Transient suppression of hepatocellular replication in the mouse liver following transduction with recombinant adeno-associated virus.

Recombinant vectors based on adeno-associated virus (AAV) are proving to be powerful tools for genetic manipulation of the liver, for both discovery and therapeutic purposes. The system can be used to deliver transgene cassettes for expression or, alternatively, DNA templates for genome editing via homologous recombination. The replicative state of target cells is known to influence the efficiency of these processes and knowledge of the host-vector interactions involved is required for optimally effective vector deployment. Here we show, for the first time in vivo, that in addition to the known effects of hepatocellular replication on AAV-mediated gene transfer, the vector itself exerts a potent, albeit transient suppressive effect on cell cycle progression that is relieved on a time course that correlates with the known rate of clearance of input single-stranded vector DNA. This finding requires further mechanistic investigation, delineates an excellent model system for such studies and further deepens our insight into the complexity of interactions between AAV vectors and the cell cycle in a clinically promising target tissue.

The global burden attributable to low bone mineral density.

INTRODUCTION: The Global Burden of Disease Study 2010 estimated the worldwide health burden of 291 diseases and injuries and 67 risk factors by calculating disability-adjusted life years (DALYs). Osteoporosis was not considered as a disease, and bone mineral density (BMD) was analysed as a risk factor for fractures, which formed part of the health burden due to falls. OBJECTIVES: To calculate (1) the global distribution of BMD, (2) its population attributable fraction (PAF) for fractures and subsequently for falls, and (3) the number of DALYs due to BMD. METHODS: A systematic review was performed seeking population-based studies in which BMD was measured by dual-energy X-ray absorptiometry at the femoral neck in people aged 50 years and over. Age- and sex-specific mean ± SD BMD values (g/cm(2)) were extracted from eligible studies. Comparative risk assessment methodology was used to calculate PAFs of BMD for fractures. The theoretical minimum risk exposure distribution was estimated as the age- and sex-specific 90th centile from the Third National Health and Nutrition Examination Survey (NHANES III). Relative risks of fractures were obtained from a previous meta-analysis. Hospital data were used to calculate the fraction of the health burden of falls that was due to fractures. RESULTS: Global deaths and DALYs attributable to low BMD increased from 103 000 and 3 125 000 in 1990 to 188 000 and 5 216 000 in 2010, respectively. The percentage of low BMD in the total global burden almost doubled from 1990 (0.12%) to 2010 (0.21%). Around one-third of falls-related deaths were attributable to low BMD. CONCLUSIONS: Low BMD is responsible for a growing global health burden, only partially representative of the real burden of osteoporosis.

AAV-encoded OTC activity persisting to adulthood following delivery to newborn spf(ash) mice is insufficient to prevent shRNA-induced hyperammonaemia.

Urea cycle defects presenting in the neonatal period with hyperammonaemia are associated with high morbidity and mortality, and necessitate liver transplantation for long-term management. Gene therapy is therefore an attractive possibility, with vectors based on adeno-associated virus (rAAV) currently showing exciting promise in liver-targeted clinical trials in adults. Successful use of rAAV vectors in infants, however, is more challenging as episomal rAAV genomes will be lost from proliferating hepatocytes during liver growth, leaving stable transgene expression dependent on the subset of vector genomes that undergo genomic integration. To explore this challenge, we exploited the partially ornithine transcarbamylase (OTC)-deficient spf(ash) mouse model and small hairpin RNA-mediated knockdown of residual endogenous OTC enzyme activity in adult mice that had received neonatal treatment with an OTC-encoding rAAV. This leaves mice reliant on vector-encoded OTC activity that has persisted from the newborn period. Despite stable transduction in approximately 8% of hepatocytes and residual vector-encoded OTC activity of up to 33% of wild-type, well above endogenous spf(ash) levels (5-7%), mice were not protected from hyperammonaemia. These data show that the distribution of OTC activity within the liver is critical and that rAAV vector re-delivery after early neonatal treatment is likely to be necessary for stable control of hyperammonaemia into adulthood.

Contrasting effects of diclofenac and ibuprofen on active imatinib uptake into leukaemic cells.

