The p38 MAPK inhibitor, SB203580, abrogates ischaemic preconditioning in rat heart but timing of administration is critical.
There is debate concerning the involvement of p38 mitogen activated protein kinase (MAPK) in the mediation of ischaemic preconditioning. Pharmacological inhibition of p38 MAPK with SB203580 has been reported to block preconditioning in some studies but not in others. We hypothesised that this divergence could be due to differences in the timing of inhibitor administration. Isolated rat hearts were perfused in the Langendorff mode and subjected to 35 min regional ischaemia followed by 120 min reperfusion. Hearts were then double stained with Evans' blue and triphenyltetrazolium chloride to determine risk (R) and infarct zones (I), expressed as I/R% ratios. Preconditioned hearts were subjected to 2 times 5 min global ischaemia with 10 min intervening reperfusion. SB203580 10 microM was perfused either during the preconditioning protocol (PC+/-SB-early),just prior to and during the first 15 min of the lethal ischaemia (PC+/-SB-late) or prior to regional ischaemia in the absence of preconditioning. Ischaemic preconditioning significantly limited infarct size (I/R 38.9 +/- 3.0% in control vs 13.4 +/- 2.4%, P < 0.01). In the PC+/-SB-early group, preconditioning was still fully protective (I/R% 14.6 +/- 1.0). However, in the PC+/-SB-late group, SB203580 completely blocked the protection afforded by preconditioning (I/R% 33.6 +/- 4.4%, P < 0.01 vs 13.4 +/- 2.4% in preconditioned hearts, p < 0.05). SB203580 alone did not affect infarct size when given prior to and during regional ischaemia (I/R 36.2 +/- 2.7%). These histological data are corroborated by a significant increase in p38 MAPK activation in the preconditioned hearts during sustained ischaemia in comparison with the controls. In conclusion the activation of p38 MAPK during lethal ischaemia, but not during the ischaemic preconditioning protocol, is essential for the mediation of protection and may resolve some of the earlier controversy surrounding the use of SB203580 in preconditioning studies.


Epidemiological and genetic characteristics of the fifth avian influenza A(H7N9) wave in Suzhou, China, from October 2016 to April 2017.
Human infections with H7N9 viruses can cause severe pneumonia and even death. To characterize the epidemiology and genetics of the H7N9 viruses circulating during from October 2016 to April 2017 in Suzhou, China, all pharyngeal swab samples were collected from severe acute respiratory infections (SARI) cases during this fifth wave of infection, and we amplified the H7N9 H7 and N9 genes using a real-time polymerase chain reaction (PCR). Positive samples were subjected to virus isolation and gene sequencing to analyze the evolution and variation of the H7N9 strains. The epidemiological features of H7N9 patients have not changed and there were no significant mutations in the key sites of the hemagglutinin (HA) gene sequence, but we identified the K526R and E627 K substitutions in the PB2 protein. In the neuraminidase (NA) protein, drug-resistant mutations (R294 K and H276Y) occurred in a few strains. Most of the H7N9 viruses isolated from Suzhou had no drug resistance mutations, but it is necessary to closely monitor and analyze the probable emergence of mutations and the spread of resistant strains. The reduction of the N-glycosylation site at position 42 of NA was observed in some strains.


Morphological and molecular features of astroblastoma, including BRAFV600E mutations, suggest an ontological relationship to other cortical-based gliomas of children and young adults.
Astroblastomas (ABs) are rare glial tumors showing overlapping features with astrocytomas, ependymomas, and sometimes other glial neoplasms, and may be challenging to diagnose. We examined clinical, histopathological, and molecular features in 28 archival formalin-fixed, paraffin-embedded AB cases and performed survival analyses using Cox proportional hazards and Kaplan-Meier methods. Unlike ependymomas and angiocentric gliomas, ABs demonstrate abundant distinctive astroblastic pseudorosettes and are usually Olig2 immunopositive. They also frequently exhibit rhabdoid cells, multinucleated cells, and eosinophilic granular material. They retain immunoreactivity to alpha thalassemia/mental retardation syndrome X-linked, are immunonegative to isocitrate dehydrogenase-1 R132H mutation, and only occasionally show MGMT promoter hypermethylation differentiating them from many diffuse gliomas. Like pleomorphic xanthoastrocytoma, ganglioglioma, supratentorial pilocytic astrocytoma, and other predominantly cortical-based glial tumors, ABs often harbor the BRAFV600E mutation, present in 38% of cases tested (n = 21), further distinguishing those tumors from ependymomas and angiocentric gliomas. Factors correlating with longer patient survival included age less than 30 years, female gender, absent BRAFV600E , and mitotic index less than 5 mitoses/10 high-power fields; however, only the latter was significant by Cox and Kaplan-Meier analyses (n = 24; P = .024 and .012, respectively). This mitotic cutoff is therefore currently the best criterion to stratify tumors into low-grade ABs and higher-grade anaplastic ABs. In addition to their own characteristic histological features, ABs share some molecular and histological findings with other, possibly ontologically related, cortical-based gliomas of mostly children and young adults. Importantly, the presence of BRAFV600E mutations in a subset of ABs suggests potential clinical utility of targeted anti-BRAF therapy.


Québec: an outbreak of HIV/AIDS-related stigma and discrimination.
In the space of a few weeks in January 2004, actions by three different institutions in Québec combined to threaten the human rights of people living with HIV/AIDS, raise the spectre of mandatory HIV testing, and create unnecessary public fears about the spread of HIV infection. In response to what they called "the worst weeks in recent history for people living with HIV/AIDS in Québec," the Canadian HIV/AIDS Legal Network and COCQ-Sida (the Québec coalition of community-based organizations fighting AIDS) called for a province-wide campaign against HIV/AIDS-related stigma and discrimination. A victory was achieved when a Montréal catholic seminary announced that it had backed down from its initial proposal to mandatorily test all applicants for priesthood for HIV, but much more is needed to fight the rapid outbreak of mandatory-testing proposals.


Gene-associated markers provide tools for tackling illegal fishing and false eco-certification.
Illegal, Unreported and Unregulated fishing has had a major role in the overexploitation of global fish populations. In response, international regulations have been imposed and many fisheries have been 'eco-certified' by consumer organizations, but methods for independent control of catch certificates and eco-labels are urgently needed. Here we show that, by using gene-associated single nucleotide polymorphisms, individual marine fish can be assigned back to population of origin with unprecedented high levels of precision. By applying high differentiation single nucleotide polymorphism assays, in four commercial marine fish, on a pan-European scale, we find 93-100% of individuals could be correctly assigned to origin in policy-driven case studies. We show how case-targeted single nucleotide polymorphism assays can be created and forensically validated, using a centrally maintained and publicly available database. Our results demonstrate how application of gene-associated markers will likely revolutionize origin assignment and become highly valuable tools for fighting illegal fishing and mislabelling worldwide.


Rhodiola rosea: A Versatile Adaptogen.
Rhodiola rosea (rose root) belonging to the family Crassulaceae is a popular medicinal plant in Russia, Scandinavia, and many other countries. Extracts of the roots of this plant have been found to favorably affect a number of physiological functions including neurotransmitter levels, central nervous system activity, and cardiovascular function. It is being used to stimulate the nervous system, decrease depression, enhance work performance, eliminate fatigue, and prevent high-altitude sickness. Most of these effects have been ascribed to constituents such as salidroside (rhodioloside), rosavins, and p-tyrosol. It has also been found to be a strong antioxidant and anticarcinogen due to the presence of several phenolic compounds. Adaptogens are plant extracts that allow an organism to counteract adverse physical, chemical, and biological stressors by generating nonspecific resistance. Adaptogens are known to increase the availability of energy during the day, reduce stressed feelings, increase endurance, and increase mental alertness. This multipurpose medicinal plant (R. rosea), with adaptogenic properties that increase the body's nonspecific resistance and normalize functions, has been traditionally grown and used in Russia and Mongolia. Due to increasing consumer demands toward natural health products and the growing interests in the secondary metabolites of plants and their application in biotechnology and therapy, much focus has been put on the rose root and its medical properties. The rose root imparts normalizing influences on adverse physical, chemical, and biological disturbances but is otherwise innocuous. In India, the plant has been growing wild in the high altitudes of the Himalayas. The Defence Research and Development Organization in India has taken on the responsibilities of its conservation, as well as the development of multiple management practices and the development of health foods, supplements, and nutraceuticals in India.


Effect of the somatostatin analog octreotide acetate on circulating insulin-like growth factor-1 and related peptides in patients with non-metastatic castration-resistant prostate cancer: results of a phase II study.
Insulin-like growth factor (IGF) mediated signaling has been implicated in the growth of many tumor types including prostate cancer, and it is hypothesized that lowering circulating IGF levels in men with castration resistant prostate cancer (CRPC) will slow tumor growth. In this study, the efficacy of depot octreotide acetate was prospectively evaluated in patients with CRPC. Eligible patients had progressive non-metastatic CRPC. Octreotide acetate 30 mg was administered intramuscularly every 28 days. Changes in PSA, IGF-1, IGF-2, IGF binding protein-1 (IGFBP-1), and IGFBP-3 were evaluated over time. Accrual was stopped early after a pre-planned interim analysis showed no prostate specific antigen (PSA) declines after 3 cycles of treatment among the first 13 patients enrolled. Median baseline PSA and IGF-1 measurements were 36.2 ng/ml and 162.6 ng/ml, respectively, and median time on treatment was 13 weeks. Radiographic progression occurred in 7 patients, PSA-only progression occurred in 5 patients, and 1 patient was taken off the study due to a grade 3 drug interaction. After 3 cycles of treatment IGF-1 significantly declined with a median -34.5% (P = 0.01) and IGFBP-1 significantly increased with a median 76.3% (P = 0.046). IGF-2 and IGFBP-3 were not significantly changed from baseline. Octreotide acetate significantly lowers IGF-1 and raises IGFBP-1 levels in patients with non-metastatic CRPC, but does not result in sustained declines in PSA. While treatment with single-agent octreotide may not be warranted, its inclusion in combination regimens directly targeting the IGF-1 receptor on tumor cells may be of interest.


