation fragmentation which led to the simultaneous loss of sugar residues, production of diagnostic oxonium ions and backbone fragmentation for glycopeptides. This feature was exploited in a new open source software tool (SpectrumFinder) developed for this work. By combining these approaches, 27 glycopeptides corresponding to six different segments of the N-terminal region of Srr1 [93-639] were identified. Our data unambiguously indicate that the same protein residue can be modified with different glycan combinations including N-acetylhexosamine, hexose, and a novel modification that was identified as O-acetylated-N-acetylhexosamine. Lectin binding and monosaccharide composition analysis strongly suggested that HexNAc and Hex correspond to N-acetylglucosamine and glucose, respectively. The same protein segment can be modified with a variety of glycans generating a wide structural diversity of Srr1. Electron transfer dissociation was used to assign glycosylation sites leading to the unambiguous identification of six serines and one threonine residues. Analysis of purified Srr1 produced in mutant strains lacking accessory glycosyltransferase encoding genes demonstrates that O-GlcNAcylation is an initial step in Srr1 glycosylation that is likely required for subsequent decoration with Hex. In summary, our data obtained by a combination of fragmentation mass spectrometry techniques associated to a new software tool, demonstrate glycosylation heterogeneity of Srr1, characterize a new protein modification, and identify six glycosylation sites located in the N-terminal region of the protein.


Kinetics of photocatalytic removal of imidacloprid from water by advanced oxidation processes with respect to nanotechnology.
In this study, the kinetics of photocatalytic removal of imidacloprid, a systemic chloronicotinoid insecticide, from water using two advanced oxidation systems (ZnO(normal)/H2O2/artificial sunlight and ZnO(nano)/H2O2/artificial sunlight) were investigated. Moreover, the effects of pH, insecticide concentration, catalyst concentration, catalyst particle size, and water type on the photocatalytic removal of imidacloprid were evaluated. Furthermore, total mineralization of imidacloprid under these advanced oxidation systems was evaluated by monitoring the decreases in dissolved organic carbon (DOC) concentrations and formation rate of inorganic ions (Cl- and NO2 -) with irradiation time using total organic carbon (TOC) analysis and ion chromatography to confirm the complete detoxification of imidacloprid in water. The degradation rate of imidacloprid was faster under the ZnO(nano)/H2O2/artificial sunlight system than the ZnO(normal)/artificial sunlight system in both pure and river water. The photocatalytic degradation of imidacloprid under both advanced oxidation systems was affected by pH, catalyst concentration, imidacloprid concentration, and water type. Almost complete mineralization of imidacloprid was only achieved in the ZnO(nano)/H2O2/artificial sunlight oxidation system. The photogeneration rate of hydroxyl radicals was higher under the ZnO(nano)/H2O2/artificial sunlight system than the ZnO(normal)/H2O2/artificial sunlight system. Advanced oxidation processes, particularly those using nanosized zinc oxide, can be regarded as an effective photocatalytic method for imidacloprid removal from water.


AAV-directed persistent expression of a gene encoding anti-nicotine antibody for smoking cessation.
Current strategies to help tobacco smokers quit have limited success as a result of the addictive properties of the nicotine in cigarette smoke. We hypothesized that a single administration of an adeno-associated virus (AAV) gene transfer vector expressing high levels of an anti-nicotine antibody would persistently prevent nicotine from reaching its receptors in the brain. To test this hypothesis, we constructed an AAVrh.10 vector that expressed a full-length, high-affinity, anti-nicotine antibody derived from the Fab fragment of the anti-nicotine monoclonal antibody NIC9D9 (AAVantiNic). In mice treated with this vector, blood concentrations of the anti-nicotine antibody were dose-dependent, and the antibody showed high specificity and affinity for nicotine. The antibody shielded the brain from systemically administered nicotine, reducing brain nicotine concentrations to 15% of those in naïve mice. The amount of nicotine sequestered in the serum of vector-treated mice was more than seven times greater than that in untreated mice, with 83% of serum nicotine bound to immunoglobulin G. Treatment with the AAVantiNic vector blocked nicotine-mediated alterations in arterial blood pressure, heart rate, and locomotor activity. In summary, a single administration of a gene transfer vector expressing a high-affinity anti-nicotine monoclonal antibody elicited persistent (18 weeks), high titers of an anti-nicotine antibody that obviated the physiologic effects of nicotine. If this degree of efficacy translates to humans, AAVantiNic could be an effective preventative therapy for nicotine addiction.


Visualization tools for vorticity transport analysis in incompressible flow.
Vortices are undesirable in many applications while indispensable in others. It is therefore of common interest to understand their mechanisms of creation. This paper aims at analyzing the transport of vorticity inside incompressible flow. The analysis is based on the vorticity equation and is performed along pathlines which are typically started in upstream direction from vortex regions. Different methods for the quantitative and explorative analysis of vorticity transport are presented and applied to CFD simulations of water turbines. Simulation quality is accounted for by including the errors of meshing and convergence into analysis and visualization. The obtained results are discussed and interpretations with respect to engineering questions are given.


Environmental levels of carbaryl impair zebrafish larvae behaviour: The potential role of ADRA2B and HTR2B.
The insecticide carbaryl is commonly found in indirectly exposed freshwater ecosystems at low concentrations considered safe for fish communities. In this study, we showed that after only 24 h of exposure to environmental concentrations of carbaryl (0.066-660 ng/L), zebrafish larvae exhibit impairments in essential behaviours. Interestingly, the observed behavioural effects induced by carbaryl were acetylcholinesterase-independent. To elucidate the molecular initiating event that resulted in the observed behavioural effects, in silico predictions were followed by in vitro validation. We identified two target proteins that potentially interacted with carbaryl, the α2B adrenoceptor (ADRA2B) and the serotonin 2B receptor (HTR2B). Using a pharmacological approach, we then tested the hypothesis that carbaryl had antagonistic interactions with both receptors. Similar to yohimbine and SB204741, which are prototypic antagonists of ADRA2B and HTR2B, respectively, carbaryl increased the heart rate of zebrafish larvae. When we compared the behavioural effects of a 24-h exposure to these pharmacological antagonists with those of carbaryl, a high degree of similarity was found. These results strongly suggest that antagonism of both ADRA2B and HTR2B is the molecular initiating event that leads to adverse outcomes in zebrafish larvae that have undergone 24 h of exposure to environmentally relevant levels of carbaryl.


Ionizing radiation selectively reduces skin regulatory T cells and alters immune function.
The skin serves multiple functions that are critical for life. The protection from pathogens is achieved by a complicated interaction between aggressive effectors and controlling functions that limit damage. Inhomogeneous radiation with limited penetration is used in certain types of therapeutics and is experienced with exposure to solar particle events outside the protection of the Earth's magnetic field. This study explores the effect of ionizing radiation on skin immune function. We demonstrate that radiation, both homogeneous and inhomogeneous, induces inflammation with resultant specific loss of regulatory T cells from the skin. This results in a hyper-responsive state with increased delayed type hypersensitivity in vivo and CD4+ T cell proliferation in vitro. The effects of inhomogeneous radiation to the skin of astronauts or as part of a therapeutic approach could result in an unexpected enhancement in skin immune function. The effects of this need to be considered in the design of radiation therapy protocols and in the development of countermeasures for extended space travel.


Anesthetic implications of epilepsy, status epilepticus, and epilepsy surgery.
Epilepsy is a clinical paroxysmal disorder of recurring seizures, excluding alcohol or drug withdrawal seizures or such recurring exogenous events as repeated insulin-induced hypoglycemia. Epilepsy has a profound impact on each individual diagnosed with this disease. Seizures have been and are thought to arise as a result of abnormalities in (a) neural circuits, (b) excitation/inhibition balance, (c) potassium, and (d) genetic abnormalities. Therapy for epilepsy is either medical, entailing the use of a variety of antiepileptic drugs, or surgical. An urgent approach to seizure control is indicated when status epilepticus occurs. When all standard therapy fails, general anesthesia can be used to control status epilepticus. Surgery is an option in the treatment of epilepsy and requires extensive preoperative evaluation. The primary concerns for the neuroanesthesiologist anesthetizing the patient with epilepsy are the capacity of anesthetics to modulate or potentiate seizure activity and the interaction of anesthetic drugs with antiepileptic drugs. Proconvulsant and anticonvulsant properties have been reported for nearly every anesthetic. If seizure spikes are to be evoked during seizure surgery, then light anesthesia with a proconvulsant anesthetic is used. Conscious analgesia can be used for awake seizure surgery. However, if electrocorticography is not planned, then a general anticonvulsant anesthetic maintenance regimen is used. The latter technique also may be useful in patients whose anesthetic management is complicated by an incidental history of epilepsy.


CD4 interacts constitutively with multiple CCR5 at the plasma membrane of living cells. A fluorescence recovery after photobleaching at variable radii approach.
The entry of human immunodeficiency virus into target cells requires successive interactions of the viral envelope glycoprotein gp120 with CD4 and the chemokine receptors CCR5 or CXCR4. We previously demonstrated, by Förster resonance energy transfer experiments, the constitutive association of CD4 and CCR5 at the surface of living cells. We therefore speculated that this interaction may correlate with compartmentalization of CD4 and CCR5 within the plasma membrane. Here, we characterize the lateral distribution, the dynamics, and the stoichiometry of these receptors in living cells stably expressing CD4 and/or CCR5 by means of fluorescence recovery after photobleaching at variable radii experiments. We found that (i) these receptors expressed alone are confined into 1-microm-sized domains, (ii) CD4-CCR5 associations occur outside and inside smaller domains, and (iii) these interactions involve multiple CCR5 molecules per CD4.


