In situ single-cell therapeutic response imaging facilitated by the TRIPODD fluorescence imaging platform.

Purpose: Small molecule drugs such as tyrosine kinase inhibitors (TKIs) targeting tumoral molecular dependencies have become standard of care for numerous cancer types. Notably, epidermal growth factor receptor (EGFR) TKIs (e.g., erlotinib, afatinib, osimertinib) are the current first-line treatment for non-small cell lung cancer (NSCLC) due to their improved therapeutic outcomes for EGFR mutated and overexpressing disease over traditional platinum-based chemotherapy. However, many NSCLC tumors develop resistance to EGFR TKI therapy causing disease progression. Currently, the relationship between in situ drug target availability (DTA), local protein expression and therapeutic response cannot be accurately assessed using existing analytical tools despite being crucial to understanding the mechanism of therapeutic efficacy. Procedure: We have previously reported development of our fluorescence imaging platform termed TRIPODD (Therapeutic Response Imaging through Proteomic and Optical Drug Distribution) that is capable of simultaneous quantification of single-cell DTA and protein expression with preserved spatial context within a tumor. TRIPODD combines two complementary fluorescence imaging techniques: intracellular paired agent imaging (iPAI) to measure DTA and cyclic immunofluorescence (cyCIF), which utilizes oligonucleotide conjugated antibodies (Ab-oligos) for spatial proteomic expression profiling on tissue samples. Herein, TRIPODD was modified and optimized to provide a downstream analysis of therapeutic response through single-cell DTA and proteomic response imaging. Results: We successfully performed sequential imaging of iPAI and cyCIF resulting in high dimensional imaging and biomarker assessment to quantify single-cell DTA and local protein expression on erlotinib treated NSCLC models. Pharmacodynamic and pharmacokinetic studies of the erlotinib iPAI probes revealed that administration of 2.5 mg/kg each of the targeted and untargeted probe 4 h prior to tumor collection enabled calculation of DTA values with high Pearson correlation to EGFR, the erlotinib molecular target, expression in the tumors. Analysis of single-cell biomarker expression revealed that a single erlotinib dose was insufficient to enact a measurable decrease in the EGFR signaling cascade protein expression, where only the DTA metric detected the presence of bound erlotinib. Conclusion: We demonstrated the capability of TRIPODD to evaluate therapeutic response imaging to erlotinib treatment as it relates to signaling inhibition, DTA, proliferation, and apoptosis with preserved spatial context.

Optimization of Zebrafish Larvae 6-OHDA Exposure for Neurotoxin Induced Dopaminergic Marker Reduction.

Parkinson's disease (PD) is a neurodegenerative disorder that is clinically assessed by motor symptoms associated with the loss of midbrain dopaminergic neurons affecting the quality of life for over 8.5 million people worldwide. The neurotoxin 6-hydroxydopamine (6-OHDA) has been used to chemically induce a PD-like state in zebrafish larvae by several laboratories; however, highly variable concentration, methodology, and reagents have resulted in conflicting results suggesting a need to investigate these issues of reproducibility. We propose a protocol that addresses the differences in methodology and induces changes in 6 days postfertilization (dpf) larvae utilizing a 24-h exposure at 3 dpf with 30 µM 6-OHDA. Despite ∼50% lethality, no morphological or development differences in surviving fish are observed. Definition of our model is defined by downregulation of the expression of th1 by reverse transcriptase-quantitative polymerase chain reaction, a marker for dopaminergic neurons and a reduction in movement. Additionally, we observed a downregulation of pink1 and an upregulation of sod1 and sod2, indicators of mitochondrial dysfunction and response to reactive oxygen species, respectively.

Ultra high content analyses of circulating and tumor associated hybrid cells reveal phenotypic heterogeneity.

