Association of solubilized angiotensin II receptors with phospholipase C-alpha in murine neuroblastoma NIE-115 cells.
The peptide angiotensin II (AngII) has been reported to stimulate phosphoinositide-specific phospholipase C (PLC) activity in the murine neuroblastoma cell line N1E-115. In the present study, polyclonal antibodies raised against a PLC isoenzyme, PLC-alpha, reacted with a 60-kDa protein present in both membrane and cytosolic fractions of differentiated N1E-115 cells. In order to examine the possible association of PLC-alpha with cell surface AngII receptors (AngII-Rs), membranes from differentiated N1E-115 cells were solubilized, using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). CHAPS (1%) solubilized AngII-Rs, from N1E-115 cells, that maintained their high affinity for agonists. Gel filtration analysis of the solubilized membranes revealed that the majority of the specific binding of 125I-AngII eluted as a large protein complex with a molecular mass of 380 kDa and that agonist binding was partially reduced by guanosine-5'-O-(3-thio)triphosphate (GTP gamma S), within this complex. CHAPS also effectively solubilized immunoreactive PLC-alpha, from N1E-115 cell membranes, that was similarly present within the 380-kDa AngII-binding complex. Anti-PLC-alpha antisera immunoprecipitated approximately 16% of the total phosphatidylinositol-4,5-bisphosphate-specific PLC activity in the 1% CHAPS extract and 40% of cytosolic PLC activity. Moreover, a 60-kDa 35S-Trans S-labeled protein, comigrating with immunoreactive PLC-alpha, was immunoprecipitated from the 1% CHAPS extract by the antisera. In addition, anti-PLC-alpha antisera immunoprecipitated approximately 20% of solubilized AngII-Rs prebound with 125I-AngII but failed to precipitate receptors prebound with the antagonist 125I-Sarc1,Ile8-AngII. The anti-PLC-alpha antisera also immunoprecipitated AngII-Rs when intact membranes were labeled with 125I-AngII before solubilization in 1% CHAPS, suggesting that the AngII-R interaction with PLC-alpha was not the result of detergent-promoted protein-protein interaction. On the other hand, monoclonal antibodies against another PLC isozyme, PLC-gamma, did not precipitate AngII-Rs in solubilized N1E-115 membranes. Finally, the formation of the immunoprecipitated AngII-R-PLC-alpha complex was disrupted by the nonhydrolyzable guanine nucleotide analog GTP gamma S, suggesting that the interaction between AngII-Rs and PLC-alpha is likely to involve a heterotrimeric guanine nucleotide-binding protein in neuron-like cells.


Involvement of environmental mercury and lead in the etiology of neurodegenerative diseases.
The incidence of neurodegenerative disease like Parkinson's disease and Alzheimer's disease (AD) increases dramatically with age; only a small percentage is directly related to familial forms. The etiology of the most abundant, sporadic forms is complex and multifactorial, involving both genetic and environmental factors. Several environmental pollutants have been associated with neurodegenerative disorders. The present article focuses on results obtained in experimental neurotoxicology studies that indicate a potential pathogenic role of lead and mercury in the development of neurodegenerative diseases. Both heavy metals have been shown to interfere with a multitude of intracellular targets, thereby contributing to several pathogenic processes typical of neurodegenerative disorders, including mitochondrial dysfunction, oxidative stress, deregulation of protein turnover, and brain inflammation. Exposure to heavy metals early in development can precondition the brain for developing a neurodegenerative disease later in life. Alternatively, heavy metals can exert their adverse effects through acute neurotoxicity or through slow accumulation during prolonged periods of life. The pro-oxidant effects of heavy metals can exacerbate the age-related increase in oxidative stress that is related to the decline of the antioxidant defense systems. Brain inflammatory reactions also generate oxidative stress. Chronic inflammation can contribute to the formation of the senile plaques that are typical for AD. In accord with this view, nonsteroidal anti-inflammatory drugs and antioxidants suppress early pathogenic processes leading to Alzheimer's disease, thus decreasing the risk of developing the disease. The effects of lead and mercury were also tested in aggregating brain-cell cultures of fetal rat telencephalon, a three-dimensional brain-cell culture system. The continuous application for 10 to 50 days of non-cytotoxic concentrations of heavy metals resulted in their accumulation in brain cells and the occurrence of delayed toxic effects. When applied at non-toxic concentrations, methylmercury, the most common environmental form of mercury, becomes neurotoxic under pro-oxidant conditions. Furthermore, lead and mercury induce glial cell reactivity, a hallmark of brain inflammation. Both mercury and lead increase the expression of the amyloid precursor protein; mercury also stimulates the formation of insoluble beta-amyloid, which plays a crucial role in the pathogenesis of AD and causes oxidative stress and neurotoxicity in vitro. Taken together, a considerable body of evidence suggests that the heavy metals lead and mercury contribute to the etiology of neurodegenerative diseases and emphasizes the importance of taking preventive measures in this regard.


Optimal packing for cascaded regenerative transmission based on phase sensitive amplifiers.
We investigate the transmission performance of advanced modulation formats in nonlinear regenerative channels based on cascaded phase sensitive amplifiers. We identify the impact of amplitude and phase noise dynamics along the transmission line and show that after a cascade of regenerators, densely packed single ring PSK constellations outperform multi-ring constellations. The results of this study will greatly simplify the design of future nonlinear regenerative channels for ultra-high capacity transmission.


An international study of hospitalized cancer patients' health status, nursing care quality, perceived individuality in care and trust in nurses: A path analysis.
Providing high quality nursing care for patients with malignancies is complex and driven by many factors. Many of the associations between nursing care quality, trust, health status and individualized care remain obscure. To empirically test a model of association linking hospitalized cancer patients' health status, nursing care quality, perceived individuality in care and trust in nurses. A cross-sectional, exploratory and correlational study design was used. This multi-site study was conducted in cancer care clinics, in-patient wards of five tertiary care hospitals in Cyprus, Finland, Greece and Sweden. Out of 876 hospitalized patients with a confirmed histopathological diagnosis of cancer approached to participate in the study in consecutive order, 599 (response rate 68%) agreed to participate and the data from 590 were used for path analysis. Data were collected in 2012-2013 with the Individualized Care Scale-Patient (ICS-Patient), the Oncology Patients' Perceptions of Quality Nursing Care Scale (OPPQNCS), the Euro-Qol (EQ-5D-3L) and the Trust in Nurses Scale. Data were analysed statistically using descriptive and inferential statistics. Mplus version 7.11 was used to determine the best Trust model with path analysis. Although the model fit indices suggested that the hypothesized model did not perfectly to the data, a slightly modified model which includes the reciprocal path between individualized care and nursing care quality demonstrated a good fit. A model of trust in nurses was developed. Health status, individualized care, and nursing care quality were found to be associated with trust. The model highlights the complexity of caring for cancer patients. Trust in nurses is influenced by the provision of individualized care. Generating and promoting trust requires interventions, which promote nursing care quality, individuality and patients' health status.


Thalassemys bruntrutana n. sp., a new coastal marine turtle from the Late Jurassic of Porrentruy (Switzerland), and the paleobiogeography of the Thalassemydidae.
Background. The Swiss Jura Mountains are a key region for Late Jurassic eucryptodiran turtles. Already in the mid 19th century, the Solothurn Turtle Limestone (Solothurn, NW Switzerland) yielded a great amount of Kimmeridgian turtles that are traditionally referred to Plesiochelyidae, Thalassemydidae, and Eurysternidae. In the past few years, fossils of these coastal marine turtles were also abundantly discovered in the Kimmeridgian of the Porrentruy region (NW Switzerland). These findings include numerous sub-complete shells, out of which we present two new specimens of Thalassemys (Thalassemydidae) in this study. Methods. We compare the new material from Porrentruy to the type species Th. hugii, which is based on a well preserved specimen from the Solothurn Turtle Limestone (Solothurn, Switzerland). In order to improve our understanding of the paleogeographic distribution of Thalassemys, anatomical comparisons are extended to Thalassemys remains from other European countries, notably Germany and England. Results. While one of the two Thalassemys specimens from Porrentruy can be attributed to Th. hugii, the other specimen represents a new species, Th. bruntrutana n. sp. It differs from Th. hugii by several features: more elongated nuchal that strongly thickens anterolaterally; wider vertebral scales; proportionally longer plastron; broader and less inclined xiphiplastron; wider angle between scapular process and acromion process. Our results show that Th. hugii and Th. bruntrutana also occur simultaneously in the Kimmeridgian of Solothurn as well as in the Kimmeridgian of England (Kimmeridge Clay). This study is an important step towards a better understanding of the paleobiogeographic distribution of Late Jurassic turtles in Europe.


The design of future pediatric mechanical ventilation trials for acute lung injury.
Pediatric practitioners face unique challenges when attempting to translate or adapt adult-derived evidence regarding ventilation practices for acute lung injury or acute respiratory distress syndrome into pediatric practice. Fortunately or unfortunately, there appears to be selective adoption of adult practices for pediatric mechanical ventilation, many of which pose considerable challenges or uncertainty when translated to pediatrics. These differences, combined with heterogeneous management strategies within pediatric critical care, can complicate clinical practice and make designing robust clinical trials in pediatric acute respiratory failure particularly difficult. These issues surround the lack of explicit ventilator protocols in pediatrics, either computer or paper based; differences in modes of conventional ventilation and perceived marked differences in the approach to high-frequency oscillatory ventilation; challenges with patient recruitment; the shortcomings of the definition of acute lung injury and acute respiratory distress syndrome; the more reliable yet still somewhat unpredictable relationship between lung injury severity and outcome; and the reliance on potentially biased surrogate outcome measures, such as ventilator-free days, for all pediatric trials. The purpose of this review is to highlight these challenges, discuss pertinent work that has begun to address them, and propose potential solutions or future investigations that may help facilitate comprehensive trials on pediatric mechanical ventilation and define clinical practice standards.


