Valid molecular dynamics simulations of human hemoglobin require a surprisingly large box size.
Recent molecular dynamics (MD) simulations of human hemoglobin (Hb) give results in disagreement with experiment. Although it is known that the unliganded (T[Formula: see text]) and liganded (R[Formula: see text]) tetramers are stable in solution, the published MD simulations of T[Formula: see text] undergo a rapid quaternary transition to an R-like structure. We show that T[Formula: see text] is stable only when the periodic solvent box contains ten times more water molecules than the standard size for such simulations. The results suggest that such a large box is required for the hydrophobic effect, which stabilizes the T[Formula: see text] tetramer, to be manifested. Even in the largest box, T[Formula: see text] is not stable unless His146 is protonated, providing an atomistic validation of the Perutz model. The possibility that extra large boxes are required to obtain meaningful results will have to be considered in evaluating existing and future simulations of a wide range of systems.


Predicting success or failure of immunotherapy for cancer: insights from a clinically applicable mathematical model.
The objective of this study was to create a clinically applicable mathematical model of immunotherapy for cancer and use it to explore differences between successful and unsuccessful treatment scenarios. The simplified predator-prey model includes four lumped parameters: tumor growth rate, g; immune cell killing efficiency, k; immune cell signaling factor, λ; and immune cell half-life decay, μ. The predator-prey equations as functions of time, t, for normalized tumor cell numbers, y, (the prey) and immunocyte numbers, ×, (the predators) are: dy/dt = gy - kx and dx/dt = λxy - μx. A parameter estimation procedure that capitalizes on available clinical data and the timing of clinically observable phenomena gives mid-range benchmarks for parameters representing the unstable equilibrium case in which the tumor neither grows nor shrinks. Departure from this equilibrium results in oscillations in tumor cell numbers and in many cases complete elimination of the tumor. Several paradoxical phenomena are predicted, including increasing tumor cell numbers prior to a population crash, apparent cure with late recurrence, one or more cycles of tumor growth prior to eventual tumor elimination, and improved tumor killing with initially weaker immune parameters or smaller initial populations of immune cells. The model and the parameter estimation techniques are easily adapted to various human cancers that evoke an immune response. They may help clinicians understand and predict certain strange and unexpected effects in the world of tumor immunity and lead to the design of clinical trials to test improved treatment protocols for patients.


Television viewing and snacking behaviors of fourth- and eighth-grade schoolchildren in Texas.
Child and adolescent overweight is a serious health issue. Both snacking and television watching have been associated with childhood overweight, but the relationships have not been well examined in a multiethnic population. The aim of this study was to examine relationships between weekday television viewing, snack consumption, consumption of foods advertised on television, and overweight status of a multiethnic sample of fourth- and eighth-grade schoolchildren in Texas. This study is a secondary analysis of data from the School Physical Activity and Nutrition monitoring system, a validated survey with objective measures of height and weight. The sample of 11,594 children in the fourth and eighth grades was weighted to provide data representative of children in Texas public schools. Children were categorized on the basis of self-reported daily television viewing, snack consumption, and consumption of foods advertised on television. Multiple logistic regression was used to analyze, by grade level, the differences in the prevalence of overweight by category. Television viewing, frequency of snack consumption, and consumption of foods advertised on television were all positively related to one another. In general, both consuming more snacks and foods advertised on television were associated with reduced odds of overweight regardless of the amount of television watched. The results suggest that the relationships between weekday snacking behaviors and television viewing in a multiethnic population are complicated. When these behaviors are clustered, overweight status may be related more to the number of snacks consumed than to the amount of television watched. To determine the exact relationship, additional research, especially among Hispanic children, is warranted.


Vaccination with adjuvanted recombinant neuraminidase induces broad heterologous, but not heterosubtypic, cross-protection against influenza virus infection in mice.
In an attempt to assess the cross-protective potential of the influenza virus neuraminidase (NA) as a vaccine antigen, different subtypes of recombinant NA were expressed in a baculovirus system and used to vaccinate mice prior to lethal challenge with homologous, heterologous, or heterosubtypic viruses. Mice immunized with NA of subtype N2 were completely protected from morbidity and mortality in a homologous challenge and displayed significantly reduced viral lung titers. Heterologous challenge with a drifted strain resulted in morbidity but no mortality. Similar results were obtained for challenge experiments with N1 NA. Mice immunized with influenza B virus NA (from B/Yamagata/16/88) displayed no morbidity when sublethally infected with the homologous strain and, importantly, were completely protected from morbidity and mortality when lethally challenged with the prototype Victoria lineage strain or a more recent Victoria lineage isolate. Upon analyzing the NA content in 4 different inactivated-virus vaccine formulations from the 2013-2014 season via Western blot assay and enzyme-linked immunosorbent assay quantification, we found that the amount of NA does indeed vary across vaccine brands. We also measured hemagglutinin (HA) and NA endpoint titers in pre- and postvaccination human serum samples from individuals who received a trivalent inactivated seasonal influenza vaccine from the 2004-2005 season; the induction of NA titers was statistically less pronounced than the induction of HA titers. The demonstrated homologous and heterologous protective capacity of recombinant NA suggests that supplementing vaccine formulations with a standard amount of NA may offer increased protection against influenza virus infection. Despite the existence of vaccine prophylaxis and antiviral therapeutics, the influenza virus continues to cause morbidity and mortality in the human population, emphasizing the continued need for research in the field. While the majority of influenza vaccine strategies target the viral hemagglutinin, the immunodominant antigen on the surface of the influenza virion, antibodies against the viral neuraminidase (NA) have been correlated with less severe disease and decreased viral shedding in humans. Nevertheless, the amount of NA is not standardized in current seasonal vaccines, and the exact breadth of NA-based protection is unknown. Greater insight into the cross-protective potential of influenza virus NA as a vaccine antigen may pave the way for the development of influenza vaccines of greater breadth and efficacy.


Undoing the 'Nordic Paradox': Factors affecting rates of disclosed violence against women across the EU.
Measuring violence against women raises methodological questions, as well as the wider question of how to understand violence and locate it in relation to a societal context. This is all the more relevant given that measurement of violence against women in the EU has made an interesting phenomenon apparent, the so-called 'Nordic Paradox', whereby prevalence is higher in more gender equal countries. This article examines this phenomenon by exploring a range of factors-methodological, demographic and societal-to contextualise disclosed levels of violence. The analysis makes use of a multilevel analytic approach to take into account how macro and micro levels contribute to the prevalence of violence. The intercepts are then used to illustrate how taking these into account might provide an alternative ranking of levels of violence against women in EU countries. The results show that the 'Nordic Paradox' disappears-and can be undone-when factors at individual and country levels are considered. We conclude that the 'Nordic Paradox' cannot be understood independently from a wider pattern of violence in society, and should be seen as connected and co-constituted in specific formations, domains or regimes of violence. Our results show that the use of multi-level models can provide new insights into the factors that may be related to disclosed prevalence of violence against women. This can generate a better understanding of how violence against women functions as a system, and in turn inform better policy responses.


Neuropsychological deficits in adolescents with conduct disorder and comorbid bipolar disorder: a pilot study.
We report pilot data on neuropsychological deficits in aggressive juvenile offenders with and without bipolar disorder compared with each other and healthy controls. We assessed 52 adolescents and their parent or guardians: 36 incarcerated juvenile offenders and 16 community controls using the Schedule for Affective Disorders and Schizophrenia for School Age Children, Present and Life-Time Version and a neuropsychological testing battery. All incarcerated subjects (n=34) met criteria for Conduct Disorder (CD); 26 are classified as Non-BD-CD, and eight with CD and Bipolar disorder (CD-BD). These subjects were compared to community controls (n=16) matched for age, gender, SES and ethnicity. Relative to controls, the Non-BD-CD subjects' impairments (p<0.05) were in cognitive ability, set shifting/inhibition, planning and verbal memory-language functioning. The CD-BD group displayed impairments (p<0.05) relative to controls in cognitive ability, set shifting, verbal memory-language functioning, and visuospatial tasks. The Non-BD-CD and CD-BD groups however did not display significant differences on most neuropsychological measures compared with each other. When we controlled for Attention Deficit Hyperactivity Disorder, the Non-CD-BP subjects continued to show deficits on Verbal measures where the CD-BD subjects maintained deficits in measures of cognitive ability, verbal measures and visual spatial tests. Juvenile offender with CD displayed a wide range of deficits on neuropsychological testing compared with controls. Although juvenile offenders with and without BD differed on their clinical presentation, differences on neuropsychological measures are not specific and may be related to comorbid diagnoses.


A general mechanistic model for admixture histories of hybrid populations.
Admixed populations have been used for inferring migrations, detecting natural selection, and finding disease genes. These applications often use a simple statistical model of admixture rather than a modeling perspective that incorporates a more realistic history of the admixture process. Here, we develop a general model of admixture that mechanistically accounts for complex historical admixture processes. We consider two source populations contributing to the ancestry of a hybrid population, potentially with variable contributions across generations. For a random individual in the hybrid population at a given point in time, we study the fraction of genetic admixture originating from a specific one of the source populations by computing its moments as functions of time and of introgression parameters. We show that very different admixture processes can produce identical mean admixture proportions, but that such processes produce different values for the variance of the admixture proportion. When introgression parameters from each source population are constant over time, the long-term limit of the expectation of the admixture proportion depends only on the ratio of the introgression parameters. The variance of admixture decreases quickly over time after the source populations stop contributing to the hybrid population, but remains substantial when the contributions are ongoing. Our approach will facilitate the understanding of admixture mechanisms, illustrating how the moments of the distribution of admixture proportions can be informative about the historical admixture processes contributing to the genetic diversity of hybrid populations.


Addition of another disease-modifying anti-rheumatic drug to methotrexate reduces the flare rate within 2 years after infliximab discontinuation in patients with rheumatoid arthritis: an open, randomized, controlled trial.
We examined whether the addition of another conventional disease-modifying anti-rheumatic drugs (DMARDs) to methotrexate (MTX) upon infliximab (IFX) discontinuation in well-controlled rheumatoid arthritis (RA) patients could suppress subsequent disease flare. RA patients maintaining DAS28-CRP (Disease Activity Score of 28 joints with C-reactive protein) scores < 2.6 for ≥ 6 months with IFX were randomized either to receive addition of bucillamine (BUC) to MTX (BUC + MTX group; n = 24) or not (MTX group; n = 31) upon discontinuing IFX. The primary endpoint was the flare rate within 2 years of IFX discontinuation. Six patients discontinuing MTX during the study were excluded from analyses. Seventeen patients (63.0%) experienced flares in the MTX group, which was significantly reduced in the BUC + MTX group (31.8%; p = 0.045). Further, the flare rates differed significantly between remission and non-remission by a Boolean definition upon IFX discontinuation in the MTX group (40.0% vs. 91.7%, respectively; p = 0.014), but they were comparable in the BUC + MTX group. BUC treatment was interrupted in seven patients due to rash, proteinuria and incompliance. DMARDs combination therapy may be a better treatment strategy than MTX monotherapy for maintaining RA control after successful discontinuation of biological agents.