Persistently high, worldwide mortality from cancer highlights the unresolved challenges of disease surveillance and detection that impact survival. Development of a non-invasive, blood-based biomarker would transform survival from cancer. We demonstrate the functionality of ultra-high content analyses of a newly identified population of tumor cells that are hybrids between neoplastic and immune cells in patient matched tumor and peripheral blood specimens. Using oligonucleotide conjugated antibodies (Ab-oligo) permitting cyclic immunofluorescence (cyCIF), we present analyses of phenotypes among tumor and peripheral blood hybrid cells. Interestingly, the majority of circulating hybrid cell (CHC) subpopulations were not identified in tumor-associated hybrids. These results highlight the efficacy of ultra-high content phenotypic analyses using Ab-oligo based cyCIF applied to both tumor and peripheral blood specimens. The combination of a multiplex phenotypic profiling platform that is gentle enough to analyze blood to detect and evaluate disseminated tumor cells represents a novel approach to exploring novel tumor biology and potential utility for developing the population as a blood-based biomarker in cancer.

Psychiatric admissions in young people after expiration of criminal justice supervision in Australia: a retrospective data linkage study.

BACKGROUND: Mental health services are available for young people involved with the criminal justice system. However, they have unmet mental health needs after the expiration of criminal justice supervision. OBJECTIVE: To determine the incidence rate and identify predictors of psychiatric hospitalisations within 24 months after the expiration of criminal justice supervision among young people involved with the New South Wales (NSW) criminal justice system. METHODS: Retrospective data from 1556 individuals aged 14-22 years who participated in four surveys of justice-involved young people in NSW were harmonised and linked to four NSW data collections. We calculated the incidence rates of psychiatric hospitalisations within 24 months postsupervision and identified predictors of these hospitalisations using a competing risks regression analysis. RESULTS: Within 24 months postsupervision, 11.4% had a psychiatric hospitalisation compared with 3.5% during supervision. 20.7% of those admitted had a known history of mental illness and engaged with community-based and outpatient mental health services postsupervision. Predictors of psychiatric hospitalisations were: female sex (adjusted subdistribution HR (asHR) 1.84, 95% CI 1.24 to 2.73); previous incarceration (highest asHR for ≥4 episodes 1.67, 95% CI 1.01 to 2.78); head injury (asHR 1.63, 95% CI 1.20 to 2.21); personality disorder (asHR 3.66, 95% CI 2.06 to 6.48) and alcohol and substance use disorder (asHR 1.89, 95% CI 1.29 to 2.77). CONCLUSION: Justice-involved youth have higher rates of psychiatric admissions after criminal justice supervision. Engagement with mental health services postsupervision is important in addressing emerging or persisting mental health needs.

Nerve Visualization using Phenoxazine-Based Near-Infrared Fluorophores to Guide Prostatectomy.

Fluorescence-guided surgery (FGS) is poised to revolutionize surgical medicine through near-infrared (NIR) fluorophores for tissue- and disease-specific contrast. Clinical open and laparoscopic FGS vision systems operate nearly exclusively at NIR wavelengths. However, tissue-specific NIR contrast agents compatible with clinically available imaging systems are lacking, leaving nerve tissue identification during prostatectomy a persistent challenge. Here, it is shown that combining drug-like molecular design concepts and fluorophore chemistry enabled the production of a library of NIR phenoxazine-based fluorophores for intraoperative nerve-specific imaging. The lead candidate readily delineated prostatic nerves in the canine and iliac plexus in the swine using the clinical da Vinci Surgical System that has been popularized for minimally invasive prostatectomy procedures. These results demonstrate the feasibility of molecular engineering of NIR nerve-binding fluorophores for ready integration into the existing surgical workflow, paving the path for clinical translation to reduce morbidity from nerve injury for prostate cancer patients.

Detection of neoplastic-immune hybrid cells with metastatic properties in uveal melanoma.

