Rexinoids Induce Differential Gene Expression in Human Glioblastoma Cells and Protein-Protein Interactions in a Yeast Two-Hybrid System.

Rexinoids are compounds that bind to the rexinoid X receptor (RXR) to modulate gene expression and have been proposed as a new class of therapeutics to treat Alzheimer's disease. Different rexinoids will initiate downstream effects that can be quite marked even though such compounds can be structurally similar and have comparable RXR binding affinities. RXR can both homo- and heterodimerize, and these protein-protein interactions and subsequent transactivating potential lead to differential gene expression, depending on the RXR dimeric partner, additional cofactors recruited, and downstream transcription factors that are up- or downregulated. Expression analysis was performed in the U87 human glioblastoma cell line treated with a panel of rexinoids, and our analysis demonstrated that rexinoids with similar RXR EC50 values can have pronounced differences in differential gene expression. Rexinoid binding likely leads to distinctive RXR conformations that cause major downstream gene expression alterations via modulation of RXR interacting proteins. Yeast two-hybrid analysis of RXR bait with two RXR interacting partners demonstrates that rexinoids drive differential binding of RXR to distinctive protein partners. Physiochemical analysis of the rexinoids reveals that the molecules cluster similarly to their gene expression patterns. Thus, rexinoids with similar RXR binding affinities drive differential gene expression by stimulating additional binding patterns in RXR and its homo- and heteropartners, driven by the physicochemical characteristics of these molecules.

UV Laser Stimulation of Ca2+ Transients in Aggressive Glioblastoma Brain Cancer Cells.

Glioblastoma (GBM) is a lethal astrocytoma being the most common highest-grade adult brain cancer. GBM tumours are highly invasive and display rapid growth to surrounding areas of the brain. Despite treatment, diagnosed patients continue to have poor prognosis with average survival time of 8 months. Calcium (Ca2+) is a main communication channel used in GBM and its understanding holds the potential to unlock new approaches to treatment. The aim of this work is to provide a first step to accurately evoking Ca2+ transients in GBM cells using single UV nanosecond laser pulses in vitro such that this communication pathway can be more reliably studied from the single-cell to the network level.

Patterning Networks of Grade IV Glioblastoma on Silicon Chip.

Glioblastoma (GBM) is the most aggressive high-grade brain cancer with a median survival time of <15 months. Due to GBMs fast and infiltrative growth patient prognosis is poor with recurrence after treatment common. Investigating GBMs ability to communicate, specifically via Ca2+ signaling, within its functional tumour networks may unlock new therapeutics to reduce the rapid infiltration and growth which currently makes treatment ineffective. This work aims to produce patterned networks of GBM cells such that the Ca2+ communication at a network level can be repeatedly and reliably investigated.

Eliciting calcium transients with UV nanosecond laser stimulation in adult patient-derived glioblastoma brain cancer cellsin vitro.

Objective.Glioblastoma (GBM) is the most common and lethal type of high-grade adult brain cancer. The World Health Organization have classed GBM as an incurable disease because standard treatments have yielded little improvement with life-expectancy being 6-15 months after diagnosis. Different approaches are now crucial to discover new knowledge about GBM communication/function in order to establish alternative therapies for such an aggressive adult brain cancer. Calcium (Ca2+) is a fundamental cell molecular messenger employed in GBM being involved in a wide dynamic range of cellular processes. Understanding how the movement of Ca2+behaves and modulates activity in GBM at the single-cell level is relatively unexplored but holds the potential to yield opportunities for new therapeutic strategies and approaches for cancer treatment.Approach.In this article we establish a spatially and temporally precise method for stimulating Ca2+transients in three patient-derived GBM cell-lines (FPW1, RN1, and RKI1) such that Ca2+communication can be studied from single-cell to larger network scales. We demonstrate that this is possible by administering a single optimized ultra-violet (UV) nanosecond laser pulse to trigger GBM Ca2+transients.Main results.We determine that 1.58µJµm-2is the optimal UV nanosecond laser pulse energy density necessary to elicit a single Ca2+transient in the GBM cell-lines whilst maintaining viability, functionality, the ability to be stimulated many times in an experiment, and to trigger further Ca2+communication in a larger network of GBM cells.Significance.Using adult patient-derived mesenchymal GBM brain cancer cell-lines, the most aggressive form of GBM cancer, this work is the first of its kind as it provides a new effective modality of which to stimulate GBM cells at the single-cell level in an accurate, repeatable, and reliable manner; and is a first step toward Ca2+communication in GBM brain cancer cells and their networks being more effectively studied.