Simian immunodeficiency virus SIVsmmPBj 1.9 induces multinucleated giant cell formation in human peripheral blood monocytes.
SIVsmmPBJ 1.9 is an extremely virulent clone of the simian immunodeficiency virus SIVsmmPBj 14 that causes an acute lethal disease in pigtail macaques, with death occurring 6 to 8 days after infection. The disease is characterized by bloody mucoid diarrhea, lymphoid hyperplasia, and giant cell pneumonia. We have developed an in vitro model for the production of multinucleated giant cells (MGCs) in which peripheral blood monocytes rapidly fuse to form MGCs when cultured in lymphocyte-conditioned medium and antibody against class II MHC. We have tested the effect of SIVsmmPBj on monocytes in our MGC model system. Peripheral blood mononuclear cells (PBMCs) from normal healthy human subjects, when cultured in the presence of anti-class II MHC monoclonal antibody and SIVsmmPBj 1.9, but not either alone, resulted in the formation of MGCs within 4 days. Experiments using Transwell chambers indicated that such MGCs are formed by fusion of monocytes, not by virus-induced fusion of lymphocytes. SIVsmmPBj 1.9 is unique in inducing MGC formation in that other SIV and HIV isolates do not induce MGCs. Whereas SIVsmmPBj 1.9 grown in PBMCs was a potent inducer of MGCs in the presence of anti-class II MHC antibody, SIVsmmPBj 1.9 grown in CEMx174 failed to do so. Antibodies against IFN-gamma and TNF-alpha significantly inhibited SIVsmmPBj/anti-class II-induced formation of MGCs. These results indicate that cytokines released in response to SIVsmmPBj 1.9, in conjunction with antibodies to class II MHC, caused fusion of monocytes.


Evaluation of the acute cardiac and central nervous system effects of the fluorocarbon trifluoromethane in baboons.
The gaseous fluorocarbon trifluoromethane has recently been investigated for its potential as an in vivo gaseous indicator for nuclear magnetic resonance studies of brain perfusion. Trifluoromethane may also have significant value as a replacement for chlorofluorocarbon fire retardants. Because of possible species-specific cardiotoxic and anesthetic properties, the toxicological evaluation of trifluoromethane in primates (Papio anubis) is necessary prior to its evaluation in humans. We report the acute cardiac and central nervous system effects of trifluoromethane in eight anesthetized baboons. A dose-response effect was established for respiratory rate, electroencephalogram, and cardiac sinus rate, which exhibited a stepwise decrease from 10% trifluoromethane. No spontaneous arrhythmias were noted, and arterial blood pressure remained unchanged at any inspired level. Intravenous epinephrine infusions (1 microgram/kg) induced transient cardiac arrhythmia in 1 animal only at 70% FC-23 (v/v) trifluoromethane. Trifluoromethane appears to induce mild dose-related physiological changes at inspired levels of 30% or more, indicative of an anesthetic effect. These data suggest that trifluoromethane may be safe to use in humans, without significant adverse acute effects, at an inspired level of 30%.


Dermoscopic and Immunohistochemical Changes in Acquired Melanocytic Nevi following Narrow-Band Ultraviolet B Therapy.
Acquired melanocytic nevi (AMN) have been reported to undergo morphological and dermoscopic changes following exposure to narrow-band ultraviolet B (NB-UVB) radiation. To study the morphological, dermoscopic and immunohistochemical changes in AMN following NB-UVB radiation. Suberythemogenic NB-UVB sessions were delivered to 40 patients with AMN. For each patient, a minimum of 2 nevi were selected. One nevus was surgically removed from each patient prior to sessions as control; for the other nevus, dermoscopic images were captured before and after NB-UVB sessions. The images were evaluated for changes. At the end, another nevus was surgically removed for immunohistochemical assessment of Ki-67 and melan-A. Our study showed a statistically significant increase in the size of AMN following NB-UVB radiation. Benign dermoscopic changes were observed. Statistically significant positive correlations were found between some dermoscopic findings and the total cumulative dose of NB-UVB. Immunohistochemical analysis did not show any significant change in the exposed AMN. AMN irradiated with repeated suberythemogenic doses of NB-UVB showed benign morphological and dermoscopic changes, and this was confirmed by our immunohistochemical study.


Cerebrospinal fluid from patients with amyotrophic lateral sclerosis inhibits sonic hedgehog function.
Sonic hedgehog (Shh) is a morphogen essential to the developing nervous system that continues to play an important role in adult life by contributing to cell proliferation and differentiation, maintaining blood-brain barrier integrity, and being cytoprotective against oxidative and excitotoxic stress, all features of importance in amyotrophic lateral sclerosis (ALS). ALS is a fatal disease characterized by selective loss of motor neurons due to poorly understood mechanisms. Evidence indicates that Shh might play an important role in ALS, and that Shh signaling might be also adversely affected in ALS. Since little is known about the functional status of Shh pathway in patients with ALS, we therefore sought to determine whether Shh protein levels or biological activity in cerebrospinal fluid (CSF) was less in ALS patients than controls, and whether these measures could be correlated with ALS disease severity and disease progression, and with other CSF analytes of biological interest in ALS. Comparing Shh levels in the CSF of normal controls (n = 13), neurological controls (n = 12), and ALS patients (n = 9) measured by ELISA, we found that CSF Shh levels were not different between controls and ALS patients. However, when assessing Shh biological activity in CSF using in vitro cell-based assays, which measure Shh activity as inducible Gli-driven luminescence, we found that in the presence of exogenous recombinant Shh or the Shh agonist, purmorphamine, the inducible activity of CSF was significantly augmented in the control groups as expected, but not in the ALS group, suggesting the presence of an inhibitor of Shh signaling in ALS CSF samples. Since purmorphamine acts on Smoothened, downstream of Shh and its receptor Patched, the inhibitory action is downstream of Smoothened. Our results also demonstrated that while the inhibitory effect of ALS CSF on Shh signaling did not correlate significantly with ALS disease characteristics, the levels of IL-1β and TNF-α did. In addition to being significantly elevated in ALS CSF, these cytokines negatively correlated with the disease duration, whereas GDF11 was a favorable predictor of ALS clinical score. We also found that TNF-α significantly inhibited Shh biological activity in vitro, potentially suggesting a novel role of TNF-α in ALS pathogenesis. Collectively, this is the first report demonstrating that Shh signaling in CSF of ALS patients is compromised.


Acute rhabdomyolysis complicating status asthmaticus in children: case series and review.
To describe a case series of 4 children who developed acute rhabdomyolysis as a complication of acute respiratory failure secondary to status asthmaticus. A retrospective review of all children who were admitted to our pediatric intensive care unit (PICU) with status asthmaticus from November 1998 through July 2004 was performed and all children who developed acute rhabdomyolysis, defined as a 5-fold increase above the upper limit of normal in the serum creatine phosphokinase (CPK) concentration (CPK > or = 1250 IU/L), were identified. Demographic and clinical data were abstracted from the medical record. During the study period, 108 children with status asthmaticus were admitted to our PICU (3.6% of all admissions). Four children (age 12-19 years) developed acute respiratory failure requiring mechanical ventilation, and all 4 of these children (3.7% of all children with status asthmaticus admitted to the PICU) developed acute rhabdomyolysis. The 4 children who developed acute rhabdomyolysis were older than the children with status asthmaticus, without rhabdomyolysis (median age 15 years vs. 5 years). Acute rhabdomyolysis complicating status asthmaticus may be more common than previously ascertained. We therefore suggest that CPK levels should be followed closely in all children with status asthmaticus and acute respiratory failure. The early presentation of rhabdomyolysis in the current series suggests that factors other than corticosteroids and neuromuscular blockers are potentially involved. Mechanical ventilation and older age seem to be significant risk factors for rhabdomyolysis, perhaps implicating a mechanism similar to the pathogenesis of severe exercise-related rhabdomyolysis. Further clinical study of the incidence and causative factors of rhabdomyolysis in this population is warranted.


Mox2 is a component of the genetic hierarchy controlling limb muscle development.
The skeletal muscles of the limbs develop from myogenic progenitors that originate in the paraxial mesoderm and migrate into the limb-bud mesenchyme. Among the genes known to be important for muscle development in mammalian embryos are those encoding the basic helix-loop-helix (bHLH) myogenic regulatory factors (MRFs; MyoD, Myf5, myogenin and MRF4) and Pax3, a paired-type homeobox gene that is critical for the development of limb musculature. Mox1 and Mox2 are closely related homeobox genes that are expressed in overlapping patterns in the paraxial mesoderm and its derivatives. Here we show that mice homozygous for a null mutation of Mox2 have a developmental defect of the limb musculature, characterized by an overall reduction in muscle mass and elimination of specific muscles. Mox2 is not needed for the migration of myogenic precursors into the limb bud, but it is essential for normal appendicular muscle formation and for the normal regulation of myogenic genes, as demonstrated by the downregulation of Pax3 and Myf5 but not MyoD in Mox2-deficient limb buds. Our findings show that the MOX2 homeoprotein is an important regulator of vertebrate limb myogenesis.