Background: Uveal melanoma is the most common non-cutaneous melanoma and is an intraocular malignancy affecting nearly 7,000 individuals per year worldwide. Of these, approximately 50% will progress to metastatic disease for which there are currently no effective therapies. Despite advances in molecular profiling and metastatic stratification of uveal melanoma tumors, little is known regarding their underlying biology of metastasis. Our group has identified a disseminated neoplastic cell population characterized by co-expression of immune and melanoma proteins, circulating hybrid cells (hybrids), in patients with uveal melanoma. Compared to circulating tumor cells, which lack expression of immune proteins, hybrids are detected at an increased prevalence in peripheral blood and can be used as a non-invasive biomarker to predict metastatic progression. Methods: To ascertain mechanisms underlying enhanced hybrid cell dissemination we identified hybrid cells within primary uveal melanoma tumors using single cell RNA sequencing and evaluated their gene expression and predicted ligand-receptor interactions in relation to other melanoma and immune cells within the primary tumor. We then verified expression of upregulated hybrid pathways within patient-matched tumor and peripheral blood hybrids using cyclic immunofluorescence and quantified their protein expression relative to other non-hybrid tumor and disseminated tumor cells. Results: Among the top upregulated genes and pathways in hybrid cells were those involved in enhanced cell motility and cytoskeletal rearrangement, immune evasion, and altered cellular metabolism. In patient-matched tumor and peripheral blood, we verified gene expression by examining concordant protein expression for each pathway category: TMSB10 (cell motility), CD74 (immune evasion) and GPX1 (metabolism). Both TMSB10 and GPX1 were expressed on significantly higher numbers of disseminated hybrid cells compared to circulating tumor cells, and CD74 and GPX1 were expressed on more disseminated hybrids than tumor-resident hybrids. Lastly, we identified that hybrid cells express ligand-receptor signaling pathways implicated in promoting metastasis including GAS6-AXL, CXCL12-CXCR4, LGALS9-P4HB and IGF1-IGFR1. Conclusion: These findings highlight the importance of TMSB10, GPX1 and CD74 for successful hybrid cell dissemination and survival in circulation. Our results contribute to the understanding of uveal melanoma tumor progression and interactions between tumor cells and immune cells in the tumor microenvironment that may promote metastasis.

Ensuring Indigenous co-leadership in health research: a Can-SOLVE CKD case example.

BACKGROUND: Indigenous people are insightful and informed about their own health and wellness, yet their visions, strengths and knowledge are rarely incorporated into health research. This can lead to subpar engagement or irrelevant research practices, which exacerbates the existing health inequities Indigenous people experience compared to the non-Indigenous population. Data consistently underscores the importance of Indigenous self-determination in research as a means to address health inequities. However, there are few formal methods to support this goal within the existing research context, which is dominated by Western perspectives. MAIN TEXT: Canadians Seeking Solutions and Innovations to Overcome Chronic Kidney Disease (Can-SOLVE CKD) is a patient-oriented research network in Canada that recognizes the need to create the space to facilitate Indigenous self-determination in research. Indigenous members of the network therefore created and evolved a unique group, called the Indigenous Peoples' Engagement and Research Council (IPERC). IPERC plays a critical role in informing Can-SOLVE CKD research priorities, as well as creating tools to support Indigenous-specific research and engagement. This approach ensures that Indigenous voices and knowledge are critical threads within the fabric of the network's operations and research projects. Here, we describe the methods taken to create a council such as IPERC, and provide examples of initiatives by the council that aim to increase Indigenous representation, participation and partnership in research. We share lessons learned on what factors contribute to the success of IPERC, which could be valuable for other organizations interested in creating Indigenous-led research councils. CONCLUSION: Indigenous self-determination in research is critical for addressing health inequities. Here, we present a unique model, led by a council of diverse Indigenous people, which could help reduce health equities and lead to a better era of research for everyone.

Analysis of uveal melanoma scRNA sequencing data identifies neoplastic-immune hybrid cells that exhibit metastatic potential.

