Developmental competence of immature and failed/abnormally fertilized human oocytes in nuclear transfer.
Somatic cell nuclear transfer holds great promise for basic studies of reprogramming human somatic cells and for the potential development of novel cell-based therapeutics. The aim of this study was to examine experimental aspects of human nuclear transfer via use of an abundant source of oocytes, those that are routinely discarded from assisted reproduction clinics. The results suggest and reinforce several findings based on the analysis of multiple parameters: first, it was observed that supplementation of commercial culture media with hormones promoted embryo development after parthenogenetic activation. Second, the use of the chemical activation reagent puromycin resulted in significant differences in cleavage rates in oocytes that were failed/abnormally fertilized after intracytoplasmic sperm injection relative to those from IVF (P < 0.05). Third, cycloheximide promoted cleavage rates >/=40% in both groups of oocytes; moreover, two blastocysts were produced following cycloheximide treatment. Finally, the use of a subset of oocytes for nuclear transfer resulted in cleaved embryos that expressed green fluorescent protein from a transgene in donor nuclei from human embryonic stem cells. In light of these results, it is suggested that the discarded oocytes can be used to investigate new human nuclear transfer protocols for embryonic stem cell derivation.


Functional expression of HLA-DP genes transfected into mouse fibroblasts.
The HLA class II antigens are a highly polymorphic family of dimeric cell-surface glycoproteins, expressed predominantly on the surface of immunocompetent cells. They are intimately involved with the induction of the T-cell response to extrinsic antigen and are important predisposing factors for a wide spectrum of autoimmune diseases. We describe here the expression of a class II product from the HLA-DP (new WHO nomenclature, formerly SB) subregion after transfer of cloned genes into mouse fibroblasts. The transfected DP antigen is recognized by several HLA class II monoclonal antibodies and, though present in a mouse cell background, is able to function in the presentation of influenza antigen to cloned DP-restricted human T lymphocytes.


Coating of adeno-associated virus with reactive polymers can ablate virus tropism, enable retargeting and provide resistance to neutralising antisera.
Copolymers based on poly-[N-(2-hydroxypropyl) methacrylamide] (HPMA) have been used previously to enable targeted delivery of adenovirus. Here we demonstrate polymer-coating techniques can also be used to modify and retarget adeno-associated virus (AAV) types 5 and 8. Three strategies for modifying transductional targeting of AAV were employed. The first involved direct reaction of AAV5 or AAV8 with amino-reactive HPMA copolymer. The second approach used carbodiimide (EDC) chemistry to increase the number of surface amino groups on the AAV5 capsid, thereby improving coating efficiency. In the third approach, the AAV5 genome was isolated from capsid proteins and delivered in a synthetic polyplex consisting of polyethylenimine (PEI) and HPMA. Efficient covalent attachment of HPMA copolymer to AAV5 could only be achieved following modification of the virus with EDC. Coating inhibited sialic acid dependent infection and provided a platform for retargeting via new ligands, including basic fibroblast growth factor. Retargeted infection was shown to be partially resistant to neutralising antisera. Delivery of AAV5 genomes using PEI and HPMA was efficient and provided absolute control of tropism and protection from antisera. In contrast AAV8 could be reacted directly with HPMA copolymer and allowed specific retargeting via the epidermal growth factor receptor, but gave no protection against neutralising antisera. Reactive HPMA polymers can be used to ablate the natural tropism of both AAV8 and EDC-modified AAV5 and enable receptor-specific infection by incorporation of targeting ligands. These data show transductional targeting strategies can be used to improve the versatility of AAV vectors.


Hospital payment based on diagnosis-related groups differs in Europe and holds lessons for the United States.
England, France, Germany, the Netherlands, and Sweden spend less as a share of gross domestic product on hospital care than the United States while delivering high-quality services. All five European countries have hospital payment systems based on diagnosis-related groups (DRGs) that classify patients of similar clinical characteristics and comparable costs. Inspired by Medicare's inpatient prospective payment system, which originated the use of DRGs, European DRG systems have implemented different design options and are generally more detailed than Medicare's system, to better distinguish among patients with less and more complex conditions. Incentives to treat more cases are often counterbalanced by volume ceilings in European DRG systems. European payments are usually broader in scope than those in the United States, including physician salaries and readmissions. These European systems, discussed in more detail in the article, suggest potential innovations for reforming DRG-based hospital payment in the United States.


The anatomy and time course of semantic priming investigated by fMRI and ERPs.
We combined complementary non-invasive brain imaging techniques with behavioural measures to investigate the anatomy and time course of brain activity associated with semantic priming in a lexical-decision task. Participants viewed pairs of stimuli, and decided whether the second item was a real word or not. There were two variables, the semantic relationship between the prime and the target (related or unrelated) and the interval between the onset of prime and target (200 or 1000 ms), to vary the degree of semantic expectancy that was possible during task performance. Behavioural results replicated the well-established finding that identification of the target is facilitated by a preceding semantically related prime. Event-related functional magnetic resonance imaging (efMRI) identified two brain areas involved in the semantic-priming effect. Activity in the anterior medial temporal cortex was diminished when target words were primed by semantically related words, suggesting involvement of this brain region during active semantic association or integration. In contrast, activity in the left supramarginal gyrus in the temporal-parietal junction was enhanced for target words primed by semantically related words. Brain areas influenced by the interval between prime and target words, and by the interaction between word interval and semantic priming were also identified. A parallel experiment using event-related potentials (ERPs) unveiled a striking difference in the time course of semantic priming as a function of expectancy. In line with previous reports, the primary effect of semantic priming on ERPs was the attenuation of the N400 component, in both short- and long-interval conditions. However, the priming effect started significantly earlier in the long-interval condition. Activity in the anterior medial temporal cortex has previously been shown to contribute to the N400 component, a finding that links the priming results obtained with efMRI and ERP methods.


Diagnostic strategies using DNA testing for hereditary haemochromatosis in at-risk populations: a systematic review and economic evaluation.
To evaluate DNA testing for detecting hereditary haemochromatosis (HHC) in subgroups of patients suspected of having the disorder and in family members of those diagnosed with HHC. Major electronic databases, searched from inception to April 2007. A systematic review was undertaken using a priori methods and a de novo model developed to assess costs and consequences of DNA testing. Eleven studies were identified for estimating the clinical validity of genotyping for the C282Y mutation for the diagnosis of HHC. No clinical effectiveness studies meeting the inclusion criteria were identified. Two North American cost-effectiveness studies of reasonable quality were identified but their generalisability to the UK is not clear. Three cohort studies met the inclusion criteria for the review of psychosocial aspects. All had methodological limitations and their generalisability is difficult to determine. The clinical sensitivity of C282Y homozygosity for HHC ranged from 28.4% to 100%, or from 91.3% to 92.4% when considering only the most relevant studies. Clinical specificity ranged from 98.8% to 100%. One study found that gene testing was a cost-effective method of screening relatives of patients with haemochromatosis, whereas the other found that genotyping the spouse of a homozygote was the most cost-efficient strategy. Genetic testing for haemochromatosis appears to be well accepted, is accompanied by few negative psychosocial outcomes and may lead to reduced anxiety. The de novo economic model showed that, in people suspected of having haemochromatosis, the DNA strategy is cost saving compared with the baseline strategy using liver biopsy (cost saved per case detected 123 pounds), largely because of the reduction in liver biopsies. For family testing of siblings the DNA strategy is not cost saving because of the costs of the DNA test (additional cost per case detected 200 pounds). If the cost of the test were to reduce from 100 pounds to 60 pounds, the DNA strategy would be the cheaper one. For family testing of offspring the DNA test strategy is cheaper than the baseline biochemical testing strategy (cost saved per case detected 7982 pounds). Sensitivity analyses showed that the conclusions in each case are robust across all reasonable parameter values. The preferred strategy in practice is DNA testing in conjunction with testing iron parameters when there is clear clinical indication of risk for haemochromatosis because of biochemical criteria or when there is familial risk for HHC. Access to genetic testing and centralisation of test provision in expert laboratories would lower the cost of testing, improve the cost-effectiveness of the strategy and improve the quality of information provided to clinicians and patients.


Synovial fluid macrophages are capable of osteoclast formation and resorption.
To determine whether synovial fluid (SF) macrophages isolated from the SF of osteoarthritis (OA), rheumatoid arthritis (RA) and pyrophosphate arthropathy (PPA) joints are capable of osteoclast formation, and to investigate the cellular and humoral factors required for this to occur, SF macrophages (CD14+) were isolated from the knee joint SF from patients with OA, RA and PPA and cultured for up to 14 days with macrophage-colony stimulating factor (M-CSF) and soluble receptor activator for nuclear factor-kappaB ligand (RANKL) or tumour-necrosis factor-alpha (TNFalpha) and interleukin-1alpha (IL-1alpha). Osteoclast differentiation was assessed by expression of tartrate-resistant acid phosphatase (TRAP) and vitronectin receptor (VNR), F-actin ring formation and lacunar resorption. Osteoclast formation and lacunar resorption was seen in RANKL-treated cultures of SF macrophages isolated from OA, RA and PPA joints with the largest amount of resorption noted in RA and PPA SF macrophage cultures. In TNFalpha/IL-1alpha-treated RA and PPA SF macrophage cultures, osteoclasts capable of lacunar resorption were also formed. Lacunar resorption was more extensive in RANKL than TNFalpha/IL-1alpha-treated cultures. These findings indicate that SF macrophages are capable of differentiating into mature osteoclasts capable of lacunar resorption. M-CSF in combination with RANKL or TNFalpha/IL-1alpha was required for osteoclast formation. As inflammatory synovial fluids contain an increase in the number of macrophages and an increase in the amounts of RANKL, TNFalpha and IL-1alpha, these findings suggest that one means whereby bone erosions may form in rheumatoid or crystal arthritis is by differentiation of synovial fluid macrophages into osteoclasts.