Protein interaction analysis of SCF ubiquitin E3 ligase subunits from Arabidopsis.
Ubiquitin E3 ligases are a diverse family of protein complexes that mediate the ubiquitination and subsequent proteolytic turnover of proteins in a highly specific manner. Among the several classes of ubiquitin E3 ligases, the Skp1-Cullin-F-box (SCF) class is generally comprised of three 'core' subunits: Skp1 and Cullin, plus at least one F-box protein (FBP) subunit that imparts specificity for the ubiquitination of selected target proteins. Recent genetic and biochemical evidence in Arabidopsis thaliana suggests that post-translational turnover of proteins mediated by SCF complexes is important for the regulation of diverse developmental and environmental response pathways. In this report, we extend upon a previous annotation of the Arabidopsis Skp1-like (ASK) and FBP gene families to include the Cullin family of proteins. Analysis of the protein interaction profiles involving the products of all three gene families suggests a functional distinction between ASK proteins in that selected members of the protein family interact generally while others interact more specifically with members of the F-box protein family. Analysis of the interaction of Cullins with FBPs indicates that CUL1 and CUL2, but not CUL3A, persist as components of selected SCF complexes, suggesting some degree of functional specialization for these proteins. Yeast two-hybrid analyses also revealed binary protein interactions between selected members of the FBP family in Arabidopsis. These and related results are discussed in terms of their implications for subunit composition, stoichiometry and functional diversity of SCF complexes in Arabidopsis.


Natural variation within the neuronal nicotinic acetylcholine receptor cluster on human chromosome 15q24: influence on heritable autonomic traits in twin pairs.
Nicotinic acetylcholine receptors (nAChRs) are combinations of subunits arranged as pentamers encircling a central cation channel. At least nine alpha and four beta subunits are expressed in the central and peripheral nervous systems; their presence in autonomic ganglia, the adrenal medulla, and central nervous system, with accompanying responses elicited by nicotinic agonists, point to their involvement in cardiovascular homeostasis. nAChRs formed by alpha3, alpha5, and beta4 subunits may regulate blood pressure (BP) by mediating release of catestatin, the endogenous nicotinic antagonist fragment of chromogranin A (CHGA) and potent inhibitor of catecholamine secretion. Genes encoding these subunits (CHRNA3, CHRNA5, and CHRNB4) are clustered on human chromosome 15q24. Because variation in this cluster may alter autonomic regulation of BP, we sequenced approximately 15 kilobase pairs in 15q24 containing their coding and 5'- and 3'-untranslated regions in 80 individuals. We identified 63 variants: 25 in coding regions of CHRNA3, CHRNA5, and CHRNB4 and 48 noncoding single-nucleotide polymorphisms (SNPs). Haplotype frequencies varied across ethnic populations. We assessed the contribution of six SNPs in the putative catestatin binding region of CHRNA3 and CHRNB4 to autonomic traits. In twins, catestatin and BP were heritable. CHRNA3 SNPs and haplotypes containing K95K (G285A) associated with circulating plasma catestatin, epinephrine levels, as well as systolic BP, suggesting altered coupling of the nAChRs to BP. Studies of chromaffin cells in vitro reveal that nicotinic agonist stimulation releases catecholamines and CHGA, a process augmented by overexpression of CHRNA3 and blocked by catestatin. These cellular events suggest a homeostatic mechanism underlying the pleiotropic actions of CHRNA3 genetic variation on autonomic function observed in twins.


The synergistic effect of homocysteine and lipopolysaccharide on the differentiation and conversion of raw264.7 macrophages.
Macrophages play pivotal roles in the progression of atherosclerosis (AS) and their heterogeneous differentiation patterns have been studied extensively. The classical subtype of activated macrophage, M1, promotes the progression of AS. Conversely, the alternative subtype of activated macrophage, M2, is regarded as a repressor of AS. Homocysteine (Hcy) may influence macrophage subtype polarization both in vivo and in vitro. Homocysteinemia (HHcy) is an independent risk factor in coronary heart disease and the effect of Hcy on macrophage differentiation has not been studied until now. Different concentrations of Hcy in combination with a fixed concentration of lipopolysaccharide (LPS, 200 ng/mL) were used to treat RAW264.7 macrophages. Real-time PCR was used to detect and quantify RNA transcripts indicative of M1 and M2 differentiation. The efficacy and specificity for each chemical stimulant in inducing macrophage differentiation were also investigated. The M2 macrophages (anti-inflammatory subtype) induced using classical methods (IL-4, 10 ng/mL) were also treated with different concentrations of Hcy complemented with LPS. The synergistic effect of Hcy and LPS in the converting the M2 subtype to M1 was also studied. Macrophages can be induced to differentiate towards M1 by a combination of Hcy with LPS, with the strongest effect observed at an Hcy concentration of 50 μmol/L. After inducing macrophages to the M2 subtype using IL-4, treatment with both Hcy and LPS could elicit conversion from the M2 to M1 subtype. Combined treatment with Hcy and LPS can induce the polarization of cultured RAW264.7 macrophages into the pro-inflammatory subtype, as well as promote subtype conversion from anti-inflammatory to pro-inflammatory.


Gene delivery systems in surgery.
Increased understanding of the genetic basis of human disease has led to a number of potential gene-based therapies for various medical and surgical disorders. The development of efficient methods for delivering genes to mammalian cells in vitro has increased the potential clinical utility of gene-based therapies; however, a major focus of research has been more efficient delivery to appropriate target cells, in vivo as well as in vitro, to establish gene therapy as an effective clinical modality for common disorders. Despite substantial progress, a number of critical technical issues to enhance and optimize not only gene transfer but also gene expression must be resolved. These future technological developments will be essential for the widespread clinical implementation of gene-based therapy.


Impact of prior biologic use on persistence of treatment in patients with psoriatic arthritis enrolled in the US Corrona registry.
Psoriatic arthritis (PsA) is a chronic condition characterized by a diverse set of symptoms, from swollen joints to nail disease to skin disease. A variety of treatment options are available, including tumor necrosis factor inhibitors (TNFis). Little is known about treatment persistence in patients with PsA who initiate TNFi therapy, with and without prior biologic use. This study assessed persistence in these subgroups of patients with PsA and identified factors associated with persistence. This retrospective study utilized data from the Corrona registry of patients with PsA-with or without prior biologic experience-who initiated TNFi therapy between October 1, 2002, and March 21, 2013. Kaplan-Meier curves estimated median time to nonpersistence (discontinuation or switch to another biologic). Cox proportional hazards models identified factors associated with TNFi nonpersistence. A total of 1241 TNFi initiations were identified: 549 by biologic-naïve and 692 by biologic-experienced patients. Through 4 years of follow-up, more biologic-naïve than biologic-experienced patients remained persistent. Biologic-naïve patients had a greater mean time to nonpersistence compared with biologic-experienced patients: 32 vs 23 months (p = 0.0002). Moderate and high disease activities based on clinical disease activity index and disease duration were associated with persistence in both biologic-naïve and biologic-experienced patients. Additionally, in the biologic-experienced patients, the number of prior medications and skin disease were associated with persistence. The majority of patients with PsA in this study were persistent with their TNFi therapy; biologic-naïve patients had greater persistence compared with biologic-experienced patients. Predictors of persistence differed slightly between biologic-naïve and biologic-experienced patients.


Changes in brain morphology in albinism reflect reduced visual acuity.
Albinism, in humans and many animal species, has a major impact on the visual system, leading to reduced acuity, lack of binocular function and nystagmus. In addition to the lack of a foveal pit, there is a disruption to the routing of the nerve fibers crossing at the optic chiasm, resulting in excessive crossing of fibers to the contralateral hemisphere. However, very little is known about the effect of this misrouting on the structure of the post-chiasmatic visual pathway, and the occipital lobes in particular. Whole-brain analyses of cortical thickness in a large cohort of subjects with albinism showed an increase in cortical thickness, relative to control subjects, particularly in posterior V1, corresponding to the foveal representation. Furthermore, mean cortical thickness across entire V1 was significantly greater in these subjects compared to controls and negatively correlated with visual acuity in albinism. Additionally, the group with albinism showed decreased gyrification in the left ventral occipital lobe. While the increase in cortical thickness in V1, also found in congenitally blind subjects, has been interpreted to reflect a lack of pruning, the decreased gyrification in the ventral extrastriate cortex may reflect the reduced input to the foveal regions of the ventral visual stream.


Coronary stent embolism to the right posterior cerebral artery.
A 57-year-old male was admitted to the emergency room with chest pain that has been present for 3 hours. His blood pressure was 70/50 mmHg and heart rate was 48 bpm. 12-lead surface electrocardiography revealed inferior myocardial infarction and third-degree atrioventricular (AV) block. An emergency coronary angiography showed a 50% stenosis in the middle segment of the left anterior descending artery and 90% in the proximal circumflex (Cx) artery. The right coronary artery was totally occluded. After the predilatation with a 2.0x15 mm compliant balloon at 10 atm, a 3.5x24 mm bare metal stent was implanted. The third-degree AV block improved and a sinus rhythm of 124 bpm was achieved, but hemodynamic stability was not attained. Percutaneous coronary intervention for the Cx artery was performed. Without predilatation, a 3.5x12 mm low profile BMS was easily advanced over the lesion. Just before the stent implantation, asystole developed, followed by convulsions. Blood pressure and heart rate recovered after the administration of 1 mg of atropine. However, during the seizure, the guidewire and coronary stent device fell to the aortic root. Stent struts were not seen on the balloon catheter in a fluoroscopic examination. Fluoroscopic scanning of the vascular system showed that the coronary stent was in the right posterior cerebral artery. There were no symptoms or signs of neurological disorder. Consultant invasive neuroradiologist recommended medical follow-up. Clopidogrel and acetylsalicylic acid were prescribed indefinitely. Two months after the primary PCI, a successful coronary artery bypass graft operation was performed. After 4 years, the patient remained without any symptoms of neurological problems.


