Systems-level discovery of quality attributes and candidate pathways for optimized production of human pluripotent stem cell-derived cardiomyocytes

Numerous protocols exist for differentiation of human pluripotent stem cells (hPSCs) to cardiomyocytes (CMs). Although these methods have improved in efficiency over the past decade, they remain highly variable in their resultant purities, not only among different source hPSC lines but also between batches in the same cell line. This substantial heterogeneity of hPSC-CM product outcomes points to poorly-understood, highly sensitive, and uncontrolled variables present within the overall process. Herein, we have undertaken a multi-omic discovery approach to identify key temporal differences in cell attributes between high- and low-purity hPSC-CM differentiations to provide systems-level insights into underlying mechanisms which drive these populations to divergent endpoints. Specifically, we are combining metabolomic, proteomic, lipidomic, and transcriptomic analyses collected throughout the differentiation process for high- and low-purity (as assessed by %cTnT+ via flow cytometry) differentiation batches. In addition to gaining fundamental insights into the underlying biology of the differentiation process, we are extending our analyses to 1) identify putative critical quality attributes for use in on- or at-line analytics for continuous process monitoring, 2) enhance process robustness through the development of protocols aimed at depressing off-target pathways and enhancing on-target ones, and 3) establish potential feedforward/feedback control schemes based on real-time analytics to respond to in-process intermediate quality attributes through rational adjustment of process parameters. To date we have identified novel putative candidate quality attributes for process monitoring and cellular pathways which may be able to be modulated to augment process robustness in a scaled manufacturing context. Beyond standard single-omic analytical workflows, ongoing work is aimed at integrating these data for deepened insight, including functional integration with systems-scale modeling and high-dimensional machine-learning methodologies to extract dynamic relationships among variables over time

Split-Brain: what we know now and why this is important for understanding consciousness

Recently, the discussion regarding the consequences of cutting the corpus callosum (“split-brain”) has regained momentum (Corballis, Corballis, Berlucchi, & Marzi, 2018; Pinto et al., 2017; Pinto, Lamme, & de Haan, 2017; Volz & Gazzaniga, 2017; Volz, Hillyard, Miller, & Gazzaniga, 2018). This collective review paper aims to summarize the empirical common ground, to delineate the different interpretations, and to identify the remaining questions. In short, callosotomy leads to a broad breakdown of functional integration ranging from perception to attention. However, the breakdown is not absolute as several processes, such as action control, seem to remain unified. Disagreement exists about the responsible mechanisms for this remaining unity. The main issue concerns the first-person perspective of a split-brain patient. Does a split-brain harbor a split consciousness or is consciousness unified? The current consensus is that the body of evidence is insufficient to answer this question, and different suggestions are made to how future studies might address this paucity. In addition, it is suggested that the answers might not be a simple yes or no but that intermediate conceptualization need to be considered

MASH Explorer: A Universal Software Environment for Top-Down Proteomics

Top-down mass spectrometry (MS)-based proteomics enable a comprehensive analysis of proteoforms with molecular specificity to achieve a proteome-wide understanding of protein functions. However, the lack of a universal software for top-down proteomics is becoming increasingly recognized as a major barrier, especially for newcomers. Here, we have developed MASH Explorer, a universal, comprehensive, and user-friendly software environment for top-down proteomics. MASH Explorer integrates multiple spectral deconvolution and database search algorithms into a single, universal platform which can process top-down proteomics data from various vendor formats, for the first time. It addresses the urgent need in the rapidly growing top-down proteomics community and is freely available to all users worldwide. With the critical need and tremendous support from the community, we envision that this MASH Explorer software package will play an integral role in advancing top-down proteomics to realize its full potential for biomedical research

Integrating data types to estimate spatial patterns of avian migration across the Western Hemisphere

