Effect of glucocorticoids for the management of immune-related adverse events on outcome in melanoma patients treated with immunotherapy-a retrospective and biomarker study.

Background: Immune-related adverse events (IRAEs) during therapy with immune checkpoint inhibitors (ICIs) are common, and their management sometimes requires glucocorticoids (GCs). Predictors for development of IRAEs and data about the impact of GCs on clinical outcome are missing. We evaluated the impact of GCs to treat IRAEs on clinical outcome, and plasmatic inflammatory proteins as predictors for IRAEs. Patients and methods: Patients with melanoma (n = 98) treated with ICIs at Karolinska University Hospital were included. Clinical information and data regarding prescription of systemic GCs were collected. Baseline plasma samples (n = 57) were analyzed for expression of 92 inflammatory proteins. Results: Forty-four patients developed at least one IRAE requiring systemic GCs and the most common was hypocortisolemia (n = 11). A median overall survival of 72.8 months for patients developing IRAEs requiring GCs, 17.7 months for those who did not, and 1.4 months for individuals receiving GCs at baseline was observed in Kaplan-Meier curves (P = 0.001). In immortal time bias adjusted analysis, patients receiving steroids to treat IRAE survived slightly longer, even though this time trend was not statistically significant. The median overall survival was 29 months for those treated with GCs within 60 days after ICIs start and was not reached for patients receiving GCs later. The number of ICI cycles was higher in subjects receiving GCs after 60 days (P = 0.0053). Hypocortisolemia occurred mainly in males (10/11) and correlated with favorable outcome. Male patients with hypocortisolemia had lower expression of interleukin 8, transforming growth factor-α, and fibroblast growth factor 5 and higher expression of Delta/Notch-like epidermal growth factor-related receptor. Conclusions: GCs may be used to treat IRAEs without major concern. GCs early during ICIs may, however, impact clinical outcome negatively. The prognostic value of hypocortisolemia and inflammation proteins as biomarkers should be further investigated.

Splice-Switching Antisense Oligonucleotides Correct Phenylalanine Hydroxylase Exon 11 Skipping Defects and Rescue Enzyme Activity in Phenylketonuria.

The PAH gene encodes the hepatic enzyme phenylalanine hydroxylase (PAH), and its deficiency, known as phenylketonuria (PKU), leads to neurotoxic high levels of phenylalanine. PAH exon 11 is weakly defined, and several missense and intronic variants identified in patients affect the splicing process. Recently, we identified a novel intron 11 splicing regulatory element where U1snRNP binds, participating in exon 11 definition. In this work, we describe the implementation of an antisense strategy targeting intron 11 sequences to correct the effect of PAH mis-splicing variants. We used an in vitro assay with minigenes and identified splice-switching antisense oligonucleotides (SSOs) that correct the exon skipping defect of PAH variants c.1199+17G>A, c.1199+20G>C, c.1144T>C, and c.1066-3C>T. To examine the functional rescue induced by the SSOs, we generated a hepatoma cell model with variant c.1199+17G>A using CRISPR/Cas9. The edited cell line reproduces the exon 11 skipping pattern observed from minigenes, leading to reduced PAH protein levels and activity. SSO transfection results in an increase in exon 11 inclusion and corrects PAH deficiency. Our results provide proof of concept of the potential therapeutic use of a single SSO for different exonic and intronic splicing variants causing PAH exon 11 skipping in PKU.

Trans-Regulation of Alternative PD-L1 mRNA Processing by CDK12 in Non-Small-Cell Lung Cancer Cells.