The impact of conditional cash transfers on health outcomes and use of health services in low and middle income countries.
Conditional cash transfers (CCT) provide monetary transfers to households on the condition that they comply with some pre-defined requirements. CCT programmes have been justified on the grounds that demand-side subsidies are necessary to address inequities in access to health and social services for poor people. In the past decade they have become increasingly popular, particularly in middle income countries in Latin America. To assess the effectiveness of CCT in improving access to care and health outcomes, in particular for poorer populations in low and middle income countries. We searched a wide range of international databases, including the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE and EMBASE, in addition to development studies and economic databases. We also searched the websites and online resources of numerous international agencies, organisations and universities to find relevant grey literature. The original searches were conducted between November 2005 and April 2006. An updated search in MEDLINE was carried out in May 2009. CCT were defined as monetary transfers made to households on the condition that they comply with some pre-determined requirements in relation to health care. Studies had to include an objective measure of at least one of the following outcomes: health care utilisation, health expenditure, health outcomes or equity outcomes. Eligible study designs were: randomised controlled trial, interrupted time series analysis, or controlled before-after study of the impact of health financing policies following criteria used by the Cochrane Effective Practice and Organisation of Care Group. We performed qualitative analysis of the evidence. We included ten papers reporting results from six intervention studies. Overall, design quality and analysis limited the risks of bias. Several CCT programmes provided strong evidence of a positive impact on the use of health services, nutritional status and health outcomes, respectively assessed by anthropometric measurements and self-reported episodes of illness. It is hard to attribute these positive effects to the cash incentives specifically because other components may also contribute. Several studies provide evidence of positive impacts on the uptake of preventive services by children and pregnant women. We found no evidence about effects on health care expenditure. Conditional cash transfer programmes have been the subject of some well-designed evaluations, which strongly suggest that they could be an effective approach to improving access to preventive services. Their replicability under different conditions - particularly in more deprived settings - is still unclear because they depend on effective primary health care and mechanisms to disburse payments. Further rigorous evaluative research is needed, particularly where CCTs are being introduced in low income countries, for example in Sub-Saharan Africa or South Asia.


The influence of acetylator phenotype on the response to sulfalene in individuals with chloroquine-resistant falciparum malaria.
The disposition of sulfalene was studied in eight individuals before and during an infection with a chloroquine-resistant strain of Plasmodium falciparum. Isoniazid acetylator phenotype was determined in each individual prior to the administration of sulfalene. Following the administration of sulfalene before infection with malaria, a significant difference in half-life of non-acetylated sulfalene and percent acetylation of sulfalene in plasma was observed between rapid and slow acetylators. When sulfalene was administered during malaria, this difference was no longer apparent. Individuals who did not respond to the therapeutic administration of sulfalene alone were treated with a combination of sulfalene and pyrimethamine. Three individuals were cured by sulfalene without pyrimethamine and one was cured by the drug combination. Three of the four individuals who were not cured by any dose of sulfalene or the drug combination were slow acetylators. There was no distinct correlation between clinical response and maximum levels or half-life of nonacetylated sulfalene. These findings suggest that acetylator phenotype does not influence the therapeutic response of individuals infected with falciparum malaria to sulfalene or to the combination of sulfalene and pyrimethamine. Further information is presented, however, to confirm the importance of an as yet unidentified host factor(s) in determining therapeutic response to these agents.


Clustering and periodicity of neurofibrillary tangles in the upper and lower cortical laminae in Alzheimer's disease.
In Alzheimer's disease (AD), neurofibrillary tangles (NFT) occur within neurons in both the upper and lower cortical laminae. Using a statistical method that estimates the size and spacing of NFT clusters along the cortex parallel to the pia mater, two hypotheses were tested: 1) that the cluster size and distribution of the NFT in gyri of the temporal lobe reflect degeneration of the feedforward (FF) and feedback (FB) cortico-cortical pathways, and 2) that there is a spatial relationship between the clusters of NFT in the upper and lower laminae. In 16 temporal lobe gyri from 10 cases of sporadic AD, NFT were present in both the upper and lower laminae in 11/16 (69%) gyri and in either the upper or lower laminae in 5/16 (31%) gyri. Clustering of the NFT was observed in all gyri. A significant peak-to-peak distance was observed in the upper laminae in 13/15 (87%) gyri and in the lower laminae in 8/12 (67%) gyri, suggesting a regularly repeating pattern of NFT clusters along the cortex. The regularly distributed clusters of NFT were between 500 and 800 microm in size, the estimated size of the cells of origin of the FF and FB cortico-cortical projections, in the upper laminae of 6/13 (46%) gyri and in the lower laminae of 2/8 (25%) gyri. Clusters of NFT in the upper laminae were spatially correlated (in phase) with those in the lower laminae in 5/16 (31%) gyri. The clustering patterns of the NFT are consistent with their formation in relation to the FF and FB cortico-cortical pathways. In most gyri, NFT clusters appeared to develop independently in the upper and lower laminae.


Understanding job satisfaction and motivation among nurses in public health facilities of Ethiopia: a cross-sectional study.
Poor job conditions and limited resources are reducing job satisfaction and motivation among nurses in low-income countries, which may affect the quality of services and attrition rates. The objective of this study was to examine job satisfaction, motivation and associated factors among nurses working in the public health facilities of Ethiopia, with the aim of improving performance and productivity in the health care system. The study employed a cross-sectional two-stage cluster sampling design. From a random sample of 125 health facilities, 424 nurses were randomly selected for face-to-face interviews in all regions of Ethiopia. Nurses responded to questions about their overall job satisfaction and job conditions, including items related to intrinsic and extrinsic motivation, using a 5-point Likert scale. Multilevel analysis was performed to adjust for different clustering effects. Satisfaction levels (percent of respondents who were satisfied) were calculated for individual items, and composite mean scores (range: 1-5) were calculated for motivational factors. Adjusted odds ratios were computed to examine the association of these factors with overall job satisfaction. Overall, 60.8% of nurses expressed satisfaction with their job. Composite mean scores for intrinsic and extrinsic motivational factors were 3.5 and 3.0, respectively. Job satisfaction levels were significantly higher for female nurses (65.6%, p = 0.04), those older than 29 years (67.8%, p = 0.048) and had over 10 years work experiences (68.8%, p = 0.007). Satisfaction with remuneration (AOR = 2.04, 95% CI = 1.36, 3.06), recognition (AOR = 2.21; 95% CI = 1.38, 3.53), professional advancement (AOR = 1.54; 95% CI = 1.06, 2.29), features of the work itself (AOR = 1.65; 95% CI = 1.20, 2.91) and nurses' work experiences from 5 to 10 years (AOR = 0.37, 95% CI = 0.17, 0.79) were significantly associated with overall job satisfaction after controlling for other predictors. The study findings are signals for the Ministry of Health to strengthen the human resource management system and practices to improve nurses' overall job satisfaction and motivation, especially among nurses with 5 to 10 years of experience on the job. Expanded recognition systems and opportunities for advancement are required to increase nurses' job satisfaction and motivation. Equitable salary and fringe benefits are also needed to reduce their dissatisfaction with the job.


The study of mentoring in the learning environment (SMILE): a randomized evaluation of the effectiveness of school-based mentoring.
The effect of providing youth school-based mentoring (SBM), in addition to other school-based support services, was examined with a sample of 516 predominately Latino students across 19 schools. Participants in a multi-component, school-based intervention program run by a youth development agency were randomly assigned to one of two conditions: (1) supportive services alone or (2) supportive services plus SBM. Compared to community-based mentoring, the duration of the SBM was brief (averaging eight meetings), partly because the agency experienced barriers to retaining mentors. Intent-to-treat (ITT) main effects of SBM were tested using hierarchical linear modeling (HLM) and revealed small, positive main effects of mentoring on self-reported connectedness to peers, self-esteem (global and present-oriented), and social support from friends, but not on several other measures, including grades and social skills. Three-way cross-level interactions of sex and school level (elementary, middle, and high school) revealed that elementary school boys and high school girls benefited the most from mentoring. Among elementary school boys, those in the mentoring condition reported higher social skills (empathy and cooperation), hopefulness, and connectedness both to school and to culturally different peers. Among high school girls, those mentored reported greater connectedness to culturally different peers, self-esteem, and support from friends. Findings suggest no or iatrogenic effects of mentoring for older boys and younger girls. Therefore, practitioners coordinating multi-component programs that include SBM would be wise to provide mentors to the youth most likely to benefit from SBM and bolster program practices that help to support and retain mentors.


Consequences of perinatal bisphenol A exposure in a mouse model of multiple sclerosis.
Multiple sclerosis (MS) is a complex disease influenced by genetic and environmental contributing factors. Endocrine disrupting compounds (EDCs) such as bisphenol A (BPA) affect gene expression and hormone-regulated systems throughout the body. We investigated the effects of BPA on Theiler's-virus induced demyelination (TVID), a mouse model of MS. Perinatal BPA exposure, combined with viral infection, resulted in a decreased level of viral antibodies, accelerated the onset of TVID symptoms, increased inflammation in both the spinal cord and digestive tract, and amplified immune-related gene expression changes induced by viral infection. These results demonstrate the effect of BPA on the trajectory of TVID, and illustrate how multiple factors collectively influence autoimmune disease.