Intimate adhesion of Neisseria meningitidis to human epithelial cells is under the control of the crgA gene, a novel LysR-type transcriptional regulator.
PilC1, a pilus-associated protein in Neisseria menin- gitidis, is a key element in initial meningococcal adhesion to target cells. A promoter element (CREN, contact regulatory element of Neisseria) is responsible for the transient induction of this gene upon cell contact. crgA (contact-regulated gene A) encodes a transcriptional regulator whose expression is also induced upon cell contact from a promoter region similar to the CREN of pilC1. CrgA shows significant sequence homologies to LysR-type transcriptional regulators. Its inactivation in meningococci provokes a dramatic reduction in bacterial adhesion to epithelial cells. Moreover, this mutant is unable to undergo intimate adhesion to epithelial cells or to provoke effacing of microvilli on infected cells. Purified CrgA is able to bind to pilC1 and crgA promoters, and CrgA seems to repress the expression of pilC1 and crgA. Our results support a dynamic model of bacteria-cell interaction involving a network of regulators acting in cascade. CrgA could be an intermediate regulator in such a network.


Prevalence, genotypes and risk factors associated with hepatitis C virus infection in hemodialysis patients in Campo Grande, MS, Brazil.
A survey was conducted among the hemodialysis units of the city of Campo Grande, located in the state of Mato Grosso do Sul in the Mid-west region of Brazil, with the aim of investigating the prevalence, risk factors, and genotypes of hepatitis C virus (HCV) infection. A total of 163 patients were interviewed in five dialysis units. Serum samples were screened for anti-HCV. Positive samples were tested for HCV RNA and genotyped. The prevalence of anti-HCV was 11% (95% CI: 6.8-17.1). A history of transfusion with blood that was not screened for anti-HCV and length of time on hemodialysis were associated with HCV infection. HCV RNA was detected in 12 samples: ten were of genotype 1, subtypes 1a (75%) and 1b (8.3%), and two were of genotype 3, subtype 3a (16.7%).


High levels of folate from supplements and fortification are not associated with increased risk of colorectal cancer.
Folate intake has been inversely associated with colorectal cancer risk in several prospective epidemiologic studies. However, no study fully assessed the influence of the high levels of folate that are frequently consumed in the United States as a result of mandatory folate fortification, which was fully implemented in 1998, and the recent increase in use of folate-containing supplements. There is evidence that consumption of high levels of folic acid, the form of folate used for fortification and in supplements, has different effects on biochemical pathways than natural folates and might promote carcinogenesis. We investigated the association between folate intake and colorectal cancer among 43,512 men and 56,011 women in the Cancer Prevention Study II (CPS-II) Nutrition Cohort; 1023 were diagnosed with colorectal cancer between 1999 and 2007, a period entirely after folate fortification began. Cox proportional hazards regression was used to calculate multivariate hazards ratios (RR) and 95% confidence interval (CI). Intake of high levels of natural folate (RRQ5vsQ1=0.86; 95% CI: 0.70-1.06; P trend=.12) or folic acid (RRQ5vsQ1=0.84; 95% CI: 0.68-1.03; P trend=.06) were not significantly associated with risk of colorectal cancer. Total folate intake was significantly associated with lower risk (RRQ5vsQ1=0.81; 95% CI: 0.66-0.99; P trend=.047). Intake of high levels of total folate reduces risk of colorectal cancer; there is no evidence that dietary fortification or supplementation with this vitamin increases colorectal cancer risk.


Early activation of arm and leg muscles following pulls to the waist during walking.
Many studies have investigated the compensatory reactions in humans elicited during walking when the support surface is perturbed. This has led to the description of characteristic responses generated in the muscles of the legs and torso, and recently the arms. The present study aimed to investigate the compensatory reactions elicited when balance was challenged by a perturbation applied to the waist, to determine to what extent balance corrective responses are generalized across perturbation modalities. A second aim was to characterize the arm responses elicited by the perturbations applied to the waist. We measured muscle activity of the left arm and leg following application of backward pulls of the waist while the subjects walked on a motorized treadmill. This resulted in robust activation of tibialis anterior and vastus lateralis, with co-activation of soleus and biceps femoris also evident when perturbations were applied at heel strike. These early responses occurred with a distal to proximal temporal organization. The responses in the leg muscles displayed a phase-dependent modulation in amplitude, decreasing in amplitude later in the stance phase. Leg muscle responses were not evident during the swing phase, except for the end of swing, just prior to heel strike. Arm muscle responses were observed in all subjects; however, the pattern of the arm responses varied considerably between subjects. Generally, shoulder muscles were more likely to respond than elbow muscles, at latencies consistent with the leg responses. Two important conclusions are drawn from the present study. First, the responses evoked in the legs with a pull to the waist are very similar to what has been reported for perturbations of the support surface, despite the very different locus of the perturbation. This suggests that balance control during walking may be achieved by preprogrammed reactions or synergies, which are triggered by multiple sensory cues. Second, rapid arm actions are integrated with these leg responses. However, the arm responses are more flexible, likely reflecting the fewer constraints imposed upon the actions of the arms, compared to the legs, during normal locomotion.


A survey of parental consent process for newborn screening in Taiwan.
Recent technological developments in newborn screening (NBS) allow many genetic/metabolic disorders to be detected pre-symptomatically even though they may be untreatable. Thus, current NBS faces ethical, legal, and social challenges. Obtaining parental consent for NBS demonstrates respect for and educates parents. The purpose of this study was to explore current practices in obtaining parental consent to conduct NBS in Taiwan. A self-administered questionnaire was mailed to obstetric clinicslhospitals (N=339) and 204 questionnaires were returned for a response rate of 60.2%. Parental consent processes for NBS were divided into three categories-informed consent, informed refusal, and no informedlconsent. Between 66.7-67.6% clinics/hospitals used an "informed consent" model for all NBS items. Although most clinics/hospitals frequently informed parents before screening was conducted, the information given was often incomplete. Moreover, a "no informedl consent" model was used for congenital adrenal hyperplasia (CAH) and rare geneticlmetabolic disorders in between 22.5% and 25.1% of the clinics/hospitals, respectively. A total of 23.1% of clinics/hospitals chose an "informed refusal" model for the five traditional genetic/metabolic disorders. Using logistic regression analysis, the parental consent process for the five traditional disorders was found to have significant associations with the classification of the hospitals and the contracted NBS center (p < 0.01). Regional hospitals less frequently used the "informed consent" model than local clinics, and the clinics/hospitals that contracted with National Taiwan University Hospital were more likely to follow an "informed consent" model rather than those contracted with Taipei Institute Pathology. Establishing specific guidelines to regulate the parental consent process and educate medical personnel is crucial.


Effect of aspirin on urinary bladder carcinogenesis initiated with N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide in rats.
N-[4-(5-Nitro-2-furyl)-2-thiazolyl]formamide (FANFT), a potent urinary bladder carcinogen, is metabolically activated in vitro by a variety of enzyme systems including aerobic cooxidation by prostaglandin H synthase which is present in the rat bladder mucosa. In a previous experiment, aspirin coadministered with FANFT for 12 weeks inhibited FANFT-induced bladder carcinogenesis and enhanced forestomach carcinogenesis. To further evaluate the effects of aspirin on FANFT carcinogenesis, male F344 rats were fed either FANFT (0.2% of the diet) for 12 weeks (Group 4), aspirin (0.5% of the diet) simultaneously with FANFT for 12 weeks (Group 2), aspirin simultaneously with FANFT for 12 weeks and then subsequently to the end of the experiment (Group 1), or FANFT only followed by aspirin (Group 3). The incidence of bladder carcinoma was significantly higher when aspirin was fed after FANFT treatment (87%) compared to FANFT followed by control diet (48%) and was higher in rats given aspirin plus FANFT followed by aspirin (73%) compared to aspirin plus FANFT followed by control diet (47%). Aspirin alone given for 13 weeks (Group 6) or throughout the experiment (68 weeks) (Group 5) did not induce bladder cancer. However, in all groups administered aspirin long-term, renal papillary necrosis and renal pelvic hyperplasia and atypia were frequently observed. Only a single forestomach tumor was observed. In the present experiment, aspirin appeared to exhibit promoting activity for bladder carcinogenesis in the rat.


Incubation of whole blood at room temperature does not alter the plasma concentrations of microRNA-16 and -223.
Plasma-derived microRNAs (miRNAs) are being used as biomarkers, and have been associated with human liver disease and function including fibrosis, inflammation, and drug-induced liver injury. They may also be biomarkers of the drug metabolism function of the liver. In order for plasma miRNA to function as a clinical biomarker, predictable variability is necessary during processing from whole blood to plasma. The current study evaluated the variability of miRNA in whole blood stored for 0.5, 1, 2, 4, 8, and 12 hours following the blood draw under clinical conditions (room temperature) prior to the separation of the plasma. Four healthy volunteers were recruited. Blood from all subjects was collected twice. MicroRNA-16 (miR-16) and miR-223 were evaluated because many studies have shown them to be reliably present in plasma and useful for normalization. miRNA concentrations were measured by real-time polymerase chain reaction. The coefficient of variability of the cycle threshold values for subjects for miR-223 and miR-16 ranged from ∼3.6 to 6.8% and ∼1.48 to 4.1%, respectively, over the 12-hour incubation. A second blood collection was performed to determine interday variability. The coefficient of variance from the initial blood draw compared with the final blood draw for each subject ranged from 0.42 to 7.9% for miR-16 and 1.7 to 8.3% for miR-223, indicating that these miRNAs have limited interday variability. We conclude that plasma miR-16 or miR-223 concentrations are stable in whole blood at room temperature for up to 12 hours.


The Transcriptional Regulator Lrp Contributes to Toxin Expression, Sporulation, and Swimming Motility in Clostridium difficile.
Clostridium difficile is a Gram-positive, spore-forming bacterium, and major cause of nosocomial diarrhea. Related studies have identified numerous factors that influence virulence traits such as the production of the two primary toxins, toxin A (TcdA) and toxin B (TcdB), as well as sporulation, motility, and biofilm formation. However, multiple putative transcriptional regulators are reportedly encoded in the genome, and additional factors are likely involved in virulence regulation. Although the leucine-responsive regulatory protein (Lrp) has been studied extensively in Gram-negative bacteria, little is known about its function in Gram-positive bacteria, although homologs have been identified in the genome. This study revealed that disruption of the lone lrp homolog in C. difficile decelerated growth under nutrient-limiting conditions, increased TcdA and TcdB production. Lrp was also found to negatively regulate sporulation while positively regulate swimming motility in strain R20291, but not in strain 630. The C. difficile Lrp appeared to function through transcriptional repression or activation. In addition, the lrp mutant was relatively virulent in a mouse model of infection. The results of this study collectively demonstrated that Lrp has broad regulatory function in C. difficile toxin expression, sporulation, motility, and pathogenesis.


