Mendelian randomization analyses clarify the effects of height on cardiovascular diseases.

An inverse correlation between stature and risk of coronary artery disease (CAD) has been observed in several epidemiologic studies, and recent Mendelian randomization (MR) experiments have suggested causal association. However, the extent to which the effect estimated by MR can be explained by cardiovascular, anthropometric, lung function, and lifestyle-related risk factors is unclear, with a recent report suggesting that lung function traits could fully explain the height-CAD effect. To clarify this relationship, we utilized a well-powered set of genetic instruments for human stature, comprising >1,800 genetic variants for height and CAD. In univariable analysis, we confirmed that a one standard deviation decrease in height (~6.5 cm) was associated with a 12.0% increase in the risk of CAD, consistent with previous reports. In multivariable analysis accounting for effects from up to 12 established risk factors, we observed a >3-fold attenuation in the causal effect of height on CAD susceptibility (3.7%, p = 0.02). However, multivariable analyses demonstrated independent effects of height on other cardiovascular traits beyond CAD, consistent with epidemiologic associations and univariable MR experiments. In contrast with published reports, we observed minimal effects of lung function traits on CAD risk in our analyses, indicating that these traits are unlikely to explain the residual association between height and CAD risk. In sum, these results suggest the impact of height on CAD risk beyond previously established cardiovascular risk factors is minimal and not explained by lung function measures.

Single-cell transcriptomics reveal synergistic and antagonistic effects of T21 and GATA1s on hematopoiesis.

Trisomy 21 (T21), or Down syndrome (DS), is associated with baseline macrocytic erythrocytosis, thrombocytopenia, and neutrophilia, and transient abnormal myelopoiesis (TAM) and myeloid leukemia of DS (ML-DS). TAM and ML-DS blasts both arise from an aberrant megakaryocyte-erythroid progenitor and exclusively express GATA1s, the truncated isoform of GATA1 , while germline GATA1s mutations in a non-T21 context lead to congenital cytopenias without a leukemic predisposition. This suggests that T21 and GATA1s perturb hematopoiesis independently and synergistically, but this interaction has been challenging to study in part due to limited human cell and murine models. To dissect the developmental impacts of GATA1s on hematopoiesis in euploid and T21 cells, we performed a single-cell RNA-sequencing timecourse on hematopoietic progenitors (HPCs) derived from isogenic human induced pluripotent stem cells differing only by chromosome 21 and/or GATA1 status. These HPCs were surprisingly heterogeneous and displayed spontaneous lineage skew apparently dictated by T21 and/or GATA1s. In euploid cells, GATA1s nearly eliminated erythropoiesis, impaired MK maturation, and promoted an immature myelopoiesis, while in T21 cells, GATA1s appeared to compete with the enhanced erythropoiesis and suppressed megakaryopoiesis driven by T21 to give rise to immature erythrocytes, MKs, and myeloid cells. T21 and GATA1s both disrupted temporal regulation of lineage-specific transcriptional programs and specifically perturbed cell cycle genes. These findings in an isogenic system can thus be attributed specifically to T21 and GATA1s and suggest that these genetic changes together enhance HPC proliferation at the expense of maturation, consistent with a pro-leukemic phenotype.

Nuclear Isolation from Cryopreserved In Vitro Derived Blood Cells.

Induced pluripotent stem cell (iPSC)-based models are excellent platforms to understand blood development, and iPSC-derived blood cells have translational utility as clinical testing reagents and transfusable cell therapeutics. The advent and expansion of multiomics analysis, including but not limited to single nucleus RNA sequencing (snRNAseq) and Assay for Transposase-Accessible Chromatin sequencing (snATACseq), offers the potential to revolutionize our understanding of cell development. This includes developmental biology using in vitro hematopoietic models. However, it can be technically challenging to isolate intact nuclei from cultured or primary cells. Different cell types often require tailored nuclear preparations depending on cellular rigidity and content. These technical difficulties can limit data quality and act as a barrier to investigators interested in pursuing multiomics studies. Specimen cryopreservation is often necessary due to limitations with cell collection and/or processing, and frozen samples can present additional technical challenges for intact nuclear isolation. In this manuscript, we provide a detailed method to isolate high-quality nuclei from iPSC-derived cells at different stages of in vitro hematopoietic development for use in single-nucleus multiomics workflows. We have focused the method development on the isolation of nuclei from iPSC-derived adherent stromal/endothelial cells and non-adherent hematopoietic progenitor cells, as these represent very different cell types with regard to structural and cellular identity. The described troubleshooting steps limited nuclear clumping and debris, allowing the recovery of nuclei in sufficient quantity and quality for downstream analyses. Similar methods may be adapted to isolate nuclei from other cryopreserved cell types.

