Interaction between differentiating cell- and niche-derived signals in hematopoietic progenitor maintenance.

Maintenance of a hematopoietic progenitor population requires extensive interaction with cells within a microenvironment or niche. In the Drosophila hematopoietic organ, niche-derived Hedgehog signaling maintains the progenitor population. Here, we show that the hematopoietic progenitors also require a signal mediated by Adenosine deaminase growth factor A (Adgf-A) arising from differentiating cells that regulates extracellular levels of adenosine. The adenosine signal opposes the effects of Hedgehog signaling within the hematopoietic progenitor cells and the magnitude of the adenosine signal is kept in check by the level of Adgf-A secreted from differentiating cells. Our findings reveal signals arising from differentiating cells that are required for maintaining progenitor cell quiescence and that function with the niche-derived signal in maintaining the progenitor state. Similar homeostatic mechanisms are likely to be utilized in other systems that maintain relatively large numbers of progenitors that are not all in direct contact with the cells of the niche.

Injury-induced inflammatory signaling and hematopoiesis in Drosophila.

Inflammatory response in Drosophila to sterile (axenic) injury in embryos and adults has received some attention in recent years, and most concentrate on the events at the injury site. Here we focus on the effect sterile injury has on the hematopoietic organ, the lymph gland, and the circulating blood cells in the larva, the developmental stage at which major events of hematopoiesis are evident. In mammals, injury activates Toll-like receptor/NF-κB signaling in macrophages, which then express and secrete secondary, proinflammatory cytokines. In Drosophila larvae, distal puncture injury of the body wall epidermis causes a rapid activation of Toll and Jun kinase (JNK) signaling throughout the hematopoietic system and the differentiation of a unique blood cell type, the lamellocyte. Furthermore, we find that Toll and JNK signaling are coupled in their activation. Secondary to this Toll/JNK response, a cytokine, Upd3, is induced as a Toll pathway transcriptional target, which then promotes JAK/STAT signaling within the blood cells. Toll and JAK/STAT signaling are required for the emergence of the injury-induced lamellocytes. This is akin to the derivation of specialized macrophages in mammalian systems. Upstream, at the injury site, a Duox- and peroxide-dependent signal causes the activation of the proteases Grass and SPE, needed for the activation of the Toll-ligand Spz, but microbial sensors or the proteases most closely associated with them during septic injury are not involved in the axenic inflammatory response.

Ketamine as an Analgesic Adjuvant in Adult Trauma Intensive Care Unit Patients With Rib Fracture.

BACKGROUND: Rib fracture associated pain is difficult to control. There are no published studies that use ketamine as a therapeutic modality to reduce the amount of opioid to control rib fracture pain. OBJECTIVE: To examine the analgesic effects of adjuvant ketamine on pain scale scores in trauma intensive care unit (ICU) rib fracture. METHODS: This retrospective, case-control cohort chart review evaluated ICU adult patients with a diagnosis of ≥1 rib fracture and an Injury Severity Score >15 during 2016. Patients received standard-of-care pain management with the physician's choice analgesics with or without ketamine as a continuous, fixed, intravenous infusion at 0.1 mg/kg/h. RESULTS: A total of 15 ketamine treatment patients were matched with 15 control standard-of-care patients. Efficacy was measured via Numeric Pain Scale (NPS)/Behavioral Pain Scale (BPS) scores, opioid use, and ICU and hospital length of stay. Safety of ketamine was measured by changes in vital signs, adverse effects, and mortality. Average NPS/BPS, severest NPS/BPS, and opioid use were lower in the ketamine group than in controls (NPS: 4.1 vs 5.8, P < 0.001; severest NPS: 7.0 vs 8.9, P = 0.004; opioid use: 2.5 vs 3.5 mg morphine equivalents/h/d, P = 0.015). No difference was found between the cohort's length of stay or mortality. Average diastolic blood pressure was higher in the treatment group versus the control group (75.3 vs 64.6 mm Hg, P = 0.014). CONCLUSION: Low-dose ketamine appears to be a safe and effective adjuvant option to reduce pain and decrease opioid use in rib fracture.

Drosophila hematopoiesis: Markers and methods for molecular genetic analysis.

