PAX Genes in Cardiovascular Development.

The mammalian heart is a four-chambered organ with systemic and pulmonary circulations to deliver oxygenated blood to the body, and a tightly regulated genetic network exists to shape normal development of the heart and its associated major arteries. A key process during cardiovascular morphogenesis is the septation of the outflow tract which initially forms as a single vessel before separating into the aorta and pulmonary trunk. The outflow tract connects to the aortic arch arteries which are derived from the pharyngeal arch arteries. Congenital heart defects are a major cause of death and morbidity and are frequently associated with a failure to deliver oxygenated blood to the body. The Pax transcription factor family is characterised through their highly conserved paired box and DNA binding domains and are crucial in organogenesis, regulating the development of a wide range of cells, organs and tissues including the cardiovascular system. Studies altering the expression of these genes in murine models, notably Pax3 and Pax9, have found a range of cardiovascular patterning abnormalities such as interruption of the aortic arch and common arterial trunk. This suggests that these Pax genes play a crucial role in the regulatory networks governing cardiovascular development.

Inhibition of CDK9 activity compromises global splicing in prostate cancer cells.

Cyclin-dependent kinase 9 (CDK9) phosphorylates RNA polymerase II to promote productive transcription elongation. Here we show that short-term CDK9 inhibition affects the splicing of thousands of mRNAs. CDK9 inhibition impairs global splicing and there is no evidence for a coordinated response between the alternative splicing and the overall transcriptome. Alternative splicing is a feature of aggressive prostate cancer (CRPC) and enables the generation of the anti-androgen resistant version of the ligand-independent androgen receptor, AR-v7. We show that CDK9 inhibition results in the loss of AR and AR-v7 expression due to the defects in splicing, which sensitizes CRPC cells to androgen deprivation. Finally, we demonstrate that CDK9 expression increases as PC cells develop CRPC-phenotype both in vitro and also in patient samples. To conclude, here we show that CDK9 inhibition compromises splicing in PC cells, which can be capitalized on by targeting the PC-specific addiction androgen receptor.

Virtual Daily Rounding for COVID-19 Facility Outbreaks: A Standardized Telehealth-Centered Approach May Reduce Hospital Transfers and Mortality.

Background: Residents of nursing homes are among the most vulnerable to COVID-19. There is no standardized approach for integration with hospitals for outbreak response. Previously, we described collaboration between a hospital and regional facilities. As a component of our COVID-19 Outbreak Response, we describe the impact of virtual daily rounds as an efficient and effective tool for facility outbreak in Central Virginia. Methods: At this facility, 82 (60%) residents were infected. Our team rapidly deployed technology to support staff at the facility. Virtual daily rounds created a systematic approach for patient care. The goals of virtual daily rounds include (1) efficiently facilitating HIPAA-compliant communication between nursing and all licensed independent providers, (2) rapid identification of clinical decline, (3) facilitation of care escalation, (4) facilitating bidirectional transfers, and (5) rapid and efficient identification of patients appropriate for telemedicine pulmonary consultation. Results: The outbreak remained active 6 weeks; 82 of 136 (60%) residents were infected, and 36 (44%) COVID-19 positive residents were seen by telemedicine consultation. Fifty-seven (70%) residents remained in-facility for treatment. Twenty-one residents died (15%); 10 in facility, 11 in hospital. Of those seen in telemedicine consultation, 24 (69%) remained on the treat-in-place protocol with goal-concordant care. These hospitalization and mortality rates are significantly lower than similar outbreaks reported. Discussion and Conclusion: We have since instituted this system at seven other facilities. A model of virtual daily rounding holds promise for decreasing mortality/hospitalization in this vulnerable population through systematically identifying patients most appropriate for telemedicine and cultivating close collaboration between hospitals and nursing homes.

Rapid Telehealth-Centered Response to COVID-19 Outbreaks in Postacute and Long-Term Care Facilities.

