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Abstract Background Every year, over 65,000 Australians experience an acute coronary syndrome (ACS) and around one-third occur in people with prior coronary heart disease. Cardiac rehabilitation (CR) aims to prevent a repeat ACS by supporting patients’ return to an active and fulfilling lifestyle. CR programs are efficacious, but audits of clinical practice show variability of program delivery, which may compromise patient outcomes. Core components, quality indicators and accreditation of programs have been introduced internationally to increase program standardisation. With Australian quality indicators (QIs) for cardiac rehabilitation recently introduced, we aimed to conduct a survey in one state of Australia to assess the extent to which programs adhere to the measurement of QIs comparing country, metropolitan, telephone and face to face programs. Methods A cross- sectional survey design with face validity testing was used to formulate questions to evaluate cardiac rehabilitation program and personnel characteristics and QI adherence. Between October 2020- December 2021, 23 cardiac rehabilitation programs across country and metropolitan areas were invited to participate. Quality improvement was defined as adherence to the Australian Quality Indicators, and we developed an objective score to calculate program performance categorised by quartiles. Significance of CR completion and time to enrolment between program type (telephone versus face to face) and location (country versus metropolitan were compared using Pearson’s Chi-square and Mann–Whitney U tests. Results Among the 23 CR programs, 15 were country and 8 metropolitan-based and 22 were face to face and 1 telephone-based. Median wait time from discharge was 27.0 days, (interquartile range 19.3–46.0) across all programs and country completions of enrolled were 76.9% versus metropolitan 56.5%, p

In vitro chemical safety assessment often relies on simple and general cytotoxicity endpoint measurements and fails to adequately predict human toxicity. To improve the in vitro chemical safety assessment, it is important to understand the underlying mechanisms of toxicity. Here we introduce ToxProfiler, a novel human-based reporter assay that quantifies the chemical-induced stress responses at a single-cell level and reveals the toxicological mode-of-action (MoA) of novel drugs and chemicals. The assay accurately measures the activation of seven major cellular stress response pathways (oxidative stress, cell cycle stress, endoplasmic reticulum stress, ion stress, protein stress, autophagy and inflammation) that play a role in the adaptive responses prior to cellular toxicity. To assess the applicability of the assay in predicting the toxicity MoA of chemicals, we tested a set of 100 chemicals with well-known in vitro and in vivo toxicological profiles. Concentration response modeling and point-of-departure estimation for each reporter protein allowed for chemical potency ranking and revealed the primary toxicological MoA of chemicals. Furthermore, the assay could effectively group chemicals based on their shared toxicity signatures and link them to specific toxicological targets, e.g. mitochondrial toxicity and genotoxicity, and different human pathologies, including liver toxicity and cardiotoxicity. Overall, ToxProfiler is a quantitative in vitro reporter assay that can accurately provide insight into the toxicological MoA of compounds, thereby assisting in the future mechanism-based safety assessment of chemicals., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Amer Jamalpoor reports a relationship with Toxys b.v. that includes: employment. Bas ter Braak reports a relationship with Toxys b.v. that includes: employment. Giel Hendriks reports a relationship with Toxys b.v. that includes: employment. Liesanne Loonstra-Wolters reports a relationship with Toxys b.v. that includes: employment. Toxys b.v. holds the exclusive commercial license for ToxProfiler reporter technology and offers the ToxProfiler assay as a service. BtB, GH and AJ are currently employed at Toxys. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: Adolescents presenting with medically unexplained symptoms (MUS) in non-mental healthcare settings, particularly Emergency Departments (EDs), pose diagnostic challenges necessitating a comprehensive bio-psycho-social approach. Amid the youth mental health crisis, recognising psychological distress is imperative. This study delved into physicians' perceptions and diagnostic tendencies regarding such cases, exploring the potential overshadowing of psychosomatic presentations by medicalized diagnoses in EDs., Methods: Our study involved 74 physicians, representing 82% of eligible respondents in the Paediatric Emergency Medicine Department, and was conducted using an online questionnaire examining perceptions of case scenarios with psychosomatic presentations., Results: Results disclosed a prevalent inclination toward medical diagnoses, with less than 10% of physicians considering psychosomatic conditions in specific scenarios. Interestingly, psychosomatic diagnoses were more probable for symptoms like headaches, shortness of breath, and chest pain. The study uncovered a possible bias among physicians towards medical diagnoses in EDs for adolescents with MUS, possibly stemming from physicians' focus on physical care, diagnostic uncertainties, cognitive biases, and concerns about stigmatisation., Conclusion: Adolescents with MUS seeking assistance in non-mental health settings may encounter delayed mental health diagnoses and interventions. Psychosomatic symptoms could signify stressors or underlying mental health disorders. Recognising psychosocial distress early on is crucial for optimal mental health outcomes. Consequently, the study advocates for a paradigm shift towards a holistic bio-psychosocial approach in both medical education and practice., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Non-invasive brain stimulation, such as repetitive transcranial magnetic stimulation (rTMS), is increasingly used in the treatment of neurological diseases and psychiatric disorders. Where rTMS is already an accepted treatment option for depression, in the Netherlands/Belgium, no consensus exist on the application of rTMS for (obsessive-compulsive disorder (OCD). People with OCD who do not respond enough to exposure therapy and serotonergic antidepressants are in great need for treatment alternatives., Aim: Consensus statement on the application of rTMS as treatment for OCD METHOD: Evaluation of the current literature on the efficacy of rTMS for OCD, mainly based on recent meta-analysis, by a broad group of experts in the field of rTMS, OCD and treatment guidelines., Results: rTMS is a potentially effective treatment for OCD with a medium effect size (g ≈ 0,5). Based on the current literature it is not yet clear which rTMS protocol is the most effective., Conclusion: based on the evidence for rTMS in OCD, and the non-invasive nature of the treatment modality, rTMS can considered in cases with OCD who do not respond enough to exposure therapy and serotonergic antidepressants, prior to the start of more invasive treatment alternatives, such as deep brain stimulation or lesion surgery.