BACKGROUND: The human organic cation transporter-1 (OCT-1) is the primary active protein for imatinib uptake into target BCR-ABL-positive cells. Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently used by chronic myeloid leukaemia (CML) patients on imatinib to manage musculoskeletal complaints. METHODS: Here we investigated the impact of NSAIDs on functional activity of the OCT-1 (OCT-1 activity; OA) in CML cells. RESULTS: Although ten of twelve NSAIDs tested had no significant impact on OA (P>0.05), we observed increased OA (27% increase in K562; 22% increase in KU812 cells, P<0.05) and reduced IC50(imatinib) when treated with diclofenac. Co-incubation with imatinib and diclofenac resulted in a significantly lower viable cell number compared with imatinib alone. In contrast, ibuprofen led to a significant decrease in OA, an increase in IC50(imatinib) and thus reduced the cytotoxicity of imatinib. In primary CML samples, diclofenac significantly increased OA, particularly in patients with low OA (<4 ng per 200 000 cells), and significantly decreased IC50(imatinib). Ibuprofen induced significant decreases in OA in CML samples and healthy donors. CONCLUSION: On the basis of the expected impact of these two drugs on OA, ibuprofen should be avoided in combination with imatinib. Further studies are warranted regarding the potential benefit of diclofenac to improve OA in a clinical setting.

Does increased sunlight exposure work as a strategy to improve vitamin D status in the elderly: a cluster randomised controlled trial.

SUMMARY: Sunlight exposure by improving vitamin D status could be a simple public health strategy in reducing falls among frail elder people. In a randomised controlled trial, adherence to sunlight exposure was low (median adherence, 26%) and no effect of increased UV exposure on falls risk was observed (incidence rate ratio (IRR) 1.06, P = 0.73). INTRODUCTION: This study aimed to determine whether increased sunlight exposure was effective to improve vitamin D status and reduce falls in the elderly. METHODS: In a cluster randomised controlled trial (NCT00322166 at ClinicalTrials.gov), 602 residents aged 70 or more (mean age, 86.4 years; 71% female) were recruited from 51 aged care facilities in Northern Sydney, Australia. Participants were randomised by facility to receive either increased sunlight exposure (additional 30-40 min/day in the early morning) with (UV+) or without (UV) calcium supplementation (600 mg/day) or neither (control) for a year. The co-primary endpoints were change in serum 25 hydroxy vitamin D (25OHD) and falls incidence after 12 months. RESULTS: Adherence to sunlight exposure was low (median adherence, 26%; IQR, 7%-45%). Serum 25OHD levels were low at baseline (median, 32.9 nmol/L) and increased only slightly depending on the number of sunlight sessions attended over 12 months (P = 0.04). During the study, 327 falls occurred in 111 (54%) subjects in the control group, 326 falls in 111 (58%) subjects in the UV only group and 335 falls in 108 (52%) subjects in the UV+ group. By intention-to-treat analysis, there was no significant effect of increased UV exposure on falls risk (IRR, 1.06; 95% CI, 0.76-1.48; P = 0.73). However, in 66 participants who attended ≥130 sessions per year (adherence, ≥50% of 260 sessions-five per week), falls were significantly reduced (IRR, 0.52; 95% CI, 0.31-0.88; P = 0.01) compared with the control group. CONCLUSIONS: Increased sunlight exposure did not reduce vitamin D deficiency or falls risk in frail older people. This public health strategy was not effective most likely due to poor adherence to the intervention.

Placental transfer of SSRI and SNRI antidepressants and effects on the neonate.

