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Role of an Adenylyl Cyclase Isoform in Alcohol\u27s Effect on Cyclic AMP Regulated Gene Expression in Mammalian Cells

Research suggests that the cyclic AMP (cAMP) signaling pathway including CREB-CRE regulated expression of various genes is implicated in the predisposition to and development of alcoholism in humans. Alcohol also induces changes in inflammatory and immune responses; these changes increase the incidence of pneumonias and other infections, which can negatively affect recovery from infections. Cyclic AMP (cAMP) is known for its immunosuppressive effects and is also required for proper development of the immune system. Previous work in our laboratory has demonstrated that ethanol enhances the activity of adenylyl cyclase (AC) in an isoform-specific manner; type 7 AC (AC7) is most enhanced by ethanol. Therefore, we hypothesize that the AC isoform expressed in the cells will play a role in ethanol’s effects on cAMP regulated gene expression. We further hypothesize that alcohol modulates cAMP signaling in immune cells by enhancing the activity of AC7; thus, AC7 may play a role in ethanol’s effects on immune function. Our objectives include: 1) evaluate the AC isoform specific effects of ethanol on cAMP regulated gene in NIH 3T3 cells by overexpressing two AC isoforms: AC3 and AC7; 2) employ immune cell lines endogenously expressing AC7, RAW 264.7 and BV-2, to further elucidate the role of AC7 in the effect of ethanol on cAMP regulated gene expression. To examine these objectives, time-lapse fluorescent resonance energy transfer (FRET) and cAMP accumulation assays were used to monitor cAMP levels within the cells. A reporter gene (luciferase) driven by an artificial promoter inducible with cAMP was utilized to evaluate the effect of ethanol on cAMP regulated gene expression. CREB phosphorylation and nuclear translocation of transducers of regulated CREB (TORCs) were examined by western blotting. Stimulation of AC activity by the addition of dopamine caused an increase in the reporter gene activity. Ethanol potentiated the increase of reporter gene activity in NIH 3T3 cells expressing AC7, while cells expressing AC3 did not respond to ethanol. Cyclic AMP pathway activation via stimulation with prostaglandin E1 (PGE1) showed an increase in cAMP and reporter gene expression in RAW 264.7 and BV-2 cells. The effect observed was potentiated in the presence of ethanol. Cyclic AMP analog, 8-Bromo-cAMP, induced luciferase activity was not significantly affected by ethanol. The level of CREB phosphorylation did not change by cAMP stimulation or in the presence of ethanol. However, there were significant changes in the TORC3 amount in nuclei depending on stimulation conditions. The results suggest that nuclear translocation of TORC3 may play a more critical role than CREB phosphorylation in the observed changes in the cAMP driven reporter gene activity. Furthermore, the ethanol effect on cAMP regulated reporter gene expression is due to a change in the amount of cAMP, which most likely results from the enhancement of AC7 activity by ethanol

Evaluation of single nucleotide polymorphisms associated with fertility and production traits in Holstein and multi-generational Angus females

The objective of this study was to test the association of single nucleotide polymorphisms (SNPs) with fertility in two populations consisting of Holstein cows and multi-generational Angus cows. The candidate gene approach was utilized and previously described SNPs were tested for possible associations with fertility. Single nucleotide polymorphisms on three genes were evaluated including leptin receptor LEPR, calpastatin CAST, and DGAT1. Fertility traits were evaluated in conjunction with production traits for Holstein females and growth traits for Angus females. One SNP was significantly associated with birth weight (P \u3c 0.05) in Angus females while a trend (P \u3c 0.10) was observed for two markers influencing birth weight performance and three markers influencing weaning weight performance. An association of two SNP for birth weight and back fat thickness in Angus females was identified.!A trend (P \u3c 0.10) was observed for one marker within LEPR influencing average services to conception, two markers within CAST influencing average days open, two markers within CAST and one marker within DGAT1 influencing average protein production, and one marker within CAST and one marker within DGAT1 influencing average milk production. One SNP within LEPR was significantly associated with average milk production (P \u3c 0.05) in Holstein females. An association of one SNP within CAST and one SNP within DGAT1 for average protein production and average milk production in Holstein females was identified. An association of one SNP within CAST for average days open and average protein production in Holstein females was also identified. The association of these markers indicates that the evaluated quantitative trait loci (QTL) region may harbor causative mutations responsible for the variation observed in fertility and production traits. Further evaluation of SNP in these regions is necessary in order to identify mutations accounting for the largest degree of variation for fertility and production traits

Trial protocol: a multicentre randomised trial of first-line treatment pathways for newly diagnosed immune thrombocytopenia: standard steroid treatment versus combined steroid and mycophenolate. The FLIGHT trial

