CALCIUM SIGNALING AND ALG-2 IN THE REGULATION OF CONSTITUTIVE SECRETION

The constitutive secretory pathway influences almost every aspect of cellular function: generation and maintenance of subcellular compartments; cell growth and differentiation; and production and deposition of the extracellular matrix. Classically, constitutive trafficking was thought to be continuous and unregulated—in contrast to regulated secretion, wherein vesicles are stored intracellularly until a Ca2+ signal causes synchronous membrane fusion. However, early studies demonstrated a Ca2+ requirement for endosome fusion, ER to Golgi, and intra- Golgi trafficking, demonstrating that Ca2+ is a regulator of constitutive trafficking steps as well. In Chapter 1, I review recent evidence for Ca2+ involvement and the specific Ca2+-related machinery implicated in trafficking events throughout the constitutive trafficking systems. Notably, I describe a series of Ca2+ pumps, channels, and Ca2+-binding effector proteins—and their trafficking machinery targets—that together regulate the flux of cargo in response to genetic alterations or baseline and agonist-dependent Ca2+ signals. Of the intracellular constitutive trafficking steps, the ER-to-Golgi transport stage is the busiest, transporting up to one-third of all eukaryotic proteins. However, the potential regulation of these steps by Ca2+ signaling events has remained largely uncharacterized. Studies indicated that the PEF protein apoptosis-linked gene 2 (ALG-2) binds Sec31A at ER-exit-sites (ERES) in a Ca2+-dependent manner, but in vitro and intact cell methodologies have been unable to adequately explain the specific role of ALG-2 and its PEF protein binding partner, peflin, in secretion. Chapter 2 describes experimental procedures, while Chapter 3 significantly expands upon the ALG-2- peflin regulatory machine. I find that although both proteins are fully dispensable for secretion, a peflin-ALG-2 heterocomplex binds to ERES via the ALG-2 subunit and confers a low, buffered transport rate, while peflin-lacking ALG-2 complexes can either enhance or inhibit transport of ER cargoes depending on expression level. This apparent bifurcated response indicates that PEF protein regulatory states may tune transport rates through expression level or Ca2+ changes, yet PEF proteins have never been observed regulating transport in response to Ca2+. In Chapter 4, I examine the roles of ALG-2 and peflin in response to physiological Ca2+-mobilizing agonists, and find that ALG-2 depresses ER export in epithelial NRK cells and enhances ER export in neuroendocrine PC12 cells. Furthermore, I find that Ca2+ signaling in the NRK cell model can produce opposing effects on secretion depending on signaling intensity and duration— phenomena that could contribute to cellular growth and intercellular communication following secretory increases or protection from excitotoxicity and infection following secretory decreases. Mechanistically, ALG-2-dependent depression of secretion involves decreased levels of the COPII outer shell and increased peflin targeting to ERES, while ALG-2-dependent enhancement of secretion involves increased COPII outer shell and decreased peflin at ERES. These data provide insights into how PEF protein dynamics affect secretion of important physiological cargoes such as collagen I and significantly impact ER stress

'It's a film' : medium specificity as textual gesture in Red road and The unloved

British cinema has long been intertwined with television. The buzzwords of the transition to digital media, 'convergence' and 'multi-platform delivery', have particular histories in the British context which can be grasped only through an understanding of the cultural, historical and institutional peculiarities of the British film and television industries. Central to this understanding must be two comparisons: first, the relative stability of television in the duopoly period (at its core, the licence-funded BBC) in contrast to the repeated boom and bust of the many different financial/industrial combinations which have comprised the film industry; and second, the cultural and historical connotations of 'film' and 'television'. All readers of this journal will be familiar – possibly over-familiar – with the notion that 'British cinema is alive and well and living on television'. At the end of the first decade of the twenty-first century, when 'the end of medium specificity' is much trumpeted, it might be useful to return to the historical imbrication of British film and television, to explore both the possibility that medium specificity may be more nationally specific than much contemporary theorisation suggests, and to consider some of the relationships between film and television manifest at a textual level in two recent films, Red Road (2006) and The Unloved (2009)

The REFLECT Statement: Methods and Processes of Creating Reporting Guidelines for Randomized Controlled Trials for Livestock and Food Safety by Modifying the CONSORT Statement