Moose body mass variation revisited: disentangling effects of environmental conditions and genetics.
Large-scale geographical variation in phenotypic traits within species is often correlated to local environmental conditions and population density. Such phenotypic variation has recently been shown to also be influenced by genetic structuring of populations. In ungulates, large-scale geographical variation in phenotypic traits, such as body mass, has been related to environmental conditions and population density, but little is known about the genetic influences. Research on the genetic structure of moose suggests two distinct genetic lineages in Norway, structured along a north-south gradient. This corresponds with many environmental gradients, thus genetic structuring provides an additional factor affecting geographical phenotypic variation in Norwegian moose. We investigated if genetic structure explained geographical variation in body mass in Norwegian moose while accounting for environmental conditions, age and sex, and if it captured some of the variance in body mass that previously was attributed to environmental factors. Genetic structuring of moose was the most important variable in explaining the geographic variation in body mass within age and sex classes. Several environmental variables also had strong explanatory power, related to habitat diversity, environmental seasonality and winter harshness. The results suggest that environmental conditions, landscape characteristics, and genetic structure should be evaluated together when explaining large-scale patterns in phenotypic characters or life history traits. However, to better understand the role of genetic and environmental effects on phenotypic traits in moose, an extended individual-based study of variation in fitness-related characters is needed, preferably in an area of convergence between different genetic lineages.


Star formation around supermassive black holes.
The presence of young massive stars orbiting on eccentric rings within a few tenths of a parsec of the supermassive black hole in the galactic center is challenging for theories of star formation. The high tidal shear from the black hole should tear apart the molecular clouds that form stars elsewhere in the Galaxy, and transport of stars to the galactic center also appears unlikely during their lifetimes. We conducted numerical simulations of the infall of a giant molecular cloud that interacts with the black hole. The transfer of energy during closest approach allows part of the cloud to become bound to the black hole, forming an eccentric disk that quickly fragments to form stars. Compressional heating due to the black hole raises the temperature of the gas up to several hundred to several thousand kelvin, ensuring that the fragmentation produces relatively high stellar masses. These stars retain the eccentricity of the disk and, for a sufficiently massive initial cloud, produce an extremely top-heavy distribution of stellar masses. This potentially repetitive process may explain the presence of multiple eccentric rings of young stars in the presence of a supermassive black hole.


In-vivo study of genotoxic and inflammatory effects of the organo-modified Montmorillonite Cloisite® 30B.
Because of the increasing use of clays and organoclays in industrial applications it is of importance to consider the toxicity of these materials. Recently it was reported that the commercially available Montmorillonite clay, Cloisite(®) 30B, which is surface-modified by organic quaternary ammonium compounds, was genotoxic in vitro. In the present study the in-vivo genotoxic and inflammatory potential of Cloisite(®) 30B was investigated as a follow-up of the in-vitro studies. Wistar rats were exposed to Cloisite(®) 30B twice 24h apart by oral gavage, at doses ranging from 250 to 1000 mg/kg body weight [indicate duration of treatment; Ed.]. There was no induction of DNA strand-breaks in colon, liver and kidney cells and there was no increase in inflammatory cytokine markers in blood-plasma samples. In order to verify the possible absorption of Cloisite(®) 30B from the gastrointestinal tract, inductively coupled plasma mass-spectrometry (ICP-MS) analysis was performed on samples of liver, kidney and faeces, with aluminium as a tracer element characteristic to clay. The results showed that aluminium could be detected in faeces, but not in the liver or kidneys. This indicated that there was no systemic exposure to clay particles from Cloisite(®) 30B. Detection and identification of free quaternary ammonium modifier in the highest dose of Cloisite(®) 30B was carried out by high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS). This analysis revealed a mixture of three quaternary ammonium analogues. The detected concentration of the organomodifier corresponded to an exposure of rats to about 5mg quaternary ammonium analogues/kg body weight.


A model for reflection for good clinical practice.
Rationale and aim The rapidly changing knowledge base of clinical practice highlights the need to keep abreast of knowledge changes that are most relevant for the practitioner. We aimed to develop a model for reflection on clinical practice that identified the key elements of medical knowledge needed for good medical practice. Method The dual theory of cognition, an integration of intuitive and analytic processes, provided the framework for the study. The design looked at the congruence between the clinical thinking process and the dual theory. A one-year study was conducted in general practice clinics in Oxfordshire, UK. Thirty-five general practitioners participated in 20-minute interviews to discuss how they worked through recently seen clinical cases. Over a one-year period 72 cases were recorded from 35 interviews. These were categorized according to emerging themes, which were manually coded and substantiated with verbatim quotations. Results There was a close fit between the dual theory and participants' clinical thinking processes. This included instant problem framing, consistent with automatic intuitive thinking, focusing on the risk and urgency of the case. Salient features accounting for these choices were recognizable. There was a second reflective phase, leading to the review of initial judgements. Conclusions The proposed model highlights the critical steps in decision making. This allows regular recalibration of knowledge that is most critical at each of these steps. In line with good practice, the model also links the crucial knowledge used in decision making, to value judgments made in relation to the patient.


Metabolic-dependent changes in plant cell redox power after ozone exposure.
The tropospheric level of the phytotoxic air pollutant ozone has increased considerably during the last century, and is expected to continue to rise. Long-term exposure of higher plants to low ozone concentrations affects biochemical processes prior to any visible symptoms of injury. The current critical level of ozone used to determine the threshold for damaging plants (biomass loss) is still based on the seasonal sum of the external concentration above 40 nl.l(-1) (AOT40). Taking into account stomatal conductance and the internal capacity of leaf defences, a more relevant concept should be based upon the 'effective ozone flux', the balance between the stomatal flux and the intensity of cellular detoxification. The large decrease in the Rubisco/PEPc ratio reflects photosynthetic damage from ozone, and a large increase in activity of cytosolic PEPc, which allows increased malate production. Although the direct detoxification of ozone (and ROS produced from its decomposition) is carried out primarily by cell wall ascorbate, the existing level of this antioxidant is not sufficient to indicate the degree of cell sensitivity. In order to regenerate ascorbate, NAD(P)H is needed as the primary supplier of reducing power. It is hypothesised that increased activity of the catabolic pathways and associated shunts (glucose-6-phosphate dehydrogenase, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, isocitrate dehydrogenase and malic enzyme) can provide sufficient NAD(P)H to maintain intracellular detoxification. Thus, measurement of the level of redox power would contribute to determination of the 'effective ozone dose', serving ultimately to improve the ozone risk index for higher plants.


Neutral glycolipids and gangliosides of concanavalin A-selected SV3T3 revertant cells and of normal and SV40-transformed Balb/c 3T3 cells.
The structural analysis of neutral glycolipids and gangliosides of the SV40 transformed Balb/c3T3 cells (SV3T3 cells) and concanavalin A-selected SV3T3 revertant cells, both compared with untransformed Balb/c3T3 cells, has shown: (i) a content of neutral glycolipids in revertant cells near to that found in the untransformed parental cells; (ii) a similar decrease of the higher gangliosides in transformed and revertant cells; (iii) a content of ganglioside GM3 in revertant cells much higher than that found in both SV3T3 and untransformed Balb/3T3 cells. The possible role of ganglioside GM3 in growth control is discussed.


Maternal multiple micronutrient supplementation and other biomedical and socioenvironmental influences on children's cognition at age 9-12 years in Indonesia: follow-up of the SUMMIT randomised trial.
Brain and cognitive development during the first 1000 days from conception are affected by multiple biomedical and socioenvironmental determinants including nutrition, health, nurturing, and stimulation. An improved understanding of the long-term influence of these factors is needed to prioritise public health investments to optimise human development. We did a follow-up study of the Supplementation with Multiple Micronutrients Intervention Trial (SUMMIT), a double-blind, cluster-randomised trial of maternal supplementation with multiple micronutrients (MMN) or iron and folic acid (IFA) in Indonesia. Of 27 356 live infants from birth to 3 months of age in 2001-04, we re-enrolled 19 274 (70%) children at age 9-12 years, and randomly selected 2879 from the 18 230 who were attending school at a known location. Of these, 574 children were oversampled from mothers who were anaemic or malnourished at SUMMIT enrolment. We assessed the effects of MMN and associations of biomedical (ie, maternal and child anthropometry and haemoglobin and preterm birth) and socioenvironmental determinants (ie, parental education, socioeconomic status, home environment, and maternal depression) on general intellectual ability, declarative memory, procedural memory, executive function, academic achievement, fine motor dexterity, and socioemotional health. The SUMMIT trial was registered, number ISRCTN34151616. Children of mothers given MMN had a mean score of 0·11 SD (95% CI 0·01-0·20, p=0·0319) higher in procedural memory than those given IFA, equivalent to the increase in scores with half a year of schooling. Children of anaemic mothers in the MMN group scored 0·18 SD (0·06-0·31, p=0·0047) higher in general intellectual ability, similar to the increase with 1 year of schooling. Overall, 18 of 21 tests showed a positive coefficient of MMN versus IFA (p=0·0431) with effect sizes from 0·00-0·18 SD. In multiple regression models, socioenvironmental determinants had coefficients of 0·00-0·43 SD and 22 of 35 tests were significant at the 95% CI level, whereas biomedical coefficients were 0·00-0·10 SD and eight of 56 tests were significant, indicating larger and more consistent impact of socioenvironmental factors (p<0·0001). Maternal MMN had long-term benefits for child cognitive development at 9-12 years of age, thereby supporting its role in early childhood development, and policy change toward MMN. The stronger association of socioenvironmental determinants with improved cognition suggests present reproductive, maternal, neonatal, and child health programmes focused on biomedical determinants might not sufficiently enhance child cognition, and that programmes addressing socioenvironmental determinants are essential to achieve thriving populations. Grand Challenges Canada Saving Brains Program.