Analyses of the Drosophila hematopoietic system are becoming more and more prevalent as developmental and functional parallels with vertebrate blood cells become more evident. Investigative work on the fly blood system has, out of necessity, led to the identification of new molecular markers for blood cell types and lineages and to the refinement of useful molecular genetic tools and analytical methods. This review briefly describes the Drosophila hematopoietic system at different developmental stages, summarizes the major useful cell markers and tools for each stage, and provides basic protocols for practical analysis of circulating blood cells and of the lymph gland, the larval hematopoietic organ.

Directed work-up of select penetrating neck injuries is safe: Hard signs continue to soften.

INTRODUCTION: Management of penetrating neck injuries (PNIs) has evolved over time, more frequently relying on increased utilization of diagnostic imaging studies. Directed work-up with computed tomography imaging has resulted in increased use of angiography and decreased operative interventions. We sought to evaluate management strategies after directed work-up, hypothesizing increased use of non-operative therapeutic interventions and lower mortality after directed work-up. METHODS: Patients with PNI from 2017 to 2022 were identified from a single-center trauma registry. Demographics, injuries, physical exam findings, diagnostic studies and interventions were collected. Patients were stratified by presence of hard signs and management strategy [directed work-up (DW) and immediate operative intervention (OR)] and compared. Outcomes included therapeutic non-operative intervention [endovascular stent, embolization, dual antiplatelet therapy (DAPT), or anticoagulation (AC)], non-therapeutic neck exploration, length of stay (LOS), and mortality. RESULTS: Of 436 patients with PNI, 143 (33%) patients had vascular and/or aerodigestive injuries. Of these, 115 (80%) patients underwent DW and 28 (20%) patients underwent OR. There were no differences in demographics or injury severity score between groups. Patients in the DW group were more likely to undergo vascular stent or embolization (p = 0.040) and had fewer non-therapeutic neck explorations (p = 0.0009), compared to the OR group. There were no differences in post-intervention stroke, leak, or mortality. Sixty percent of patients with vascular hard signs and 78% of patients with aerodigestive hard signs underwent DW. CONCLUSIONS: Directed work-up in select patients with PNI is associated with fewer non-therapeutic neck explorations. There was no difference in mortality. Selective use of endovascular management, AC and DAPT is safe.

Management of Extraperitoneal Bladder Injuries in Patients With Pelvic Fractures.

BACKGROUND: The management of extraperitoneal bladder injuries (EBIs) when present with concomitant pelvic fractures is controversial. Current evidence is divided between supporting non-operative management with catheter drainage compared to operative management of bladder injury. The purpose of this study was to evaluate current management of EBI in the setting of pelvic fractures at our institution. We hypothesize there is no difference between operative and non-operative groups. METHODS: Retrospective review of patients with concomitant bladder injuries and pelvic fractures at a level 1 trauma center from 2017 to 2022 was performed. Demographics, injury characteristics, management strategies, and complications were collected. Patients were stratified by management (cystorrhaphy vs non-operative) and compared. RESULTS: Of 90 patients with bladder injuries and pelvic fractures, 50 patients (56%) presented with EBI, 26 patients (29%) presented with only intraperitoneal injuries, and 14 patients (16%) presented with a combined injury. Of patients with EBI, 18 (36%) underwent cystorrhaphy and 32 (64%) underwent non-operative management. There was no difference in demographics, orthopedic pelvic operative intervention, length of stay, or mortality between groups. Patients in the operative cohort had more bladder leaks [7 (39%) vs 4 (13%), P = .0406], compared to those in the non-operative cohort. Composite complications [7 (39%) vs 7 (22%), P = .1984] were similar between groups. CONCLUSIONS: Patients with EBI and pelvic fractures who underwent cystorrhaphy had more bladder leaks on follow-up imaging, although there was no difference in composite complications, when compared to those who underwent non-operative management.

Clinical Implications of Over- and Under-Triage Using Need for Trauma Intervention and Cribari Indices.