INTRODUCTION: We investigated placental transfer and neurobehavioural effects in neonates exposed to citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine or sertraline (SSRI's), or to venlafaxine (an SNRI). METHODS: Women receiving antidepressants during pregnancy and their neonates were studied. Cord and maternal drug concentrations were measured at birth and in the neonates plasma on day 3. Neonates were also assessed using a range of neurobehavioral tests and compared to controls. RESULTS: Median cord/maternal distribution ratio was 0.7-0.86 (range) for SSRIs, 0.72 for the SNRI venlafaxine and 1.08 for the O-desmethyl metabolite. Neonatal abstinence scores were significantly higher (p<0.05) in exposed infants than controls on day 1. Brazelton scores for habituation, social-interactive, motor and autonomic clusters, and serotonin scores were significantly greater (p<0.05) in exposed infants. DISCUSSION: Transfer of SSRIs and SNRIs across the placenta was substantial. Neonates developed mild behavioral symptoms in the early perinatal period but these were self-limiting and similar for both SSRIs and the SNRI venlafaxine.

Fluid flow characteristics within an oscillating lower spherical surface and a stationary concentric upper surface for application to the artificial hip joint.

The synovial fluid motion in an artificial hip joint is important in understanding the thermo-fluids effects that can affect the reliability of the joint, although it is difficult to be studied theoretically, as the modelling involves the viscous fluid interacting with a moving surface. A new analytical solution has been derived for the maximum induced fluid motion within a spherical gap with an oscillating lower surface and a stationary upper surface, assuming one-dimensional incompressible laminar Newtonian flow with constant properties, and using the Navier-Stokes equation. The resulting time-dependent motion is analysed in terms of two dimensionless parameters R and ß, which are functions of geometry, fluid properties and the oscillation rate. The model is then applied to the conditions of the synovial fluid enclosed in the artificial hip joint and it is found that the motion may be described by a simpler velocity variation, whereby laying the foundation to thermal studies in the joint.

A novel, somatic, transforming mutation in the extracellular domain of Epidermal Growth Factor Receptor identified in myeloproliferative neoplasm.

We describe a novel ERBB1/EGFR somatic mutation (p. C329R; c.985 T > C) identified in a patient with JAK2V617F Polycythaemia Vera (PV). This substitution affects a conserved cysteine residue in EGFR domain 2 and leads to the formation of a ligand-independent covalent receptor dimer, associated with increased transforming potential. Aberrant signalling from the EGFRC329R receptor is cell type-dependent and in the TF1.8 erythroid cell line expression of this mutant suppresses EPO-induced differentiation. Clonal analysis shows that the dominant JAK2V617F-positive clone in this PV patient harbors EGFRC329R, thus this mutation may contribute to clonal expansion. Somatic mutations affecting other ERBB and related receptor tyrosine kinases are observed in myeloproliferative neoplasms (MPN), and we show elevated EGFR levels in MPN samples, consistent with previous reports. Thus activation of this group of receptors, via multiple mechanisms, may contribute to clonal growth and survival of the JAK2V617F disease clone in MPN.

Mechanical properties of type I and type IIB single porcine muscle fibres.

Single muscle fibres were isolated from longissimus dorsi (LD) and vastus intermedius muscles at 1 or 8 days post-mortem. Fibres were classified as type I or IIB and their mechanical properties determined. In LD day 1, individual type IIB fibres fractured at higher (P<0.001) loads and lower (P<0.001) stress values than type I fibres. The strength of type I fibres did not differ between muscles. Storage for 8 days decreased (P<0.001) breaking stress of type I fibres, whereas the breaking stress of type IIB fibres did not change. In conclusion, type IIB fibres were weaker per cross-sectional area than type I fibres in LD. The mechanical properties of type I fibres did not differ between muscles and the change in strength with post-mortem storage were more marked in type I than in type IIB fibres.

Novel feeding system to promote establishment of breastfeeds after preterm birth: a randomized controlled trial.

OBJECTIVE: We aimed to determine if a novel feeding system where milk only flowed when the preterm infant created a vacuum would influence time to full oral feeds, the length of stay (LOS) in hospital and breastfeeding at discharge. STUDY DESIGN: This was a randomized controlled trial in the tertiary neonatal intensive care unit at King Edward Memorial Hospital, Perth, Australia. Eligibility criteria were: preterm infants of gestational age 25 to 34 weeks receiving >75% human milk by gastric tube. Infants were randomly assigned to being fed with a novel teat (NT) or conventional teat (CT). Intention to treat analysis was performed. RESULT: Time to full suck feeds was not different between groups. LOS was shorter (mean: 2.5 days; P=0.026) and less formula was fed at discharge in the NT group (P=0.036). CONCLUSION: Use of a NT that releases milk when the infant applies vacuum while establishing breastfeeding reduces duration of hospitalization of preterm infants.

