A One-Step Staining Probe for Phosphatidylethanolamine.

Phosphatidylethanolamine (PE) is an abundant phospholipid in cellular membranes, but relatively little is known about the kinetics of PE in biological membrane systems. Characterizing PE on a cellular level has been challenging owing to a lack of proper molecular tools. The lantibiotic duramycin and its structural analogue, cinnamycin, are currently the only known polypeptides that have an established stereospecific structure for binding membrane PE with high affinity and high specificity. These lantibiotics are recognized for their potential as molecular probes for studying PE kinetics in various membranes. However, owing to their antibiotic nature, duramycin and cinnamycin exhibit appreciable levels of cytotoxicity at low micromolar concentrations in cultured mammalian cells by inducing membrane distortion and possible PE redistribution. These issues can potentially complicate study design and data interpretation. Here, we report the construction of a molecular probe consisting of duramycin attached to the C terminus of green fluorescent protein (GFP) by a PEG linker at a stoichiometry of 1. The construct retained specific binding toward PE and essentially no cytotoxicity compared to native duramycin. The biological utilities of this probe were demonstrated in a number of cellular staining studies involving PE dynamics. The availability of a one-step, nontoxic molecular probe for PE will enable characterization of the biology of this important phospholipid.

In Vivo Mapping of Myocardial Injury Outside the Infarct Zone: Tissue at an Intermediate Pathological State.

BACKGROUND: The goal was to determine the feasibility of mapping the injured-but-not-infarcted myocardium using 99mTc-duramycin in the postischemic heart, with spatial information for its characterization as a pathophysiologically intermediate tissue, which is neither normal nor infarcted. METHODS AND RESULTS: Coronary occlusion was conducted in Sprague Dawley rats with preconditioning and 30-minute ligation. In vivo single-photon emission computed tomography was acquired after 3 hours (n=6) using 99mTc-duramycin, a phosphatidylethanolamine-specific radiopharmaceutical. The 99mTc-duramycin+ areas were compared with infarct and area-at-risk (n=8). Cardiomyocytes and endothelial cells were isolated for gene expression profiling. Cardiac function was measured with echocardiography (n=6) at 4 weeks. In vivo imaging with 99mTc-duramycin identified the infarct (3.9±2.4% of the left ventricle and an extensive area 23.7±2.2% of the left ventricle) with diffuse signal outside the infarct, which is pathologically between normal and infarcted (apoptosis 1.8±1.6, 8.9±4.2, 13.6±3.8%; VCAM-1 [vascular cell adhesion molecule 1] 3.2±0.8, 9.8±4.1, 15.9±4.2/mm2; tyrosine hydroxylase 14.9±2.8, 8.6±4.4, 5.6±2.2/mm2), with heterogeneous changes including scattered micronecrosis, wavy myofibrils, hydropic change, and glycogen accumulation. The 99mTc-duramycin+ tissue is quantitatively smaller than the area-at-risk (26.7% versus 34.4% of the left ventricle, P=0.008). Compared with infarct, gene expression in the 99mTc-duramycin+-noninfarct tissue indicated a greater prosurvival ratio (BCL2/BAX [B-cell lymphoma 2/BCL2-associated X] 7.8 versus 5.7 [cardiomyocytes], 3.7 versus 3.2 [endothelial]), and an upregulation of ion channels in electrophysiology. There was decreased contractility at 4 weeks (regional fractional shortening -8.6%, P<0.05; circumferential strain -52.9%, P<0.05). CONCLUSIONS: The injured-but-not-infarcted tissue, being an intermediate zone between normal and infarct, is mapped in vivo using phosphatidylethanolamine-based imaging. The intermediate zone contributes significantly to cardiac dysfunction.

Target detection with randomized thresholds for lidar applications.

Light detection and ranging (lidar) systems use binary hypothesis tests to detect the presence of a target in a range interval. For systems that count photon detections, hypothesis test thresholds are normally set so that a target detection is declared if the number of detections exceeds a particular number. When this method is employed, the false alarm probability can not be selected arbitrarily. In this paper, a hypothesis test that uses randomized thresholds is described. This randomized method of thresholding allows lidar operation at any false alarm probability. When there is a maximum allowable false alarm probability, the hypothesis test that uses randomized thresholds generally produces higher target detection probabilities than the conventional (nonrandom) hypothesis test.

Applying a Modern Pedagogy to Emergency Residency Ultrasound Education.