For many avian species, spatial migration patterns remain largely undescribed, especially across hemispheric extents. Recent advancements in tracking technologies and high-resolution species distribution models (i.e., eBird Status and Trends products) provide new insights into migratory bird movements and offer a promising opportunity for integrating independent data sources to describe avian migration. Here, we present a three-stage modeling framework for estimating spatial patterns of avian migration. First, we integrate tracking and band re-encounter data to quantify migratory connectivity, defined as the relative proportions of individuals migrating between breeding and nonbreeding regions. Next, we use estimated connectivity proportions along with eBird occurrence probabilities to produce probabilistic least-cost path (LCP) indices. In a final step, we use generalized additive mixed models (GAMMs) both to evaluate the ability of LCP indices to accurately predict (i.e., as a covariate) observed locations derived from tracking and band re-encounter data sets versus pseudo-absence locations during migratory periods and to create a fully integrated (i.e., eBird occurrence, LCP, and tracking/band re-encounter data) spatial prediction index for mapping species-specific seasonal migrations. To illustrate this approach, we apply this framework to describe seasonal migrations of 12 bird species across the Western Hemisphere during pre- and postbreeding migratory periods (i.e., spring and fall, respectively). We found that including LCP indices with eBird occurrence in GAMMs generally improved the ability to accurately predict observed migratory locations compared to models with eBird occurrence alone. Using three performance metrics, the eBird + LCP model demonstrated equivalent or superior fit relative to the eBird-only model for 22 of 24 species–season GAMMs. In particular, the integrated index filled in spatial gaps for species with over-water movements and those that migrated over land where there were few eBird sightings and, thus, low predictive ability of eBird occurrence probabilities (e.g., Amazonian rainforest in South America). This methodology of combining individual-based seasonal movement data with temporally dynamic species distribution models provides a comprehensive approach to integrating multiple data types to describe broad-scale spatial patterns of animal movement. Further development and customization of this approach will continue to advance knowledge about the full annual cycle and conservation of migratory birds

The discovery, distribution, and evolution of viruses associated with drosophila melanogaster

Drosophila melanogaster is a valuable invertebrate model for viral infection and antiviral immunity, and is a focus for studies of insect-virus coevolution. Here we use a metagenomic approach to identify more than 20 previously undetected RNA viruses and a DNA virus associated with wild D. melanogaster. These viruses not only include distant relatives of known insect pathogens, but also novel groups of insect-infecting viruses. By sequencing virus-derived small RNAs we show that the viruses represent active infections of Drosophila. We find that the RNA viruses differ in the number and properties of their small RNAs, and we detect both siRNAs and a novel miRNA from the DNA virus. Analysis of small RNAs also allows us to identify putative viral sequences that lack detectable sequence similarity to known viruses. By surveying >2000 individually collected wild adult Drosophila we show that more than 30% of D. melanogaster carry a detectable virus, and more than 6% carry multiple viruses. However, despite a high prevalence of the Wolbachia endosymbiont—which is known to be protective against virus infections in Drosophila—we were unable to detect any relationship between the presence of Wolbachia and the presence of any virus. Using publicly available RNA-seq datasets we show that the community of viruses in Drosophila laboratories is very different from that seen in the wild, but that some of the newly discovered viruses are nevertheless widespread in laboratory lines and are ubiquitous in cell culture. By sequencing viruses from individual wild-collected flies we show that some viruses are shared between D. melanogaster and D. simulans. Our results provide an essential evolutionary and ecological context for host-virus interaction in Drosophila, and the newly reported viral sequences will help develop D. melanogaster further as a model for molecular and evolutionary virus research

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

GRK5 Controls SAP97-Dependent Cardiotoxic β1 Adrenergic Receptor-CaMKII Signaling in Heart Failure

RationaleCardiotoxic β1 adrenergic receptor (β1AR)-CaMKII (calmodulin-dependent kinase II) signaling is a major and critical feature associated with development of heart failure. SAP97 (synapse-associated protein 97) is a multifunctional scaffold protein that binds directly to the C-terminus of β1AR and organizes a receptor signalosome.ObjectiveWe aim to elucidate the dynamics of β1AR-SAP97 signalosome and its potential role in chronic cardiotoxic β1AR-CaMKII signaling that contributes to development of heart failure.Methods and resultsThe integrity of cardiac β1AR-SAP97 complex was examined in heart failure. Cardiac-specific deletion of SAP97 was developed to examine β1AR signaling in aging mice, after chronic adrenergic stimulation, and in pressure overload hypertrophic heart failure. We show that the β1AR-SAP97 signaling complex is reduced in heart failure. Cardiac-specific deletion of SAP97 yields an aging-dependent cardiomyopathy and exacerbates cardiac dysfunction induced by chronic adrenergic stimulation and pressure overload, which are associated with elevated CaMKII activity. Loss of SAP97 promotes PKA (protein kinase A)-dependent association of β1AR with arrestin2 and CaMKII and turns on an Epac (exchange protein directly activated by cAMP)-dependent activation of CaMKII, which drives detrimental functional and structural remodeling in myocardium. Moreover, we have identified that GRK5 (G-protein receptor kinase-5) is necessary to promote agonist-induced dissociation of SAP97 from β1AR. Cardiac deletion of GRK5 prevents adrenergic-induced dissociation of β1AR-SAP97 complex and increases in CaMKII activity in hearts.ConclusionsThese data reveal a critical role of SAP97 in maintaining the integrity of cardiac β1AR signaling and a detrimental cardiac GRK5-CaMKII axis that can be potentially targeted in heart failure therapy. Graphical Abstract: A graphical abstract is available for this article