TGF-α and IL-6 plasma levels selectively identify CML patients who fail to achieve an early molecular response or progress in the first year of therapy.

Early molecular response (EMR, BCR-ABL1 (IS)⩽10% at 3 months) is a strong predictor of outcome in imatinib-treated chronic phase chronic myeloid leukemia (CP-CML) patients, but for patients who transform early, 3 months may be too late for effective therapeutic intervention. Here, we employed multiplex cytokine profiling of plasma samples to test newly diagnosed CP-CML patients who subsequently received imatinib treatment. A wide range of pro-inflammatory and angiogenesis-promoting cytokines, chemokines and growth factors were elevated in the plasma of CML patients compared with that of healthy donors. Most of these normalized after tyrosine kinase inhibitor treatment while others remained high in remission samples. Importantly, we identified TGF-α and IL-6 as novel biomarkers with high diagnostic plasma levels strongly predictive of subsequent failure to achieve EMR and deep molecular response, as well as transformation to blast crisis and event-free survival. Interestingly, high TGF-α alone can also delineate a poor response group raising the possibility of a pathogenic role. This suggests that the incorporation of these simple measurements to the diagnostic work-up of CP-CML patients may enable therapy intensity to be individualized early according to the cytokine-risk profile of the patient.

COSMIC: High-Resolution Cancer Genetics Using the Catalogue of Somatic Mutations in Cancer.

COSMIC (http://cancer.sanger.ac.uk) is an expert-curated database of somatic mutations in human cancer. Broad and comprehensive in scope, recent releases in 2016 describe over 4 million coding mutations across all human cancer disease types. Mutations are annotated across the entire genome, but expert curation is focused on over 400 key cancer genes. Now encompassing the majority of molecular mutation mechanisms in oncogenetics, COSMIC additionally describes 10 million non-coding mutations, 1 million copy-number aberrations, 9 million gene-expression variants, and almost 8 million differentially methylated CpGs. This information combines a consistent interpretation of the data from the major cancer genome consortia and cancer genome literature with exhaustive hand curation of over 22,000 gene-specific literature publications. This unit describes the graphical Web site in detail; alternative protocols overview other ways the entire database can be accessed, analyzed, and downloaded. © 2016 by John Wiley & Sons, Inc.

Mitochondrial DNA variants in inclusion body myositis.

Mitochondrial DNA variants have been shown to be associated with many diseases. Mutations at mitochondrial DNA nucleotide positions 3192, 3196, 3397 and 4336 have been described in association with late-onset Alzheimer's disease. The pathological similarities between inclusion body myositis and Alzheimer's disease prompted an analysis of the relationship between the reported mutations and sporadic inclusion body myositis. The 4336G variant was not significantly increased in patients with inclusion body myositis or Alzheimer's disease when compared to controls. None of the patients with inclusion body myositis carried mutations at nucleotide positions 3192, 3196 and 3397. A transition at nucleotide position 4580 was detected in some patients with inclusion body myositis and Alzheimer's disease but was not significantly higher in frequency when compared to controls. Phylogenetic analysis showed that the 4336G and 4580A variants clustered together in their respective group. A group of patients with inclusion body myositis also clustered together on a separate branch of the phylogenetic tree. Closer investigation of this group revealed a common polymorphism at nucleotide position 16311. The frequency of the 16311C variant was higher in inclusion body myositis than in Alzheimer's disease and controls, although when only caucasian patients were considered the increased frequency was not statistically significant. Further studies will be required to determine whether this variant plays a role in the pathogenesis of inclusion body myositis.

Lesions in the hypothalamus after active immunisation against GnRH in the pig.