Measurement of stress-induced sympathetic nervous activity using multi-wavelength PPG.

The onset of stress triggers sympathetic arousal (SA), which causes detectable changes to physiological parameters such as heart rate, blood pressure, dilation of the pupils and sweat release. The objective quantification of SA has tremendous potential to prevent and manage psychological disorders. Photoplethysmography (PPG), a non-invasive method to measure skin blood flow changes, has been used to estimate SA indirectly. However, the impact of various wavelengths of the PPG signal has not been investigated for estimating SA. In this study, we explore the feasibility of using various statistical and nonlinear features derived from peak-to-peak (AC) values of PPG signals of different wavelengths (green, blue, infrared and red) to estimate stress-induced changes in SA and compare their performances. The impact of two physical stressors: and Hand Grip are studied on 32 healthy individuals. Linear (Mean, s.d.) and nonlinear (Katz, Petrosian, Higuchi, SampEn, TotalSampEn) features are extracted from the PPG signal's AC amplitudes to identify the onset, continuation and recovery phases of those stressors. The results show that the nonlinear features are the most promising in detecting stress-induced sympathetic activity. TotalSampEn feature was capable of detecting stress-induced changes in SA for all wavelengths, whereas other features (Petrosian, AvgSampEn) are significant (AUC ≥ 0.8) only for IR and Red wavelengths. The outcomes of this study can be used to make device design decisions as well as develop stress detection algorithms.

Critical Spatial-Temporal Dynamics and Prominent Shape Collapse of Calcium Waves Observed in Human hNT Astrocytes in Vitro.

Networks of neurons are typically studied in the field of Criticality. However, the study of astrocyte networks in the brain has been recently lauded to be of equal importance to that of the neural networks. To date criticality assessments have only been performed on networks astrocytes from healthy rats, and astrocytes from cultured dissociated resections of intractable epilepsy. This work, for the first time, presents studies of the critical dynamics and shape collapse of calcium waves observed in cultures of healthy human astrocyte networks in vitro, derived from the human hNT cell line. In this article, we demonstrate that avalanches of spontaneous calcium waves display strong critical dynamics, including power-laws in both the size and duration distributions. In addition, the temporal profiles of avalanches displayed self-similarity, leading to shape collapse of the temporal profiles. These findings are significant as they suggest that cultured networks of healthy human hNT astrocytes self-organize to a critical point, implying that healthy astrocytic networks operate at a critical point to process and transmit information. Furthermore, this work can serve as a point of reference to which other astrocyte criticality studies can be compared.

SARS-CoV-2 Delta Variant N Gene Mutations Reduce Sensitivity to the TaqPath COVID-19 Multiplex Molecular Diagnostic Assay.

As the SARS-CoV-2 virus evolves, mutations may result in diminished sensitivity to qRT-PCR diagnostic assays. We investigated four polymorphisms circulating in the SARS-CoV-2 Delta lineage that result in N gene target failure (NGTF) on the TaqPath COVID-19 Combo Kit. These mutations were detected from the SARS-CoV-2 genome sequences that matched with the diagnostic assay results of saliva specimens. Full length N genes from the samples displaying NGTF were cloned into plasmids and assayed using three SARS-CoV-2 qRT-PCR assays. These constructs resulted in reduced sensitivity to the TaqPath COVID-19 Combo Kit compared to the controls (mean Ct differences of 3.06, 7.70, 12.46, and 14.12), but were detected equivalently on the TaqPath COVID-19 Fast PCR Combo 2.0 or CDC 2019_nCoV_N2 assays. This work highlights the importance of genomic sequencing to monitor circulating mutations and provide guidance in improving diagnostic assays.

Genomic Sequencing of SARS-CoV-2 E484K Variant B.1.243.1, Arizona, USA.