BACKGROUND: Need for Trauma Intervention (NFTI) score was proposed to help identify injured trauma patients while minimizing under (UT) and over triage (OT). Using a national database, we aimed to describe UT and OT of NFTI vs standard Cribari method (CM) and hypothesized triage sensitivity remains poor. METHODS: The 2021 Trauma Quality Improvement Program (TQIP) database was queried. Demographics, mechanism, verification level, interfacility transfer (IF), and level of activation were collected. Patients were stratified by both NFTI [+ vs -] and CM [Injury severity score (ISS) < 15 vs > 15]. UT was defined as NFTI + or ISS >15 without full trauma activation. RESULTS: 1,030,526 patients were identified in TQIP. 84,969 were UT and 97,262 were OT using NFTI while 94,020 were UT and 108,823 were OT using CM. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of NFTI is 49%, 89%, 45%, and 90%, respectively vs 43%, 87%, 39%, and 89% of CM, respectively. Age was higher in the UT group using both scores (52 vs 42, P < .0001 and 54 vs 42, P < .0001, respectively). Using MLR, level 2 and 3 verification, blunt mechanism, female, IF, and older age were associated with UT in both NFTI and CM. Level 1 verification, penetrating mechanism, male, no IF, and younger age were associated with OT. CONCLUSIONS: Current prehospital triage criteria have poor sensitivity for identifying severely injured trauma patients by both NFTI and CM. UT increases as age of the patient increases. Further studies are needed to improve triage.

A Hedgehog- and Antennapedia-dependent niche maintains Drosophila haematopoietic precursors.

The Drosophila melanogaster lymph gland is a haematopoietic organ in which pluripotent blood cell progenitors proliferate and mature into differentiated haemocytes. Previous work has defined three domains, the medullary zone, the cortical zone and the posterior signalling centre (PSC), within the developing third-instar lymph gland. The medullary zone is populated by a core of undifferentiated, slowly cycling progenitor cells, whereas mature haemocytes comprising plasmatocytes, crystal cells and lamellocytes are peripherally located in the cortical zone. The PSC comprises a third region that was first defined as a small group of cells expressing the Notch ligand Serrate. Here we show that the PSC is specified early in the embryo by the homeotic gene Antennapedia (Antp) and expresses the signalling molecule Hedgehog. In the absence of the PSC or the Hedgehog signal, the precursor population of the medullary zone is lost because cells differentiate prematurely. We conclude that the PSC functions as a haematopoietic niche that is essential for the maintenance of blood cell precursors in Drosophila. Identification of this system allows the opportunity for genetic manipulation and direct in vivo imaging of a haematopoietic niche interacting with blood precursors.

Risk factors for clostridium difficile infection in general surgery patients.

BACKGROUND: Clostridium Difficile Infection (CDI) is a significant cause of mortality. This study aims to identify predictors of CDI in general surgery patients. METHODS: Patients who underwent general surgery operations in the 2019 National Surgical Quality Improvement Program database were identified with demographic, intervention, and outcome data abstracted. Patients with CDI and no CDI were compared by univariate analysis. Multivariable logistic regression (MLR) was performed to determine independent predictors of CDI. RESULTS: Of 436,831 surgical patients, 1,840 patients were diagnosed with CDI (0.4%). Patients with CDI have a higher mortality (2.1% vs 0.76%,p < 0.0001), longer length of stay (7 days vs 1 day, p < 0.0001), and are less likely to undergo a laparoscopic procedure (29.9% vs 37.5%, p < 0.0001). MLR identified older age, emergent operation, increased time to operation, surgical site infection, deep organ space infection, steroid use, metastatic cancer, smoking, and decreased body mass index (BMI) as independent predictors of CDI. CONCLUSIONS: CDI is rare following general surgery. Infections, delay to operation, and emergency operations are associated with CDI.

Fly-CURE, a Multi-institutional CURE using Drosophila, Increases Students' Confidence, Sense of Belonging, and Persistence in Research.

The Fly-CURE is a genetics-focused multi-institutional Course-Based Undergraduate Research Experience (CURE) that provides undergraduate students with hands-on research experiences within a course. Through the Fly-CURE, undergraduate students at diverse types of higher education institutions across the United States map and characterize novel mutants isolated from a genetic screen in Drosophila melanogaster. To evaluate the impact of the Fly-CURE experience on students, we developed and validated assessment tools to identify students' perceived research self-efficacy, sense of belonging in science, and intent to pursue additional research opportunities. Our data show gains in these metrics after completion of the Fly-CURE across all student subgroups analyzed, including comparisons of gender, academic status, racial and ethnic groups, and parents' educational background. Importantly, our data also show differential gains in the areas of self-efficacy and interest in seeking additional research opportunities between Fly-CURE students with and without prior research experience, illustrating the positive impact of research exposure (dosage) on student outcomes. Altogether, our data indicate that the Fly-CURE experience has a significant impact on students' efficacy with research methods, sense of belonging to the scientific community, and interest in pursuing additional research experiences.