The Lupus Autoantigen La Prevents Mis-channeling of tRNA Fragments into the Human MicroRNA Pathway.
The Lupus autoantigen La is an RNA-binding protein that stabilizes RNA polymerase III (Pol III) transcripts and supports RNA folding and has in addition been implicated in the mammalian microRNA (miRNA) pathway. Here, we have analyzed effects of La depletion on Argonaute (Ago)-bound small RNAs in human cells. We find that in the absence of La, distinct tRNA fragments are loaded into Ago proteins. Thus, La functions as gatekeeper ensuring correct tRNA maturation and protecting the miRNA pathway from potentially functional tRNA fragments. However, one specific isoleucin pre-tRNA produces both a functional tRNA and a miRNA even when La is present. We demonstrate that the fully complementary 5' leader and 3' trailer of the pre-tRNA-Ile form a double-stranded RNA molecule that has low affinity to La. Instead, Exportin-5 (Xpo5) recognizes it as miRNA precursor and transports it into the cytoplasm for Dicer processing and Ago loading.


Macromolecular assemblage of aminoacyl-tRNA synthetases: identification of protein-protein interactions and characterization of a core protein.
In eukaryotes, from fly to human, nine aminoacyl-tRNA synthetases contribute a multienzyme complex of defined and conserved structural organization. This ubiquitous multiprotein assemblage comprises a unique bifunctional aminoacyl-tRNA synthetase, glutamyl-prolyl-tRNA synthetase, as well as the monospecific isoleucyl, leucyl, glutaminyl, methionyl, lysyl, arginyl, and aspartyl-tRNA synthetases. Three auxiliary proteins of apparent molecular masses of 18, 38 and 43 kDa are invariably associated with the nine tRNA synthetase components of the complex. As part of an inquiry into the molecular and functional organization of this macromolecular assembly, we isolated the cDNA encoding the p38 non-synthetase component and determined its function. The 320 amino acid residue encoded protein has been shown to have no homolog in yeast, bacteria and archaea, according to the examination of the complete genomic sequences available. The p38 protein is a moderately hydrophobic protein, displays a putative leucine-zipper motif, and shares a sequence pattern with protein domains that are involved in protein-protein interactions. We used the yeast two-hybrid system to register protein connections between components of the complex. We performed an exhaustive search of interactive proteins, involving 10 of the 11 components of the complex. Twenty-one protein pairs have been unambiguously identified, leading to a global view of the topological arrangement of the subunits of the multisynthetase complex. In particular, p38 was found to associate with itself to form a dimer, but also with p43, with the class I tRNA synthetases ArgRS and GlnRS, with the class II synthetases AspRS and LysRS, and with the bifunctional GluProRS. We generated a series of deletion mutants to localize the regions of p38 mediating the identified interactions. Mapping the interactive domains in p38 showed the specific association of p38 with its different protein partners. These findings suggest that p38, for which no homologous protein has been identified to date in organisms devoid of multisynthetase complexes, plays a pivotal role for the assembly of the subunits of the eukaryotic tRNA synthetase complex.


Role of the multidrug resistance protein-1 in hypertension and vascular dysfunction caused by angiotensin II.
Human endothelial cells use the multidrug resistance protein-1 (MRP1) to export glutathione disulfide (GSSG). This can promotes thiol loss during states of increased glutathione oxidation. We investigated how MRP1 modulates blood pressure and vascular function during angiotensin II-induced hypertension. Angiotensin II-induced hypertension altered vascular glutathione flux by increasing GSSG export and decreasing vascular levels of glutathione in wild-type (FVB) but not in MRP1-/- mice. Aortic endothelium-dependent vasodilatation was reduced in FVB after angiotensin II infusion, but unchanged in MRP1-/- mice. Aortic superoxide (O2*-) production and expression of several NADPH oxidase subunits were increased by angiotensin II in FVB. These effects were markedly blunted in MRP1-/- vessels. The increase in O2*- production in FVB vessels caused by angiotensin II was largely inhibited by L-NAME, suggesting eNOS uncoupling. Accordingly, aortic tetrahydrobiopterin and levels of NO were decreased by angiotensin II in FVB but were unchanged in MRP1-/-. Finally, the hypertension caused by angiotensin II was markedly blunted in MRP1-/- mice (137+/-4 versus 158+/-6 mm Hg). MRP1 plays a crucial role in the genesis of multiple vascular abnormalities that accompany hypertension and its presence is essential for the hypertensive response to angiotensin II.


Phenotypic consequences of rearranging the P, M, and G genes of vesicular stomatitis virus.
The nonsegmented negative-strand RNA viruses (order Mononegavirales) include many important human pathogens. The order of their genes, which is highly conserved, is the major determinant of the relative levels of gene expression, since genes that are close to the single promoter site at the 3' end of the viral genome are transcribed at higher levels than those that occupy more distal positions. We manipulated an infectious cDNA clone of the prototypic vesicular stomatitis virus (VSV) to rearrange three of the five viral genes, using an approach which left the viral nucleotide sequence otherwise unaltered. The central three genes in the gene order, which encode the phosphoprotein P, the matrix protein M, and the glycoprotein G, were rearranged into all six possible orders. Viable viruses were recovered from each of the rearranged cDNAs. The recovered viruses were examined for their levels of gene expression, growth potential in cell culture, and virulence in mice. Gene rearrangement changed the expression levels of the encoded proteins in concordance with their distance from the 3' promoter. Some of the viruses with rearranged genomes replicated as well or slightly better than wild-type virus in cultured cells, while others showed decreased replication. All of the viruses were lethal for mice, although the time to symptoms and death following inoculation varied. These data show that despite the highly conserved gene order of the Mononegavirales, gene rearrangement is not lethal or necessarily even detrimental to the virus. These findings suggest that the conservation of the gene order observed among the Mononegavirales may result from immobilization of the ancestral gene order due to the lack of a mechanism for homologous recombination in this group of viruses. As a consequence, gene rearrangement should be irreversible and provide an approach for constructing viruses with novel phenotypes.


NUDT2 Disruption Elevates Diadenosine Tetraphosphate (Ap4A) and Down-Regulates Immune Response and Cancer Promotion Genes.
Regulation of gene expression is one of several roles proposed for the stress-induced nucleotide diadenosine tetraphosphate (Ap4A). We have examined this directly by a comparative RNA-Seq analysis of KBM-7 chronic myelogenous leukemia cells and KBM-7 cells in which the NUDT2 Ap4A hydrolase gene had been disrupted (NuKO cells), causing a 175-fold increase in intracellular Ap4A. 6,288 differentially expressed genes were identified with P < 0.05. Of these, 980 were up-regulated and 705 down-regulated in NuKO cells with a fold-change ≥ 2. Ingenuity® Pathway Analysis (IPA®) was used to assign these genes to known canonical pathways and functional networks. Pathways associated with interferon responses, pattern recognition receptors and inflammation scored highly in the down-regulated set of genes while functions associated with MHC class II antigens were prominent among the up-regulated genes, which otherwise showed little organization into major functional gene sets. Tryptophan catabolism was also strongly down-regulated as were numerous genes known to be involved in tumor promotion in other systems, with roles in the epithelial-mesenchymal transition, proliferation, invasion and metastasis. Conversely, some pro-apoptotic genes were up-regulated. Major upstream factors predicted by IPA® for gene down-regulation included NFκB, STAT1/2, IRF3/4 and SP1 but no major factors controlling gene up-regulation were identified. Potential mechanisms for gene regulation mediated by Ap4A and/or NUDT2 disruption include binding of Ap4A to the HINT1 co-repressor, autocrine activation of purinoceptors by Ap4A, chromatin remodeling, effects of NUDT2 loss on transcript stability, and inhibition of ATP-dependent regulatory factors such as protein kinases by Ap4A. Existing evidence favors the last of these as the most probable mechanism. Regardless, our results suggest that the NUDT2 protein could be a novel cancer chemotherapeutic target, with its inhibition potentially exerting strong anti-tumor effects via multiple pathways involving metastasis, invasion, immunosuppression and apoptosis.


Early influence of the steppe tribes in the peopling of Siberia.
The Yakuts, Middle Age Turkic speakers (15th-16th centuries), are widely accepted as the first settlers of the Altai-Baikal area in eastern Siberia. They are supposed to have introduced horses and developed metallurgy in this geographic area during the 15th or 16th century A.D. The analysis of the Siberian grave of Pokrovsk, recently discovered near the Lena River (61 degrees 29' N) and dated by accelerator mass spectrometry from 2,400 to 2,200 years B.P., may provide new elements to test this hypothesis. The exceptional combination of various artifacts and the mitochondrial DNA data extracted from the bone remains of the Pokrovsk man might prove the existence of previous contacts between autochthonous hunters of Oriental Siberia and the nomadic horse breeders from the Altai-Baikal area (Mongolia and Buryatia). Indeed, the stone arrowhead and the harpoons relate this Pokrovsk man to the traditional hunters of the Taiga. Some artifacts made of horse bone and the pieces of armor, however, are related to the tribes of Mongolia and Buryatia of the Xiongnu period (3rd century B.C.). This affinity has been confirmed by the match of the mitochondrial haplotype of this subject with a woman of the Egyin Gol necropolis (Mongolia, 2nd/3rd century A.D.) as well as with two modern Buryats. This result allows us to postulate that contacts between southern steppe populations and Siberian tribes occurred before the 15th century.


Coking- and sintering-resistant palladium catalysts achieved through atomic layer deposition.
We showed that alumina (Al(2)O(3)) overcoating of supported metal nanoparticles (NPs) effectively reduced deactivation by coking and sintering in high-temperature applications of heterogeneous catalysts. We overcoated palladium NPs with 45 layers of alumina through an atomic layer deposition (ALD) process that alternated exposures of the catalysts to trimethylaluminum and water at 200°C. When these catalysts were used for 1 hour in oxidative dehydrogenation of ethane to ethylene at 650°C, they were found by thermogravimetric analysis to contain less than 6% of the coke formed on the uncoated catalysts. Scanning transmission electron microscopy showed no visible morphology changes after reaction at 675°C for 28 hours. The yield of ethylene was improved on all ALD Al(2)O(3) overcoated Pd catalysts.


