Tensor modeling of MRSA bacteremia cytokine and transcriptional patterns reveals coordinated, outcome-associated immunological programs.

Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is a common and life-threatening infection that imposes up to 30% mortality even when appropriate therapy is used. Despite in vitro efficacy determined by minimum inhibitory concentration breakpoints, antibiotics often fail to resolve these infections in vivo, resulting in persistent MRSA bacteremia. Recently, several genetic, epigenetic, and proteomic correlates of persistent outcomes have been identified. However, the extent to which single variables or their composite patterns operate as independent predictors of outcome or reflect shared underlying mechanisms of persistence is unknown. To explore this question, we employed a tensor-based integration of host transcriptional and cytokine datasets across a well-characterized cohort of patients with persistent or resolving MRSA bacteremia outcomes. This method yielded high correlative accuracy with outcomes and immunologic signatures united by transcriptomic and cytokine datasets. Results reveal that patients with persistent MRSA bacteremia (PB) exhibit signals of granulocyte dysfunction, suppressed antigen presentation, and deviated lymphocyte polarization. In contrast, patients with resolving bacteremia (RB) heterogeneously exhibit correlates of robust antigen-presenting cell trafficking and enhanced neutrophil maturation corresponding to appropriate T lymphocyte polarization and B lymphocyte response. These results suggest that transcriptional and cytokine correlates of PB vs. RB outcomes are complex and may not be disclosed by conventional modeling. In this respect, a tensor-based integration approach may help to reveal consensus molecular and cellular mechanisms and their biological interpretation.

Genes and gene networks underlying spatial cognition in food-caching chickadees.

Substantial progress has been made in understanding the genetic architecture of phenotypes involved in a variety of evolutionary processes. Behavioral genetics remains, however, among the least understood. We explore the genetic architecture of spatial cognitive abilities in a wild passerine bird, the mountain chickadee (Poecile gambeli). Mountain chickadees cache thousands of seeds in the fall and require specialized spatial memory to recover these caches throughout the winter. We previously showed that variation in spatial cognition has a direct effect on fitness and has a genetic basis. It remains unknown which specific genes and developmental pathways are particularly important for shaping spatial cognition. To further dissect the genetic basis of spatial cognitive abilities, we combine experimental quantification of spatial cognition in wild chickadees with whole-genome sequencing of 162 individuals, a new chromosome-scale reference genome, and species-specific gene annotation. We have identified a set of genes and developmental pathways that play a key role in creating variation in spatial cognition and found that the mechanism shaping cognitive variation is consistent with selection against mildly deleterious non-coding mutations. Although some candidate genes were organized into connected gene networks, about half do not have shared regulation, highlighting that multiple independent developmental or physiological mechanisms contribute to variation in spatial cognitive abilities. A large proportion of the candidate genes we found are associated with synaptic plasticity, an intriguing result that leads to the hypothesis that certain genetic variants create antagonism between behavioral plasticity and long-term memory, each providing distinct benefits depending on ecological context.

Dissecting signaling regulators driving AXL-mediated bypass resistance and associated phenotypes by phosphosite perturbations.

Receptor tyrosine kinase (RTK)-targeted therapies are often effective but invariably limited by drug resistance. A major mechanism of acquired resistance involves "bypass" switching to alternative pathways driven by non-targeted RTKs that restore proliferation. One such RTK is AXL whose overexpression, frequently observed in bypass resistant tumors, drives both cell survival and associated malignant phenotypes such as epithelial-to-mesenchymal (EMT) transition and migration. However, the signaling molecules and pathways eliciting these responses have remained elusive. To explore these coordinated effects, we generated a panel of mutant lung adenocarcinoma PC9 cell lines in which each AXL intracellular tyrosine residue was mutated to phenylalanine. By integrating measurements of phosphorylation signaling and other phenotypic changes associated with resistance through multivariate modeling, we mapped signaling perturbations to specific resistant phenotypes. Our results suggest that AXL signaling can be summarized into two clusters associated with progressive disease and poor clinical outcomes in lung cancer patients. These clusters displayed favorable Abl1 and SFK motifs and their phosphorylation was consistently decreased by dasatinib. High-throughput kinase specificity profiling showed that AXL likely activates the SFK cluster through FAK1 which is known to complex with Src. Moreover, the SFK cluster overlapped with a previously established focal adhesion kinase (FAK1) signature conferring EMT-mediated erlotinib resistance in lung cancer cells. Finally, we show that downstream of this kinase signaling, AXL and YAP form a positive feedback loop that sustains drug tolerant persister cells. Altogether, this work demonstrates an approach for dissecting signaling regulators by which AXL drives erlotinib resistance-associated phenotypic changes.

