The ATR inhibitor ceralasertib potentiates cancer checkpoint immunotherapy by regulating the tumor microenvironment.

The Ataxia telangiectasia and Rad3-related (ATR) inhibitor ceralasertib in combination with the PD-L1 antibody durvalumab demonstrated encouraging clinical benefit in melanoma and lung cancer patients who progressed on immunotherapy. Here we show that modelling of intermittent ceralasertib treatment in mouse tumor models reveals CD8+ T-cell dependent antitumor activity, which is separate from the effects on tumor cells. Ceralasertib suppresses proliferating CD8+ T-cells on treatment which is rapidly reversed off-treatment. Ceralasertib causes up-regulation of type I interferon (IFNI) pathway in cancer patients and in tumor-bearing mice. IFNI is experimentally found to be a major mediator of antitumor activity of ceralasertib in combination with PD-L1 antibody. Improvement of T-cell function after ceralasertib treatment is linked to changes in myeloid cells in the tumor microenvironment. IFNI also promotes anti-proliferative effects of ceralasertib on tumor cells. Here, we report that broad immunomodulatory changes following intermittent ATR inhibition underpins the clinical therapeutic benefit and indicates its wider impact on antitumor immunity.

Comparative exploration of mammalian deafness gene homologues in the Drosophila auditory organ shows genetic correlation between insect and vertebrate hearing.

Johnston's organ, the Drosophila auditory organ, is anatomically very different from the mammalian organ of Corti. However, recent evidence indicates significant cellular and molecular similarities exist between vertebrate and invertebrate hearing, suggesting that Drosophila may be a useful platform to determine the function of the many mammalian deafness genes whose underlying biological mechanisms are poorly characterized. Our goal was a comprehensive screen of all known orthologues of mammalian deafness genes in the fruit fly to better understand conservation of hearing mechanisms between the insect and the fly and ultimately gain insight into human hereditary deafness. We used bioinformatic comparisons to screen previously reported human and mouse deafness genes and found that 156 of them have orthologues in Drosophila melanogaster. We used fluorescent imaging of T2A-GAL4 gene trap and GFP or YFP fluorescent protein trap lines for 54 of the Drosophila genes and found 38 to be expressed in different cell types in Johnston's organ. We phenotypically characterized the function of strong loss-of-function mutants in three genes expressed in Johnston's organ (Cad99C, Msp-300, and Koi) using a courtship assay and electrophysiological recordings of sound-evoked potentials. Cad99C and Koi were found to have significant courtship defects. However, when we tested these genes for electrophysiological defects in hearing response, we did not see a significant difference suggesting the courtship defects were not caused by hearing deficiencies. Furthermore, we used a UAS/RNAi approach to test the function of seven genes and found two additional genes, CG5921 and Myo10a, that gave a statistically significant delay in courtship but not in sound-evoked potentials. Our results suggest that many mammalian deafness genes have Drosophila homologues expressed in the Johnston's organ, but that their requirement for hearing may not necessarily be the same as in mammals.

Experiences of participating in a group-based sensory modulation intervention for mental health service users.

BACKGROUND: People with mental health issues often experience difficulties with sensory modulation affecting occupational engagement. Research conducted in inpatient units has shown positive effects of individual sensory modulation interventions, however, research on experiences of group-based interventions in outpatient units is limited. Hence, a group-based sensory modulation intervention was adapted and tested within Swedish mental health outpatient units. AIM: To explore the experiences of participating in a group-based sensory modulation intervention for service users in mental health outpatient units. MATERIAL AND METHODS: This qualitative study involved interviews with 25 informants who had participated in the intervention. The interview data were analysed using reflexive thematic analysis. RESULTS: Synthesis of the interviews resulted in one overarching theme of 'Embodied awareness facilitates improved coping and sense of self' organised into four themes: (1) 'Developing embodied awareness and strategies', (2) 'Taking control of everyday life', (3) 'Creating a stronger sense of self', and (4) 'From alienation to belonging'. CONCLUSION AND SIGNIFICANCE: The informants experienced the intervention to provide new embodied coping strategies that had previously been neglected. This understanding may enrich occupational therapy practice in new ways to support service users' engagement in occupations.