Insulin-like growth factor-1 and TNF-alpha regulate autophagy through c-jun N-terminal kinase and Akt pathways in human atherosclerotic vascular smooth cells.
A balance between programmed cell death and survival of vascular smooth muscle cells (VSMC) in the fibrous cap, which is primarily composed of VSMC and extracellular matrix, appears to best correlate with plaque instability or stability and is controlled by growth factors and cytokines. Autophagy is also involved in programmed cell death. We assessed the effect of TNF-alpha and insulin-like growth factor-1 (IGF-1) on the expression of autophagic genes, microtubule-associated protein 1 light chain 3 (MAPLC-3) and Beclin-1 in VSMC isolated from atherosclerotic plaques. Transmission electron microscopy showed a significantly higher number of vacuolated cells in the TNF-alpha-treated VSMC and a markedly lower number in the IGF-1-treated VSMC when compared with the untreated control group. TNF-alpha-induced MAPLC-3 mRNA expression through c-jun N-terminal kinase and protein kinase B pathways and induced Beclin-1 protein expression through the c-jun N-terminal kinase pathway. Expression of MAPLC-3 and Beclin-1 correlated with autophagic cell death of plaque VSMC. IGF-1 inhibited MAPLC-3 mRNA transcripts through the Akt pathway. These findings suggest that the expression of autophagy genes can be influenced by IGF-1 and TNF-alpha through c-jun N-terminal kinase or Akt pathways and autophagy might be involved in the regulation of plaque stability.


High dietary NaCl early in development enhances mean arterial pressure of adult rats.
We investigated the long-term influence of early dietary NaCl on adult mean arterial pressure (MAP) and heart rate (HR), assessed continuously for 7 weeks when fed water and chow containing 1% NaCl (weeks 1, 4, 7), 0% NaCl (weeks 2-3), and 3% NaCl (weeks 5-6) while on a 12:12 light/dark cycle. Subjects were offspring of female Sprague-Dawley rats fed solid chow consisting of either 0.1% (basal), 1% (intermediate), or 3% (high) NaCl throughout pregnancy and lactation. After weaning on postnatal day (PD) 21, offspring were fed the same NaCl diet of their mother until PD 30, at which time all offspring were given Purina 5001 diet (1% NaCl) as their solid chow. On PD 60, 22 adult male offspring (eight basals, six intermediates, eight highs) were implanted with an aortic electronic sensor for transmitting blood-pressure signals by telemetry. MAP and HR varied significantly across the 7-week testing period in association with dietary NaCl levels. The three perinatal salt groups had similar HR levels on normal 1% NaCl chow. However, HR increased in all groups during 0% NaCl and decreased in all groups during 3% NaCl. In contrast, the average MAP levels were significantly greater in the highs compared to both the intermediate and basal groups during both dark and light periods. MAP increased in high and basal groups during both 0% and 3%; however, the increase was more pronounced in the highs. In conclusion, exposure to high levels of dietary NaCl early in development led to a persistent increase in MAP in adulthood.


Advances in multiplex PCR: balancing primer efficiencies and improving detection success.
1. Multiplex PCR is a valuable tool in many biological studies but it is a multifaceted procedure that has to be planned and optimised thoroughly to achieve robust and meaningful results. In particular, primer concentrations have to be adjusted to assure an even amplification of all targeted DNA fragments. Until now, total DNA extracts were used for balancing primer efficiencies; however, the applicability for comparisons between taxa or different multiple-copy genes was limited owing to the unknown number of template molecules present per total DNA. 2. Based on a multiplex system developed to track trophic interactions in high Alpine arthropods, we demonstrate a fast and easy way of generating standardised DNA templates. These were then used to balance the amplification success for the different targets and to subsequently determine the sensitivity of each primer pair in the multiplex PCR. 3. In the current multiplex assay, this approach led to an even amplification success for all seven targeted DNA fragments. Using this balanced multiplex PCR, methodological bias owing to variation in primer efficiency will be avoided when analysing field-derived samples. 4. The approach outlined here allows comparing multiplex PCR sensitivity, independent of the investigated species, genome size or the targeted genes. The application of standardised DNA templates not only makes it possible to optimise primer efficiency within a given multiplex PCR, but it also offers to adjust and/or to compare the sensitivity between different assays. Along with other factors that influence the success of multiplex reactions, and which we discuss here in relation to the presented detection system, the adoption of this approach will allow for direct comparison of multiplex PCR data between systems and studies, enhancing the utility of this assay type.


Factors influencing disease phenotype and penetrance in HFE haemochromatosis.
Haemochromatosis is predominantly associated with the HFE p.C282Y homozygous genotype, which is present in approximately 1 in 200 people of Northern European origin. However, not all p.C282Y homozygotes develop clinical features of haemochromatosis, and not all p.C282Y homozygotes even present abnormal iron parameters justifying venesection therapy. This situation was not apparent from initial genotype/phenotype correlation studies as there was a selection bias of patients. Only those patients with a significant iron burden were included in these early studies. It is now largely accepted that the p.C282Y/p.C282Y genotype is necessary for the development of HFE haemochromatosis. However, this genotype provides few clues as to why certain symptoms are associated with the disease. Expression of iron overload in people with this genotype depends on the complex interplay of environmental factors and modifier genes. In this review, we restrict our discussion to work done in humans giving examples of animal models where this has helped clarify our understanding. We discuss penetrance, explaining that this concept normally does not apply to autosomal recessive disorders, and discuss the usefulness of different biochemical markers in ascertaining iron burden. Hepcidin, a peptide synthesized primarily by the liver, has been identified as the central regulator in iron homeostasis. Consequently, understanding its regulation is the key. We conclude that the main goal now is to identify important modifiers that have a significant and unambiguous effect on iron storage.


Dramatis Personae in β-Cell Mass Regulation: Enter SerpinB1.
How is β-cell mass adjusted to changes in the functional insulin requirements? The answer to this question is central to the understanding of the causes and (potentially) the therapy of type 2 diabetes. In this issue of Cell Metabolism, El Ouaamari et al. (2016) report that increased production of the protease inhibitor SerpinB1 in the liver links insulin resistance to stimulation of β-cell proliferation.


The transcription factors islet and Lim3 combinatorially regulate ion channel gene expression.
Expression of appropriate ion channels is essential to allow developing neurons to form functional networks. Our previous studies have identified LIM-homeodomain (HD) transcription factors (TFs), expressed by developing neurons, that are specifically able to regulate ion channel gene expression. In this study, we use the technique of DNA adenine methyltransferase identification (DamID) to identify putative gene targets of four such TFs that are differentially expressed in Drosophila motoneurons. Analysis of targets for Islet (Isl), Lim3, Hb9, and Even-skipped (Eve) identifies both ion channel genes and genes predicted to regulate aspects of dendritic and axonal morphology. Significantly, some ion channel genes are bound by more than one TF, consistent with the possibility of combinatorial regulation. One such gene is Shaker (Sh), which encodes a voltage-dependent fast K(+) channel (Kv1.1). DamID reveals that Sh is bound by both Isl and Lim3. We used body wall muscle as a test tissue because in conditions of low Ca(2+), the fast K(+) current is carried solely by Sh channels (unlike neurons in which a second fast K(+) current, Shal, also contributes). Ectopic expression of isl, but not Lim3, is sufficient to reduce both Sh transcript and Sh current level. By contrast, coexpression of both TFs is additive, resulting in a significantly greater reduction in both Sh transcript and current compared with isl expression alone. These observations provide evidence for combinatorial activity of Isl and Lim3 in regulating ion channel gene expression.


The end-systolic pressure-volume relationship in conscious dogs.
The end-systolic pressure-volume relationship (ESPVR) has been shown to be an afterload-insensitive descriptor of ventricular inotropic state in the isolated heart. The purpose of this study was to examine the effects of changes in afterload, heart rate, intravascular volume, autonomic tone, and inotropic state on the ESPVR in conscious dogs. In 30 dogs, left ventricular and pleural pressures were measured with micromanometers, and left ventricular volume was assessed with global ultrasonic crystals. The ESPVR was obtained during vena caval occlusions in each dog during pharmacologic afterload interventions at control and after autonomic blockade. Analysis of variance techniques were used to compare the slopes (Emax) and intercepts (Vd) of ESPVR regression lines in a given study. All estimates of the ESPVR in conscious dogs involved large extrapolations to obtain estimates of Vd. Repeat determinations of Emax at control in the unblocked state were significantly different in six of eight dogs (p less than .05). After autonomic blockade, these differences were significant in only one of eight dogs. Changes in heart rate and volume loading had minimal effects on the ESPVR. In the absence of autonomic blockade, increases in inotropic state with either calcium or dobutamine tended to cause parallel shifts in the ESPVR. After autonomic blockade, Emax increased with augmentation of inotropic state, while Vd was unchanged. ESPVRs obtained at different afterloads showed statistically significant differences in Emax and in Vd in 12 of 14 dogs. However, no statistically significant relationship of Emax to afterload was observed. Thus, the ESPVR is probably valid in conscious dogs, but measurement with an intact cardiovascular system is hampered by statistically significant variability in Emax and Vd with changes in afterload. Baseline variability is magnified by the autonomic nervous system, probably mediated through sympathetic reflexes.


Uses and abuses of digital imaging in plastic surgery.
Surgeons extensively rely on photographic communication for documenting surgical results, teaching and research, and obtaining informed consent from patients. With the advent of digital photography and widespread availability of sophisticated image manipulation software, the potential for committing digital fraud cannot be discounted. Ten 'before' and 'after' plastic surgical photographs were selected, and a number of them were digitally enhanced using a standard desktop software by a non-expert in digital photography. A panel of 10 consultant plastic surgeons was asked to judge which, if any of the images had been digitally manipulated. Expert assessment had a sensitivity of only 12% in identifying digitally manipulated images. Furthermore, there was poor interobserver agreement with an Intraclass Correlation Coefficient of 0.39. Digital fraud is easy to commit and difficult to detect. Furthermore, a number of inadvertent and simple image manipulation functions can also amount to misrepresentation. There may be scope for cooperation within editorial circles to set standards for the submission of digital photographs. Surgeons need also to be aware of the potential for misrepresentation of information through digital image manipulation and exercise caution in the communication of digital photographic information.


Detection of waterborne parasites using field-portable and cost-effective lensfree microscopy.
Protection of human health and well-being through water quality management is an important goal for both the developed and the developing parts of the world. In the meantime, insufficient disinfection techniques still fail to eliminate pathogenic contaminants in freshwater as well as recreational water resources. Therefore, there is a significant need for screening of water quality to prevent waterborne outbreaks and incidents of water-related diseases. Toward this end, here we investigate the use of a field-portable and cost-effective lensfree holographic microscope to image and detect pathogenic protozoan parasites such as Giardia Lamblia and Cryptosporidium Parvum at low concentration levels. This compact lensless microscope (O. Mudanyali et al., Lab Chip, 2010, 10, 1417-1428), weighing approximately 46 grams, achieves a numerical aperture of approximately 0.1-0.2 over an imaging field of view that is more than an order of magnitude larger than a typical 10X objective lens, and therefore may provide an important high-throughput analysis tool for combating waterborne diseases especially in resource limited settings.


