The PD-Ballet study: study protocol for a randomised controlled single-blind hybrid type 2 clinical trial evaluating the effects of ballet dancing on motor and non-motor symptoms in Parkinson's disease.

BACKGROUND: To date, beneficial effects of multimodal exercise programmes on Parkinson's disease (PD) have focused on motor symptoms and little attention has been paid to the potential effects of such programmes on the non-motor symptoms of PD, which are now universally known as one of the key drivers of quality of life and a key unmet need. We aim to explore clinical effectiveness of a ballet-based dance programme in addressing non-motor and motor symptoms of Parkinson's disease across all stages of progression. METHODS: A randomised, single-blind, controlled trial of 160 people with Parkinson's across all motor stages (Participants will be stratified into three groups of motor advancement: Hoehn and Yahr (HY) stages I and II being Mild Group, HY Stage III being Moderate Group and HY Stages IV and V being Severe Group) will be randomly allocated to either an intervention or a control group using an independent randomisation body. The primary outcome is an improvement in non-motor symptoms as measured by the Movement Disorders Society Non-Motor Scale (MDS-NMS). The intervention protocol consists of 12 one-weekly dance sessions led by English National Ballet. Each session is followed by a 'tea and biscuit' social time. Control group follows standard clinical pathway and joins the 'tea and biscuit' to control for any positive effects of social interactions. All participants are assessed at baseline, immediately after completion of the intervention and 3-6 months later to explore any potential longitudinal effects. DISCUSSION: To our knowledge, no adequately powered study has explored the effects of a dance-based intervention on non-motor symptoms of Parkinson's disease, assessing these on both holistic and granular levels. We also aim to stratify participants in accordance with their motor state as assessed by. HY staging to explore specific effects on the symptoms at the initial, moderate and complex stages of the disease. If successful, this trial provides first evidence on clinical effectiveness of a ballet-based dance intervention for symptoms of Parkinson's disease, assessed in a robust, rigorous manner. TRIAL REGISTRATION: NCT04719468.

Experiences of acquired brain injury survivors participating in online and hybrid performance arts programmes: an ethnographic study.

BACKGROUND: Performance arts can benefit people with acquired brain injury (ABI). This study explored the online delivery during COVID-19 restrictions, of a performance art intervention through the experiences of participants, artists and facilitators. METHODS: Two community-based programmes were delivered. Online ethnographic observations and semi-structured interviews with participants, artists and facilitators were completed. RESULTS: The programmes benefited participants by addressing loneliness and isolation; building confidence through peer support; improving physical limitations through movement; improving communication through music and vocal work; and using poetry, visual arts, metaphor and performance to make sense of participants' experiences. Participants had mixed experiences of participation, but it was an acceptable alternative to in-person arts interventions for those who overcame digital challenges. CONCLUSIONS: ABI survivors can engage in online performance art programmes and find participation valuable for their health, well-being, and recovery. More work is needed to explore the generalisability of these findings, especially given digital poverty.

Feasibility, clinical efficacy, and well-being outcomes of an online singing intervention for postnatal depression in the UK: SHAPER-PNDO, a single-arm clinical trial.

BACKGROUND: Postnatal depression (PND) affects over 12% of mothers, with numbers rising during COVID-19. Singing groups can support mothers with PND; however, online delivery has never been evaluated. SHAPER-PNDO, a single-arm clinical trial, evaluated the feasibility, clinical efficacy, and well-being outcomes of a 6-week online version of Breathe Melodies for Mums (M4M) singing intervention developed for mothers with PND during COVID-19 lockdowns. METHODS: The primary objective of this study was to assess the feasibility of a group online singing intervention for new mothers with postnatal depression. This was ascertained through recruitment rates, study retention rates, attendance rates to the singing sessions, and study completion rates. The secondary objective of the study was to assess the clinical efficacy and well-being outcomes of the singing intervention. Specifically, we measured change in Edinburgh Postnatal Depression Scale (EPDS), State-Trait Anxiety Inventory (STAI), Perceived Stress Scale (PSS), and Office for National Statistics Wellbeing Scale (ONS) scores from baseline to end-of-intervention (week 6); follow-up assessments were completed at weeks 3, 16, and 32. Mothers were eligible if they scored ≥10 on the baseline EPDS. RESULTS: Eighty-seven percent of the 37 recruited mothers completed the study, attending, on average, 5 of the 6 group singing sessions. With regard to secondary outcomes, at end-of-treatment, mothers experienced significant reductions in depression (EPDS, 16.6 ± 3.7 to 11.2 ± 5.3, 95% CI [0.79,1.65]), anxiety (STAI-S, 48.4 ± 27.1 to 41.7 ± 26.8, 95% CI [4.96, 17.65]) and stress (PSS, 29.0 ± 5.7 to 19.7 ± 5.3, 95% CI [1.33, 7.07]); and, furthermore, significant improvements in life satisfaction (ONS, 50.5 ± 23.0 to 72.8 ± 11.7, 95% CI [- 39.86, - 4.64]) and feelings of worthwhileness (ONS, 51.7 ± 30.4 to 78.6 ± 15.1, 95% CI [- 52.79, - 0.85]). Reduction on the EPDS correlated with a reduction on the BDI and the STAI-S and maternal childhood maltreatment was predictive of a smaller treatment response. CONCLUSIONS: M4M online was feasible to mothers who partook in the programme. Furthermore, M4M online supports the mental health and well-being of new mothers experiencing PND, especially when barriers to in-person treatment are present. TRIAL REGISTRATION: ClinicalTrials.gov NCT04857593 . Registered 22 April 2021, retrospectively registered.