Moderate alcohol intake and cancer incidence in women.
With the exception of breast cancer, little is known about the effect of moderate intakes of alcohol, or of particular types of alcohol, on cancer risk in women. A total of 1,280,296 middle-aged women in the United Kingdom enrolled in the Million Women Study were routinely followed for incident cancer. Cox regression models were used to calculate adjusted relative risks and 95% confidence intervals (CIs) for 21 site-specific cancers according to amount and type of alcoholic beverage consumed. All statistical tests were two-sided. A quarter of the cohort reported drinking no alcohol; 98% of drinkers consumed fewer than 21 drinks per week, with drinkers consuming an average of 10 g alcohol (1 drink) per day. During an average 7.2 years of follow-up per woman 68,775 invasive cancers occurred. Increasing alcohol consumption was associated with increased risks of cancers of the oral cavity and pharynx (increase per 10 g/d = 29%, 95% CI = 14% to 45%, Ptrend < .001), esophagus (22%, 95% CI = 8% to 38%, Ptrend = .002), larynx (44%, 95% CI = 10% to 88%, Ptrend = .008), rectum (10%, 95% CI = 2% to 18%, Ptrend = .02), liver (24%, 95% CI = 2% to 51%, Ptrend = .03), breast (12%, 95% CI = 9% to 14%, Ptrend < .001), and total cancer (6%, 95% CI = 4% to 7%, Ptrend < .001). The trends were similar in women who drank wine exclusively and other consumers of alcohol. For cancers of the upper aerodigestive tract, the alcohol-associated risk was confined to current smokers, with little or no effect of alcohol among never and past smokers (P(heterogeneity) < .001). Increasing levels of alcohol consumption were associated with a decreased risk of thyroid cancer (Ptrend = .005), non-Hodgkin lymphoma (Ptrend = .001), and renal cell carcinoma (Ptrend = .03). Low to moderate alcohol consumption in women increases the risk of certain cancers. For every additional drink regularly consumed per day, the increase in incidence up to age 75 years per 1000 for women in developed countries is estimated to be about 11 for breast cancer, 1 for cancers of the oral cavity and pharynx, 1 for cancer of the rectum, and 0.7 each for cancers of the esophagus, larynx and liver, giving a total excess of about 15 cancers per 1000 women up to age 75.


Abuse of recombinant human growth hormone: studies in two different dog models.
The search for inappropriately high growth hormone (GH) titers in plasma has been widely used to detect GH abuse, despite many shortcomings especially related to the pulsatile nature of GH secretion. Hence, the need for new anti-doping strategies. In the present study dogs were used to evaluate the ability of recombinant human GH (rhGH) to affect canine GH (cGH) release ensuing after somatostatin (SS) infusion withdrawal (SSIW) - a purported stimulus for the release of endogenous GH-releasing hormone (GHRH) - or the cGH response to administration of a GH-releasing peptide (GHRP). In the SSIW experiments, 8 beagle dogs of either gender (4-6 years old) were given a subcutaneous bolus injection of physiological saline (0.1 ml/kg) or, alternatively, rhGH (0.2 IU/kg s.c.) 60 min before the starting a continuous infusion of SS (4 microg/kg g h i.v.) of 1.5 h duration. In the dogs given a saline bolus, SSIW was followed by a 'rebound' rise in plasma cGH levels. In contrast, in dogs which had received the bolus injection of rhGH, the cGH rise elicited by SSIW was completely abrogated. In the set of experiments with a GHRP challenge, 13 dogs of either gender (3-12 years old) received the following treatment schedule at least 15 days apart: (1) a single bolus injection of rhGH (0.2 IU/kg s.c.); (2) rhGH (0.05 IU/kg s.c.) daily for 12 days; (3) rhGH (0.2 IU/kg s.c.) on alternate days for 12 days, and (4) rhGH (0.2 IU/kg s.c.) daily for 12 days. For each treatment schedule, before treatment, during treatment (24 h from the previous rhGH injection) and 1, 5 and 10 days after treatment, all dogs received an intravenous injection of a GHRP, EP51216 (125 microg/kg). In all treatments under baseline conditions, a single injection of EP51216 elicited an abrupt rise in plasma cGH. Twenty-four hours after the injection of an acute bolus of rhGH, the C(max) and AUC(0-90) of the GHRP-stimulated cGH response were significantly lower than the baseline cGH response. Five days later, there was a trend in the C(max) and AUC(0-90) towards complete restoration of the original values. One, 5 and 10 days after the end of the daily treatment with rhGH (0.05 IU/kg s.c.), no significant changes in the GHRP-stimulated cGH responses vs. the baseline GH response were recorded. In contrast, treatment with rhGH at a dose of 0.2 IU/kg s.c., on either alternate or daily administration, markedly reduced the GHRP-stimulated cGH responses evaluated after 3 and 5 rhGH injections. One day after the last rhGH injection, the EP51216-stimulated cGH response was still significantly reduced when compared with that present under baseline conditions. Five and 10 days following termination of rhGH treatment on alternate days, no significant differences in the C(max) and AUC(0-90) of the cGH responses to EP51216 were present. Differently, following the end of daily rhGH treatment, a marked inhibition in the C(max) of the cGH response to EP51216 was still present at 1 and 5 days, though not at 10 days. In conclusion, these studies show that a single administration of rhGH can abrogate the cGH response ensuing SSIW or acute stimulation by a GHRP. The inhibitory effect of rhGH on the cGH response to GHRP is present even 5 days after termination of a short-lived treatment with rhGH at a dose (0.2 IU/kg) which, in the dog, is undoubtedly lower than that used in humans for doping purposes. Extrapolation of these preclinical results to humans may pave the way for the development of a new rhGH anti-doping test.


Ceramide and activated Bax act synergistically to permeabilize the mitochondrial outer membrane.
A critical step in apoptosis is mitochondrial outer membrane permeabilization (MOMP), releasing proteins critical to downstream events. While the regulation of this process by Bcl-2 family proteins is known, the role of ceramide, which is known to be involved at the mitochondrial level, is not well-understood. Here, we demonstrate that Bax and ceramide induce MOMP synergistically. Experiments were performed on mitochondria isolated from both rat liver and yeast (lack mammalian apoptotic machinery) using both a protein release assay and real-time measurements of MOMP. The interaction between activated Bax and ceramide was also studied in a defined isolated system: planar phospholipid membranes. At concentrations where ceramide and activated Bax have little effects on their own, the combination induces substantial MOMP. Direct interaction between ceramide and activated Bax was demonstrated both by using yeast mitochondria and phospholipid membranes. The apparent affinity of activated Bax for ceramide increases with ceramide content indicating that activated Bax shows enhanced propensity to permeabilize in the presence of ceramide. An agent that inhibits ceramide-induced but not activated Bax induced permeabilization blocked the enhanced MOMP, suggesting that ceramide is the key permeabilizing entity, at least when ceramide is present. These and previous findings that anti-apoptotic proteins disassemble ceramide channels suggest that ceramide channels, regulated by Bcl-2-family proteins, may be responsible for the MOMP during apoptosis.


Warfarin-fluconazole. II. A metabolically based drug interaction: in vivo studies.
Consistent with expectations based on human in vitro microsomal experiments, administration of fluconazole (400 mg/day) for 6 days to six human volunteers significantly reduced the cytochrome P450 (P450)-dependent metabolic clearance of the warfarin enantiomers. In particular, P4502C9 catalyzed 6- and 7-hydroxylation of (S)-warfarin, the pathway primarily responsible for termination of warfarin's anticoagulant effect, was inhibited by approximately 70%. The change in (S)-warfarin pharmacokinetics caused by fluconazole dramatically increased the magnitude and duration of warfarin's hypoprothrombinemic effect. These observations indicate that co-administration of fluconazole and warfarin will result in a clinically significant metabolically based interaction The major P450-dependent, in vivo pathways of (R)-warfarin clearance were also strongly inhibited by fluconazole. 10-Hydroxylation, a metabolic pathway catalyzed exclusively by P4503A4, was inhibited by 45% whereas 6-, 7-, and 8-hydroxylations were inhibited by 61, 73, and 88%, respectively. The potent inhibition of the phenolic metabolites suggests that enzymes other than P4501A2 (weakly inhibited by fluconazole in vitro) are primarily responsible for the formation of these metabolites in vivo as predicted from in vitro kinetic studies. These data suggest that fluconazole can be expected to interact with any drug whose clearance is dominated by P450s 2C9, 3A4, and other as yet undefined isoforms. Overall, the results strongly support the hypothesis that metabolically based in vivo drug interactions may be predicted from human in vitro microsomal data.


Human histamine N-methyltransferase pharmacogenetics: cloning and expression of kidney cDNA.
Histamine N-methyltransferase (HNMT) catalyzes the NT-methylation of histamine. The level of HNMT activity in human red blood cells is controlled by a common genetic polymorphism. We set out to clone and express a cDNA for HNMT from human tissue as a first step toward a determination of the molecular basis for this genetic polymorphism. The cloning strategy was based on possible sequence homology between rat and human kidney HNMT. Human kidney cDNA libraries were screened with the 885-nucleotide open reading frame of rat kidney HNMT cDNA. A 1.4-kilobase cDNA clone was isolated that contained two potential translation initiation codons, both in the same reading frame. The longest open reading frame of the human kidney cDNA clone contained 876 nucleotides and encoded a protein 292 amino acids in length. The amino acid sequence of this protein was 84% identical to that of rat kidney HNMT. The human kidney cDNA clone was transcribed in vitro and translated in a rabbit reticulocyte lystate system to yield a protein with an apparent molecular mass of 33 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The human kidney cDNA was also subcloned into the eukaryotic expression vector p91023(B). Partially purified HNMT isolated from the cytosol of GOS-1 cells transfected with this expression construct had biochemical properties similar to those of human kidney HNMT. Human renal cortical HNMT, partially purified human renal cortical HNMT, and partially purified transfected COS-1 cell HNMT had Km values for histamine and S-adenosyl-L-methionine, the two cosubstrates for the enzyme reaction, of 20, 13, and 14 microM and 2.0, 3.0, and 6.2 microM, respectively. IC50 values for the HNMT inhibitor amodiaquine were 0.50, 0.48, and 0.40 microM, respectively, for enzyme from these same three sources. Northern blot analyses performed with poly(A)+ RNA from a series of human tissues including kidney demonstrated three transcripts, approximately 1.3, 3.8, and 4.0 kilobases in length. Cloning of a cDNA for HNMT may now make it possible to determine the molecular basis for the HNMT genetic polymorphism in humans.