Comparison of transvaginal ultrasound utilization between radiology and point of care ultrasound in first trimester pregnancy.

BACKGROUND: Transvaginal (TVUS) and transabdominal ultrasound (TAUS) are both utilized in the evaluation of early pregnancy patients. While many practitioners using point of care ultrasound (POCUS) will generally not pursue TVUS in cases where an intrauterine pregnancy (IUP) is visualized on TAUS, this may not be true in Radiology performed ultrasound. OBJECTIVES: To evaluate for differences in transvaginal ultrasound (TVUS) utilization between Radiology performed (RP) ultrasound and point of care ultrasound (POCUS) by Emergency Department (ED) physicians in early pregnancy patients. Secondarily, to assess length of stay (LOS) differences and the impact of specialized emergency ultrasound training on TVUS utilization. METHODS: This was a retrospective study at a single academic ED. Study population was all ED patients who underwent first trimester ultrasound during the one year period of March 1, 2021 to February 28, 2022. Variables evaluated were chief complaint, gestational age, LOS, TAUS and TVUS utilization, ultrasound findings, and ultrasound specialty training of the ED physician. RESULTS: There were 133 cases of POCUS ultrasound and 254 cases of RP ultrasound. All cases had TAUS imaging performed. Median LOS for patients when POCUS was utilized was 207 min (IQR 151-294) and 258 min (IQR 208-328) for those only using RP ultrasound, p ≤ 0.001. In the POCUS cohort, 38% (95% CI 30%-46%) received TVUS, while 94% received TVUS in the RP cohort (95% CI 90%-96%), p ≤ 0.001. Patients seen by ED faculty with ultrasound specialty training had TVUS 53% of the time (95% CI 41%-65%), while those seen by other ED faculty had TVUS 79% (95% CI 74%-83%) of the time, p = 0.035. CONCLUSION: POCUS in early pregnancy is associated with a significant reduction in TVUS usage. We suspect that POCUS users elect not to pursue TVUS after an IUP is identified on TAUS, while technicians perform protocol-based TVUS irrespective of TAUS findings. Patients seen by ultrasound trained ED physicians are less likely to receive TVUS.

Modeling primitive and definitive erythropoiesis with induced pluripotent stem cells.

ABSTRACT: During development, erythroid cells are produced through at least 2 distinct hematopoietic waves (primitive and definitive), generating erythroblasts with different functional characteristics. Human induced pluripotent stem cells (iPSCs) can be used as a model platform to study the development of red blood cells (RBCs) with many of the differentiation protocols after the primitive wave of hematopoiesis. Recent advances have established that definitive hematopoietic progenitors can be generated from iPSCs, creating a unique situation for comparing primitive and definitive erythrocytes derived from cell sources of identical genetic background. We generated iPSCs from healthy fetal liver (FL) cells and produced isogenic primitive or definitive RBCs which were compared directly to the FL-derived RBCs. Functional assays confirmed differences between the 2 programs, with primitive RBCs showing a reduced proliferation potential, larger cell size, lack of Duffy RBC antigen expression, and higher expression of embryonic globins. Transcriptome profiling by scRNA-seq demonstrated high similarity between FL- and iPSC-derived definitive RBCs along with very different gene expression and regulatory network patterns for primitive RBCs. In addition, iPSC lines harboring a known pathogenic mutation in the erythroid master regulator KLF1 demonstrated phenotypic changes specific to definitive RBCs. Our studies provide new insights into differences between primitive and definitive erythropoiesis and highlight the importance of ontology when using iPSCs to model genetic hematologic diseases. Beyond disease modeling, the similarity between FL- and iPSC-derived definitive RBCs expands potential applications of definitive RBCs for diagnostic and transfusion products.