A Protocol to Understand the Implementation and Experiences of an Online Community-Based Performance Arts Programme Through and Beyond the COVID-19 Pandemic, Brain Waves.

Introduction: Individuals living with acquired brain injury experience numerous psychological, physical, and social challenges. Since the COVID-19 pandemic, many have experienced additional isolation, mental health issues and have had limited access to social and physical activities otherwise available in the community. Materials and Methods: Brain Waves is a 12-week online performance arts programme developed during the COVID-19 pandemic, for people with acquired brain injury (ABI). The research component of Brain Waves is a qualitative study, using Interpretative Phenomenological Analysis (IPA) and ethnographic methods (Observations and Interviews). The study will recruit two distinct populations: individuals living with acquired brain injury (including people who have experienced traumatic brain injury and stroke who are participating in the programme) and stakeholders (facilitators, involved in the delivery of Brain Waves). This paper presents the protocol for a project which aims to gain an understanding of the implementation and experiences of creating and participating in an online community-based performance arts programme.

Online singing interventions for postnatal depression in times of social isolation: a feasibility study protocol for the SHAPER-PNDO single-arm trial.

BACKGROUND: Postnatal depression (PND) affects 13% of new mothers, with numbers rising during the COVID-19 pandemic. Despite this prevalence, many women have difficulty with or hesitancy towards accessing pharmacological and/or psychological interventions. Group-based mother-baby activities, however, have a good uptake, with singing improving maternal mental health and the mother-infant relationship. The recent lockdowns highlight the importance of adapting activities to an online platform that is wide-reaching and accessible. AIMS: The SHAPER-PNDO study will primarily analyse the feasibility of a 6-week online singing intervention, Melodies for Mums (M4M), for mothers with PND who are experiencing barriers to treatment. The secondary aim of the SHAPER-PNDO study will be to analyse the clinical efficacy of the 6-week M4M intervention for symptoms of postnatal depression. METHODS: A total of 120 mothers and their babies will be recruited for this single-arm study. All dyads will attend 6 weekly online singing sessions, facilitated by Breathe Arts Health Research. Assessments will be conducted on Zoom at baseline and week 6, with follow-ups at weeks 16 and 32, and will contain interviews for demographics, mental health, and social circumstances, and biological samples will be taken for stress markers. Qualitative interviews will be undertaken to understand the experiences of women attending the sessions and the facilitators delivering them. Finally, data will be collected on recruitment, study uptake and attendance of the programme, participant retention, and acceptability of the intervention. DISCUSSION: The SHAPER-PNDO study will focus on the feasibility, alongside the clinical efficacy, of an online delivery of M4M, available to all mothers with PND. We hope to provide a more accessible, effective treatment option for mothers with PND that can be available both during and outside of the pandemic for mothers who would otherwise struggle to attend in-person sessions, as well as to prepare for a subsequent hybrid RCT. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04857593 . Registered retrospectively on 22 April 2021. The first participants were recruited on 27 January 2021, and the trial is ongoing.

Evaluation of a community-based performance arts programme for people who have experienced stroke in the UK: protocol for the SHAPER-Stroke Odysseys study.