Targeting macrophage activation for the prevention and treatment of Staphylococcus aureus biofilm infections.
Biofilm infections often lead to significant morbidity due to their chronicity and recalcitrance to antibiotics. We have demonstrated that methicillin-resistant Staphylococcus aureus (MRSA) biofilms can evade macrophage (MΦ) antibacterial effector mechanisms by skewing MΦs toward an alternatively activated M2 phenotype. To overcome this immune evasion, we have used two complementary approaches. In the first, a proinflammatory milieu was elicited by local administration of classically activated M1 MΦs and in the second by treatment with the C5a receptor (CD88) agonist EP67, which invokes MΦ proinflammatory activity. Early administration of M1-activated MΦs or EP67 significantly attenuated biofilm formation in a mouse model of MRSA catheter-associated infection. Several proinflammatory mediators were significantly elevated in biofilm-infected tissues from MΦ- and EP67-treated animals, revealing effective reprogramming of the biofilm environment to a proinflammatory milieu. A requirement for MΦ proinflammatory activity was demonstrated by the fact that transfer of MyD88-deficient MΦs had minimal impact on biofilm growth. Likewise, neutrophil administration had no effect on biofilm formation. Treatment of established biofilm infections with M1-activated MΦs also significantly reduced catheter-associated biofilm burdens compared with antibiotic treatment. Collectively, these results demonstrate that targeting MΦ proinflammatory activity can overcome the local immune inhibitory environment created during biofilm infections and represents a novel therapeutic strategy.


Fibroblasts can express glial fibrillary acidic protein (GFAP) in vivo.
Neuropathologists use anti-glial fibrillary acidic protein (GFAP) antibodies as specific markers for glial cells, and neurobiologists use GFAP for targeting transgenes to glial cells. Since GFAP has also been detected in non-glial cells, we systematically analyzed GFAP expression in human and murine non-CNS tissues using a panel of anti-GFAP antibodies. In human tissues we confirm previously observed GFAP expression in Schwann cells, myoepithelial cells, and chondrocytes, and show for the first time GFAP expression in fibroblasts of epiglottic and auricular perichondrium, ligamentum flavum, and cardiac valves. In mice we show GFAP expression in Schwann cells, bone marrow stromal cells, chondrocytes, and in fibroblasts of dura mater, skull and spinal perichondrium, and periosteum, connective stroma of oral cavity, dental pulp, and cardiac valves. Anti-GFAP immunoblotting of human non-CNS tissues reveals protein bands with a molecular mass ranging between approximately 35 and approximately 42 kDa. In GFAP-v-src transgenic mice, whose oncogenic v-src transgene transforms GFAP expressing cells, non-CNS tumors originate from fibroblasts. We conclude that human and murine fibroblasts can express GFAP in vivo. The somatic distribution of GFAP expressing fibroblasts indicates origin from the neural crest. Development of non-CNS tumors from fibroblasts in GFAP-v-src mice functionally confirms GFAP expression in these cells.


Living with treatment decisions: regrets and quality of life among men treated for metastatic prostate cancer.
To examine variation in men's long-term regret of treatment decisions, ie, surgical versus chemical castration, for metastatic prostate cancer and its associations with quality of life. Survey of previously treated patients to assess treatment decisions and quality of life, supplemented with focus groups. Two items addressing whether a patient wished he could change his mind and the belief that he would have been better off with the treatment not chosen were combined in classifying survey respondents as either satisfied or regretful. Chi(2) and t tests were used to test associations between regret and treatment history, complications, and quality of life. Survey respondents included 201 men aged 45 to 93 years (median, 71 years), who had begun treatment (71% chemical castration, 29% orchiectomy) a median of 2 years previously. Most reported complications: hot flashes (70%), nausea (34%), and erectile dysfunction (81%). Most were satisfied with the treatment decision, but 23% expressed regret. Regretful men more frequently reported surgical (43%) versus chemical (36%) castration (P: = .030) and nausea in the past week (54% v 32%; P: = .010) but less frequently reported erectile dysfunction (56% v 72%; P: = .048). Regretful men indicated poorer scores on every measure of generic and prostate cancer-related quality of life. Qualitative analyses revealed substantial uncertainty about the progress of their disease and the quality of the decisions in which patients participated. Regret was substantial and associated with treatment choice and quality of life. It may derive from underlying psychosocial distress and problematic communication with physicians when decisions are being reached and over subsequent years.


Effect of myricetin on 1,2 dimethylhydrazine induced rat colon carcinogenesis.
Colorectal cancer (CRC) is a leading cause of cancer mortality especially in the western world. The incidence of CRC in Asia is now increasing at an alarming rate from the previously negligible levels. The pathogenesis of CRC is a multistep process wherein pre cancer lesions accumulate in the mucosal cells finally resulting in cancer. Diet plays an important role in its aetio-pathogenesis--the high levels of dietary fat correlates to the increased incidence of CRC. This along with hereditary, environmental factors and singular lack of physical exercise provides a potent combination in its pathogenesis. Besides CRC isfrequently associated with persistent oxidative stress which results not only in DNA damage but also in mutation of cancer related genes besides the epigenetic silencing of the tumor suppressor gene. An important approach in the prevention of cancer is chemoprevention. Variety of plant products have been found to be highly effective in retarding the pathogenesis of the colorectal cancer. Myricetin is a well known bioflavonoid that is claimed to have anti cancer action particularly in colorectal cancer. Myricetin not only brought about significant decrease in the incidence of number of tumor bearing rats but also the tumor incidence. Myricetin supplementation significantly reduced liver TBARS. Further the anti oxidant enzymes like Catalase, Glutathione peroxidase and GSH were significantly rejuvenated following myricetin supplementation in a dose dependent manner. The fecal and colonic bacterial enzyme activity was also significantly decreased with the supplementation of myricetin 50 and 100 mg/kg. There was no additional benefit with the supplementation of 200 mg/kg of myricetin.


Abortion legislation, maternal healthcare, fertility, female literacy, sanitation, violence against women and maternal deaths: a natural experiment in 32 Mexican states.
To test whether there is an association between abortion legislation and maternal mortality outcomes after controlling for other factors thought to influence maternal health. Population-based natural experiment. Official maternal mortality data from 32 federal states of Mexico between 2002 and 2011. Maternal mortality ratio (MMR), MMR with any abortive outcome (MMRAO) and induced abortion mortality ratio (iAMR). Abortion legislation grouped as less (n=18) or more permissive (n=14); constitutional amendment protecting the unborn (n=17); skilled attendance at birth; all-abortion hospitalisation ratio; low birth weight rate; contraceptive use; total fertility rates (TFR); clean water; sanitation; female literacy rate and intimate-partner violence. Over the 10-year period, states with less permissive abortion legislation exhibited lower MMR (38.3 vs 49.6; p<0.001), MMRAO (2.7 vs 3.7; p<0.001) and iAMR (0.9 vs 1.7; p<0.001) than more permissive states. Multivariate regression models estimating effect sizes (β-coefficients) for mortality outcomes showed independent associations (p values between 0.001 and 0.055) with female literacy (β=-0.061 to -1.100), skilled attendance at birth (β=-0.032 to -0.427), low birth weight (β=0.149 to 2.166), all-abortion hospitalisation ratio (β=-0.566 to -0.962), clean water (β=-0.048 to -0.730), sanitation (β=-0.052 to -0.758) and intimate-partner violence (β=0.085 to 0.755). TFR showed an inverse association with MMR (β=-14.329) and MMRAO (β=-1.750) and a direct association with iAMR (β=1.383). Altogether, these factors accounted for (R(2)) 51-88% of the variance among states in overall mortality rates. No statistically independent effect was observed for abortion legislation, constitutional amendment or other covariates. Although less permissive states exhibited consistently lower maternal mortality rates, this finding was not explained by abortion legislation itself. Rather, these differences were explained by other independent factors, which appeared to have a more favourable distribution in these states.


Evaluating the impact of sequencing depth on transcriptome profiling in human adipose.
Recent advances in RNA sequencing (RNA-Seq) have enabled the discovery of novel transcriptomic variations that are not possible with traditional microarray-based methods. Tissue and cell specific transcriptome changes during pathophysiological stress in disease cases versus controls and in response to therapies are of particular interest to investigators studying cardiometabolic diseases. Thus, knowledge on the relationships between sequencing depth and detection of transcriptomic variation is needed for designing RNA-Seq experiments and for interpreting results of analyses. Using deeply sequenced Illumina HiSeq 2000 101 bp paired-end RNA-Seq data derived from adipose of a healthy individual before and after systemic administration of endotoxin (LPS), we investigated the sequencing depths needed for studies of gene expression and alternative splicing (AS). In order to detect expressed genes and AS events, we found that ∼100 to 150 million (M) filtered reads were needed. However, the requirement on sequencing depth for the detection of LPS modulated differential expression (DE) and differential alternative splicing (DAS) was much higher. To detect 80% of events, ∼300 M filtered reads were needed for DE analysis whereas at least 400 M filtered reads were necessary for detecting DAS. Although the majority of expressed genes and AS events can be detected with modest sequencing depths (∼100 M filtered reads), the estimated gene expression levels and exon/intron inclusion levels were less accurate. We report the first study that evaluates the relationship between RNA-Seq depth and the ability to detect DE and DAS in human adipose. Our results suggest that a much higher sequencing depth is needed to reliably identify DAS events than for DE genes.


The time and place of European admixture in Ashkenazi Jewish history.
The Ashkenazi Jewish (AJ) population is important in genetics due to its high rate of Mendelian disorders. AJ appeared in Europe in the 10th century, and their ancestry is thought to comprise European (EU) and Middle-Eastern (ME) components. However, both the time and place of admixture are subject to debate. Here, we attempt to characterize the AJ admixture history using a careful application of new and existing methods on a large AJ sample. Our main approach was based on local ancestry inference, in which we first classified each AJ genomic segment as EU or ME, and then compared allele frequencies along the EU segments to those of different EU populations. The contribution of each EU source was also estimated using GLOBETROTTER and haplotype sharing. The time of admixture was inferred based on multiple statistics, including ME segment lengths, the total EU ancestry per chromosome, and the correlation of ancestries along the chromosome. The major source of EU ancestry in AJ was found to be Southern Europe (≈60-80% of EU ancestry), with the rest being likely Eastern European. The inferred admixture time was ≈30 generations ago, but multiple lines of evidence suggest that it represents an average over two or more events, pre- and post-dating the founder event experienced by AJ in late medieval times. The time of the pre-bottleneck admixture event, which was likely Southern European, was estimated to ≈25-50 generations ago.