The absence of treatment studies for obsessive compulsive disorder (OCD) in older adults and the fact that OCD typically starts at a young age and often follows a chronic, fluctuating course quickly leads to therapeutic nihilism for older adults with OCD. In this case report, we present a 72-year-old man with OCD symptoms from the age of 35, who has only been treated with medication and psychotherapy for a recurrent depressive disorder. After a short, intensive exposure and response prevention treatment (four days in two weeks), the OCD symptoms and the depressive symptoms were fully in remission and all medications (venlafaxine, olanzapine, depakine) were discontinued. Treatment gains were maintained with persistent remission until 18 months follow up. This case report shows that a comorbid depressive disorder may lead to undertreatment of OCD. It also shows that long standing OCD can be successfully treated in older adults.

ToxTracker is a mammalian cell reporter assay that predicts the genotoxic properties of compounds with high accuracy. By evaluating induction of various reporter genes that play a key role in relevant cellular pathways, it provides insight into chemical mode-of-action (MoA), thereby supporting discrimination of direct-acting genotoxicants and cytotoxic chemicals. A comprehensive interlaboratory validation trial was conducted, in which the principles outlined in OECD Guidance Document 34 were followed, with the primary objectives of establishing transferability and reproducibility of the assay and confirming the ability of ToxTracker to correctly classify genotoxic and non-genotoxic compounds. Reproducibility of the assay to predict genotoxic MoA was confirmed across participating laboratories and data were evaluated in terms of concordance with in vivo genotoxicity outcomes. Seven laboratories tested a total of 64 genotoxic and non-genotoxic chemicals that together cover a broad chemical space. The within-laboratory reproducibility (WLR) was up to 98% (73%-98% across participants) and the overall between-laboratory reproducibility (BLR) was 83%. This trial confirmed the accuracy of ToxTracker to predict in vivo genotoxicants with a sensitivity of 84.4% and a specificity of 91.2%. We concluded that ToxTracker is a robust in vitro assay for the accurate prediction of in vivo genotoxicity. Considering ToxTracker's robust standalone accuracy and that it can provide important information on the MoA of chemicals, it is seen as a valuable addition to the regulatory in vitro genotoxicity battery that may even have the potential to replace certain currently used in vitro battery assays., (© 2024 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals LLC on behalf of Environmental Mutagenesis and Genomics Society.)