INTRODUCTION: Stroke survivors, once in the community, face challenges with their long-term rehabilitation care and present higher levels of loneliness, depression and anxiety than the rest of the population. A community-based performance arts programme, Stroke Odysseys (SO), has been devised to tackle the challenges of living with stroke in the UK. In this study, we aim to evaluate the implementation, impact and experiences of SO for stroke survivors. METHODS AND ANALYSIS: Scaling-up Health Arts Programmes: Implementation and Effectiveness Research (SHAPER)-SO aims to scale-up SO to 75 participants and 47 stakeholders, while simultaneously evaluating the effectiveness and implementation of the programme. The main research aim is to evaluate the implementation, effectiveness, impact and experiences of a community-based performance arts programme (SO for stroke survivors). This mixed-methods study will evaluate the experience and impact of SO on those participating using mixed methods (interviews, observations and surveys) before and after each stage and carry out non-participant observations during a percentage of the workshops, training and tour. Data will be analysed using quantitative and qualitative approaches. This is a study within the SHAPER programme. ETHICS AND DISSEMINATION: Ethical approval has been granted by the King's College London PNM Research Ethics Panel, REC reference: LRS/DP-20/21-21549. Written informed consent will be sought for participants and stakeholders. The results of the study will be reported and disseminated at international conferences and in peer-reviewed scientific journals. TRIAL REGISTRATION NUMBER: NCT04864470.

Study protocol: randomised controlled hybrid type 2 trial evaluating the scale-up of two arts interventions for postnatal depression and Parkinson's disease.

INTRODUCTION: Research on the benefits of 'arts' interventions to improve individuals' physical, social and psychological well-being is growing, but evidence on implementation and scale-up into health and social care systems is lacking. This protocol reports the SHAPER-Implement programme (Scale-up of Health-Arts Programmes Effectiveness-Implementation Research), aimed at studying the impact, implementation and scale-up of: Melodies for Mums (M4M), a singing intervention for postnatal depression; and Dance for Parkinson's (PD-Ballet) a dance intervention for Parkinson's disease. We examine how they could be embedded in clinical pathways to ensure their longer-term sustainability. METHODS AND ANALYSIS: A randomised two-arm effectiveness-implementation hybrid type 2 trial design will be used across M4M/PD-Ballet. We will assess the implementation in both study arms (intervention vs control), and the cost-effectiveness of implementation. The design and measures, informed by literature and previous research by the study team, were refined through stakeholder engagement. Participants (400 in M4M; 160 in PD-Ballet) will be recruited to the intervention or control group (2:1 ratio). Further implementation data will be collected from stakeholders involved in referring to, delivering or supporting M4M/PD-Ballet (N=25-30 for each intervention).A mixed-methods approach (surveys and semi-structured interviews) will be employed. 'Acceptability' (measured by the 'Acceptability Intervention Measure') is the primary implementation endpoint for M4M/PD-Ballet. Relationships between clinical and implementation outcomes, implementation strategies (eg, training) and outcomes will be explored using generalised linear mixed models. Qualitative data will assess factors affecting the acceptability, feasibility and appropriateness of M4M/PD-Ballet, implementation strategies and longer-term sustainability. Costs associated with implementation and future scale-up will be estimated. ETHICS AND DISSEMINATION: SHAPER-PND (the M4M trial) and SHAPER-PD (the PD trial) are approved by the West London and GTAC (20/PR/0813) and the HRA and Health and Care Research Wales (REC Reference: 20/WA/0261) Research Ethics Committees. Study findings will be disseminated through scientific peer-reviewed journals and scientific conferences. TRIAL REGISTRATION NUMBERS: Both trials are registered with NIH US National Library of Medicine, ClinicalTrials.gov. The trial registration numbers, URLs of registry records, and dates of registration are: (1) PD-Ballet: URL: NCT04719468 (https://eur03.safelinks.protection. OUTLOOK: com/?url=https%3A%2F%2Fwww.clinicaltrials.gov%2Fct2%2Fshow%2FNCT04719468%3Fterm%3DNCT04719468%26draw%3D2%26rank%3D1&data=04%7C01%7Crachel.davis%40kcl.ac.uk%7C11a7c5142782437919f808d903111449%7C8370cf1416f34c16b83c724071654356%7C0%7C0%7C6375441942616) (date of registration: 22 Jan 2021). (2) Melodies for Mums: NCT04834622 (https://clinicaltrials.gov/ct2/show/NCT04834622?term=shaper-pnd&draw=2&rank=1) (date of registration: 8 Apr 2021).