Speciation of arsenic in a thermoacidophilic iron-oxidizing archaeon, Acidianus brierleyi, and its culture medium by inductively coupled plasma-optical emission spectroscopy combined with flow injection pretreatment using an anion-exchange mini-column.
The thermoacidophilic iron-oxidizing archaeon Acidianus brierleyi is a microorganism that could be useful in the removal of inorganic As from wastewater, because it simultaneously oxidizes As(III) and Fe(II) to As(V) and Fe(III) in an acidic culture medium, resulting in the immobilization of As(V) as FeAsO₄. To investigate the oxidation mechanism, speciation of the As species in both the cells and its culture media is an important issue. Here we describe the successive determination of As(III), As(V), and total As in A. brierleyi and its culture medium via a facile method based on inductively coupled plasma-optical emission spectroscopy (ICP-OES) with a flow injection pretreatment system using a mini-column packed with an anion-exchange resin. The flow-injection pretreatment system consisted of a syringe pump, a selection valve, and a switching valve, which were controlled by a personal computer. Sample solutions with the pH adjusted to 5 were flowed into the mini-column to retain the anionic As(V), whereas As(III) was introduced into ICP-OES with no adsorption on the mini-column due to its electrically neutral form. An acidic solution (1 M HNO₃) was then flowed into the mini-column to elute As(V) followed by ICP-OES measurement. The same sample was also subjected to ICP-OES without being passed through the mini-column in order to determine the total amounts of As(III) and As(V). The method was verified by comparing the results of the total As with the sum of As(III) and As(V). The calibration curves showed good linearity with limits of detection of 158, 86, and 211 ppb for As(III), As(V), and total As, respectively. The method was successfully applicable to the determination of the As species contained in the pellets of A. brierleyi and their culture media. The results suggested that the oxidation of As(III) was influenced by the presence of Fe(II) in the culture medium, i.e., Fe(II) enhanced the oxidation of As(III) in A. brierleyi. In addition, we found that no soluble As species was contained in the cell pellets and more than 60% of the As(III) in the culture medium was oxidized by A. brierleyi after a 6-day incubation.


Merkel cell carcinoma and multiple primary cancers.
Merkel cell carcinoma (MCC) is an aggressive neuroendocrine tumor of the skin for which causative factors remain largely unknown. The site-specific risks of multiple primary cancers associated with MCC, which may provide insight into etiologic influences, have not been quantified in large population-based studies. We estimated the long-term risk of subsequent primary tumors after a first primary MCC (1,306 patients) and the risk of second primary MCC following other first primary cancers (2,048,739 patients) within 11 population-based cancer registries which report to the National Cancer Institute's Surveillance, Epidemiology, and End Results Program (1986-2002). Patients with first primary MCC were at significantly increased risk of developing a subsequent cancer [standardized incidence ratio (SIR), 1.22; 95% confidence intervals (95% CI), 1.01-1.45; observed (O = 122)], with significant excesses restricted to the first year after diagnosis (SIR, 1.71; 95% CI, 1.21-2.33; O = 39). Significantly elevated site-specific risks were observed for cancers of salivary gland (SIR, 11.55; 95% CI, 2.32-33.76; O = 3), biliary sites other than liver and gallbladder (SIR, 7.24; 95% CI, 1.46-21.16; O = 3), and non-Hodgkin lymphoma (SIR, 2.56; 95% CI, 1.23-4.71; O = 10). Nonsignificantly increased risks of 2-fold or higher were seen for chronic lymphocytic leukemia, and cancers of the small intestine and brain. A significantly increased 1.36-fold risk (95% CI, 1.19-1.55; O = 221) of MCC as a second primary malignancy was observed among patients with all other first primary cancers taken together. In particular, significant 3- to 7-fold excesses of MCC followed multiple myeloma (SIR, 3.70; 95% CI, 1.01-9.47; O = 4), chronic lymphocytic leukemia (SIR, 6.89; 95% CI, 3.77-11.57; O = 14), non-Hodgkin lymphoma (SIR, 3.37; 95% CI, 1.93-5.47; O = 16), and malignant melanoma (SIR, 3.05; 95% CI, 1.74-4.95; O = 16). Although enhanced medical surveillance may play a role, increased reciprocal risks suggest that MCC may share etiologic influences with other malignancies. Heightened awareness of the associations of lymphohematopoietic malignancies with MCC may facilitate early clinical recognition.


The extracellular matrix: Structure, composition, age-related differences, tools for analysis and applications for tissue engineering.
The extracellular matrix is a structural support network made up of diverse proteins, sugars and other components. It influences a wide number of cellular processes including migration, wound healing and differentiation, all of which is of particular interest to researchers in the field of tissue engineering. Understanding the composition and structure of the extracellular matrix will aid in exploring the ways the extracellular matrix can be utilised in tissue engineering applications especially as a scaffold. This review summarises the current knowledge of the composition, structure and functions of the extracellular matrix and introduces the effect of ageing on extracellular matrix remodelling and its contribution to cellular functions. Additionally, the current analytical technologies to study the extracellular matrix and extracellular matrix-related cellular processes are also reviewed.


A genome-wide role for CHD remodelling factors and Nap1 in nucleosome disassembly.
Chromatin remodelling factors and histone chaperones were previously shown to cooperatively affect nucleosome assembly and disassembly processes in vitro. Here, we show that Schizosaccharomyces pombe CHD remodellers, the Hrp1 and Hrp3 paralogs physically interact with the histone chaperone Nap1. Genome-wide analysis of Hrp1, Hrp3 and Nap1 occupancy, combined with nucleosome density measurements revealed that the CHD factors and Nap1 colocalized in particular to promoter regions where they remove nucleosomes near the transcriptional start site. Hrp1 and Hrp3 also regulate nucleosome density in coding regions, where they have redundant roles to stimulate transcription. Previously, DNA replication-dependent and -independent nucleosome disassembly processes have been described. We found that nucleosome density increased in the hrp1 mutant in the absence of DNA replication. Finally, regions where nucleosome density increased in hrp1, hrp3 and nap1 mutants also showed nucleosome density and histone modification changes in HDAC and HAT mutants. Thus, this study revealed an important in vivo role for CHD remodellers and Nap1 in nucleosome disassembly at promoters and coding regions, which are linked to changes in histone acetylation.


Non-invasive detection of coronary inflammation using computed tomography and prediction of residual cardiovascular risk (the CRISP CT study): a post-hoc analysis of prospective outcome data.
Coronary artery inflammation inhibits adipogenesis in adjacent perivascular fat. A novel imaging biomarker-the perivascular fat attenuation index (FAI)-captures coronary inflammation by mapping spatial changes of perivascular fat attenuation on coronary computed tomography angiography (CTA). However, the ability of the perivascular FAI to predict clinical outcomes is unknown. In the Cardiovascular RISk Prediction using Computed Tomography (CRISP-CT) study, we did a post-hoc analysis of outcome data gathered prospectively from two independent cohorts of consecutive patients undergoing coronary CTA in Erlangen, Germany (derivation cohort) and Cleveland, OH, USA (validation cohort). Perivascular fat attenuation mapping was done around the three major coronary arteries-the proximal right coronary artery, the left anterior descending artery, and the left circumflex artery. We assessed the prognostic value of perivascular fat attenuation mapping for all-cause and cardiac mortality in Cox regression models, adjusted for age, sex, cardiovascular risk factors, tube voltage, modified Duke coronary artery disease index, and number of coronary CTA-derived high-risk plaque features. Between 2005 and 2009, 1872 participants in the derivation cohort underwent coronary CTA (median age 62 years [range 17-89]). Between 2008 and 2016, 2040 patients in the validation cohort had coronary CTA (median age 53 years [range 19-87]). Median follow-up was 72 months (range 51-109) in the derivation cohort and 54 months (range 4-105) in the validation cohort. In both cohorts, high perivascular FAI values around the proximal right coronary artery and left anterior descending artery (but not around the left circumflex artery) were predictive of all-cause and cardiac mortality and correlated strongly with each other. Therefore, the perivascular FAI measured around the right coronary artery was used as a representative biomarker of global coronary inflammation (for prediction of cardiac mortality, hazard ratio [HR] 2·15, 95% CI 1·33-3·48; p=0·0017 in the derivation cohort, and 2·06, 1·50-2·83; p<0·0001 in the validation cohort). The optimum cutoff for the perivascular FAI, above which there is a steep increase in cardiac mortality, was ascertained as -70·1 Hounsfield units (HU) or higher in the derivation cohort (HR 9·04, 95% CI 3·35-24·40; p<0·0001 for cardiac mortality; 2·55, 1·65-3·92; p<0·0001 for all-cause mortality). This cutoff was confirmed in the validation cohort (HR 5·62, 95% CI 2·90-10·88; p<0·0001 for cardiac mortality; 3·69, 2·26-6·02; p<0·0001 for all-cause mortality). Perivascular FAI improved risk discrimination in both cohorts, leading to significant reclassification for all-cause and cardiac mortality. The perivascular FAI enhances cardiac risk prediction and restratification over and above current state-of-the-art assessment in coronary CTA by providing a quantitative measure of coronary inflammation. High perivascular FAI values (cutoff ≥-70·1 HU) are an indicator of increased cardiac mortality and, therefore, could guide early targeted primary prevention and intensive secondary prevention in patients. British Heart Foundation, and the National Institute of Health Research Oxford Biomedical Research Centre.