Immunotherapy using checkpoint inhibitors targeting the interaction between PD-1 on T cells and PD-L1 on cancer cells has shown significant results in non-small-cell lung cancer (NSCLC). Not all patients respond to the therapy, and PD-L1 expression heterogeneity is proposed to be one determinant for this. The alternative processing of PD-L1 RNA, which depends on an alternative poly-A site in intron 4, generates a shorter mRNA variant (PD-L1v4) encoding soluble PD-L1 (sPD-L1), relative to the canonical PD-L1v1 mRNA encoding membrane-associated PD-L1 (mPD-L1). This study aimed to identify factors influencing the ratio between these two PD-L1 mRNAs in NSCLC cells. First, we verified the existence of the alternative PD-L1 RNA processing in NSCLC cells, and from in silico analyses, we identified a candidate list of regulatory factors. Examining selected candidates showed that CRISPR/Cas9-generated loss-of-function mutations in CDK12 increased the PD-L1v4/PD-L1v1 mRNA ratio and, accordingly, the sPD-L1/mPD-L1 balance. The CDK12/13 inhibitor THZ531 could also increase the PD-L1v4/PD-L1v1 mRNA ratio and impact the PD-L1 transcriptional response to IFN-γ stimulation. The fact that CDK12 regulates PD-L1 transcript variant formation in NSCLC cells is consistent with CDK12's role in promoting transcriptional elongation over intron-located poly-A sites. This study lays the groundwork for clinical investigations to delineate the implications of the CDK12-mediated balancing of sPD-L1 relative to mPD-L1 for immunotherapeutic responses in NSCLC.

Clinical, splicing, and functional analysis to classify BRCA2 exon 3 variants: Application of a points-based ACMG/AMP approach.

Skipping of BRCA2 exon 3 (∆E3) is a naturally occurring splicing event, complicating clinical classification of variants that may alter ∆E3 expression. This study used multiple evidence types to assess pathogenicity of 85 variants in/near BRCA2 exon 3. Bioinformatically predicted spliceogenic variants underwent mRNA splicing analysis using minigenes and/or patient samples. ∆E3 was measured using quantitative analysis. A mouse embryonic stem cell (mESC) based assay was used to determine the impact of 18 variants on mRNA splicing and protein function. For each variant, population frequency, bioinformatic predictions, clinical data, and existing mRNA splicing and functional results were collated. Variant class was assigned using a gene-specific adaptation of ACMG/AMP guidelines, following a recently proposed points-based system. mRNA and mESC analysis combined identified six variants with transcript and/or functional profiles interpreted as loss of function. Cryptic splice site use for acceptor site variants generated a transcript encoding a shorter protein that retains activity. Overall, 69/85 (81%) variants were classified using the points-based approach. Our analysis shows the value of applying gene-specific ACMG/AMP guidelines using a points-based approach and highlights the consideration of cryptic splice site usage to appropriately assign PVS1 code strength.

Down-Regulation of CK2α Leads toUp-Regulation of the Cyclin-Dependent Kinase Inhibitor p27KIP1 in Conditions Unfavorable for the Growth of Myoblast Cells.

BACKGROUND/AIMS: Compelling evidence indicates that CK2α, which is one of the two catalytic isoforms of protein kinase CK2, is required for cell viability and plays an important role in cell proliferation and differentiation. While much is known on CK2 in the context of disease states, particularly cancer, its critical role in non-cancerous cell growth has not been extensively investigated. METHODS: In the present study, we have employed a cell line derived from rat heart with inducible down-regulation of CK2α and CK2α-knockout mouse tissue to identify CK2-mediated molecular mechanisms regulating cell growth. For this, we have performed Incucyte® live-cell analysis and applied flow cytometry, western blot, immunoprecipitation, immunohistochemistry, RT-qPCR and luciferase-based methods. RESULTS: Here, we show that lack of CK2α results in significantly delayed cell cycle progression through G1, inhibition of cyclin E-CDK2 complex, decreased phosphorylation of Rb protein at S795, and inactivation of E2F transcription factor. These events are accompanied by nuclear accumulation and up-regulation of the cyclin-dependent kinase inhibitor p27KIP1 in cells and CK2α-knockout mouse tissues. We found that increased levels of p27KIP1 are mainly attributable to post-translational modifications, namely phosphorylation at S10 and T197 amino acid residues catalyzed by Dyrk1B and AMPK, respectively, as silencing of FoxO3A transcription factor, which activates CDKN1B the gene coding for p27KIP1, does not result in markedly decreased expression levels of the corresponding protein. Interestingly, simultaneous silencing of CK2α and p27KIP1 significantly impairs cell cycle progression without increasing cell death. CONCLUSION: Taken together, our study sheds light on the molecular mechanisms controlling cell cycle progression through G1 phase when myoblasts proliferation potential is impaired by CK2α depletion. Our results suggest that elevated levels of p27KIP1, which follows CK2α depletion, contribute to delay the G1-to-S phase transition. Effects seen when p27KIP1 is down-regulated are independent of CK2α and reflect the protective role exerted by p27KIP1 under unfavorable cell growth conditions.