Human embryonic stem cells as models for aneuploid chromosomal syndromes.
Syndromes caused by chromosomal aneuploidies are widely recognized genetic disorders in humans and often lead to spontaneous miscarriage. Preimplantation genetic screening is used to detect chromosomal aneuploidies in early embryos. Our aim was to derive aneuploid human embryonic stem cell (hESC) lines that may serve as models for human syndromes caused by aneuploidies. We have established 25 hESC lines from blastocysts diagnosed as aneuploid on day 3 of their in vitro development. The hESC lines exhibited morphology and expressed markers typical of hESCs. They demonstrated long-term proliferation capacity and pluripotent differentiation. Karyotype analysis revealed that two-third of the cell lines carry a normal euploid karyotype, while one-third remained aneuploid throughout the derivation, resulting in eight hESC lines carrying either trisomy 13 (Patau syndrome), 16, 17, 21 (Down syndrome), X (Triple X syndrome), or monosomy X (Turner syndrome). On the basis of the level of single nucleotide polymorphism heterozygosity in the aneuploid chromosomes, we determined whether the aneuploidy originated from meiotic or mitotic chromosomal nondisjunction. Gene expression profiles of the trisomic cell lines suggested that all three chromosomes are actively transcribed. Our analysis allowed us to determine which tissues are most affected by the presence of a third copy of either chromosome 13, 16, 17 or 21 and highlighted the effects of trisomies on embryonic development. The results presented here suggest that aneuploid embryos can serve as an alternative source for either normal euploid or aneuploid hESC lines, which represent an invaluable tool to study developmental aspects of chromosomal abnormalities in humans.


Igf2 ligand dependency of Pten(+/-) developmental and tumour phenotypes in the mouse.
The tumour suppressor PTEN is a key negative regulator of the PI3K-Akt pathway, and is frequently either reduced or lost in human tumours. Murine genetic studies have confirmed that reduction of Pten promotes tumourigenesis in multiple organs, and demonstrated dependency of tumour development on the activation of downstream components such as Akt. Insulin-like growth factors (IGFs) act via IGF1R to activate the PI3K-Akt pathway, and are commonly upregulated in cancer. A context-dependent interplay between IGFs and PTEN exists in normal tissue and tumours; increased IGF2 ligand supply induces Pten expression creating an autoregulatory negative feedback loop, whereas complete loss of PTEN may either cooperate with IGF overexpression in tumour promotion, or result in desensitisation to IGF ligand. However, it remains unknown whether neoplasia associated with Pten loss is dependent on upstream IGF ligand supply in vivo. We evaluated this by generation of Pten(+/-) mice with differing allelic dosage of Igf2, an imprinted gene encoding the potent embryonic and tumour growth factor Igf2. We show that biallelic Igf2 supply potentiates a previously unreported Pten(+/-) placental phenotype and results in strain-dependent cardiac hyperplasia and neonatal lethality. Importantly, we also show that the effects of Pten loss in vivo are modified by Igf2 supply, as lack of Igf2 results in extended survival and delayed tumour development while biallelic supply is associated with reduced lifespan and accelerated neoplasia in females. Furthermore, we demonstrate that reduction of PTEN protein to heterozygote levels in human MCF7 cells is associated with increased proliferation in response to IGF2, and does not result in desensitisation to IGF2 signalling. These data indicate that the effects of Pten loss at heterozygote levels commonly observed in human tumours are modified by Igf2 ligand, and emphasise the importance of the evaluation of upstream pathways in tumours with Pten loss.


Mitochondrial role in the neonatal predisposition to developing nonalcoholic fatty liver disease.
Nonalcoholic fatty liver disease (NAFLD) is a global epidemic in obese children and adults, and the onset might have fetal origins. A growing body of evidence supports the role of developmental programming, whereby the maternal environment affects fetal and infant development, altering the risk profile for disease later in life. Human and nonhuman primate studies of maternal obesity demonstrate that risk factors for pediatric obesity and NAFLD begin in utero. The pathologic mechanisms for NAFLD are multifactorial but have centered on altered mitochondrial function/dysfunction that might precede insulin resistance. Compared with the adult liver, the fetal liver has fewer mitochondria, low activity of the fatty acid metabolic enzyme carnitine palmitoyl-CoA transferase-1, and little or no gluconeogenesis. Exposure to excess maternal fuels during fetal life uniquely alters hepatic fatty acid oxidation, tricarboxylic acid cycle activity, de novo lipogenesis, and mitochondrial health. These events promote increased oxidative stress and excess triglyceride storage, and, together with altered immune function and epigenetic changes, they prime the fetal liver for NAFLD and might drive the risk for nonalcoholic steatohepatitis in the next generation.


Hypoxia, hypocapnia and spirometry at altitude.
1. Both hypoxia and hypocapnia can cause broncho-constriction in humans, and this could have a bearing on performance at high altitude or contribute to altitude sickness. We studied the relationship between spirometry, arterial oxygen saturation and end-tidal carbon dioxide (ETCO2) concentration in a group of healthy lowland adults during a stay at high altitude, and then evaluated the response to supplementary oxygen and administration of a beta 2 agonist. 2. We collected spirometric data from 51 members of the 1994 British Mount Everest Medical Expedition at sea level (barometric pressure 101.2-101.6 kPa) and at Mount Everest Base Camp in Nepal (altitude 5300 m, barometric pressure 53-54.7 kPa) using a pocket turbine spirometer. A total of 205 spirometric measurements were made on the 51 subjects during the first 6 days after arrival at Base Camp. Further measurements were made before and after inhalation of oxygen (n = 47) or a beta 2 agonist (n = 39). ETCO2 tensions were measured on the same day as spirometric measurements in 30 of these subjects. 3. In the first 6 days after arrival at 5300 m, lower oxygen saturations were associated with lower forced expiratory volume in 1 s (FEV1; P < 0.02) and forced vital capacity (FVC; P < 0.01), but not with peak expiratory flow (PEF). Administration of supplementary oxygen for 5 min increased oxygen saturation from a mean of 81%-94%, but there was no significant change in FEV1 or FVC, whilst PEF fell by 2.3% [P < 0.001; 95% confidence intervals (CI) -4 to -0.7%]. After salbutamol administration, there was no significant change in PEF, FEV1 or FVC in 35 non-asthmatic subjects. Mean ETCO2 at Everest Base Camp was 26 mmHg, and a low ETCO2 was weakly associated with a larger drop in FVC at altitude compared with sea level (r = 0.38, P < 0.05). There was no correlation between either ETCO2 or oxygen saturation and changes in FEV1 or PEF compared with sea-level values. 4. In this study, in normal subjects who were acclimatized to hypobaric hypoxia at an altitude of 5300 m, we found no evidence of hypoxic broncho-constriction. Individuals did not have lower PEF when they were more hypoxic, and neither PEF nor FEV1 were increased by either supplementary oxygen or salbutamol. FVC fell at altitude, and there was a greater fall in FVC for subjects with lower oxygen saturations and probably lower ETCO2.


A germline mutation of CDKN2A and a novel RPLP1-C19MC fusion detected in a rare melanotic neuroectodermal tumor of infancy: a case report.
Melanotic neuroectodermal tumor of infancy (MNTI) is exceptionally rare and occurs predominantly in the head and neck (92.8 % cases). The patient reported here is only the eighth case of MNTI presenting in an extremity, and the first reported in the fibula. A 2-month-old female presented with a mass arising in the fibula. Exhaustive genomic, transcriptomic, epigenetic and pathological characterization was performed on the excised primary tumor and a derived cell line. Whole-exome analysis of genomic DNA from both the tumor and blood indicated no somatic, non-synonymous coding mutations within the tumor, but a heterozygous, unique germline, loss of function mutation in CDKN2A (p16(INK4A), D74A). SNP-array CGH on DNA samples revealed the tumor to be euploid, with no detectable gene copy number variants. Multiple chromosomal translocations were identified by RNA-Seq, and fusion genes included RPLP1-C19MC, potentially deregulating the C19MC cluster, an imprinted locus containing microRNA genes reactivated by gene fusion in embryonal tumors with multilayered rosettes. Since the presumed cell of origin of MNTI is from the neural crest, we also compared gene expression with a dataset from human neural crest cells and identified 185 genes with significantly different expression. Consistent with the melanotic phenotype of the tumor, elevated expression of tyrosinase was observed. Other highly expressed genes encoded muscle proteins and modulators of the extracellular matrix. A derived MNTI cell line was sensitive to inhibitors of lysine demethylase, but not to compounds targeting other epigenetic regulators. In the absence of somatic copy number variations or mutations, the fully transformed phenotype of the MNTI may have arisen in infancy because of the combined effects of a germline CDKN2A mutation, tumor promoting somatic fusion genes and epigenetic deregulation. Very little is known about the etiology of MNTI and this report advances knowledge of these rare tumors by providing the first comprehensive genomic, transcriptomic and epigenetic characterization of a case.


The effects of mutational processes and selection on driver mutations across cancer types.
Epidemiological evidence has long associated environmental mutagens with increased cancer risk. However, links between specific mutation-causing processes and the acquisition of individual driver mutations have remained obscure. Here we have used public cancer sequencing data from 11,336 cancers of various types to infer the independent effects of mutation and selection on the set of driver mutations in a cancer type. First, we detect associations between a range of mutational processes, including those linked to smoking, ageing, APOBEC and DNA mismatch repair (MMR) and the presence of key driver mutations across cancer types. Second, we quantify differential selection between well-known alternative driver mutations, including differences in selection between distinct mutant residues in the same gene. These results show that while mutational processes have a large role in determining which driver mutations are present in a cancer, the role of selection frequently dominates.