The association between first abortion and first-time non-fatal suicide attempt: a longitudinal cohort study of Danish population registries.
Suicidal ideation due to abortion has been used to justify restrictive US abortion policies. Much research examining abortion and mental health has relied on self-report, has had low participation rates, and did not consider confounding factors. In the present study, we used data that do not rely on self-report and are not affected by low participation rates to examine the association between abortion and non-fatal suicide attempts, adjusting for confounding factors. In this longitudinal cohort study of Danish population registries, we linked data on a cohort of women born in Denmark between Jan 1, 1980, and Dec 30, 1998, who did not die or emigrate from Denmark before their 18th birthday or before study entry. Follow-up started on the woman's 18th birthday or Jan 1, 2000, whichever came last. Follow-up ended at the date of first suicide attempt, date of emigration from Denmark, date of death, or Dec 31, 2016, whichever came first. Women were between the ages of 18 and 36 years during the study period. We used a survival analysis to examine the risk of first suicide attempts or self-harm associated with a first abortion compared with no abortion, in the complete study cohort. To examine incidence rate ratios (IRRs) associated with abortion, we used Poisson regression with the logarithm of woman-years at risk as an offset. We also examined whether the risk of suicide attempts changed before and after the abortion, adjusting for age, calendar year, socioeconomic status, and history of childbirth, mental health, parental mental health, and physical health. Data on 523 280 women were included in this study. Of these, 48 990 (9·4%) women had a record of at least one first-trimester abortion, and 10 216 (2·0%) had a suicide attempt during the study period. Among 48 990 women who had an abortion, 1402 (2·9%) had a first suicide attempt after the first abortion. In our fully-adjusted model which adjusted for all covariates, the risk of first-time non-fatal suicide attempts was similar in the year before an abortion (IRR 2·46 [95% CI 2·22-2·72]) and the year after an abortion (IRR 2·54 [2·29-2·81], p=0·509) compared with women who had not had an abortion, and decreased with increasing time since the abortion (1-5 years IRR 1·90 [1·75-2·06]; ≥5 years IRR 1·73 [1·53-1·96]). We found that women who had abortions had a higher risk of non-fatal suicide attempts compared with women who did not have an abortion. However, because the increased risk was the same both the year before and after the abortion, it is not attributable to the abortion. Thus, policies based on the notion that abortion increases women's risk of suicide attempts are misinformed. Society of Family Planning, American Foundation for Suicide Prevention, and The Lundbeck Foundation Initiative for Integrative Psychiatric Research.


Retinopathy of prematurity and risk factors: a prospective cohort study.
Increased survival of extremely low birth infants due to advances in antenatal and neonatal care has resulted in a population of infants at high risk of developing retinopathy of prematurity (ROP). Therapeutic interventions include the use of antenatal and postnatal steroids however, their effects on the severity of ROP is in dispute. In addition, it has not been investigated whether severe ROP is due to therapeutic interventions or due to the severity of illness. The aim of the present study was to assess the association between the incidence of severe retinopathy of prematurity (greater than stage 2 - International classification of ROP) and mechanical ventilation, oxygen therapy, gestational age, antenatal and postnatal steroids in extremely low birth weight infants. Neonates admitted to the neonatal intensive care unit in Lansing, Michigan, during 1993-2000 were followed to determine factors influencing the development of severe retinopathy of prematurity. Ophthalmologic examinations were started at 6 weeks and followed until resolution. We used logistic regression to estimate the relative risk (odds ratio) associated with risk factors of ROP. Of the neonates with <or= 1500 g birth weight, admitted to the neonatal intensive care unit, 85% (616/725) survived. Severe retinopathy of prematurity was detected in 7.8% of 576 neonates who had eye examinations. Neonates of lower gestational age (<or= 25 weeks and 26-28 weeks) had an increased odds ratio of 8.49 and 3.19 for the development of severe retinopathy of prematurity, respectively, compared to those 29 weeks and older. Late postnatal steroid treatment starting after 3 weeks of life showed 2.9-fold increased odds ratio, in particular administration for two weeks and more (OR: 4.09, 95% CI: 1.52-11.03). With increasing antenatal steroids courses the risk of severe retinopathy of prematurity decreased, however, it was not significant. Lower gestational age, dependence on ventilation, and use of postnatal steroids were intertwined. Simultaneous presence of these factors seems to indicate severe disease status. Prolonged and late postnatal steroids treatment in very low birth weight infants may pose an increased risk for the development of severe retinopathy of prematurity; however, use of postnatal steroids may also be a marker for severity of illness. Further studies need to focus on biologic markers in the pathogenesis of retinopathy of prematurity and to better understand the influence of therapies.


Pneumatic pressure bioreactor for cyclic hydrostatic stress application: mechanobiology effects on periodontal ligament cells.
A bioreactor system was developed to provide high-amplitude cyclic hydrostatic compressive stress (cHSC) using compressed air mixed commercially as needed to create partial pressures of oxygen and carbon dioxide appropriate for the cells under investigation. Operating pressures as high as 300 psi are achievable in this system at cyclic speeds of up to 0.2 Hz. In this study, ligamentous fibroblasts from human periodontal ligaments (n = 6) were compressed on two consecutive days at 150 psi for 3 h each day, and the mRNA for families of extracellular matrix protein and protease isoforms was evaluated by real-time PCR array. Several integrins were significantly upregulated, most notably alpha-3 (6.4-fold), as was SPG7 (12.1-fold). Among the collagens, Col8a1 was highly upregulated at 53.5-fold, with Col6a1, Col6a2, and Col7a1 also significantly upregulated 4.4- to 8.5-fold. MMP-1 was the most affected at 122.9-fold upregulation. MMP-14 likewise increased 17.8-fold with slight reductions for the gelatinases and a significant increase of TIMP-2 at 5.8-fold. The development of this bioreactor system and its utility in characterizing periodontal ligament fibroblast mechanobiology in intermediate-term testing hold promise for better simulating the conditions of the musculoskeletal system and the large cyclic compressive stresses joints may experience in gait, exertion, and mastication.


Burden of Disease in Coastal Areas of South Korea: An Assessment Using Health Insurance Claim Data.
While measuring and monitoring disease morbidity, it is essential to focus on regions experiencing inequitable health outcomes, especially coastal populations. However, no research investigating population health outcomes in coastal areas has been conducted. Therefore, we aimed to investigate the burden of disease morbidity in coastal areas of South Korea. Using an administrative division map and the ArcGIS, we identified and included 496 coastal districts. In this observational study, years lived with disability (YLDs) were estimated using incidence-based approaches to calculate the burden of disease in 2015. Incidence and prevalence cases were collected using National Health Insurance Service (NHIS) medical claim data using a specialized algorithm. Age-standardized years lived with disability (ASYLDs) in the coastal areas were 24,398 per 100,000 population, which is greater than the 22,613 YLDs observed nationwide. In coastal areas, the burden of disease morbidity was higher in females than in males. Diabetes mellitus was the leading specific disease of total YLDs per 100,000 population, followed by low back pain, chronic obstructive pulmonary disease, osteoarthritis, and ischemic stroke. In this study, the coastal areas of South Korea carry a higher burden than the national population. Additionally, chronic diseases compose the majority of the health burden in coastal areas. Despite the limitation of data, YLD was the best tool available for evaluating the health outcomes in specific areas, and has the advantage of simplicity and timely analysis.


Necrotic enteritis in chickens: development of a straightforward disease model system.
The interaction between Eimeria species and Clostridium perfringens was investigated in two different necrotic enteritis (NE) models: 120-day-old broilers were used in two separate experiments consisting of six groups (n=10) each. Besides controls, chickens were infected with coccidia on study day (SD) 18 (Eimeria maxima and Eimeria acervulina (experiment 1) or Eimeria tenella and Eimeria brunetti (experiment 2) and/or a NetB toxin positive C perfringens strain (both experiments: SD 14 or SD 22, respectively)). Body weight, feed intake, mortality rate, clinical disease, Eimeria species oocyst excretion and C perfringens counts were recorded. NE and coccidiosis specific lesion scores were assessed (SD 24 and SD 30). In coinfected groups, NE-typical clinical signs occurred. Coccidiosis-specific lesions were most severe in coinfected groups (significant for E tenella, P<0.05). Most pronounced NE lesions occurred in coinfected chickens compared with C perfringens monoinfected groups (experiment 2, C perfringens infections on SD 22: P<0.05). In experiment 2, E tenella antibody levels were (non-significantly) higher in coinfected groups than in Eimeria species monoinfected groups. Thus, infection with E tenella and Eimeria brunetti followed by C perfringens inoculation is regarded as an easy to handle and suitable model for investigations into NE of chickens.


Age-related spontaneous intracerebral hematoma in a German community.
We investigated incidence, age distribution in relation to etiology, and localization of spontaneous intracerebral hematoma in 100 consecutive cases. Incidence in the total population of the Giessen area was estimated to be greater than 11/100,000 inhabitants/yr and increased with age. There was a trend toward higher incidence in males. Overall mortality was 27%, 22% of 58 patients aged less than 70 years and 33% of 42 patients aged greater than or equal to 70 years. Hypertensive putaminal hematoma showed the highest mortality rate (42%, 10 of 24 cases). Chronic alcoholism and anticoagulant medication influenced the mortality rate unfavourably. We found the following localizations and etiologies to have a specific relation with age: 1) lobar hematomas from vascular malformations, group aged less than 40 years; 2) hypertensive putaminal hematomas and hypertensive thalamic hematomas, group aged 40-69 years; and 3) lobar hematomas, group aged greater than or equal to 70 years. Alcoholism was an additional factor in 38% of the 13 middle-aged men with hypertensive putaminal hematomas. Fourteen cases of spontaneous intracerebral hematoma were possibly due to cerebral amyloid angiopathy. Six of these 14 patients had recurrent lobar hematomas, but only three of the six could be histologically investigated. In these three cases, cerebral amyloid angiopathy was proven.


Immunohistochemical detection of EGFR, fibrillin-2, P-cadherin and AP2beta as biomarkers for rhabdomyosarcoma diagnostics.
Subclassification of rhabdomyosarcoma (RMS) has clinical relevance, as the two major subclasses embryonal (ERMS) and alveolar (ARMS) rhabdomyosarcoma differ greatly in terms of aggressiveness and prognosis. However, histological analysis is not always sufficient for an unequivocal subclassification of RMS. Furthermore, clinical presentation of ARMS has been reported to mimic other tumour types, specifically lymphoma. The aim was to determine the role of four biomarkers in the diagnosis of rhabdomyosarcoma. Recently, we identified four potential biomarkers to subclassify RMS with high sensitivity and specificity. These included epidermal growth factor receptor (EGFR) and fibrillin-2 as markers for ERMS, and AP2beta and P-cadherin as markers for translocation-positive ARMS. Here, we further validate the potential of these four markers in a second, independent patient cohort by immunohistochemistry on 80 sections of RMS biopsy specimens as well as a tissue microarray representing 18 different additional tumour types, including seven lymphomas. The combination of EGFR and fibrillin-2 was able to detect ERMS with a specificity of 76% and sensitivity of 90%. The combination of AP2beta and P-cadherin detected ARMS with a specificity of 97% and sensitivity of 90%, data very similar to our previous study. Furthermore, all lymphomas were clearly negative for AP2beta and P-cadherin. These four biomarkers are suitable for clinical implementation in the future diagnosis of RMS.