SHAPER-PND trial: clinical effectiveness protocol of a community singing intervention for postnatal depression.

INTRODUCTION: Postnatal depression (PND) affects approximately 13% of new mothers. Community-based activities are sought after by many mothers, especially mothers that prefer not to access pharmacological or psychological interventions. Singing has shown positive effects in maternal mood and mother-child bonding. The Scaling-Up Health-Arts Programmes: Implementation and Effectiveness Research-Postnatal Depression study will analyse the clinical and implementation effectiveness of 10-week singing sessions for PND in new mothers. This protocol paper will focus on the clinical effectiveness of this trial. METHODS AND ANALYSIS: A total of 400 mothers with PND (with a score of at least 10 on the Edinburgh Postnatal Depression Scale) and their babies will be recruited for this hybrid type II randomised controlled trial. The intervention group will attend 10 weekly singing sessions held at community venues or online, facilitated by the arts organisation, Breathe Arts Health Research (Breathe). A control group will be encouraged to attend non-singing sessions in the community or online for 10 weeks. A package of assessments will be collected from participants for clinical, mechanistic and implementation outcomes, at different stages of the trial. Clinical assessments will include questionnaires and interviews for demographics, mental health and social measures, together with biological samples for measurement of stress markers; the study visits are at baseline, week 6 (mid-trial) and week 10 (end of trial), with follow ups at weeks 20 and 36. Multiple imputation will be used to deal with possible missing data and multivariable models will be fitted to assess differences between groups in the outcomes of the study. ETHICS AND DISSEMINATION: Ethical approval has been granted by the London-West London and GTAC Research Ethics Committee, REC reference: 20/PR/0813. TRIAL REGISTRATION NUMBER: NCT04834622; Pre-results.

CD36 promotes vasculogenic mimicry in melanoma by mediating adhesion to the extracellular matrix.

BACKGROUND: The formation of blood vessels within solid tumors directly contributes to cancer growth and metastasis. Until recently, tumor vasculature was thought to occur exclusively via endothelial cell (EC) lined structures (i.e. angiogenesis), but a second source of tumor vasculature arises from the cancer cells themselves, a process known as vasculogenic mimicry (VM). While it is generally understood that the function of VM vessels is the same as that of EC-lined vessels (i.e. to supply oxygen and nutrients to the proliferating cancer cells), the molecular mechanisms underpinning VM are yet to be fully elucidated. METHODS: Human VM-competent melanoma cell lines were examined for their VM potential using the in vitro angiogenesis assays (Matrigel), together with inhibition studies using small interfering RNA and blocking monoclonal antibodies. Invasion assays and adhesion assays were used to examine cancer cell function. RESULTS: Herein we demonstrate that CD36, a cell surface glycoprotein known to promote angiogenesis by ECs, also supports VM formation by human melanoma cancer cells. In silico analysis of CD36 expression within the melanoma cohort of The Cancer Genome Atlas suggests that melanoma patients with high expression of CD36 have a poorer clinical outcome. Using in vitro 'angiogenesis' assays and CD36-knockdown approaches, we reveal that CD36 supports VM formation by human melanoma cells as well as adhesion to, and invasion through, a cancer derived extracellular matrix substrate. Interestingly, thrombospondin-1 (TSP-1), a ligand for CD36 on ECs that inhibits angiogenesis, has no effect on VM formation. Further investigation revealed a role for laminin, but not collagen or fibronectin, as ligands for CD36 expressing melanoma cells. CONCLUSIONS: Taken together, this study suggests that CD36 is a novel regulator of VM by melanoma cancer cells that is facilitated, at least in part, via integrin-α3 and laminin. Unlike angiogenesis, VM is not perturbed by the presence of TSP-1, thus providing new information on differences between these two processes of tumor vascularization which may be exploited to combat cancer progression.

Development of the venom delivery system in Elapidae snake species: Naja siamensis and Oxyuranus microlepidotus.

Many advanced snakes possess a unique venom delivery system which they utilise to subdue prey and for defence. Despite extensive efforts, the evolutionary differences in this key system between advanced snake families remains enigmatic. The current study has investigated the development of the venom delivery system using two oviparous Elapidae models, Naja siamensis and Oxyuranus microlepidotus. The development stages of the embryos in both models were detailed using previously standardised characterisation. Variations in the days post-oviposition between these stages was observed, despite a continuous development trajectory. These differences also translated to the development of the venom delivery system.