Gain- and loss-of-function approaches in the chick embryo.
The chicken embryo has been used as a classical embryological model for studying developmental events because of its ready availability, similarity to the human embryos, and amenability to embryological and surgical manipulations. With the arrival of the molecular era, however, avian embryos presented distinct experimental limitations, largely because of the difficulty of performing targeted mutagenesis or transgenic studies. However, in the last decade and a half, a number of new methods for transient transgenesis have been developed that allow efficient alteration of gene function during early embryonic development. These techniques have made it possible to study the effects of gene inactivation or overexpression on downstream transcriptional regulation as well as on embryonic derivatives. This, together with sequencing of the chicken genome, has allowed the chicken embryo to enter the genomic era. While attempts to establish germ line transgenesis are ongoing, methods for rapid, transient spatiotemporally targeted gene alterations have thus again re-established the chick embryo as an important experimental niche by making it possible to apply genetics in concert with classical embryological techniques. This provides a unique tool to explore the role of developmentally important genes (Ishii and Mikawa, 2005; Itasaki et al., 1999; Krull, 2004; Ogura, 2002; Swartz et al., 2001). Transient transfection methods have allowed for efficient mis- and overexpression of transgenes. For long-term analyses, retrovirally mediated gene transfer has particular advantage. For short-term experiments, electroporation and adenoviral-mediated gene transfer methods provide transient expression, largely because of the short persistence time of the transgene within the cell. More recently, Tol2 transposon-mediated constructs have been employed, allowing for integration into the genome and prolonged expression of the transgene (Sato et al., 2007), see Chapter 14 by Takahashi et al., this volume). These methods today are routinely used for gain-of-function analysis, to overexpress or ectopically express genes of interest (Arber et al., 1999; Barembaum and Bronner-Fraser, 2007; Bel-Vialar et al., 2002). Loss-of-function experiments are also possible using electroporation of dominant-negative constructs that act as competitive inhibitors (Bel-Vialar et al., 2002; Renzi et al., 2000; Suzuki-Hirano et al., 2005), morpholino antisense oligos (Basch et al., 2006; Kos et al., 2001; Sheng et al., 2003) that block translation or splicing, or constructs expressing small interfering or small hairpin RNAs (siRNAs or shRNAs) (Chesnutt and Niswander, 2004; Das et al., 2006; Katahira and Nakamura, 2003). Electroporation as the most popular method of the transient transfection into the chick embryos. Electroporation of chicken embryos involves application of an electric field to the exposed tissue that transiently disrupts the stability of the cell plasma membrane, creating reversible pores through which nucleic acids or their analogues can be readily transported into the cytosol. The use of this method for transfection into the vertebrate embryos has been facilitated by adapting the voltage parameters and the type and the duration of the electric pulse. By applying several successive square pulses at a very low voltage, with long rest periods in between, one can successfully deliver a DNA construct or another small charged particle into the cytoplasm, with minimal cell death, high efficiency of the uptake and good embryonic survival rate. The size limit of the DNA molecule that can be transfected in such a way is not yet known, though it is more likely that the size limitation in this procedure (if any) lies within the practical problems of cloning large fragments into the plasmid. We routinely overexpress constructs containing 3-4 kb inserts and coharboring a GFP or RFP reporter whose translation is initiated from an internal ribosomal entry site (IRES), thus allowing easy detection of the electroporated cells.


CYP-dependent metabolism of PF9601N, a new monoamine oxidase-B inhibitor, by C57BL/6 mouse and human liver microsomes.
The selective monoamine oxidase-B (MAO-B) inhibitor, l-deprenyl, is still used for treating Parkinson's patients, however, a disadvantage of its use lies in the formation of l-amphetamine and l-methamphetamine. Subsequently, this has promoted the design of a novel, more potent, MAO-B inhibitor PF9601N, which also has neuroprotective and antioxidant properties. The aim of this work was to investigate the effect of treatment with PF9601N on its own phase I hepatic metabolism. Kinetic parameters of PF9601N CYP-dependent N-dealkylation reaction was also studied and compared with those of l-deprenyl. C57BL/6 mice were treated with PF9601N for 4 days. After CYP content and related monooxygenase activities were assayed in liver microsomes of control and treated animals. CYP activities, cytochrome b5 content, NADPH-cytochrome P450 reductase and various monooxygenase activities were unaffected by in vivo PF9601N treatment. With microsomes from both control and treated mice, the PF9601N-dealkylation product, FA72, was the only detected metabolite with its formation rate following an hyperbolic, Michaelis-Menten curve. Among various inhibitors, only ketoconazole inhibited the FA72 formation rate, indicating a major involvement for CYP3A. Apparent Km and Vmax values generated by human liver microsomes were similar to those found with mouse microsomes. Ketoconazole inhibition indicates that CYP3A is one of the major enzymes involved in PF9601N metabolism also by human liver microsomes. In mouse liver microsomes, the intrinsic clearance of PF9601N was significantly lower than that of l-deprenyl suggestive of an improved bioavailability for the former. The observed favourable metabolic profile may suggest suitability of PF9601N for clinical use.


Unique transcriptome signatures and GM-CSF expression in lymphocytes from patients with spondyloarthritis.
Spondyloarthritis encompasses a group of common inflammatory diseases thought to be driven by IL-17A-secreting type-17 lymphocytes. Here we show increased numbers of GM-CSF-producing CD4 and CD8 lymphocytes in the blood and joints of patients with spondyloarthritis, and increased numbers of IL-17A+GM-CSF+ double-producing CD4, CD8, γδ and NK cells. GM-CSF production in CD4 T cells occurs both independently and in combination with classical Th1 and Th17 cytokines. Type 3 innate lymphoid cells producing predominantly GM-CSF are expanded in synovial tissues from patients with spondyloarthritis. GM-CSF+CD4+ cells, isolated using a triple cytokine capture approach, have a specific transcriptional signature. Both GM-CSF+ and IL-17A+GM-CSF+ double-producing CD4 T cells express increased levels of GPR65, a proton-sensing receptor associated with spondyloarthritis in genome-wide association studies and pathogenicity in murine inflammatory disease models. Silencing GPR65 in primary CD4 T cells reduces GM-CSF production. GM-CSF and GPR65 may thus serve as targets for therapeutic intervention of spondyloarthritis.


An Exploration of Rhythmic Grouping of Speech Sequences by French- and German-Learning Infants.
Rhythm in music and speech can be characterized by a constellation of several acoustic cues. Individually, these cues have different effects on rhythmic perception: sequences of sounds alternating in duration are perceived as short-long pairs (weak-strong/iambic pattern), whereas sequences of sounds alternating in intensity or pitch are perceived as loud-soft, or high-low pairs (strong-weak/trochaic pattern). This perceptual bias-called the Iambic-Trochaic Law (ITL)-has been claimed to be an universal property of the auditory system applying in both the music and the language domains. Recent studies have shown that language experience can modulate the effects of the ITL on rhythmic perception of both speech and non-speech sequences in adults, and of non-speech sequences in 7.5-month-old infants. The goal of the present study was to explore whether language experience also modulates infants' grouping of speech. To do so, we presented sequences of syllables to monolingual French- and German-learning 7.5-month-olds. Using the Headturn Preference Procedure (HPP), we examined whether they were able to perceive a rhythmic structure in sequences of syllables that alternated in duration, pitch, or intensity. Our findings show that both French- and German-learning infants perceived a rhythmic structure when it was cued by duration or pitch but not intensity. Our findings also show differences in how these infants use duration and pitch cues to group syllable sequences, suggesting that pitch cues were the easier ones to use. Moreover, performance did not differ across languages, failing to reveal early language effects on rhythmic perception. These results contribute to our understanding of the origin of rhythmic perception and perceptual mechanisms shared across music and speech, which may bootstrap language acquisition.


Cortical glutathione levels in young people with bipolar disorder: a pilot study using magnetic resonance spectroscopy.
Glutathione (GSH) is a key scavenger for cellular free radicals, and patients with bipolar disorder may have lowered GSH levels in plasma and in post-mortem brain tissue. The objective of the current study was to use magnetic resonance spectroscopy (MRS) to measure cortical GSH levels in young people with bipolar disorder to determine if lowered GSH might be a useful biomarker of vulnerability to the illness. We studied 13 patients with DSM-IV bipolar disorder and 11 healthy age-matched controls using proton MRS in conjunction with the SPECIAL acquisition technique. Voxels were placed in prefrontal and occipital cortex. All patients were clinically euthymic at the time of study and unmedicated. GSH and other relevant neurometabolites were measured relative to creatinine. There was no difference in GSH levels between bipolar participants and controls in either prefrontal or occipital cortex. Similarly, participants showed no difference from controls in other measured cortical metabolites including γ-aminobutyric acid, glutamate and N-acetylaspartate. This pilot study suggests that levels of cortical GSH are unlikely to be a useful trait biomarker of bipolar disorder in young people with a history of relatively mild mood instability at an early stage of illness. Lowered GSH levels may be relevant to bipolar pathophysiology in more severely ill patients, particular those with significant current mood disturbance.


Fat depot-specific mRNA expression of novel loci associated with waist-hip ratio.
We hypothesized that genes within recently identified loci associated with waist-hip ratio (WHR) exhibit fat depot-specific mRNA expression, which correlates with obesity-related traits. Adipose tissue (AT) mRNA expression of 6 genes (TBX15/WARS2, STAB1, PIGC, ZNRF3 and GRB14) within these loci showing coincident cis-expression quantitative trait loci was measured in 222 paired samples of human visceral (vis) and subcutaneous (sc) AT. The relationship of mRNA expression levels with obesity-related quantitative traits was assessed by Pearson's correlation analyses. Multivariate linear relationships were assessed by generalized linear regression models. Whereas only PIGC, ZNFR3 and STAB1 mRNA expression in sc AT correlated nominally with WHR (P<0.05, adjusted for age and sex), mRNA expression of all studied genes in at least one of the fat depots correlated significantly with vis and/or sc fat area (P ranging from 0.05 to 4.0 × 10(6), adjusted for age and sex). Consistently, the transcript levels of WARS, PIGC and GRB14 were nominally associated with body mass index (BMI) (P ranging from 0.02 to 9.2 × 10(5), adjusted for age and sex). Moreover, independent of sex, obesity and diabetes status, differential expression between vis and sc AT was observed for all tested genes (P<0.01). Finally, the rs10195252 T-allele was nominally associated with increased GRB14 sc mRNA expression (P=0.025 after adjusting for age, sex and BMI). Our data including the inter-depot variability of mRNA expression suggests that genes within the WHR-associated loci might be involved in the regulation of fat distribution.


Increased flux through the mevalonate pathway mediates fibrotic repair without injury.
Macrophages are important in mounting an innate immune response to injury as well as in repair of injury. Gene expression of Rho proteins is known to be increased in fibrotic models; however, the role of these proteins in idiopathic pulmonary fibrosis (IPF) is not known. Here, we show that BAL cells from patients with IPF have a profibrotic phenotype secondary to increased activation of the small GTPase Rac1. Rac1 activation requires a posttranslational modification, geranylgeranylation, of the C-terminal cysteine residue. We found that by supplying more substrate for geranylgeranylation, Rac1 activation was substantially increased, resulting in profibrotic polarization by increasing flux through the mevalonate pathway. The increased flux was secondary to greater levels of acetyl-CoA from metabolic reprogramming to β oxidation. The polarization mediated fibrotic repair in the absence of injury by enhancing macrophage/fibroblast signaling. These observations suggest that targeting the mevalonate pathway may abrogate the role of macrophages in dysregulated fibrotic repair.