Uveal melanoma (UM) is the most common non-cutaneous melanoma and is an intraocular malignancy that affects nearly 7,000 individuals per year worldwide. Of these, nearly 50% will progress to metastatic disease for which there are currently no effective therapies. Despite advances in the molecular profiling and metastatic stratification of class 1 and 2 UM tumors, little is known regarding the underlying biology of UM metastasis. Our group has identified a disseminated tumor cell population characterized by co-expression of immune and melanoma proteins, (circulating hybrid cells (CHCs), in patients with UM. Compared to circulating tumor cells, CHCs are detected at an increased prevalence in peripheral blood and can be used as a non-invasive biomarker to predict metastatic progression. To identify mechanisms underlying enhanced hybrid cell dissemination we sought to identify hybrid cells within a primary UM single cell RNA-seq dataset. Using rigorous doublet discrimination approaches, we identified UM hybrids and evaluated their gene expression, predicted ligand-receptor status, and cell-cell communication state in relation to other melanoma and immune cells within the primary tumor. We identified several genes and pathways upregulated in hybrid cells, including those involved in enhancing cell motility and cytoskeleton rearrangement, evading immune detection, and altering cellular metabolism. In addition, we identified that hybrid cells express ligand-receptor signaling pathways implicated in promoting cancer metastasis including IGF1-IGFR1, GAS6-AXL, LGALS9-P4HB, APP-CD74 and CXCL12-CXCR4. These results contribute to our understanding of tumor progression and interactions between tumor cells and immune cells in the UM microenvironment that may promote metastasis.

Self-reported traumatic brain injury in a sample of impulsive violent offenders: neuropsychiatric correlates and possible "dose effects".

Background: Traumatic brain injury (TBI) is a major public health problem that may be associated with numerous behavioral problems, including impulsivity, aggression and violence. Rates of self-reported TBI are high within offender populations, but the extent to which TBI is causally implicated in causing illegal behavior is unclear. This study examined the psychological and functional correlates of histories of traumatic brain injury in a sample of impulsive violent offenders. Methods: Study participants, all men, had been recruited to participate in a randomized controlled trial of sertraline to reduce recidivism. Study entry criteria were an age of at least 18 years, a documented history of two or more violent offenses and a score of 70 or above on the Barratt Impulsiveness Scale. An extensive list of standardized questionnaires was administered to obtain information on previous TBI and other neuropsychiatric conditions or symptoms. Results: In the sample of 693 men, 66% were aged between 18 and 35 years old, and 55% gave a history of TBI ("TBI+"). Overall, 55% of study participants reported at least one TBI. High levels of neuropsychiatric symptomatology were reported. In 75% of TBI+ individuals, their most severe TBI (by self-report) was associated with loss of consciousness (LOC) < 30 min. Compared to TBI- (those without history of TBI) participants, TBI+ individuals were more impulsive (Eysenck Impulsivity), irritable, angry, and reported higher levels of assaultive behavior, depressive symptomology, alcohol use disorder, suicidal ideation, suicide attempts, and lower quality of life. Potential "dose effects" of TBI severity and frequency in terms of neuropsychiatric symptomatology were identified. Conclusion: Like other studies of offender populations, single and multiple TBIs were very common. The associations of TBI, TBI severity, and TBI frequency (i.e., TBI "burden") with adverse neuropsychiatric phenomena suggest TBI contributes importantly to offender morbidity but the select nature of the sample and cross-sectional study design constrain the interpretation of these findings.

Cumulative incidence of child protection system contacts among a cohort of Western Australian Aboriginal children born 2000 to 2013.

BACKGROUND: Reducing the over-representation of Aboriginal children in the child protection system is a key target for the Australian government. OBJECTIVE: We aimed to provide more recent evidence on the population-level cumulative incidence of contacts for Aboriginal children with child protective services (CPS) in Western Australia (WA). PARTICIPANTS AND SETTING: Linked administrative data was provided for WA CPS between 2000 and 2015 for 33,709 Aboriginal children born in WA between 2000 and 2013. METHODS: Descriptive summaries and cumulative incidence estimates were used to examine changes in CPS contact trends over time and within sibling groups. RESULTS: There was an increase in early-childhood contacts for children born more recently, with 7.6 % and 2.3 % of children born in 2000-2001 having a notification and placement in out-of-home care by age one, respectively, compared to 15.1 % and 4.3 % of children born in 2012-2013. Among sibling groups where at least one sibling had a CPS contact, approximately half of children had their first contacts on the same date as another sibling. For children born after one of their siblings had been placed in out-of-home care, 31.9 % had themselves been placed in out-of-home care by age one. CONCLUSIONS: Multiple children tend to be placed into out-of-home care when at least one sibling is, which is likely to have a significant impact on families affected. The additional risk of placement also carries over to children born after the first removal in a sibling group, highlighting the need for further support to prevent future removals.