Background Modern technology has revolutionized pedagogy in medicine. With the availability of high-quality resources in the palm of our hands, the flipped classroom model has gained widespread support. Additionally, devices such as "clickers" allow for the ability to interact much more directly with lecture participants. Objective We aimed to investigate the impact of a modern refresh to our emergency point of care ultrasound (POCUS) curriculum on resident exam performance and satisfaction. Methods In 2021, we conducted a single-site pre-post interventional study with Emergency residents as eligible participants. The interventions included two modifications to the POCUS curriculum. First, residents prepared and delivered mini-lectures in lieu of formal didactic sessions. Second, weekly image review was reimplemented with more active participation. Our primary outcome was end-of-rotation exam performance and our secondary outcome was learner satisfaction. Results During the study period, 19 residents participated in the curriculum. Exam scores were similar, 90.84% +/- 2.27% and 89.34% +/- 3.43% (p = 0.105) for pre and post-intervention scores respectively. Satisfaction surveys were completed by 12 of the 19 participants. On a scale of 1-5, average satisfaction was fair (3.9 to 4.1) regarding the mini-lecture intervention and high (4.3 to 4.6) for the active weekly image review modification. Conclusions The POCUS curriculum refresh was well-received by emergency medicine residents. However, there was no clear competency benefit when compared to the traditional approach. The modified active weekly review was particularly well-received amongst our residents and maybe a strong model for emergency POCUS programs across the country.

Examining D-dimer and Empiric Anti-coagulation in COVID-19-Related Thrombosis.

Objective Thrombosis is thought to occur frequently in the setting of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We aimed to elucidate the relationship between macro/microvascular thrombosis, D-dimer levels, and empiric anticoagulation in coronavirus disease 2019 (COVID-19). Methods This was an exploratory prospective, single-site, observational study. Adult emergency department patients with COVID-19 requiring hospitalization received a point-of-care lower extremity venous duplex ultrasound. The primary endpoint was thromboembolism and associated D-dimer level. Secondary endpoints included rates of micro and macro thrombotic complications as well as empiric anticoagulant use. Results Between January 13th and April 12th 2021, 52 patients were enrolled. Median D-dimer at presentation was 650 ng/mL (range 250-10,000 ng/mL) among patients with negative duplex studies. During hospitalization, 18 patients underwent 20 additional studies assessing for venous thromboembolism (VTE). These studies yielded one deep vein thrombosis (DVT) diagnosis. Among patients with negative studies median D-dimer was 1,246 ng/mL (range 329-10,000 ng/mL). Two patients experienced microvascular complications. Seven patients were started on empiric full dose anticoagulation. Conclusion While VTE remains a major concern amongst patients with COVID-19, the normal D-dimer cut off of >500 ng/mL likely should not be used to initiate further VTE workup. Additionally, moderately elevated D-dimer did not correlate strongly with microvascular complications and may not be relevant in the decision to initiate empiric anticoagulation.

Targeted design of a recombinant tracer for SPECT renal imaging.

Rationale: A robust radiopharmaceutical has high uptake in the target and low retention in non-target tissues. However, traditional tracers for renal imaging that chemically chelate 99mTc are excreted through the renal route with transient resident time in the kidney. Following a rational design approach, we constructed a protein-based radiotracer, designated PBT-Fc, to sequentially bind tubular neonatal Fc-receptor and subsequently proximal tubular basement membrane for its targeted sequestration in kidney parenchyma. In this process, the tracer participates in physiologic glomerular filtration and tubular reabsorption while escaping lysosomal catabolism and urinary clearance. Methods: To specifically target renal receptors in navigating the urinary passage in the kidney, we produced a recombinant fusion protein with two separate functional parts: a polybasic PBT segment derived from human Vascular Endothelial Growth Factor and Fc segment of IgG1. The chimeric fusion of PBT-Fc was labeled with radionuclide 99mTc and tested in rodent models of kidney diseases. Planar scintigraphy and single-photon emission computerized tomography (SPECT) were performed to evaluate renal-specificity of the tracer. Results: When injected in mouse and rat, following a brief 10 - 15 min dynamic redistribution phase in circulation, ~ 95% of the [99mTc]-PBT-Fc signal was concentrated in the kidney and lasted for hours without urinary loss or surrounding tissue activities. Long-lasting tracer signals in the kidney cortex in conjunction with SPECT greatly augmented the image quality in detecting pathological lesions in a variety of disease models, including ischemic acute kidney injury, drug-induced renal toxicity, and chronic kidney disease from renin-angiotensin system (RAS) overactivation. Conclusion: Exclusive renal retention of the recombinant radiotracer greatly facilitated static-phase signal acquisition by SPECT and achieved submillimeter spatial resolution of kidney alternations in glomerular and tubular disease models.

Cramer-Rao lower bound on range error for LADARs with Geiger-mode avalanche photodiodes.

The Cramer-Rao lower bound (CRLB) on range error is calculated for laser detection and ranging (LADAR) systems using Geiger-mode avalanche photodiodes (GMAPDs) to detect reflected laser pulses. For the cases considered, the GMAPD range error CRLB is greater than the CRLB for a photon-counting device. It is also shown that the GMAPD range error CRLB is minimized when the mean energy in the received laser pulse is finite. Given typical LADAR system parameters, a Gaussian-envelope received pulse, and a noise detection rate of less than 4 MHz, the GMAPD range error CRLB is minimized when the quantum efficiency times the mean number of received laser pulse photons is between 2.2 and 2.3.