Quantitative label-free mass spectrometry reveals content and signaling differences between neonatal and adult platelets.

BACKGROUND: Recent clinical studies have shown that transfusions of adult platelets increase morbidity and mortality in preterm infants. Neonatal platelets are hyporesponsive to agonist stimulation, and emerging evidence suggests developmental differences in platelet immune functions. OBJECTIVES: This study was designed to compare the proteome and phosphoproteome of resting adult and neonatal platelets. METHODS: We isolated resting umbilical cord blood-derived platelets from healthy full-term neonates (n = 8) and resting blood platelets from healthy adults (n = 6) and compared protein and phosphoprotein contents using data-independent acquisition mass spectrometry. RESULTS: We identified 4770 platelet proteins with high confidence across all samples. Adult and neonatal platelets were clustered separately by principal component analysis. Adult platelets were significantly enriched in immunomodulatory proteins, including ß2 microglobulin and CXCL12, whereas neonatal platelets were enriched in ribosomal components and proteins involved in metabolic activities. Adult platelets were enriched in phosphorylated GTPase regulatory enzymes and proteins participating in trafficking, which may help prime them for activation and degranulation. Neonatal platelets were enriched in phosphorylated proteins involved in insulin growth factor signaling. CONCLUSION: Using label-free data-independent acquisition mass spectrometry, our findings expanded the known neonatal platelet proteome and identified important differences in protein content and phosphorylation between neonatal and adult platelets. These developmental differences suggested enhanced immune functions for adult platelets and presence of molecular machinery related to platelet activation. These findings are important to understanding mechanisms underlying key platelet functions as well as the harmful effects of adult platelet transfusions given to preterm infants.

Tropomyosin 1 deficiency facilitates cell state transitions to enhance hemogenic endothelial cell specification during hematopoiesis.

Tropomyosins coat actin filaments and impact actin-related signaling and cell morphogenesis. Genome-wide association studies have linked Tropomyosin 1 (TPM1) with human blood trait variation. Prior work suggested that TPM1 regulated blood cell formation in vitro, but it was unclear how or when TPM1 affected hematopoiesis. Using gene-edited induced pluripotent stem cell (iPSC) model systems, TPM1 knockout was found to augment developmental cell state transitions, as well as TNFα and GTPase signaling pathways, to promote hemogenic endothelial (HE) cell specification and hematopoietic progenitor cell (HPC) production. Single-cell analyses showed decreased TPM1 expression during human HE specification, suggesting that TPM1 regulated in vivo hematopoiesis via similar mechanisms. Indeed, analyses of a TPM1 gene trap mouse model showed that TPM1 deficiency enhanced the formation of HE during embryogenesis. These findings illuminate novel effects of TPM1 on developmental hematopoiesis.

Phosphoproteomics reveals content and signaling differences between neonatal and adult platelets.

Background and Objective: Recent clinical studies have shown that transfusions of adult platelets increase morbidity and mortality in preterm infants. Neonatal platelets are hyporesponsive to agonist stimulation, and emerging evidence suggests developmental differences in platelet immune functions. This study was designed to compare the proteome and phosphoproteome of resting adult and neonatal platelets. Methods: We isolated resting umbilical cord blood-derived platelets from healthy full term neonates (n=9) and resting blood platelets from healthy adults (n=7), and compared protein and phosphoprotein contents using data independent acquisition mass spectrometry. Results: We identified 4745 platelet proteins with high confidence across all samples. Adult and neonatal platelets clustered separately by principal component analysis. Adult platelets were significantly enriched for immunomodulatory proteins, including ß2 microglobulin and CXCL12, whereas neonatal platelets were enriched for ribosomal components and proteins involved in metabolic activities. Adult platelets were enriched for phosphorylated GTPase regulatory enzymes and proteins participating in trafficking, which may help prime them for activation and degranulation. Neonatal platelets were enriched for phosphorylated proteins involved in insulin growth factor signaling. Conclusions: Using state-of-the-art mass spectrometry, our findings expanded the known neonatal platelet proteome and identified important differences in protein content and phosphorylation compared with adult platelets. These developmental differences suggested enhanced immune functions for adult platelets and presence of a molecular machinery related to platelet activation. These findings are important to understanding mechanisms underlying key platelet functions as well as the harmful effects of adult platelet transfusions given to preterm infants.