Associations between maternal physical activity in early and late pregnancy and offspring birth size: remote federated individual level meta-analysis from eight cohort studies.

OBJECTIVE: Evidence on the impact of leisure time physical activity (LTPA) in pregnancy on birth size is inconsistent. We aimed to examine the association between LTPA during early and late pregnancy and newborn anthropometric outcomes. DESIGN: Individual level meta-analysis, which reduces heterogeneity across studies. SETTING: A consortium of eight population-based studies (seven European and one US) comprising 72 694 participants. METHODS: Generalised linear models with consistent inclusion of confounders (gestational age, sex, parity, maternal age, education, ethnicity, BMI, smoking, and alcohol intake) were used to test associations between self-reported LTPA at either early (8-18 weeks gestation) or late pregnancy (30+ weeks) and the outcomes. Results were pooled using random effects meta-analyses. MAIN OUTCOME MEASURES: Birth weight, large-for-gestational age (LGA), macrosomia, small-for-gestational age (SGA), % body fat, and ponderal index at birth. RESULTS: Late, but not early, gestation maternal moderate to vigorous physical activity (MVPA), vigorous activity, and LTPA energy expenditure were modestly inversely associated with BW, LGA, macrosomia, and ponderal index, without heterogeneity (all: I2  = 0%). For each extra hour/week of MVPA, RR for LGA and macrosomia were 0.97 (95% CI: 0.96, 0.98) and 0.96 (95% CI: 0.94, 0.98), respectively. Associations were only modestly reduced after additional adjustments for maternal BMI and gestational diabetes. No measure of LTPA was associated with risk for SGA. CONCLUSIONS: Physical activity in late, but not early, pregnancy is consistently associated with modestly lower risk of LGA and macrosomia, but not SGA. TWEETABLE ABSTRACT: In an individual participant meta-analysis, late pregnancy moderate to vigorous physical activity modestly reduced birth size outcomes.

Next generation sequencing of RNA reveals novel targets of resveratrol with possible implications for Canavan disease.

Resveratrol (RSV) is a small compound first identified as an activator of sirtuin 1 (SIRT1), a key factor in mediating the effects of caloric restriction. Since then, RSV received great attention for its widespread beneficial effects on health and in connection to many diseases. RSV improves the metabolism and the mitochondrial function, and more recently it was shown to restore fatty acid ß-oxidation (FAO) capacities in patient fibroblasts harboring mutations with residual enzyme activity. Many of RSV's beneficial effects are mediated by the transcriptional coactivator PGC-1α, a direct target of SIRT1 and a master regulator of the mitochondrial fatty acid oxidation. Despite numerous studies RSV's mechanism of action is still not completely elucidated. Our aim was to investigate the effects of RSV on gene regulation on a wide scale, possibly to detect novel genes whose up-regulation by RSV may be of interest with respect to disease treatment. We performed Next Generation Sequencing of RNA on normal fibroblasts treated with RSV. To investigate whether the effects of RSV are mediated through SIRT1 we expanded the analysis to include SIRT1-knockdown fibroblasts. We identified the aspartoacylase (ASPA) gene, mutated in Canavan disease, to be strongly up-regulated by RSV in several cell lines, including Canavan disease fibroblasts. We further link RSV to the up-regulation of other genes involved in myelination including the glial specific transcription factors POU3F1, POU3F2, and myelin basic protein (MBP). We also observe a strong up-regulation by RSV of the riboflavin transporter gene SLC52a1. Mutations in SLC52a1 cause transient multiple acyl-CoA dehydrogenase deficiency (MADD). Our analysis of alternative splicing identified novel metabolically important genes affected by RSV, among which is particularly interesting the α subunit of the stimulatory G protein (Gsα), which regulates the cellular levels of cAMP through adenylyl cyclase. We conclude that in fibroblasts RSV stimulates the PGC-1α and p53 pathways, and up-regulates genes affecting the glucose metabolism, mitochondrial ß-oxidation, and mitochondrial biogenesis. We further confirm that RSV might be a relevant treatment in the correction of FAO deficiencies and we suggest that treatment in other metabolic disorders including Canavan disease and MADD might be also beneficial.

