Large genome-mapping consortia and thousands of genome-wide association studies have identified non-protein-coding elements in the genome as having a central role in various biological processes. ...However, decoding the functions of the millions of putative regulatory elements discovered in these studies remains challenging. CRISPR-Cas9-based epigenome editing technologies have enabled precise perturbation of the activity of specific regulatory elements. Here we describe CRISPR-Cas9-based epigenomic regulatory element screening (CERES) for improved high-throughput screening of regulatory element activity in the native genomic context. Using dCas9
repressor and dCas9
activator constructs and lentiviral single guide RNA libraries to target DNase I hypersensitive sites surrounding a gene of interest, we carried out both loss- and gain-of-function screens to identify regulatory elements for the β-globin and HER2 loci in human cells. CERES readily identified known and previously unidentified regulatory elements, some of which were dependent on cell type or direction of perturbation. This technology allows the high-throughput functional annotation of putative regulatory elements in their native chromosomal context.
Animals inhabiting extreme environments allow the powerful opportunity to examine adaptive evolution in response to diverse pressures. One such pressure is reduced oxygen, commonly present at ...high-altitude and subterranean environments. Cave-dwelling animals must also deal with darkness and starvation, both of which have been rigorously studied as key forces driving the evolution of cave-associated traits. Interestingly, hypoxia as an environmental pressure has received less attention. Here we examined putatively adaptive phenotypes evolving in a freshwater teleost fish, Astyanax mexicanus, which includes both surface- and cave-dwelling forms. This model system also provides the opportunity to identify convergent responses to hypoxia, owing to the presence of numerous natural and independently-colonised cave populations, alongside closely-related surface conspecifics. The focus of this study is hemoglobin, an essential molecule for oxygen transport and delivery. We found that multiple cave populations harbor a higher concentration of hemoglobin in their blood, which is coincident with an increase in cave morph erythrocyte size compared to surface fish. Interestingly, both cave and surface morphs have comparable numbers of erythrocytes per unit of blood, suggesting elevated hemoglobin is not due to overproduction of red blood cells. Alternatively, owing to an increased cell area of erythrocytes in cavefish, we reason that they contain more hemoglobin per erythrocyte. These findings support the notion that cavefish have adapted to hypoxia in caves through modulation of both hemoglobin production and erythrocyte size. This work reveals an additional adaptive feature of Astyanax cavefish, and demonstrates that coordinated changes between cellular architecture and molecular changes are necessary for organisms evolving under intense environmental pressure.
Extracellular fatty acid incorporation into the phospholipids of Staphylococcus aureus occurs via fatty acid phosphorylation. We show that fatty acid kinase (Fak) is composed of two dissociable ...protein subunits encoded by separate genes. FakA provides the ATP binding domain and interacts with two distinct FakB proteins to produce acyl-phosphate. The FakBs are fatty acid binding proteins that exchange bound fatty acid/acyl-phosphate with fatty acid/acyl-phosphate presented in detergent micelles or liposomes. The Δ fakA and Δ fakB1 Δ fakB2 strains were unable to incorporate extracellular fatty acids into phospholipid. FakB1 selectively bound saturated fatty acids whereas FakB2 preferred unsaturated fatty acids. Affymetrix array showed a global perturbation in the expression of virulence genes in the Δ fakA strain. The severe deficiency in α-hemolysin protein secretion in Δ fakA and Δ fakB1 Δ fakB2 mutants coupled with quantitative mRNA measurements showed that fatty acid kinase activity was required to support virulence factor transcription. These data reveal the function of two conserved gene families, their essential role in the incorporation of host fatty acids by Gram-positive pathogens, and connects fatty acid kinase to the regulation of virulence factor transcription in S. aureus .