Purpose: The vulnerability of postacute and long-term care (PA/LTC) facility residents to COVID-19 has manifested across the world with increasing facility outbreaks associated with high hospitalization and mortality rates. Systematic protocols to guide telehealth-centered interventions in response to COVID-19 outbreaks have yet to be delineated. This article is intended to inform PA/LTC facilities and neighboring health care partners how to collaboratively utilize telehealth-centered strategies to improve outcomes in facility outbreaks. Methods: The University of Virginia rapidly developed a multidisciplinary telehealth-centered COVID-19 facility outbreak strategy in response to a LTC facility outbreak in which 41 (out of 48) facility residents and 7 staff members tested positive. This strategy focused on supporting the facility team remotely using rapidly deployed technologic solutions. Goals included (1) early identification of patients who need their care escalated, (2) monitoring and treating patients deemed safe to remain in the facility, (3) care coordination to facilitate bidirectional transfers between the skilled nursing facility (SNF) and hospital, and (4) daily facility needs assessment related to technology, infection control, and staff well-being. To achieve these goals, a standardized approach centered on daily multidisciplinary virtual rounds and telemedicine consultation was provided. Results: Over a month since the outbreak began, 18 out of 48 (38%) facility residents required hospitalization and 6 (12.5%) died. Eleven facility residents have since returned back to the SNF after recovering from their hospitalization. No staff required hospitalization. Conclusions: Interventions that reduce hospitalizations and mortality are a critical need during the COVID-19 pandemic. The mortality and hospitalization rates seen in this PA/LTC facility outbreak are significantly lower than has been documented in other facility outbreaks. Our multidisciplinary approach centered on telemedicine should be considered as other PA/LTC facilities partner with neighboring health care systems in responding to COVID-19 outbreaks. We have begun replicating these services to additional PA/LTC facilities facing COVID-19 outbreaks.

New applications and perspectives of fast field cycling NMR relaxometry.

The field cycling NMR relaxometry method (also known as fast field cycling (FFC) when instruments employing fast electrical switching of the magnetic field are used) allows determination of the spin-lattice relaxation time (T1 ) continuously over five decades of Larmor frequency. The method can be exploited to observe the T1 frequency dependence of protons, as well as any other NMR-sensitive nuclei, such as (2) H, (13) C, (31) P, and (19) F in a wide range of substances and materials. The information obtained is directly correlated with the physical/chemical properties of the compound and can be represented as a 'nuclear magnetic resonance dispersion' curve. We present some recent academic and industrial applications showing the relevance of exploiting FFC NMR relaxometry in complex materials to study the molecular dynamics or, simply, for fingerprinting or quality control purposes. The basic nuclear magnetic resonance dispersion features are outlined in representative examples of magnetic resonance imaging (MRI) contrast agents, porous media, proteins, and food stuffs. We will focus on the new directions and perspectives for the FFC technique. For instance, the introduction of the latest Wide Bore FFC NMR relaxometers allows probing, for the first time, of the dynamics of confined surface water contained in the macro-pores of carbonate rock cores. We also evidence the use of the latest field cycling technology with a new cryogen-free variable-field electromagnet, which enhances the range of available frequencies in the 2D T1 -T2 correlation spectrum for separating oil and water in crude oil. Copyright © 2015 John Wiley & Sons, Ltd.

Modulating the unfolded protein response with ONC201 to impact on radiation response in prostate cancer cells.

Prostate cancer (PCa) is the most common non-cutaneous cancer in men and a notable cause of cancer mortality when it metastasises. The unfolded protein response (UPR) can be cytoprotective but when acutely activated can lead to cell death. In this study, we sought to enhance the acute activation of the UPR using radiation and ONC201, an UPR activator. Treating PCa cells with ONC201 quickly increased the expression of all the key regulators of the UPR and reduced the oxidative phosphorylation, with cell death occurring 72 h later. We exploited this time lag to sensitize prostate cancer cells to radiation through short-term treatment with ONC201. To understand how priming occurred, we performed RNA-Seq analysis and found that ONC201 suppressed the expression of cell cycle and DNA repair factors. In conclusion, we have shown that ONC201 can prime enhanced radiation response.