Type-D personality and body image in men: the role of exercise status.
The 'Distressed' or Type-D personality is described by the interaction between high levels of negative affectivity and social inhibition. This study investigated the prevalence of Type-D personality in men of different exercise status, the association between Type-D and body image perceptions, and the moderating effect of exercise status. Participants were 564 British males aged between 18 and 55 years. Of these 200 were classified as sedentary, 148 as active and 216 as weight trainers. Participants completed the DS14 and Multidimensional Body-Self Relations Questionnaire. Results showed that more individuals were classified as Type-D in the sedentary group (45%) than the two active groups, and in the weight training (24.5%) than the active (14.2%) group. Both Type-D and a sedentary lifestyle were associated with a significantly poorer body image. However, exercise mode was not associated with body image differences. Sedentary Type-D men scored significantly lower in Body Areas Satisfaction and higher in Self-Classified Weight than both active groups. Regular exercise might provide a pathway for Type-D men to develop a more positive body image.


Authentication analysis of red fruit (Pandanus Conoideus Lam) oil using FTIR spectroscopy in combination with chemometrics.
Red fruit (Pandanus conoideus Lam) is endemic plant of Papua, Indonesia and Papua New Guinea. The price of its oil (red fruit oil, RFO) is 10-15 times higher than that of common vegetable oils; consequently, RFO is subjected to adulteration with lower price oils. Among common vegetable oils, canola oil (CaO) and rice bran oil (RBO) have similar fatty acid profiles to RFO as indicated by the score plot of principal component analysis; therefore, CaO and RBO are potential adulterants in RFO. To develop FTIR spectroscopy in combination with chemometrics of partial least square regression (PLSR) and discriminant analysis (DA) for authentication of RFO from CaO and RBO. The presence of CaO in RFO was better determined at frequency regions of 1200-1050 cm⁻¹; meanwhile, the combined frequency ranges of 1207-1078 and 1747-1600 cm⁻¹ were exploited for quantitative analysis of RBO with acceptable values of coefficient of determination (R²) and errors in calibration, prediction and during cross validation. DA based on Mahalanobis distance was able to discriminate between RFO and RFO adulterated with CaO and RBO. FTIR spectroscopy combined with PLSR and DA can be successfully used for quantification and classification of oil adulterants in RFO. The developed method is rapid and environmentally friendly and sample preparation is easy.


Minimum requirements for the diagnosis of psychogenic nonepileptic seizures: a staged approach: a report from the International League Against Epilepsy Nonepileptic Seizures Task Force.
An international consensus group of clinician-researchers in epilepsy, neurology, neuropsychology, and neuropsychiatry collaborated with the aim of developing clear guidance on standards for the diagnosis of psychogenic nonepileptic seizures (PNES). Because the gold standard of video electroencephalography (vEEG) is not available worldwide, or for every patient, the group delineated a staged approach to PNES diagnosis. Using a consensus review of the literature, this group evaluated key diagnostic approaches. These included: history, EEG, ambulatory EEG, vEEG/monitoring, neurophysiologic, neurohumoral, neuroimaging, neuropsychological testing, hypnosis, and conversation analysis. Levels of diagnostic certainty were developed including possible, probable, clinically established, and documented diagnosis, based on the availability of history, witnessed event, and investigations, including vEEG. The aim and hope of this report is to provide greater clarity about the process and certainty of the diagnosis of PNES, with the intent to improve the care for people with epilepsy and nonepileptic seizures.


Mitochondrial and Peroxisomal Alterations Contribute to Energy Dysmetabolism in Riboflavin Transporter Deficiency.
Riboflavin transporter deficiency (RTD) is a childhood-onset neurodegenerative disorder characterized by progressive pontobulbar palsy, sensory and motor neuron degeneration, sensorineural hearing loss, and optic atrophy. As riboflavin (RF) is the precursor of FAD and FMN, we hypothesize that both mitochondrial and peroxisomal energy metabolism pathways involving flavoproteins could be directly affected in RTD, thus impacting cellular redox status. In the present work, we used induced pluripotent stem cells (iPSCs) from RTD patients to investigate morphofunctional features, focusing on mitochondrial and peroxisomal compartments. Using this model, we document the following RTD-associated alterations: (i) abnormal colony-forming ability and loss of cell-cell contacts, revealed by light, electron, and confocal microscopy, using tight junction marker ZO-1; (ii) mitochondrial ultrastructural abnormalities, involving shape, number, and intracellular distribution of the organelles, as assessed by focused ion beam/scanning electron microscopy (FIB/SEM); (iii) redox imbalance, with high levels of superoxide anion, as assessed by MitoSOX assay accompanied by abnormal mitochondrial polarization state, evaluated by JC-1 staining; (iv) altered immunofluorescence expression of antioxidant systems, namely, glutathione, superoxide dismutase 1 and 2, and catalase, as assessed by quantitatively evaluated confocal microscopy; and (v) peroxisomal downregulation, as demonstrated by levels and distribution of fatty acyl β-oxidation enzymes. RF supplementation results in amelioration of cell phenotype and rescue of redox status, which was associated to improved ultrastructural features of mitochondria, thus strongly supporting patient treatment with RF, to restore mitochondrial- and peroxisomal-related aspects of energy dysmetabolism and oxidative stress in RTD syndrome.


Four Ia invariant chain forms derive from a single gene by alternate splicing and alternate initiation of transcription/translation.
We determined the structural basis for the presence of electrophoretically-distinct, antigenically-related forms of invariant chains in Ia oligomers, and established the mechanisms by which they can be expressed from a single gene. S1 nuclease protection assays indicated that, in B cells, transcription of this gene initiates at a minimum of three sites. Thus, unlike previously thought, invariant chain mRNAs have heterogeneous 5' untranslated segments that may differentially affect initiation of translation. Further, restriction mapping and nucleotide sequencing of cDNAs revealed two kinds of invariant chain mRNAs differing by an internal coding segment of 192 bp. This segment represents an alternatively spliced exon, as demonstrated by nucleotide sequencing of corresponding genomic regions. The exon (exon X) encodes a cysteine-rich stretch of 64 amino acids near the COOH terminus that displays a striking and surprising homology to an internal amino acid repeat of thyroglobulin, suggesting an evolutionary mechanism of exon shuffling. Transient expression of cDNAs indicated that both types of alternatively spliced mRNAs contain two in-frame AUGs functioning as alternate start sites for translation. Thus, transfections with exon X-lacking cDNAs resulted in the expression of Mr 33,000 and 31,000 proteins, detected by immunoprecipitation with anti-invariant chain antisera, and identical by two-dimensional gel (2-D) analyses to the B cell invariant-chain forms gamma 1 (Mr 31,000), gamma 2, and gamma 3 (Mr 33,000). Similarly, exon X-containing cDNAs expressed Mr 43,000 and 41,000 proteins, also identical by 2-D migration to Ia-associated proteins. Thus, human Ia molecules contain four forms of invariant chain of closely related but nonidentical primary structure that are generated from a single gene by a complex pattern of alternate transcriptional start, exon splicing, and translational start.


Effects of morphine on the human sphincter of Oddi.
The effects of morphine on intraluminal pressures recorded from the sphincter of Oddi (SO) at endoscopic retrograde cholangiopancreatography in 19 patients who were without evidence of biliary or pancreatic disease were studied. Morphine was given in four successive doses of 2.5, 2.5, 5, and 10 micrograms/kg iv at five minute intervals. Morphine in subanalgesic doses increased the frequency of SO phasic pressure waves to a maximum of 10-12/min, caused the phasic waves to occur simultaneously along the sphincter segment, increased phasic wave amplitude from 72 (26) (SE) to 136 (31) mmHg, and increased SO basal pressure from 10 (1) to 29 (9) mmHg (p less than 0.05). The effects of morphine on the SO are mediated by more than one opioid receptor type, as naloxone competitively antagonised the increase in phasic wave frequency induced by morphine, but did not affect the increase in SO basal pressure elicited by morphine. When given after naloxone, morphine decreased phasic wave amplitude, an inhibitory effect that is normally masked by morphine's dominant naloxone sensitive excitatory effect. Mu receptors do not appear to be involved in control of spontaneous SO motor function, as naloxone alone did not affect SO motor activity. The excitatory effects of morphine on the SO are not mediated by cholinergic nerves, as they were not blocked by atropine. Cholinergic nerves, however, may have a role in regulating spontaneous SO motor function because atropine alone depressed phasic wave activity and basal pressure. Although morphine does cause 'spasm' of the human SO, its effects are more complex than is commonly believed.


Clinician responses to cannabis use during pregnancy and lactation: a systematic review and integrative mixed-methods research synthesis.
Perinatal cannabis use is increasing, and clinician counselling is an important aspect of reducing the potential harm of cannabis use during pregnancy and lactation. To understand current counselling practices, we conducted a systematic review and integrative mixed-methods synthesis to determine "how do perinatal clinicians respond to pregnant and lactating patients who use cannabis?" We searched 6 databases up until 2021-05-31. Eligible studies described the attitudes, perceptions, or beliefs of perinatal clinician about cannabis use during pregnancy or lactation. Eligible clinicians were those whose practice particularly focusses on pregnant and postpartum patients. The search was not limited by study design, geography, or year. We used a convergent integrative analysis method to extract relevant findings for inductive analysis. Thirteen studies were included; describing perspectives of 1,366 clinicians in 4 countries. We found no unified approach to screening and counselling. Clinicians often cited insufficient evidence around the effects of perinatal cannabis use and lacked confidence in counselling about use. At times, this meant clinicians did not address cannabis use with patients. Most counselled for cessation and there was little recognition of the varied reasons that patients might use cannabis, and an over-reliance on counselling focussed on the legal implications of use. Current approaches to responding to cannabis use might result in inadequate counselling. Counselling may be improved through increased education and training, which would facilitate conversations to mitigate the potential harm of perinatal cannabis use while recognizing the benefits patients perceive.