High ambient glucose augments angiotensin II-induced proinflammatory gene mRNA expression in human mesangial cells: effects of valsartan and simvastatin.
Hyperglycemia may potentiate the adverse renal effects of angiotensin II (AII). In the kidney, the major target of AII action is the glomerular mesangial cell, where its hemodynamic and proinflammatory action contributes to renal injury. AII action is mediated by several types of cell receptors. Among those, the AT1 receptor has been best studied using specific AII receptor blockers (ARBs). These agents have emerged as major new modalities in the prevention and amelioration of renal disease where the ARB renoprotective anti-inflammatory properties could be more important than previously appreciated. Like the ARBs, statins may also modulate inflammatory responses that are renoprotective and complement their cholesterol-lowering effects. The aim of this project was to (i) identify a repertoire of proinflammatory mesangial cell AII-inducible mRNAs; (ii) determine if the AII-induced proinflammatory mRNA responses depend on ambient glucose, and (iii) test the anti-inflammatory effectiveness of an ARB, valsartan, either alone or in combination with a statin, simvastatin. Using high-density microarrays and real-time PCR we identified several AII-inducible proinflammatory mesangial genes that exhibited augmented mRNA responses in high-glucose milieu. Valsartan blocked the AII-induced mRNA expression of proinflammatory genes (i.e. MCP-1, LIF and COX-2) maintained in normal and high glucose. These observations add to the mounting evidence that ARBs have anti-inflammatory effects in the kidney, a beneficial effect that may be more important in protecting renal function in diabetic patients. While simvastatin inhibited expression of some mRNAs encoding chemokines/cytokines, it enhanced expression of mRNA encoding COX-2, a key mediator of inflammation. Thus, the non-cholesterol effects of statins on inflammatory responses appear complex.


Improving the practice of child death overview panels: a paediatric perspective.
In England, every death in childhood is reviewed by a local multidisciplinary Child Death Overview Panel (CDOP) with the intention of understanding causation and implementing interventions to reduce future deaths. This study aimed to establish how well panels work from the perspective of the paediatricians involved and to ascertain whether they deliver good value and identify areas for improvement. A questionnaire was sent to every CDOP paediatrician in the country (n=93). Questions focused on the quality of CDOP case discussions as well as examples of effective and significant recommendations. Responses were analysed using simple quantitative and qualitative methods. 84/93 (90%) of the paediatricians responded. Among the respondents, 60 (71%) believe that investment in CDOPs is offering good value, 73 (87%) feel that case discussions are rigorous and consistent and over 90% believe that the correct issues are emerging from discussions. However, responders noted many areas for improvement: 40 (48%) suggested devolving the discussion of specialist deaths (eg, neonates) to hospital-based review meetings or holding themed meetings with invited specialists, 11 (13%) suggested filtering out cases where learning is unlikely before full CDOP meetings and 13 (15%) called for national integration and analysis of data. In this time of economic austerity it is vital that the CDOPs add value to the invested resources. Although CDOP paediatricians feel that panels are working well, there is scope for improvement through enhancing relationships with commissioning bodies, aggregate review and analysis of CDOP data at a national level and consideration of specialist and/or network review of certain categories of deaths such as cardiac surgery, oncology and neonates.


Helicobacter pylori infection in family members of patients with gastroduodenal symptoms. A cross-sectional analytical study.
Primary Helicobacter pylori (H. pylori) infection is acquired predominantly in childhood in the family setting. We aimed to investigate the presence of intrafamilial concurrent H. pylori infection. Cross-sectional analytical study with a control group, conducted in a tertiary care hospital. Fifty adult patients with gastroduodenal symptoms who underwent gastroscopy (index parents), their spouses and their children were enrolled in the study. Blood samples were collected from all of the study subjects to test for immunoglobulin G (IgG) antibody response. H. pylori antigen was investigated in the stool specimens of children only. The participants were divided into two groups: Group 1 consisted of the 40 patients in whom H. pylori infection was demonstrated via endoscopy, their spouses and their children. Group 2 included the remaining 10 patients who underwent endoscopy revealing negative results for H. pylori, their spouses and their children. IgG antibodies were present in all of the index parents, 95% of their spouses and 93% of their children in group 1; 13 of the children (9%) were also positive for H. pylori stool antigen (HpSA). However, IgG antibodies were present in only 2 of the 10 index parents in group 2. One of their spouses and one of their children had a positive antibody response. All of their children had negative stool antigen test results. H. pylori infections exhibit intrafamilial clustering. Parental infection, age ≥ years and having three or more siblings are the major risk factors for H. pylori infection in children.


Unraveling the relative role of light and water competition between lianas and trees in tropical forests: A vegetation model analysis.
Despite their low contribution to forest carbon stocks, lianas (woody vines) play an important role in the carbon dynamics of tropical forests. As structural parasites, they hinder tree survival, growth and fecundity; hence, they negatively impact net ecosystem productivity and long-term carbon sequestration.Competition (for water and light) drives various forest processes and depends on the local abundance of resources over time. However, evaluating the relative role of resource availability on the interactions between lianas and trees from empirical observations is particularly challenging. Previous approaches have used labour-intensive and ecosystem-scale manipulation experiments, which are infeasible in most situations.We propose to circumvent this challenge by evaluating the uncertainty of water and light capture processes of a process-based vegetation model (ED2) including the liana growth form. We further developed the liana plant functional type in ED2 to mechanistically simulate water uptake and transport from roots to leaves, and start the model from prescribed initial conditions. We then used the PEcAn bioinformatics platform to constrain liana parameters and run uncertainty analyses.Baseline runs successfully reproduced ecosystem gas exchange fluxes (gross primary productivity and latent heat) and forest structural features (leaf area index, aboveground biomass) in two sites (Barro Colorado Island, Panama and Paracou, French Guiana) characterized by different rainfall regimes and levels of liana abundance.Model uncertainty analyses revealed that water limitation was the factor driving the competition between trees and lianas at the drier site (BCI), and during the relatively short dry season of the wetter site (Paracou). In young patches, light competition dominated in Paracou but alternated with water competition between the wet and the dry season on BCI according to the model simulations.The modelling workflow also identified key liana traits (photosynthetic quantum efficiency, stomatal regulation parameters, allometric relationships) and processes (water use, respiration, climbing) driving the model uncertainty. They should be considered as priorities for future data acquisition and model development to improve predictions of the carbon dynamics of liana-infested forests. Synthesis. Competition for water plays a larger role in the interaction between lianas and trees than previously hypothesized, as demonstrated by simulations from a process-based vegetation model.


Protection of rabbits against challenge with rabbit papillomaviruses by immunization with the N terminus of human papillomavirus type 16 minor capsid antigen L2.
Current L1 virus-like particle (VLP) vaccines provide type-restricted protection against a small subset of the human papillomavirus (HPV) genotypes associated with cervical cancer, necessitating continued cytologic screening of vaccinees. Cervical cancer is most problematic in countries that lack the resources for screening or highly multivalent HPV VLP vaccines, suggesting the need for a low-cost, broadly protective vaccinogen. Here, N-terminal L2 polypeptides comprising residues 1 to 88 or 11 to 200 derived from HPV16, bovine papillomavirus type 1 (BPV1), or cottontail rabbit papillomavirus (CRPV) were produced in bacteria. Rabbits were immunized with these N-terminal L2 polypeptides and concurrently challenged with CRPV and rabbit oral papillomavirus (ROPV). Vaccination with either N-terminal L2 polypeptides of CRPV effectively protected rabbits from CRPV challenge but not from papillomas induced by cutaneous challenge with CRPV genomic DNA. Furthermore, papillomas induced by CRPV genomic DNA deficient for L2 expression grew at the same rate as those induced by wild-type CRPV genomic DNA, further suggesting that the L2 polypeptide vaccines lack therapeutic activity. Neutralizing serum antibody titers of >15 correlated with protection (P < 0.001), a finding consistent with neutralizing antibody-mediated protection. Surprisingly, a remarkable degree of protection against heterologous papillomavirus types was observed after vaccination with N-terminal L2 polypeptides. Notably, vaccination with HPV16 L2 11-200 protected against cutaneous and mucosal challenge with CRPV and ROPV, respectively, papillomaviruses that are evolutionarily divergent from HPV16. Further, vaccination with HPV16 L2 11-200 generates broadly cross-neutralizing serum antibody, suggesting the potential of L2 as a second-generation preventive HPV vaccine antigen.


What do we really assess with organisational culture tools in healthcare? An interpretive systematic umbrella review of tools in healthcare.
A toxic organisational culture (OC) is a major contributing factor to serious failings in healthcare delivery. Poor OC with its consequences of unprofessional behaviour, unsafe attitudes of professionals and its impact on patient care still need to be addressed. Although various tools have been developed to determine OC and improve patient safety, it remains a challenge to decide on the suitability of tools for uncovering the underlying factors which truly impact OC, such as behavioural norms, or the unwritten rules. A better understanding of the underlying dimensions that these tools do and do not unravel is required. The aim of this study is to provide an overview of existing tools to assess OC and the tangible and intangible OC dimensions these tools address. An interpretive umbrella review was conducted. Literature reviews were considered for inclusion if they described multiple tools and their dimensional characteristics in the context of OC, organisational climate, patient safety culture or climate. OC tools and the underlying dimensions were extracted from the reviews. A qualitative data analysis software program (MAX.QDA 2007) was used for coding the dimensions, which resulted in tangible and intangible themes. Fifteen reviews met our inclusion criteria. A total of 127 tools were identified, which were mainly quantitative questionnaires covering tangible key dimensions. Qualitative analyses distinguished nine intangible themes (commitment, trust, psychological safety, power, support, communication openness, blame and shame, morals and valuing ethics, and cohesion) and seven tangible themes (leadership, communication system, teamwork, training and development, organisational structures and processes, employee and job attributes, and patient orientation). This umbrella review identifies the essential tangible and intangible themes of OC tools. OC tools in healthcare do not seem to be designed to determine deeper underlying dimensions of culture. We suggest approaching complex underlying OC problems by focusing on the intangible dimensions, rather than putting the tangible dimensions up front.


Lentiviral vectors as a gene delivery system in the mouse midbrain: cellular and behavioral improvements in a 6-OHDA model of Parkinson's disease using GDNF.
Local delivery of therapeutic molecules represents one of the limiting factors for the treatment of neurodegenerative disorders. In vivo gene transfer using viral vectors constitutes a powerful strategy to overcome this limitation. The aim of the present study was to validate the lentiviral vector as a gene delivery system in the mouse midbrain in the perspective of screening biotherapeutic molecules in mouse models of Parkinson's disease. A preliminary study with a LacZ-encoding vector injected above the substantia nigra of C57BL/6j mice indicated that lentiviral vectors can infect approximately 40,000 cells and diffuse over long distances. Based on these results, glial cell line-derived neurotrophic factor (GDNF) was assessed as a neuroprotective molecule in a 6-hydroxydopamine model of Parkinson's disease. Lentiviral vectors carrying the cDNA for GDNF or mutated GDNF were unilaterally injected above the substantia nigra of C57BL/6j mice. Two weeks later, the animals were lesioned ipsilaterally with 6-hydroxydopamine into the striatum. Apomorphine-induced rotation was significantly decreased in the GDNF-injected group compared to control animals. Moreover, GDNF efficiently protected 69.5% of the tyrosine hydroxylase-positive cells in the substantia nigra against 6-hydroxydopamine-induced toxicity compared to 33.1% with control mutated GDNF. These data indicate that lentiviral vectors constitute a powerful gene delivery system for the screening of therapeutic molecules in mouse models of Parkinson's disease.