An optimized velocity selective arterial spin labeling module with reduced eddy current sensitivity for improved perfusion quantification.
Velocity-selective (VS) arterial spin labeling is a promising method for measuring perfusion in areas of slow or collateral flow by eliminating the bolus arrival delay associated with other spin labeling techniques. However, B(0) and B(1) inhomogeneities and eddy currents during the VS preparation hinder accurate quantification of perfusion with VS arterial spin labeling. In this study, it is demonstrated through simulations and experiments in healthy volunteers that eddy currents cause erroneous tagging of static tissue. Consequently, mean gray matter perfusion is overestimated by up to a factor of 2, depending on the VS preparation used. A novel eight-segment B(1) insensitive rotation VS preparation is proposed to reduce eddy current effects while maintaining the B(0) and B(1) insensitivity of previous preparations. Compared to two previous VS preparations, the eight-segment B(1) insensitive rotation is the most robust to eddy currents and should improve the quality and reliability of VS arterial spin labeling measurements in future studies.


DOMINO-AD protocol: donepezil and memantine in moderate to severe Alzheimer's disease - a multicentre RCT.
Alzheimer's disease (AD) is the commonest cause of dementia. Cholinesterase inhibitors, such as donepezil, are the drug class with the best evidence of efficacy, licensed for mild to moderate AD, while the glutamate antagonist memantine has been widely prescribed, often in the later stages of AD. Memantine is licensed for moderate to severe dementia in AD but is not recommended by the England and Wales National Institute for Health and Clinical Excellence. However, there is little evidence to guide clinicians as to what to prescribe as AD advances; in particular, what to do as the condition progresses from moderate to severe. Options include continuing cholinesterase inhibitors irrespective of decline, adding memantine to cholinesterase inhibitors, or prescribing memantine instead of cholinesterase inhibitors. The aim of this trial is to establish the most effective drug option for people with AD who are progressing from moderate to severe dementia despite treatment with donepezil. DOMINO-AD is a pragmatic, 15 centre, double-blind, randomized, placebo controlled trial. Patients with AD, currently living at home, receiving donepezil 10 mg daily, and with Standardized Mini-Mental State Examination (SMMSE) scores between 5 and 13 are being recruited. Each is randomized to one of four treatment options: continuation of donepezil with memantine placebo added; switch to memantine with donepezil placebo added; donepezil and memantine together; or donepezil placebo with memantine placebo. 800 participants are being recruited and treatment continues for one year. Primary outcome measures are cognition (SMMSE) and activities of daily living (Bristol Activities of Daily Living Scale). Secondary outcomes are non-cognitive dementia symptoms (Neuropsychiatric Inventory), health related quality of life (EQ-5D and DEMQOL-proxy), carer burden (General Health Questionnaire-12), cost effectiveness (using Client Service Receipt Inventory) and institutionalization. These outcomes are assessed at baseline, 6, 18, 30 and 52 weeks. All participants will be subsequently followed for 3 years by telephone interview to record institutionalization. There is considerable debate about the clinical and cost effectiveness of anti-dementia drugs. DOMINO-AD seeks to provide clear evidence on the best treatment strategies for those managing patients at a particularly important clinical transition point. Current controlled trials ISRCTN49545035.


Individual differences in amygdala and ventromedial prefrontal cortex activity are associated with evaluation speed and psychological well-being.
Using functional magnetic resonance imaging, we examined whether individual differences in amygdala activation in response to negative relative to neutral information are related to differences in the speed with which such information is evaluated, the extent to which such differences are associated with medial prefrontal cortex function, and their relationship with measures of trait anxiety and psychological well-being (PWB). Results indicated that faster judgments of negative relative to neutral information were associated with increased left and right amygdala activation. In the prefrontal cortex, faster judgment time was associated with relative decreased activation in a cluster in the ventral anterior cingulate cortex (ACC, BA 24). Furthermore, people who were slower to evaluate negative versus neutral information reported higher PWB. Importantly, higher PWB was strongly associated with increased activation in the ventral ACC for negative relative to neutral information. Individual differences in trait anxiety did not predict variation in judgment time or in amygdala or ventral ACC activity. These findings suggest that people high in PWB effectively recruit the ventral ACC when confronted with potentially aversive stimuli, manifest reduced activity in subcortical regions such as the amygdala, and appraise such information as less salient as reflected in slower evaluative speed.


Loss of major histocompatibility complex-encoded transporter associated with antigen presentation (TAP) in colorectal cancer.
Presentation of endogenous antigenic peptides to cytotoxic T lymphocytes (CTLs) is mediated by the major histocompatibility complex (MHC) class I molecules. These antigenic peptides derived from the cytoplasmic protein pool are transported by the recently described MHC-encoded transporters (TAP1 and TAP2) into a pre-Golgi region where they take part in the assembly of MHC class I molecules. Using an affinity-purified polyclonal antibody (AK1.7) for TAP1, we analyzed 81 colorectal carcinomas, 32 adenomas, and the respective nonneoplastic mucosa. Loss of the transporter molecule (TAP1) was observed in 14% (11 of 81) of the carcinomas, either complete (7 of 11) or focal (4 of 11), whereas adenomas and normal mucosa were always positive. This study adds further information to the understanding of the mechanisms related to the defective presentation of the MHC class I molecules by tumor cells.


Knee replacement.
Knee-replacement surgery is frequently done and highly successful. It relieves pain and improves knee function in people with advanced arthritis of the joint. The most common indication for the procedure is osteoarthritis. We review the epidemiology of and risk factors for knee replacement. Because replacement is increasingly considered for patients younger than 55 years, improved decision making about whether a patient should undergo the procedure is needed. We discuss assessment of surgery outcomes based on data for revision surgery from national joint-replacement registries and on patient-reported outcome measures. Widespread surveillance of existing implants is urgently needed alongside the carefully monitored introduction of new implant designs. Developments for the future are improved delivery of care and training for surgeons and clinical teams. In an increasingly ageing society, the demand for knee-replacement surgery will probably rise further, and we predict future trends. We also emphasise the need for new strategies to treat early-stage osteoarthritis, which will ultimately reduce the demand for joint-replacement surgery.


A study of preslaughter pig handling and stunning in selected South African Highveld Region abattoirs.
The study hypothesis was that pre-slaughter handling is not conducted in such a way that pigs can be considered humanely slaughtered, nor is it conducive to promoting those properties of fresh meat that could enhance pork quality. The 1st phase of the investigation was aimed at confirming the present compliance level within the prescribed norms. To this end the importance of stunning induction requirements for effective electrical stunning, the welfare implications relating to the pre-slaughter handling of pigs and the stunning and sticking techniques were investigated. Pre-slaughter welfare of the pigs at the abattoir was found to be influenced at the outset by the origin and type of pig slaughtered, the daily throughput range of pigs and the type of abattoir involved. A disappointing pre-arrival aspect was that deficiencies in road motor vehicles were observed while off-loading pigs. Through poor design or lack of maintenance, another factor that hampered free movement of pigs was the off-loading facilities. The nature of animal behaviour in the pens, in the passages and when going into the stunning area was directly related to the pig handling efficiency and to the nature and extent of design and maintenance problems with equipment. None of the abattoirs had a well designed in-feed to a well planned stunning area/facility, and a very high level of pig pre-slaughter stress prevailed. The optimum position of the electrodes is virtually impossible to attain under practical conditions and the practice of repeat application of electrical stunning is common. Of the total sample size (n = 1175), 34% were difficult to shackle, 90% of these being from the abattoirs tending to slaughter more routinely a wide range of different sized pigs. In this survey 149 pigs (13%) required more than a single sticking attempt to bleed out properly, considerably higher than in other published reports.


Characterization and propagation of uncertainty in diffusion-weighted MR imaging.
A fully probabilistic framework is presented for estimating local probability density functions on parameters of interest in a model of diffusion. This technique is applied to the estimation of parameters in the diffusion tensor model, and also to a simple partial volume model of diffusion. In both cases the parameters of interest include parameters defining local fiber direction. A technique is then presented for using these density functions to estimate global connectivity (i.e., the probability of the existence of a connection through the data field, between any two distant points), allowing for the quantification of belief in tractography results. This technique is then applied to the estimation of the cortical connectivity of the human thalamus. The resulting connectivity distributions correspond well with predictions from invasive tracer methods in nonhuman primate.


Pre-synaptic NO-cGMP pathway modulates vagal control of heart rate in isolated adult guinea pig atria.
The role of nitric oxide (NO) in the vagal modulation of heart rate (HR) is controversial. We tested the hypothesis that NO acts via a pre-synaptic, guanylyl cyclase (GC) dependent pathway. The effects of inhibiting NO synthase (NOS) and GC were evaluated in isolated atrial/right vagal nerve preparations from adult (550-750 g) and young (150-250 g) female guinea pigs. Levels of NOS protein were quantified in right atria using Western blotting and densitometry. The non-specific NOS inhibitor N- omega -nitro- L -arginine (L -NA, 100 microM, n=5) significantly reduced the negative chronotropic response to vagal nerve stimulation (VNS) at 3 and 5 Hz in the adult guinea pig. This effect was reversed with 1 m ML -arginine. Similar results were observed with the specific neuronal NOS inhibitor vinyl-N5-(1-imino-3-butenyl)- L -ornithine (L -VNIO, 100 microM, n=7). Inhibition of GC with 1H-(1,2,4)-oxadiazolo-(4, 3-a)-quinoxalin-1-one (ODQ, 10 microM, n=7) also significantly reduced the negative chronotropic response to VNS at 3 and 5 Hz in adult guinea pigs. Neither L -NA (n=6), L -VNIO (n=5) nor ODQ (n=6) changed the HR response to cumulative doses of carbamylcholine in adult guinea pig atria suggesting that the action of NO is pre-synaptic. The HR response to VNS was unaffected by L -NA (n=7) or ODQ (n=7) in young guinea pigs and Western blot analysis showed significantly lower levels of nNOS protein in right atria from young animals. These results suggest a pre-synaptic NO-cGMP pathway modulates cardiac cholinergic transmission, although this may depend on the developmental stage of the guinea pig.