Benign Schwannoma of the Lateral Plantar Nerve: A Case Report.

Schwannomas are benign, encapsulated soft-tissue tumors that rarely present to the foot and ankle. These tumors are usually asymptomatic unless an increase in size or disruption of the nerve causes pain. We report a case of a painful mass along the lateral plantar nerve near the fourth metatarsal base that was surgically excised and confirmed as a benign schwannoma by means of histopathologic analysis. At the final follow-up of over 2 years, the patient reported no pain, neurologic deficits, or signs of recurrence. This case demonstrates an unusual location of a schwannoma arising from the lateral plantar nerve.

COVID-19 Collaborative Model for an Academic Hospital and Long-Term Care Facilities.

The COVID-19 pandemic is devastating post-acute and long-term care (PA/LTC). As geriatricians practicing in PA/LTC and a regional academic medical center, we created this program for collaboration between academic medical centers and regional PA/LTC facilities. The mission of the Geriatric Engagement and Resource Integration in Post-Acute and Long-Term Care Facilities (GERI-PaL) program is to support optimal care of residents in PA/LTC facilities during the COVID-19 pandemic. There are 5 main components of our program: (1) Project ECHO; (2) nursing liaisons; (3) infection advisory consultation; (4) telemedicine consultation; and (5) resident social contact remote connections. Implementation of this program has had positive response from our local PA/LTC facilities. A key component of our program is our interprofessional team, which includes physicians and nursing, emergency response, and public health experts. With diverse professional backgrounds, our team members have created a new model for academic medical centers to collaborate with local PA/LTC facilities.

Inhibition of O-GlcNAc Transferase Renders Prostate Cancer Cells Dependent on CDK9.

O-GlcNAc transferase (OGT) is a nutrient-sensitive glycosyltransferase that is overexpressed in prostate cancer, the most common cancer in males. We recently developed a specific and potent inhibitor targeting this enzyme, and here, we report a synthetic lethality screen using this compound. Our screen identified pan-cyclin-dependent kinase (CDK) inhibitor AT7519 as lethal in combination with OGT inhibition. Follow-up chemical and genetic approaches identified CDK9 as the major target for synthetic lethality with OGT inhibition in prostate cancer cells. OGT expression is regulated through retention of the fourth intron in the gene and CDK9 inhibition blunted this regulatory mechanism. CDK9 phosphorylates carboxy-terminal domain (CTD) of RNA Polymerase II to promote transcription elongation. We show that OGT inhibition augments effects of CDK9 inhibitors on CTD phosphorylation and general transcription. Finally, the combined inhibition of both OGT and CDK9 blocked growth of organoids derived from patients with metastatic prostate cancer, but had minimal effects on normal prostate spheroids. We report a novel synthetic lethal interaction between inhibitors of OGT and CDK9 that specifically kills prostate cancer cells, but not normal cells. Our study highlights the potential of combining OGT inhibitors with other treatments to exploit cancer-specific vulnerabilities. IMPLICATIONS: The primary contribution of OGT to cell proliferation is unknown, and in this study, we used a compound screen to indicate that OGT and CDK9 collaborate to sustain a cancer cell-specific pro-proliferative program. A better understanding of how OGT and CDK9 cross-talk will refine our understanding of this novel synthetic lethal interaction.

Investigating Radiotherapy Response in a Novel Syngeneic Model of Prostate Cancer.