Fly-CURE, a multi-institutional CURE using Drosophila, increases students' confidence, sense of belonging, and persistence in research.

The Fly-CURE is a genetics-focused multi-institutional Course-Based Undergraduate Research Experience (CURE) that provides undergraduate students with hands-on research experiences within a course. Through the Fly-CURE, undergraduate students at diverse types of higher education institutions across the United States map and characterize novel mutants isolated from a genetic screen in Drosophila melanogaster. To date, more than 20 mutants have been studied across 20 institutions, and our scientific data have led to eleven publications with more than 500 students as authors. To evaluate the impact of the Fly-CURE experience on students, we developed and validated assessment tools to identify students' perceived research self-efficacy, sense of belonging in science, and intent to pursue additional research opportunities. Our data, collected over three academic years and involving 14 institutions and 480 students, show gains in these metrics after completion of the Fly-CURE across all student subgroups analyzed, including comparisons of gender, academic status, racial and ethnic groups, and parents' educational background. Importantly, our data also show differential gains in the areas of self-efficacy and interest in seeking additional research opportunities between Fly-CURE students with and without prior research experience, illustrating the positive impact of research exposure (dosage) on student outcomes. Altogether, our data indicate that the Fly-CURE experience has a significant impact on students' efficacy with research methods, sense of belonging to the scientific research community, and interest in pursuing additional research experiences.

G-TRACE: rapid Gal4-based cell lineage analysis in Drosophila.

We combined Gal4-UAS and the FLP recombinase-FRT and fluorescent reporters to generate cell clones that provide spatial, temporal and genetic information about the origins of individual cells in Drosophila melanogaster. We named this combination the Gal4 technique for real-time and clonal expression (G-TRACE). The approach should allow for screening and the identification of real-time and lineage-traced expression patterns on a genomic scale.

The I.3.2 developmental mutant has a single nucleotide deletion in the gene centromere identifier.

The mutation I.3.2 was previously identified in a FLP/FRT screen of chromosome 2R for conditional growth regulators. Here we report the phenotypic characterization and genetic mapping of I.3.2 by undergraduate students participating in Fly-CURE, a pedagogical program that teaches the science of genetics through a classroom research experience. We find that creation of I.3.2 cell clones in the developing eye-antennal imaginal disc causes a headless adult phenotype, suggestive of both autonomous and non-autonomous effects on cell growth or viability. We also identify the I.3.2 mutation as a loss-of-function allele of the gene centromere identifier ( cid ), which encodes centromere-specific histone H3 variant critical for chromosomal segregation.

A functional genomics screen identifying blood cell development genes in Drosophila by undergraduates participating in a course-based research experience.

Undergraduate students participating in the UCLA Undergraduate Research Consortium for Functional Genomics (URCFG) have conducted a two-phased screen using RNA interference (RNAi) in combination with fluorescent reporter proteins to identify genes important for hematopoiesis in Drosophila. This screen disrupted the function of approximately 3500 genes and identified 137 candidate genes for which loss of function leads to observable changes in the hematopoietic development. Targeting RNAi to maturing, progenitor, and regulatory cell types identified key subsets that either limit or promote blood cell maturation. Bioinformatic analysis reveals gene enrichment in several previously uncharacterized areas, including RNA processing and export and vesicular trafficking. Lastly, the participation of students in this course-based undergraduate research experience (CURE) correlated with increased learning gains across several areas, as well as increased STEM retention, indicating that authentic, student-driven research in the form of a CURE represents an impactful and enriching pedagogical approach.

Clinical Decision Support Intervention for Rib Fracture Treatment.