Synthetic Light-Activated Ion Channels for Optogenetic Activation and Inhibition.
Optogenetic manipulation of cells or living organisms became widely used in neuroscience following the introduction of the light-gated ion channel channelrhodopsin-2 (ChR2). ChR2 is a non-selective cation channel, ideally suited to depolarize and evoke action potentials in neurons. However, its calcium (Ca2+) permeability and single channel conductance are low and for some applications longer-lasting increases in intracellular Ca2+ might be desirable. Moreover, there is need for an efficient light-gated potassium (K+) channel that can rapidly inhibit spiking in targeted neurons. Considering the importance of Ca2+ and K+ in cell physiology, light-activated Ca2+-permeant and K+-specific channels would be welcome additions to the optogenetic toolbox. Here we describe the engineering of novel light-gated Ca2+-permeant and K+-specific channels by fusing a bacterial photoactivated adenylyl cyclase to cyclic nucleotide-gated channels with high permeability for Ca2+ or for K+, respectively. Optimized fusion constructs showed strong light-gated conductance in Xenopus laevis oocytes and in rat hippocampal neurons. These constructs could also be used to control the motility of Drosophila melanogaster larvae, when expressed in motoneurons. Illumination led to body contraction when motoneurons expressed the light-sensitive Ca2+-permeant channel, and to body extension when expressing the light-sensitive K+ channel, both effectively and reversibly paralyzing the larvae. Further optimization of these constructs will be required for application in adult flies since both constructs led to eclosion failure when expressed in motoneurons.


Comparative chemistry of propolis from eight brazilian localities.
Propolis is a complex honeybee product with resinous aspect, containing plant exudates and beeswax. Their color, texture, and chemical composition vary, depending on the location of the hives and local flora. The most studied Brazilian propolis is the green (alecrim-do-campo) type, which contains mainly prenylated phenylpropanoids and caffeoylquinic acids. Other types of propolis are produced in Brazil, some with red color, others brown, grey, or black. The aim of the present work was to determine the chemical profiles of alcohol and chloroform extracts of eight samples of propolis, corresponding to six Brazilian regions. Methanol and chloroform extracts were obtained and analyzed by HPLC/DAD/ESI/MS and GC/MS. Two chemical profiles were recognized among the samples analyzed: (1) black Brazilian propolis, characterized chiefly by flavanones and glycosyl flavones, stemming from Picos (Piauí state) and Pirenópolis (Goiás state); (2) green Brazilian propolis, characterized by prenylated phenylpropanoids and caffeoylquinic acids, stemming from Cabo Verde (Bahia state), Lavras and Mira Bela (Minas Gerais state), Pariquera-Açu and Bauru (São Paulo state), and Ponta Grossa (Paraná state). The present work represents the first report of prenylated flavonoids in Brazilian propolis and schaftoside (apigenin-8-C-glucosyl-6-C-arabinose) in green propolis.


A survival analysis of 1084 knees of the Oxford unicompartmental knee arthroplasty: a comparison between consultant and trainee surgeons.
The aim of this to study was to compare the previously unreported long-term survival outcome of the Oxford medial unicompartmental knee arthroplasty (UKA) performed by trainee surgeons and consultants. We therefore identified a previously unreported cohort of 1084 knees in 947 patients who had a UKA inserted for anteromedial knee arthritis by consultants and surgeons in training, at a tertiary arthroplasty centre and performed survival analysis on the group with revision as the endpoint. The ten-year cumulative survival rate for revision or exchange of any part of the prosthetic components was 93.2% (95% confidence interval (CI) 86.1 to 100, number at risk 45). Consultant surgeons had a nine-year cumulative survival rate of 93.9% (95% CI 90.2 to 97.6, number at risk 16). Trainee surgeons had a cumulative nine-year survival rate of 93.0% (95% CI 90.3 to 95.7, number at risk 35). Although there was no differences in implant survival between consultants and trainees (p = 0.30), there was a difference in failure pattern whereby all re-operations performed for bearing dislocation (n = 7), occurred in the trainee group. This accounted for 0.6% of the entire cohort and 15% of the re-operations. This is the largest single series of the Oxford UKA ever reported and demonstrates that good results can be achieved by a heterogeneous group of surgeons, including trainees, if performed within a high-volume centre with considerable experience with the procedure. Cite this article: Bone Joint J 2016;(10 Suppl B):22-7.


Phase4: automatic evaluation of database search methods.
It has become standard to evaluate newly devised database search methods in terms of sensitivity and selectivity and to compare them with existing methods. This involves the construction of a suitable evaluation scenario, the execution of the methods, the assessment of their performances, and the presentation of the results. Each of these four phases and their smooth connection usually imposes formidable work. To relieve the evaluator of this burden, a system has been designed with which evaluations can be effected rapidly. It is implemented in the programming language Python whose object-oriented features are used to offer a great flexibility in changing the evaluation design. A graphical user interface is provided which offers the usual amenities such as radio- and checkbuttons or file browsing facilities.


Heterosexual anal intercourse among men in Long Beach, California.
Anal intercourse poses a greater risk for human immunodeficiency virus (HIV) transmission than vaginal intercourse, and in recent years there has been a growing understanding that heterosexual anal intercourse (HAI) is not uncommon. However, the majority of the anal intercourse literature has focused on men who have sex with men. The little research on HAI has mostly looked at women, with limited work among men. This analysis examined the association between HAI and high-risk behaviors (N = 1,622) and sexual sensation seeking (N = 239) in a sample of men recruited from 2001 to 2012 in Long Beach, California. Almost half of the sample was non-Hispanic Black. The median age was 42 years, 42% were homeless, and 20% reported recent HAI. Men who reported HAI were likely to be Hispanic, were likely to be homeless, had a male partner, engaged in sex exchange, and used cocaine or amphetamines during sex. Men who reported HAI scored higher on the Sexual Sensation Seeking scale. This research supports other work showing the relationship between HAI and high-risk behaviors. More important, it contributes new knowledge by demonstrating the association between HAI and sexual sensation seeking. This research highlights the importance of personality traits when trying to understand sexual behavior and when developing HIV prevention interventions.


Development of drugs for severe malaria in children.
Over 90% of deaths attributable to malaria are in African children under 5 years old. Yet, new treatments are often tested primarily in adult patients and extrapolations have proven to be sometimes invalid, especially in dosing regimens. For studies in severe malaria an additional complication is that the decline in severe malaria in adult patients precludes sufficiently powered trials in adults, before the intervention can be tested in the ultimate target group, paediatric severe malaria. In this paper we propose an alternative pathway to the development of drugs for use in paediatric severe malaria. We argue that following the classical phase I and II studies, small safety and efficacy studies using well-chosen surrogate endpoints in adult severe malaria be conducted, instead of larger mortality endpoint trials. If the drug appears safe and promising small pilot studies in paediatric severe malaria using the same endpoints can follow. Finally, with carefully observed safeguards in place to ensure high ethical standards, promising candidate interventions can be taken forward into mortality endpoint, well-powered, large paediatric studies in African children with severe malaria. Given the available research capacity, limited numbers of prudently selected interventions can be studied in phase III trials, and adaptive designs should be considered.


Standard claims and appeals forms. Final rule.
The Department of Veterans Affairs (VA) amends its adjudication regulations and the appeals regulations and rules of practice of the Board of Veterans' Appeals (Board) to require that all claims governed by VA's adjudication regulations be filed on standard forms prescribed by the Secretary, regardless of the type of claim or posture in which the claim arises. This rulemaking also eliminates the constructive receipt of VA reports of hospitalization or examination and other medical records as informal claims for increase or to reopen while retaining the retroactive effective date assignment for awards for claims for increase which are filed on a standard form within 1 year of such hospitalization, examination, or treatment. This final rule also implements the concept of an intent to file a claim for benefits, which operates similarly to the current informal claim process, but requires that the submission establishing a claimant's effective date of benefits must be received in one of three specified formats. Finally, these amendments will provide that VA will accept an expression of dissatisfaction or disagreement with an adjudicative determination by the agency of original jurisdiction(AOJ) as a Notice of Disagreement (NOD) only if it is submitted on a standardized form provided by VA for the purpose of appealing the decision, in cases where such a form is provided. Although a standardized NOD form will only initially be provided in connection with decisions on compensation claims, VA may require a standard NOD form for any type of claim for VA benefits if, in the future, it develops and provides a standardized NOD form for a particular benefit. The purpose of these amendments is to improve the quality and timeliness of the processing of veterans' claims for benefits by standardizing the claims and appeals processes through the use of forms.


Microglial activation, increased TNF and SERT expression in the prefrontal cortex define stress-altered behaviour in mice susceptible to anhedonia.
A chronic stress paradigm comprising exposure to predation, tail suspension and restraint induces a depressive syndrome in C57BL/6J mice that occurs in some, but not all, animals. Here, we sought to extend our behavioural studies to investigate how susceptibility (sucrose preference<65%) or resilience (sucrose preference>65%) to stress-induced anhedonia affects the 5HT system and the expression of inflammation-related genes. All chronically stressed animals, displayed increased level of anxiety, but susceptible mice exhibited an increased propensity to float in the forced swim test and demonstrate hyperactivity under stressful lighting conditions. These changes were not present in resilient or acutely stressed animals. Compared to resilient animals, susceptible mice showed elevated expression of tumour necrosis factor alpha (TNF) and the 5-HT transporter (SERT) in the pre-frontal area. Enhanced expression of 5HT(2A) and COX-1 in the pre-frontal area was observed in all stressed animals. In turn, indoleamine-2,3-dioxygenase (IDO) was significantly unregulated in the raphe of susceptible animals. At the cellular level, increased numbers of Iba-1-positive microglial cells were also present in the prefrontal area of susceptible animals compared to resilient animals. Consequently, the susceptible animals display a unique molecular profile when compared to resilient, but anxious, animals. Unexpectedly, this altered profile provides a rationale for exploring anti-inflammatory, and possibly, TNF-targeted therapy for major depression.