ADAR1p150 prevents MDA5 and PKR activation via distinct mechanisms to avert fatal autoinflammation.

Effective immunity requires the innate immune system to distinguish foreign nucleic acids from cellular ones. Cellular double-stranded RNAs (dsRNAs) are edited by the RNA-editing enzyme ADAR1 to evade being recognized as viral dsRNA by cytoplasmic dsRNA sensors, including MDA5 and PKR. The loss of ADAR1-mediated RNA editing of cellular dsRNA activates MDA5. Additional RNA-editing-independent functions of ADAR1 have been proposed, but a specific mechanism has not been delineated. We now demonstrate that the loss of ADAR1-mediated RNA editing specifically activates MDA5, whereas loss of the cytoplasmic ADAR1p150 isoform or its dsRNA-binding activity enabled PKR activation. Deleting both MDA5 and PKR resulted in complete rescue of the embryonic lethality of Adar1p150-/- mice to adulthood, contrasting with the limited or no rescue by removing MDA5 or PKR alone. Our findings demonstrate that MDA5 and PKR are the primary in vivo effectors of fatal autoinflammation following the loss of ADAR1p150.

Anthropogenic Change and the Process of Speciation.

Anthropogenic impacts on the environment alter speciation processes by affecting both geographical contexts and selection patterns on a worldwide scale. Here we review evidence of these effects. We find that human activities often generate spatial isolation between populations and thereby promote genetic divergence but also frequently cause sudden secondary contact and hybridization between diverging lineages. Human-caused environmental changes produce new ecological niches, altering selection in diverse ways that can drive diversification; but changes also often remove niches and cause extirpations. Human impacts that alter selection regimes are widespread and strong in magnitude, ranging from local changes in biotic and abiotic conditions to direct harvesting to global climate change. Altered selection, and evolutionary responses to it, impacts early-stage divergence of lineages, but does not necessarily lead toward speciation and persistence of separate species. Altogether, humans both promote and hinder speciation, although new species would form very slowly relative to anthropogenic hybridization, which can be nearly instantaneous. Speculating about the future of speciation, we highlight two key conclusions: (1) Humans will have a large influence on extinction and "despeciation" dynamics in the short term and on early-stage lineage divergence, and thus potentially speciation in the longer term, and (2) long-term monitoring combined with easily dated anthropogenic changes will improve our understanding of the processes of speciation. We can use this knowledge to preserve and restore ecosystems in ways that promote (re-)diversification, increasing future opportunities of speciation and enhancing biodiversity.

Multivalent, asymmetric IL-2-Fc fusions show enhanced selectivity for regulatory T cells.

The cytokine interleukin-2 (IL-2) has the potential to treat autoimmune disease but is limited by its modest specificity toward immunosuppressive regulatory T (Treg) cells. IL-2 receptors consist of combinations of α, ß, and γ chains of variable affinity and cell specificity. Engineering IL-2 to treat autoimmunity has primarily focused on retaining binding to the relatively Treg-selective, high-affinity receptor while reducing binding to the less selective, low-affinity receptor. However, we found that refining the designs to focus on targeting the high-affinity receptor through avidity effects is key to optimizing Treg selectivity. We profiled the dynamics and dose dependency of signaling responses in primary human immune cells induced by engineered fusions composed of either wild-type IL-2 or mutant forms with altered affinity, valency, and fusion to the antibody Fc region for stability. Treg selectivity and signaling response variations were explained by a model of multivalent binding and dimer-enhanced avidity-a combined measure of the strength, number, and conformation of interaction sites-from which we designed tetravalent IL-2-Fc fusions that had greater Treg selectivity in culture than do current designs. Biasing avidity toward IL2Rα with an asymmetrical multivalent design consisting of one α/ß chain-binding and one α chain-binding mutant further enhanced Treg selectivity. Comparative analysis revealed that IL2Rα was the optimal cell surface target for Treg selectivity, indicating that avidity for IL2Rα may be the optimal route to producing IL-2 variants that selectively target Tregs.