The conduct of randomized controlled trials in livestock with production, health and food-safety outcomes presents unique challenges that may not be adequately reported in trial reports. The objective of this project was to modify the CONSORT (Consolidated Standards of Reporting Trials) statement to reflect the unique aspects of reporting these livestock trials. A 2-day consensus meeting was held on 18–19 November 2008 in Chicago, IL, USA, to achieve the objective. Prior to the meeting, a Web-based survey was conducted to identify issues for discussion. The 24 attendees were biostatisticians, epidemiologists, food-safety researchers, livestock-production specialists, journal editors, assistant editors and associate editors. Prior to the meeting, the attendees completed a Web-based survey indicating which CONSORT statement items may need to be modified to address unique issues for livestock trials. The consensus meeting resulted in the production of the REFLECT (Reporting Guidelines for Randomized Control Trials) statement for livestock and food safety and 22-item checklist. Fourteen items were modified from the CONSORT checklist and an additional sub-item was proposed to address challenge trials. The REFLECT statement proposes new terminology, more consistent with common usage in livestock production, to describe study subjects. Evidence was not always available to support modification to or inclusion of an item. The use of the REFLECT statement, which addresses issues unique to livestock trials, should improve the quality of reporting and design for trials reporting production, health and food-safety outcomes

The Cancer/Testis Antigen Prostate-associated Gene 4 (PAGE4) Is a Highly Intrinsically Disordered Protein

The cancer/testis antigens (CTAs) are an important group of heterogeneous proteins that are predominantly expressed in the testis in the normal human adult but are aberrantly expressed in several types of cancers. Prostate-associated gene 4 (PAGE4) is a member of the CT-X family of CTAs that in addition to testis, is highly expressed in the fetal prostate, and may also play an important role both in benign and malignant prostate diseases. However, the function of this gene remains poorly understood. Here, we show that PAGE4 is a highly (100%) intrinsically disordered protein (IDP). The primary protein sequence conforms to the features of a typical IDP sequence and the secondary structure prediction algorithm metaPrDOS strongly supported this prediction. Furthermore, SDS-gel electrophoresis and analytical size exclusion chromatography of the recombinant protein revealed an anomalous behavior characteristic of IDPs. UV circular dichroism (CD) and NMR spectroscopy confirmed that PAGE4 is indeed a highly disordered protein. In further bioinformatic analysis, the PredictNLS algorithm uncovered a potential nuclear localization signal, whereas the algorithm DBS-Pred returned a 99.1% probability that PAGE4 is a DNA-binding protein. Consistent with this prediction, biochemical experiments showed that PAGE4 preferentially binds a GC-rich sequence. Silencing PAGE4 expression induced cell death via apoptosis and in mice carrying PCa xenografts, siRNA-mediated knockdown of the PAGE4 mRNA attenuated tumor growth in vivo. Furthermore, overexpressing PAGE4 protected cells from stress-induced death. To our knowledge, PAGE4 is the first example of a CTA that is an IDP with an anti-apoptotic function

Australia\u27s health 2002 : the eighth biennial report of the Australian Institute of Health and Welfare

Australia\u27s Health 2002 is the eighth biennial health report of the Australian Institute of Health and Welfare. It is the nation\u27s authoritative source of information on patterns of health and illness, determinants of health, the supply and use of health services, and health service costs and performance. Australia\u27s Health 2002 is an essential reference and information resource for all Australians with an interest in health

The Novel Deacetylase Inhibitor AR-42 Demonstrates Pre-Clinical Activity in B-Cell Malignancies In Vitro and In Vivo