Exploring the pattern and neural correlates of neuropsychological impairment in late-life depression.
Neuropsychological impairment is a key feature of late-life depression, with deficits observed across multiple domains. However, it is unclear whether deficits in multiple domains represent relatively independent processes with specific neural correlates or whether they can be explained by cognitive deficits in executive function or processing speed. We examined group differences across five domains (episodic memory; executive function; language skills; processing speed; visuospatial skills) in a sample of 36 depressed participants and 25 control participants, all aged ≥ 60 years. The influence of executive function and processing speed deficits on other neuropsychological domains was also investigated. Magnetic resonance imaging correlates of executive function, processing speed and episodic memory were explored in the late-life depression group. Relative to controls, the late-life depression group performed significantly worse in the domains of executive function, processing speed, episodic memory and language skills. Impairments in executive function or processing speed were sufficient to explain differences in episodic memory and language skills. Executive function was correlated with anisotropy of the anterior thalamic radiation and uncinate fasciculus; processing speed was correlated with anisotropy of genu of the corpus callosum. Episodic memory was correlated with anisotropy of the anterior thalamic radiation, the genu and body of the corpus callosum and the fornix. Executive function and processing speed appear to represent important cognitive deficits in late-life depression, which contribute to deficits in other domains, and are related to reductions in anisotropy in frontal tracts.


Individual Differences in the Alignment of Structural and Functional Markers of the V5/MT Complex in Primates.
Extrastriate visual area V5/MT in primates is defined both structurally by myeloarchitecture and functionally by distinct responses to visual motion. Myelination is directly identifiable from postmortem histology but also indirectly by image contrast with structural magnetic resonance imaging (sMRI). First, we compared the identification of V5/MT using both sMRI and histology in Rhesus macaques. A section-by-section comparison of histological slices with in vivo and postmortem sMRI for the same block of cortical tissue showed precise correspondence in localizing heavy myelination for V5/MT and neighboring MST. Thus, sMRI in macaques accurately locates histologically defined myelin within areas known to be motion selective. Second, we investigated the functionally homologous human motion complex (hMT+) using high-resolution in vivo imaging. Humans showed considerable intersubject variability in hMT+ location, when defined with myelin-weighted sMRI signals to reveal structure. When comparing sMRI markers to functional MRI in response to moving stimuli, a region of high myelin signal was generally located within the hMT+ complex. However, there were considerable differences in the alignment of structural and functional markers between individuals. Our results suggest that variation in area identification for hMT+ based on structural and functional markers reflects individual differences in human regional brain architecture.


A Validated Quantification of Sudan Red Dyes in Spicery using TLC and a 16-bit Flatbed Scanner.
We present a video-densitometric quantification method for Sudan red dyes in spices and spice mixtures, separated by TLC. Application was done band-wise in small dots using a 5 μL glass pipette. For separation, the RP-18 plates (20 × 20 cm with fluorescent dye; Merck, Germany, 1.05559) were developed in a vertical developing chamber without vapor saturation from the starting point to a distance of 70 mm by using acetonitrile, methanol, and aqueous ammonia solution (25%; 8 + 1.8 + 0.2, v/v) as mobile phase. The quantification is based on direct measurements using an inexpensive 16-bit flatbed scanner for color measurements (in red, green, and blue). Evaluation of only the green channel makes the measurements very specific. For linearization, an extended Kubelka-Munk expression for data transformation was used. The range of linearity covers more than two magnitudes and lies between 20 and 500 ng. The extraction from a 2 g sample with acetonitrile, evaporation, and reconstitution to 200 μL with methanol and the band-wise application (7 mm) of a 10 μL sample allows a statistically defined LOD of less than 500 ppb of Sudan red dyes. To perform the analysis, a separation chamber, RP-18 plates, 5 μL glass pipettes, and a 16-bit flatbed scanner for 105 € are needed; therefore, the separation method is inexpensive, fast, and reliable.


Measurement of Power in Selectorized Strength Training Equipment.
We derived the exact analytical expression of the instantaneous joint power in exercises with single-joint, variable-resistance, selectorized strength-training equipment, taking into account all the relevant geometric, kinematic and dynamic variables of both the movable equipment elements (resistance input lever, cam-pulley system, weight stack) and of the user's exercising limb. A numerical algorithm was also designed to express, in the presence of a cam, the rectilinear kinematic variables of the weight stack as a function of the rotational kinematic variables of the resistance input lever, and vice versa. Given that information, one can measure the value of the instantaneous and mean joint power exclusively by means of a linear encoder placed on the weight stack or, alternatively, only by the use of an angular encoder placed on the rotational axis of the resistance lever. The results highlight that, for knee extension exercises with leg extension equipment, the real values of both instantaneous and mean joint power may differ by more than 50% in comparison with the values obtained by taking into account only the mass and velocity of the weight stack. These differences are notable not only in explosive exercises, but whenever considerable joint velocities/accelerations occur within the range of motion.


Multifunctional T-cell analyses to study response and progression in adoptive cell transfer immunotherapy.
Adoptive cell transfer (ACT) of genetically engineered T cells expressing cancer-specific T-cell receptors (TCR) is a promising cancer treatment. Here, we investigate the in vivo functional activity and dynamics of the transferred cells by analyzing samples from 3 representative patients with melanoma enrolled in a clinical trial of ACT with TCR transgenic T cells targeted against the melanosomal antigen MART-1. The analyses included evaluating 19 secreted proteins from individual cells from phenotypically defined T-cell subpopulations, as well as the enumeration of T cells with TCR antigen specificity for 36 melanoma antigens. These analyses revealed the coordinated functional dynamics of the adoptively transferred, as well as endogenous, T cells, and the importance of highly functional T cells in dominating the antitumor immune response. This study highlights the need to develop approaches to maintaining antitumor T-cell functionality with the aim of increasing the long-term efficacy of TCR-engineered ACT immunotherapy. A longitudinal functional study of adoptively transferred TCR–engineered lymphocytes yielded revealing snapshots for understanding the changes of antitumor responses over time in ACT immunotherapy of patients with advanced melanoma.


Construction of Functional Coatings with Durable and Broad-Spectrum Antibacterial Potential Based on Mussel-Inspired Dendritic Polyglycerol and in Situ-Formed Copper Nanoparticles.
A novel surface coating with durable broad-spectrum antibacterial ability was prepared based on mussel-inspired dendritic polyglycerol (MI-dPG) embedded with copper nanoparticles (Cu NPs). The functional surface coating is fabricated via a facile dip-coating process followed by in situ reduction of copper ions with a MI-dPG coating to introduce Cu NPs into the coating matrix. This coating has been demonstrated to possess efficient long-term antibacterial properties against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and kanamycin-resistant E. coli through an "attract-kill-release" strategy. The synergistic antibacterial activity of the coating was shown by the combination of two functions of the contact killing, reactive oxygen species production and Cu ions released from the coating. Furthermore, this coating inhibited biofilm formation and showed good compatibility to eukaryotic cells. Thus, this newly developed Cu NP-incorporated MI-dPG surface coating may find potential application in the design of antimicrobial coating, such as implantable devices.


Effect of radical scavenger N-tert-butyl-alpha-phenylnitrone on stroke in a rat model using a telemetric system.
We used malignant stroke-prone spontaneously hypertensive rats (M-SHRSP) as a stroke model to explore the effects of the radical scavenger N-tert-butyl-alpha-phenylnitrone (PBN) on stroke. PBN was administrated in drinking water to M-SHRSP. Circadian rhythms in heart rate, blood pressure, and locomotive activity in M-SHRSP were monitored with a telemetric system, in addition to measurement of water intake and body weight. Stroke-onset was assessed by changes in neurological symptoms, water intake, and body weight. Circadian rhythms were basically the same between PBN-treated and control rats several days after stroke onset. Significant differences were seen in blood pressure, relative weight of brain and water intake, heart rate, and locomotive activity between two groups. As a result, no significant difference in age of stroke onset was seen between PBN-treated and control rats, but PBN-treated rats displayed prolonged mean life spans. PBN might be effective in prolonging life span.


Assessing reading difficulties: the validity and utility of current measures of reading skill.
Accurate assessment of reading difficulties is clearly important if appropriate support and remediation is to be provided. Many different reading tests are routinely used yet it is not clear to what extent different tests tap the same underlying skills. The nature of the relationships between different tests of reading accuracy, reading comprehension and linguistic comprehension is investigated in this paper. SAMPLES, METHODS AND RESULTS: In study 1, 184 7-10 year old children completed a listening comprehension test, three tests of reading accuracy (reading of nonwords, single words and text) and two tests of reading comprehension (test comprehension and sentence completion). While sentence completion was well accounted for by individual differences in word recognition, text comprehension was more heavily dependent on listening comprehension. Study 2 compared the performance of children with poor comprehension skills with controls matched for age, nonverbal ability and decoding skill. The poor comprehenders had greatest difficulty with those tests most heavily dependent on linguistic comprehension and least difficulty on purer measures of decoding. These findings show that different reading tests measure different aspects of the reading process and that caution should be exercised when selecting tests for the assessment of reading difficulties.


Reliability of administrative data to identify sexually transmitted infections for population health: a systematic review.
International Classification of Diseases (ICD) codes in administrative health data are used to identify cases of disease, including sexually transmitted infections (STIs), for population health research. The purpose of this review is to examine the extant literature on the reliability of ICD codes to correctly identify STIs. We conducted a systematic review of empirical articles in which ICD codes were validated with respect to their ability to identify cases of chlamydia, gonorrhoea, syphilis or pelvic inflammatory disease (PID). Articles that included sensitivity, specificity and positive predictive value of ICD codes were the target. In addition to keyword searches in PubMed and Scopus databases, we further examined bibliographies of articles selected for full review to maximise yield. From a total of 1779 articles identified, only two studies measured the reliability of ICD codes to identify cases of STIs. Both articles targeted PID, a serious complication of chlamydia and gonorrhoea. Neither article directly assessed the validity of ICD codes to identify cases of chlamydia, gonorrhoea or syphilis independent of PID. Using ICD codes alone, the positive predictive value for PID was mixed (range: 18%-79%). While existing studies have used ICD codes to identify STI cases, their reliability is unclear. Further, available evidence from studies of PID suggests potentially large variation in the accuracy of ICD codes indicating the need for primary studies to evaluate ICD codes for use in STI-related public health research.