Neonatal Thrombocytopenia as a Presenting Finding in de novo Pyruvate Kinase Deficiency.

Thrombocytopenia is a common laboratory abnormality encountered in critically ill neonates. The broad differential for thrombocytopenia, and its association with potentially severe neonatal pathology, often presents a diagnostic dilemma prompting extensive evaluation. Hemolysis due to red cell enzymopathies is a rare cause of neonatal thrombocytopenia that is typically brief and self-limiting. Here, we present a case of thrombocytopenia, refractory to transfusion, associated with anemia and hyperbilirubinemia in a neonate with pyruvate kinase deficiency (PKD) arising from compound heterozygous PKLR mutations. The nature of the thrombocytopenia in this patient created considerable diagnostic uncertainty, which was ultimately resolved by whole-exome sequencing. This case emphasizes that inherited red cell defects, such as PKD, are important to consider in cases of neonatal thrombocytopenia.

Generation of a human Tropomyosin 1 knockout iPSC line.

The CHOPWT17_TPM1KOc28 iPSC line was generated to interrogate the functions of Tropomyosin 1 (TPM1) in primary human cell development. This line was reprogrammed from a previously published wild type control iPSC line.

Focused Ultrasonography in Cardiac Arrest.

Rapid diagnostic tools available to the emergency physician caring for cardiac arrest patients are limited. Focused ultrasound (US), and in particular, focused echocardiography, is a useful tool in the evaluation of patients in cardiac arrest. It can help identify possible causes of cardiac arrest like tamponade and pulmonary embolism, which can guide therapy. US can also yield prognostic information, with lack of cardiac activity being highly specific for failure to achieve return of spontaneous circulation. US may also be used to aid in procedural guidance. Recently, focused transesophageal echocardiography has been used in the emergency department setting.

The Utility of Transvaginal Ultrasound After Intrauterine Pregnancy Identification on Transabdominal Ultrasound in Emergency Department Patients.

Introduction: Ultrasonography has an important role in the evaluation of Emergency Department (ED) patients presenting with early pregnancy complaints. Both transabdominal (TAUS) and transvaginal ultrasound (TVUS) can be utilized. While TVUS generally allows for greater detail, it is unclear how much added benefit exists in performing TVUS once an intrauterine pregnancy (IUP) has been identified on TAUS. Methods: This was a retrospective study utilizing Radiology Department ultrasound examinations obtained in first trimester pregnancy ED patients during a consecutive four month period in 2019. Studies wherein both TAUS and TVUS were both performed were included. Two ED physicians with specialized training in point of care ultrasound reviewed only the TAUS images from these studies. Their findings were compared to the Radiologist interpretation, which was inclusive of both TAUS and TVUS components of the study. Results: 108 studies met inclusion criteria. Amongst these, 82 had IUP's identified on the radiologist report. 69 studies had an IUP identified by ED physician review of the TAUS images, with 1 false positive. Each case of intrauterine fetal demise (IUFD) was identified on ED physician review of TAUS. Two ectopic pregnancies were present, neither of which was mistaken for IUP on ED physician TAUS review. There were 15 studies with subchorionic hemorrhage and 3 studies with an ovarian cyst noted on the radiologist report. Conclusion: Following the identification of an IUP on TAUS, the added diagnostic value of TVUS amongst this cohort of ED patients was low. Given the added time and cost of TVUS, selective instead of routine usage should be encouraged.