The prostate cancer (PCa) field lacks clinically relevant, syngeneic mouse models which retain the tumour microenvironment observed in PCa patients. This study establishes a cell line from prostate tumour tissue derived from the Pten-/-/trp53-/- mouse, termed DVL3 which when subcutaneously implanted in immunocompetent C57BL/6 mice, forms tumours with distinct glandular morphology, strong cytokeratin 8 and androgen receptor expression, recapitulating high-risk localised human PCa. Compared to the commonly used TRAMP C1 model, generated with SV40 large T-antigen, DVL3 tumours are immunologically cold, with a lower proportion of CD8+ T-cells, and high proportion of immunosuppressive myeloid derived suppressor cells (MDSCs), thus resembling high-risk PCa. Furthermore, DVL3 tumours are responsive to fractionated RT, a standard treatment for localised and metastatic PCa, compared to the TRAMP C1 model. RNA-sequencing of irradiated DVL3 tumours identified upregulation of type-1 interferon and STING pathways, as well as transcripts associated with MDSCs. Upregulation of STING expression in tumour epithelium and the recruitment of MDSCs following irradiation was confirmed by immunohistochemistry. The DVL3 syngeneic model represents substantial progress in preclinical PCa modelling, displaying pathological, micro-environmental and treatment responses observed in molecular high-risk disease. Our study supports using this model for development and validation of treatments targeting PCa, especially novel immune therapeutic agents.

Propagation of human prostate tissue from induced pluripotent stem cells.

Primary culture of human prostate organoids and patient-derived xenografts is inefficient and has limited access to clinical tissues. This hampers their use for translational study to identify new treatments. To overcome this, we established a complementary approach where rapidly proliferating and easily handled induced pluripotent stem cells enabled the generation of human prostate tissue in vivo and in vitro. By using a coculture technique with inductive urogenital sinus mesenchyme, we comprehensively recapitulated in situ 3D prostate histology, and overcame limitations in the primary culture of human prostate stem, luminal and neuroendocrine cells, as well as the stromal microenvironment. This model now unlocks new opportunities to undertake translational studies of benign and malignant prostate disease.

Nitric oxide-donating carbonic anhydrase inhibitors for the treatment of open-angle glaucoma.

Novel bi-functional compounds with a nitric oxide (NO)-releasing moiety bound to a dorzolamide scaffold were investigated. Several compounds were synthesized and their activity as selective carbonic anhydrase inhibitors (CAI) evaluated in vitro on recombinant hCA type I, II and IV enzyme isoforms where they showed different degrees of potency and selectivity to hCA II. A high resolution X-ray crystal structure for the CA II adduct with 8 confirmed the high affinity of this class of compounds for the enzyme. Compounds 4, 6, and 8 showed highly potent and efficacious NO-mediated properties as assessed by their vascular relaxant effect on methoxamine-precontracted rabbit aortic rings. Finally, compounds 4 and 6 exerted potent intraocular pressure (IOP) lowering effects in vivo in normotensive rabbits thereby anticipating their potential for the treatment of hypertensive glaucoma.

The impact of transcription on metabolism in prostate and breast cancers.

Metabolic dysregulation is regarded as an important driver in cancer development and progression. The impact of transcriptional changes on metabolism has been intensively studied in hormone-dependent cancers, and in particular, in prostate and breast cancer. These cancers have strong similarities in the function of important transcriptional drivers, such as the oestrogen and androgen receptors, at the level of dietary risk and epidemiology, genetics and therapeutically. In this review, we will focus on the function of these nuclear hormone receptors and their downstream impact on metabolism, with a particular focus on lipid metabolism. We go on to discuss how lipid metabolism remains dysregulated as the cancers progress. We conclude by discussing the opportunities that this presents for drug repurposing, imaging and the development and testing of new therapeutics and treatment combinations.

Increasing radiology capacity within the lung cancer pathway: centralised work-based support for trainee chest X-ray reporting radiographers.

INTRODUCTION: Diagnostic capacity and time to diagnosis are frequently identified as a barrier to improving cancer patient outcomes. Maximising the contribution of the medical imaging workforce, including reporting radiographers, is one way to improve service delivery. METHODS: An efficient and effective centralised model of workplace training support was designed for a cohort of trainee chest X-ray (CXR) reporting radiographers. A comprehensive schedule of tutorials was planned and aligned with the curriculum of a post-graduate certificate in CXR reporting. Trainees were supported via a hub and spoke model (centralised training model), with the majority of education provided by a core group of experienced CXR reporting radiographers. Trainee and departmental feedback on the model was obtained using an online survey. RESULTS: Fourteen trainees were recruited from eight National Health Service Trusts across London. Significant efficiencies of scale were possible with centralised support (48 h) compared to traditional workplace support (348 h). Trainee and manager feedback overall was positive. Trainees and managers both reported good trainee support, translation of learning to practice and increased confidence. Logistics, including trainee travel and release, were identified as areas for improvement. CONCLUSION: Centralised workplace training support is an effective and efficient method to create sustainable diagnostic capacity and support improvements in the lung cancer pathway.