Sexually transmissible infections (STI) and HIV testing and diagnosis among Aboriginal and non-Aboriginal adolescents in contact with the Australian justice system: a cross-sectional study.

BACKGROUND: It is unclear what factors are associated with sexually transmissible infections (STI) and HIV testing and diagnosis among justice-involved adolescents, and if these differ for Aboriginal or Torres Strait Islander peoples. METHODS: A cross-sectional survey of 465 justice-involved adolescents (aged 14-17years) from Australia was conducted between 2016 and 2018. Participants were asked about sexual behaviours, STI/HIV knowledge, and prior STI diagnoses and testing. RESULTS: Approximately 38% (n =130) of those sexually active had ever been screened for STI/HIV and 17.8% (n =23) had been diagnosed with an STI. No participant reported living with HIV. For Aboriginal participants, being male (aOR 3.6, 95% CI 1.3-10.1) and having under three sexual partners in the past 12months (aOR 3.1, 95% CI 1.2-8.0) was associated with never having had an STI/HIV test. For non-Aboriginal participants, being male (aOR 2.7, 95%CI 1.2-5.7), single (aOR 2.4, 95% CI 1.2-4.9), attending school (aOR 2.4, 95% CI 1.1-5.1), not having sought sexual health information (aOR 2.8, 95% CI 1.4-5.8), and having a lower STI/HIV knowledge score (aOR 2.3, 95% CI 1.1-5.0) were associated with never having had an STI/HIV test. Factors associated with STI diagnosis were non-heterosexual sexual orientation (aOR 5.6, 95% CI 1.1-28.2), transactional sex (aOR 11.2, 95% CI 3.0-41.3), and having sought sexual health information (aOR 3.5, 95% CI 1.0-12.5). CONCLUSIONS: Males, particularly Aboriginal male adolescents, should be engaged with sexual health promotion and testing services as soon as they come into contact with the justice system. Approaches should consider different cultural, gender and sexual orientations.

Dual probe difference specimen imaging for prostate cancer margin assessment.

Significance: Positive margin status due to incomplete removal of tumor tissue during radical prostatectomy for high-risk localized prostate cancer requires reoperation or adjuvant therapy, which increases morbidity and mortality. Adverse effects of prostate cancer treatments commonly include erectile dysfunction, urinary incontinence, and bowel dysfunction, making successful initial curative prostatectomy imperative. Aim: Current intraoperative tumor margin assessment is largely limited to frozen section analysis, which is a lengthy, labor-intensive process that is obtrusive to the clinical workflow within the operating room (OR). Therefore, a rapid method for prostate cancer margin assessment in the OR could improve outcomes for patients. Approach: Dual probe difference specimen imaging (DDSI), which uses paired antibody-based probes that are labeled with spectrally distinct fluorophores, was shown herein for prostate cancer margin assessment. The paired antibody-based probes consisted of a targeted probe to prostate-specific membrane antigen (PSMA) and an untargeted probe, which were used as a cocktail to stain resected murine tissue specimens including prostate tumor, adipose, muscle, and normal prostate. Ratiometric images (i.e., DDSI) of the difference between targeted and untargeted probe uptake were calculated and evaluated for accuracy using receiver operator characteristic curve analysis with area under the curve values used to evaluate the utility of the DDSI method to detect PSMA positive prostate cancer. Results: Targeted and untargeted probe uptake was similar between the high and low PSMA expressing tumor due to nonspecific probe uptake after topical administration. The ratiometric DDSI approach showed substantial contrast difference between the PSMA positive tumors and their respective normal tissues (prostate, adipose, muscle). Furthermore, DDSI showed substantial contrast difference between the high PSMA expressing tumors and the minimally PSMA expressing tumors due to the ratiometric correction for the nonspecific uptake patterns in resected tissues. Conclusions: Previous work has shown that ratiometic imaging has strong predictive value for breast cancer margin status using topical administration. Translation of the ratiometric DDSI methodology herein from breast to prostate cancers demonstrates it as a robust, ratiometric technique that provides a molecularly specific imaging modality for intraoperative margin detection. Using the validated DDSI protocol on resected prostate cancers permitted rapid and accurate assessment of PSMA status as a surrogate for prostate cancer margin status. Future studies will further evaluate the utility of this technology to quantitatively characterize prostate margin status using PSMA as a biomarker.