Synthesis of type III collagen by fibroblasts from the embryonic chick cornea.
Synthesis of collagen types I, II, III, and IV in cells from the embryonic chick cornea was studied using specific antibodies and immunofluorescence. Synthesis of radioactively labeled collagen types I and III was followed by fluorographic detection of cyanogen bromide peptides on polyacrylamide slab gels and by carboxymethylcellulose chromatography followed by disc gel electrophoresis. Type III collagen had been detected previously by indirect immunofluorescence in the corneal epithelial cells at Hamburger-Hamilton stages 20--30 but not in the stroma at any age. Intact corneas from embryos older than stage 30 contain and synthesize type I collagen but no detectable type III collagen. However, whole stromata subjected to collagenase treatment and scraping (to remove epithelium and endothelium) and stromal fibroblasts from such corneas inoculated in vitro begin synthesis of type III collagen within a few hours while continuing to synthesize type I collagen. As demonstrated by double-antibody staining, most corneal fibroblasts contain collagen types I and III simultaneously. Collagen type III was identified biochemically in cell layers and media after chromatography on carboxymethylcellulose be detection of disulfide-linked alpha l (III)3 by SDS gel electrophoresis. The conditions under which the corneal fibroblasts gain the ability to synthesize type III collagen are the same as those under which they lose the ability to synthesize the specific proteoglycan of the cornea: the presence of corneal-type keratan sulfate.


Acylcarnitines: potential implications for skeletal muscle insulin resistance.
Insulin resistance may be linked to incomplete fatty acid β-oxidation and the subsequent increase in acylcarnitine species in different tissues including skeletal muscle. It is not known if acylcarnitines participate in muscle insulin resistance or simply reflect dysregulated metabolism. The aims of this study were to determine whether acylcarnitines can elicit muscle insulin resistance and to better understand the link between incomplete muscle fatty acid β-oxidation, oxidative stress, inflammation, and insulin-resistance development. Differentiated C2C12, primary mouse, and human myotubes were treated with acylcarnitines (C4:0, C14:0, C16:0) or with palmitate with or without carnitine acyltransferase inhibition by mildronate. Treatment with C4:0, C14:0, and C16:0 acylcarnitines resulted in 20-30% decrease in insulin response at the level of Akt phosphorylation and/or glucose uptake. Mildronate reversed palmitate-induced insulin resistance concomitant with an ∼25% decrease in short-chain acylcarnitine and acetylcarnitine secretion. Although proinflammatory cytokines were not affected under these conditions, oxidative stress was increased by 2-3 times by short- or long-chain acylcarnitines. Acylcarnitine-induced oxidative stress and insulin resistance were reversed by treatment with antioxidants. Results are consistent with the conclusion that incomplete muscle fatty acid β-oxidation causes acylcarnitine accumulation and associated oxidative stress, raising the possibility that these metabolites play a role in muscle insulin resistance.


Glucose-responsive neurons of the paraventricular thalamus control sucrose-seeking behavior.
Feeding behavior is governed by homeostatic needs and motivational drive to obtain palatable foods. Here, we identify a population of glutamatergic neurons in the paraventricular thalamus of mice that express the glucose transporter Glut2 (encoded by Slc2a2) and project to the nucleus accumbens. These neurons are activated by hypoglycemia and, in freely moving mice, their activation by optogenetics or Slc2a2 inactivation increases motivated sucrose-seeking but not saccharin-seeking behavior. These neurons may control sugar overconsumption in obesity and diabetes.


A SAM oligomerization domain shapes the genomic binding landscape of the LEAFY transcription factor.
Deciphering the mechanisms directing transcription factors (TFs) to specific genome regions is essential to understand and predict transcriptional regulation. TFs recognize short DNA motifs primarily through their DNA-binding domain. Some TFs also possess an oligomerization domain suspected to potentiate DNA binding but for which the genome-wide influence remains poorly understood. Here we focus on the LEAFY transcription factor, a master regulator of flower development in angiosperms. We have determined the crystal structure of its conserved amino-terminal domain, revealing an unanticipated Sterile Alpha Motif oligomerization domain. We show that this domain is essential to LEAFY floral function. Moreover, combined biochemical and genome-wide assays suggest that oligomerization is required for LEAFY to access regions with low-affinity binding sites or closed chromatin. This finding shows that domains that do not directly contact DNA can nevertheless have a profound impact on the DNA binding landscape of a TF.


Network incorporation of intermediate filament molecules differs between preexisting and newly assembling filaments.
When studying the way in which intermediate filaments assemble in vivo, it is important to distinguish between the incorporation of intermediate filament proteins into an existing intermediate filament network and the ability to form a new network within cells. To distinguish between these alternatives, we have made a hybrid construct consisting of the rod and tail domains of murine glial fibrillary acidic protein (GFAP) coupled to the head domain of bovine keratin 19, called K19GFAP. The assembly characteristics of K19GFAP were analyzed in vitro and in vivo. Replacement of the head domain with the bovine K19 sequence did not prevent the incorporation of K19GFAP into the existing network of vimentin intermediate filaments in NIH 3T3 cells but it was incompatible with de novo formation of filament networks in the epithelial cell line MCF-7, which lacks an endogenous vimentin network. By in vitro assembly studies, it was confirmed that K19GFAP was unable to assemble into typical intermediate filaments. We also investigated the ability of an appropriate type II keratin partner to rescue K19GFAP from incorporation into a vimentin network and initiate de novo filament assembly, using the fibroblast cell line KF-K8(3), an NIH 3T3 fibroblast cell line expressing a single human keratin, K8. The results confirm the importance of the coiled coil interactions in determining the fate of intermediate filament proteins. The results also emphasize that filament networks can not only tolerate but also incorporate assembly-deficient intermediate filament protein subunits.


CD34 expression on long-term repopulating hematopoietic stem cells changes during developmental stages.
The CD34 antigen serves as an important marker for primitive hematopoietic cells in therapeutic transplantation of hematopoietic stem cells (HSC) and gene therapy, but it has remained an open question as to whether or not most HSC express CD34. Using a competitive long-term reconstitution assay, the results of this study confirm developmental changes in CD34 expression on murine HSC. In fetuses and neonates, CD34 was expressed on Lin(-)c-Kit(+) long-term repopulating HSC of bone marrow (BM), liver, and spleen. However, CD34 expression on HSC decreased with aging, and in mice older than 10 weeks, HSC were most enriched in the Lin(-)c-Kit(+)CD34(-) marrow cell fraction. A second transplantation was performed from primary recipients who were transplanted with neonatal Lin(-)c-Kit(+) CD34(high) HSC marrow. Although donor-type HSC resided in CD34-expressing cell fraction in BM cells of the first recipients 4 weeks after the first transplantation, the stem cell activity had shifted to Lin(-)c-Kit(+)CD34(-) cells after 16 weeks, indicating that adult Lin(-)c-Kit(+)CD34(-) HSC are the progeny of neonatal CD34-expresssing HSC. Assays for colony-forming cells showed that hematopoietic progenitor cells, unlike HSC, continue to express CD34 throughout murine development. The present findings are important because the clinical application of HSC can be extended, in particular as related to CD34-enriched HSC and umbilical cord blood HSC.


Use of modified OSCEs to verify technical skill performance and competency of lay caregivers.
The demands on lay caregivers of blood and marrow stem-cell transplant recipients are complex. Effective caregiver education that clearly delineates and demonstrates what needs to be learned is needed, as well as a method to evaluate caregiver's skill-based competencies. A pilot study was conducted to determine the feasibility of adapting objective structured clinical examinations (OSCEs) for teaching and evaluation in lay caregiver educational programs. A modified OSCE, a performance-based assessment process, with seven practice stations for the care of tunneled central lines was developed and tested with 13 laypersons. Inter-rater techniques were used to establish accuracy and reproducibility of the examination. Reproducibility of station scenarios and accuracy in performance evaluation were supported by an overall kappa coefficient of 0.94, indicating excellent agreement. Participants' overall mean performance scores were 80% for knowledge and 94% for skills. The use of modified OSCEs for teaching and evaluating lay caregivers' competencies for complex care demands is feasible, accurate, and warrants further investigation.


Climate change impacts on mass movements--case studies from the European Alps.
This paper addresses the current knowledge on climate change impacts on mass movement activity in mountain environments by illustrating characteristic cases of debris flows, rock slope failures and landslides from the French, Italian, and Swiss Alps. It is expected that events are likely to occur less frequently during summer, whereas the anticipated increase of rainfall in spring and fall could likely alter debris-flow activity during the shoulder seasons (March, April, November, and December). The magnitude of debris flows could become larger due to larger amounts of sediment delivered to the channels and as a result of the predicted increase in heavy precipitation events. At the same time, however, debris-flow volumes in high-mountain areas will depend chiefly on the stability and/or movement rates of permafrost bodies, and destabilized rock glaciers could lead to debris flows without historic precedents in the future. The frequency of rock slope failures is likely to increase, as excessively warm air temperatures, glacier shrinkage, as well as permafrost warming and thawing will affect and reduce rock slope stability in the direction that adversely affects rock slope stability. Changes in landslide activity in the French and Western Italian Alps will likely depend on differences in elevation. Above 1500 m asl, the projected decrease in snow season duration in future winters and springs will likely affect the frequency, number and seasonality of landslide reactivations. In Piemonte, for instance, 21st century landslides have been demonstrated to occur more frequently in early spring and to be triggered by moderate rainfalls, but also to occur in smaller numbers. On the contrary, and in line with recent observations, events in autumn, characterized by a large spatial density of landslide occurrences might become more scarce in the Piemonte region.