Proceedings of the fifth international RASopathies symposium: When development and cancer intersect.

This report summarizes and highlights the fifth International RASopathies Symposium: When Development and Cancer Intersect, held in Orlando, Florida in July 2017. The RASopathies comprise a recognizable pattern of malformation syndromes that are caused by germ line mutations in genes that encode components of the RAS/mitogen-activated protein kinase (MAPK) pathway. Because of their common underlying pathogenetic etiology, there is significant overlap in their phenotypic features, which includes craniofacial dysmorphology, cardiac, cutaneous, musculoskeletal, gastrointestinal and ocular abnormalities, neurological and neurocognitive issues, and a predisposition to cancer. The RAS pathway is a well-known oncogenic pathway that is commonly found to be activated in somatic malignancies. As in somatic cancers, the RASopathies can be caused by various pathogenetic mechanisms that ultimately impact or alter the normal function and regulation of the MAPK pathway. As such, the RASopathies represent an excellent model of study to explore the intersection of the effects of dysregulation and its consequence in both development and oncogenesis.

Intronic PAH gene mutations cause a splicing defect by a novel mechanism involving U1snRNP binding downstream of the 5' splice site.

Phenylketonuria (PKU), one of the most common inherited diseases of amino acid metabolism, is caused by mutations in the phenylalanine hydroxylase (PAH) gene. Recently, PAH exon 11 was identified as a vulnerable exon due to a weak 3' splice site, with different exonic mutations affecting exon 11 splicing through disruption of exonic splicing regulatory elements. In this study, we report a novel intron 11 regulatory element, which is involved in exon 11 splicing, as revealed by the investigated pathogenic effect of variants c.1199+17G>A and c.1199+20G>C, identified in PKU patients. Both mutations cause exon 11 skipping in a minigene system. RNA binding assays indicate that binding of U1snRNP70 to this intronic region is disrupted, concomitant with a slightly increased binding of inhibitors hnRNPA1/2. We have investigated the effect of deletions and point mutations, as well as overexpression of adapted U1snRNA to show that this splicing regulatory motif is important for regulation of correct splicing at the natural 5' splice site. The results indicate that U1snRNP binding downstream of the natural 5' splice site determines efficient exon 11 splicing, thus providing a basis for development of therapeutic strategies to correct PAH exon 11 splicing mutations. In this work, we expand the functional effects of non-canonical intronic U1 snRNP binding by showing that it may enhance exon definition and that, consequently, intronic mutations may cause exon skipping by a novel mechanism, where they disrupt stimulatory U1 snRNP binding close to the 5' splice site. Notably, our results provide further understanding of the reported therapeutic effect of exon specific U1 snRNA for splicing mutations in disease.

VLCAD deficiency: Follow-up and outcome of patients diagnosed through newborn screening in Victoria.

Very long chain acyl-CoA dehydrogenase (VLCAD) deficiency is an inherited metabolic disorder of fatty acid oxidation. Treatment practices of the disorder have changed over the past 10-15years since this disorder was included in newborn screening programs and patients were diagnosed pre-symptomatically. A genotype-phenotype correlation has been suggested but the discovery of novel mutations make this knowledge limited. Herein, we describe our experience in treating patients (n=22) diagnosed through newborn screening and mutational confirmation and followed up over a median period of 104months. We report five novel mutations. In 2013 we formalised our treatment protocol, which essentially follows a European consensus paper from 2009 and our own experience. The prescribed low natural fat diet is relaxed for patients who are asymptomatic when reaching age 5years but medium-chain triglyceride oil is recommended before and after physical activity regardless of age. Metabolic stability, growth, development and cardiac function are satisfactory in all patients. There were no episodes of encephalopathy or hypoglycaemia but three patients had episodes of muscle pain with our without rhabdomyolysis. Body composition studies showed a negative association between dietary protein intake and percent body fat. Larger patient cohort and longer follow up time are required for further elucidation of genotype-phenotype correlations and for establishing the role of dietary protein in metabolic stability and long-term healthier body composition in patients with VLCAD deficiency.