BACKGROUND: Rib fractures are associated with significant morbidity and mortality. Despite the publication of management guidelines and national outcomes benchmarking, there is significant variation in evidence-based (EB) adherence and outcomes. Systems for clinical decision support intervention (CDSI) allow rapid ordering of bundled disease-specific EB treatments. We developed an EB rib fracture protocol and CDSI at our institution. The purpose of the current study was to evaluate implementation and clinical outcomes using this CDSI. STUDY DESIGN: A rib fracture care CDSI was developed, disseminated, and implemented in July 2018. Implementation outcomes were evaluated using the Proctor framework. Adherence was tracked monthly via run charts and acceptance was evaluated on a 7-point Likert scale using the Unified Theory of Acceptance and Use of Technology questionnaire. Propensity score matching was used to compare in-hospital morbidity and mortality in pre-implementation (January 1, 2016 through December 31, 2016) vs post-implementation (September 1, 2018 through April 30, 2019) cohorts. RESULTS: A total of 197 patients were eligible for the intervention. Provider CDSI adherence was 83% at 1 month and reached 100% after 7 months. Acceptance of CDSI using the Unified Theory of Acceptance and Use of Technology had a mean Likert score higher than 6 (range 6.1 to 6.8, SD 0.5 to 1.5), indicating high acceptance. A significant reduction in hospital length of stay was found post implementation (incident rate ratio 0.80; 95% CI, 0.66 to 0.98; p = 0.03) comparing propensity-matched subjects. CONCLUSIONS: The development and use of a CDSI resulted in improved provider delivery of EB practice and was associated with reduced hospital length of stay.

Long-term functional outcomes after traumatic popliteal artery injury: A 20-year experience.

BACKGROUND: Traumatic popliteal arterial injury (TPAI) is associated with a risk of both limb loss and long-term morbidity due to prolonged ischemia and the often-associated musculoskeletal injuries. Long-term functional outcome following this injury has not been adequately studied. We evaluated patients with TPAI to determine if there was an improvement in functional outcome over time. We hypothesized that both the initial severity of ischemia and the associated injuries limited the ability of patients to improve functional outcome. METHODS: Patients with TPAI for 20 years were identified. All patients had at least a 2-year follow-up. Functional outcomes were measured using the Boston University Activity Measure for Post-Acute Care to assess basic mobility (BM) and daily activity (DA). Multiple linear regression, adjusted for age, severity of injury and shock, operative complexity, associated injuries, ischemic time, and length of follow-up were used to identify predictors of functional outcome after TPAI. RESULTS: A total of 214 patients were identified: 123 penetrating (57%) and 91 blunt (43%). Overall mortality was 1.9% (all in-hospital), and amputation occurred in 10%. Of the 210 survivors, follow-up was obtained in 145 patients (69%). Median follow-up was 9.2 years (interquartile range, 5.7-15.7 years). Mean Activity Measure for Post-Acute Care scores for BM and DA were 78 and 75, respectively, both signifying mild impairment (normal, >84). Multiple linear regression failed to identify increasing length of follow-up as a predictor of improved functional outcomes. Only age, lower extremity fracture, and ischemic time were identified as predictors of decreased BM and DA. CONCLUSION: Increasing age, lower extremity fracture, and prolonged ischemic time worsened long-term functional outcomes. Functional outcome did not improve over time, suggesting that maximal recovery may be achieved within the first 2 years postinjury. Thus, early and effective revascularization remains the only potentially modifiable risk factor for improving functional outcomes following TPAI. LEVEL OF EVIDENCE: Prognostic, level III.

Thicker than blood: conserved mechanisms in Drosophila and vertebrate hematopoiesis.

Blood development in Drosophila melanogaster shares several interesting features with hematopoiesis in vertebrates, including spatiotemporal regulation as well as the use of similar transcriptional regulators and signaling pathways. In this review, we describe what is known about hematopoietic development in Drosophila and the various cell types generated and their functions. Additionally, the molecular genetic mechanisms of hematopoietic cell fate determination and commitment within Drosophila blood cell lineages are discussed and compared to vertebrate mechanisms.

Expression-Based Cell Lineage Analysis in Drosophila Through a Course-Based Research Experience for Early Undergraduates.