Person-centred critical care for a person with learning disability and COVID-19: case study of positive risk taking.

People with learning disabilities are known to experience a wide range of health inequalities and have a lower life expectancy than the general population. During the COVID-19 pandemic this extended to higher mortality rates following infection with the novel coronavirus. This case study presents an example of a positive outcome for Jade, a 21-year-old woman with learning disabilities and autism who required a long period in intensive care following COVID-19 infection. It demonstrates the impact of effective multidisciplinary collaboration involving the acute hospital learning disability liaison nurse and Jade's family, leading to a wide range of reasonable and achievable adjustments to her care.

Thermal acclimation in a non-migratory songbird occurs via changes to thermogenic capacity, but not conductance.

Thermoregulatory performance can be modified through changes in various subordinate traits, but the rate and magnitude of change in these traits is poorly understood. We investigated flexibility in traits that affect thermal balance between black-capped chickadees (Poecile atricapillus) acclimated for 6 weeks to cold (-5°C) or control (23°C) environments (n=7 per treatment). We made repeated measurements of basal and summit metabolic rates via flow-through respirometry and of body composition using quantitative magnetic resonance of live birds. At the end of the acclimation period, we measured thermal conductance of the combined feathers and skins. Cold-acclimated birds had a higher summit metabolic rate, reflecting a greater capacity for endogenous heat generation, and an increased lean mass. However, birds did not alter their thermal conductance. These results suggest that chickadees respond to cold stress by increasing their capacity for heat production rather than increasing heat retention, an energetically expensive strategy.

Quantitative Analyses of Coupling in Hybrid Zones.

In hybrid zones, whether barrier loci experience selection mostly independently or as a unit depends on the ratio of selection to recombination as captured by the coupling coefficient. Theory predicts a sharper transition between an uncoupled and coupled system when more loci affect hybrid fitness. However, the extent of coupling in hybrid zones has rarely been quantified. Here, we use simulations to characterize the relationship between the coupling coefficient and variance in clines across genetic loci. We then reanalyze 25 hybrid zone data sets and find that cline variances and estimated coupling coefficients form a smooth continuum from high variance and weak coupling to low variance and strong coupling. Our results are consistent with low rates of hybridization and a strong genome-wide barrier to gene flow when the coupling coefficient is much greater than 1, but also suggest that this boundary might be approached gradually and at a near constant rate over time.

The phenotype of the most common human ADAR1p150 Zα mutation P193A in mice is partially penetrant.

ADAR1 -mediated A-to-I RNA editing is a self-/non-self-discrimination mechanism for cellular double-stranded RNAs. ADAR mutations are one cause of Aicardi-Goutières Syndrome, an inherited paediatric encephalopathy, classed as a "Type I interferonopathy." The most common ADAR1 mutation is a proline 193 alanine (p.P193A) mutation, mapping to the ADAR1p150 isoform-specific Zα domain. Here, we report the development of an independent murine P195A knock-in mouse, homologous to human P193A. The Adar1P195A/P195A mice are largely normal and the mutation is well tolerated. When the P195A mutation is compounded with an Adar1 null allele (Adar1P195A/- ), approximately half the animals are runted with a shortened lifespan while the remaining Adar1P195A/- animals are normal, contrasting with previous reports. The phenotype of the Adar1P195A/- animals is both associated with the parental genotype and partly non-genetic/environmental. Complementation with an editing-deficient ADAR1 (Adar1P195A/E861A ), or the loss of MDA5, rescues phenotypes in the Adar1P195A/- mice.

ADAR1p150 Prevents MDA5 and PKR Activation via Distinct Mechanisms to Avert Fatal Autoinflammation.