Initiating NTD programs targeting schistosomiasis and soil-transmitted helminthiasis in two provinces of the Democratic Republic of the Congo: Establishment of baseline prevalence for mass drug administration.
Schistosomiasis (SCH) and soil-transmitted helminthiasis (STH) are widely distributed in the Democratic Republic of the Congo (DRC) and constitute a serious public health problem. As recommended by the World Health Organization (WHO), before launching mass chemotherapy to control these diseases, parasitological surveys were conducted in sentinel sites in six health zones (HZs) in Bandundu and Maniema provinces. Baseline prevalence and intensity of infection for SCH and STH were determined to establish the appropriate treatment plan using Praziquantel (PZQ) and Albendazole (ALB). Parasitological surveys were conducted from April to May 2015 in twenty-six selected sampling units (schools) for baseline mapping in six HZs: Fifty school children (25 females and 25 males) aged 9-15 years were randomly selected per sampling unit. A total of 1300 samples (urine and stool) were examined using haematuria dipsticks, parasite-egg filtration and the point-of-care Circulating Cathodic Antigen (POC-CCA) assay for urine samples and the Kato-Katz technique for stool specimens. Three species of schistosomes (S. mansoni, S. haematobium and S. intercalatum) and three groups of STH (hookworm, Ascaris and Trichuris) were detected at variable prevalence and intensity among the schools, the HZs and the provinces. In Bandundu, no SCH was detected by either Kato-Katz or the POC-CCA technique, despite a high prevalence of STH with 68% and 80% at Kiri and Pendjua HZs, respectively. In Maniema, intestinal schistosomiasis was detected by both Kato-Katz and POC-CCA with an average prevalence by Kato-Katz of 32.8% and by POC-CCA of 42.1%. Comparative studies confirmed the greater sensitivity (and operational feasibility) of the POC-CCA test on urine compared to Kato-Katz examination of stool for diagnosing intestinal schistosomiasis even in areas of comparatively light infections. STH was widely distributed and present in all HZs with a mean prevalence (95% CI) of 59.62% (46.00-65.00%). The prevalence of hookworm, roundworm and whipworm were 51.62% (32.40%-71.50%), 15.77% (0.50%-39.60%) and 13.46 (0.50%-33.20%), respectively. This study provided the evidence base for implementing programs targeting SCH and STH in these Health Zones. Observations also reinforce the operational value and feasibility of the POC-CCA test to detect S. mansoni and, for the first time, S. intercalatum infections in a routine NTD program setting.


Why won't they come? Stakeholder perspectives on collaborative national forest planning by participation level.
Collaboration has taken root in national forest planning, providing expanded opportunities for stakeholder participation in decision-making, but are these processes considered meaningful by key stakeholders? Do the processes result in increased participation by key stakeholders? We present results of a study of stakeholder perspectives of a collaborative planning process on the Grand Mesa, Uncompahgre, and Gunnison National Forests in Western Colorado, U.S.A. The stakeholders were stratified by participation levels in order to explore a possible relationship between participation and perceptions of the collaborative process. We used a Q-methodology approach to compare and contrast perspectives across participant levels in the North Fork Valley Landscape Working Group process. The results demonstrate four distinct perspectives on the collaborative process: 1) The collaborative process is valued by the Forest Service and will directly influence planning decisions; 2) The Forest Service, the collaborative process, and other stakeholders are not to be trusted; 3) The collaborative process is most effective when emphasizing place-specific dialogue that primarily involves stakeholders educating the Forest Service about issues; and 4) Forest planning involves issues requiring the application of scientific knowledge and expertise rather than collaboration. These perspectives were not strongly associated with participation levels, with time constraint being the primary mediating factor affecting participation. There are several possible actions policymakers and planners can take to enhance participation and overcome high rates of nonparticipation.


Psychotic symptoms, functioning and coping in adolescents with mental illness.
Psychotic symptoms in the context of psychiatric disorders are associated with poor functional outcomes. Environmental stressors are important in the development of psychosis; however, distress may only be pathogenic when it exceeds an individual's ability to cope with it. Therefore, one interesting factor regarding poor functional outcomes in patients with psychotic symptoms may be poor coping. This paper aimed to address the question whether 1) psychotic symptoms are associated with poorer functioning and 2) whether poor coping moderated the association. In a clinical case-clinical control study of 106 newly-referred adolescent patients with non-psychotic psychiatric disorders, coping was investigated using the Adolescents Coping Scale. Severity of impairment in socio-occupational functioning was assessed with the Children's Global Assessment Scale. Patients with non-psychotic psychiatric disorders and additional psychotic symptoms (N = 50) had poorer functioning and were more likely to use avoidance-oriented coping compared to patients with non-psychotic psychiatric disorders without psychotic symptoms (N = 56). No differences were found with respect to approach-oriented coping. When stratifying for poor/good coping, only those adolescent patients with psychotic symptoms who applied poor coping (i.e. less use of approach-oriented coping styles [OR 0.24, p < 0.015] and more use of avoidance-oriented coping [OR 0.23, p < 0.034]) had poorer functioning. However, these interactions were not significant. Non-adaptive coping and poorer functioning were more often present in adolescents with non-psychotic psychiatric disorders and additional psychotic symptoms. Due to small subgroups, our analyses could not give definitive conclusions about the question whether coping moderated the association between psychotic symptoms and functioning. Improvement of coping skills may form an important target for intervention that may contribute to better clinical and functional outcomes in patients with psychotic symptoms.


CT diagnosis of circumcaval ureter.
Variability in the normal course of the ureter makes it difficult to differentiate a normal variant from a congenital anomaly or deviation caused by a mass. CT can be useful in making this distinction. We present five cases of circumcaval ureter in which contrast-enhanced CT, by showing the ureter passing posterior and medial to the inferior vena cava (IVC), provided the definitive diagnosis. In addition, we studied the position of the inferior vena cava in the midlumbar region in 100 consecutive contrast-enhanced CT scans and compared the results with the position of the IVC in our five cases of circumcaval ureter plus one case of circumcaval ureter taken from the literature. Whereas only 6% of the normal patients had an IVC lateral to the right pedicle of the third lumbar vertebra, all of the patients with a circumcaval ureter and the one case in the literature had the IVC lateral to the pedicle. Therefore, the finding of a laterally placed IVC on CT is suggestive but not pathognomonic of circumcaval ureter. Contrast-enhanced CT can be used to prove the diagnosis of circumcaval ureter, and lateral placement of the IVC on non-contrast-enhanced CT suggests the diagnosis.


NRSF-dependent epigenetic mechanisms contribute to programming of stress-sensitive neurons by neonatal experience, promoting resilience.
Resilience to stress-related emotional disorders is governed in part by early-life experiences. Here we demonstrate experience-dependent re-programming of stress-sensitive hypothalamic neurons, which takes place through modification of neuronal gene expression via epigenetic mechanisms. Specifically, we found that augmented maternal care reduced glutamatergic synapses onto stress-sensitive hypothalamic neurons and repressed expression of the stress-responsive gene, Crh. In hypothalamus in vitro, reduced glutamatergic neurotransmission recapitulated the repressive effects of augmented maternal care on Crh, and this required recruitment of the transcriptional repressor repressor element-1 silencing transcription factor/neuron restrictive silencing factor (NRSF). Increased NRSF binding to chromatin was accompanied by sequential repressive epigenetic changes which outlasted NRSF binding. chromatin immunoprecipitation-seq analyses of NRSF targets identified gene networks that, in addition to Crh, likely contributed to the augmented care-induced phenotype, including diminished depression-like and anxiety-like behaviors. Together, we believe these findings provide the first causal link between enriched neonatal experience, synaptic refinement and induction of epigenetic processes within specific neurons. They uncover a novel mechanistic pathway from neonatal environment to emotional resilience.


Rejection-associated Mitochondrial Impairment After Heart Transplantation.
Mitochondrial dysfunction is associated with poor allograft prognosis. Mitochondrial-related gene expression (GE) in endomyocardial biopsies (EMBs) could be useful as a nonimmune functional marker of rejection. We hypothesize that acute cardiac allograft rejection is associated with decreased mitochondrial-related GE in EMBs. We collected 64 routines or clinically indicated EMB from 47 patients after heart transplant. The EMBs were subjected to mRNA sequencing. We conducted weighted gene coexpression network analysis to construct module-derived eigengenes. The modules were assessed by gene ontology enrichment and hub gene analysis. Modules were correlated with the EMBs following the International Society of Heart and Lung Transplantation histology-based criteria and a classification based on GE alone; we also correlated with clinical parameters. The modules enriched with mitochondria-related and immune-response genes showed the strongest correlation to the clinical traits. Compared with the no-rejection samples, rejection samples had a decreased activity of mitochondrial-related genes and an increased activity of immune-response genes. Biologic processes and hub genes in the mitochondria-related modules were primarily involved with energy generation, substrate metabolism, and regulation of oxidative stress. Compared with International Society of Heart and Lung Transplantation criteria, GE-based classification had stronger correlation to the weighted gene coexpression network analysis-derived functional modules. The brain natriuretic peptide level, ImmuKnow, and Allomap scores had negative relationships with the expression of mitochondria-related modules and positive relationships with immune-response modules. During acute cardiac allograft rejection, there was a decreased activity of mitochondrial-related genes, related to an increased activity of immune-response genes, and depressed allograft function manifested by brain natriuretic peptide elevation. This suggests a rejection-associated mitochondrial impairment.


The peripheral cannabinoid receptor Cb2, frequently expressed on AML blasts, either induces a neutrophilic differentiation block or confers abnormal migration properties in a ligand-dependent manner.
Cb2, the gene encoding the peripheral cannabinoid receptor, is located in a common virus integration site and is overex-pressed in retrovirally induced murine myeloid leukemias. Here we show that this G protein-coupled receptor (GPCR) is also aberrantly expressed in a high percentage of human acute myeloid leukemias. We investigated the mechanism of transformation by Cb2 and demonstrate that aberrant expression of this receptor on hematopoietic precursor cells results in distinct effects depending on the ligand used. Cb2-expressing myeloid precursors migrate upon stimulation by the endocannabinoid 2-arachidonoylglycerol and are blocked in neutrophilic differentiation upon exposure to another ligand, CP55940. Both effects depend on the activation of G(alphai) proteins and require the mitogen-induced extracellular kinase/extracellular signal-regulated kinase (MEK/ERK) pathway. Down-regulation of cyclic adenosine monophosphate (cAMP) levels upon G(alphai) activation is important for migration induction but is irrelevant for the maturation arrest. Moreover, the highly conserved G protein-interacting DRY motif, present in the second intracellular loop of GPCRs, is critical for migration but unimportant for the differentiation block. This suggests that the Cb2-mediated differentiation block requires interaction of G(alphai) proteins with other currently unknown motifs. This indicates a unique mechanism by which a transforming GPCR, in a ligand-dependent manner, causes 2 distinct oncogenic effects: altered migration and block of neutrophilic development.