GRK5 Controls SAP97-Dependent Cardiotoxic β1 Adrenergic Receptor-CaMKII Signaling in Heart Failureβ

Rationale: Cardiotoxic β1 adrenergic receptor (β1AR)-CaMKII signaling is a major and critical feature associated with development of heart failure. Synapse-associated protein 97 (SAP97) is a multi-functional scaffold protein that binds directly to the C-terminus of β1AR and organizes a receptor signalosome. Objective: We aim to elucidate the dynamics of β1AR-SAP97 signalosome and its potential role in chronic cardiotoxic β1AR-CaMKII signaling that contributes to development of heart failure. Methods and Results: The integrity of cardiac β1AR-SAP97 complex was examined in heart failure. Cardiac specific deletion of SAP97 was developed to examine β1AR signaling in ageing mice, after chronic adrenergic stimulation, and in pressure overload hypertrophic heart failure. We show that the β1AR-SAP97 signaling complex is reduced in heart failure. Cardiac specific deletion of SAP97 yields an ageing-dependent cardiomyopathy and exacerbates cardiac dysfunction induced by chronic adrenergic stimulation and pressure overload, which are associated with elevated CaMKII activity. Loss of SAP97 promotes PKA-dependent association of β1AR with arrestin2 and CaMKII and turns on an Epac-dependent activation of CaMKII, which drives detrimental functional and structural remodeling in myocardium. Moreover, we have identified that GRK5 is necessary to promote agonist-induced dissociation of SAP97 from β1AR. Cardiac deletion of GRK5 prevents adrenergic-induced dissociation of β1AR-SAP97 complex and increases in CaMKII activity in hearts. Conclusions: These data reveal a critical role of SAP97 in maintaining the integrity of cardiac β1AR signaling and a detrimental cardiac GRK5-CaMKII axis that can be potentially targeted in heart failure therapy

Phylogenetic analysis of viruses not closely related to known insect pathogens.

<p>Although the majority of viruses were closely related to known insect pathogens (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002210#pbio.1002210.t001" target="_blank">Table 1</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002210#pbio.1002210.s012" target="_blank">S3 Fig</a>), a minority were not, and may represent novel insect-infecting lineages. These include (tree A) six sequences similar to the Partitiviridae; (tree B) the BLAST-candidate Motts Mill Virus, distantly related to Luteoviridae and Sobemoviruses; (tree C) the siRNA-candidate Galbut Virus; and (tree D) the siRNA-candidate Chaq Virus. Trees are mid-point rooted, maximum clade credibility trees inferred from the Bayesian posterior sample, and the scale is given in amino-acid substitutions per site. Node support and sequence accession identifiers are given in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002210#pbio.1002210.s012" target="_blank">S3 Fig</a>. <i>Drosophila</i>-associated sequences discovered here are shown in red, Transcriptome Shotgun Assemblies sequences in blue. The Partitivirus-like sequences are all closely related to sequences present in insect transcriptome datasets, suggesting they may form a novel clade of insect pathogens. Motts Mill Virus similarly clusters with invertebrate transcriptome sequences and two recently reported viruses from <i>Ixodes</i> ticks [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002210#pbio.1002210.ref058" target="_blank">58</a>], again suggesting a new clade of pathogens. Neither Chaq Virus nor Galbut Virus has high sequence similarity to known viruses, but both also cluster with invertebrate transcriptome-derived sequences. These may represent new virus lineages, or be weakly conserved genes in a known virus group. Alignments with MrBayes command blocks are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002210#pbio.1002210.s003" target="_blank">S3 Data</a>, and maximum clade credibility trees are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002210#pbio.1002210.s004" target="_blank">S4 Data</a>.</p