Emergency-physician Performed, Ultrasound-guided Lateral Femoral Cutaneous Nerve Block for Meralgia Paresthetica: A Report of Two Cases.

INTRODUCTION: Neuropathy of the lateral femoral cutaneous nerve, also known as meralgia paresthetica, causes pain and paresthesia to the anterolateral thigh. It commonly results from nerve irritation from extrinsic compression; however, it may occur spontaneously. Symptoms from this condition can be debilitating, and the pain may be ascribed to other conditions leading to delays in diagnosis. Peripheral nerve blockade can be useful both diagnostically and therapeutically for meralgia paresthetica. CASE REPORT: Two female patients in their sixties presented to the emergency department for chronic, atraumatic, left upper thigh pain. In both cases the patients had hyperalgesia and paresthesia to the anterolateral, upper thigh. The emergency physician performed an ultrasound-guided nerve block of the lateral femoral cutaneous nerve for each patient, which resulted in temporary, complete resolution of their pain. CONCLUSION: Meralgia paresthetica is an uncommon but painful condition that can elude diagnosis. Physical exam findings such as allodynia and hyperalgesia of the anterolateral thigh in the absence of back pain is suggestive of the diagnosis. Utrasound-guided nerve blockade can be helpful to the emergency physician to confirm the diagnosis and provide non-opioid pain relief to the patient.

Genetically influenced tobacco and alcohol use behaviors impact erythroid trait variation.

Genome wide association studies (GWAS) have associated thousands of loci with quantitative human blood trait variation. Blood trait associated loci and related genes may regulate blood cell-intrinsic biological processes, or alternatively impact blood cell development and function via systemic factors and disease processes. Clinical observations linking behaviors like tobacco or alcohol use with altered blood traits can be subject to bias, and these trait relationships have not been systematically explored at the genetic level. Using a Mendelian randomization (MR) framework, we confirmed causal effects of smoking and drinking that were largely confined to the erythroid lineage. Using multivariable MR and causal mediation analyses, we confirmed that an increased genetic predisposition to smoke tobacco was associated with increased alcohol intake, indirectly decreasing red blood cell count and related erythroid traits. These findings demonstrate a novel role for genetically influenced behaviors in determining human blood traits, revealing opportunities to dissect related pathways and mechanisms that influence hematopoiesis.

Generation of a human Tropomyosin 1 knockout iPSC line.

The CHOPWT17_TPM1KOc28 iPSC line was generated to interrogate the functions of Tropomyosin 1 ( TPM1 ) in primary human cell development. This line was reprogrammed from a previously published wild type control iPSC line.

Mendelian randomization analyses clarify the effects of height on cardiovascular diseases.

An inverse correlation between stature and risk of coronary artery disease (CAD) has been observed in several epidemiologic studies, and recent Mendelian randomization (MR) experiments have suggested causal association. However, the extent to which the effect estimated by MR can be explained by established cardiovascular risk factors is unclear, with a recent report suggesting that lung function traits could fully explain the height-CAD effect. To clarify this relationship, we utilized a well-powered set of genetic instruments for human stature, comprising >1,800 genetic variants for height and CAD. In univariable analysis, we confirmed that a one standard deviation decrease in height (~6.5 cm) was associated with a 12.0% increase in the risk of CAD, consistent with previous reports. In multivariable analysis accounting for effects from up to 12 established risk factors, we observed a >3-fold attenuation in the causal effect of height on CAD susceptibility (3.7%, p = 0.02). However, multivariable analyses demonstrated independent effects of height on other cardiovascular traits beyond CAD, consistent with epidemiologic associations and univariable MR experiments. In contrast with published reports, we observed minimal effects of lung function traits on CAD risk in our analyses, indicating that these traits are unlikely to explain the residual association between height and CAD risk. In sum, these results suggest the impact of height on CAD risk beyond previously established cardiovascular risk factors is minimal and not explained by lung function measures.

POINT-OF-CARE ULTRASOUND FOR GUIDANCE OF CLOSED REDUCTION OF FIFTH METACARPAL NECK (BOXER'S) FRACTURE.