IMPORTANCE: Both military and civilian clinical practice guidelines include early plasma transfusion to achieve a plasma to red cell ratio approaching 1:1 to 1:2. However, it was not known how early ...plasma should be given for optimal benefit. Two recent randomized clinical trials were published, with apparently contradictory results. The Prehospital Air Medical Plasma (PAMPer) clinical trial showed a nearly 30% reduction in mortality with plasma transfusion in the prehospital environment, while the Control of Major Bleeding After Trauma (COMBAT) clinical trial showed no survival improvement. OBJECTIVE: To facilitate a post hoc combined analysis of the COMBAT and PAMPer trials to examine questions that could not be answered by either clinical trial alone. We hypothesized that prehospital transport time influenced the effects of prehospital plasma on 28-day mortality. DESIGN, SETTING, AND PARTICIPANTS: A total of 626 patients in the 2 clinical trials were included. Patients with trauma and hemorrhagic shock were randomly assigned to receive either standard care or 2 U of thawed plasma followed by standard care in the prehospital environment. Data analysis was performed between September 2018 and January 2019. INTERVENTIONS: Prehospital transfusion of 2 U of plasma compared with crystalloid-based resuscitation. MAIN OUTCOMES AND MEASURES: The main outcome was 28-day mortality. RESULTS: In this post hoc analysis of 626 patients (467 men 74.6% and 159 women 25.4%; median interquartile range age, 42 27-57 years) who had trauma with hemorrhagic shock, a Cox regression analysis showed a significant overall survival benefit for plasma (hazard ratio HR, 0.65; 95% CI, 0.47-0.90; P = .01) after adjustment for injury severity, age, and clinical trial cohort (COMBAT or PAMPer). A significant association with prehospital transport time was detected (from arrival on scene to arrival at the trauma center). Increased mortality was observed in patients in the standard care group when prehospital transport was longer than 20 minutes (HR, 2.12; 95% CI, 1.05-4.30; P = .04), while increased mortality was not observed in patients in the prehospital plasma group (HR, 0.78; 95% CI, 0.40-1.51; P = .46). No serious adverse events were associated with prehospital plasma transfusion. CONCLUSIONS AND RELEVANCE: These data suggest that prehospital plasma is associated with a survival benefit when transport times are longer than 20 minutes and that the benefit-risk ratio is favorable for use of prehospital plasma. TRIAL REGISTRATION: ClinicalTrials.gov identifiers: NCT01838863 (COMBAT) and NCT01818427 (PAMPer)
Abstract
Many arachnids use internal hemolymph pressure to actuate extension in their leg joints. The inherent large foot displacement‐to‐body length ratio that arachnids can achieve through ...hydraulics relative to muscle‐based actuators is both energy and volumetrically efficient. Until recent advances in nano/microscale 3D printing with two‐photon polymerization (2PP), the physical realization of synthetic complex ‘soft’ joints would have been impossible to replicate and fill with a hydraulic fluid into a sealed sub‐millimeter system. Inspired by nature, the smallest scale 3D‐printed hydraulic actuator (4.9 × 10
−4
mm
3
) by more than an order of magnitude is demonstrated. The use of stiff 2PP polymers with micron‐scale dimensions enable compliant membranes similar to exoskeletons seen in nature without the requirement for low‐modulus materials. The bio‐inspired system is designed to mimic similar hydraulic pressure‐activated mechanisms in arachnid joints utilized for large displacement motions relative to body length. Using variations on this actuator design, the ability to transmit forces with relatively large magnitudes (milliNewtons) in 3D space is demonstrated, as well as the ability to direct motion that is useful towards microrobotics and medical applications. Microscale hydraulic actuation provides a promising approach toward the transmission of large forces and 3D motions at small scales, previously unattainable in wafer‐level 2D microelecromechanical systems (MEMS).
MINIHemorrhage is the primary cause of preventable trauma death. Secondary analyses of scene patients from the PAMPer trial demonstrated that prehospital packed red blood cell and plasma had the ...greatest reduction in 30-day mortality compared with crystalloid-only resuscitation. Patients with hemorrhagic shock should receive prehospital blood products when available, preferably packed red blood cell and plasma.
OBJECTIVE:The aim of this study was to determine whether prehospital blood products reduce 30-day mortality in patients at risk for hemorrhagic shock compared with crystalloid only resuscitation.
SUMMARY OF BACKGROUND DATA:Hemorrhage is the primary cause of preventable death after injury. Large volume crystalloid resuscitation can be deleterious. The benefits of prehospital packed red blood cells (PRBCs), plasma, or transfusion of both products among trauma patients is unknown compared with crystalloid.
METHODS:Secondary analysis of the multicenter PAMPer trial was performed on hypotensive injured patients from the scene. The trial randomized 27 helicopter bases to prehospital plasma or standard resuscitation. Standard resuscitation at the sites was equally divided between crystalloid and crystalloid + PRBC. This led to 4 prehospital resuscitation groupscrystalloid only; PRBC; plasma; and PRBC+plasma. Cox regression determined the association between resuscitation groups and risk-adjusted 30-day mortality. The dose effect of resuscitation fluids was also explored.