Regulation of neurotrophin receptor expression during embryonic and postnatal development.
Members of the NGF family of proteins act as neurotrophic agents for defined populations of peripheral and central neurons during embryonic and postnatal development. We have studied the presence of receptors for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 and -4/5 (NT-3, NT-4/5) by cross-linking radioiodinated neurotrophins to specific cell surface receptors. We have identified neurotrophin receptors representing full-length TrkB and TrkC and their truncated forms (lacking a functional cytoplasmic kinase domain) in neuronal as well as in non-neuronal tissues. During chicken embryonic and early postnatal brain development, expression of full-length TrkB and TrkC proteins preceded the onset of the truncated forms of these receptors. A similar pattern was also observed in mouse embryonic and early postnatal brain. The relative levels of neurotrophin receptors in the basal forebrain and in the hippocampus did not change significantly with age in mice. High levels of receptors for the three neurotrophins were detected in the nigrostriatal system. Full-length TrkB and TrkC receptors were found in chicken and rat embryonic ventral spinal cord, as well as on purified motoneurons. Again, truncated TrkB appeared significantly later than the full-length form on spinal motoneurons. In chicken embryonic retina and optic tectum we detected full-length TrkB and TrkC; however, the optic tectum also expressed large amounts of the truncated form of TrkB. TrkC but not TrkB was detected in chicken embryonic skeletal muscle, suggesting that NT-3 may have a novel function in this tissue. The presence of neurotrophin receptors in a wide variety of embryonic and postnatal tissues underlines the significant role of BDNF, NT-3, and NT-4/5 in embryonic and postnatal development. The regulation of the ratio of full-length versus truncated neurotrophin receptors may play an important role in the development, maturation, and maintenance of various neuronal networks.


The toxicity of antiprion antibodies is mediated by the flexible tail of the prion protein.
Prion infections cause lethal neurodegeneration. This process requires the cellular prion protein (PrP(C); ref. 1), which contains a globular domain hinged to a long amino-proximal flexible tail. Here we describe rapid neurotoxicity in mice and cerebellar organotypic cultured slices exposed to ligands targeting the α1 and α3 helices of the PrP(C) globular domain. Ligands included seven distinct monoclonal antibodies, monovalent Fab1 fragments and recombinant single-chain variable fragment miniantibodies. Similar to prion infections, the toxicity of globular domain ligands required neuronal PrP(C), was exacerbated by PrP(C) overexpression, was associated with calpain activation and was antagonized by calpain inhibitors. Neurodegeneration was accompanied by a burst of reactive oxygen species, and was suppressed by antioxidants. Furthermore, genetic ablation of the superoxide-producing enzyme NOX2 (also known as CYBB) protected mice from globular domain ligand toxicity. We also found that neurotoxicity was prevented by deletions of the octapeptide repeats within the flexible tail. These deletions did not appreciably compromise globular domain antibody binding, suggesting that the flexible tail is required to transmit toxic signals that originate from the globular domain and trigger oxidative stress and calpain activation. Supporting this view, various octapeptide ligands were not only innocuous to both cerebellar organotypic cultured slices and mice, but also prevented the toxicity of globular domain ligands while not interfering with their binding. We conclude that PrP(C) consists of two functionally distinct modules, with the globular domain and the flexible tail exerting regulatory and executive functions, respectively. Octapeptide ligands also prolonged the life of mice expressing the toxic PrP(C) mutant, PrP(Δ94-134), indicating that the flexible tail mediates toxicity in two distinct PrP(C)-related conditions. Flexible tail-mediated toxicity may conceivably play a role in further prion pathologies, such as familial Creutzfeldt-Jakob disease in humans bearing supernumerary octapeptides.


The effects of stress during early postnatal periods on behavior and hippocampal neuroplasticity markers in adult male mice.
Infancy is a critical period for brain development. Emerging evidence indicates that stress experienced during that period can have long-term programming effects on the brain and behavior. However, whether different time periods represent different vulnerabilities to the programming of different neurobehavioral domains is not yet known. Disrupted maternal care is known to interfere with neurodevelopmental processes and may lead to the manifestation of behavioral abnormalities in adulthood. Mouse dams confronted with insufficient bedding/nesting material have been shown to provide fragmented maternal care to their offspring. Here, we compared the impact of this model of early-life stress (ELS) during different developmental periods comprising either postnatal days (PNDs) 2-9 (ELS-early) or PND 10-17 (ELS-late) on behavior and hippocampal cell adhesion molecules in male mice in adulthood. ELS-early treatment caused a permanent reduction in bodyweight, whereas this reduction only occurred transiently during juvenility in ELS-late mice. Anxiety was only affected in ELS-late mice, while cognition and sociability were equally impaired in both ELS-treated groups. We analyzed hippocampal gene expression of the γ2 subunit of the GABAa receptor (Gabrg2) and of genes encoding cell adhesion molecules. Gabrg2 expression was increased in the ventral hippocampus in ELS-late-treated animals and was correlated with anxiety-like behavior in the open-field (OF) test. ELS-early-treated animals exhibited an increase in nectin-1 expression in the dorsal hippocampus, and this increase was associated with the social deficits seen in these animals. Our findings highlight the relevance of developmental age on stress-induced long-term behavioral alterations. They also suggest potential links between early stress-induced alterations in hippocampal Gabrg2 expression and the developmental programming of anxiety and between changes in hippocampal nectin-1 expression and stress-induced social impairments.


Long-term health-related outcomes in survivors of childhood cancer treated with HSCT versus conventional therapy: a report from the Bone Marrow Transplant Survivor Study (BMTSS) and Childhood Cancer Survivor Study (CCSS).
HSCT is being increasingly offered as a curative option for children with hematologic malignancies. Although survival has improved, the long-term morbidity ascribed to the HSCT procedure is not known. We compared the risk of chronic health conditions and adverse health among children with cancer treated with HSCT with survivors treated conventionally, as well as with sibling controls. HSCT survivors were drawn from BMTSS (N = 145), whereas conventionally treated survivors (N = 7207) and siblings (N = 4020) were drawn from CCSS. Self-reported chronic conditions were graded with CTCAEv3.0. Fifty-nine percent of HSCT survivors reported ≥ 2 conditions, and 25.5% reported severe/life-threatening conditions. HSCT survivors were more likely than sibling controls to have severe/life-threatening (relative risk [RR] = 8.1, P < .01) and 2 or more (RR = 5.7, P < .01) conditions, as well as functional impairment (RR = 7.7, P < .01) and activity limitation (RR = 6.3, P < .01). More importantly, compared with CCSS survivors, BMTSS survivors demonstrated significantly elevated risks (severe/life-threatening conditions: RR = 3.9, P < .01; multiple conditions: RR = 2.6, P < .01; functional impairment: RR = 3.5, P < .01; activity limitation: RR = 5.8, P < .01). Unrelated donor HSCT recipients were at greatest risk. Childhood HSCT survivors carry a significantly greater burden of morbidity not only compared with noncancer populations but also compared with conventionally treated cancer patients, providing evidence for close monitoring of this high-risk population.


Pharmacist-initiated general practitioner referral of patients with suboptimal asthma management.
To assess the impact of an intervention initiated by community pharmacists, involving the provision of educational material and general practitioner (GP) referral, on asthma knowledge and self-reported asthma control and asthma-related quality of life (QOL) in patients who may have suboptimal management of their asthma, as evidenced by pharmacy dispensing records. Setting Community pharmacies throughout Tasmania, Australia. Forty-two pharmacies installed a software application that data mined dispensing records and generated a list of patients with suboptimal asthma management, as indicated by having three or more canisters of inhaled short-acting beta-2-agonists dispensed in the preceding 6 months. Identified patients were randomised to an intervention or control group. At baseline, intervention patients were mailed intervention packs consisting of a letter encouraging them to see their GP for a review, educational material, asthma knowledge, asthma control and asthma-related QOL questionnaires, and a letter with a dispensing history to give to their GP. Pharmacists were blinded to the control patients' identities for 6 months, after which time intervention patients were sent repeat questionnaires, and control patients were sent intervention packs. Asthma knowledge, asthma control and asthma-related QOL scores. Thirty-five pharmacies completed the study, providing 706 intervention and 427 control patients who were eligible to receive intervention packs. Intervention patients' asthma control and asthma-related QOL scores at 6 months were significantly higher compared to the control patients (P < 0.01 and P < 0.05, respectively) and to the intervention patients' baseline scores (P < 0.001 and P < 0.05, respectively). Symptom-related QOL was significantly higher compared to the control patients (P < 0.01) and activities-related QOL significantly improved compared to baseline (P < 0.05). No significant change was observed in asthma knowledge. The results suggest that community pharmacists are ideally placed to identify patients with suboptimal asthma management and refer such patients for a review by their GP. This type of collaborative intervention can significantly improve self-reported asthma control and asthma-related QOL in patients identified as having suboptimal management of their asthma. A larger trial is needed to confirm the effects are real and sustained.


Evaluation of the anesthetic and cardiorespiratory effects of intramuscular alfaxalone administration and isoflurane in budgerigars (Melopsittacus undulatus) and comparison with manual restraint.
OBJECTIVE To evaluate the anesthetic and cardiorespiratory effects of IM alfaxalone and isoflurane administration in budgerigars (Melopsittacus undulatus) and compare use of these agents with use of manual restraint. ANIMALS 42 healthy budgerigars. PROCEDURES For dose comparison, birds received alfaxalone (5 or 10 mg/kg [2.27 or 4.54 mg/lb], IM; groups A5 and A10, respectively; n = 6/group). For treatment comparison, birds received alfaxalone (10 mg/kg, IM) or isoflurane (via face mask) or were manually restrained (groups A, I, and M, respectively; n = 10/group). Data were obtained on onset, degree, and duration of sedation or anesthesia; heart and respiratory rates; and recovery times. Birds in the treatment comparison underwent physical examination and blood gas analysis. RESULTS All group A5 birds became sedate, but not recumbent. In group A10, 5 of 6 birds lost the righting reflex; however, none lost the noxious stimulus response. Median time to initial effects was significantly shorter and mean time to complete recovery was significantly longer in group A10 than in group A5. Heart and respiratory rates in group A10 remained clinically acceptable; however, some birds had signs of excitement during induction and recovery. Times to initial effects, recumbency, and complete recovery were significantly longer, yet clinically practical, in group A than in group I. Plasma lactate concentrations were significantly higher in group M than in groups A and I. CONCLUSIONS AND CLINICAL RELEVANCE Alfaxalone administered IM at 10 mg/kg produced effective sedation in healthy budgerigars and may be a viable alternative to isoflurane and manual restraint for brief, minimally invasive procedures. Brief manual restraint resulted in a significant increase in plasma lactate concentration.