BACKGROUND: Ultrasound has been used previously in fracture identification, analgesia delivery, and fracture reduction for patients in the emergency department. It has not been previously described as a tool for the guidance of closed fracture reduction in fifth metacarpal neck fractures ("boxer's fractures"). CASE REPORT: A 28-year-old man presented with hand pain and swelling after punching a wall. Point-of-care ultrasound revealed a significantly angulated fifth metacarpal fracture, which was confirmed with a subsequent hand x-ray study. After an ultrasound-guided ulnar nerve block, closed reduction was performed. Ultrasound was used to assess reduction and ensure improvement in bony angulation during the closed reduction attempts. Post-reduction x-ray study confirmed improved angulation and adequate alignment. Why Should an Emergency Physician Be Aware of This? Point-of-care ultrasound has previously had efficacy in fracture diagnosis and anesthesia delivery for fifth metacarpal fractures. Ultrasound can also be used at the bedside to assist in the determination of adequate fracture reduction when performing closed reduction of a boxer's fracture.

Comparison of the Quality of Echocardiography Imaging Between the Left Lateral Decubitus and Supine Positions.

Introduction It is commonly taught that positioning the patient in the left lateral decubitus (LLD) position will improve transthoracic echocardiography (TTE) image quality. Despite this, no previous studies have been performed that study this practice. Our goal was to quantify the difference in image quality of TTE views between the supine and LLD positions.  Methods This was a prospective study in a single academic Emergency Department (ED) of a convenience sample of 30 patients. Three separate ED physicians performed TTE views in both the supine and LLD position on each patient. The order of position was randomized. Images were then reviewed on a previously validated TTE image quality scale by two blinded ED physicians with specialized training in ultrasound. The scale used a 0 to 5 (highest quality) metric for quality assessment. Interpretability of right ventricular and left ventricular function was also assessed. Results The mean image quality for the supine position was 2.85 (standard deviation {SD} 1.1) and 3.05 (SD 1.2) for the LLD position (p=0.044). In the subset of parasternal and apical windows, the mean quality for the supine position was 2.87 (SD 1.1) and 3.23 (SD 1.1) for the LLD position (p=0.003). The number of studies in which right ventricular function was interpretable was significantly higher in the LLD position (62% versus 42%, p=0.044). Conclusions There was a statistically significant increase in image quality when TTE was performed in the LLD position as compared to supine. This was especially pronounced in the apical four and parasternal windows.

A Narrative Review of the Uses of Ultrasound in the Evaluation, Analgesia, and Treatment of Distal Forearm Fractures.

BACKGROUND: Distal forearm fractures are a commonly encountered injury in the emergency department (ED), accounting for 500,000 to 1.5 million visits and 17% of ED fractures. The evaluation and management of these fractures frequently employs x-ray studies, conscious sedation, closed reduction, and splinting. Point-of-care ultrasound (POCUS) can offer significant benefit in the diagnosis and management of these common injuries. OBJECTIVE OF THE REVIEW: To review the clinical utility of POCUS in the diagnosis of distal forearm fractures, as well as to demonstrate the performance of ultrasound-guided analgesia delivery and ultrasound-guided reduction technique. DISCUSSION: The initial evaluation of forearm injuries frequently includes x-ray studies. However, multiple studies have shown ultrasound to be sensitive and specific for distal radius fractures, with the added value of detecting soft tissue injuries missed by conventional radiography. POCUS may also facilitate analgesia through the use of ultrasound-guided hematoma blocks, which removes the need for conscious sedation prior to manipulation. Finally, POCUS can be used after manipulation to assess cortical realignment of the bone fragments and spare the patient multiple reduction attempts and repeat radiographs. CONCLUSION: Distal forearm fractures are common, and the emergency physician should be adept with the evaluation and management of these injuries. POCUS can be a reliable modality in the detection of these fractures and can be used to facilitate analgesia and augment success of reduction attempts. These techniques may decrease length of stay, improve patient pain, and decrease reduction attempts.