Scaling-up Health-Arts Programmes: the largest study in the world bringing arts-based mental health interventions into a national health service.

The Scaling-up Health-Arts Programme: Implementation and Effectiveness Research (SHAPER) project is the world's largest hybrid study on the impact of the arts on mental health embedded into a national healthcare system. This programme, funded by the Wellcome Trust, aims to study the impact and the scalability of the arts as an intervention for mental health. The programme will be delivered by a team of clinicians, research scientists, charities, artists, patients and healthcare professionals in the UK's National Health Service (NHS) and the community, spanning academia, the NHS and the charity sector. SHAPER consists of three studies - Melodies for Mums, Dance for Parkinson's, and Stroke Odysseys - which will recruit over 800 participants, deliver the interventions and draw conclusions on their clinical impact, implementation effectiveness and cost-effectiveness. We hope that this work will inspire organisations and commissioners in the NHS and around the world to expand the remit of social prescribing to include evidence-based arts interventions.

Platelets disrupt vasculogenic mimicry by cancer cells.

Tumour vasculature supports the growth and progression of solid cancers with both angiogenesis (endothelial cell proliferation) and vasculogenic mimicry (VM, the formation of vascular structures by cancer cells themselves) predictors of poor patient outcomes. Increased circulating platelet counts also predict poor outcome for cancer patients but the influence of platelets on tumour vasculature is incompletely understood. Herein, we show with in vitro assays that platelets did not influence angiogenesis but did actively inhibit VM formation by cancer cell lines. Both platelet sized beads and the releasates from platelets were partially effective at inhibiting VM formation suggesting that direct contact maximises the effect. Platelets also promoted cancer cell invasion in vitro. B16F10 melanomas in Bcl-xPlt20/Plt20 thrombocytopenic mice showed a higher content of VM than their wildtype counterparts while angiogenesis did not differ. In a xenograft mouse model of breast cancer with low-dose aspirin to inactivate the platelets, the burden of MDA-MB-231-LM2 breast cancer cells was reduced and the gene expression profile of the cancer cells was altered; but no effect on tumour vasculature was observed. Taken together, this study provides new insights into the action of platelets on VM formation and their involvement in cancer progression.

Contributions of patient and citizen researchers to 'Am I the right way up?' study of balance in posterior cortical atrophy and typical Alzheimer's disease.

The current report describes the journey from the sharing of a single, extraordinary experience during a support group conversation to the development of a novel scientific investigation of balance problems in a rarer form of dementia. The story centres around the involvement of people living with or caring for someone with posterior cortical atrophy (often referred to as the visual variant of Alzheimer's disease) in highlighting hitherto under-appreciated consequences of their condition upon their ability to know 'Am I the right way up?'. We describe how comments and descriptions of these balance symptoms were collated and communicated, and the involvement of people with posterior cortical atrophy in shaping a series of scientific hypotheses and developing and adapting appropriate experimental materials and procedures. We also reflect more broadly on how we might better recognise, acknowledge and encourage different forms of involvement, and describe several engagement-inspired extensions to the research involving people living with dementia, scientists and artists.

Regulation of expression of venom toxins: silencing of prothrombin activator trocarin D by AG-rich motifs.

Trocarin D (TroD), a venom prothrombin activator from Tropidechis carinatus, shares similar structure and function with blood coagulation factor Xa [Tropidechis carinatus FX (TrFX) a]. Their distinct physiologic roles are due to their distinct expression patterns. The genes of TroD and TrFX are highly similar, except for promoter and intron 1, indicating that TroD has probably evolved by duplication of FX, the plasma counterpart. The promoter insertion in TroD accounts for the elevated but not venom gland-specific expression. Here we examined the roles of 3 insertions and 2 deletions in intron 1 of TroD in the regulation of expression using luciferase as a reporter. By systematic deletions, we showed that a 209 bp region within the second insertion silences expression in mammalian and unmilked venom gland cells. Through bioinformatics analysis, we identified 5 AG-rich motifs in this region. All except the 5th motif are important for silencing function. YY1, Sp3 and HMGB2 were identified to bind these AG-rich motifs and silence gene expression in mammalian cells. Similar AG-rich motif clusters are also found in other toxin genes but not in their physiologic counterparts. Thus, AG-rich motifs contribute to regulation of expression of TroD, and probably other toxin genes.-Han, S. X., Kwong, S., Ge, R., Kolatkar, P. R., Woods, A. E., Blanchet, G., Kini, R. M. Regulation of expression of venom toxins: silencing of prothrombin activator trocarin D by AG-rich motifs.