Effective immunity requires the innate immune system to distinguish foreign (non-self) nucleic acids from cellular (self) nucleic acids. Cellular double-stranded RNAs (dsRNAs) are edited by the RNA editing enzyme ADAR1 to prevent their dsRNA structure pattern being recognized as viral dsRNA by cytoplasmic dsRNA sensors including MDA5, PKR and ZBP1. A loss of ADAR1-mediated RNA editing of cellular dsRNA activates MDA5. However, additional RNA editing-independent functions of ADAR1 have been proposed, but a specific mechanism has not been delineated. We now demonstrate that the loss of ADAR1-mediated RNA editing specifically activates MDA5, while loss of the cytoplasmic ADAR1p150 isoform or its dsRNA binding activity enabled PKR activation. Deleting both MDA5 and PKR resulted in complete rescue of the embryonic lethality of Adar1p150 -/- mice to adulthood, contrasting with the limited or no rescue by removing MDA5, PKR or ZBP1 alone, demonstrating that this is a species conserved function of ADAR1p150. Our findings demonstrate that MDA5 and PKR are the primary in vivo effectors of fatal autoinflammation following the loss of ADAR1p150.

Survey of haemosporidian parasite infections in an endangered high alpine bird.

The Brown-capped Rosy-Finch is an endangered high alpine specialist that breeds on cliffs in the Rocky Mountains of North America. We know little about the parasites that infect these birds but filling this knowledge gap is important for understanding their population decline. The aim of this study was to survey haemosporidian parasite infections in Brown-capped Rosy-Finches. We sampled 104 Brown-capped Rosy-Finches during their breeding season at six sampling sites spanning the Colorado Rocky Mountains where they are nearly endemic. We used nested PCR methods to screen birds for parasite infections, and Sanger sequencing data were used to identify parasite lineages. Four of the sampling sites had birds with infections. Females were more often infected than males (prevalence = 15.6% and 9.7%, respectively). We observed twice as many infected birds sampled in July compared to birds sampled in June (prevalence = 12.5% and 6.25%, respectively). The percent of infected birds by sampling site ranged from 0 to over 20%. In total we identified eight different genetic lineages of haemosporidian parasites infecting Brown-capped Rosy-Finches-seven were in the genus Leucocytozoon, and one was in the genus Haemoproteus. Network analysis clearly separates Haemoproteus from Leucocytozoon, with Leucocytozoon lineages comprising two major clusters. Based on reports made to the MalAvi database, all lineages in Cluster 1 have the same reported insect vector, Simulium silvestre. We report that Brown-capped Rosy-Finches experience infections with haemosporidian parasites and that the percentage of infected birds differed markedly between sampling sites. We hypothesize that vector ecology and associated variation in climate at sampling sites explain our observations.

The genetic basis of plumage coloration and elevation adaptation in a clade of recently diverged alpine and arctic songbirds.

Trait genetic architecture plays an important role in the probability that variation in that trait leads to divergence and speciation. In some cases, speciation may be driven by the generation of novel phenotypes through the recombination of genes associated with traits that are important for local adaptation or sexual selection. Here, we investigate the genetic basis of three plumage color traits, and one ecological trait, breeding elevation, in a recent avian radiation, the North American rosy-finches (Leucosticte spp.). We identify unique genomic regions associated with each trait and highlight 11 candidate genes. Among these are well-characterized melanogenesis genes, including Mitf and Tyrp1, and previously reported hypoxia-related genes including Egln1. Additionally, we use mitochondrial data to date the divergence of rosy-finch clades which appear to have diverged within the past 250 ky. Given the low levels of genome-wide differentiation among rosy-finch taxa, and evidence for extensive introgression in North America, plumage coloration and adaptation to high elevations have likely played large roles in generating the observed patterns of lineage divergence. The relative independence of these candidate regions across the genome suggests that recombination might have led to multiple phenotypes, and subsequent rosy-finch speciation, over short periods of time.

Hybridization between closely related songbirds is related to human habitat disturbance.