Elevated cardiac troponin measurements in critically ill patients.
The clinical significance of elevated cardiac troponin (cTn) level in patients in the intensive care unit (ICU) is uncertain. We reviewed the frequency of cTn elevation and its association with mortality and length of ICU stay in these patients. Studies were identified using MEDLINE, EMBASE, and reference list review. We included observational studies of critically ill patients that measured cTn at least once and reported the frequency of elevated cTn or outcome (mortality and length of ICU or hospital stay). We pooled the odds ratios (ORs) using the inverse variance method in studies that conducted multivariable analysis to examine the relationship between elevated cTn and mortality (adjusted analysis). We calculated the weighted mean difference in length of stay between patients with and without elevated cTn and pooled the results using the inverse variance method (unadjusted analysis). A total of 23 studies involving 4492 critically ill patients were included. In 20 studies, elevated cTn was found in a median of 43% (interquartile range, 21% to 59%) of 3278 patients. In adjusted analysis (6 studies comprising 1706 patients), elevated cTn was associated with an increased risk of death (OR, 2.5; 95% confidence interval [CI], 1.9 to 3.4; P < .001). In the unadjusted analysis (8 studies comprising 1019 patients), elevated cTn was associated with an increased length of ICU stay of 3.0 days (95% CI, 1.0 to 5.1 days; P = .004) and an increased length of hospital stay of 2.2 days (95% CI, -0.6 to 4.9; P = .12). Elevated cTn measurements among critically ill patients are associated with increased mortality and ICU length of stay. Research is needed to clarify the underlying causes of elevated cTn in this population and to examine their clinical significance.


Comprehensive characterization and optimization of anti-LRRK2 (leucine-rich repeat kinase 2) monoclonal antibodies.
Missense mutations in LRRK2 (leucine-rich repeat kinase 2) are a major cause of PD (Parkinson's disease). Several antibodies against LRRK2 have been developed, but results using these polyclonal antibodies have varied widely leading to conflicting conclusions. To address this challenge, the Michael J. Fox Foundation for Parkinson's Research generated a number of monoclonal antibodies targeting epitopes across the LRRK2 protein. In the present paper, we report optimized protocols and results for ten monoclonal antibodies for immunoblotting, immunohistochemistry, immunoprecipitation and kinase activity assays, in rat, mouse and human brain tissue. Several efficacious antibodies were identified, but results demonstrate that the mouse monoclonal N241A/34 is suitable for most applications, with the best overall rabbit monoclonal antibody being c41-2. These antibodies produced a dominant band of the expected size via immunoblotting and a lack of labelling in tissue derived from LRRK2-knockout animals under optimized conditions. A significant proportion of LRRK2 protein localizes to insoluble fractions and no evidence of truncated LRRK2 protein was detected in any fraction from rodent or human tissues. An assay was developed for the robust detection of LRRK2 kinase activity directly from frozen mouse and human brain tissue, but precipitous declines in activity were observed that corresponded to increasing post-mortem intervals and processing times. Finally, we demonstrate the highest levels of brain-localized LRRK2 in the striatum, but note differential expression patterns between rat and mouse in both striatum and cortex. Anti-LRRK2 monoclonal antibodies that are unlimited in availability together with the proposed standardized protocols should aid in the definition of LRRK2 function in both health and disease.


Combined tests of prostate specific antigen and testosterone will improve diagnosis and monitoring the progression of prostate cancer.
Prostate-specific antigen (PSA) testing has been widely used to screen men for prostate cancer (PCa) and to monitor PCa progression. However, more studies have shown that around 15% of men with low or normal PSA levels have PCa. In this study, we aimed to investigate the relationship of androgen and PSA levels and to better understand the reason that some PCa patients have low serum PSA values. The in vitro data demonstrated that cultured LNCaP cells ceased to produce PSA after androgen withdrawal and resumed PSA production after androgen was re-added. The in vivo experiment results showed that 48% of PCa xenografts carrying mice have serum PSA level lower than 4 ng ml-1 . The serum PSA levels increased significantly with rises in testosterone (T) levels 1 week after T pellet implantation. These data indicated that the androgen is a key factor controlling the production of PSA. Low serum PSA levels in mice with PCa xenografts are associated with low serum T levels. Raising serum T levels in tumor caring mice will also significantly increase serum PSA level. This may have clinical implications when screening PSA in men, who have occult PCa.


Treating hepatitis C virus in children: time for a new paradigm.
Hepatitis C virus infection is a leading cause of liver-related morbidity and mortality. In the paediatric population, HCV infection is underdiagnosed and undertreated in the absence of robust screening policies worldwide, and a lack of tolerable, effective treatment. The recent advances in HCV drug development allow for optimism, a change in outcomes for the millions of children infected with this virus and a unique opportunity for strategies aiming at HCV eradication. The rapid development of the new compounds has been followed by a welcome shift in the regulatory processes; however, strategies aiming at improving diagnosis, selecting the best combinations and addressing mother-to-child transmission issues are required for the new therapeutic agents to be introduced safely and effectively in the paediatric population and to contribute to the goal of virus eradication.


Genomic rearrangements in OPA1 are frequent in patients with autosomal dominant optic atrophy.
Autosomal dominant optic atrophy (ADOA) is considered as the most common form of hereditary optic neuropathy. Although genetic linkage studies point to the OPA1 locus on chromosome 3q28-q29 as by far the most common gene locus, previous screening studies-based on sequencing of the coding exons-detected OPA1 mutations in only 32-70% of ADOA patients. We therefore hypothesised that larger deletions or duplications that remained undetected in previous screening approaches may substantially contribute to the prevalence of OPA1 mutations in ADOA. 42 independent ADOA patients were analysed for the presence of genomic rearrangements in OPA1 by means of multiplex ligation probe amplification (MLPA). Deletions or duplications were confirmed either by long distance polymerase chain reaction (PCR) and breakpoint sequencing or loss of heterozygosity analyses with flanking microsatellite markers. Patients underwent ophthalmological examination including visual acuity, colour vision testings, perimetry and funduscopy. We identified genomic rearrangements in 8 of 42 patients, including single exon deletions of exon 9 and exon 24, respectively, a deletion of exons 1-5, two different deletions of the complete OPA1 gene as well as a duplication of the exons 7-9, with the latter being present in three unrelated families. Patients' phenotypes were highly variable, similar to patients with point mutation in OPA1. Our findings show that gross genomic aberrations at the OPA1 gene locus are frequent in ADOA and substantially contribute to the spectrum and prevalence of OPA1 mutations in ADOA patients. They further strengthen the hypothesis that haploinsufficiency is a major pathomechanism in OPA1 associated ADOA.


Discovery, Characterization, and Effects on Renal Fluid and Electrolyte Excretion of the Kir4.1 Potassium Channel Pore Blocker, VU0134992.
The inward rectifier potassium (Kir) channel Kir4.1 (KCNJ10) carries out important physiologic roles in epithelial cells of the kidney, astrocytes in the central nervous system, and stria vascularis of the inner ear. Loss-of-function mutations in KCNJ10 lead to EAST/SeSAME syndrome, which is characterized by epilepsy, ataxia, renal salt wasting, and sensorineural deafness. Although genetic approaches have been indispensable for establishing the importance of Kir4.1 in the normal function of these tissues, the availability of pharmacological tools for acutely manipulating the activity of Kir4.1 in genetically normal animals has been lacking. We therefore carried out a high-throughput screen of 76,575 compounds from the Vanderbilt Institute of Chemical Biology library for small-molecule modulators of Kir4.1. The most potent inhibitor identified was 2-(2-bromo-4-isopropylphenoxy)-N-(2,2,6,6-tetramethylpiperidin-4-yl)acetamide (VU0134992). In whole-cell patch-clamp electrophysiology experiments, VU0134992 inhibits Kir4.1 with an IC50 value of 0.97 µM and is 9-fold selective for homomeric Kir4.1 over Kir4.1/5.1 concatemeric channels (IC50 = 9 µM) at -120 mV. In thallium (Tl+) flux assays, VU0134992 is greater than 30-fold selective for Kir4.1 over Kir1.1, Kir2.1, and Kir2.2; is weakly active toward Kir2.3, Kir6.2/SUR1, and Kir7.1; and is equally active toward Kir3.1/3.2, Kir3.1/3.4, and Kir4.2. This potency and selectivity profile is superior to Kir4.1 inhibitors amitriptyline, nortriptyline, and fluoxetine. Medicinal chemistry identified components of VU0134992 that are critical for inhibiting Kir4.1. Patch-clamp electrophysiology, molecular modeling, and site-directed mutagenesis identified pore-lining glutamate 158 and isoleucine 159 as critical residues for block of the channel. VU0134992 displayed a large free unbound fraction (fu) in rat plasma (fu = 0.213). Consistent with the known role of Kir4.1 in renal function, oral dosing of VU0134992 led to a dose-dependent diuresis, natriuresis, and kaliuresis in rats. Thus, VU0134992 represents the first in vivo active tool compound for probing the therapeutic potential of Kir4.1 as a novel diuretic target for the treatment of hypertension.


Clinical and Genomic Characterization of Treatment-Emergent Small-Cell Neuroendocrine Prostate Cancer: A Multi-institutional Prospective Study.
Purpose The prevalence and features of treatment-emergent small-cell neuroendocrine prostate cancer (t-SCNC) are not well characterized in the era of modern androgen receptor (AR)-targeting therapy. We sought to characterize the clinical and genomic features of t-SCNC in a multi-institutional prospective study. Methods Patients with progressive, metastatic castration-resistant prostate cancer (mCRPC) underwent metastatic tumor biopsy and were followed for survival. Metastatic biopsy specimens underwent independent, blinded pathology review along with RNA/DNA sequencing. Results A total of 202 consecutive patients were enrolled. One hundred forty-eight (73%) had prior disease progression on abiraterone and/or enzalutamide. The biopsy evaluable rate was 79%. The overall incidence of t-SCNC detection was 17%. AR amplification and protein expression were present in 67% and 75%, respectively, of t-SCNC biopsy specimens. t-SCNC was detected at similar proportions in bone, node, and visceral organ biopsy specimens. Genomic alterations in the DNA repair pathway were nearly mutually exclusive with t-SCNC differentiation ( P = .035). Detection of t-SCNC was associated with shortened overall survival among patients with prior AR-targeting therapy for mCRPC (hazard ratio, 2.02; 95% CI, 1.07 to 3.82). Unsupervised hierarchical clustering of the transcriptome identified a small-cell-like cluster that further enriched for adverse survival outcomes (hazard ratio, 3.00; 95% CI, 1.25 to 7.19). A t-SCNC transcriptional signature was developed and validated in multiple external data sets with > 90% accuracy. Multiple transcriptional regulators of t-SCNC were identified, including the pancreatic neuroendocrine marker PDX1. Conclusion t-SCNC is present in nearly one fifth of patients with mCRPC and is associated with shortened survival. The near-mutual exclusivity with DNA repair alterations suggests t-SCNC may be a distinct subset of mCRPC. Transcriptional profiling facilitates the identification of t-SCNC and novel therapeutic targets.