Flexible Cyclic Immunofluorescence (cyCIF) Using Oligonucleotide Barcoded Antibodies.

Advances in our understanding of the complex, multifaceted interactions between tumor epithelia, immune infiltrate, and tumor microenvironmental cells have been driven by highly multiplexed imaging technologies. These techniques are capable of labeling many more biomarkers than conventional immunostaining methods. However, multiplexed imaging techniques suffer from low detection sensitivity, cell loss-particularly in fragile samples-, and challenges with antibody labeling. Herein, we developed and optimized an oligonucleotide antibody barcoding strategy for cyclic immunofluorescence (cyCIF) that can be amplified to increase the detection efficiency of low-abundance antigens. Stained fluorescence signals can be readily removed using ultraviolet light treatment, preserving tissue and fragile cell sample integrity. We also extended the oligonucleotide barcoding strategy to secondary antibodies to enable the inclusion of difficult-to-label primary antibodies in a cyCIF panel. Using both the amplification oligonucleotides to label DNA barcoded antibodies and in situ hybridization of multiple fluorescently labeled oligonucleotides resulted in signal amplification and increased signal-to-background ratios. This procedure was optimized through the examination of staining parameters including staining oligonucleotide concentration, staining temperature, and oligonucleotide sequence design, resulting in a robust amplification technique. As a proof-of-concept, we demonstrate the flexibility of our cyCIF strategy by simultaneously imaging with the original oligonucleotide conjugated antibody (Ab-oligo) cyCIF strategy, the novel Ab-oligo cyCIF amplification strategy, as well as direct and indirect immunofluorescence to generate highly multiplexed images.

Perspectives of health service providers in delivering best-practice care for Aboriginal mothers and their babies during the postnatal period.

BACKGROUND: Evidence suggests that Aboriginal babies in Western Australia are not receiving adequate primary health care in their first 3 months of life, leading to questions about enablers and constraints to delivering such care. This paper presents findings from a qualitative research project investigating health providers' perceptions and experiences of best and current practice in discharge planning, postnatal care and health education for Aboriginal mothers and their newborn babies. METHODS: Constructivist grounded theory guided this research involving 58 semi-structured interviews conducted with health providers who deliver care to Aboriginal mothers and infants. Participants were recruited from hospital-based and primary health sites in metropolitan Perth, and regional and remote locations in Western Australia. RESULTS: Structural factors enabling best practice in discharge planning, postnatal care, and health education for mothers included health providers following best practice guidelines and adequate staffing levels. Organisational enablers included continuity of care throughout pregnancy, birth and postnatally. In particular, good communication between services around discharge planning, birth notifications, and training in culturally respectful care. Structural and organisational constraints to delivering best practice and compromising continuity of care were identified as beyond individual control. These included poor communication between different health and social services, insufficient hospital staffing levels leading to early discharge, inadequate cultural training, delayed receipt of birth notifications and discharge summaries received by Aboriginal primary health services. CONCLUSION: Findings highlight the importance of examining current policies and practices to promote best practice in postnatal care to improve health outcomes for mothers and their Aboriginal babies.

Oligonucleotide conjugated antibody strategies for cyclic immunostaining.