While deacetylase (DAC) inhibitors show promise for the treatment of B-cell malignancies, those introduced to date are weak inhibitors of class I and II DACs or potent inhibitors of class I DAC only, and have shown suboptimal activity or unacceptable toxicities. We therefore investigated the novel DAC inhibitor AR-42 to determine its efficacy in B-cell malignancies.In mantle cell lymphoma (JeKo-1), Burkitt's lymphoma (Raji), and acute lymphoblastic leukemia (697) cell lines, the 48-hr IC(50) (50% growth inhibitory concentration) of AR-42 is 0.61 microM or less. In chronic lymphocytic leukemia (CLL) patient cells, the 48-hr LC(50) (concentration lethal to 50%) of AR-42 is 0.76 microM. AR-42 produces dose- and time-dependent acetylation both of histones and tubulin, and induces caspase-dependent apoptosis that is not reduced in the presence of stromal cells. AR-42 also sensitizes CLL cells to TNF-Related Apoptosis Inducing Ligand (TRAIL), potentially through reduction of c-FLIP. AR-42 significantly reduced leukocyte counts and/or prolonged survival in three separate mouse models of B-cell malignancy without evidence of toxicity.Together, these data demonstrate that AR-42 has in vitro and in vivo efficacy at tolerable doses. These results strongly support upcoming phase I testing of AR-42 in B-cell malignancies

Bush the transnationalist: a reappraisal of the unilateralist impulse in US foreign policy, 2001-2009

This article challenges the common characterisation of George W. Bush’s foreign policy as “unilateral.” It argues that the Bush administration developed a new post-9/11 understanding of terrorism as a transnational, networked phenomenon shaped by the forces of globalisation. This led to a new strategic emphasis on bi- and multilateral security co-operation and counterterrorism operations, especially outside of Afghanistan and Iraq, driven by the perceived need to counter a transnational security challenge present in multiple locations. This (flawed) attempt to engage with transnational security challenges supplemented the existing internationalist pillar of the Bush administration’s foreign policy. Highlighting the transnational realm of international relations and the ways in which the Bush administration was able to co-opt other states to tackle perceived transnational challenges also shows the high importance the administration attached to concerted action even as it frequented eschewed institutional multilateralism

Semaglutide and cardiovascular outcomes in patients with obesity and prevalent heart failure: a prespecified analysis of the SELECT trial

Background: Semaglutide, a GLP-1 receptor agonist, reduces the risk of major adverse cardiovascular events (MACE) in people with overweight or obesity, but the effects of this drug on outcomes in patients with atherosclerotic cardiovascular disease and heart failure are unknown. We report a prespecified analysis of the effect of once-weekly subcutaneous semaglutide 2·4 mg on ischaemic and heart failure cardiovascular outcomes. We aimed to investigate if semaglutide was beneficial in patients with atherosclerotic cardiovascular disease with a history of heart failure compared with placebo; if there was a difference in outcome in patients designated as having heart failure with preserved ejection fraction compared with heart failure with reduced ejection fraction; and if the efficacy and safety of semaglutide in patients with heart failure was related to baseline characteristics or subtype of heart failure. Methods: The SELECT trial was a randomised, double-blind, multicentre, placebo-controlled, event-driven phase 3 trial in 41 countries. Adults aged 45 years and older, with a BMI of 27 kg/m2 or greater and established cardiovascular disease were eligible for the study. Patients were randomly assigned (1:1) with a block size of four using an interactive web response system in a double-blind manner to escalating doses of once-weekly subcutaneous semaglutide over 16 weeks to a target dose of 2·4 mg, or placebo. In a prespecified analysis, we examined the effect of semaglutide compared with placebo in patients with and without a history of heart failure at enrolment, subclassified as heart failure with preserved ejection fraction, heart failure with reduced ejection fraction, or unclassified heart failure. Endpoints comprised MACE (a composite of non-fatal myocardial infarction, non-fatal stroke, and cardiovascular death); a composite heart failure outcome (cardiovascular death or hospitalisation or urgent hospital visit for heart failure); cardiovascular death; and all-cause death. The study is registered with ClinicalTrials.gov, NCT03574597. Findings: Between Oct 31, 2018, and March 31, 2021, 17 604 patients with a mean age of 61·6 years (SD 8·9) and a mean BMI of 33·4 kg/m2 (5·0) were randomly assigned to receive semaglutide (8803 [50·0%] patients) or placebo (8801 [50·0%] patients). 4286 (24·3%) of 17 604 patients had a history of investigator-defined heart failure at enrolment: 2273 (53·0%) of 4286 patients had heart failure with preserved ejection fraction, 1347 (31·4%) had heart failure with reduced ejection fraction, and 666 (15·5%) had unclassified heart failure. Baseline characteristics were similar between patients with and without heart failure. Patients with heart failure had a higher incidence of clinical events. Semaglutide improved all outcome measures in patients with heart failure at random assignment compared with those without heart failure (hazard ratio [HR] 0·72, 95% CI 0·60-0·87 for MACE; 0·79, 0·64-0·98 for the heart failure composite endpoint; 0·76, 0·59-0·97 for cardiovascular death; and 0·81, 0·66-1·00 for all-cause death; all pinteraction>0·19). Treatment with semaglutide resulted in improved outcomes in both the heart failure with reduced ejection fraction (HR 0·65, 95% CI 0·49-0·87 for MACE; 0·79, 0·58-1·08 for the composite heart failure endpoint) and heart failure with preserved ejection fraction groups (0·69, 0·51-0·91 for MACE; 0·75, 0·52-1·07 for the composite heart failure endpoint), although patients with heart failure with reduced ejection fraction had higher absolute event rates than those with heart failure with preserved ejection fraction. For MACE and the heart failure composite, there were no significant differences in benefits across baseline age, sex, BMI, New York Heart Association status, and diuretic use. Serious adverse events were less frequent with semaglutide versus placebo, regardless of heart failure subtype. Interpretation: In patients with atherosclerotic cardiovascular diease and overweight or obesity, treatment with semaglutide 2·4 mg reduced MACE and composite heart failure endpoints compared with placebo in those with and without clinical heart failure, regardless of heart failure subtype. Our findings could facilitate prescribing and result in improved clinical outcomes for this patient group. Funding: Novo Nordisk