The king cobra genome reveals dynamic gene evolution and adaptation in the snake venom system.

Snakes are limbless predators, and many species use venom to help overpower relatively large, agile prey. Snake venoms are complex protein mixtures encoded by several multilocus gene families that function synergistically to cause incapacitation. To examine venom evolution, we sequenced and interrogated the genome of a venomous snake, the king cobra (Ophiophagus hannah), and compared it, together with our unique transcriptome, microRNA, and proteome datasets from this species, with data from other vertebrates. In contrast to the platypus, the only other venomous vertebrate with a sequenced genome, we find that snake toxin genes evolve through several distinct co-option mechanisms and exhibit surprisingly variable levels of gene duplication and directional selection that correlate with their functional importance in prey capture. The enigmatic accessory venom gland shows a very different pattern of toxin gene expression from the main venom gland and seems to have recruited toxin-like lectin genes repeatedly for new nontoxic functions. In addition, tissue-specific microRNA analyses suggested the co-option of core genetic regulatory components of the venom secretory system from a pancreatic origin. Although the king cobra is limbless, we recovered coding sequences for all Hox genes involved in amniote limb development, with the exception of Hoxd12. Our results provide a unique view of the origin and evolution of snake venom and reveal multiple genome-level adaptive responses to natural selection in this complex biological weapon system. More generally, they provide insight into mechanisms of protein evolution under strong selection.

The effects of selected Australian snake venoms on tumour-associated microvascular endothelial cells (TAMECs) in vitro.

The effects of various viperid and elapid venoms on the cellular biology of tumour-associated microvascular endothelial cells (TAMECs) were determined in the current study using cells isolated from a rat mammary adenocarcinoma. Previous studies to determine the effects of snake venoms on endothelial cells in vitro have in the main been performed on either human umbilical vein endothelial cells (HUVECs), bovine aortic endothelial cells (BAECs) or endothelial cell lines. These cell populations are accessible and easy to maintain in culture, however, it is well established that endothelial cells display vast heterogeneity depending upon the local microenvironment of the tissue from which they are isolated. Vascular targeting agents have been isolated from a variety of snake venoms, particularly from snakes of the Viperidae family, but it is yet to be established to what extent the venoms from Australian elapids possess similar vascular targeting properties. The present study used endothelial cells (ECs) isolated from the microvasculature of a rat mammary adenocarcinoma to determine the effects of a panel of snake venoms, including viperid venoms with known apoptotic activity and elapid venoms (both exotic and indigenous to Australia), on endothelial morphology and viability, paying specific attention to apoptotic responses. Three of the five Australian snake venoms investigated in this study elicited significant apoptotic responses in ECs which were in many ways similar to responses elicited by the selected viperid venoms. This suggests that these Australian elapids may possess vascular targeting components similar to those found within viperid venoms.

The effects of insufflation conditions on rat mesothelium.

Aim. The aim of this investigation was to examine the alterations in the peritoneum after cold dry CO2, heated dry CO2, and humidified heated CO2 at pressures equivalent to intraperitoneal pressures used in human laparoscopy. Methods. Eighteen rats were divided into 4 treatment groups-group 1: untreated control; group 2: insufflation with cold dry CO2; group 3: insufflation with heated, dry CO2; group 4: insufflation with heated and humidified CO2. The abdomen was insufflated to 5 mm/Hg (flow rate 50 mL/min) for 2 h. Twelve hours later, tissue samples were collected for analysis by light microscopy (LM) and scanning electron microscopy (SEM). Results. Group 1: no abnormalities were detected. Group 2: specimens revealed an inflammatory response with loss of mesothelium and mesothelial cell nuclei showing lytic change. Cells were rounded with some areas of cell flattening and separation. Group 3: some animals showed little or no alteration, while others had a mild inflammatory response. Mesothelial cells were rounded and showed crenation on the exposed surface. Group 4: specimens showed little change from the control group. Conclusions. The LM results indicate that insufflations with heated, humidified CO2 are the least likely to induce mesothelial damage.