Clustering Patterns Connecting COVID-19 Dynamics and Human Mobility Using Optimal Transport.
Social distancing and stay-at-home are among the few measures that are known to be effective in checking the spread of a pandemic such as COVID-19 in a given population. The patterns of dependency between such measures and their effects on disease incidence may vary dynamically and across different populations. We described a new computational framework to measure and compare the temporal relationships between human mobility and new cases of COVID-19 across more than 150 cities of the United States with relatively high incidence of the disease. We used a novel application of Optimal Transport for computing the distance between the normalized patterns induced by bivariate time series for each pair of cities. Thus, we identified 10 clusters of cities with similar temporal dependencies, and computed the Wasserstein barycenter to describe the overall dynamic pattern for each cluster. Finally, we used city-specific socioeconomic covariates to analyze the composition of each cluster.


Imaging macropinosomes during Shigella infections.
Macropinocytosis is the uptake of extracellular fluid within vesicles of varying size that takes place during numerous cellular processes in a large variety of cells. A growing number of pathogens, including viruses, parasites, and bacteria are known to induce macropinocytosis during their entry into targeted host cells. We have recently discovered that the human enteroinvasive, bacterial pathogen Shigella causes in situ macropinosome formation during its entry into epithelial cells. These infection-associated macropinosomes are not generated to ingest the bacteria, but are instead involved in Shigella's intracellular niche formation. They make contacts with the phagocytosed shigellae to promote vacuolar membrane rupture and their cytosolic release. Here, we provide an overview of the different imaging approaches that are currently used to analyze macropinocytosis during infectious processes with a focus on Shigella entry. We detail the advantages and disadvantages of genetically encoded reporters as well as chemical probes to trace fluid phase uptake. In addition, we report how such reporters can be combined with ultrastructural approaches for correlative light electron microscopy either in thin sections or within large volumes. The combined imaging techniques introduced here provide a detailed characterization of macropinosomes during bacterial entry, which, apart from Shigella, are relevant for numerous other ones, including Salmonella, Brucella or Mycobacteria.


A computed tomography-based spatial normalization for the analysis of [18F] fluorodeoxyglucose positron emission tomography of the brain.
We developed a new computed tomography (CT)-based spatial normalization method and CT template to demonstrate its usefulness in spatial normalization of positron emission tomography (PET) images with [(18)F] fluorodeoxyglucose (FDG) PET studies in healthy controls. Seventy healthy controls underwent brain CT scan (120 KeV, 180 mAs, and 3 mm of thickness) and [(18)F] FDG PET scans using a PET/CT scanner. T1-weighted magnetic resonance (MR) images were acquired for all subjects. By averaging skull-stripped and spatially-normalized MR and CT images, we created skull-stripped MR and CT templates for spatial normalization. The skull-stripped MR and CT images were spatially normalized to each structural template. PET images were spatially normalized by applying spatial transformation parameters to normalize skull-stripped MR and CT images. A conventional perfusion PET template was used for PET-based spatial normalization. Regional standardized uptake values (SUV) measured by overlaying the template volume of interest (VOI) were compared to those measured with FreeSurfer-generated VOI (FSVOI). All three spatial normalization methods underestimated regional SUV values by 0.3-20% compared to those measured with FSVOI. The CT-based method showed slightly greater underestimation bias. Regional SUV values derived from all three spatial normalization methods were correlated significantly (p < 0.0001) with those measured with FSVOI. CT-based spatial normalization may be an alternative method for structure-based spatial normalization of [(18)F] FDG PET when MR imaging is unavailable. Therefore, it is useful for PET/CT studies with various radiotracers whose uptake is expected to be limited to specific brain regions or highly variable within study population.


Wideband slow light with low dispersion in asymmetric slotted photonic crystal waveguides.
A new procedure of designing slotted photonic crystal waveguides is proposed to achieve slow light with improved normalized delay-bandwidth product and low group velocity dispersion that is suitable for both the W1 defect mode and the slot mode. The lateral symmetry of the waveguide in our study is broken by shifting the air holes periodically along the slot axis. The conversion of the "flat band" from band-up slow light to band-down slow light is achieved for the W1 defect mode. The group index curves of the W1 mode change from U-like to step-like and the group indices of 47, 67 and 130 are obtained with the bandwidth over 7.2, 4.8, and 2.3 nm around 1550 nm, respectively. We also obtain the group indices of 42, 55, and 108 for the slot mode with the bandwidth over 6.2, 5.6, and 2.2 nm, respectively. Then the low dispersion slow light propagation is numerically demonstrated by the finite-difference time-domain method.


The posterior parietal cortex remaps touch into external space.
Localizing tactile events in external space is required for essential functions such as orienting, haptic exploration, and goal-directed action in peripersonal space. In order to map somatosensory input into a spatiotopic representation, information about skin location must be integrated with proprioceptive information about body posture. We investigated the neural bases of this tactile remapping mechanism in humans by disrupting neural activity in the putative human homolog of the monkey ventral intraparietal area (hVIP), within the right posterior parietal cortex (rPPC), which is thought to house external spatial representations. Participants judged the elevation of touches on their (unseen) forearm relative to touches on their face. Arm posture was passively changed along the vertical axis, so that elevation judgments required the use of an external reference frame. Single-pulse transcranial magnetic stimulation (TMS) over the rPPC significantly impaired performance compared to a control site (vertex). Crucially, proprioceptive judgments of arm elevation or tactile localization on the skin remained unaffected by rPPC TMS. This selective disruption of tactile remapping suggests a distinct computational process dissociable from pure proprioceptive and somatosensory localization. Furthermore, this finding highlights the causal role of human PPC, putatively VIP, in remapping touch into external space.


Direct T cell activation via CD40 ligand generates high avidity CD8+ T cells capable of breaking immunological tolerance for the control of tumors.
CD40 and CD40 ligand (CD40L) are costimulatory molecules that play a pivotal role in the proinflammatory immune response. Primarily expressed by activated CD4+ T cells, CD40L binds to CD40 on antigen presenting cells (APCs), thereby inducing APC activation. APCs, in turn, prime cytotoxic T lymphocytes (CTLs). Here, two tumor-associated antigen (TAA) animal models, p53-based and GP100-based, were utilized to examine the ability of CD40-CD40L to improve antigen-specific CTL-mediated antitumor immune responses. Although p53 and GP100 are self-antigens that generate low affinity antigen-specific CD8+ T cells, studies have shown that their functional avidity can be improved with CD40L-expressing APCs. Therefore, in the current study, we immunized mice with a DNA construct encoding a TAA in conjunction with another construct encoding CD40L via intramuscular injection followed by electroporation. We observed a significant increase in the antigen-specific CTL-mediated immune responses as well as the potent antitumor effects in both models. Antibody depletion experiments demonstrated that CD8+ T cells play a crucial role in eliciting antitumor effects in vaccinated mice. Furthermore, we showed that in vitro stimulation with irradiated tumor cells expressing both TAA and CD40L improved the functional avidity of antigen-specific CD8+ T cells. Thus, our data show that vaccination with TAA/CD40L DNA can induce potent antitumor effects against TAA-expressing tumors through the generation of better functioning antigen-specific CD8+ T cells. Our study serves as an important foundation for future clinical translation.


MALAT1 inhibits the Wnt/β-catenin signaling pathway in colon cancer cells and affects cell proliferation and apoptosis.
Metastasis associated lung adenocarcinoma transcript 1 (MALAT1) is a highly conserved long noncoding RNA, which has been related to various pathological processes, including cancer. The role and mechanism of MALAT1 in colon cancer are not clear. We investigated MALAT1 expression in colon cancer tissues, the effect of MALAT1 on proliferation and apoptosis of SW480 cells, and the signaling pathway involved in the MALAT1 effects. MALAT1 expression was determined in 60 colon cancer and para-carcinoma tissues using reverse transcription polymerase chain reaction (RT-PCR). Knockdown of MALAT1 in SW480 cells was induced by small interfering RNA (siRNA), and the cells were divided into three groups: untreated control, nonsense siRNA-treated control, and MALAT1 siRNA-treated group. SW480 cell apoptosis was assessed using TUNEL assay and flow cytometry. Apoptosis-related and Wnt/β-catenin signaling pathway-related proteins were detected by Western blotting in SW480 cells. SW480 cell proliferation was assessed by CCK-8 assay. MALAT1 expression was significantly higher in colon cancer vs. para-carcinoma tissues. Knockdown of MALAT1 by siRNA increased the number of apoptotic cells and the apoptosis rate at 24 h post-transfection in SW480 cells. Bcl2 associated X protein (Bax) expression was increased, B-cell lymphoma 2 (Bcl-2) expression was decreased, and the ratio of cleaved caspase-3 to truncated caspase-3 was increased in MALAT1 siRNA-transfected SW480 cells. MALAT1 knockdown decreased the proliferation of SW480 cells at 24 h, 48 h, and 72 h post-transfection. Wnt and β-catenin expression was inhibited in MALAT1 siRNA-transfected SW480 cells. Inhibition of MALAT1 expression in colon cancer may promote apoptosis and hinder cell proliferation by suppressing the activation of Wnt/β-catenin signaling pathway.


Exome sequencing-based copy-number variation and loss of heterozygosity detection: ExomeCNV.
The ability to detect copy-number variation (CNV) and loss of heterozygosity (LOH) from exome sequencing data extends the utility of this powerful approach that has mainly been used for point or small insertion/deletion detection. We present ExomeCNV, a statistical method to detect CNV and LOH using depth-of-coverage and B-allele frequencies, from mapped short sequence reads, and we assess both the method's power and the effects of confounding variables. We apply our method to a cancer exome resequencing dataset. As expected, accuracy and resolution are dependent on depth-of-coverage and capture probe design. CRAN package 'ExomeCNV'. firstname.lastname@example.com; email@example.com Supplementary data are available at Bioinformatics online.


Long-term cognitive sequelae of acyclovir-treated herpes simplex encephalitis.
Survival from untreated herpes simplex type 1 encephalitis is well known to be accompanied by severe cognitive impairments. Recently, acyclovir has been proven to be the most effective available treatment for this disease, with the expectation that it would appreciably reduce morbidity. We performed detailed assessments of four consecutive patients who received acyclovir in the early stages of biopsy-proven herpes encephalitis and who now have been followed up for 1.5 to 4 years. All four patients showed definite residual on either clinical or formal neuropsychological testing, most commonly dysnomia and impaired new learning for both verbal and visual material, even though three had normal performance on a standard clinical mental status test. All four patients were unable to function at their prior level of achievement. Therefore, despite early administration of acyclovir in herpes encephalitis, long-lasting neuropsychological residua are likely. Furthermore, cognitive deficits of prognostic importance may not be detected by clinical screening.