Prevalence of and risk factors for perioperative arrhythmias in neonates and children after cardiopulmonary bypass: continuous holter monitoring before and for three days after surgery.
A comprehensive evaluation of postoperative arrhythmias following surgery for congenital heart disease by continuous Holter monitoring has not been carried out. We aimed, firstly, to establish the time course of pre- and early postoperative arrhythmias by beat-to-beat analysis following cardiopulmonary bypass and, secondly, to examine which surgical procedures present risk factors for specific arrhythmias. 494 consecutive patients, including 96 neonates, were studied with serial 24-hour Holter electrocardiograms before as well as uninterruptedly during the first 72 hours after surgery and prior to discharge. Within 24 hours of surgery 59% of the neonates and 79% of the older children developed arrhythmias. Junctional ectopic tachycardia occurred in 9% of neonates and 5% of non-neonates and ventricular tachycardia in 3% and 15%, respectively.For neonates, male sex and longer cross-clamping time independently increased the risk for arrhythmias (odds ratios 2.83 and 1.96/minute, respectively). Ventricular septal defect repair was a strong risk factor for junctional ectopic tachycardia in neonates and in older children (odds ratios 18.8 and 3.69, respectively). For infants and children, older age (odds ratio 1.01/month) and closure of atrial septal defects (odds ratio 2.68) predisposed to arrhythmias of any type. We present the largest cohort of neonates, infants and children that has been prospectively studied for the occurrence of arrhythmias after cardiac surgery. Postoperative arrhythmias are a frequent and transient phenomenon after cardiopulmonary bypass, provoked both by mechanical irritation of the conduction system and by humoral factors.


Inference of trustworthiness from intuitive moral judgments.
Moral judgments play a critical role in motivating and enforcing human cooperation, and research on the proximate mechanisms of moral judgments highlights the importance of intuitive, automatic processes in forming such judgments. Intuitive moral judgments often share characteristics with deontological theories in normative ethics, which argue that certain acts (such as killing) are absolutely wrong, regardless of their consequences. Why do moral intuitions typically follow deontological prescriptions, as opposed to those of other ethical theories? Here, we test a functional explanation for this phenomenon by investigating whether agents who express deontological moral judgments are more valued as social partners. Across 5 studies, we show that people who make characteristically deontological judgments are preferred as social partners, perceived as more moral and trustworthy, and are trusted more in economic games. These findings provide empirical support for a partner choice account of moral intuitions whereby typically deontological judgments confer an adaptive function by increasing a person's likelihood of being chosen as a cooperation partner. Therefore, deontological moral intuitions may represent an evolutionarily prescribed prior that was selected for through partner choice mechanisms. (PsycINFO Database Record


MLP accumulation and remodelling in the infarcted rat heart.
Mutation of cytoskeletal protein genes results in abnormal protein function and causes cardiomyopathy. We hypothesised that cardiac levels of cytoskeletal proteins, such as dystrophin, desmin and muscle LIM protein (MLP), would be altered during remodelling caused by myocardial infarction (MI). We measured left-ventricular morphology, function and cytoskeletal protein levels 10 weeks after coronary artery ligation or sham operation in male Wistar rats. Two-dimensional echocardiography revealed significant impairment of systolic function and decreased ejection fraction in infarcted hearts compared with sham (47+/-5% versus 73+/-4%), commensurate with the development of heart failure. Western blotting was used to measure levels of beta-myosin heavy chain (beta-MyHC), a marker of hypertrophy, and levels of dystrophin, desmin, MLP, beta-tubulin, utrophin and syncoilin, using GAPDH for normalization. Relative to shams, beta-MyHC and MLP levels were increased 1.9-fold and 1.7-fold, respectively, in infarcted rat hearts, whereas the levels of other cytoskeletal proteins were unchanged. Both MLP and desmin protein levels correlated negatively with ejection fraction, with the strongest relation between MLP and ejection fraction (r=-0.95, n=13, p<0.0001). This work suggests that MLP may play an important compensatory role in cardiac remodelling following MI.


Antibodies to the N-methyl-D-aspartate receptor and other synaptic proteins in psychosis.
This review concentrates on the evidence for autoantibodies to cell surface synaptic proteins in psychosis and schizophrenia. We and others have recently found antibodies to the N-methyl-D-aspartate receptor in first-episode psychosis. We describe the evidence for pathogenicity and disease-relevance of these antibodies, which builds on the novel field in neuroimmunology of cell surface antibody-associated central nervous system disorders. Relevant autoantibodies in psychosis and schizophrenia are likely to be those directed to cell surface proteins, in which the likelihood of pathogenicity is greater. We discuss the evidence for this from the field of paraneoplastic neurologic syndromes and the discovery of novel cell surface antigen central nervous system autoimmune syndromes.


Monitoring modulators of platelet aggregation in a microtiter plate assay.
Platelets play a central role in maintaining biological hemostasis. Inappropriate platelet activation is responsible for thrombotic diseases such as myocardial infarction and stroke. Therefore, novel agents that can inhibit platelet activation are necessary. However, assays that monitor platelet aggregation are generally time-consuming and require high volumes of blood and specialized equipment. Therefore, a medium- to high-throughput assay that can monitor platelet aggregation would be considered useful. Such an assay should be sensitive, comparable to the "gold standard" assay of platelet aggregometry, and able to monitor multiple samples simultaneously but with low assay volumes. We have developed such a microtiter assay. It can assay an average of 60 independent treatments per 60 ml blood donation and demonstrates greater sensitivity than the current gold standard assay, namely platelet aggregation in stirring conditions in a platelet aggregometer. The microtiter plate (MTP) assay can detect known inhibitors of platelet function such as indomethacin, aspirin, and ReoPro. It is highly reproducible when using standard doses of agonists such as thrombin receptor-activating peptide (20 microM) and collagen (0.19 mg/ml). Finally, the MTP assay is rapid and sensitive and can detect unknown platelet-modulating agents from a library of compounds.


Mice overexpressing the 5-hydroxytryptamine transporter show no alterations in feeding behaviour and increased non-feeding responses to fenfluramine.
The 5-HT transporter (5-HTT) is implicated in the regulation of appetite. Expression of the 5-HTT varies in the human population, and this variation may determine both individual differences in feeding and abnormal feeding behaviours such as eating disorders. The effects of 5-HTT expression on feeding and satiety were examined in a transgenic mouse model of 5-HTT overexpression. We measured free-feeding food intake and observed the behavioural satiety sequence (BSS) after food deprivation in mice at baseline and after administration of the anorectic drug fenfluramine. 5-HTT overexpressing mice were both lighter and shorter than their wildtype littermates. Despite this size difference, food intake by transgenic and wildtype mice did not differ. There was no effect of genotype on the BSS or on food intake during the test at baseline. Increasing doses of fenfluramine reduced food intake in a similar manner in both transgenic and wildtype mice. After 0.3 and 1 mg/kg fenfluramine, the temporal pattern of the BSS was the same for both groups, whereas 3 and 10 mg/kg fenfluramine disrupted the BSS. In transgenic mice, this disruption was evident at the 3 mg/kg dose, while in wildtypes, it emerged only at the 10-mg/kg dose. These data suggest that overexpression of the 5-HTT does not lead to alterations in feeding or satiety in food-deprived mice but does increase the occurrence of other non-feeding behaviours in response to the 5-HT releasing agent fenfluramine.


The promise of layer-specific neuroimaging for testing predictive coding theories of psychosis.
Predictive coding potentially provides an explanatory model for understanding the neurocognitive mechanisms of psychosis. It proposes that cognitive processes, such as perception and inference, are implemented by a hierarchical system, with the influence of each level being a function of the estimated precision of beliefs at that level. However, predictive coding models of psychosis are insufficiently constrained-any phenomenon can be explained in multiple ways by postulating different changes to precision at different levels of processing. One reason for the lack of constraint in these models is that the core processes are thought to be implemented by the function of specific cortical layers, and the technology to measure layer specific neural activity in humans has until recently been lacking. As a result, our ability to constrain the models with empirical data has been limited. In this review we provide a brief overview of predictive processing models of psychosis and then describe the potential for newly developed, layer specific neuroimaging techniques to test and thus constrain these models. We conclude by discussing the most promising avenues for this research as well as the technical and conceptual challenges which may limit its application.


5' AMP-activated protein kinase activation causes GLUT4 translocation in skeletal muscle.
It has previously been reported that exercise causes an increase in glucose uptake in skeletal muscle and also an increase in 5' AMP-activated protein kinase (AMPK) activity. 5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICA-riboside), an analog of adenosine, is taken up into cells and phosphorylated to form AICA-riboside monophosphate (ZMP), which can also activate AMPK. This study was designed to determine whether the increase in glucose uptake observed with AMPK activation by AICA-riboside is due to GLUT4 translocation from an intracellular location to the plasma membranes, similar to that seen in response to contraction. Rat hindlimbs were perfused with Krebs-Henseleit bicarbonate containing 4% bovine serum albumin, washed bovine erythrocytes, 8 mmol/l glucose, and +/-2 mmol/AICA-riboside or +/-60 nmol/l insulin. Perfusion medium containing AICA-riboside was found to significantly increase AMPK activity, glucose uptake, and GLUT4 translocation in skeletal muscle above basal levels. Insulin-perfused muscles showed significant increases in glucose uptake and GLUT4 translocation, but AMPK activation was not significantly changed from basal levels. These results provide evidence that the increased glucose uptake observed with AMPK activation by AICA-riboside in perfused rat hindlimb muscles is due to an increase in the translocation of GLUT4 to surface membranes.


Subventricular zone cytoarchitecture changes in autism.
Autism is thought to be a neurodevelopmental disorder with symptoms developing during neonatal neurogenesis in the subventricular zone (SVZ). Autism associated genes alter SVZ proliferation and cytoarchitecture, yet the response of the human SVZ in autism is unknown. Epilepsy drives neurogenesis in rodents, but it is unclear how epilepsy interacts with autism in SVZ responses. The striatal and septal SVZ derive from separate lineages in rodents and generate different interneuron types. Yet it is unclear if autism unevenly regulates the striatal and septal SVZ. The human SVZ was immunohistochemically examined post-mortem from individuals with autism (n = 11) and controls (n = 11). Autism showed a lower cell density in the septal, but not striatal, SVZ hypocellular gap only in the absence of epilepsy. There was a decline in septal hypocellular gap cells with age in autism, but no correlation with age in controls. In contrast, PCNA+ cell numbers increased only in autism with epilepsy both in the hypocellular gap and in the ependymal layer on the septal but not striatal side. Ependymal cells also became GFAP immunoreactive in autism irrespective of epilepsy co-morbidity; however, this only occurred on the striatal side. In examining these questions we also discovered a subset of ependymal, astrocyte ribbon and RMS cells which express PCNA and Ki67, PLP, and α-tubulin. These results are the first example of a neuropsychiatric disease differentially affecting the septal and striatal SVZ. Altered cell density in the hypocellular gap and proliferation marker expression suggest individuals with autism may follow a different growth-trajectory.