Impact of periictal interventions on respiratory dysfunction, postictal EEG suppression, and postictal immobility.
Sudden unexpected death in epilepsy (SUDEP) is the leading cause of epilepsy-related mortality. Seizure-related respiratory dysfunction (RD), the duration of postictal generalized electroencephalography (EEG) suppression (PGES), and duration of postictal immobility (PI) may be important in the pathophysiology of SUDEP. Periictal interventions may reduce the risk of SUDEP. We assessed the impact of periictal nursing interventions on RD, PGES, and PI duration in patients with localization-related epilepsy and secondarily generalized convulsions (GCs) recorded during video-EEG telemetry in the epilepsy monitoring unit. Video-EEG data were retrospectively reviewed. Interventions including administration of supplemental oxygen, oropharyngeal suction, and patient repositioning were evaluated. Interventions were performed based on nursing clinical judgment at the bedside and were not randomized. The two-sided Wilcoxon rank-sum test was used to compare GCs with and those without intervention. Robust simple linear regression was used to assess the association between timing of intervention and duration of hypoxemia (SaO(2) < 90%), PGES, and PI using data from only the first GC for each patient. Data from 39 patients with 105 GCs were analyzed. PGES >2 s occurred following 31 GCs in 16 patients. There were 21 GCs with no intervention (NOINT) and 84 GC with interventions (INT). In the INT group, the duration of hypoxemia was shorter (p = 0.0014) when intervention occurred before hypoxemia onset (mean duration 53.1 s) than when intervention was delayed (mean duration 132.42 s). Linear regression indicated that in GCs with nursing interventions, earlier intervention was associated with shorter duration of hypoxemia (p < 0.0001) and shorter duration of PGES (p = 0.0012). Seizure duration (p < 0.0001) and convulsion duration (p = 0.0457) were shorter with earlier intervention. PI duration was longer for GCs with PGES than GCs without PGES (p < 0.0001). The mean delay to first active nonrespiratory movement following GCs with PGES was 251.96 s and for GC without PGES was 66.06 s. The duration of PI was positively associated with lower SaO(2) nadir (p = 0.003) and longer duration of oxygen desaturation (p = 0.0026). There was no association between PI duration and seizure duration (p = 0.773), between PI duration and PGES duration (p = 0.758), or between PI duration and the timing of first intervention relative to seizure onset (p = 0.823). PGES did not occur in the NOINT group. The mean duration of desaturation was longer (110.9 vs. 49.9 s) (p < 0.0001), mean SaO(2) nadir was lower (72.8% vs. 79.7%) (p = 0.0086), and mean end-tidal CO(2) was higher (58.6 vs. 50.3 mmHg) (p = 0.0359) in the INT group compared with the NOINT group. The duration of the seizure or of the convulsive component was not significantly different between the INT and NOINT groups. Early periictal nursing intervention was associated with reduced duration of RD and reduced duration of PGES. These findings suggest the possibility that such interventions may be effective in reducing the risk of SUDEP in the outpatient setting. Validation of these preliminary data with a prospective study is needed before definitive conclusions can be reached regarding the efficacy of periictal interventions in reducing the risk of SUDEP.


Effects on caregiver burden of a donepezil hydrochloride dosage increase to 10 mg/day in patients with Alzheimer's disease.
In this study, we evaluated changes in functioning and caregiver burden in Alzheimer's disease (AD) patients after a dosage increase that was made based on pharmacists' evaluation of AD patients' behavior in daily life. Pharmacists used a checklist, a questionnaire, and the Repetitive Saliva Swallowing Test (RSST) to gather data on the daily life of AD patients taking donepezil 5 mg/day and their caregivers. In 27 cases, pharmacists suggested a dosage change to 10 mg/day to AD patients' physicians. Pharmacists then evaluated these patients for 16 weeks after the increase to determine changes in functional assessment staging, caregiver burden, and swallowing function. During the 16-week study, 20 of the 27 patients showed at least one-stage improvement in relation to the five assessed aspects of daily life (time/place, speech, bathing, dressing, and toileting). The mean score for caregiver burden due to personal strain was significantly lower after the dosage increase than before (5.15±3.76 at baseline; from 3.89±3.42 at week 4 to 3.59±3.90 at week 16; P<0.05), as was the mean score due to role strain (2.19±2.80 at baseline; 1.56±2.64 at week 8; P<0.05). After the dosage increase, the impaired swallowing function that accompanies AD was improved in the patients with swallowing problems, as indicated by a higher mean RSST score (1.22±0.67 at baseline; from 2.78±1.72 at week 4 to 2.78±1.79 at week 16; P<0.05). The dosage increase not only decreased caregiver burden, but also appeared to improve impaired swallowing function. Medication therapy management by pharmacists of AD patients, including the use of a checklist, contributed to the correct use of donepezil and improved quality of life for caregivers.


Mapping the organizational readiness to change assessment to the Consolidated Framework for Implementation Research.
Implementation researchers recognize the influential role of organizational factors and, thus, seek to assess these factors using quantitative measurement instruments. However, researchers are hindered by instruments that measure similar constructs but rely on different nomenclature and/or definitions. The Consolidated Framework for Implementation Research (CFIR) provides a taxonomy of constructs derived from prior frameworks and empirical studies of implementation-related constructs. The CFIR includes constructs based on the original Promoting Action on Research Implementation in Health Services (PARiHS) framework which highlights the key roles of strength of evidence for a specific evidence-based intervention (EBI), favorability of organizational context for change, and capacities to facilitate implementation of the EBI. Although the CFIR is among the most frequently used implementation frameworks, it does not include quantitative measures. The Organizational Resource and Context Assessment (ORCA) is a quantitative measurement instrument that was developed based on PARiHS, assessing its three domains. Factors within these three domains are conceptually similar to constructs in the CFIR but do not match directly. The aim of this work was to map ORCA survey items to CFIR constructs to enable direct comparisons and syntheses of findings across studies using the CFIR and/or ORCA. Two distinct, independent research teams, each used rigorous constant comparative techniques with deliberation and consensus to map individual items from the ORCA to the five domains and 39 constructs of CFIR. ORCA items were mapped primarily to three of five CFIR domains: Inner Setting, Process, and Intervention Characteristics. The two research teams agreed on 88% of mappings at the higher domain level; at the lower construct level, their mappings aligned for 62.2% of the ORCA items. Mapping results reveal that the ORCA focuses measurement prominently on Inner Setting, Process, and Intervention Characteristics. This mapping guide can help improve consistency in measurement and reporting, enabling more efficient comparison and synthesis of findings that use either the ORCA instrument or the CFIR framework. The guide helps advance implementation science utilizing mixed methods by providing CFIR users with quantitative measures for selected constructs and enables ORCA users to map their findings to CFIR constructs.


Identification, cloning and functional characterization of novel sperm associated antigen 11 (SPAG11) isoforms in the rat.
Sperm binding proteins and their C-terminal peptides of the Sperm Associated Antigen 11 (SPAG11) family were found to play an important role in epididymal innate immunity in addition to their role in sperm maturation. However, the expression of Spag11 transcripts in rodents is not well documented. Computational analysis was employed to identify novel Spag11 isoforms in the rat. RT-PCR analyses were carried out on RNAs isolated from the male reproductive tract tissues of rat using gene specific primers for Spag11c and Spag11t. The identities of PCR products were confirmed by sequencing. Tissue distribution, developmental expression and androgen regulation of Spag11t and Spag11c were studied using RT-PCR. The antimicrobial activities of recombinant Spag11t and Spag11c were tested against E coli in a colony forming unit assay. In this study, we identified two novel Spag11 transcripts, namely, Spag11t and Spag11c derived from the long arm of chromosome 16 in the rat (Rattus norvegicus), using both in silico and molecular biology approaches. Spag11c is expressed in all three regions of the epididymis, in testis and in ovary but is absent from the seminal vesicle. Spag11t expression is confined to the caput and it is not expressed in the testis, seminal vesicle or ovary. Age dependent expression of Spag11t and Spag11c was observed in the epididymides of rats (10-60 day old). Their expression was found to be most abundant in the adult rat (60 day) suggesting roles in mature reproductive function. Further, both Spag11t and Spag11c expression was down regulated in castrated rat epididymides and the expression was maintained in the testosterone replaced castrated rats. SPAG11C is a potent antibacterial agent. SPAG11T also displayed bactericidal capacity although weaker than SPAG11C and SPAG11E. The abundant expression of Spag11t and Spag11c in the male reproductive tract suggests an important role in male reproductive tract immunity. Their expression is developmentally regulated and androgen dependent. Characterization of novel SPAG11 isoforms will contribute to our understanding of the role of epididymal proteins in sperm maturation and innate immunity.


Reinforcement Learning with Limited Reinforcement: Using Bayes Risk for Active Learning in POMDPs.
Partially Observable Markov Decision Processes (POMDPs) have succeeded in planning domains that require balancing actions that increase an agent's knowledge and actions that increase an agent's reward. Unfortunately, most POMDPs are defined with a large number of parameters which are difficult to specify only from domain knowledge. In this paper, we present an approximation approach that allows us to treat the POMDP model parameters as additional hidden state in a "model-uncertainty" POMDP. Coupled with model-directed queries, our planner actively learns good policies. We demonstrate our approach on several POMDP problems.