The Next-Generation Oral Selective Estrogen Receptor Degrader Camizestrant (AZD9833) Suppresses ER+ Breast Cancer Growth and Overcomes Endocrine and CDK4/6 Inhibitor Resistance.

Oral selective estrogen receptor degraders (SERD) could become the backbone of endocrine therapy (ET) for estrogen receptor-positive (ER+) breast cancer, as they achieve greater inhibition of ER-driven cancers than current ETs and overcome key resistance mechanisms. In this study, we evaluated the preclinical pharmacology and efficacy of the next-generation oral SERD camizestrant (AZD9833) and assessed ER-co-targeting strategies by combining camizestrant with CDK4/6 inhibitors (CDK4/6i) and PI3K/AKT/mTOR-targeted therapy in models of progression on CDK4/6i and/or ET. Camizestrant demonstrated robust and selective ER degradation, modulated ER-regulated gene expression, and induced complete ER antagonism and significant antiproliferation activity in ESR1 wild-type (ESR1wt) and mutant (ESR1m) breast cancer cell lines and patient-derived xenograft (PDX) models. Camizestrant also delivered strong antitumor activity in fulvestrant-resistant ESR1wt and ESR1m PDX models. Evaluation of camizestrant in combination with CDK4/6i (palbociclib or abemaciclib) in CDK4/6-naive and -resistant models, as well as in combination with PI3Kαi (alpelisib), mTORi (everolimus), or AKTi (capivasertib), indicated that camizestrant was active with CDK4/6i or PI3K/AKT/mTORi and that antitumor activity was further increased by the triple combination. The response was observed independently of PI3K pathway mutation status. Overall, camizestrant shows strong and broad antitumor activity in ER+ breast cancer as a monotherapy and when combined with CDK4/6i and PI3K/AKT/mTORi. SIGNIFICANCE: Camizestrant, a next-generation oral SERD, shows promise in preclinical models of ER+ breast cancer alone and in combination with CDK4/6 and PI3K/AKT/mTOR inhibitors to address endocrine resistance, a current barrier to treatment.

Combining the AKT inhibitor capivasertib and SERD fulvestrant is effective in palbociclib-resistant ER+ breast cancer preclinical models.

Combining the selective AKT inhibitor, capivasertib, and SERD, fulvestrant improved PFS in a Phase III clinical trial (CAPItello-291), treating HR+ breast cancer patients following aromatase inhibitors, with or without CDK4/6 inhibitors. However, clinical data suggests CDK4/6 treatment may reduce response to subsequent monotherapy endocrine treatment. To support understanding of trials such as CAPItello-291 and gain insight into this emerging population of patients, we explored how CDK4/6 inhibitor treatment influences ER+ breast tumour cell function and response to fulvestrant and capivasertib after CDK4/6 inhibitor treatment. In RB+, RB- T47D and MCF7 palbociclib-resistant cells ER pathway ER and Greb-1 expression were reduced versus naïve cells. PI3K-AKT pathway activation was also modified in RB+ cells, with capivasertib less effective at reducing pS6 in RB+ cells compared to parental cells. Expression profiling of parental versus palbociclib-resistant cells confirmed capivasertib, fulvestrant and the combination differentially impacted gene expression modulation in resistant cells, with different responses seen in T47D and MCF7 cells. Fulvestrant inhibition of ER-dependent genes was reduced. In resistant cells, the combination was less effective at reducing cell cycle genes, but a consistent reduction in cell fraction in S-phase was observed in naïve and resistant cells. Despite modified signalling responses, both RB+ and RB- resistant cells responded to combination treatment despite some reduction in relative efficacy and was effective in vivo in palbociclib-resistant PDX models. Collectively these findings demonstrate that simultaneous inhibition of AKT and ER signalling can be effective in models representing palbociclib resistance despite changes in pathway dependency.