[Typology, values and preferences of people with HIV and imaginaries of infection: a qualitative research: Spain, 2010].
Therapeutic advance achieved over the last fifteen years in addressing HIV and AIDS correlate with changes in the subjective experiences of persons with HIV and in the sociocultural imaginary of HIV infection. This paper reports findings on two areas: the current typology of HIV patients in the Spanish case and the evolution of the perception of HIV among PLWHA. Qualitative study in eight Spanish cities (October 2010-March 2011). We conducted 9 focus groups and 30 in-depth interviews with PLWHA of different ages and length of treatment, all of them under medical care. Interviews and focus group were transcribed and narrative and discursive data were subjected to sociological analysis. We suggest a classification of 7 different collectives of persons with HIV in the Spanish context, which reflect different historical periods in the epidemiology of the infection as well as the evolution of treatments and of the experiences of those infected. In recent times there have been significant changes in the social system of images associated with HIV, which point to an increasingly debilitated image of chronicity and to a decline in the perception of its danger to health. But this new imaginary does not distribute equally among all persons with HIV. On the contrary, there is a certain polarization of images, essentially between the two sub-groups most epidemiologically relevant among newly diagnosed HIV patients in Spain: younger men who have sex with men (<30-35 years) and immigrants.


Mindfulness-based stress reduction in relation to quality of life, mood, symptoms of stress, and immune parameters in breast and prostate cancer outpatients.
This study investigated the relationships between a mindfulness-based stress reduction meditation program for early stage breast and prostate cancer patients and quality of life, mood states, stress symptoms, lymphocyte counts, and cytokine production. Forty-nine patients with breast cancer and 10 with prostate cancer participated in an 8-week MBSR program that incorporated relaxation, meditation, gentle yoga, and daily home practice. Demographic and health behavior variables, quality of life (EORTC QLQ C-30), mood (POMS), stress (SOSI), and counts of NK, NKT, B, T total, T helper, and T cytotoxic cells, as well as NK and T cell production of TNF, IFN-gamma, IL-4, and IL-10 were assessed pre- and postintervention. Fifty-nine and 42 patients were assessed pre- and postintervention, respectively. Significant improvements were seen in overall quality of life, symptoms of stress, and sleep quality. Although there were no significant changes in the overall number of lymphocytes or cell subsets, T cell production of IL-4 increased and IFN-gamma decreased, whereas NK cell production of IL-10 decreased. These results are consistent with a shift in immune profile from one associated with depressive symptoms to a more normal profile. MBSR participation was associated with enhanced quality of life and decreased stress symptoms in breast and prostate cancer patients. This study is also the first to show changes in cancer-related cytokine production associated with program participation.


Downregulation of ASPP1 in gestational trophoblastic disease: correlation with hypermethylation, apoptotic activity and clinical outcome.
Gestational trophoblastic disease encompasses a spectrum of trophoblastic lesions including true neoplasms such as choriocarcinomas and the potentially malignant hydatidiform moles, which may develop persistent disease requiring chemotherapy. ASPP1, a member of apoptosis-stimulating proteins of p53 (ASPPs), is a proapoptotic protein that can stimulate apoptosis through its interaction with p53. We evaluated the promoter methylation and expression profiles of ASPP1 in different trophoblastic tissues and its in vitro functional effect on two choriocarcinoma cell lines, namely JEG-3 and JAR. Significant downregulation of ASPP1 mRNA and protein levels was demonstrated in hydatidiform moles and choriocarcinomas, when compared with normal placentas by quantitative-PCR and immunohistochemistry. The ASPP1 mRNA level was significantly correlated with its hypermethylation status, evaluated with methylation-specific PCR, in placenta and gestational trophoblastic disease samples (P=0.024). Moreover, lower ASPP1 immunoreactivity was shown in hydatidiform moles that progressed to persistent gestational trophoblastic neoplasms than in those that regressed (P=0.045). A significant correlation was also found between expression of ASPP1 and proliferative indices (assessed by Ki67 and MCM7), apoptotic activity (M30 CytoDeath antibody), p53 and caspase-8 immunoreactivities. An in vitro study showed that ectopic expression of ASPP1 could trigger apoptosis through intrinsic and extrinsic pathways as indicated by an increase in cleaved caspase-9 and Fas ligand protein expression. The latter suggests a hitherto unreported novel link between ASPP1 and the extrinsic pathway of apoptosis. Our findings suggest that downregulation of ASPP1 by hypermethylation may be involved in the pathogenesis and progress of gestational trophoblastic disease, probably through its effect on apoptosis.


Associations of adiponectin and leptin with stage and grade of PSA-detected prostate cancer: the ProtecT study.
Obesity has been associated with an increased risk of advanced and fatal prostate cancer; adipokines may mediate this association. We examined associations of the adipokines leptin and adiponectin with the stage and grade of PSA-detected prostate cancer. We conducted a nested case-control study comparing 311 men with mainly locally advanced (≥T3, N1, or M1 cases) vs. 413 men with localized (T ≤2 & NX-0 & M0 controls) PSA-detected prostate cancer, recruited 2001-2009 from 9 UK regions to the ProtecT study. Associations of body mass index and adipokine levels with prostate cancer stage were determined by conditional logistic regression and with grade (Gleason score ≥7 vs. ≤6) by unconditional logistic regression. Adiponectin was inversely associated with prostate cancer stage in overweight and obese men (OR 0.62; 95 % CI 0.42-0.90; p = 0.01), but not in normal weight men (OR 1.48; 0.77-2.82; p = 0.24) (p for interaction 0.007), or all men (OR 0.86; 0.66-1.11; p = 0.24). There was no compelling evidence of associations between leptin or leptin to adiponectin ratio and prostate cancer stage. No strong associations of adiponectin, leptin, or leptin:adiponectin ratio with grade were seen. This study provides some evidence that adiponectin levels may be associated with prostate cancer stage, dependent on the degree of adiposity of the man. Our results are consistent with adiponectin countering the adverse effects of obesity on prostate cancer progression.


Healthcare Access and Quality Index based on mortality from causes amenable to personal health care in 195 countries and territories, 1990-2015: a novel analysis from the Global Burden of Disease Study 2015.
National levels of personal health-care access and quality can be approximated by measuring mortality rates from causes that should not be fatal in the presence of effective medical care (ie, amenable mortality). Previous analyses of mortality amenable to health care only focused on high-income countries and faced several methodological challenges. In the present analysis, we use the highly standardised cause of death and risk factor estimates generated through the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) to improve and expand the quantification of personal health-care access and quality for 195 countries and territories from 1990 to 2015. We mapped the most widely used list of causes amenable to personal health care developed by Nolte and McKee to 32 GBD causes. We accounted for variations in cause of death certification and misclassifications through the extensive data standardisation processes and redistribution algorithms developed for GBD. To isolate the effects of personal health-care access and quality, we risk-standardised cause-specific mortality rates for each geography-year by removing the joint effects of local environmental and behavioural risks, and adding back the global levels of risk exposure as estimated for GBD 2015. We employed principal component analysis to create a single, interpretable summary measure-the Healthcare Quality and Access (HAQ) Index-on a scale of 0 to 100. The HAQ Index showed strong convergence validity as compared with other health-system indicators, including health expenditure per capita (r=0·88), an index of 11 universal health coverage interventions (r=0·83), and human resources for health per 1000 (r=0·77). We used free disposal hull analysis with bootstrapping to produce a frontier based on the relationship between the HAQ Index and the Socio-demographic Index (SDI), a measure of overall development consisting of income per capita, average years of education, and total fertility rates. This frontier allowed us to better quantify the maximum levels of personal health-care access and quality achieved across the development spectrum, and pinpoint geographies where gaps between observed and potential levels have narrowed or widened over time. Between 1990 and 2015, nearly all countries and territories saw their HAQ Index values improve; nonetheless, the difference between the highest and lowest observed HAQ Index was larger in 2015 than in 1990, ranging from 28·6 to 94·6. Of 195 geographies, 167 had statistically significant increases in HAQ Index levels since 1990, with South Korea, Turkey, Peru, China, and the Maldives recording among the largest gains by 2015. Performance on the HAQ Index and individual causes showed distinct patterns by region and level of development, yet substantial heterogeneities emerged for several causes, including cancers in highest-SDI countries; chronic kidney disease, diabetes, diarrhoeal diseases, and lower respiratory infections among middle-SDI countries; and measles and tetanus among lowest-SDI countries. While the global HAQ Index average rose from 40·7 (95% uncertainty interval, 39·0-42·8) in 1990 to 53·7 (52·2-55·4) in 2015, far less progress occurred in narrowing the gap between observed HAQ Index values and maximum levels achieved; at the global level, the difference between the observed and frontier HAQ Index only decreased from 21·2 in 1990 to 20·1 in 2015. If every country and territory had achieved the highest observed HAQ Index by their corresponding level of SDI, the global average would have been 73·8 in 2015. Several countries, particularly in eastern and western sub-Saharan Africa, reached HAQ Index values similar to or beyond their development levels, whereas others, namely in southern sub-Saharan Africa, the Middle East, and south Asia, lagged behind what geographies of similar development attained between 1990 and 2015. This novel extension of the GBD Study shows the untapped potential for personal health-care access and quality improvement across the development spectrum. Amid substantive advances in personal health care at the national level, heterogeneous patterns for individual causes in given countries or territories suggest that few places have consistently achieved optimal health-care access and quality across health-system functions and therapeutic areas. This is especially evident in middle-SDI countries, many of which have recently undergone or are currently experiencing epidemiological transitions. The HAQ Index, if paired with other measures of health-system characteristics such as intervention coverage, could provide a robust avenue for tracking progress on universal health coverage and identifying local priorities for strengthening personal health-care quality and access throughout the world. Bill & Melinda Gates Foundation.