The terminals of the hypothalamic gonadotrophin hormone-releasing hormone (GnRH) neurons are located within the median eminence and thereby extend beyond the protection of the blood-brain barrier. Thus, these terminals may be subjected to direct autoimmune action in animals that are actively immunised against GnRH. Boars (male pigs) (n = 108) were actively immunised against GnRH by two successive injections with synthetic GnRH, covalently coupled to KLH and dissolved in CFA or IFA. They were killed at 26 weeks of age. Immunised boars were selected on the basis of the resultant testes size, which indicates the effectiveness of the immunisation. The hypothalami of 25 selected animals were studied by histological and immunocytochemical techniques and compared with the hypothalami of three sham- and nine control animals. In the immunised animals, changes in the GnRH system had taken place. These comprised dystrophy of the perikarya and a sharp decrease of the GnRH immunocytochemical reactivity in the terminals within the median eminence. In addition, various degrees of inflammatory reactions were present, particularly within the median eminence. These consisted of tissue disruption by edema, collapse of the capillaries, fibrosis and infiltration with fibroblasts. In addition, accumulations of neurosecretum within the median eminence in combination with hypertrophy of magnocellular neurons within the hypothalamus were present. The reactions were restricted to the median eminence and did not involve other neurohemal organs or other parts of the hypothalamus. A correlation could be established between the incidence of the lesions and the effectiveness of the GnRH autoimmunity (as indicated by the size and endocrine function of the gonads and the anti-GnRH titres). Changes in extra- and intracellular IgG immunocytochemical reactivity within the median eminence indicated the involvement of IgG. The effects were absent from control and sham vaccinated animals and after vaccinations with other compositions of the vaccine. Thus, hypothalamic lesions have been observed in this selected group of animals, vaccinated against GnRH with this particular vaccine.

The supraoptic nucleus in the pig hypothalamus: postnatal development and the effect of gonadal steroids.

Postnatal development of the supraoptic nucleus (SON) in the pig hypothalamus was studied morphometrically. The volume of the SON increased from 6.2 +/- 0.45 (S.E.M.) mm3 at 7 weeks postnatally to 18.5 +/- 1.35 mm3 at 2.5 years of age. A sex difference was found at the development point when the SON volume increased, with earlier SON enlargement in males. This sex difference was 30% at 30 weeks and 50% at 1 year of age. At 2.5 years of age no difference in volume was apparent between the sexes. The number of SON neurones was similar for all age groups concerned (43,500 +/- 1475), except for the 2.5-year-old females where 40% more were found (55,500 +/- 3285). No significant difference was found in neurone number between gonadectomized and sham-operated animals, but the operation caused a 30% reduction in the number of neurones and SON volume. Testosterone supplementation following gonadectomy, during the first 4 weeks postnatally, resulted in sexual dimorphism, the males having more SON neurones than the females. The volume showed only a trend in the same direction. Testosterone supplementation at other ages did not result in any difference when compared with controls. The results of this study show that the postnatal development of the SON of the pig is sexually dimorphic, and that it continues after puberty in females. In contrast to the vasopressin- and oxytocin-containing nucleus, the development of the SON was not influenced by gonadectomy and only slightly by gonadal steroids.

Reproductive and hormonal factors in male and female colon cancer.

We analysed data from a case-control study in the Netherlands in order to investigate whether reproductive events and hormonal factors are similarly related to colon cancer risk in men and women after adjustment for dietary factors. In total, 232 colon cancer cases (102 women, 130 men) and 259 controls (123 women, 136 men) were interviewed about life style, medical conditions and usual dietary patterns, using a structured dietary history technique. In women, age at first childbirth was positively associated with colon cancer risk (odds ratio (OR) age > or = 26 vs < 26 years, 1.7; 95% confidence interval (CI), 0.9-3.3). Women with three or more children were at reduced risk compared with women with one or two children (OR, 0.6; 95% CI, 0.3-1.1). When women had had their first child after the age of 26 years, parity was observed to be important (for one or two children vs > or = three children: OR, 2.8; 95% CI, 1.1-7.0). For men, opposite but non-significant associations were found. Adjustment for dietary patterns and other risk factors did not change the estimates markedly. Of the hormonal factors, late age at menarche decreased risk (OR, 0.5; 95% CI, 0.3-0.9) while late age at natural menopause slightly increased risk. Our study provides additional support for the role of reproductive status in the aetiology of colon cancer in women, independently of dietary factors.