Genomic surveillance can provide early insights into new circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. While conducting genomic surveillance (1,663 cases) from December 2020-April 2021 in Arizona, USA, we detected an emergent E484K-harboring variant, B.1.243.1. This finding demonstrates the importance of real-time SARS-CoV-2 surveillance to better inform public health responses.

Disruption of transverse-tubular network reduces energy efficiency in cardiac muscle contraction.

AIM: Altered organization of the transverse-tubular network is an early pathological event occurring even prior to the onset of heart failure. Such t-tubular remodelling disturbs the synchrony and signalling between membranous and intracellular ion channels, exchangers, receptors and ATPases essential in the dynamics of excitation-contraction coupling, leading to ionic abnormality and mechanical dysfunction in heart disease progression. In this study, we investigated whether a disrupted t-tubular network has a direct effect on cardiac mechano-energetics. Our aim was to understand the fundamental link between t-tubular remodelling and impaired energy metabolism, both of which are characteristics of heart failure. We thus studied healthy tissue preparations in which cellular processes are not altered by any disease event. METHODS: We exploited the "formamide-detubulation" technique to acutely disrupt the t-tubular network in rat left-ventricular trabeculae. We assessed the energy utilization by cellular Ca2+ cycling and by crossbridge cycling, and quantified the change of energy efficiency following detubulation. For these measurements, trabeculae were mounted in a microcalorimeter where force and heat output were simultaneously measured. RESULTS: Following structural disorganization from detubulation, muscle heat output associated with Ca2+ cycling was reduced, indicating impaired intracellular Ca2+ homeostasis. This led to reduced force production and heat output by crossbridge cycling. The reduction in force-length work was not paralleled by proportionate reduction in the heat output and, as such, energy efficiency was reduced. CONCLUSIONS: These results reveal the direct energetic consequences of disrupted t-tubular network, linking the energy disturbance and the t-tubular remodelling typically observed in heart failure.

Health Worker Focused Distributed Simulation for Improving Capability of Health Systems in Liberia.

INTRODUCTION: The main goal of this study was to produce an adaptable learning platform using virtual learning and distributed simulation, which can be used to train health care workers, across a wide geographical area, key safety messages regarding infection prevention control (IPC). METHODS: A situationally responsive agile methodology, Scrum, was used to develop a distributed simulation module using short 1-week iterations and continuous synchronous plus asynchronous communication including end users and IPC experts. The module contained content related to standard IPC precautions (including handwashing techniques) and was structured into 3 distinct sections related to donning, doffing, and hazard perception training. OUTCOME: Using Scrum methodology, we were able to link concepts applied to best practices in simulation-based medical education (deliberate practice, continuous feedback, self-assessment, and exposure to uncommon events), pedagogic principles related to adult learning (clear goals, contextual awareness, motivational features), and key learning outcomes regarding IPC, as a rapid response initiative to the Ebola outbreak in West Africa. Gamification approach has been used to map learning mechanics to enhance user engagement. CONCLUSIONS: The developed IPC module demonstrates how high-frequency, low-fidelity simulations can be rapidly designed using scrum-based agile methodology. Analytics incorporated into the tool can help demonstrate improved confidence and competence of health care workers who are treating patients within an Ebola virus disease outbreak region. These concepts could be used in a range of evolving disasters where rapid development and communication of key learning messages are required.

Experience of Using Simulation Technology and Analytics During the Ebola Crisis to Empower Frontline Health Workers and Improve the Integrity of Public Health Systems.

The Ebola outbreak highlighted the challenge of health security and particularly of how best to give frontline workers the knowledge, confidence and competence to respond effectively. The goal was to develop a tool to improve infection prevention and control through local capacity building within the context of an emergency response. The research showed that digital technology could be a powerful 'force multiplier' allowing much greater access to high fidelity training during an outbreak and keeping it current as protocols evolved or new safety critical steps were identified. Tailoring training to the local context was crucial to its relevance and accessibility. This initiative used a novel approach to the development of the training tool - ebuddi. It used agile development to co-create the tool with active participation of local communities. A further pilot showed how it could be extended to meet the longer term needs of triage training and ensure better quality assurance. In the longer term it may have the potential to improve compliance with International Health Regulations, be adapted for future emergencies, and contribute to global health security.