The Splicing Efficiency of Activating HRAS Mutations Can Determine Costello Syndrome Phenotype and Frequency in Cancer.

Costello syndrome (CS) may be caused by activating mutations in codon 12/13 of the HRAS proto-oncogene. HRAS p.Gly12Val mutations have the highest transforming activity, are very frequent in cancers, but very rare in CS, where they are reported to cause a severe, early lethal, phenotype. We identified an unusual, new germline p.Gly12Val mutation, c.35_36GC>TG, in a 12-year-old boy with attenuated CS. Analysis of his HRAS cDNA showed high levels of exon 2 skipping. Using wild type and mutant HRAS minigenes, we confirmed that c.35_36GC>TG results in exon 2 skipping by simultaneously disrupting the function of a critical Exonic Splicing Enhancer (ESE) and creation of an Exonic Splicing Silencer (ESS). We show that this vulnerability of HRAS exon 2 is caused by a weak 3' splice site, which makes exon 2 inclusion dependent on binding of splicing stimulatory proteins, like SRSF2, to the critical ESE. Because the majority of cancer- and CS- causing mutations are located here, they affect splicing differently. Therefore, our results also demonstrate that the phenotype in CS and somatic cancers is not only determined by the different transforming potentials of mutant HRAS proteins, but also by the efficiency of exon 2 inclusion resulting from the different HRAS mutations. Finally, we show that a splice switching oligonucleotide (SSO) that blocks access to the critical ESE causes exon 2 skipping and halts proliferation of cancer cells. This unravels a potential for development of new anti-cancer therapies based on SSO-mediated HRAS exon 2 skipping.

The Fourth International Symposium on Genetic Disorders of the Ras/MAPK pathway.

The RASopathies are a group of disorders due to variations of genes associated with the Ras/MAPK pathway. Some of the RASopathies include neurofibromatosis type 1 (NF1), Noonan syndrome, Noonan syndrome with multiple lentigines, cardiofaciocutaneous (CFC) syndrome, Costello syndrome, Legius syndrome, and capillary malformation-arteriovenous malformation (CM-AVM) syndrome. In combination, the RASopathies are a frequent group of genetic disorders. This report summarizes the proceedings of the 4th International Symposium on Genetic Disorders of the Ras/MAPK pathway and highlights gaps in the field. © 2016 Wiley Periodicals, Inc.

The descriptive epidemiology of accelerometer-measured physical activity in older adults.

BACKGROUND: Objectively measured physical activity between older individuals and between populations has been poorly described. We aimed to describe and compare the variation in accelerometry data in older UK (EPIC-Norfolk) and American (NHANES) adults. METHODS: Physical activity was measured by uniaxial accelerometry in 4,052 UK (49-91 years) and 3459 US older adults (49-85 years). We summarized physical activity as volume (average counts/minute), its underlying intensity distribution, and as time spent <100counts/minute, ≥809counts/minute and ≥2020counts/minute both for total activity and that undertaken in ≥10-min bouts. RESULTS: In EPIC-Norfolk 65% of wear-time was spent at <100 counts/minute and 20% spent in the range 100-500 counts/minute. Only 4.1% of this cohort accumulated more than 30 min/day of activity above 2020 counts/minute in 10-min bouts. If a cut-point of >809 counts/minute is used 18.7% of people reached the 30 min/day threshold. By comparison, 2.5% and 9.5% of American older adults accumulated activity at these levels, respectively. CONCLUSION: As assessed by objectively measured physical activity, the majority of older adults in this UK study did not meet current activity guidelines. Older adults in the UK were more active overall, but also spent more time being sedentary than US adults.

Objectively measured sedentary time, physical activity and kidney function in people with recently diagnosed Type 2 diabetes: a prospective cohort analysis.

