A pharmacological cocktail for arresting actin dynamics in living cells.

The actin cytoskeleton is regulated by factors that influence polymer assembly, disassembly, and network rearrangement. Drugs that inhibit these events have been used to test the role of actin dynamics in a wide range of cellular processes. Previous methods of arresting actin rearrangements take minutes to act and work well in some contexts, but can lead to significant actin reorganization in cells with rapid actin dynamics, such as neutrophils. In this paper, we report a pharmacological cocktail that not only arrests actin dynamics but also preserves the structure of the existing actin network in neutrophil-like HL-60 cells, human fibrosarcoma HT1080 cells, and mouse NIH 3T3 fibroblast cells. Our cocktail induces an arrest of actin dynamics that initiates within seconds and persists for longer than 10 min, during which time cells maintain their responsivity to external stimuli. With this cocktail, we demonstrate that actin dynamics, and not simply morphological polarity or actin accumulation at the leading edge, are required for the spatial persistence of Rac activation in HL-60 cells. Our drug combination preserves the structure of the existing cytoskeleton while blocking actin assembly, disassembly, and rearrangement, and should prove useful for investigating the role of actin dynamics in a wide range of cellular signaling contexts

Maternal Employment, Marital Status, and Religiosity and the Social Adaptational Status of First Grade Children in Selected Oregon Schools

This study attempted to examine whether certain maternal variables (i.e., maternal employment, marital status, and religiosity) could be used to accurately predict first-graders\u27 social adaptational status (SAS), as measured by the Teacher Observation of Classroom Adaptation Scale (TOCA). One-hundred-and-fifty volunteering mothers (or mother surrogates) and their first-grade child(ren) participated in this study. Familial and individual data were obtained through a structured mother interview. The participating children\u27s teachers rated the children\u27s SAS on the TOCA during the 9th and 15th weeks of the academic school year. Five Christian-oriented private schools and two public schools participated in this study

Elucidating the Role of Endocytosis in cAMP-dependent Transcription

There is a growing body of evidence from the last decade that supports the ability of G protein-coupled receptors (GPCRs) to signal from the endosome. With examples of different subtypes (Gs, Gi, Gq) of GPCRs now recognized with this ability, we still have much to learn about the role of the endosomal signal. This work explores how endocytosis selectively promotes cAMP-dependent transcription by studying a Gs-coupled GPCR, the beta-2 adrenergic receptor (β2AR). To begin to investigate the mechanism of how the endosome signal is selective for transcription, I utilized recently developed tools including a cAMP fluorescence biosensor, and a knock-in cell line endogenously expressing fluorescently-labeled protein kinase A catalytic subunit (PKAcat). I examined the effects of endocytic blockade on each step of the cAMP signaling cascade using primarily spinning disk confocal microscopy, genetic manipulations, biochemical and molecular biology methods. In this study we found that endocytosis greatly affected PKAcat nuclear accumulation, and that the accumulation in the nucleus was necessary for cAMP-dependent transcription of PCK1.Because this is still a relatively new area of GPCR cell biology, many questions remain unanswered, including how does endocytosis affect the functional relationship between two receptors? β2ARs, known to signal from the endosome, have an antagonistic relationship with the M2 muscarinic acetylcholine receptor (M2R). These Gs- and Gi-coupled receptors, respectively, control the overall cAMP produced in the cell by stimulating or inhibiting the cAMP producing enzyme. The functional relationship between these two receptors are a great example for studying the effects of endocytosis on integrative cellular signaling. This work explored the relationship between the β2AR and M2R both by examining the trafficking of each receptor and by registering the endosomal signal of the β2AR, cAMP-dependent transcription. I utilized already established imaging and molecular biology methods to probe the effect of M2R inhibition on β2AR-stimulated cAMP signaling. I additionally generated modified constructs complementary to existing nanobody biosensors to detect activated states of the M2R and Gαi protein. This work found that it was not necessary for M2R to undergo endocytosis in order to inhibit β2AR cAMP-dependent transcription

Implementation of uplink network-coded modulation for two-hop networks

With the fast growing number of wireless devices and demand of user data, the backhaul load becomes a bottleneck in wireless networks. Physical layer network coding (PNC) allows Access Points (APs) to relay compressed, network coded user data, therefore reducing the backhaul traffic. In this paper, an implementation of uplink Network Coded Modulation (NetCoM) with PNC is presented. A 5-node prototype NetCoM system is established using Universal Software Radio Peripherals (USRPs) and a practical PNC scheme designed for binary systems is utilised. An orthogonal frequency division multiplexing (OFDM) waveform implementation and the practical challenges (e.g. device synchronisation and clock drift) of applying OFDM to NetCoM are discussed. To the best of our knowledge this is the first PNC implementation in an uplink scenario in radio access networks and our prototype provides an industrially-applicable implementation of the proposed NetCoM with PNC approach