in vitro screening platforms to assess teratogenic potential of compounds are emerging rapidly. ReproTracker is a human induced pluripotent stem cells (hiPSCs)-based biomarker assay that is shown to identify the teratogenicity potential of new pharmaceuticals and chemicals reliably. In its current state, the assay is limited to identifying the potential teratogenic effects and does not immediately quantify a clinical dose relevant to the exposure of chemicals or drugs observable in mothers or fetuses. The goal of this study was to evaluate whether the ReproTracker assay can be extrapolated in vivo and quantitatively predict developmental toxicity exposure levels of two known human teratogens, thalidomide, and carbamazepine. Here, we utilized Physiologically Based Pharmacokinetic (PBPK) modeling to describe the pharmacokinetic behavior of these compounds and conducted an in vitro to in vivo extrapolation (IVIVE) approach to predict human equivalent effect doses (HEDs) that correspond with in vitro concentrations potentially associated with adverse outcomes in ReproTracker. The HEDs derived from the ReproTracker concentration predicted to cause developmental toxicity were close to the reported teratogenic human clinical doses and the HED derived from the rat or rabbit developmental toxicity study. The ReproTracker derived-HED revealed to be sensitive and protective of humans. Overall, this pilot study demonstrated the importance of integrating PBPK model in extrapolating and assessing developmental toxicity in vitro. The combination of these tools demonstrated that they could improve the safety assessment of drugs and chemicals without animal testing., Competing Interests: Declaration of Competing Interest The authors declared no potential conflicts of interest with respect to the research authorship, and/or publication of this article. Amer Jamalpoor, Sabine Hartvelt, and Giel Hendriks are employed by Toxys, a Dutch company that offers ReproTracker® as a commercial service. Both Marjory Moreau and Amer Jamalpoor contributed equally to this study and have the right to list their name first in their CV., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Anxiety-related disorders are characterized by high levels of avoidance, but experimental research into avoidance learning in patients is scarce. To fill this gap, we compared healthy controls (HC, n = 47) with patients with obsessive-compulsive disorder (OCD, n = 33), panic disorder with agoraphobia (PDA, n = 40), and post-traumatic stress disorder (PTSD, n = 66) in a computer-based avoidance learning task, in order to examine (1) differences in rates of avoidance responses, (2) differences in action-safety learning during avoidance, and (3) differences in subjective relief following successful avoidance. The task comprised aversive negative pictures (unconditional stimulus, US) that followed pictures of two colored lamps (conditional stimuli, CS+), but not a third colored lamp (safety stimulus, CS-), and could be avoided by pressing a button during one CS+ (CS+ avoidable) but not the other (CS+ unavoidable). Participants rated their US-expectancy and level of relief on a trial-by-trial basis. Compared to the HC group, patient groups displayed higher levels of avoidance to the safety stimulus, and higher levels of US-expectancy and relief following the safety and avoidable danger stimulus. We propose that patients with anxiety disorders have low confidence in the safety consequences of avoidance actions, which induces increased relief during US omissions that reinforce the avoidance action., Competing Interests: Conflict of interest All authors declare to have no conflict of interest related to the presented work., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