The effect of footwear on preoperative gait mechanics in a group of total ankle replacement patients.
Debate exists within the current clinical literature as to whether patients should be tested barefoot or in shoes either before or following surgical interventions. The objective of this study was to determine the effect of shod versus barefoot walking on spatiotemporal gait parameters before total ankle arthroplasty (TAA). Twenty patients who were scheduled for TAA were asked to walk four trials in shoes and four barefoot trials over a GaitRite mat while spatiotemporal variables were recorded. A series of repeated measures analysis of variance were performed to determine significant differences (alpha = 0.05). Step length, stride length, double support time, and walking velocity were significantly increased in the shod condition. However, step width, swing time, and support time were significantly decreased in the shod condition. The results of this study indicate the importance of testing clinical populations barefoot in order to more accurately assess gait pathology.


A monoclonal antibody to a human neutrophil-specific plasma membrane antigen. Effect of the antibody on the C3bi-mediated adherence by neutrophils and expression of the antigen during myelopoiesis.
We have used mice selectively tolerized to antigens of human lymphocytes by treatment with cyclophosphamide to raise an mAb, BH2-C6, that reacts with a plasma membrane antigen specific for human neutrophils. This specificity is demonstrated by indirect immunofluorescence microscopy, cytochemical analysis of fluorescence-positive and -negative cell populations separated by flow cytometry, and by the selective, complement-mediated killing of mAb BH2-C6-treated neutrophils. Additional evidence for the neutrophil specificity of mAb BH2-C6 is shown by immunoelectron microscopy, which demonstrates a lack of reactivity with human eosinophils. Immunoblotting of SDS-PAGE-separated proteins of polymorphonuclear leukocytes with 125I-labeled BH2-C6 identifies protein with an average molecular mass of 157 kD. Binding studies show that, at saturation, neutrophils bind 214,000 molecules of 125I-BH2-C6 per cell. Addition of mAb BH2-C6 to neutrophils significantly reduces the number of C3bi-opsonized sheep erythrocytes (EIgMC3bi) bound by these cells. This reduction is partly reversed by the presence of soybean trypsin inhibitor (SBTI), indicating that at least one part of this inhibition is due to BH2-C6-stimulated secretion of a serine protease that may affect ligand binding. Cytochemical analysis of normal human bone marrow cells sorted by cytofluorimetry identifies the promyelocyte as the precursor cell that first expresses BH2-Ag on the plasma membrane. Using the leukemic cell line HL-60, we demonstrate that only inducers of granulocytic differentiation, cis-retinoic acid, and dimethyloxazolidine stimulate the expression of BH2-Ag. These results show that the expression of BH2-Ag during myelomonocytic differentiation is a property uniquely possessed by cells committed to the neutrophilic lineage.


Myocardial infarction increases ACE2 expression in rat and humans.
Angiotensin converting enzyme (ACE) 2 catalyses the cleavage of angiotensin (Ang) I to Ang 1-9 and of Ang II to Ang 1-7. ACE2 deficiency impairs cardiac contractility and upregulates hypoxia-induced genes, suggesting a link with myocardial ischaemia. We studied the expression of ACE2 after myocardial infarction (MI) in the rat as well as in human failing hearts. Rats were killed at days 1, 3, and 28 after MI, or treated for 4 weeks with the ACE inhibitor ramipril (1 mg/kg). Cardiac gene and protein expression of ACE and ACE2 were assessed by quantitative real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry/activity assays/in vitro autoradiography, respectively. Both ACE (P = 0.022) and ACE2 (P = 0.015) mRNA increased in the border/infarct area compared with the viable area at day 3 after MI. By day 28, increases in ACE (P = 0.005) and ACE2 (P = 0.006) mRNA were also seen in the viable myocardium of MI rats compared with myocardium of control rats. ACE2 protein localized to macrophages, vascular endothelium, smooth muscle, and myocytes. Ramipril attenuated cardiac hypertrophy and inhibited cardiac ACE. In contrast, ramipril had no effect on cardiac ACE2 mRNA, which remained elevated in all areas of the MI rat heart. Immunoreactivity of both ACE and ACE2 increased in failing human hearts. The increase in ACE2 after MI suggests that it plays an important role in the negative modulation of the renin angiotensin system in the generation and degradation of angiotensin peptides after cardiac injury.


Does Iliopsoas Lengthening Adversely Affect Clinical Outcomes After Hip Arthroscopy? A Multicenter Comparative Study.
Iliopsoas fractional lengthening (IFL) continues to be a controversial procedure in hip arthroscopy. Patients who underwent arthroscopic surgery for femoroacetabular impingement (FAI) and a labral tear either with or without IFL would experience favorable outcomes, and there would be no difference in postoperative patient-reported outcomes (PROs) between the 2 groups at minimum 2-year follow-up. Cohort study; Level of evidence, 3. Data from July 2009 and April 2015 were retrospectively reviewed. Patients were eligible if they had hip arthroscopy for both FAI and labral tear treatment with IFL and without IFL. IFL was indicated for painful internal snapping. Minimum postoperative follow-up was set to 2 years. The authors calculated the modified Harris Hip Score, International Hip Outcome Tool-12, Hip Outcome Score-Activity of Daily Living Score, Hip Outcome Score-Sports Specific Subscale, Non-Arthritic Hip Score, visual analog scale for pain, patient satisfaction, minimal clinically important difference (MCID), and the percentage of patients who achieved patient acceptable symptomatic state (PASS). Revision surgeries and conversions to total hip arthroplasty (THA) were documented. 351 hips (307 patients) met the necessary inclusion criteria in the IFL cohort, with a mean ± SD follow-up time of 42.5 ± 18.1 months. For the control cohort, 392 hips (354 patients) were included, with a mean ± SD follow-up time of 43.9 ± 19.6 months. Both groups showed significant postoperative improvement in 2-year follow-up PROs. The group with iliopsoas lengthening showed comparable results to the control group with respect to PRO improvement, MCID, PASS, and rates of revision or THA conversion. This comparative cohort study demonstrated that treatment of painful internal snapping syndrome with arthroscopic IFL, in the setting of FAI and a labral tear, is a safe procedure with good short- to mid-term follow-up results and associated improvement in PROs. Patients who underwent IFL showed similar outcomes compared with a control group treated for FAI and labral tear without IFL. In appropriately selected patients, arthroscopic IFL did not adversely affect clinical outcomes compared with patients who did not need IFL.


BM-SNP: A Bayesian Model for SNP Calling Using High Throughput Sequencing Data.
A single-nucleotide polymorphism (SNP) is a sole base change in the DNA sequence and is the most common polymorphism. Detection and annotation of SNPs are among the central topics in biomedical research as SNPs are believed to play important roles on the manifestation of phenotypic events, such as disease susceptibility. To take full advantage of the next-generation sequencing (NGS) technology, we propose a Bayesian approach, BM-SNP, to identify SNPs based on the posterior inference using NGS data. In particular, BM-SNP computes the posterior probability of nucleotide variation at each covered genomic position using the contents and frequency of the mapped short reads. The position with a high posterior probability of nucleotide variation is flagged as a potential SNP. We apply BM-SNP to two cell-line NGS data, and the results show a high ratio of overlap ( >95 percent) with the dbSNP database. Compared with MAQ, BM-SNP identifies more SNPs that are in dbSNP, with higher quality. The SNPs that are called only by BM-SNP but not in dbSNP may serve as new discoveries. The proposed BM-SNP method integrates information from multiple aspects of NGS data, and therefore achieves high detection power. BM-SNP is fast, capable of processing whole genome data at 20-fold average coverage in a short amount of time.


SOD3 induces a HIF-2α-dependent program in endothelial cells that provides a selective signal for tumor infiltration by T cells.
Tumor-infiltrating lymphocytes (TILs), mainly CD8+ cytotoxic T lymphocytes (CTL), are linked to immune-mediated control of human cancers and response to immunotherapy. Tumors have nonetheless developed specific mechanisms that selectively restrict T cell entry into the tumor microenvironment. The extracellular superoxide dismutase (SOD3) is an anti-oxidant enzyme usually downregulated in tumors. We hypothesize that upregulation of SOD3 in the tumor microenvironment might be a mechanism to boost T cell infiltration by normalizing the tumor-associated endothelium. Here we show that SOD3 overexpression in endothelial cells increased in vitro transmigration of naïve and activated CD4+ and CD8+ T cells, but not of myeloid cells. Perivascular expression of SOD3 also specifically increased CD4+ and CD8+ effector T cell infiltration into tumors and improved the effectiveness of adoptively transferred tumor-specific CD8+ T cells. SOD3-induced enhanced transmigration in vitro and tumor infiltration in vivo were not associated to upregulation of T cell chemokines such as CXCL9 or CXCL10, nor to changes in the levels of endothelial adhesion receptors such as intercellular adhesion molecule-1 (ICAM-1) or vascular cell adhesion molecule-1 (VCAM-1). Instead, SOD3 enhanced T cell infiltration via HIF-2α-dependent induction of specific WNT ligands in endothelial cells; this led to WNT signaling pathway activation in the endothelium, FOXM1 stabilization, and transcriptional induction of laminin-α4 (LAMA4), an endothelial basement membrane component permissive for T cell infiltration. In patients with stage II colorectal cancer, SOD3 was associated with increased CD8+ TIL density and disease-free survival. SOD3 expression was also linked to a T cell-inflamed gene signature using the COAD cohort from The Cancer Genome Atlas program. Our findings suggest that SOD3-induced upregulation of LAMA4 in endothelial cells boosts selective tumor infiltration by T lymphocytes, thus transforming immunologically "cold" into "hot" tumors. High SOD3 levels are associated with human colon cancer infiltration by CD8+ T cells, with potential consequences for the clinical outcome of these patients. Our results also uncover a cell type-specific, distinct activity of the WNT pathway for the regulation of T cell infiltration into tumors.