Assessment of AAV9 distribution and transduction in rats after administration through Intrastriatal, Intracisterna magna and Lumbar Intrathecal routes.

Challenges in obtaining efficient transduction of brain and spinal cord following systemic AAV delivery have led to alternative administration routes being used in clinical trials that directly infuse the virus into the CNS. However, data comparing different direct AAV injections into the brain remain limited making it difficult to choose optimal routes. Here we tested both AAV9-egfp and AAV9-fLuc delivery via intrastriatal (IST), intracisterna magna (ICM) and lumbar intrathecal (LIT) routes in adult rats and assessed vector distribution and transduction in brain, spinal cord and peripheral tissues. We find that IST infusion leads to robust transgene expression in the striatum, thalamus and cortex with lower peripheral tissue transduction and anti-AAV9 capsid titers compared to ICM or LIT. ICM delivery provided strong GFP and luciferase expression across more brain regions than the other routes and similar expression in the spinal cord to LIT injections, which itself largely failed to transduce the rat brain. Our data highlight the strengths and weaknesses of each direct CNS delivery route which will help with future clinical targeting.

Multi-modal molecular imaging maps the correlation between tumor microenvironments and nanomedicine distribution.

Gaining insight into the heterogeneity of nanoparticle drug distribution within tumors would improve both design and clinical translation of nanomedicines. There is little data showing the spatio-temporal behavior of nanomedicines in tissues as current methods are not able to provide a comprehensive view of the nanomedicine distribution, released drug or its effects in the context of a complex tissue microenvironment. Methods: A new experimental approach which integrates the molecular imaging and bioanalytical technologies MSI and IMC was developed to determine the biodistribution of total drug and drug metabolite delivered via PLA-PEG nanoparticles and to overlay this with imaging of the nanomedicine in the context of detailed tumor microenvironment markers. This was used to assess the nanomedicine AZD2811 in animals bearing three different pre-clinical PDX tumors. Results: This new approach delivered new insights into the nanoparticle/drug biodistribution. Mass spectrometry imaging was able to differentiate the tumor distribution of co-dosed deuterated non-nanoparticle-formulated free drug alongside the nanoparticle-formulated drug by directly visualizing both delivery approaches within the same animal or tissue. While the IV delivered free drug was uniformly distributed, the nanomedicine delivered drug was heterogeneous. By staining for multiple biomarkers of the tumor microenvironment on the same tumor sections using imaging mass cytometry, co-registering and integrating data from both imaging modalities it was possible to determine the features in regions with highest nanomedicine distribution. Nanomedicine delivered drug was associated with regions higher in macrophages, as well as more stromal regions of the tumor. Such a comparison of complementary molecular data allows delineation of drug abundance in individual cell types and in stroma. Conclusions: This multi-modal imaging solution offers researchers a better understanding of drug and nanocarrier distribution in complex tissues and enables data-driven drug carrier design.

Test-Retest Reliability of the Isometric Soleus Strength Test in Elite Male Academy Footballers.