To determine the utility of the ToxTracker assay in animal alternative testing strategies, the genotoxic potential of four fragrance materials (2-octen-4-one, lauric aldehyde, veratraldehyde, and p-methoxy cinnamaldehyde) were tested in the ToxTracker assay. These materials have been previously evaluated in an in vitro as well as in vivo micronucleus assay, conducted as per OECD guidelines. In addition to these studies, reconstructed human skin micronucleus studies were conducted on all four materials. All four materials were positive in an in vitro micronucleus assay but were negative in both in vivo and 3D skin micronucleus assays. The ToxTracker assay, in combination with in silico methods to predict metabolism was used to identify mechanisms for the misleading positive outcomes observed in the in vitro micronucleus assays. The results show that the ToxTracker assay, in conjunction with in silico predictions, can provide the information needed to aid in the identification of an appropriate animal alternative follow-up assay, for substances with positive results in the standard in vitro test battery. Thus, the ToxTracker assay is a valuable tool to identify the genotoxic potential of fragrance materials and can aid with replacing animal-based follow-up testing with appropriate animal alternative assay(s)., (© 2023 RIFM Inc. Environmental and Molecular Mutagenesis published by Wiley Periodicals LLC on behalf of Environmental Mutagen Society.)

ToxTracker is an in vitro mammalian stem cell-based reporter assay that detects activation of specific cellular signaling pathways (DNA damage, oxidative stress, and/or protein damage) upon chemical exposure using flow cytometry. Here we used quantitative methods to empirically analyze historical control data, and dose-response data across a wide range of reference chemicals. First, we analyzed historical control data to define a fold-change threshold for identification of a significant positive response. Next, we used the benchmark dose (BMD) combined-covariate approach for potency ranking of a set of more than 120 compounds; the BMD values were used for comparative identification of the most potent inducers of each reporter. Lastly, we used principal component analysis (PCA) to investigate functional and statistical relationships between the ToxTracker reporters. The PCA results, based on the BMD results for all substances, indicated that the DNA damage (Rtkn, Bscl2) and p53 (Btg2) reporters are functionally complementary and indicative of genotoxic stress. The oxidative stress (Srxn1 and Blvrb) and protein stress (Ddit3) reporters are independent indicators of cellular stress, and essential for toxicological profiling using the ToxTracker assay. Overall, dose-response modeling of multivariate ToxTracker data can be used for potency ranking and mode-of-action determination. In the future, IVIVE (in vitro to in vivo extrapolation) methods can be employed to determine in vivo AED (administered equivalent dose) values that can in turn be used for human health risk assessment., (© 2023 Environmental Mutagen Society.)

Genotoxicity assessment is a critical component in the development and evaluation of chemicals. Traditional genotoxicity assays (i.e., mutagenicity, clastogenicity, and aneugenicity) have been limited to dichotomous hazard classification, while other toxicity endpoints are assessed through quantitative determination of points-of-departures (PODs) for setting exposure limits. The more recent higher-throughput in vitro genotoxicity assays, many of which also provide mechanistic information, offer a powerful approach for determining defined PODs for potency ranking and risk assessment. In order to obtain relevant human dose context from the in vitro assays, in vitro to in vivo extrapolation (IVIVE) models are required to determine what dose would elicit a concentration in the body demonstrated to be genotoxic using in vitro assays. Previous work has demonstrated that application of IVIVE models to in vitro bioactivity data can provide PODs that are protective of human health, but there has been no evaluation of how these models perform with in vitro genotoxicity data. Thus, the Genetic Toxicology Technical Committee, under the Health and Environmental Sciences Institute, conducted a case study on 31 reference chemicals to evaluate the performance of IVIVE application to genotoxicity data. The results demonstrate that for most chemicals considered here (20/31), the PODs derived from in vitro data and IVIVE are health protective relative to in vivo PODs from animal studies. PODs were also protective by assay target: mutations (8/13 chemicals), micronuclei (9/12), and aneugenicity markers (4/4). It is envisioned that this novel testing strategy could enhance prioritization, rapid screening, and risk assessment of genotoxic chemicals., (© 2022 His Majesty the King in Right of Canada and The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals LLC on behalf of Environmental Mutagen Society. Reproduced with the permission of the Minister of Health Canada. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Background: Testing for developmental toxicity according to the current regulatory guidelines requires large numbers of animals, making these tests very resource intensive, time-consuming, and ethically debatable. Over the past decades, several alternative in vitro assays have been developed, but these often suffered from low predictability and the inability to provide a mechanistic understanding of developmental toxicity., Methods: To identify embryotoxic compounds, we developed a human induced pluripotent stem cells (hiPSCs)-based biomarker assay. The assay is based on the differentiation of hiPSCs into functional cardiomyocytes and hepatocytes. Proper stem cell differentiation is investigated by morphological profiling and assessment of time-dependent expression patterns of cell-specific biomarkers. In this system, a decrease in the expression of the biomarker genes and morphology disruption of the differentiated cells following compound treatment indicated teratogenicity., Results: The hiPSCs-based biomarker assay was validated with 21 well-established in vivo animal teratogenic and non-teratogenic compounds during cardiomyocyte and hepatocyte differentiation. The in vivo teratogenic compounds (e.g., thalidomide and valproic acid) markedly disrupted morphology, functionality, and the expression pattern of the biomarker genes in either one or both cell types. Non-teratogenic chemicals generally had no effect on the morphology of differentiated cells, nor on the expression of the biomarker genes. Compared to the in vivo classification, the assay achieved high accuracy (91%), sensitivity (91%), and specificity (90%)., Conclusion: The assay, which we named ReproTracker®, is a state-of-the-art in vitro method that can identify the teratogenicity potential of new pharmaceuticals and chemicals and signify the outcome of in vivo test systems., (© 2022 Wiley Periodicals LLC.)