A coiled-coil-repeat protein 'Ccrp' in Bdellovibrio bacteriovorus prevents cellular indentation, but is not essential for vibroid cell morphology.
Bdellovibrio bacteriovorus are small, vibroid, predatory bacteria that grow within the periplasmic space of a host Gram-negative bacterium. The intermediate-filament (IF)-like protein crescentin is a member of a broad class of IF-like, coiled-coil-repeat-proteins (CCRPs), discovered in Caulobacter crescentus, where it contributes to the vibroid cell shape. The B. bacteriovorus genome has a single ccrp gene encoding a protein with an unusually long, stutter-free, coiled-coil prediction; the inactivation of this did not alter the vibriod cell shape, but caused cell deformations, visualized as chiselled insets or dents, near the cell poles and a general 'creased' appearance, under the negative staining preparation used for electron microscopy, but not in unstained, frozen, hydrated cells. Bdellovibrio bacteriovorus expressing 'teal' fluorescent protein (mTFP), as a C-terminal tag on the wild-type Ccrp protein, did not deform under negative staining, suggesting that the function was not impaired. Localization of fluorescent Ccrp-mTFP showed some bias to the cell poles, independent of the cytoskeleton, as demonstrated by the addition of the MreB-specific inhibitor A22. We suggest that the Ccrp protein in B. bacteriovorus contributes as an underlying scaffold, similar to that described for the CCRP protein FilP in Streptomyces coelicolor, preventing cellular indentation, but not contributing to the vibroid shape of the B. bacteriovorus cells.


Symbiosis Specificity of the Preceding Host Plant Can Dominate but Not Obliterate the Association Between Wheat and Its Arbuscular Mycorrhizal Fungal Partners.
The symbiosis established between arbuscular mycorrhizal fungi (AMF) and roots of most land plants plays a key role in plant nutrient acquisition and alleviation of environmental stresses. Despite the ubiquity of the symbiosis, AMF and host species display significant specificity in their interactions. To clarify preferential associations between wheat (Triticum aestivum) and AMF, we characterized root AMF communities in the transition from two first host species, ryegrass (Lolium rigidum) and yellow-serradella (Ornithopus compressus), grown separately or together, to a second host (wheat), by sequencing the large subunit ribosomal DNA (LSU rDNA) gene. The response of AMF communities in wheat to prior soil disturbance - and consequently of the mycelial network [intact extraradical mycelium (ERM) vs. disrupted mycelium] established with either of the first hosts - was also investigated. Since the outcome of a specific host-symbiont interaction depends on the molecular responses of the host plant upon microbial colonization, we studied the expression of six key symbiosis-related genes in wheat roots. AMF communities on L. rigidum and O. compressus roots were clearly distinct. Within an undisturbed ERM, wheat AMF communities were similar to that of previous host, and O. compressus-wheat-AMF interactions supported a greater growth of wheat than L. rigidum-wheat-AMF interactions. This effect declined when ERM was disrupted, but generated a greater activation of symbiotic genes in wheat, indicating that plant symbiotic program depends on some extent on the colonizing symbiont propagule type. When a mixture of L. rigidum and O. compressus was planted, the wheat colonization pattern resembled that of O. compressus, although this was not reflected in a greater growth. These results show a lasting effect of previous hosts in shaping wheat AMF communities through an efficient use of the established ERM, although not completely obliterating host-symbiont specificity.


Cancer statistics for Hispanics/Latinos, 2015.
Cancer is the leading cause of death among Hispanics/Latinos, who represent the largest racial/ethnic minority group in the United States, accounting for 17.4% (55.4 million/318 million) of the total US population in 2014. Every 3 years, the American Cancer Society reports on cancer statistics for Hispanics based on incidence data from the National Cancer Institute, the Centers for Disease Control and Prevention, and the North American Association of Central Cancer Registries and mortality data from the National Center for Health Statistics. Among Hispanics in 2015, there will be an estimated 125,900 new cancer cases diagnosed and 37,800 cancer deaths. For all cancers combined, Hispanics have 20% lower incidence rates and 30% lower death rates compared with non-Hispanic whites (NHWs); however, death rates are slightly higher among Hispanics during adolescence (aged 15-19 years). Hispanic cancer rates vary by country of origin and are generally lowest in Mexicans, with the exception of infection-associated cancers. Liver cancer incidence rates in Hispanic men, which are twice those in NHW men, doubled from 1992 to 2012; however, rates in men aged younger than 50 years declined by 43% since 2003, perhaps a bellwether of future trends for this highly fatal cancer. Variations in cancer risk between Hispanics and NHWs, as well as between subpopulations, are driven by differences in exposure to cancer-causing infectious agents, rates of screening, and lifestyle patterns. Strategies for reducing cancer risk in Hispanic populations include increasing the uptake of preventive services (e.g., screening and vaccination) and targeted interventions to reduce obesity, tobacco use, and alcohol consumption.


Proposal to transfer some members of the genera Haemobartonella and Eperythrozoon to the genus Mycoplasma with descriptions of 'Candidatus Mycoplasma haemofelis', 'Candidatus Mycoplasma haemomuris', 'Candidatus Mycoplasma haemosuis' and 'Candidatus Mycoplasma wenyonii'.
Cell-wall-less uncultivated parasitic bacteria that attach to the surface of host erythrocytes currently are classified in the order Rickettsiales, family Anaplasmataceae, in the genera Haemobartonella and Eperythrozoon. Recently 16S rRNA gene sequences have been determined for four of these species: Haemobartonella felis and Haemobartonella muris and Eperythrozoon suis and Eperythrozoon wenyonii. Phylogenetic analysis of these sequence data shows that these haemotrophic bacteria are closely related to species in the genus Mycoplasma (class Mollicutes). These haemotrophic bacteria form a new phylogenetic cluster within the so-called pneumoniae group of Mycoplasma and share properties with one another as well as with other members of the pneumoniae group. These studies clearly indicate that the classification of these taxa should be changed to reflect their phylogenetic affiliation and the following is proposed: (i) that Haemobartonella felis and Haemobartonella muris should be transferred to the genus Mycoplasma as 'Candidatus Mycoplasma haemofelis' and 'Candidatus Mycoplasma haemomuris' and (ii) that Eperythrozoon suis and Eperythrozoon wenyonii should be transferred to the genus Mycoplasma as 'Candidatus Mycoplasma haemosuis' and 'Candidatus Mycoplasma wenyonii'. The former Haemobartonella and Eperythrozoon species described here represent a new group of parasitic mycoplasmas that possess a pathogenic capacity previously unrecognized among the mollicutes. These haemotrophic mycoplasmas have been given the trivial name haemoplasmas. These results call into question the affiliation of the remaining officially named species of Haemobartonella and Eperythrozoon which should be considered species of uncertain affiliation pending the resolution of their phylogenetic status.


In-stent restenosis limitation with stent-based controlled-release nitric oxide: initial results in rabbits.
To evaluate effect of controlled stent-based release of an NO donor to limit in-stent restenosis in rabbits. Bioerodable microspheres containing NO donor or biodegradable polymer (polylactide-co-glycolide-polyethylene glycol) were prepared and loaded in channeled stents. Daily concentrations of NO release from NO-containing microspheres were assayed in vitro. NO- and polymer-containing (control) microsphere-loaded stents were deployed in aortas of New Zealand white rabbits (n = 8). Aortas with stents were harvested at 7 (n = 5) and 28 days (n = 3) and evaluated for cyclic guanosine monophosphate (cGMP) levels (7 days), number of proliferating cell nuclear antigen-positive cells (7 days), and intima-to-media ratio (7 and 28 days), with statistical significance evaluated by using one-way analysis of variance. NO-containing microspheres released NO with an initial bolus in the 1st week, followed by sustained release for the remaining 3 weeks. Significant increase in cGMP levels and decrease in proliferating cell nuclear antigen-positive cells were found at 7 days for the NO-treated group relative to controls (P <.05). Intima-to-media ratio in the NO-treated group was reduced by 46% and 32% relative to controls at 7 and 28 days, respectively (mean, 0.14 +/- 0.01 [standard error] vs 0.26 +/- 0.02 at 7 days, P <.01; 1.34 +/- 0.05 vs 1.98 +/- 0.08 at 28 days, P <.01). Stent-based controlled release of NO donor significantly reduces in-stent restenosis and is associated with increase in vascular cGMP and suppression of proliferation.


The epidemiology of rotavirus diarrhea in Latin America. Anticipating rotavirus vaccines.
To assess the disease burden and characterize the epidemiology of rotavirus diarrhea in Latin America. We conducted a literature review of studies of children < 5 years of age who were hospitalized or seen as outpatients for diarrhea and for whom rotavirus was sought as the etiologic agent of the diarrhea. This review included inpatient and outpatient studies published since 1998 that included at least 100 children and reported surveillance activities lasting at least 12 consecutive months. A total of 18 inpatient and 10 outpatient studies met the criteria for inclusion in this review. Rotavirus was detected in a median of 31% of inpatients (range, 16%-52%) and 30.5% of outpatients (range, 4%-42%). The median detection rate was higher in studies that used an enzyme-linked immunosorbent assay (ELISA) (inpatients 38%, outpatients 33%) versus less sensitive methods of detection. The age distribution of rotavirus disease varied among countries, with 65%-85% of children hospitalized in the first year of life. Most countries had rotavirus admissions year round, and rotavirus generally exhibited a winter seasonal peak in both temperate and tropical climates. The heavy burden of disease attributable to rotavirus in Latin America suggests that vaccines currently being tested could have considerable impact in preventing hospitalizations, clinic visits, and deaths. The findings of the young age distribution of patients highlight the importance of early immunization for the success of a vaccine program. The data suggest that future surveillance for rotavirus diarrhea in Latin America should use a standardized surveillance protocol with an ELISA for detection. Data from surveillance studies will be critical to monitor the impact of the future introduction of vaccines.


Alcohol Use Severity and Sexual Risk Behavior Among Female Substance Users.
Substance use has been identified as one of the leading factors related to HIV transmission in the United States. The association of problematic drinking with sexual risk behavior puts individuals at greater risk for HIV transmission. This may be of particular concern for women given that approximately 66% of new HIV infections occurring through heterosexual transmission are female. To investigate alcohol use severity and sexual risk behavior among females who use heavy, illicit drugs. Female substances users (N = 251; Mage = 31.90, SD = 7.67; 63.7% Black) self-reported past month alcohol use and lifetime sexual risk behaviors with both casual and steady sex partners. Problematic alcohol users were more likely to use noninjection drugs and less likely to use injection drugs than abstainers and more likely than moderate alcohol users to use alcohol before/during sex with a steady partner. White problematic alcohol users were less likely to use injection drugs before/during sex with a steady partner than abstainers. Black problematic alcohol users were more likely to use non-injection and alcohol than moderate alcohol users before/during sex with steady partners. The current study extends the existing literature by taking a closer look at the role of alcohol use severity in sexual risk taking behavior of Black and White female substance users, a particularly vulnerable group for HIV transmission.