AIM: To assess the prospective association between objectively measured physical activity and kidney function over 4 years in people with Type 2 diabetes. METHODS: Individuals (120 women and 206 men) participating in the ADDITION-Plus trial underwent assessment of sedentary time (SED-time), time spent in moderate-to-vigorous-intensity physical activity (MVPA) and total physical activity energy expenditure (PAEE) using a combined heart rate and movement sensor, and kidney function [estimated glomerular filtration rate (eGFR), serum creatinine and urine albumin-to-creatinine ratio (ACR)] at baseline and after 4 years of follow-up. Multivariate regression was used to quantify the association between change in SED-time, MVPA and PAEE and kidney measures at four-year follow-up, adjusting for change in current smoking status, waist circumference, HbA1c , systolic blood pressure, triglycerides and medication usage. RESULTS: Over 4 years, there was a decline in eGFR values from 87.3 to 81.7 ml/min/1.73m(2) (P < 0.001); the prevalence of reduced eGFR (eGFR < 60 ml/min/1.73m(2) ) increased from 6.1 to 13.2% (P < 0.001). There were small increases in serum creatinine (median: 81-84 µmol/l, P < 0.001) and urine ACR (median: 0.9-1.0 mg/mmol, P = 0.005). Increases in SED-time were associated with increases in serum creatinine after adjustment for MVPA and cardiovascular risk factors (ß = 0.013, 95% CI: 0.001, 0.03). Conversely, increases in PAEE were associated with reductions in serum creatinine (ß = -0.001, 95% CI: -0.003, -0.0001). CONCLUSION: Reducing time spent sedentary and increasing overall physical activity may offer intervention opportunities to improve kidney function among individuals with diabetes. (Trial Registry no. ISRCTN 99175498).

Molecular subtype and tumor characteristics of breast cancer metastases as assessed by gene expression significantly influence patient post-relapse survival.

BACKGROUND: We and others have recently shown that tumor characteristics are altered throughout tumor progression. These findings emphasize the need for re-examination of tumor characteristics at relapse and have led to recommendations from ESMO and the Swedish Breast Cancer group. Here, we aim to determine whether tumor characteristics and molecular subtypes in breast cancer metastases confer clinically relevant prognostic information for patients. PATIENTS AND METHODS: The translational aspect of the Swedish multicenter randomized trial called TEX included 111 patients with at least one biopsy from a morphologically confirmed locoregional or distant breast cancer metastasis diagnosed from December 2002 until June 2007. All patients had detailed clinical information, complete follow-up, and metastasis gene expression information (Affymetrix array GPL10379). We assessed the previously published gene expression modules describing biological processes [proliferation, apoptosis, human epidermal receptor 2 (HER2) and estrogen (ER) signaling, tumor invasion, immune response, and angiogenesis] and pathways (Ras, MAPK, PTEN, AKT-MTOR, PI3KCA, IGF1, Src, Myc, E2F3, and ß-catenin) and the intrinsic subtypes (PAM50). Furthermore, by contrasting genes expressed in the metastases in relation to survival, we derived a poor metastasis survival signature. RESULTS: A significant reduction in post-relapse breast cancer-specific survival was associated with low-ER receptor signaling and apoptosis gene module scores, and high AKT-MTOR, Ras, and ß-catenin module scores. Similarly, intrinsic subtyping of the metastases provided statistically significant post-relapse survival information with the worst survival outcome in the basal-like [hazard ratio (HR) 3.7; 95% confidence interval (CI) 1.3-10.9] and HER2-enriched (HR 4.4; 95% CI 1.5-12.8) subtypes compared with the luminal A subtype. Overall, 25% of the metastases were basal-like, 32% HER2-enriched, 10% luminal A, 28% luminal B, and 5% normal-like. CONCLUSIONS: We show that tumor characteristics and molecular subtypes of breast cancer metastases significantly influence post-relapse patient survival, emphasizing that molecular investigations at relapse provide prognostic and clinically relevant information. CLINICALTRIALS.GOV: This is the translational part of the Swedish multicenter and randomized trial TEX, clinicaltrials.gov identifier nct01433614 (http://www.clinicaltrials.gov/ct2/show/nct01433614).

Physical activity, sedentary time and gain in overall and central body fat: 7-year follow-up of the ProActive trial cohort.