Comparative Analysis of Conformational Dynamics and Systematic Characterization of Cryptic Pockets in the SARS-CoV-2 Omicron BA.2, BA.2.75 and XBB.1 Spike Complexes with the ACE2 Host Receptor: Confluence of Binding and Structural Plasticity in Mediating Networks of Conserved Allosteric Sites

In the current study, we explore coarse-grained simulations and atomistic molecular dynamics together with binding energetics scanning and cryptic pocket detection in a comparative examination of conformational landscapes and systematic characterization of allosteric binding sites in the SARS-CoV-2 Omicron BA.2, BA.2.75 and XBB.1 spike full-length trimer complexes with the host receptor ACE2. Microsecond simulations, Markov state models and mutational scanning of binding energies of the SARS-CoV-2 BA.2 and BA.2.75 receptor binding domain complexes revealed the increased thermodynamic stabilization of the BA.2.75 variant and significant dynamic differences between these Omicron variants. Molecular simulations of the SARS-CoV-2 Omicron spike full-length trimer complexes with the ACE2 receptor complemented atomistic studies and enabled an in-depth analysis of mutational and binding effects on conformational dynamic and functional adaptability of the Omicron variants. Despite considerable structural similarities, Omicron variants BA.2, BA.2.75 and XBB.1 can induce unique conformational dynamic signatures and specific distributions of the conformational states. Using conformational ensembles of the SARS-CoV-2 Omicron spike trimer complexes with ACE2, we conducted a comprehensive cryptic pocket screening to examine the role of Omicron mutations and ACE2 binding on the distribution and functional mechanisms of the emerging allosteric binding sites. This analysis captured all experimentally known allosteric sites and discovered networks of inter-connected and functionally relevant allosteric sites that are governed by variant-sensitive conformational adaptability of the SARS-CoV-2 spike structures. The results detailed how ACE2 binding and Omicron mutations in the BA.2, BA.2.75 and XBB.1 spike complexes modulate the distribution of conserved and druggable allosteric pockets harboring functionally important regions. The results are significant for understanding the functional roles of druggable cryptic pockets that can be used for allostery-mediated therapeutic intervention targeting conformational states of the Omicron variants

HRP's Healthcare Spin-Offs Through Computational Modeling and Simulation Practice Methodologies

Spaceflight missions expose astronauts to novel operational and environmental conditions that pose health risks that are currently not well understood, and perhaps unanticipated. Furthermore, given the limited number of humans that have flown in long duration missions and beyond low Earth-orbit, the amount of research and clinical data necessary to predict and mitigate these health and performance risks are limited. Consequently, NASA's Human Research Program (HRP) conducts research and develops advanced methods and tools to predict, assess, and mitigate potential hazards to the health of astronauts. In this light, NASA has explored the possibility of leveraging computational modeling since the 1970s as a means to elucidate the physiologic risks of spaceflight and develop countermeasures. Since that time, substantial progress has been realized in this arena through a number of HRP funded activates such as the Digital Astronaut Project (DAP) and the Integrated Medical Model (IMM). Much of this success can be attributed to HRP's endeavor to establish rigorous verification, validation, and credibility (VV&C) processes that ensure computational models and simulations (M&S) are sufficiently credible to address issues within their intended scope. This presentation summarizes HRP's activities in credibility of modeling and simulation, in particular through its outreach to the community of modeling and simulation practitioners. METHODS: The HRP requires all M&S that can have moderate to high impact on crew health or mission success must be vetted in accordance to NASA Standard for Models and Simulations, NASA-STD-7009 (7009) [5]. As this standard mostly focuses on engineering systems, the IMM and DAP have invested substantial efforts to adapt the processes established in this standard for their application to biological M&S, which is more prevalent in human health and performance (HHP) and space biomedical research and operations [6,7]. These methods have also generated substantial interest by the broader medical community though institutions like the National Institutes of Health (NIH) and the Food and Drug Administration (FDA) to develop similar standards and guidelines applicable to the larger medical operations and research community. DISCUSSION: Similar to NASA, many leading government agencies, health institutions and medical product developers around the world are recognizing the potential of computational M&S to support clinical research and decision making. In this light, substantial investments are being made in computational medicine and notable discoveries are being realized [8]. However, there is a lack of broadly applicable practice guidance for the development and implementation of M&S in clinical care and research in a manner that instills confidence among medical practitioners and biological researchers [9,10]. In this presentation, we will give an overview on how HRP is working with the NIH's Interagency Modeling and Analysis Group (IMAG), the FDA and the American Society of Mechanical Engineers (ASME) to leverage NASA's biomedical VV&C processes to establish a new regulatory standard for Verification and Validation in Computational Modeling of Medical Devices, and Guidelines for Credible Practice of Computational Modeling and Simulation in Healthcare