Examination of genitalia in children: 'the remaining taboo'.
Trained student observers rated 123 physical examinations by pediatric house staff during health care maintenance visits to assess the frequency of examinations done on male and female genitalia compared with examination of ears, heart, and abdomen. Sixty-five observations were made of 21 male house staff and 58 observations of 18 female house staff. One to five examinations per physician were observed. Whereas physicians examined the ears, heart, and abdomen of pediatric patients greater than or equal to 97% of the time regardless of the sex or age of the child, female genitalia were examined 39% of the time and male genitalia 84% of the time. Female genitalia were examined approximately half as frequently as male genitalia at all ages with a trend to less frequent genital examinations on older children. Both male and female physicians examined female genitalia less frequently than male genitalia. It is concluded that increased emphasis is needed in house staff training programs on the examination of genitalia.


Genotype-phenotype association and biochemical analyses of glucose-6-phosphate dehydrogenase variants: Implications for the hemolytic risk of using 8-aminoquinolines for radical cure.
Background: Plasmodium vivax remains the malaria species posing a major threat to human health worldwide owing to its relapse mechanism. Currently, the only drugs of choice for radical cure are the 8-aminoquinolines (primaquine and tafenoquine), which are capable of killing hypnozoites and thus preventing P. vivax relapse. However, the therapeutic use of primaquine and tafenoquine is restricted because these drugs can cause hemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. This study aimed to assess and understand the hemolytic risk of using 8-aminoquinolines for radical treatment in a malaria endemic area of Thailand. Methods: The prevalence of G6PD deficiency was determined using a quantitative test in 1,125 individuals. Multiplexed high-resolution meltinging (HRM) assays were developed and applied to detect 12 G6PD mutations. Furthermore, biochemical and structural characterization of G6PD variants was carried out to understand the molecular basis of enzyme deficiency. Results: The prevalence of G6PD deficiency was 6.76% (76/1,125), as assessed by a phenotypic test. Multiplexed HRM assays revealed G6PD Mahidol in 15.04% (77/512) of males and 28.38% (174/613) of females, as well as G6PD Aures in one female. G6PD activity above the 30% cut-off was detected in those carrying G6PD Mahidol, even in hemizygous male individuals. Two variants, G6PD Murcia Oristano and G6PD Songklanagarind + Viangchan, were identified for the first time in Thailand. Biochemical characterization revealed that structural instability is the primary cause of enzyme deficiency in G6PD Aures, G6PD Murcia Oristano, G6PD Songklanagarind + Viangchan, and G6PD Chinese 4 + Viangchan, with double G6PD mutations causing more severe enzyme deficiency. Conclusion: In western Thailand, up to 22% of people may be ineligible for radical cure. Routine qualitative tests may be insufficient for G6PD testing, so quantitative tests should be implemented. G6PD genotyping should also be used to confirm G6PD status, especially in female individuals suspected of having G6PD deficiency. People with double G6PD mutations are more likely to have hemolysis than are those with single G6PD mutations because the double mutations significantly reduce the catalytic activity as well as the structural stability of the protein.


Attention deficit hyperactivity disorder, other mental health problems, substance use, and driving: examination of a population-based, representative canadian sample.
The purpose of this study is to examine the relationships among self-reported screening measures of attention deficit hyperactivity disorder (ADHD), other psychiatric problems, and driving-related outcomes in a provincially representative sample of adults 18 years and older living in the province of Ontario, Canada. The study examined the results of the Centre for Addictions and Mental Health (CAMH) Ontario Monitor, an ongoing repeated cross-sectional telephone survey of Ontario adults over a 2-year period. Measures included ADHD measures (Adult ADHD Self-Report Scale-V1.1 [ASRS-V1.1], previous ADHD diagnosis, ADHD medication use); psychiatric distress measures (General Health Questionnaire [GHQ12], use of pain, anxiety, and depression medication); antisocial behavior measure (The Antisocial Personality Disorder Scale from the Mini-International Neuropsychiatric Interview [APD]); substance use and abuse measures (alcohol, cannabis, and cocaine), Alcohol Use Disorders Identification Test (AUDIT), Alcohol, Smoking and Substance Involvement Screening Test (ASSIST), driving-related outcomes (driving after drinking, driving after cannabis use, street racing, collisions in past year), and sociodemographics (gender, age, vehicle-kilometers traveled). A total of 4,014 Ontario residents were sampled, of which 3,485 reported having a valid driver's license. Overall, 3.22% screened positive for ADHD symptoms on the ASRS-V1.1 screening tool. A greater percentage of those who screened positive were younger, reported previous ADHD diagnosis and medication use, distress, antisocial behavior, anti-anxiety and antidepressant medication use, substance use, and social problems compared to those who screened negative. However, there were no statistically significant differences between those who screened positive or negative for ADHD symptoms on self-reported driving after having 2 or more drinks in the previous hour; within an hour of using cannabis, marijuana, or hash; or in a street race or collision involvement as a driver in the past year. When a sequential regression was conducted to predict self-reported collisions, younger age and higher weekly kilometers driven showed higher odds of collision involvement, and the odds ratio for cannabis use ever approached statistical significance. This study is the first population-based study of a representative sample of adults 18 years and older living in Ontario, Canada. These results showed no relationship between the ADHD screen and collision when age, sex, and kilometers driven are controlled for. However, these analyses are based on self-report screeners and not psychiatric diagnoses and a limited sample of ADHD respondents. Thus, these results should be interpreted with caution.


Estimation of indices of health service readiness with a principal component analysis of the Tanzania Service Provision Assessment Survey.
Service Provision Assessment (SPA) surveys have been conducted to gauge primary health care and family planning clinical readiness throughout East and South Asia as well as sub-Saharan Africa. Intended to provide useful descriptive information on health system functioning to supplement the Demographic and Health Survey data, each SPA produces a plethora of discrete indicators that are so numerous as to be impossible to analyze in conjunction with population and health survey data or to rate the relative readiness of individual health facilities. Moreover, sequential SPA surveys have yet to be analyzed in ways that provide systematic evidence that service readiness is improving or deteriorating over time. This paper presents an illustrative analysis of the 2006 Tanzania SPA with the goal of demonstrating a practical solution to SPA data utilization challenges using a subset of variables selected to represent the six building blocks of health system strength identified by the World Health Organization (WHO) with a focus on system readiness to provide service. Principal Components Analytical (PCA) models extract indices representing common variance of readiness indicators. Possible uses of results include the application of PCA loadings to checklist data, either for the comparison of current circumstances in a locality with a national standard, for the ranking of the relative strength of operation of clinics, or for the estimation of trends in clinic service quality improvement or deterioration over time. Among hospitals and health centers in Tanzania, indices representing two components explain 32% of the common variance of 141 SPA indicators. For dispensaries, a single principal component explains 26% of the common variance of 86 SPA indicators. For hospitals/HCs, the principal component is characterized by preventive measures and indicators of basic primary health care capabilities. For dispensaries, the principal component is characterized by very basic newborn care as well as preparedness for delivery. PCA of complex facility survey data generates composite scale coefficients that can be used to reduce indicators to indices for application in comparative analyses of clinical readiness, or for multi-level analysis of the impact of clinical capability on health outcomes or on survival.


Reflex parasympathetic coronary vasodilation elicited from cardiac receptors in the dog.
Veratrum alkaloids injected into the coronary circulation stimulate myocardial receptors to produce reflex bradycardia and arterial hypotension (the Bezold-Jarisch reflex). This study investigated the hypothesis that parasympathetic coronary vasodilation occurs as part of the Bezold-Jarisch reflex. Blood flow in the circumflex coronary artery was measured in chloralose-anesthetized, closed-chest dogs with a newly developed cannula-tip flow transducer. Alpha-receptor blockade with Dibozane (2 mg/kg) was used to prevent peripheral vasodilation, and beta-receptor blockade with propranolol (1 mg/kg) was used to prevent adrenergic cardiac effects. Electrical pacing was used to maintain a constant heart rate. Under these conditions, veratridine injected into the anterior descending coronary artery but not into the circumflex coronary artery produced a 63% increase in circumflex coronary blood flow and an 88% increase in diastolic coronary conductance. The effect was abolished when the reflex arc was interrupted by either vagotomy or atropine administration. It is concluded that a cardiocoronary reflex parasympathetic coronary vasodilation can be elicited by stimulating cardiac receptors with veratridine.


Comparison of regional cerebral blood flow responses to hypoglycemia using pulsed arterial spin labeling and positron emission tomography.
Different brain regions sense and modulate the counterregulatory responses that can occur in response to declining plasma glucose levels. The aim of this study was to determine if changes in regional cerebral blood flow (rCBF) during hypoglycemia relative to euglycemia are similar for two imaging modalities-pulsed arterial spin labeling magnetic resonance imaging (PASL-MRI) and positron emission tomography (PET). Nine healthy non-diabetic participants underwent a hyperinsulinemic euglycemic (92±3 mg/dL) - hypoglycemic (53±1 mg/dL) clamp. Counterregulatory hormone levels were collected at each of these glycemic levels and rCBF measurements within the previously described network of hypoglycemia-responsive regions (thalamus, medial prefrontal cortex and globus pallidum) were obtained using PASL-MRI and [(15)O] water PET. In response to hypoglycemia, rCBF was significantly increased in the thalamus, medial prefrontal cortex, and globus pallidum compared to euglycemia for both PASL-MRI and PET methodologies. Both imaging techniques found similar increases in rCBF in the thalamus, medial prefrontal cortex, and globus pallidum in response to hypoglycemia. These brain regions may be involved in the physiologic and symptom responses to hypoglycemia. Compared to PET, PASL-MRI may provide a less invasive, less expensive method for assessing changes in rCBF during hypoglycemia without radiation exposure.