RESULTS:Four hundred seven patients were included. PRBC+plasma had the greatest benefit hazard ratio (HR) 0.38; 95% confidence interval (95% CI) 0.26–0.55, P < 0.001, followed by plasma (HR 0.57; 95% CI 0.36–0.91, P = 0.017) and PRBC (HR 0.68; 95% CI 0.49–0.95, P = 0.025) versus crystalloid only. Mortality was lower per-unit of PRBC (HR 0.69; 95% CI 0.52–0.92, p = 0.009) and plasma (HR 0.68; 95% CI 0.54–0.88, P = 0.003). Crystalloid volume was associated with increased mortality among patients receiving blood products (HR 1.65; 95% CI 1.17–2.32, P = 0.004).
CONCLUSION:Patients receiving prehospital PRBC+plasma had the greatest mortality benefit. Crystalloid only had the worst survival. Patients with hemorrhagic shock should receive prehospital blood products when available, preferably PRBC+plasma. Prehospital whole blood may be ideal in this population.
Technologies to reprogram cell-type specification have revolutionized the fields of regenerative medicine and disease modeling. Currently, the selection of fate-determining factors for cell ...reprogramming applications is typically a laborious and low-throughput process. Therefore, we use high-throughput pooled CRISPR activation (CRISPRa) screens to systematically map human neuronal cell fate regulators. We utilize deactivated Cas9 (dCas9)-based gene activation to target 1,496 putative transcription factors (TFs) in the human genome. Using a reporter of neuronal commitment, we profile the neurogenic activity of these factors in human pluripotent stem cells (PSCs), leading to a curated set of pro-neuronal factors. Activation of pairs of TFs reveals neuronal cofactors, including E2F7, RUNX3, and LHX8, that improve conversion efficiency, subtype specificity, and maturation of neuronal cell types. Finally, using multiplexed gene regulation with orthogonal CRISPR systems, we demonstrate improved neuronal differentiation with concurrent activation and repression of target genes, underscoring the power of CRISPR-based gene regulation for programming complex cellular phenotypes.
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•CRISPRa screens identify factors regulating human neuronal fate specification•Paired gRNA screens reveal synergistic transcription factor interactions•Identified factors influence conversion rate, subtype profile, and maturation•Gene regulation with orthogonal CRISPR systems enables improved differentiation
Black et al. perform pooled CRISPR activation screens to identify factors that regulate neuronal fate specification of human pluripotent stem cells. The identified factors improve conversion efficiencies and modulate neuronal subtype profiles and maturation. Overall, this approach provides a broad framework for programming complex cellular phenotypes.
Postcopulatory sexual selection results from variation in competitive fertilization success among males and comprises powerful evolutionary forces that operate after the onset of mating.1,2 ...Theoretical advances in the field of sexual selection addressing the buildup and coevolutionary consequences of genetic coupling3–5 motivate the hypothesis that indirect postcopulatory sexual selection may promote evolution of male secondary sexual traits—those traits traditionally ascribed to mate choice and male fighting.6,7 A crucial prediction of this hypothesis is genetic covariance between trait expression and competitive fertilization success, which has been predicted to arise, for example, when traits subject to pre- and postcopulatory sexual selection are under positive correlational selection.8 We imposed bidirectional artificial selection on male ornament (sex comb) size in Drosophila bipectinata and demonstrated increased competitive fertilization success as a correlated evolutionary response to increasing ornament size. Transcriptional analyses revealed that levels of specific seminal fluid proteins repeatedly shifted in response to this selection, suggesting that properties of the ejaculate, rather than the enlarged sex comb itself, contributed fertilizing capacity. We used ultraprecise laser surgery to reduce ornament size of high-line males and found that their fertilizing superiority persisted despite the size reduction, reinforcing the transcriptional results. The data support the existence of positive genetic covariance between a male secondary sexual trait and competitive fertilization success, and suggest the possibility that indirect postcopulatory sexual selection may, under certain conditions, magnify net selection on ornamental trait expression.