A number of highly multiplexed immunostaining and imaging methods have advanced spatial proteomics of cancer for improved treatment strategies. While a variety of methods have been developed, the most widely used methods are limited by harmful signal removal techniques, difficulties with reagent production and antigen sensitivity. Multiplexed immunostaining employing oligonucleotide (oligos)-barcoded antibodies is an alternative approach that is growing in popularity. However, challenges remain in consistent conjugation of oligos to antibodies with maintained antigenicity as well as non-destructive, robust and cost-effective signal removal methods. Herein, a variety of oligo conjugation and signal removal methods were evaluated in the development of a robust oligo conjugated antibody cyclic immunofluorescence (Ab-oligo cyCIF) methodology. Both non- and site-specific conjugation strategies were assessed to label antibodies, where site-specific conjugation resulted in higher retained binding affinity and antigen-specific staining. A variety of fluorescence signal removal methods were also evaluated, where incorporation of a photocleavable link (PCL) resulted in full fluorescence signal removal with minimal tissue disruption. In summary, this work resulted in an optimized Ab-oligo cyCIF platform capable of generating high dimensional images to characterize the spatial proteomics of the hallmarks of cancer.

Sertraline hydrochloride for reducing impulsive behaviour in male, repeat-violent offenders (ReINVEST): protocol for a phase IV, double-blind, placebo-controlled, randomised clinical trial.

INTRODUCTION: Considerable evidence supports an association between poor impulse control (impulsivity) and violent crime. Furthermore, impulsivity and aggression has been associated with reduced levels of serotonergic activity in the brain. Selective serotonin reuptake inhibitors (SSRIs) are a class of anti-depressants that aim to regulate brain serotonin concentrations. Several small studies in psychiatric populations have administered SSRIs to impulsive--aggressive individuals, resulting in reduced impulsivity, anger, aggression and depression. However, no clinical trial has been undertaken in a criminal justice population. This protocol describes the design and implementation of the first systematic study of the potential benefits of SSRIs in impulsive---violent offenders who are at high risk of reoffending. METHODS AND ANALYSIS: A randomised, double-blinded, multicentre trial to test the clinical efficacy of an SSRI, sertraline hydrochloride, compared with placebo on recidivism and behavioural measures (including impulsivity, anger, aggression, depression and self-reported offending) over 12 months. 460 participants with histories of violence and screening positive for impulsivity are recruited at several local courts and correctional service offices in New South Wales, Australia. ETHICS AND DISSEMINATION: Results will be submitted for publication in a peer-reviewed journal. Possible implications of the effectiveness of this pharmacological intervention include economic benefits of reducing prison costs and societal benefits of improving safety. This study has received ethical approval from the University of New South Wales, Aboriginal Health & Medical Research Council, Corrective Services NSW and the NSW Justice Health and Forensic Mental Health Network. TRIAL REGISTRATION NUMBER: ACTRN12613000442707.

Identifying research priorities to improve the health of incarcerated populations: results of citizens' juries in Australian prisons.

Health disparities in incarcerated populations should guide investment in the health care and research of these communities. Although users of health-care services are important in providing input into decisions about research, the voices of people in prison are absent regarding research into their health. In this Health Policy paper, we present priorities for research into the health of people in prison according to people in prison themselves. By use of a deliberative research approach, citizens' juries were conducted in six prisons (three men's and three women's prisons) in Australia. Participants were selected following submissions of expression of interest forms that were distributed within the prisons. Prerecorded information by experts in the health of incarcerated people was shown to participants. Participants deliberated for up to 4 h before agreeing on five research priorities. All citizens' juries endorsed mental health as a number one research priority. Prison health-care services, alcohol and other drug use, education, and infectious diseases were identified as research priorities by most citizens' juries. Focal points within priorities included serious mental illness; grief and trauma; medication management; health-care service access, quality, and resources; drug withdrawal and peer support; prison-based needle and syringe programmes; and health and life skills education. If endeavours in research priority setting are to consider health equity goals, the views of our most health affected citizens need to be included.

TRIPODD: a Novel Fluorescence Imaging Platform for In Situ Quantification of Drug Distribution and Therapeutic Response.