BACKGROUND: Currently there is no reliability data available for the isometric soleus strength test (ISST), commonly used as a monitoring tool in elite football settings. Isometric strength testing for other muscle groups, most notably the hamstrings, is utilized to identify injury risk and readiness to train/play. To profile athletes efficiently, performance practitioners require optimal measures that are reliable. The aim of this study was to investigate the test-retest reliability of the isometric strength test of the soleus and propose a standardized protocol for its use within an elite male football population. STUDY DESIGN: Test-retest reliability single cohort study. METHODS: Thirty elite male footballers (age = 22.8±5.0 years, height = 180.0±0.08 cm, weight = 70.57±4.0 kg) performed the ISST, through three maximum 3-second hold efforts with one minute rest between repetitions and 48 hours between tests, in each test. The test was performed mid-competitive season. All data bilaterally were checked for normality using the Shapiro-Wilk test before a Pearson's Correlations and Bland-Altman's analyses were performed. RESULTS: Test-retest reliability demonstrated high reliability for ISST bilaterally (Right: r = 0.89; Left: r = 0.79, p<0.05). The standard error of measurement (SEM) (%) was 9.09 - 12.47% and minimal detectable change (MDC) was 25.19 - 34.56 (N) for Peak Force (PF) measures of the ISST. Bilateral levels of agreement were found to be +/- 2 standard deviations (SD) of the interval of agreement bilaterally for ISST (Levels of agreement (LOA): Right: Upper 352.49 - Lower -494.76; Left: Upper 523.82 - Lower -591.30. Bilaterally no significant difference was detected between values (Right: p=0.09, CI: -153.21-10.95; Left: p=0.52, CI: -139.81-72.33). CONCLUSION: The results of this study demonstrate high reliability for the ISST. The ISST displays a high test-retest reliability for assessing PF characteristics of the soleus in elite male academy footballers. This test may be beneficial for performance practitioners for profiling soleus function of athletes.

Method To Visualize the Intratumor Distribution and Impact of Gemcitabine in Pancreatic Ductal Adenocarcinoma by Multimodal Imaging.

Gemcitabine (dFdC) is a common treatment for pancreatic cancer; however, it is thought that treatment may fail because tumor stroma prevents drug distribution to tumor cells. Gemcitabine is a pro-drug with active metabolites generated intracellularly; therefore, visualizing the distribution of parent drug as well as its metabolites is important. A multimodal imaging approach was developed using spatially coregistered mass spectrometry imaging (MSI), imaging mass cytometry (IMC), multiplex immunofluorescence microscopy (mIF), and hematoxylin and eosin (H&E) staining to assess the local distribution and metabolism of gemcitabine in tumors from a genetically engineered mouse model of pancreatic cancer (KPC) allowing for comparisons between effects in the tumor tissue and its microenvironment. Mass spectrometry imaging (MSI) enabled the visualization of the distribution of gemcitabine (100 mg/kg), its phosphorylated metabolites dFdCMP, dFdCDP and dFdCTP, and the inactive metabolite dFdU. Distribution was compared to small-molecule ATR inhibitor AZD6738 (25 mg/kg), which was codosed. Gemcitabine metabolites showed heterogeneous distribution within the tumor, which was different from the parent compound. The highest abundance of dFdCMP, dFdCDP, and dFdCTP correlated with distribution of endogenous AMP, ADP, and ATP in viable tumor cell regions, showing that gemcitabine active metabolites are reaching the tumor cell compartment, while AZD6738 was located to nonviable tumor regions. The method revealed that the generation of active, phosphorylated dFdC metabolites as well as treatment-induced DNA damage primarily correlated with sites of high proliferation in KPC PDAC tumor tissue, rather than sites of high parent drug abundance.

A Suture Anchor-Based Repair Plus Reconstruction Using Acellular Human Dermal Allograft for Recurrent Sports-Related Patellar Tendon Rupture.

Recurrent traumatic patellar tendon rupture following early repair of a primary rupture is exceedingly rare; there is little technical literature on how to manage this potentially devastating injury. We describe here a suture anchor-based technique for revision repair augmented with an extensor reconstruction using acellular human dermal allograft.

The experience and meaning of recovery-oriented practice for nurses working in acute mental health services.