Human habitat disturbances can promote hybridization between closely related, but typically reproductively isolated, species. We explored whether human habitat disturbances are related to hybridization between two closely related songbirds, black-capped and mountain chickadees, using both genomic and citizen science data sets. First, we genotyped 409 individuals from across both species' ranges using reduced-representation genome sequencing and compared measures of genetic admixture to a composite measure of human landscape disturbance. Then, using eBird observations, we compared human landscape disturbance values for sites where phenotypically diagnosed hybrids were observed to locations where either parental species was observed to determine whether hybrid chickadees are reported in more disturbed areas. We found that hybridization between black-capped and mountain chickadees positively correlates with human habitat disturbances. From genomic data, we found that (1) hybrid index (HI) significantly increased with habitat disturbance, (2) more hybrids were sampled in disturbed habitats, (3) mean HIs were higher in disturbed habitats versus wild habitats, and (4) hybrids were detected in habitats with significantly higher disturbance values than parentals. Using eBird data, we found that both hybrid and black-capped chickadees were significantly more disturbance-associated than mountain chickadees. Surprisingly, we found that nearly every black-capped chickadee we sampled contained some proportion of hybrid ancestry, while we detected very few mountain chickadee backcrosses. Our results highlight that hybridization between black-capped and mountain chickadees is widespread, but initial hybridization is rare (few F1s were detected). We conclude that human habitat disturbances can erode pre-zygotic reproductive barriers between chickadees and that post-zygotic isolation is incomplete. Understanding what becomes of recently hybridizing species following large-scale habitat disturbances is a new, but pressing, consideration for successfully preserving genetic biodiversity in a rapidly changing world.

The Ethics and Politics of Academic Knowledge Production: Thoughts on the Future of Business Ethics.

To commemorate 40 years since the founding of the Journal of Business Ethics, the editors in chief of the journal have invited the editors to provide commentaries on the future of business ethics. This essay comprises a selection of commentaries aimed at creating dialogue around the theme The Ethics and Politics of Academic Knowledge Production. Questions of who produces knowledge about what, and how that knowledge is produced, are inherent to editing and publishing academic journals. At the Journal of Business Ethics, we understand the ethical responsibility of academic knowledge production as going far beyond conventions around the integrity of the research content and research processes. We are deeply aware that access to resources, knowledge of the rules of the game, and being able to set those rules, are systematically and unequally distributed. One could ask the question "for whom is knowledge now ethical'"? (See the Burrell commentary.) We have a responsibility to address these inequalities and open up our journal to lesser heard voices, ideas, and ways of being. Our six commentators pursue this through various aspects of the ethics and politics of academic knowledge production. Working with MacIntyre's scheme of practices and institutions, Andrew West provides commentary on the internal good of business ethics learning and education. Inviting us to step out of the cave, Christopher Michaelson urges a clear-eyed, unblinking focus on the purposes and audiences of business ethics scholarship. As developmental editor, Scott Taylor uncovers some of the politics of peer review with the aim of nurturing of unconventional research. Mike Hyman presents his idiosyncratic view of marketing ethics. In the penultimate commentary, Julie Nelson attributes difficulties in the academic positioning of the Business Ethics field to the hegemony of a masculine-centric model of the firm. And finally, Gibson Burrell provides a powerful provocation to go undercover as researcher-investigators in a parallel ethics of the research process.

Effect of Lipid Length and Cationic Residues on the Antibacterial and Hemolytic Activities of Paenibacterin.

Paenibacterin A1 (PA1) is a broad-spectrum, cationic cyclic lipodepsipeptide antibiotic isolated from Paenibacillus thiaminolyticus. In this study, the roles of the cationic residues and lipid tail length on the in vitro antibacterial and hemolytic activities of PA1 was examined in the context of an active PA1 analogue, called PAK, in which the two D-Orn residues in PA1 were converted to D-Lys residues. The effect of reducing the length of the lipid tail in PAK from 15 to 12-10 carbons on the minimum inhibitory concentration (MIC) depended upon the bacteria. This change had little effect on the MIC against Escherichia coli and Bacillus subtilis but resulted in a reduction in activity against most of the ESKAPE pathogens tested with the exception of Klebsiella pneumoniae. Any one of the four cationic residues in PAK could be replaced with alanine with only a minimal effect on its MIC against B. subtilis, E.coli, K. pneumoniae, Acinetobacter baumannii, and MSSA. For Pseudomonas aeruginosa and the two MRSA strains tested, the presence of cationic residues at positions 7 and 12 are not important for activity, while the cationic residues at positions 1 and 4 are important. While PAK exhibited some hemolysis at 8 µg/mL and 70% hemolysis at 128 µg/mL, its C-12 and C-10 analogues were not hemolytic up to 128 µg/mL. All PAK analogues that had one or two cationic residues replaced with alanine were as hemolytic as or more hemolytic than PAK.