Order-disorder transition in thin films of horizontally-oriented cylinder-forming block copolymers: thermal fluctuations vs. preferential wetting.
We present experimental and theoretical investigations of the order-disorder transition (ODT) in thin films of cylinder-forming diblock copolymers with asymmetric wetting conditions. Grazing incidence small-angle X-ray scattering (GISAXS) was implemented to determine the ODT temperatures (TODT) for poly(styrene-b-2-vinyl pyridine) (PS-P2VP) block copolymer thin films on a P2VP-preferential silicon substrate. Specifically, films consisting of multilayers of horizontally-oriented cylindrical structures (from 1- to 9-layers) were tested. We find that films with more than 2 cylindrical layers have a TODT comparable to the bulk case. However, TODT decreases as the film becomes thinner and the monolayer system has an ODT 30 °C below the bulk. Using self-consistent field theory (SCFT), we studied the ordering in corresponding thin films with asymmetric (top and bottom surface) wetting conditions. Surprisingly, SCFT is found to predict an opposite trend in TODT with film thickness than observed experimentally. Field-theoretic simulations with complex Langevin sampling were employed to resolve this discrepancy and demonstrate that thermal fluctuations in the PS-P2VP thin-film system dominate its ordering behavior in monolayer and bilayer films.


Hearing aid use in everyday life: managing contextual variability.
We investigated usage of and satisfaction with hearing aid devices in everyday life among older adults with hearing loss. Our research further advances the role of hearing contexts for hearing aid use and satisfaction. A central assumption was that hearing aid owners adapt the usage of the hearing aid devices to contextual demands of hearing depending on their personal resources. In a sample of 158 hearing aid owners aged 50-88 years, we examined proactive hearing aid use in everyday life and its association with the usage of, and satisfaction with, hearing aids. The study was administered online. Using an adapted version of the day-reconstruction method, participants reported hearing episodes during the preceding day. Hearing aid use was assessed via the proportion of waking time with hearing aids. Satisfaction with hearing aids was measured via the Satisfaction with Daily Amplification scale. When using the hearing aids more often, and when reporting greater satisfaction, older adults indicated more diverse listening situations and experienced less variability in hearing quality with hearing aids. Our findings suggest that hearing aid owners may proactively use the hearing devices to master situation-specific demands and difficulties in hearing quality. Such findings underline that hearing aid use and satisfaction depend on the extent to which the devices help to manage everyday life successfully.


Direct healthcare costs of sedentary behaviour in the UK.
Growing evidence indicates that prolonged sedentary behaviour increases the risk of several chronic health conditions and all-cause mortality. Sedentary behaviour is prevalent among adults in the UK. Quantifying the costs associated with sedentary behaviour is an important step in the development of public health policy. National Health Service (NHS) costs associated with prolonged sedentary behaviour (≥6 hours/day) were estimated over a 1-year period in 2016-2017 costs. We calculated a population attributable fraction (PAF) for five health outcomes (type 2 diabetes, cardiovascular disease [CVD], colon cancer, endometrial cancer and lung cancer). Adjustments were made for potential double-counting due to comorbidities. We also calculated the avoidable deaths due to prolonged sedentary behaviour using the PAF for all-cause mortality. The total NHS costs attributable to prolonged sedentary behaviour in the UK in 2016-2017 were £0.8 billion, which included expenditure on CVD (£424 million), type 2 diabetes (£281 million), colon cancer (£30 million), lung cancer (£19 million) and endometrial cancer (£7 million). After adjustment for potential double-counting, the estimated total was £0.7 billion. If prolonged sedentary behaviour was eliminated, 69 276 UK deaths might have been avoided in 2016. In this conservative estimate of direct healthcare costs, prolonged sedentary behaviour causes a considerable burden to the NHS in the UK. This estimate may be used by decision makers when prioritising healthcare resources and investing in preventative public health programmes.


Effects of near-future ocean acidification, fishing, and marine protection on a temperate coastal ecosystem.
Understanding ecosystem responses to global and local anthropogenic impacts is paramount to predicting future ecosystem states. We used an ecosystem modeling approach to investigate the independent and cumulative effects of fishing, marine protection, and ocean acidification on a coastal ecosystem. To quantify the effects of ocean acidification at the ecosystem level, we used information from the peer-reviewed literature on the effects of ocean acidification. Using an Ecopath with Ecosim ecosystem model for the Wellington south coast, including the Taputeranga Marine Reserve (MR), New Zealand, we predicted ecosystem responses under 4 scenarios: ocean acidification + fishing; ocean acidification + MR (no fishing); no ocean acidification + fishing; no ocean acidification + MR for the year 2050. Fishing had a larger effect on trophic group biomasses and trophic structure than ocean acidification, whereas the effects of ocean acidification were only large in the absence of fishing. Mortality by fishing had large, negative effects on trophic group biomasses. These effects were similar regardless of the presence of ocean acidification. Ocean acidification was predicted to indirectly benefit certain species in the MR scenario. This was because lobster (Jasus edwardsii) only recovered to 58% of the MR biomass in the ocean acidification + MR scenario, a situation that benefited the trophic groups lobsters prey on. Most trophic groups responded antagonistically to the interactive effects of ocean acidification and marine protection (46%; reduced response); however, many groups responded synergistically (33%; amplified response). Conservation and fisheries management strategies need to account for the reduced recovery potential of some exploited species under ocean acidification, nonadditive interactions of multiple factors, and indirect responses of species to ocean acidification caused by declines in calcareous predators.


Signaling mechanisms of angiotensin II-induced attenuation of GABAergic input to hypothalamic presympathetic neurons.
The hypothalamic paraventricular nucleus (PVN) is an important site for the regulation of sympathetic outflow. Angiotensin II (Ang II) can activate AT(1) receptors to stimulate PVN presympathetic neurons through inhibition of GABAergic input. However, little is known about the downstream pathway involved in this presynaptic action of Ang II in the PVN. In this study, using whole cell recording from retrogradely labeled PVN neurons in rat brain slices, we determined the signaling mechanisms responsible for the effect of Ang II on synaptic GABA release to spinally projecting PVN neurons. Bath application of Ang II reproducibly decreased the frequency of GABAergic miniature postsynaptic inhibitory currents (mIPSCs) in fluorescence-labeled PVN neurons. Ang II failed to change the frequency of mIPSCs in labeled PVN neurons treated with pertussis toxin. However, Ang II-induced inhibition of mIPSCs persisted in the presence of either CdCl(2), a voltage-gated Ca(2+) channel blocker, or 4-aminopyridine, a blocker of voltage-gated K(+) channels. Interestingly, inhibition of superoxide with superoxide dismutase or Mn(III) tetrakis (4-benzoic acid) prophyrin completely blocked Ang II-induced decrease in mIPSCs. By contrast, inhibition of hydroxyl radical formation with the ion chelator deferoxamine did not significantly alter the effect of Ang II. These findings suggest that the presynaptic action of Ang II on synaptic GABA release in the PVN is mediated by the pertussis toxin-sensitive G(i/o) proteins but not by voltage-gated Ca(2+) and K(+) channels. Ang II attenuates GABAergic input to PVN presympathetic neurons through reactive oxygen species, especially superoxide anions.


The validity and reliability of continuous-wave near-infrared spectroscopy for the assessment of leg blood volume during an orthostatic challenge.
Near-infrared spectroscopy (NIRS) can potentially be used to assess the cardiovascular autonomic system by monitoring orthostatic challenge-induced shifts in lower limb blood volume. However, in order to be of clinical utility the test must be valid, reliable, and relatively simple to conduct. To induce lower limb blood volume shifts using a 10 min 70° head-up tilt, and: (1) in the soleus, determine the validity of an inexpensive continuous wave (cw)-NIRS device by comparing to a criterion frequency-domain (fd-) NIRS device, (2) determine the between-day reliability of soleus assessments obtained from cw-NIRS and fd-NIRS; and, (3) compare the between-day reliability for fd-NIRS assessments obtained at the soleus (standard) and gastrocnemius (simpler alternative). Fifteen non-smoking healthy adults were tested on 3 different mornings, under standardized conditions, separated by a maximum of 7 days. Total haemoglobin concentration (tHb) was continuously monitored bi-laterally in the medial soleus using cw-NIRS and fd-NIRS. For site comparison, tHb was measured in the medial gastrocnemius using fd-NIRS. (1) The area under the curve (AUC) for cw-NIRS and fd-NIRS assessments at the soleus were not significantly different (p = 0.619). (2) The criterion (0.75) intra-class correlation coefficient (ICC) was exceeded for both cw-NIRS and fd-NIRS. (3) The criterion ICC was exceeded for both soleus and gastrocnemius assessments. Continuous-wave NIRS can be used to monitor orthostatic stress-induced shifts in lower leg blood volume with acceptable validity and reliability. This orthostatic test may present a relatively simple and inexpensive approach for assessing the cardiovascular autonomic nervous system.


Variability of total step activity in children with cerebral palsy: influence of definition of a day on participant retention within the study.
Activity monitoring is important to establish accurate daily physical activity levels in children with cerebral palsy (CP). However, few studies address issues around inclusion or exclusion of step count data; in particular, how a valid day should be defined and what impact different lengths of monitoring have on retention of participant data within a study. This study assessed how different 'valid day' definitions influenced inclusion of participant data in final analyses and the subsequent variability of the data. Sixty-nine children with CP were fitted with a StepWatch™ Activity Monitor and instructed to wear it for a week. Data analysis used two broad definitions of a day, based on either number of steps in a 24 h monitoring period or the number of hours of recorded activity in a 24 h monitoring period. Eight children either did not use the monitor, or used it for only 1 day. The remaining 61 children provided 2 valid days of monitoring defined as >100 recorded steps per 24 h period and 55 (90 %) completed 2 valid days of monitoring with ≥10 h recorded activity per 24 h period. Performance variability in daily step count was lower across 2 days of monitoring when a valid day was defined as ≥10 h recorded activity per 24 h period (ICC = 0.765) and, higher when the definition >100 recorded steps per 24 h period (ICC = 0.62). Only 46 participants (75 %) completed 5 days of monitoring with >100 recorded steps per 24 h period and only 23 (38 %) achieved 5 days of monitoring with ≥10 h recorded activity per 24 h period. Datasets of participants who functioned at GMFCS level II were differentially excluded when the criteria for inclusion in final analysis was 5 valid days of ≥10 h recorded activity per 24 h period, leaving datasets available for only 8 of 32 participant datasets retained in the study. We conclude that changes in definition of a valid day have significant impacts on both inclusion of participant data in final analysis and measured variability of total step count.