Since the 1990s, New Zealand mental health policy has shifted from a focus on the management of symptoms and risk to the recovery of psychological, social, cultural, and physical well-being. Despite a vision for recovery-oriented services being integrated within national and regional policies, there is growing concern that barriers to recovery-oriented service provision continue to exist. Such barriers include the attitudes, skills, and knowledge of front-line staff, as well as system issues. This study explored the experience and meaning of recovery-oriented practice for 10 nurses working in an acute inpatient mental health service. A phenomenological and hermeneutic lens was used to explore the nurses' experience of working in a recovery-focused manner alongside service users. Stories of practice were collected from participants through open-ended conversational interviews. Transcribed narratives were analysed to explore taken-for-granted aspects of working in acute mental health care and to uncover the meaning of being recovery-oriented in this setting. Findings revealed that although the experience and meaning of recovery-focused care varied among nurses, there were common elements in the practice accounts. The accounts highlighted the nurses' role in creating different therapeutic spaces to promote safety, relational commitment, and healing for service users. However, the nurses faced challenges to recovery-oriented care within the team hierarchical culture and the broader service systems. The nurses were, at times, fearless in advocating for service users and recognized that this was essential for developing recovery-focused services. The findings have implications for nursing practice, as well as training and service development.

Deep Learning-Based Annotation Transfer between Molecular Imaging Modalities: An Automated Workflow for Multimodal Data Integration.

An ever-increasing array of imaging technologies are being used in the study of complex biological samples, each of which provides complementary, occasionally overlapping information at different length scales and spatial resolutions. It is important to understand the information provided by one technique in the context of the other to achieve a more holistic overview of such complex samples. One way to achieve this is to use annotations from one modality to investigate additional modalities. For microscopy-based techniques, these annotations could be manually generated using digital pathology software or automatically generated by machine learning (including deep learning) methods. Here, we present a generic method for using annotations from one microscopy modality to extract information from complementary modalities. We also present a fast, general, multimodal registration workflow [evaluated on multiple mass spectrometry imaging (MSI) modalities, matrix-assisted laser desorption/ionization, desorption electrospray ionization, and rapid evaporative ionization mass spectrometry] for automatic alignment of complex data sets, demonstrating an order of magnitude speed-up compared to previously published work. To demonstrate the power of the annotation transfer and multimodal registration workflows, we combine MSI, histological staining (such as hematoxylin and eosin), and deep learning (automatic annotation of histology images) to investigate a pancreatic cancer mouse model. Neoplastic pancreatic tissue regions, which were histologically indistinguishable from one another, were observed to be metabolically different. We demonstrate the use of the proposed methods to better understand tumor heterogeneity and the tumor microenvironment by transferring machine learning results freely between the two modalities.

Method to Investigate the Distribution of Water-Soluble Drug-Delivery Systems in Fresh Frozen Tissues Using Imaging Mass Cytometry.

Imaging mass cytometry (IMC) offers the opportunity to image metal- and heavy halogen-containing xenobiotics in a highly multiplexed experiment with other immunochemistry-based reagents to distinguish uptake into different tissue structures or cell types. However, in practice, many xenobiotics are not amenable to this analysis, as any compound which is not bound to the tissue matrix will delocalize during aqueous sample-processing steps required for IMC analysis. Here, we present a strategy to perform IMC experiments on a water-soluble polysarcosine-modified dendrimer drug-delivery system (S-Dends). This strategy involves two consecutive imaging acquisitions on the same tissue section using the same instrumental platform, an initial laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MSI) experiment followed by tissue staining and a standard IMC experiment. We demonstrated that settings can be found for the initial ablation step that leave sufficient residual tissue for subsequent antibody staining and visualization. This workflow results in lateral resolution for the S-Dends of 2 µm followed by imaging of metal-tagged antibodies at 1 µm.

Experiences of sensory input in daily occupations for people with serious mental illness.

BACKGROUND: There is growing evidence that people with serious mental illness have impaired capacity for processing sensory inputs which affects daily occupation. Although this is known, research regarding the target groups experiences of sensory inputs in daily occupations is lacking. AIM: To investigate the experience of sensory input and strategies used in daily occupations among people with serious mental illness. MATERIAL AND METHODS: Fourteen people with serious mental illness were interviewed regarding their experiences of sensory processing and strategies for managing sensory inputs in daily occupations. Data were analysed using content analysis. RESULTS: Sensory processing issues affected occupational engagement and strategies to control inputs were intuitively used to cope with sensory challenges. Informants either ignored, reduced or avoided sensory inputs. Informants also enabled daily life through strategies such as creating a home that provides rest, finding a safe place, using nature and animals for relaxing and using effects of calming and alerting occupations. DISCUSSION: Specific sensory inputs were difficult to process, which was experienced as stressful and affected occupations negatively. The results imply a need for further research exploring the management of sensory input and the use of sensory modulation approaches to enable engagement in daily activities.