The recruitment of blood coagulation factor X into snake venom gland as a toxin: the role of promoter cis-elements in its expression.

Trocarin D is a prothrombin activator from the Tropidechis carinatus venom. It is a functional and structural homologue to mammalian blood coagulation factor Xa. Trocarin D is hypothesised to have evolved from its factor X counterpart (TrFX) through gene duplication and recruitment. The genes of trocarin D and TrFX have significant sequence identities, except for insertions/deletions in their intron 1 and promoter regions. In trocarin D intron 1 region, there are three insertions and two deletions. In trocarin D promoter region, there is a novel 264 bp insertion which has potential cis-elements. This insertion is termed as Venom Recruitment/Switch Element (VERSE) and is hypothesised to account for switching the low-level constitutive expression of factor X in the liver to the high-level inducible expression of trocarin D in the venom gland. To understand the role of VERSE in the trocarin D expression, its cis-elements were characterised by luciferase assays in mammalian cell lines as well as snake venom gland cells. The ability of VERSE to drive luciferase expression is comparable to that of the trocarin D promoter. The predicted cis-elements are important in promoting expression as their mutagenesis resulted in lower luciferase expression. VERSE minimal core promoter and three novel cis-elements (two up-regulatory and one suppressor elements) were identified using deletion/site-directed mutagenesis studies. VERSE is primarily responsible for the increase of trocarin D expression. The insertions/deletions within trocarin D intron 1 need to be characterised for their role in tissue-specific and inducible expression of trocarin D.

Localisation of estrogen responsive genes in the liver and testis of Murray rainbowfish Melanotaenia fluviatilis exposed to 17beta-estradiol.

The localisation of estrogen receptors (ERalpha and ERbeta) and vitellogenin (VTG) transcripts were examined in the liver and testis in male rainbowfish exposed to 17beta-estradiol (E2; 0, 50 and 500 ng/L) via the water for up to 7 days. The ER transcripts were localised within the perinuclear region of the hepatocytes and were up-regulated with E2 exposure. A parallel induction of liver VTG transcripts and protein was observed within 24h, followed by a time-dependent increase in VTG protein. In the testis, both ERs were up-regulated in the germ and epithelial cells, while VTG protein was detected in the cellular space surrounding the spermatids and in association with the connective tissue of the sperm tubules. These results indicate that the ERs are positively auto-regulated in the liver and testis of male rainbowfish. The cellular localisation of VTG within the testis may suggest implication in the mediation of adverse effects of endocrine disrupting chemicals such as testicular growth inhibition, testis-ova and sex reversal.

Transforming growth factor-beta-mediated signaling in T lymphocytes impacts on prostate-specific immunity and early prostate tumor progression.

T cells are in general tolerant of prostate-specific tumor antigens. That prostate tumor tissue makes transforming growth factor-beta (TGFbeta) is thought to play a role in the induction of T-cell tolerance within the host and to contribute to tumor progression itself. Here we sought to investigate the influence of TGFbeta signaling on prostate antigen-specific T-cell responses as well as prostate tumorogenesis in an autochthonous murine model of the disease. The response of naive and activated ovalbumin (OVA) antigen-specific T cells, which had been rendered incapable of responding to TGFbeta through T-cell-specific transgenic expression of a dominant-negative variant of the TGFbeta receptor II (dnTGFRII), was analyzed after adoptive transfer into prostate OVA-expressing transgenic (POET) mice. The role of TGFbeta signaling in endogenous T cells in mice, which spontaneously form tumors, was also assessed by monitoring prostate tumor formation and progression in F1 progeny of productive matings between transgenic adenocarcinoma of the mouse prostate (TRAMP) and dnTGFRII mice. TGFbeta-resistant CD8(+) T cells proliferated more and produced IFNgamma more readily after OVA stimulation in vitro. OVA-specific T cells did not damage the prostate gland of POET mice irrespective of TGFbeta responsiveness. However, ex vivo activation facilitated entry of TGFbeta-insensitive T cells into the prostate and was associated with prostate tissue damage. Early tumor progression was delayed in TRAMP mice that carried endogenous TGFbeta-insensitive T cells. Together, these results suggest that TGFbeta-signaling represses CD8(+) T-cell responses to a prostate-specific antigen. TGFbeta-mediated repression of T-cell function may include production of IFNgamma, which is known to contribute to tumor immunosurveillance.