It has been reported that incorporation of fire retardants into home furnishings and electronics increases the toxicity of smoke produced during combustion in house fires. Studies have been limited to exercises in analytical chemistry but the biological effects of emissions, particularly regarding chronic toxicity, have not been investigated. The combustion of furnishings with and without chemical flame retardants (FR) regarding (1) ignition resistance and fire progression, (2) chemical composition of smoke (analytical chemistry), and (3) toxicity was compared. Data demonstrated that flame retarded furnishings slowed the generation of toxic levels of acutely toxic gases. The potential chronic toxicity of smoke was assessed using the ToxTracker® assay. Smoke samples from rooms with less flame retarded furnishings exhibited a lesser response in this assay than smoke samples from rooms with flame retarded furnishings. Chemicals associated with activation of the aryl hydrocarbon receptor (AHR), namely benzo[b]fluoranthene, benzo[a]anthracene, benzo[a]pyrene, chrysene, and indeno[1,2,3-cd]pyrene, were not found in smoke from more flame retarded furnished rooms, but were present only in smoke from rooms with less flame retarded furnishings. In conclusion, smoke resulting from combustion of flame retarded furnishings did not increase indicators of potential chronic toxicity hazards relative to non-flame retarded furnishings.

Nucleoside analogues have long been designed and tested in cancer treatment and against viral infections. However, several early compounds were shown to have mutagenic properties as a consequence of their mode-of-action. This limited their use, and several have been discontinued for lengthy treatments or altogether. Nonetheless, nucleoside analogues remain an attractive modality for virally driven diseases, of which many still are without proper treatment options. To quantitatively assess the genotoxic mode-of-action of a panel of nucleoside analogues, we applied the ToxTracker® reporter assay. Many of the early nucleoside analogues showed a genotoxic response. The more recently developed nucleoside analogues, Remdesivir and Molnupiravir that are currently being repurposed for Covid-19 treatment, had a different profile in ToxTracker and did not induce the genotoxicity reporters. Our analyses support the metabolite GS-441524 over the parent analogue Remdesivir. In contrast, Molnupiravir was devoid of clear cellular toxicity while its active metabolite (EIDD-1931) was cytotoxic and induced several biomarkers. Nucleoside analogues continue to be attractive treatment options upon viral infections. ToxTracker readily distinguished between the genotoxic analogues and those with different profiles and provides a basis for clustering and potency ranking, offering a comprehensive tool to assess the toxicity of nucleoside analogues., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Aneuploidy is characterized by the presence of an abnormal number of chromosomes and is a common hallmark of cancer. However, exposure to aneugenic compounds does not necessarily lead to cancer. Aneugenic compounds are mainly identified using the in vitro micronucleus assay but this assay cannot standardly discriminate between aneugens and clastogens and cannot be used to identify the exact mode-of-action (MOA) of aneugens; tubulin stabilization, tubulin destabilization, or inhibition of mitotic kinases. To improve the classification of aneugenic substances and determine their MOA, we developed and validated the TubulinTracker assay that uses a green fluorescent protein-tagged tubulin reporter cell line to study microtubule stability using flow cytometry. Combining the assay with a DNA stain also enables cell cycle analysis. Substances whose exposure resulted in an accumulation of cells in G2/M phase, combined with increased or decreased tubulin levels, were classified as tubulin poisons. All known tubulin poisons included were classified correctly. Moreover, we correctly classified compounds, including aneugens that did not affect microtubule levels. However, the MOA of aneugens not affecting tubulin stability, such as Aurora kinase inhibitors, could not be identified. Here, we