PURPOSE: Personalized medicine has largely failed to produce curative therapies in advanced cancer patients. Evaluation of in situ drug target availability (DTA) concomitant with local protein expression is critical to an accurate assessment of therapeutic efficacy, but tools capable of both are currently lacking. PROCEDURE: We developed and optimized a fluorescence imaging platform termed TRIPODD (Therapeutic Response Imaging through Proteomic and Optical Drug Distribution), resulting in the only methodology capable of simultaneous quantification of single-cell DTA and protein expression with preserved spatial context within a tumor. Using TRIPODD, we demonstrate the feasibility of combining two complementary fluorescence imaging techniques, intracellular paired agent imaging (iPAI) and cyclic immunofluorescence (cyCIF), conducted with oligonucleotide-conjugated antibodies (Ab-oligos) on tissue samples. RESULTS: We successfully performed sequential imaging on a single tissue section of iPAI to capture single-cell DTA and local protein expression heterogeneity using Ab-oligo cyCIF. Fluorescence imaging data acquisition was followed by spatial registration resulting in high dimensional data correlating DTA to protein expression at the single-cell level where uptake of a targeted probe alone was not well correlated to protein expression. CONCLUSION: Herein, we demonstrated the utility of TRIPODD as a powerful imaging platform capable of interpreting tumor heterogeneity for a mechanistic understanding of therapeutic response and resistance through quantification of drug target availability and proteomic response with preserved spatial context at single-cell resolution.

Substance use and help-seeking among justice-involved young people in Queensland and Western Australia: A cross-sectional survey of 14-17-year-olds.

INTRODUCTION: This study investigated substance use and help-seeking among justice-involved young people to inform and improve service provision during and after contact with the justice system. METHODS: Young people (14-17 years) in the community with current or prior contact with the justice system were recruited in Queensland and Western Australia, Australia using purposive sampling between 2016 and 2018. A cross-sectional survey was delivered by computer-assisted telephone interview. Information was collected on sociodemographic and health factors; lifetime and frequency of use of alcohol, tobacco and other drugs; and use of health services related to substance use and mental health. RESULTS: Of the 465 justice-involved young people surveyed, most had used alcohol (89%), tobacco (86%) or other drugs (81%). Of the latter, cannabis use was most prevalent (79%), followed by ecstasy (26%) and amphetamine (22%). Young people engaging in higher risk drug use (daily use, injecting use) were more likely to also have an alcohol use disorder, be disengaged from education, unemployed, have attempted suicide and experienced incarceration. Of the cohort, 24% had received treatment at an alcohol and drug service in the past year and 30% had seen a health professional about emotional/behavioural problems. Males and Aboriginal and Torres Strait Islander young people were less likely to have sought professional help. CONCLUSION: The high levels of substance use and disproportionate levels of help-seeking observed in this study illustrate the importance of delivering tailored, comprehensive and coordinated trauma-informed and culturally safe alcohol and drug services to justice-involved young people.

The sexual behaviours of adolescents aged between 14 and 17 years involved with the juvenile justice system in Australia: A community-based survey.

OBJECTIVES: To overcome key knowledge gaps in relation to justice involved and vulnerable young people and their sexual health and to compare this group with their peers from other youth health surveys in Australia to determine the extent of the issues. METHODS: Young people, aged between 14 and 17 years, who had ever been or were currently involved with the criminal justice system were purposively sampled. The survey was anonymous and delivered using Computer Assisted Telephone Interview (CATI). RESULTS: A total of 465 justice involved MeH-JOSH young people, aged between 14 and 17 years, participated in the study: 44% Aboriginal and/or Torres Strait Islander (Indigenous) and 37% not attending school. Of the total valid responses, 76% (n = 348) reported having ever had sex, with sexual initiation at a median age of 14 years. We compared these data with their peers in other Australian surveys and found that young people in our study had a higher engagement in sex and start having sex at a younger age, reporting more sexual partners at all ages. CONCLUSIONS: The sexual behaviours of young people involved in the justice system in this study suggest they may be at a greater risk for sexually transmissible infections than their age-matched peers in the general population. Policymakers should elevate them to a priority population for targeting sexual health services and health promotion.