Introduction Immune thrombocytopenia (ITP) is an autoimmune condition that may cause thrombocytopenia-related bleeding. Current first-line ITP treatment is with high-dose corticosteroids but frequent side effects, heterogeneous responses and high relapse rates are significant problems with only 20% remaining in sustained remission with this approach. Mycophenolate mofetil (MMF) is often used as the next treatment with efficacy in 50%–80% of patients and good tolerability but can take up to 2 months to work. Objective To test the hypothesis that MMF combined with corticosteroid is a more effective first-line treatment for immune thrombocytopenia (ITP) than current standard of corticosteroid alone. Design Multicentre, UK-based, open-label, randomised controlled trial. Setting Haematology departments in secondary care. Participants We plan to recruit 120 patients >16 years old with a diagnosis of ITP and a platelet count <30x109/L who require first-line treatment. Patients will be followed up for a minimum of 12 months following randomisation. Primary outcome Time from randomisation to treatment failure defined as platelets <30x109/L and a need for second-line treatment. Secondary outcomes Side effects, bleeding events, remission rates, time to relapse, time to next therapy, cumulative corticosteroid dose, rescue therapy, splenectomy, socioeconomic costs, patient-reported outcomes (quality of life, fatigue, impact of bleeding, care costs). Analysis The sample size of 120 achieves a 91.5% power to detect a doubling of the median time to treatment failure from 5 to 10 months. This will be expressed as an HR with 95% CI, median time to event if more than 50% have had an event and illustrated with Kaplan-Meier curves. Cost-effectiveness will be based on the first 12 months from diagnosis

Agreement between telehealth and in-person assessment of patients with chronic musculoskeletal conditions presenting to an advanced-practice physiotherapy screening clinic

Objective: To determine the level of agreement between a telehealth and in-person assessment of a representative sample of patients with chronic musculoskeletal conditions referred to an advanced-practice physiotherapy screening clinic. Design: Repeated-measures study design. Participants: 42 patients referred to the Neurosurgical & Orthopaedic Physiotherapy Screening Clinic (Queensland, Australia) for assessment of their chronic lumbar spine, knee or shoulder condition. Intervention: Participants underwent two consecutive assessments by different physiotherapists within a single clinic session. In-person assessments were conducted as per standard clinical practice. Telehealth assessments took place remotely via videoconferencing. Six Musculoskeletal Physiotherapists were paired together to perform both assessment types. Main outcome measures: Clinical management decisions including (i) recommended management pathways, (ii) referral to allied health professions, (iii) clinical diagnostics, and (iv) requirement for further investigations were compared using reliability and agreement statistics. Results: There was substantial agreement (83.3%; 35/42 cases) between in-person and telehealth assessments for recommended management pathways. Moderate to near perfect agreement (AC1 = 0.58–0.9) was reached for referral to individual allied health professionals. Diagnostic agreement was 83.3% between the two delivery mediums, whilst there was substantial agreement (81%; AC1 = 0.74) when requesting further investigations. Overall, participants were satisfied with the telehealth assessment. Conclusion: There is a high level of agreement between telehealth and in-person assessments with respect to clinical management decisions and diagnosis of patients with chronic musculoskeletal conditions managed in an advanced-practice physiotherapy screening clinic. Telehealth can be considered as a viable and effective medium to assess those patients who are unable to attend these services in person

Influence of glyphosate on Rhizoctonia and Fusarium root rot in sugar beet.

Abstract: This study tests the effect of glyphosate application on disease severity in glyphosate-resistant sugar beet, and examines whether the increase in disease is fungal or plant mediated. In greenhouse studies of glyphosateresistant sugar beet, increased disease severity was observed following glyphosate application and inoculation with certain isolates of Rhizoctonia solani Kuhn and Fusarium oxysporum Schlecht. f. sp. betae Snyd. &amp; Hans. Significant increases in disease severity were noted for R. solani AG-2-2 isolate R-9 and moderately virulent F. oxysporum isolate FOB13 on both cultivars tested, regardless of the duration between glyphosate application and pathogen challenge, but not with highly virulent F. oxysporum isolate F-19 or an isolate of R. solani AG-4. The increase in disease does not appear to be fungal mediated, since in vitro studies showed no positive impact of glyphosate on fungal growth or overwintering structure production or germination for either pathogen. Studies of glyphosate impact on sugar beet physiology showed that shikimic acid accumulation is tissue specific and the rate of accumulation is greatly reduced in resistant cultivars when compared with a susceptible cultivar. The results indicate that precautions need to be taken when certain soil-borne diseases are present if weed management for sugar beet is to include post-emergence glyphosate treatments

Educational Attainment at Age 10–11 Years Predicts Health Risk Behaviors and Injury Risk During Adolescence

Purpose: To examine the effect of educational attainment in primary school on later adolescent health. Methods: Education data attainments at age 7 and 11 were linked with (1) primary and secondary care injury consultation/admissions and (2) the Health Behaviour in School-aged Children survey. Cox regression was carried out to examine if attainment in primary school predicts time to injury in adolescence. Results: Pupils that achieve attainment at age 7 but not at age 11 (i.e., declining attainment over time in primary school) are more likely to have an injury during adolescence. These children are also more likely to self-report drinking in adolescence. Conclusions: Interventions aimed at children with declining attainment in primary school could help to improve adolescent health