A variety of genetic techniques have been devised to determine cell lineage relationships during tissue development. Some of these systems monitor cell lineages spatially and/or temporally without regard to gene expression by the cells, whereas others correlate gene expression with the lineage under study. The GAL4 Technique for Real-time and Clonal Expression (G-TRACE) system allows for rapid, fluorescent protein-based visualization of both current and past GAL4 expression patterns and is therefore amenable to genome-wide expression-based lineage screens. Here we describe the results from such a screen, performed by undergraduate students of the University of California, Los Angeles (UCLA) Undergraduate Research Consortium for Functional Genomics (URCFG) and high school summer scholars as part of a discovery-based education program. The results of the screen, which reveal novel expression-based lineage patterns within the brain, the imaginal disc epithelia, and the hematopoietic lymph gland, have been compiled into the G-TRACE Expression Database (GED), an online resource for use by the Drosophila research community. The impact of this discovery-based research experience on student learning gains was assessed independently and shown to be greater than that of similar programs conducted elsewhere. Furthermore, students participating in the URCFG showed considerably higher STEM retention rates than UCLA STEM students that did not participate in the URCFG, as well as STEM students nationwide.

Evolution of the total syntheses of ustiloxin natural products and their analogues.

Ustiloxins A-F are antimitotic heterodetic cyclopeptides containing a 13-membered cyclic core structure with a synthetically challenging chiral tertiary alkyl-aryl ether linkage. The first total synthesis of ustiloxin D was achieved in 31 linear steps using an S(N)Ar reaction. An NOE study of this synthetic product showed that ustiloxin D existed as a single atropisomer. Subsequently, highly concise and convergent syntheses of ustiloxins D and F were developed by utilizing a newly discovered ethynyl aziridine ring-opening reaction in a longest linear sequence of 15 steps. The approach was further optimized to achieve a better macrolactamization strategy. Ustiloxins D, F, and eight analogues (14-MeO-ustiloxin D, four analogues with different amino acid residues at the C-6 position, and three (9R,10S)-epi-ustiloxin analogues) were prepared via the second-generation route. Evaluation of these compounds as inhibitors of tubulin polymerization demonstrated that variation at the C-6 position is tolerated to a certain extent. In contrast, the S configuration of the C-9 methylamino group and a free phenolic hydroxyl group are essential for inhibition of tubulin polymerization.

Keeping it Simple: Impact of a Restrictive Antibiotic Policy for Ventilator-Associated Pneumonia in Trauma Patients on Incidence and Sensitivities of Causative Pathogens.

BACKGROUND: An integral part of ventilator-associated pneumonia (VAP) therapy is the appropriate choice of empiric antibiotics. Our previous experience demonstrated adherence to an empiric therapy pathway was associated with only modest changes in organisms causing VAP. The purpose of the current study was to evaluate the impact of a restrictive antibiotic policy for VAP in trauma patients on the incidence and sensitivities of causative pathogens since the previous study. PATIENTS AND METHODS: Patients with VAP diagnosed on bronchoalveolar lavage since the previous study were stratified by age, gender, mechanism of injury, and injury severity. All patients received empiric antibiotics based on duration of intensive care unit (ICU) stay using a unit-specific pathway. The incidence and sensitivities of causative pathogens in the current study were documented. The adequacy of the VAP pathway was evaluated for all VAP episodes. The current study was then compared with the previous study. RESULTS: Over a 10-year period, 1,474 episodes of VAP were diagnosed with 2,387 causative pathogens isolated. Overall incidence of gram-positive and gram-negative VAP pathogens was unchanged between the study periods. The current study experienced an increase in the incidence of Staphylococcus aureus (23% vs. 17%, p = 0.001) and methicillin-resistant Staphylococcus aureus (10% vs. 6%, p = 0.002) compared with the previous study. The pathway for empiric antibiotics resulted in adequate empiric coverage in 85% of VAP episodes, which was improved compared with the previous study (76%, p = 0.024). Furthermore, despite the increased incidence of early methicillin-resistant Staphylococcus aureus (MRSA) VAP, adequacy of the pathway improved for both the early period (91% vs. 86%, p = 0.001) as well as the late period (86% vs. 63%, p < 0.001) in the current study compared with the previous study. CONCLUSIONS: A comprehensive protocol for the diagnosis and management of VAP, along with antibiotic stewardship, can prevent the development of bacterial resistance to empiric therapy.