OBJECTIVE: The objective of this study is to examine the independent associations of time spent in moderate-to-vigorous physical activity (MVPA) and sedentary (SED-time), with total and abdominal body fat (BF), and the bidirectionality of these associations in adults at high risk of type 2 diabetes. DESIGN AND SUBJECTS: We measured MVPA (min per day) and SED-time (h per day) by accelerometry, and indices of total (body weight, fat mass (FM), BF% and FM index) and abdominal BF (waist circumference (WC)) using standard procedures in 231 adults (41.3 ± 6.4 years) with parental history of type 2 diabetes (ProActive UK) at baseline, 1-year and 7-year follow-up. Mixed effects models were used to quantify the independent associations (expressed as standardised ß-coefficients (95% confidence interval (CI))) of MVPA and SED-time with fat indices, using data from all three time points. All models were adjusted for age, sex, intervention arm, monitor wear time, follow-up time, smoking status, socioeconomic status and MVPA/SED-time. RESULTS: MVPA was inversely and independently associated with all indices of total BF (for example, 1 s.d. higher MVPA was associated with a reduction in FM, ß = -0.09 (95% CI: -0.14, -0.04) s.d.) and abdominal BF (for example, WC: ß = -0.07 (-0.12, -0.02)). Similarly, higher fat indices were independently associated with a reduction in MVPA (for example, WC: ß = -0.25 (-0.36, -0.15); FM: ß = -0.27 (-0.36, -0.18)). SED-time was positively and independently associated with most fat indices (for example, WC: ß = 0.03 (-0.04, 0.09); FM: ß = 0.10 (0.03, 0.17)). Higher values of all fat indices independently predicted longer SED-time (for example, WC: ß = 0.10 (0.02, 0.18), FM: ß = 0.15 (0.07, 0.22)). CONCLUSIONS: The associations of MVPA and SED-time with total and abdominal BF are bidirectional and independent among individuals at high risk for type 2 diabetes. The association between BF and MVPA is stronger than the reciprocal association, highlighting the importance of considering BF as a determinant of decreasing activity and a potential consequence. Promoting more MVPA and less SED-time may reduce total and abdominal BF.

Proteomics analysis of melanoma metastases: association between S100A13 expression and chemotherapy resistance.

BACKGROUND: Disseminated cutaneous malignant melanoma (CMM) is commonly unresponsive to standard chemotherapies, and there are as yet no predictive markers of therapy response. METHODS: In the present study we collected fresh-frozen pretreatment lymph-node metastasis samples (n=14) from melanoma patients with differential response to dacarbazine (DTIC) or temozolomide (TMZ) chemotherapy, to identify proteins with an impact on treatment response. We performed quantitative protein profiling using tandem mass spectrometry and compared the proteome differences between responders (R) and non-responders (NR), matched for age, gender and histopathological type of CMM. RESULTS: Biological pathway analyses showed several signalling pathways differing between R vs NR, including Rho signalling. Gene expression profiling data was available for a subset of the samples, and the results were compared with the proteomics data. Four proteins with differential expression between R and NR were selected for technical validation by immunoblotting (ISYNA1, F13A1, CSTB and S100A13), and CSTB and S100A13 were further validated on a larger sample set by immunohistochemistry (n=48). The calcium binding protein S100A13 was found to be significantly overexpressed in NR compared with R in all analyses performed. CONCLUSIONS: Our results suggest that S100A13 is involved in CMM resistance to DTIC/TMZ.

The ETFDH c.158A>G variation disrupts the balanced interplay of ESE- and ESS-binding proteins thereby causing missplicing and multiple Acyl-CoA dehydrogenation deficiency.

Multiple acyl-CoA dehydrogenation deficiency is a disorder of fatty acid and amino acid oxidation caused by defects of electron transfer flavoprotein (ETF) or its dehydrogenase (ETFDH). A clear relationship between genotype and phenotype makes genotyping of patients important not only diagnostically but also for prognosis and for assessment of treatment. In the present study, we show that a predicted benign ETFDH missense variation (c.158A>G/p.Lys53Arg) in exon 2 causes exon skipping and degradation of ETFDH protein in patient samples. Using splicing reporter minigenes and RNA pull-down of nuclear proteins, we show that the c.158A>G variation increases the strength of a preexisting exonic splicing silencer (ESS) motif UAGGGA. This ESS motif binds splice inhibitory hnRNP A1, hnRNP A2/B1, and hnRNP H proteins. Binding of these inhibitory proteins prevents binding of the positive splicing regulatory SRSF1 and SRSF5 proteins to nearby and overlapping exonic splicing enhancer elements and this causes exon skipping. We further suggest that binding of hnRNP proteins to UAGGGA is increased by triggering synergistic hnRNP H binding to GGG triplets located upstream and downsteam of the UAGGGA motif. A number of disease-causing exonic elements that induce exon skipping in other genes have a similar architecture as the one in ETFDH exon 2.