The influence of selected water quality parameters on the decay rate and exoenzymatic activity of detritus of Nymphaea alba L. floating leaf blades in laboratory experiments.

A chemostat study was conducted to investigate the influence of water quality parameters related to acidification processes on the decomposition of floating leaves of Nymphaea alba L. HCO inf3sup- and total Al concentration and pH influenced the decay rate. The activity of four cell-wall decaying exoenzymes was measured in the detritus. The activity of two types of pectic enzyme and xylanase was low under acid conditions. Cellulase activity was little affected. The exoenzyme activity seemed to be influenced by the pH of the water within the detritus. Inhibition of pectic enzymes under acid conditions may be an important factor causing the slow decay of macrophyte remains in acid waters.

The influence of pH on concentrations of protein and phenolics and resource quality of decomposing floating leaf material of Nymphaea alba L. (Nymphaeaceae) for the detritivore Asellus aquaticus (L.).

Senescent floating leaf material of Nymphaea alba L., collected in an acidified moorland pool, was used in decomposition studies in two aquatic systems that differed greatly in pH, alkalinity and nutrient concentration. Concentrations of extractable protein and phenolics in the decomposing leaf material were measured during the incubation period. Protein levels were not significantly different in the leaf material from the two study sites, whereas the concentrations of phenolics in the degrading leaf blades from the acid site remained higher than in the material from the alkaline site. The resource quality of the decomposing leaf material was estimated by feeding tests using Asellus aquaticus (L.) in the laboratory. The effect of an artificially increased level of tannin on the feeding activity of A. aquaticus was also studied. Material from the acid system was consumed at a lower rate than material from the other system. The phenolic content of the material was found to be the most important feeding cue. The protein level of the leaf blade detritus seems to be of less importance. The structure of the decomposing leaf blades may have influenced the resource quality in the later stages of the experiment.

DSM-IV Cluster A personality disorder diagnoses among young adults with a 2-7-8 MMPI profile.

The present study evaluated the presence of DSM-IV personality disorders among young adults from a nonclinical setting who produced an MMPI 2-7-8 profile in comparison to a group of MMPI-defined controls. Categorical and dimensional analyses of personality disorders were evaluated. Participants in the 2-7-8 group (n = 20) received significantly more personality disorder diagnoses than did controls (n = 29), and 85% of these individuals received at least one Cluster A (Paranoid, Schizoid, Schizotypal) diagnosis in contrast to only 6.9% of controls (categorical analysis). The 2-7-8 group also received significantly more Cluster A diagnoses than Cluster B or C diagnoses. When dimensional analyses were applied (subclinical diagnoses), 95% of the 2-7-8 group evidenced Cluster A features. Comorbidity patterns were also evaluated; the most frequent comorbid diagnosis for the 2-7-8 group was Avoidant Personality Disorder (n = 8), consistent with Meehl s (1962, 1989, 1990) conceptualization of schizotypy. These results support the use of the MMPI 2-7-8 profile as an indicator of schizophrenia-related pathology within nonclinical samples of young adults.

Antimicrobial resistance and conjugative R plasmids in Escherichia coli strains isolated from animals in Peninsular Malaysia.

Fifteen independent E. coli strains of avian, bovine and porcine origin in Peninsular Malaysia were tested for antibiotic resistance and conjugative R plasmids. Eight (53%) isolates were found to be antibiotic resistant. Among them, 37.5% were mono-resistant and 62.5% were resistant to three or more antibiotics, i.e., multi-resistant. All of them were resistant to Tc and sensitive to Gm and Nx. Three of the eight antibiotic resistant strains were able to transfer all or part of their resistance to an E. coli K12 recipient by conjugation. The transfer frequencies of Km, Sm and Tc resistance of the three donors varied between 4.5 X 10(-8) to 6.8 X 10(-7). Analysis of the plasmid profiles of all the three donors and their respective transconjugants after agarose gel electrophoresis provided conclusive evidence that the transferable resistance traits were plasmid-mediated.