Genome, Functional Gene Annotation, and Nuclear Transformation of the Heterokont Oleaginous Alga \u3ci\u3eNannochloropsis oceanica\u3c/i\u3e CCMP1779

Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of N. oceanica CCMP1779. RNA sequencing data from nitrogen-replete and nitrogendepleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the N. oceanica CCMP1779 gene repertoire with the recently published N. gaditana genome identified 2,649 genes likely specific to N. oceanica CCMP1779. Many of these N. oceanica–specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of N. oceanica CCMP1779 are provided. The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing academic community focused on this genus

Genome-Wide Association Study of Susceptibility to Idiopathic Pulmonary Fibrosis

Rationale: Idiopathic pulmonary fibrosis (IPF) is a complex lung disease characterised by scarring of the lung that is believed to result from an atypical response to injury of the epithelium. Genome-wide association studies have reported signals of association implicating multiple pathways including host defence, telomere maintenance, signalling and cell-cell adhesion. Objectives: To improve our understanding of factors that increase IPF susceptibility by identifying previously unreported genetic associations. Methods and measurements: We conducted genome-wide analyses across three independent studies and meta-analysed these results to generate the largest genome-wide association study of IPF to date (2,668 IPF cases and 8,591 controls). We performed replication in two independent studies (1,456 IPF cases and 11,874 controls) and functional analyses (including statistical fine-mapping, investigations into gene expression and testing for enrichment of IPF susceptibility signals in regulatory regions) to determine putatively causal genes. Polygenic risk scores were used to assess the collective effect of variants not reported as associated with IPF. Main results: We identified and replicated three new genome-wide significant (P<5×10−8) signals of association with IPF susceptibility (associated with altered gene expression of KIF15, MAD1L1 and DEPTOR) and confirmed associations at 11 previously reported loci. Polygenic risk score analyses showed that the combined effect of many thousands of as-yet unreported IPF susceptibility variants contribute to IPF susceptibility. Conclusions: The observation that decreased DEPTOR expression associates with increased susceptibility to IPF, supports recent studies demonstrating the importance of mTOR signalling in lung fibrosis. New signals of association implicating KIF15 and MAD1L1 suggest a possible role of mitotic spindle-assembly genes in IPF susceptibility

BHPR research: qualitative1. Complex reasoning determines patients' perception of outcome following foot surgery in rheumatoid arhtritis

Background: Foot surgery is common in patients with RA but research into surgical outcomes is limited and conceptually flawed as current outcome measures lack face validity: to date no one has asked patients what is important to them. This study aimed to determine which factors are important to patients when evaluating the success of foot surgery in RA Methods: Semi structured interviews of RA patients who had undergone foot surgery were conducted and transcribed verbatim. Thematic analysis of interviews was conducted to explore issues that were important to patients. Results: 11 RA patients (9 ♂, mean age 59, dis dur = 22yrs, mean of 3 yrs post op) with mixed experiences of foot surgery were interviewed. Patients interpreted outcome in respect to a multitude of factors, frequently positive change in one aspect contrasted with negative opinions about another. Overall, four major themes emerged. Function: Functional ability & participation in valued activities were very important to patients. Walking ability was a key concern but patients interpreted levels of activity in light of other aspects of their disease, reflecting on change in functional ability more than overall level. Positive feelings of improved mobility were often moderated by negative self perception ("I mean, I still walk like a waddling duck”). Appearance: Appearance was important to almost all patients but perhaps the most complex theme of all. Physical appearance, foot shape, and footwear were closely interlinked, yet patients saw these as distinct separate concepts. Patients need to legitimize these feelings was clear and they frequently entered into a defensive repertoire ("it's not cosmetic surgery; it's something that's more important than that, you know?”). Clinician opinion: Surgeons' post operative evaluation of the procedure was very influential. The impact of this appraisal continued to affect patients' lasting impression irrespective of how the outcome compared to their initial goals ("when he'd done it ... he said that hasn't worked as good as he'd wanted to ... but the pain has gone”). Pain: Whilst pain was important to almost all patients, it appeared to be less important than the other themes. Pain was predominately raised when it influenced other themes, such as function; many still felt the need to legitimize their foot pain in order for health professionals to take it seriously ("in the end I went to my GP because it had happened a few times and I went to an orthopaedic surgeon who was quite dismissive of it, it was like what are you complaining about”). Conclusions: Patients interpret the outcome of foot surgery using a multitude of interrelated factors, particularly functional ability, appearance and surgeons' appraisal of the procedure. While pain was often noted, this appeared less important than other factors in the overall outcome of the surgery. Future research into foot surgery should incorporate the complexity of how patients determine their outcome Disclosure statement: All authors have declared no conflicts of interes