Enabling 1536-Well High-Throughput Cell-Based Screening through the Application of Novel Centrifugal Plate Washing.

Cell-based assays have long been important within hit discovery paradigms; however, improving the disease relevance of the assay system can positively affect the translation of small-molecule drug discovery, especially if adopted in the initial hit identification assay. Consequently, there is an increasing need for disease-relevant assay systems capable of running at large scale, including the use of induced pluripotent stem cells and donor-derived primary cells. Major hurdles to adopting these assays for high-throughput screening are the cost, availability of cells, and complex protocols. Miniaturization of such assays to 1536-well format is an approach that can reduce costs and increase throughput. Adaptation of these complex cell assays to 1536-well format brings major challenges in liquid handling for high-content assays requiring washing steps and coating of plates. In addition, problematic edge effects and reduced assay quality are frequently encountered. In this study, we describe the novel application of a centrifugal plate washer to facilitate miniaturization of a range of 1536-well cell assays and techniques to reduce edge effects, all of which improved throughput and data quality. Cell assays currently limited in throughput because of cost and complex protocols may be enabled by the techniques presented in this study.

Silencing Mist1 Gene Expression Is Essential for Recovery from Acute Pancreatitis.

Acinar cells of the exocrine pancreas are tasked with synthesizing, packaging and secreting vast quantities of pro-digestive enzymes to maintain proper metabolic homeostasis for the organism. Because the synthesis of high levels of hydrolases is potentially dangerous, the pancreas is prone to acute pancreatitis (AP), a disease that targets acinar cells, leading to acinar-ductal metaplasia (ADM), inflammation and fibrosis-events that can transition into the earliest stages of pancreatic ductal adenocarcinoma. Despite a wealth of information concerning the broad phenotype associated with pancreatitis, little is understood regarding specific transcriptional regulatory networks that are susceptible to AP and the role these networks play in acinar cell and exocrine pancreas responses. In this study, we examined the importance of the acinar-specific maturation transcription factor MIST1 to AP damage and organ recovery. Analysis of wild-type and Mist1 conditional null mice revealed that Mist1 gene transcription and protein accumulation were dramatically reduced as acinar cells underwent ADM alterations during AP episodes. To test if loss of MIST1 function was primarily responsible for the damaged status of the organ, mice harboring a Cre-inducible Mist1 transgene (iMist1) were utilized to determine if sustained MIST1 activity could alleviate AP damage responses. Unexpectedly, constitutive iMist1 expression during AP led to a dramatic increase in organ damage followed by acinar cell death. We conclude that the transient silencing of Mist1 expression is critical for acinar cells to survive an AP episode, providing cells an opportunity to suppress their secretory function and regenerate damaged cells. The importance of MIST1 to these events suggests that modulating key pancreas transcription networks could ease clinical symptoms in patients diagnosed with pancreatitis and pancreatic cancer.

Highly enantioselective oxidations of ketene dithioacetals leading to trans bis-sulfoxides.

Ketene dithioacetals undergo a Sharpless-type asymmetric oxidation using (+)-DET, Ti(O(i)()Pr)(4), and cumene hydroperoxide to give the trans bis-sulfoxides 4a-f with essentially complete control of enantioselectivity and diastereoselectivity. The high enantioselectivity is a consequence of carrying out two asymmetric processes on the same substrate. However, this should lead to the formation of a small amount of the meso isomer but none was isolated. From monitoring the enantioselectivity of the monoxide over time, it was concluded that small amounts of the meso isomer must be formed. The inability to isolate this compound could be because it acted as a ligand on titanium and remained tightly bound even upon workup.