Dietary extra virgin olive oil attenuates kidney injury in pristane-induced SLE model via activation of HO-1/Nrf-2 antioxidant pathway and suppression of JAK/STAT, NF-κB and MAPK activation.
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a widespread organ involvement. Recent studies have suggested that extra virgin olive oil (EVOO) might possess preventive effects on this immunoinflammation-related disease. However, its role in SLE remained unknown. In this work, we evaluated the effects of EVOO diet in a pristane-induced SLE model in mice. Three-month-old mice received an injection of pristane or saline solution and were fed with different experimental diets: sunflower oil diet or EVOO diet. After 24weeks, mice were sacrificed, spleens were collected and kidneys were removed for immunoinflammatory detections. The kidney expression of microsomal prostaglandin E synthase 1, heme oxygenase 1 (HO-1), nuclear factor E2-related factor 2 (Nrf-2), mitogen-activated protein kinases (MAPKs), Janus kinase/signal transducer and activator of transcription (JAK/STAT) and nuclear transcription factor-kappa B (NF-κB) pathways were studied by western blotting. In addition to macroscopic and histological analyses, serum matrix metalloproteinase 3 (MMP-3) levels and proinflammatory cytokines production in splenocytes were evaluated by enzyme-linked immunoassay. We have demonstrated that EVOO diet significantly reduced renal damage and decreased MMP-3 serum and PGE2 kidney levels as well as the proinflammatory cytokines production in splenocytes. Our data indicate that Nrf-2 and HO-1 protein expressions were up-regulated in those mice fed with EVOO and the activation of JAK/STAT, MAPK and NF-κB pathways were drastically ameliorated. These results support the interest of EVOO as a beneficial functional food exerting a preventive/palliative role in the management of SLE.


Evidence of genetic heterogeneity in the autosomal recessive adult forms of limb-girdle muscular dystrophy following linkage analysis with 15q probes in Brazilian families.
The autosomal recessive limb-girdle muscular dystrophies (LGMD) represent a heterogeneous group of diseases which may be characterised by one or more autosomal loci. A gene at 15q has recently been found to be responsible for a mild form of LGMD in a group of families from the isolated island of Réunion, now classified as LGMD2. Based on results of eight out of 11 large Brazilian LGMD families of different racial background (which were informative for the closest available probe to the LGMD2 gene), we confirmed linkage to the LGMD2 gene at 15q in two of these families and exclusion in six others. These data provide the first evidence of genetic heterogeneity for the autosomal recessive limb-girdle muscular dystrophies.


Investigation of the Syncytial Nature of Detrusor Smooth Muscle as a Determinant of Action Potential Shape.
Unlike most excitable cells, certain syncytial smooth muscle cells are known to exhibit spontaneous action potentials of varying shapes and sizes. These differences in shape are observed even in electrophysiological recordings obtained from a single cell. The origin and physiological relevance of this phenomenon are currently unclear. The study presented here aims to test the hypothesis that the syncytial nature of the detrusor smooth muscle tissue contributes to the variations in the action potential profile by influencing the superposition of the passive and active signals. Data extracted from experimental recordings have been compared with those obtained through simulations. The feature correlation studies on action potentials obtained from the experimental recordings suggest the underlying presence of passive signals, called spontaneous excitatory junction potentials (sEJPs). Through simulations, we are able to demonstrate that the syncytial organization of the cells, and the variable superposition of the sEJPs with the "native action potential", contribute to the diversity in the action potential profiles exhibited. It could also be inferred that the fraction of the propagated action potentials is very low in the detrusor. It is proposed that objective measurements of spontaneous action potential profiles can lead to a better understanding of bladder physiology and pathology.


Incidence and prevalence of Systemic Sclerosis (SSc) in Poland - differences between rural and urban regions.
[b] Abstract Introduction.[/b] Systemic sclerosis (SSc) is a rare and potentially severe connective tissue disease, characterized by skin fibrosis and involvement of internal organs. Because of its rarity and heterogeneous clinical presentation, reliable epidemiological studies on SSc have been particularly difficult to carry out. [b]Objectives[/b]. The purpose of this study was to present SSc epidemiology among hospitalized patients in Poland. The analysis was based on population-based administrative data, taken from a Polish hospital morbidity study carried out by the National Institute of Public Health between January 2008 - December 2012. [b]Results[/b]. Analyzed data covered 9,049 hospitalization records. The final sample comprised 3,653 patients with first-time hospitalizations for SSc. The average age of the sample was 53 years (SD 16.2; range 0-91 years); 84% of patients were female and 16% male. Based on hospitalization registers, the average SSc incidence was estimated to be 1.9/100,000 per year and peak age of incidence was 55 years. The point prevalence was estimated to be 9.4/100,000 at the end of 2012. SSc was more common in females, with F:M ratio ranging from 6.2:1-4.6:1 depending on the year. Analysis of hospitalization trends revealed that overall numbers of SSc hospitalizations increased, while first-time hospitalizations decreased between 2008-2012. Clusters of higher incidence were observed in more rural regions vs. urban regions. [b]Conclusion[/b]. Estimated incidence of SSc in Poland was comparable to reported incidence in other European countries. Analysis of demographic factors and reports of clusters of higher incidence may suggest the existence of environmental risk factors for the development of SSc. These observations may require further research.


The Absence of C-5 DNA Methylation in Leishmania donovani Allows DNA Enrichment from Complex Samples.
Cytosine C5 methylation is an important epigenetic control mechanism in a wide array of eukaryotic organisms and generally carried out by proteins of the C-5 DNA methyltransferase family (DNMTs). In several protozoans, the status of this mechanism remains elusive, such as in Leishmania, the causative agent of the disease leishmaniasis in humans and a wide array of vertebrate animals. In this work, we showed that the Leishmania donovani genome contains a C-5 DNA methyltransferase (DNMT) from the DNMT6 subfamily, whose function is still unclear, and verified its expression at the RNA level. We created viable overexpressor and knock-out lines of this enzyme and characterized their genome-wide methylation patterns using whole-genome bisulfite sequencing, together with promastigote and amastigote control lines. Interestingly, despite the DNMT6 presence, we found that methylation levels were equal to or lower than 0.0003% at CpG sites, 0.0005% at CHG sites, and 0.0126% at CHH sites at the genomic scale. As none of the methylated sites were retained after manual verification, we conclude that there is no evidence for DNA methylation in this species. We demonstrated that this difference in DNA methylation between the parasite (no detectable DNA methylation) and the vertebrate host (DNA methylation) allowed enrichment of parasite vs. host DNA using methyl-CpG-binding domain columns, readily available in commercial kits. As such, we depleted methylated DNA from mixes of Leishmania promastigote and amastigote DNA with human DNA, resulting in average Leishmania:human enrichments from 62× up to 263×. These results open a promising avenue for unmethylated DNA enrichment as a pre-enrichment step before sequencing Leishmania clinical samples.


The independence of the infundibular building blocks in the setting of double-outlet right ventricle.
It has long been contentious as to whether the presence of bilateral infundibulums, or conuses, is a prerequisite for the diagnosis of double-outlet right ventricle. As the use of such a criterion would abrogate the so-called "morphological method", which correctly states that one variable entity should not be defined on the basis of another entity that is itself variable, it is now accepted that double outlet can exist in the setting of fibrous continuity between the leaflets of the atrioventricular and arterial valves. Although this debate has now been resolved, there are other contentious areas still requiring clarification in the setting of hearts unified because of the presence of this particular ventriculo-arterial connection - for example, it is questionable whether the channel between the ventricles should be described as a "ventricular septal defect", whereas it is equally arguable that the mere presence of fibrous continuity between the leaflets of the arterial valves does not necessarily place the channel in a doubly committed location. In this review, we describe a series of autopsied hearts in which the anatomical features serve to illuminate these various topics. We then discuss recent findings regarding cardiac development that point to the individuality of the building blocks of the ventricular outflow tracts, specifically the outlet septum, the inner heart curvature, or ventriculo-infundibular fold, and the septomarginal trabeculation, or septal band.


Impact of functional decline on outcome in elderly patients with acute coronary syndromes.
Management of acute coronary syndromes in elderly patients is poorly defined. To assess the impact of functional decline on all-cause mortality in elderly patients with acute coronary syndromes. Clinical data, including the Global Registry of Acute Coronary Events score and assessment of functional status obtained by using the Katz scale, were prospectively collected on 272 patients 70 years or older hospitalized for acute coronary syndromes. All-cause mortality was assessed at 6 months, and longer term outcome data were obtained. Mean age of the patients was 78 years (SD, 6), and 58% were men. A total of 28% had functional decline. Six months after the index hospitalization, 38 patients had died. Another 29 patients died during a median follow-up of 611 days after the initial 6 months. Functional decline was associated with both 6-month (hazards ratio, 3.63; 95% CI, 1.91-6.88; P < .001) and long-term (hazards ratio, 2.69; 95% CI, 1.28-5.64; P = .009) outcomes. Functional decline remained associated with both 6-month and long-term outcomes in multivariate analysis and was systematically selected in the most predictive multivariate models for 6-month and long-term mortality. The multivariate model including the Global Registry of Acute Coronary Events score and functional decline was predictive of 6-month mortality, but the combination of functional decline and biological data was more predictive of long-term mortality than was a model combining functional decline and the Global Registry score. Functional decline in elderly patients with acute coronary syndromes is predictive of poor outcomes.