Iliac artery pseudoaneurysm presenting as a paravertebral collection: a potentially fatal mimic.
Pseudoaneurysms of the iliac arteries are extremely rare and can complicate trauma, surgical or interventional procedures. We report a case of pseudoaneurysm arising from the common iliac artery in a 37-year-old man which presented as a paravertebral collection. As the MRI appearance mimicked spondylodiscitis, a CT guided biopsy was requested. However, the presence of a signal void centre guided the radiologist to the correct diagnosis, which was later confirmed on contrast-enhanced CT. Following unsuccessful treatment by percutaneous thrombin injection, open surgery was performed which revealed a leaking aneurysm associated with a huge retroperitoneal haematoma. We present this case to highlight the importance of considering vascular lesions as a differential diagnosis in patients presenting with a mass or pressure symptoms in different parts of the body. Misdiagnosing such lesions as soft tissue tumours or abscesses may result in serious consequences.


Anti-Hepatitis C Virus Activity of a Crude Extract from Longan (Dimocarpus longan Lour.) Leaves.
Infection with hepatitis C virus (HCV) results in hepatitis C, a disease characterized by chronic infection, cirrhosis, and hepatocellular carcinoma. Currently, the standard therapy is a combination of pegylated interferon-α plus ribavirin with NS3 protease inhibitors. Addition of NS3 protease inhibitors to the standard therapy improves response rates; however, use of NS3 protease inhibitors is also associated with significant adverse effects and an increase in the overall cost of treatment. Therefore, there is a need to develop safe and inexpensive drugs for the treatment of HCV infections. In this study, we examined the antiviral activity of a crude extract from Dimocarpus longan leaves against HCV (genotype 2a strain JFH1). The D. longan crude extract (DL-CE) exhibited anti-HCV activity with a 50% effective concentration (EC50) of 19.4 μg/ml without cytotoxicity. A time-of-addition study demonstrated that DL-CE has anti-HCV activity at both the entry and post-entry steps and markedly blocks the viral entry step through direct virucidal activity with marginal inhibition of virion assembly. Co-treatment of DL-CE with cyclosporine A, an immunosuppressant or telaprevir, an NS3 protease inhibitor, resulted in additive and synergistic antiviral effects, respectively. Our findings suggest that DL-CE may be useful as an add-on therapy candidate for treating HCV infections.


Immobilisation of the knee and ankle and its impact on drivers' braking times: a driving simulator study.
The purpose of this study was to investigate the effects of right leg restriction at the knee, ankle or both, on a driver's braking times. Previous studies have not investigated the effects of knee restriction on braking performance. A total of 23 healthy drivers performed a series of emergency braking tests in a driving simulator in either an above-knee plaster cast, a below-knee cast, or in a knee brace with an increasing range of restriction. The study showed that total braking reaction time was significantly longer when wearing an above-knee plaster cast, a below-knee plaster cast or a knee brace fixed at 0°, compared with braking normally (p < 0.001). Increases in the time taken to move the foot from the accelerator to the brake accounted for some of the increase in the total braking reaction time. Unexpectedly, thinking time also increased with the level of restriction (p < 0.001). The increase in braking time with an above-knee plaster cast in this study would increase the stopping distance at 30 miles per hour by almost 3 m. These results suggest that all patients wearing any lower-limb plaster cast or knee brace are significantly impaired in their ability to perform an emergency stop. We suggest changes to the legislation to prevent patients from driving with lower-limb plaster casts or knee braces.


Persistence of improvement in insulin sensitivity following a dietary weight loss programme.
Short-term dietary weight loss can improve insulin resistance but long-term studies are lacking. We sought to quantify the degree to which maintenance of weight loss after a short-term dietary intervention was associated with persistent metabolic benefits. Fifty-seven insulin-resistant obese subjects had insulin-mediated glucose disposal quantified through the steady-state plasma glucose (SSPG) test, and associated metabolic risk markers quantified at baseline, after a 16-week dietary weight loss intervention, and in 25 subjects, at follow-up of 28.8 +/- 13 months. Changes in metabolic variables over time were analysed and correlation with weight loss ascertained. Those with greatest vs. least long-term SSPG response (responders vs. non-responders) were compared. Multivariate analysis was performed for predictors of persistent SSPG response. At follow-up, the 25 subjects who returned for metabolic testing had, on average, maintained their weight loss. Insulin-mediated glucose disposal remained significantly improved vs. baseline, as did plasma triglyceride and HDL cholesterol (HDL-C) concentrations, and improvement correlated with total amount of weight lost. Comparison of SSPG responders to non-responders showed no difference in amount of weight lost and SSPG change during the 16-week dietary intervention; however, SSPG non-responders regained 2.6% of weight lost, whereas responders lost an additional 1.5% at follow-up (p < 0.05 vs. non-responders). Non-responders had baseline characteristics consistent with more severe insulin resistance, including higher fasting plasma glucose (p = 0.03). Long-term SSPG change was independently predicted by both total weight loss (p = 0.005) and baseline fasting plasma glucose (p = 0.007). Improvement in insulin sensitivity is maintained for 2-3 years following dietary weight loss if weight is not regained. Triglyceride and HDL-C concentrations also remain improved over time, consistent with improvement in insulin sensitivity. Fasting glucose and weight regain predict less long-term response in insulin sensitivity. These results highlight the potential long-term benefits of weight loss and importance of preventing weight regain among high-risk individuals.


Induction of cyclooxygenase-2 and invasiveness by transforming growth factor-beta(1) in immortalized mouse colonocytes expressing oncogenic Ras.
Cyclooxygenase-2 (COX-2) expression appears to be important in colorectal carcinogenesis. Elevated COX-2 expression and activity have been observed in several different transformed cell types. Prior studies implicating involvement of the Ras oncogene and growth factors on COX-2 expression were largely derived from rat small intestinal cell lines. We have investigated whether mouse colonocyte COX-2 levels are regulated by oncogenic Ras or transforming growth factor-beta(1) (TGF-beta(1)), and whether these factors also serve to regulate cellular invasiveness. Young adult mouse colonocyte cells are colonocytes derived from the "Immortomouse" and immortalized by the SV40 large T antigen. Young adult mouse colonocyte Ras cells were derived by transfection of young adult mouse colonocyte cells with oncogenic Ha-Ras and are known to be tumorigenic. We found that the induction of COX-2 and eicosanoid release were augmented in the presence of activated Ras and that TGF-beta(1) caused a further increase in COX-2 in the Ras-transformed mouse colonocytes. Increased COX-2 expression was correlated with increased release of prostaglandins E(2) and I(2). Activated Ras and TGF-beta increased the invasiveness of the young adult mouse colonocyte cells, but treatment with a COX-2 inhibitor did not inhibit invasiveness. Thus we found that transforming growth factor-beta collaborates to increase COX-2 expression, protaglandin release, and invasiveness in mouse colonocytes, but the increased COX-2 activity does not appear to contribute to the invasive response.


Home assessment of activities of daily living in patients with severe chronic obstructive pulmonary disease on long-term oxygen therapy.
In patients with severe chronic obstructive pulmonary disease (COPD) using long-term oxygen therapy (LTOT), few studies have investigated activities of daily living (ADL). We examined the relationships between ADL, quality of life, mood state and airways obstruction in patients using long-term oxygen therapy (LTOT) and in patients not requiring LTOT. We studied 23 patients (14 males, 9 females; median age 71, range 60-84 yrs) with COPD who received LTOT using oxygen concentrators (LTOT group). We also studied a control group of 19 patients (14 males, 5 females; median age 72, range 62-75 yrs) with COPD but without severe hypoxaemia (non-LTOT group). We found no significant difference between groups in health status using the St George's Respiratory Questionnaire (SGRQ). Median Nottingham Extended Activities of Daily Living (EADL) total scores were: LTOT group 10, non-LTOT 17; (p=0.01). Significant correlations (p<0.001) with Nottingham EADL score were found for Hospital Anxiety and Depression (HAD) score (rho=0.59), SGRQ Total score (rho=0.65) and percentage predicted forced expiratory volume in one second (FEV1) (rho=0.66). In conclusion, in patients with severe chronic obstructive pulmonary disease and broadly similar health status, those using long-term oxygen therapy were less independent in activities of daily living than those not requiring long-term oxygen therapy. Reduced independence in activities of daily living is, however, associated with the extent of airflow limitation, depression and poor health status, and does not, therefore, appear to be simply a result of restriction in movements imposed by the stationary device.


The in vivo formation and fate of antigen-antibody complexes formed by fragments and polypeptide chains of rabbit gamma-G-antibodies.
The in vivo formation and subsequent fate of complexes formed between specific rabbit gammaG-antibody subunits and circulating protein antigens was studied in rabbits and guinea pigs. Subunits obtained from purified antibodies were injected immediately after an injection of antigen, and the elimination from the circulation of either I*-labeled gammaG-subunits or labeled antigen determined. In the absence of antigen, all gammaG-subunits which lack the Fc fragment were rapidly eliminated. In the presence of excess antigen, F (ab')(2), Fab and Fd fragments reacted with antigen and remained in the circulation as complexes which were eliminated at the same rate as the antigen. Fab hybrids containing a specific Fd fragment and a nonspecific L chain similarly reacted with antigen and remained in the circulation complexed to antigen. In contrast, L chains and Fab hybrids containing a specific L chain and a nonspecific Fd fragment did not react with antigen in vivo and were rapidly eliminated in both presence and absence of antigen. H chains remained in the circulation of rabbits in the absence of antigen, however, in the presence of antigen, more H chains which formed complexes with antigen remained in the intravascular space and were rapidly eliminated when the immune elimination of the antigen by the host occurred. Nonspecific H chains were rapidly eliminated from the circulaion of guinea pigs, whereas specific H chains remained in the circulation with the antigen. F (ab')(2) fragments formed complexes with antigen near antibody equivalence and in antibody excess which were rapidly eliminated, however, less effectively than complexes formed near antibody equivalence with intact gammaG. Complexes formed in antibody excess between Fab fragments and H chains remained in the circulation at all concentrations studied and were eliminated at the rate of antigen. The role of the Fc fragment in the immune elimination of antigen-antibody complexes is discussed.