The impact of lysyl-phosphatidylglycerol on the interaction of daptomycin with model membranes.

Daptomycin is an important clinical antibiotic for which resistance is rising. Daptomycin resistant strains of S. aureus often have increased 1,2-diacyl-sn-glycero-3-[phospho-1-(3-lysyl(1-glycerol))] (lysyl-PG) and mutations to the proteins directly involved in the synthesis and translocation of lysyl-PG are implicated in resistance mechanisms. To study the interaction of daptomycin with lysyl-DMPG-containing model membranes a new stereospecific and regioselective synthesis of lysyl-DMPG was developed. Studies on model membranes containing lysyl-DMPG demonstrate that: (1) daptomycin is not significantly repelled by the cationic charge of lysyl-DMPG; (2) daptomycin binds less avidly to lysyl-DMPG compared to DMPG; (3) the presence of lysyl-DMPG does not impact the membrane bound backbone conformation of daptomycin in a significant way; (4) lysyl-DMPG increases oligomer formation; (5) lysyl-DMPG does not impact model membrane fluidity at lysyl-PG : PG ratios that are relevant to daptomycin resistance. The results of these studies suggest that increased lysyl-PG content does not confer resistance to daptomycin by altering membrane fluidity or reducing membrane affinity but may confer resistance by altering the structure of daptomycin oligomers.

A54145 Factor D Is Not Less Susceptible to Inhibition by Lung Surfactant than Daptomycin.

A54145 factor D (A5D) is a cyclic lipopeptide antibiotic that shares several structural and mechanistic features with the clinically important antibiotic daptomycin, such as their requirement for calcium and phosphatidylglycerol (PG) for activity. Studies by others have suggested that daptomycin's activity is strongly inhibited by lung surfactant while A5D's activity is not. This finding has inspired efforts, albeit unsuccessful, to develop an A5D analogue that is highly active in the presence of lung surfactant and can be used for treating community acquired pneumonia (CAP). Here we demonstrate that A5D, like daptomycin, has a strong preference for the 1,2-diacyl-sn-glycero-3-phospho-1'-sn-glycerol stereoisomer (2R,2'S configuration) of PG. This PG stereoisomer was determined to be the only stereoisomer of PG in lung surfactant. Both antibiotics are completely antagonized by approximately 1-2 mol equiv of 2R,2'S-PG. Studies performed in the presence of lung surfactant revealed that the antagonism of these peptides by surfactant is mainly due to their interaction with PG and that A5D is not significantly less susceptible to inhibition by lung surfactant than daptomycin.

Establishing the Structure-Activity Relationship between Phosphatidylglycerol and Daptomycin.

Daptomycin is a clinical antibiotic used to treat serious infections caused by Gram-positive bacteria. Although there is debate about the action mechanism of daptomycin, it is known that daptomycin requires both calcium and phosphatidylglycerol (PG) to exert its antibacterial effect. Despite the importance and uniqueness of the interaction of daptomycin with PG, very little is known about this interaction or the nascent daptomycin-PG complex. In this work, we establish a structure-activity relationship between daptomycin and PG through the synthesis of PG analogues. In total, nine PGs were synthesized using a divergent approach employing phosphoramidite chemistry. The interaction between daptomycin and these PGs was studied using fluorescence, circular dichroism, and isothermal titration calorimetry. It was determined that daptomycin is highly sensitive to the modification of the headgroup of PG and both hydroxyl groups influence membrane binding, oligomerization, and backbone structure. Methylation of each hydroxyl in the headgroup suggests that the binding pocket envelops both hydroxyl groups. A PG acyl tail chain length of at least 7-8 carbons is required for stoichiometric binding at micromolar peptide concentrations. Daptomycin binds to PG having 8-carbon, linear, unsaturated acyl groups (C8PGs) at the micromolar concentration and interacts with C8PG in essentially the same manner as when the PG is incorporated into a liposome, and thus, preassembly of individual PG moieties is not a prerequisite for binding, structural transition, and oligomerization.