30-Day Outcomes of Operative Versus Nonoperative Management for Humeral Diaphyseal Fractures in Patients with Concomitant Hip Fractures.

Every year over 330,000 people are hospitalized for hip fractures in the US. Of those patients, approximately 4-5% of patients with hip fractures present with a concomitant upper extremity fracture. Upper extremity (UE) fractures account for an estimated 2 million fractures a year. The number of hip fractures is projected to double by 2050. There is evidence that the presence of a concomitant upper extremity fracture results in an increased hospital length of stay (LOS), lower functional capacity upon discharge and higher mortality rates than seen with hip fractures in isolation. Additionally, hip fractures pose a significant economic burden and are expected to increase to over $62 billion in 2040. As such, hip fractures are a public health crisis. This study seeks to evaluate the differences in perioperative outcomes between nonoperative and operative management of humeral diaphyseal fractures in patients with concomitant hip fractures. (Journal of Surgical Orthopaedic Advances 29(2):99-102, 2020).

Empowerment, self and engagement in day center occupations: A longitudinal study among people with long-term mental illness.

BACKGROUND: Day centers are a common form of mental health service internationally. They are aimed at enhancing occupational engagement and social relations, but there is a need to clarify the outcomes of day center programs, including the impact on important aspects of recovery such as empowerment. AIMS: The aim of this study was to explore whether perceived empowerment changed over time among Swedish day center users and whether self-esteem, quality of life, socio-demographic data and self-reported diagnosis, type of program and level of engagement in day center occupations could predict future empowerment. METHOD: The study involved a re-analysis of longitudinal data from 14 day centers, where measures of perceived empowerment and other individual factors were collected over a 15-month period. Non-parametric statistics were used, including Wilcoxon's signed-rank test and logistic regression analysis. RESULTS: There were no significant changes in day center attendee empowerment scores. Self-esteem and level of engagement in day center occupations were found to be predictors of empowerment, together explaining 34% of the variation. CONCLUSIONS: Developing empowerment in the day center context involves a complex interaction of individual, social and material factors. Potential barriers to empowerment are discussed along with considerations related to measuring empowerment as an outcome of day center programs.

Harnessing molecular motors for nanoscale pulldown in live cells.

Protein-protein interactions (PPIs) regulate assembly of macromolecular complexes, yet remain challenging to study within the native cytoplasm where they normally exert their biological effect. Here we miniaturize the concept of affinity pulldown, a gold-standard in vitro PPI interrogation technique, to perform nanoscale pulldowns (NanoSPDs) within living cells. NanoSPD hijacks the normal process of intracellular trafficking by myosin motors to forcibly pull fluorescently tagged protein complexes along filopodial actin filaments. Using dual-color total internal reflection fluorescence microscopy, we demonstrate complex formation by showing that bait and prey molecules are simultaneously trafficked and actively concentrated into a nanoscopic volume at the tips of filopodia. The resulting molecular traffic jams at filopodial tips amplify fluorescence intensities and allow PPIs to be interrogated using standard epifluorescence microscopy. A rigorous quantification framework and software tool are provided to statistically evaluate NanoSPD data sets. We demonstrate the capabilities of NanoSPD for a range of nuclear and cytoplasmic PPIs implicated in human deafness, in addition to dissecting these interactions using domain mapping and mutagenesis experiments. The NanoSPD methodology is extensible for use with other fluorescent molecules, in addition to proteins, and the platform can be easily scaled for high-throughput applications.