show that the TubulinTracker assay can be used to classify microtubule stabilizing and destabilizing compounds in living cells. This insight into the MOA of aneugenic agents is important, eg, to support a weight-of-evidence approach for risk assessment, and the classification as an aneugen as opposed to a clastogen or mutagen, has a big impact on the assessment., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The Genetic Toxicology Technical Committee (GTTC) of the Health and Environmental Sciences Institute (HESI) is developing adverse outcome pathways (AOPs) that describe modes of action leading to potentially heritable genomic damage. The goal was to enhance the use of mechanistic information in genotoxicity assessment by building empirical support for the relationships between relevant molecular initiating events (MIEs) and regulatory endpoints in genetic toxicology. Herein, we present an AOP network that links oxidative DNA damage to two adverse outcomes (AOs): mutations and chromosomal aberrations. We collected empirical evidence from the literature to evaluate the key event relationships between the MIE and the AOs, and assessed the weight of evidence using the modified Bradford-Hill criteria for causality. Oxidative DNA damage is constantly induced and repaired in cells given the ubiquitous presence of reactive oxygen species and free radicals. However, xenobiotic exposures may increase damage above baseline levels through a variety of mechanisms and overwhelm DNA repair and endogenous antioxidant capacity. Unrepaired oxidative DNA base damage can lead to base substitutions during replication and, along with repair intermediates, can also cause DNA strand breaks that can lead to mutations and chromosomal aberrations if not repaired adequately. This AOP network identifies knowledge gaps that could be filled by targeted studies designed to better define the quantitative relationships between key events, which could be leveraged for quantitative chemical safety assessment. We anticipate that this AOP network will provide the building blocks for additional genotoxicity-associated AOPs and aid in designing novel integrated testing approaches for genotoxicity., (© 2022 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals LLC on behalf of Environmental Mutagen Society.)

The introduction of ultrafast ultrasound and spatiotemporal filtering has significantly improved the sensitivity of Doppler ultrasound imaging. This work describes the development of a 3D power Doppler imaging technique which uses a 1D-array ultrasound probe that mechanically translates at a constant speed. The continuous translation allows for a fast scan of a large 3D volume without requiring complex hardware. The technique was realized in a prototype and its feasibility illustrated using phantom and in vivo kidney and breast lesion experiments. Although this 3D Doppler imaging technique is limited in some aspects, it enables power Doppler imaging of a large volume in a short acquisition time with less computational costs.

Cobalt metal and cobalt sulfate are carcinogenic in rodents following inhalation exposure. The pre-carcinogenic effects associated with exposure to these cobalt substances include oxidative stress and genotoxicity. Some, but not all, cobalt substances induce in vitro clastogenicity or an increase in micronuclei. As a result, these substances are classified genotoxic carcinogens, having major impacts on their risk assessment, e.g. assumption of a non-thresholded dose response. Here, we investigated the potential of nine cobalt substances to cause genotoxicity and oxidative stress using the ToxTracker assay, with an extension to measure biomarkers of hypoxia. None of the nine tested substances activated the DNA damage markers in ToxTracker, and five substances activated the oxidative stress response reporters. The same five substances also activated the expression of several hypoxia target genes. Consistent with the lower tier of testing found in the preceding paper of this series, these compounds can be grouped based on their ability to release bioavailable cobalt ion and to trigger subsequent key events., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)