T-type and L-type Ca2+ currents in canine bronchial smooth muscle: characterization and physiological roles.
We examined the voltage-dependent Ca2+ currents in freshly dissociated smooth muscle cells obtained from canine bronchi (3rd to 5th order). When cells were depolarized from -40 mV, we observed an inward current that 1) exhibited threshold and peak activation at approximately -35 mV and +10 mV, respectively; 2) inactivated slowly with half-inactivation at -20 mV; 3) deactivated rapidly (time constant < 1 ms) upon repolarization; and 4) was abolished by nifedipine and suppressed by cholinergic agonist. These characteristics are typical of L-type Ca2+ current. During depolarization from -70 or -80 mV, however, many cells exhibited a second inward current superimposed on the L-type Ca2+ current. Activation of this other current was first noted at -60 mV, was maximal at approximately -20 mV, and was very rapid (reaching a peak within 10 ms). Inactivation of the other current was also rapid (time constant approximately 3 ms) and half-maximal at approximately -70 mV. There was a persistent "window" current over the physiologically relevant range of potentials (i.e., -60 to -30 mV). This current was also sensitive to nifedipine (although less so than the L-type current) and to Ni2+, but not to cholinergic agonist. Finally, the tail currents evoked upon repolarization to the holding potential decayed approximately 10 times more slowly than did L-type tail currents. These characteristics are all typical of T-type Ca2+ current. We conclude that there is a prominent T-type Ca2+ current in canine bronchial smooth muscle; this current may play a central role in excitation-contraction coupling, in refilling of the internal Ca2+ pool, and in electrical slow waves. Because airflow resistance is determined primarily by the smaller airways and not the trachea, these findings may have important implications with respect to airway physiology and the mechanisms underlying airway hyperreactivity and asthma.


Liposome-nanoparticle hybrids for multimodal diagnostic and therapeutic applications.
Liposomes have a decade-long clinical presence as nanoscale delivery systems of encapsulated anthracycline molecules. However, their use as delivery systems of nanoparticles is still in the preclinical development stages. Liposome-nanoparticle hybrid constructs present great opportunities in terms of nanoscale delivery system engineering for combinatory therapeutic-imaging modalities. Moreover, many novel materials are being developed in nanotechnology laboratories that often require methodologies to enhance their compatibility with the biological milieu in vitro and in vivo. Liposomes are structurally suitable to make nanoparticles biocompatible and offer a clinically proven, versatile platform for the further enhancement of pharmacological efficacy. Small iron oxide nanoparticles, quantum dots, liposomes, silica and polystyrene nanoparticles have been incorporated into liposomes for a variety of different applications. In this review, all such liposome-nanoparticle hybrid systems are described, both in terms of their structural characteristics and the potential they offer as diagnostic and therapeutic multimodality agents.


A non-fatal intoxication and seven deaths involving the dissociative drug 3-MeO-PCP.
3-methoxyphencyclidine (3-MeO-PCP) appeared on the illicit drug market in 2011 and is an analogue of phencyclidine, which exhibits anesthetic, analgesic and hallucinogenic properties. In this paper, we report data from a non-fatal intoxication and seven deaths involving 3-MeO-PCP in Sweden during the period March 2014 until June 2016. The non-fatal intoxication case, a 19-year-old male with drug problems and a medical history of depression, was found awake but tachycardic, hypertensive, tachypnoeic and catatonic at home. After being hospitalized, his condition worsened as he developed a fever and lactic acidosis concomitant with psychomotor agitation and hallucinations. After 22h of intensive care, the patient had made a complete recovery. During his hospitalization, a total of four blood samples were collected at different time points. The seven autopsy cases, six males and one female, were all in their twenties to thirties with psychiatric problems and/or an ongoing drug abuse. 3-MeO-PCP was identified with liquid chromatography (LC)/time-of-flight technology and quantified using LC-tandem mass spectrometry. In the clinical case, the concentration of 3-MeO-PCP was 0.14μg/g at admission, 0.08μg/g 2.5h after admission, 0.06μg/g 5h after admission and 0.04μg/g 17h after admission. The half-life of 3-MeO-PCP was estimated to 11h. In the autopsy cases, femoral blood concentrations ranged from 0.05μg/g to 0.38μg/g. 3-MeO-PCP was the sole finding in the case with the highest concentration and the cause of death was established as intoxication with 3-MeO-PCP. In the remaining six autopsy cases, other medications and drugs of abuse were present as well. Despite being scheduled in January 2015, 3-MeO-PCP continues to be abused in Sweden. Exposure to 3-MeO-PCP may cause severe adverse events and even death, especially if the user does not receive life-supporting treatment.


The interaction between L1-type proteins and ankyrins--a master switch for L1-type CAM function.
L1-type cell adhesion molecules (CAMs) are important mediators of neural differentiation, including axonal outgrowth and pathfinding and also of synapse formation and maintenance. In addition, their interactions with cytoskeletal components are highly conserved and regulated. How these different aspects of CAM functionality relate to each other is not well understood. Based on results from our and other laboratories we propose that ankyrin-binding to L1-type CAMs provides a master switch. The interaction with ankyrins directs L1-type adhesive proteins into different functional contexts, either ankyrin-independent functions, such as neurite outgrowth and axonal pathfinding or into ankyrin-dependent functions, such as L1's role at axon initial segments (AIS), paranodal regions, synapses and in dendrites.


Ankaferd Blood Stopper for controlling gastrointestinal bleeding due to distinct benign lesions refractory to conventional antihemorrhagic measures.
To assess the hemostatic efficacy of the Ankaferd Blood Stopper (ABS, Ankaferd Health Products Ltd, Turkey) hemostatic agent for controlling gastrointestinal bleeding associated with various benign lesions refractory to conventional antihemorrhagic measures. The records of all patients who underwent upper and lower endoscopy procedures at the Turkiye Yuksek Ihtisas Teaching and Research Hospital (Ankara, Turkey) between April 2008 and June 2009 were reviewed. Patients in whom ABS was used as a primary or adjuvant hemostatic agent were included in the study. Rates of bleeding control and postprocedural complications were documented. Hemostasis with no immediate complications was achieved in all patients within seconds of endoscopic application of ABS. ABS may have a role as a primary treatment or as an adjuvant to conventional modalities used to control gastrointestinal bleeding. Prospective controlled studies are needed to help establish its efficacy and, perhaps, offer a comparison with conventional hemostatic interventions.


Phylogenetic relationships within cation transporter families of Arabidopsis.
Uptake and translocation of cationic nutrients play essential roles in physiological processes including plant growth, nutrition, signal transduction, and development. Approximately 5% of the Arabidopsis genome appears to encode membrane transport proteins. These proteins are classified in 46 unique families containing approximately 880 members. In addition, several hundred putative transporters have not yet been assigned to families. In this paper, we have analyzed the phylogenetic relationships of over 150 cation transport proteins. This analysis has focused on cation transporter gene families for which initial characterizations have been achieved for individual members, including potassium transporters and channels, sodium transporters, calcium antiporters, cyclic nucleotide-gated channels, cation diffusion facilitator proteins, natural resistance-associated macrophage proteins (NRAMP), and Zn-regulated transporter Fe-regulated transporter-like proteins. Phylogenetic trees of each family define the evolutionary relationships of the members to each other. These families contain numerous members, indicating diverse functions in vivo. Closely related isoforms and separate subfamilies exist within many of these gene families, indicating possible redundancies and specialized functions. To facilitate their further study, the PlantsT database (http://plantst.sdsc.edu) has been created that includes alignments of the analyzed cation transporters and their chromosomal locations.


Time course of nitric oxide synthases, nitrosative stress, and poly(ADP ribosylation) in an ovine sepsis model.
Different isoforms of nitric oxide synthases (NOS) and determinants of oxidative/nitrosative stress play important roles in the pathophysiology of pulmonary dysfunction induced by acute lung injury (ALI) and sepsis. However, the time changes of these pathogenic factors are largely undetermined. Twenty-four chronically instrumented sheep were subjected to inhalation of 48 breaths of cotton smoke and instillation of live Pseudomonas aeruginosa into both lungs and were euthanized at 4, 8, 12, 18, and 24 hours post-injury. Additional sheep received sham injury and were euthanized after 24 hrs (control). All animals were mechanically ventilated and fluid resuscitated. Lung tissue was obtained at the respective time points for the measurement of neuronal, endothelial, and inducible NOS (nNOS, eNOS, iNOS) mRNA and their protein expression, calcium-dependent and -independent NOS activity, 3-nitrotyrosine (3-NT), and poly(ADP-ribose) (PAR) protein expression. The injury induced severe pulmonary dysfunction as indicated by a progressive decline in oxygenation index and concomitant increase in pulmonary shunt fraction. These changes were associated with an early and transient increase in eNOS and an early and profound increase in iNOS expression, while expression of nNOS remained unchanged. Both 3-NT, a marker of protein nitration, and PAR, an indicator of DNA damage, increased early but only transiently. Identification of the time course of the described pathogenetic factors provides important additional information on the pulmonary response to ALI and sepsis in the ovine model. This information may be crucial for future studies, especially when considering the timing of novel treatment strategies including selective inhibition of NOS isoforms, modulation of peroxynitrite, and PARP.


Tooth decay in alcohol and tobacco abusers.
Alcohol and tobacco abuse are detrimental to general and oral health. Though the effects of these harmful habits on oral mucosa had been demonstrated, their independent and combined effect on the dental caries experience is unknown and worthy of investigation. We compared 268 alcohol-only abusers with 2426 alcohol and tobacco abusers in chewing and smoking forms to test the hypothesis that various components of their dental caries experience are significantly different due to plausible sociobiological explanations. Clinical examination, Decay, Missing, Filled Teeth (DMFT) Index and Oral Hygiene Index - Simplified were measured in a predetermined format. Descriptive statistics, Chi-square test and one-way ANOVA analysis were done using SPSS Version 16.0. The mean DMFT were 3.31, 3.24, 4.09, 2.89 for alcohol-only abusers, alcohol and chewing tobacco abusers, smoking tobacco and alcohol abusers, and those who abused tobacco in smoke and smokeless forms respectively. There was no significant difference between the oral hygiene care measures between the study groups. Presence of attrition among chewers and those with extrinsic stains experienced less caries than others. The entire study population exhibited a higher incidence of caries experience. Use of tobacco in any form appears to substantially increase the risk for dental caries. Attrition with use of chewing tobacco and presence of extrinsic stains with tobacco use appear to provide a protective effect from caries. The changes in oral micro-flora owing to tobacco use and alcohol may play a critical role in the initiation and progression of dental caries.