Rate of weight gain predicts change in physical activity levels: a longitudinal analysis of the EPIC-Norfolk cohort.

OBJECTIVE: To investigate the relationship of body weight and its changes over time with physical activity (PA). DESIGN: Population-based prospective cohort study (Norfolk cohort of the European Prospective Investigation into Cancer and Nutrition, EPIC-Norfolk, United Kingdom). SUBJECTS: A total of 25 639 men and women aged 39-79 years at baseline. PA was self-reported. Weight and height were measured by standard clinical procedures at baseline and self-reported at 18-month and 10-year follow-ups (calibrated against clinical measures). Main outcome measure was PA at the 10-year follow-up. RESULTS: Body weight and PA were inversely associated in cross-sectional analyses. In longitudinal analyses, an increase in weight was associated with higher risk of being inactive 10 years later, after adjusting for baseline activity, 18-month activity, sex, baseline age, prevalent diseases, socioeconomic status, education, smoking, total daily energy intake and alcohol intake. Compared with stable weight, a gain in weight of >2 kg per year in the short-, medium- and long-term was consistently and significantly associated with greater likelihood of physical inactivity after 10 years, with the most pronounced effect for long-term weight gain, OR=1.89 (95% CI: 1.30-2.70) in fully adjusted analysis. Weight gain of 0.5-2 kg per year over long-term was substantially associated with physical inactivity after full adjustment, OR=1.26 (95% CI: 1.11-1.41). CONCLUSION: Weight gain (during short-, medium- and long-term) is a significant determinant of future physical inactivity independent of baseline weight and activity. Compared with maintaining weight, moderate (0.5-2 kg per year) and large weight gain (>2 kg per year) significantly predict future inactivity; a potentially vicious cycle including further weight gain, obesity and complications associated with a sedentary lifestyle. On the basis of current predictions of obesity trends, we estimate that the prevalence of inactivity in England would exceed 60% in the year 2020.

Physical activity reduces the risk of incident type 2 diabetes in general and in abdominally lean and obese men and women: the EPIC-InterAct Study.

AIMS/HYPOTHESIS: We examined the independent and combined associations of physical activity and obesity with incident type 2 diabetes in men and women. METHODS: The InterAct case-cohort study consists of 12,403 incident type 2 diabetes cases and a randomly selected subcohort of 16,154 individuals, drawn from a total cohort of 340,234 participants with 3.99 million person-years of follow-up. Physical activity was assessed by a four-category index. Obesity was measured by BMI and waist circumference (WC). Associations between physical activity, obesity and case-ascertained incident type 2 diabetes were analysed by Cox regression after adjusting for educational level, smoking status, alcohol consumption and energy intake. In combined analyses, individuals were stratified according to physical activity level, BMI and WC. RESULTS: A one-category difference in physical activity (equivalent to approximately 460 and 365 kJ/day in men and women, respectively) was independently associated with a 13% (HR 0.87, 95% CI 0.80, 0.94) and 7% (HR 0.93, 95% CI 0.89, 0.98) relative reduction in the risk of type 2 diabetes in men and women, respectively. Lower levels of physical activity were associated with an increased risk of diabetes across all strata of BMI. Comparing inactive with active individuals, the HRs were 1.44 (95% CI 1.11, 1.87) and 1.38 (95% CI 1.17, 1.62) in abdominally lean and obese inactive men, respectively, and 1.57 (95% CI 1.19, 2.07) and 1.19 (95% CI 1.01, 1.39) in abdominally lean and obese inactive women, respectively. CONCLUSIONS/INTERPRETATION: Physical activity is associated with a reduction in the risk of developing type 2 diabetes across BMI categories in men and women, as well as in abdominally lean and obese men and women.