Hunter-gatherer multilevel sociality accelerates cumulative cultural evolution.
Although multilevel sociality is a universal feature of human social organization, its functional relevance remains unclear. Here, we investigated the effect of multilevel sociality on cumulative cultural evolution by using wireless sensing technology to map inter- and intraband social networks among Agta hunter-gatherers. By simulating the accumulation of cultural innovations over the real Agta multicamp networks, we demonstrate that multilevel sociality accelerates cultural differentiation and cumulative cultural evolution. Our results suggest that hunter-gatherer social structures [based on (i) clustering of families within camps and camps within regions, (ii) cultural transmission within kinship networks, and (iii) high intercamp mobility] may have allowed past and present hunter-gatherers to maintain cumulative cultural adaptation despite low population density, a feature that may have been critical in facilitating the global expansion of Homo sapiens.


Neural representations of location composed of spatially periodic bands.
The mammalian hippocampal formation provides neuronal representations of environmental location, but the underlying mechanisms are poorly understood. Here, we report a class of cells whose spatially periodic firing patterns are composed of plane waves (or bands) drawn from a discrete set of orientations and wavelengths. The majority of cells recorded in parasubicular and medial entorhinal cortices of freely moving rats belonged to this class and included grid cells, an important subset that corresponds to three bands at 60° orientations and has the most stable firing pattern. Occasional changes between hexagonal and nonhexagonal patterns imply a common underlying mechanism. Our results indicate a Fourier-like spatial analysis underlying neuronal representations of location, and suggest that path integration is performed by integrating displacement along a restricted set of directions.


Sequential expressions of Notch1, Jagged2 and Math1 in molar tooth germ of mouse.
The Notch signaling pathway is an evolutionary conserved mechanism that plays an important role in cell-cell communication and cell fate in a wide range of tissues. The mammalian family of Notch receptors consists of 4 members: Notch1/2/3/4. The Notch ligand family consists of 5 members: Delta1/3/4 and Jagged1/2. Math1 encodes a murine basic helix-loop-helix (bHLH) transcription factor that acts as positive regulator of cell differentiation. Recently, links between Notch and Math1 pathways were demonstrated in various tissues. Expression of Notch1, Jagged2 and Math1 were analyzed in the mouse molar tooth germ during embryonic stage (E) 13 and E15 and during postnatal stage (PN) 1, PN3, PN5, PN10 and PN14 by using in situ hybridization. Positive Notch1 expression was found at the tooth bud during embryonic stages, but its expression was absent from the basal cells in contact with the dental mesenchyme. Jagged2 and Math1 were strongly expressed in differentiated ameloblasts and odontoblasts and Math1 strong expression was even maintained until PN14 stage. Math1 showed the strongest expression. Our results suggest that the Notch1 signaling pathway through Jagged2 could be importantly related to Math1, directing the process of odontogenesis toward cell differentiation.


Usefulness of N-Terminal Pro-Brain Natriuretic Peptide to Predict Mortality in Adults With Congenital Heart Disease.
Natriuretic peptides are often elevated in congenital heart disease (CHD); however, the clinical impact on mortality is unclear. The aim of our study was to evaluate the prognostic value of N-terminal pro-brain natriuretic peptide (NT-proBNP) in the prediction of all-cause mortality in adults with different CHD. In this prospective longitudinal mortality study, we evaluated NT-proBNP in 1,242 blood samples from 646 outpatient adults with stable CHD (mean age 35 ± 12 years; 345 women). Patients were followed up for 6 ± 3 (1 to 10) years. The mortality rate was 5% (35 patients, mean age 40 ± 14 years, 17 women). Median NT-proBNP (pg/ml) was 220 in the whole cohort, 203 in survivors, and 1,548 in deceased patients. The best discrimination value for mortality prediction was 630 pg/ml with 74% sensitivity and 84% specificity. During the follow-up, the survival rate was 65% for those with median NT-proBNP ≥630 pg/ml and 94% for NT-proBNP <630 pg/ml; p <0.0001. There was only 1% mortality among 388 patients with at least 1 NT-proBNP value ≤220 pg/ml compared with 41% mortality among 54 patients with at least 1 NT-proBNP value >1,548 pg/ml. Even the first (baseline) measurements of NT-proBNP were strongly associated with a high risk of death (log10 NT-proBNP had hazard ratio 7, p <0.0001). In conclusion, NT-proBNP assessment is a useful and simple tool for the prediction of mortality in long-term follow-up of adults with CHD.


Microcomputed tomographic and histomorphometric analyses of novel titanium mesh membranes for guided bone regeneration: a study in rat calvarial defects.
The objective of this study was to evaluate the optimal thickness and porosity of novel titanium mesh membranes to enhance bone augmentation, prevent soft tissue ingrowth, and prevent membrane exposure. Six types of novel titanium meshes with different thicknesses and pore sizes, along with three commercially available membranes, were used to cover surgically created calvarial defects in 6-week-old Sprague-Dawley rats. The animals were killed after 4 or 8 weeks. Microcomputed tomographic analyses were performed to analyze the three-dimensional bone volume and bone mineral density. Soft tissue ingrowth was also evaluated histologically and histomorphometrically. The novel titanium membranes used in this study were as effective at augmenting bone in the rat calvarial defect model as the commercially available membranes. The greatest bone volume was observed on 100-μm-thick membranes with larger pores, although these membranes promoted growth of bone with lower mineral density. Soft tissue ingrowth when 100-μm membranes were used was increased at 4 weeks but decreased again by 8 weeks to a level not statistically significantly different from other membranes. Membrane thickness affects the total amount of new bone formation, and membrane porosity is an essential factor for guided bone regeneration, especially during the initial healing period, although the final bone volume obtained is essentially the same. Newly developed titanium mesh membranes of 100 μm in thickness and with large pores appear to be optimal for guided bone regeneration.


Proteasome inhibition with bortezomib induces cell death in GBM stem-like cells and temozolomide-resistant glioma cell lines, but stimulates GBM stem-like cells' VEGF production and angiogenesis.
Recurrent malignant gliomas have inherent resistance to traditional chemotherapy. Novel therapies target specific molecular mechanisms involved in abnormal signaling and resistance to apoptosis. The proteasome is a key regulator of multiple cellular functions, and its inhibition in malignant astrocytic lines causes cell growth arrest and apoptotic cell death. The proteasome inhibitor bortezomib was reported to have very good in vitro activity against malignant glioma cell lines, with modest activity in animal models as well as in clinical trials as a single agent. In this paper, the authors describe the multiple effects of bortezomib in both in vitro and in vivo glioma models and offer a novel explanation for its seeming lack of activity. Glioma stem-like cells (GSCs) were obtained from resected glioblastomas (GBMs) at surgery and expanded in culture. Stable glioma cell lines (U21 and D54) as well as temozolomide (TMZ)-resistant glioma cells derived from U251 and D54-MG were also cultured. GSCs from 2 different tumors, as well as D54 and U251 cells, were treated with bortezomib, and the effect of the drug was measured using an XTT cell viability assay. The activity of bortezomib was then determined in D54-MG and/or U251 cells using apoptosis analysis as well as caspase-3 activity and proteasome activity measurements. Human glioma xenograft models were created in nude mice by subcutaneous injection. Bevacizumab was administered via intraperitoneal injection at a dose of 5 mg/kg daily. Bortezomib was administered by intraperitoneal injection 1 hour after bevacizumab administration in doses of at a dose of 0.35 mg/kg on days 1, 4, 8, and 11 every 21 days. Tumors were measured twice weekly. Bortezomib induced caspase-3 activation and apoptotic cell death in stable glioma cell lines and in glioma stem-like cells (GSCs) derived from malignant tumor specimens Furthermore, TMZ-resistant glioma cell lines retained susceptibility to the proteasome inhibition. The bortezomib activity was directly proportional with the cells' baseline proteasome activity. The proteasome inhibition stimulated both hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) production in malignant GSCs. As such, the VEGF produced by GSCs stimulated endothelial cell growth, an effect that could be prevented by the addition of bevacizumab (VEGF antibody) to the media. Similarly, administration of bortezomib and bevacizumab to athymic mice carrying subcutaneous malignant glioma xenografts resulted in greater tumor inhibition and greater improvement in survival than administration of either drug alone. These data indicate that simultaneous proteasome inhibition and VEGF blockade offer increased benefit as a strategy for malignant glioma therapy. The results of this study indicate that combination therapies based on bortezomib and bevacizumab might offer an increased benefit when the two agents are used in combination. These drugs have a complementary mechanism of action and therefore can be used together to treat TMZ-resistant malignant gliomas.


Designing theoretically-informed implementation interventions: fine in theory, but evidence of effectiveness in practice is needed.
The Improved Clinical Effectiveness through Behavioural Research Group (ICEBeRG) authors assert that a key weakness in implementation research is the unknown applicability of a given intervention outside its original site and problem, and suggest that use of explicit theory offers an effective solution. This assertion is problematic for three primary reasons. First, the presence of an underlying theory does not necessarily ease the task of judging the applicability of a piece of empirical evidence. Second, it is not clear how to translate theory reliably into intervention design, which undoubtedly involves the diluting effect of "common sense." Thirdly, there are many theories, formal and informal, and it is not clear why any one should be given primacy. To determine whether explicitly theory-based interventions are, on average, more effective than those based on implicit theories, pragmatic trials are needed. Until empirical evidence is available showing the superiority of theory-based interventions, the use of theory should not be used as a basis for assessing the value of implementation studies by research funders, ethics committees, editors or policy decision makers.


Inhibition of nitric oxide synthase produces hypothermia and depresses lipopolysaccharide fever.
The labile gas nitric oxide (NO) mediates a wide variety of thermoregulatory processes including vasomotor control, brown fat thermogenesis, and neuroendocrine regulation. Additionally, during endotoxemia, NO modulates the release of cytokines and hypothalamic peptides. To determine the role of NO in thermoregulation and fever, we intravenously injected the NO synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) and measured its effects on body temperature during normal thermoregulation and endotoxemia in awake, unrestrained rats. L-NAME produced a stereoselective, dose-dependent hypothermia that lasted up to 4 h after bolus intravenous injection. Intravenous lipopolysaccharide (LPS) produced fever in a dose-dependent manner, which was preceded by hypothermia at higher doses alpha-LPS. NOS inhibition reduced the febrile response to LPS and produced marked hypothermia with a low dose of LPS. These findings indicate that NO may play an important role in thermoregulation and suggest that NO is required for the production of fever.