•Genetic covariance among traits is a requirement for indirect selection to operate•In Drosophila bipectinata, ejaculate traits coevolve with a secondary sexual trait•Sexual trait size is positively genetically correlated with fertilizing ability•Postcopulatory sexual selection may magnify net selection on sexual trait size
Using artificial selection, transcriptomic profiling, and laser phenotypic engineering, Polak et al. document a positive genetic correlation between a Darwinian precopulatory “ornament,” the male sex comb in Drosophila bipectinata, and competitive fertilizing ability. Two models potentially explain how such genetic covariance may evolve.
The lack of consistent, accurate information on evapotranspiration (ET) and consumptive use of water by irrigated agriculture is one of the most important data gaps for water managers in the western ...United States (U.S.) and other arid agricultural regions globally. The ability to easily access information on ET is central to improving water budgets across the West, advancing the use of data‐driven irrigation management strategies, and expanding incentive‐driven conservation programs. Recent advances in remote sensing of ET have led to the development of multiple approaches for field‐scale ET mapping that have been used for local and regional water resource management applications by U.S. state and federal agencies. The OpenET project is a community‐driven effort that is building upon these advances to develop an operational system for generating and distributing ET data at a field scale using an ensemble of six well‐established satellite‐based approaches for mapping ET. Key objectives of OpenET include: Increasing access to remotely sensed ET data through a web‐based data explorer and data services; supporting the use of ET data for a range of water resource management applications; and development of use cases and training resources for agricultural producers and water resource managers. Here we describe the OpenET framework, including the models used in the ensemble, the satellite, meteorological, and ancillary data inputs to the system, and the OpenET data visualization and access tools. We also summarize an extensive intercomparison and accuracy assessment conducted using ground measurements of ET from 139 flux tower sites instrumented with open path eddy covariance systems. Results calculated for 24 cropland sites from Phase I of the intercomparison and accuracy assessment demonstrate strong agreement between the satellite‐driven ET models and the flux tower ET data. For the six models that have been evaluated to date (ALEXI/DisALEXI, eeMETRIC, geeSEBAL, PT‐JPL, SIMS, and SSEBop) and the ensemble mean, the weighted average mean absolute error (MAE) values across all sites range from 13.6 to 21.6 mm/month at a monthly timestep, and 0.74 to 1.07 mm/day at a daily timestep. At seasonal time scales, for all but one of the models the weighted mean total ET is within ±8% of both the ensemble mean and the weighted mean total ET calculated from the flux tower data. Overall, the ensemble mean performs as well as any individual model across nearly all accuracy statistics for croplands, though some individual models may perform better for specific sites and regions. We conclude with three brief use cases to illustrate current applications and benefits of increased access to ET data, and discuss key lessons learned from the development of OpenET.
Developmental patterning is a complex biological phenomenon, involving integrated cellular and molecular signaling across diverse tissues. In Astyanax cavefish, the lateral line sensory system is ...dramatically expanded in a region of the cranium marked by significant bone abnormalities. This system provides the opportunity to understand how facial bone patterning can become altered through sensory system changes. Here we investigate a classic postulation that mechanosensory receptor neuromasts seed intramembranous facial bones in aquatic vertebrates. Using an in vivo staining procedure across individual life history, we observed infraorbital canal neuromasts serving as sites of ossification for suborbital bones. The manner in which cavefish departed from the stereotypical and symmetrical canal neuromast patterns of closely-related surface-dwelling fish were associated with specific changes to the suborbital bone complex. For instance, bony fusion, rarely observed in surface fish, was associated with shorter distances between canal neuromasts in cavefish, suggesting that closer canal neuromasts result in bony fusions. Additionally, cavefish lacking the sixth suborbital bone (SO6) uniformly lacked the associated (sixth) canal neuromast. This study suggests that patterning of canal neuromasts may impact spatial position of suborbital bones across development. The absence of an eye and subsequent orbital collapse in cavefish appears to influence positional information normally inherent to the infraorbital canal. These alterations result in coordinated changes to adult neuromast and bone structures. This work highlights complex interactions between visual, sensory and bony tissues during development that explain certain abnormal craniofacial features in cavefish.
•Canal neuromasts serve as ossification sites for suborbital bones in Astyanax fish.•Cavefish display asymmetrical patterning of neuromasts and facial bones.•Neuromast patterning impacts spatial positioning of suborbital bones in cavefish.
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