Distribution of Genes Encoding Resistance to Macrolides Among Staphylococci Isolated From the Nasal Cavity of Hospital Employees in Khorramabad, Iran.
Epidemiological data on antibiotic susceptibility of Staphylococcus strains isolated from nasal carriers in each region can be helpful to select appropriate drugs to eradicate carriage states, control nosocomial infections and also treat patients. The current study aimed to investigate the antibiotic resistance profile and the molecular prevalence of the ermA, ermB, ermC and msrA genes among Staphylococcus strains isolated from the anterior nares of hospital employees. In this cross-sectional study, a total of 100 Staphylococcus isolates, 51 Staphylococcus aureus, 49 coagulase-negative staphylococci (CoNS) were isolated from the anterior nares of hospital employees in Khorramabad, Iran. Susceptibility pattern to macrolide antibiotics were determined using the disk diffusion method. The polymerase chain reaction (PCR) assay was applied to determine the major erythromycin-resistant genes (ermA, ermB, ermC and msrA). Fifty-three (53%) isolates were simultaneously resistant to erythromycin, azithromycin and clarithromycin (cross-resistance); while 8 (8%) isolates had variable macrolide susceptibility pattern. Among the S. aureus isolates, the difference in prevalence of resistance to erythromycin between males and females was significant (P = 0.011). The frequency of ermA, ermB, ermC, and msrA genes were 3%, 5%, 33% and 20%, respectively. It was also found that out of 53 isolates resistant to erythromycin, 44 (83%) isolates (eight S. aureus and thirty-six CoNS strains) carried at least one of the four tested genes. Eight (8%) isolates had intermediate phenotype to erythromycin, in which 4 (50%) isolates carried ermB or ermC genes. In addition, out of 39 erythromycin-susceptible isolates, 3 (7.7%) isolates were positive for ermB or ermC genes. No entire association was found between genotype and phenotype methods to detect macrolides-resistant isolates. In addition, distribution of genetically erythromycin-resistant isolates is geographically different among staphylococci. It is recommend removing S. aureus from nasal carriers by proved approaches such as local or systemic administration of effective antibiotics or bacterial interference.


Evaluation of two commercial global miRNA expression profiling platforms for detection of less abundant miRNAs.
microRNAs (miRNA) are short, endogenous transcripts that negatively regulate the expression of specific mRNA targets. miRNAs are found both in tissues and body fluids such as plasma. A major perspective for the use of miRNAs in the clinical setting is as diagnostic plasma markers for neoplasia. While miRNAs are abundant in tissues, they are often scarce in plasma. For quantification of miRNA in plasma it is therefore of importance to use a platform with high sensitivity and linear performance in the low concentration range. This motivated us to evaluate the performance of three commonly used commercial miRNA quantification platforms: GeneChip miRNA 2.0 Array, miRCURY Ready-to-Use PCR, Human panel I+II V1.M, and TaqMan Human MicroRNA Array v3.0. Using synthetic miRNA samples and plasma RNA samples spiked with different ratios of 174 synthetic miRNAs we assessed the performance characteristics reproducibility, recovery, specificity, sensitivity and linearity. It was found that while the qRT-PCR based platforms were sufficiently sensitive to reproducibly detect miRNAs at the abundance levels found in human plasma, the array based platform was not. At high miRNA levels both qRT-PCR based platforms performed well in terms of specificity, reproducibility and recovery. At low miRNA levels, as in plasma, the miRCURY platform showed better sensitivity and linearity than the TaqMan platform. For profiling clinical samples with low miRNA abundance, such as plasma samples, the miRCURY platform with its better sensitivity and linearity would probably be superior.


Differential patterns of intronic and exonic DNA regions with respect to RNA polymerase II occupancy, nucleosome density and H3K36me3 marking in fission yeast.
The generation of mature mRNAs involves interconnected processes, including transcription by RNA polymerase II (Pol II), modification of histones, and processing of pre-mRNAs through capping, intron splicing, and polyadenylation. These processes are thought to be integrated, both spatially and temporally, but it is unclear how these connections manifest at a global level with respect to chromatin patterns and transcription kinetics. We sought to clarify the relationships between chromatin, transcription and splicing using multiple genome-wide approaches in fission yeast. To investigate these functional interdependencies, we determined Pol II occupancy across all genes using high-density tiling arrays. We also performed ChIP-chip on the same array platform to globally map histone H3 and its H3K36me3 modification, complemented by formaldehyde-assisted isolation of regulatory elements (FAIRE). Surprisingly, Pol II occupancy was higher in introns than in exons, and this difference was inversely correlated with gene expression levels at a global level. Moreover, introns showed distinct distributions of histone H3, H3K36me3 and FAIRE signals, similar to those at promoters and terminators. These distinct transcription and chromatin patterns of intronic regions were most pronounced in poorly expressed genes. Our findings suggest that Pol II accumulates at the 3' ends of introns, leading to substantial transcriptional delays in weakly transcribed genes. We propose that the global relationship between transcription, chromatin remodeling, and splicing may reflect differences in local nuclear environments, with highly expressed genes being associated with abundant processing factors that promote effective intron splicing and transcriptional elongation.


Thermosulfuriphilus ammonigenes gen. nov., sp. nov., a thermophilic, chemolithoautotrophic bacterium capable of respiratory ammonification of nitrate with elemental sulfur.
An extremely thermophilic, anaerobic, chemolithoautotrophic bacterium (strain ST65T) was isolated from a deep-sea hydrothermal vent chimney located on the Eastern Lau Spreading Centre in the south-western Pacific Ocean, at a depth of 1870 m. Cells of strain ST65T were non-motile straight or slightly curved short rods, 0.5-0.6 µm in diameter and 0.8-1.5 µm in length. The temperature range for growth was 47-75 °C, with an optimum at 65 °C. The pH range for growth was 5.5-7.5, with an optimum at pH 6.5. Growth of strain ST65T was observed at NaCl concentrations ranging from 1.5 to 4.5 % (w/v), with an optimum at 2.0-2.5 %. Strain ST65T grew anaerobically with inorganic carbon as a carbon source and with elemental sulfur as an electron donor and nitrate as an electron acceptor producing sulfate and ammonium. It was also able to grow by disproportionation of elemental sulfur, thiosulfate and sulfite. Sulfate was not utilized as an electron acceptor. Analysis of the 16S rRNA gene sequence revealed that the isolate belongs to a deep lineage in the phylum Thermodesulfobacteria. On the basis of its physiological properties and results of phylogenetic analyses, it is proposed that the isolate represents a novel species of a new genus, Thermosulfuriphilus ammonigenes gen. nov., sp. nov. ST65T (=DSM 102941T=VKM B-2855T) is the type strain of the type species.


Identification of common and cell type specific LXXLL motif EcR cofactors using a bioinformatics refined candidate RNAi screen in Drosophila melanogaster cell lines.
During Drosophila development, titers of the steroid ecdysone trigger and maintain temporal and tissue specific biological transitions. Decades of evidence reveal that the ecdysone response is both unique to specific tissues and distinct among developmental timepoints. To achieve this diversity in response, the several isoforms of the Ecdysone Receptor, which transduce the hormone signal to the genome level, are believed to interact with tissue specific cofactors. To date, little is known about the identity of these cofactor interactions; therefore, we conducted a bioinformatics informed, RNAi luciferase reporter screen against a subset of putative candidate cofactors identified through an in silico proteome screen. Candidates were chosen based on criteria obtained from bioinformatic consensus of known nuclear receptor cofactors and homologs, including amino acid sequence motif content and context. The bioinformatics pre-screen of the Drosophila melanogaster proteome was successful in identifying an enriched putative candidate gene cohort. Over 80% of the genes tested yielded a positive hit in our reporter screen. We have identified both cell type specific and common cofactors which appear to be necessary for proper ecdysone induced gene regulation. We have determined that certain cofactors act as co-repressors to reduce target gene expression, while others act as co-activators to increase target gene expression. Interestingly, we find that a few of the cofactors shared among cell types have a reversible roles to function as co-repressors in certain cell types while in other cell types they serve as co-activators. Lastly, these proteins are highly conserved, with higher order organism homologs also harboring the LXXLL steroid receptor interaction domains, suggesting a highly conserved mode of steroid cell target specificity. In conclusion, we submit these cofactors as novel components of the ecdysone signaling pathway in order to further elucidate the dynamics of steroid specificity.


Machine Learning Applied to Registry Data: Development of a Patient-Specific Prediction Model for Blood Transfusion Requirements During Craniofacial Surgery Using the Pediatric Craniofacial Perioperative Registry Dataset.
Craniosynostosis is the premature fusion of ≥1 cranial sutures and often requires surgical intervention. Surgery may involve extensive osteotomies, which can lead to substantial blood loss. Currently, there are no consensus recommendations for guiding blood conservation or transfusion in this patient population. The aim of this study is to develop a machine-learning model to predict blood product transfusion requirements for individual pediatric patients undergoing craniofacial surgery. Using data from 2143 patients in the Pediatric Craniofacial Surgery Perioperative Registry, we assessed 6 machine-learning classification and regression models based on random forest, adaptive boosting (AdaBoost), neural network, gradient boosting machine (GBM), support vector machine, and elastic net methods with inputs from 22 demographic and preoperative features. We developed classification models to predict an individual's overall need for transfusion and regression models to predict the number of blood product units to be ordered preoperatively. The study is reported according to the Transparent Reporting of a multivariable prediction model for Individual Prognosis Or Diagnosis (TRIPOD) checklist for prediction model development. The GBM performed best in both domains, with an area under receiver operating characteristic curve of 0.87 ± 0.03 (95% confidence interval) and F-score of 0.91 ± 0.04 for classification, and a mean squared error of 1.15 ± 0.12, R-squared (R) of 0.73 ± 0.02, and root mean squared error of 1.05 ± 0.06 for regression. GBM feature ranking determined that the following variables held the most information for prediction: platelet count, weight, preoperative hematocrit, surgical volume per institution, age, and preoperative hemoglobin. We then produced a calculator to show the number of units of blood that should be ordered preoperatively for an individual patient. Anesthesiologists and surgeons can use this continually evolving predictive model to improve clinical care of patients presenting for craniosynostosis surgery.