Case Reports1. A Late Presentation of Loeys-Dietz Syndrome: Beware of TGFβ Receptor Mutations in Benign Joint Hypermobility

Background: Thoracic aortic aneurysms (TAA) and dissections are not uncommon causes of sudden death in young adults. Loeys-Dietz syndrome (LDS) is a rare, recently described, autosomal dominant, connective tissue disease characterized by aggressive arterial aneurysms, resulting from mutations in the transforming growth factor beta (TGFβ) receptor genes TGFBR1 and TGFBR2. Mean age at death is 26.1 years, most often due to aortic dissection. We report an unusually late presentation of LDS, diagnosed following elective surgery in a female with a long history of joint hypermobility. Methods: A 51-year-old Caucasian lady complained of chest pain and headache following a dural leak from spinal anaesthesia for an elective ankle arthroscopy. CT scan and echocardiography demonstrated a dilated aortic root and significant aortic regurgitation. MRA demonstrated aortic tortuosity, an infrarenal aortic aneurysm and aneurysms in the left renal and right internal mammary arteries. She underwent aortic root repair and aortic valve replacement. She had a background of long-standing joint pains secondary to hypermobility, easy bruising, unusual fracture susceptibility and mild bronchiectasis. She had one healthy child age 32, after which she suffered a uterine prolapse. Examination revealed mild Marfanoid features. Uvula, skin and ophthalmological examination was normal. Results: Fibrillin-1 testing for Marfan syndrome (MFS) was negative. Detection of a c.1270G > C (p.Gly424Arg) TGFBR2 mutation confirmed the diagnosis of LDS. Losartan was started for vascular protection. Conclusions: LDS is a severe inherited vasculopathy that usually presents in childhood. It is characterized by aortic root dilatation and ascending aneurysms. There is a higher risk of aortic dissection compared with MFS. Clinical features overlap with MFS and Ehlers Danlos syndrome Type IV, but differentiating dysmorphogenic features include ocular hypertelorism, bifid uvula and cleft palate. Echocardiography and MRA or CT scanning from head to pelvis is recommended to establish the extent of vascular involvement. Management involves early surgical intervention, including early valve-sparing aortic root replacement, genetic counselling and close monitoring in pregnancy. Despite being caused by loss of function mutations in either TGFβ receptor, paradoxical activation of TGFβ signalling is seen, suggesting that TGFβ antagonism may confer disease modifying effects similar to those observed in MFS. TGFβ antagonism can be achieved with angiotensin antagonists, such as Losartan, which is able to delay aortic aneurysm development in preclinical models and in patients with MFS. Our case emphasizes the importance of timely recognition of vasculopathy syndromes in patients with hypermobility and the need for early surgical intervention. It also highlights their heterogeneity and the potential for late presentation. Disclosures: The authors have declared no conflicts of interes