Whole-body Imaging of Cell Death Provides a Systemic, Minimally Invasive, Dynamic, and Near-real Time Indicator for Chemotherapeutic Drug Toxicity.

PURPOSE: Response to toxicity in chemotherapies varies considerably from tissue to tissue and from patient to patient. An ability to monitor the tissue damage done by chemotherapy may have a profound impact on treatment and prognosis allowing for a proactive management in understanding and mitigating such events. For the first time, we investigated the feasibility of using whole-body imaging to map chemotherapeutic drug-induced toxicity on an individual basis. EXPERIMENTAL DESIGN: In a preclinical proof-of-concept, rats were treated with a single clinical dose of cyclophosphamide, methotrexate, or cisplatin. In vivo whole-body imaging data were acquired using 99mTc-duramycin, which identifies dead and dying cells as an unambiguous marker for tissue injury in susceptible organs. Imaging results were cross-validated using quantitative ex vivo measurements and histopathology and compared with standard blood and serum panels for toxicology. RESULTS: The in vivo whole-body imaging data detected widespread changes, where spatially heterogeneous toxic effects were identified across different tissues, within substructures of organs, as well as among different individuals. The signal changes were consistent with established toxicity profiles of these chemotherapeutic drugs. Apart from generating a map of susceptible tissues, this in vivo imaging approach was more sensitive compared with conventional blood and serum markers used in toxicology. Also, repeated imaging during the acute period after drug treatment captured different kinetics of tissue injury among susceptible organs in males and females. CONCLUSIONS: This novel and highly translational imaging approach shows promise in optimizing therapeutic decisions by detecting and managing drug toxicity on a personalized basis.Toxicity to normal tissues is a significant limitation in chemotherapies. This work demonstrated an in vivo imaging-based approach for characterizing toxicity-induced tissue injury in a systemic, dynamic, and near-real time fashion. This novel approach shows promise in optimizing therapeutic decisions by monitoring drug toxicity on a personalized basis.

Decreased bone turnover with balanced resorption and formation prevent cortical bone loss during disuse (hibernation) in grizzly bears (Ursus arctos horribilis).

Disuse uncouples bone formation from resorption, leading to increased porosity, decreased bone geometrical properties, and decreased bone mineral content which compromises bone mechanical properties and increases fracture risk. However, black bear bone properties are not adversely affected by aging despite annual periods of disuse (i.e., hibernation), which suggests that bears either prevent bone loss during disuse or lose bone and subsequently recover it at a faster rate than other animals. Here we show decreased cortical bone turnover during hibernation with balanced formation and resorption in grizzly bear femurs. Hibernating grizzly bear femurs were less porous and more mineralized, and did not demonstrate any changes in cortical bone geometry or whole bone mechanical properties compared to active grizzly bear femurs. The activation frequency of intracortical remodeling was 75% lower during hibernation than during periods of physical activity, but the normalized mineral apposition rate was unchanged. These data indicate that bone turnover decreases during hibernation, but osteons continue to refill at normal rates. There were no changes in regional variation of porosity, geometry, or remodeling indices in femurs from hibernating bears, indicating that hibernation did not preferentially affect one region of the cortex. Thus, grizzly bears prevent bone loss during disuse by decreasing bone turnover and maintaining balanced formation and resorption, which preserves bone structure and strength. These results support the idea that bears possess a biological mechanism to prevent disuse osteoporosis.

The feasibility of imaging myocardial ischemic/reperfusion injury using (99m)Tc-labeled duramycin in a porcine model.

UNLABELLED: When pathologically externalized, phosphatidylethanolamine (PE) is a potential surrogate marker for detecting tissue injuries. (99m)Tc-labeled duramycin is a peptide-based imaging agent that binds PE with high affinity and specificity. The goal of the current study was to investigate the clearance kinetics of (99m)Tc-labeled duramycin in a large animal model (normal pigs) and to assess its uptake in the heart using a pig model of myocardial ischemia-reperfusion injury. METHODS: The clearance and distribution of intravenously injected (99m)Tc-duramycin were characterized in sham-operated animals (n=5). In a closed chest model of myocardial ischemia, coronary occlusion was induced by balloon angioplasty (n=9). (99m)Tc-duramycin (10-15mCi) was injected intravenously at 1hour after reperfusion. SPECT/CT was acquired at 1 and 3hours after injection. Cardiac tissues were analyzed for changes associated with acute cellular injuries. Autoradiography and gamma counting were used to determine radioactivity uptake. For the remaining animals, (99m)Tc-tetrafosamin scan was performed on the second day to identify the infarct site. RESULTS: Intravenously injected (99m)Tc-duramycin cleared from circulation predominantly via the renal/urinary tract with an α-phase half-life of 3.6±0.3minutes and ß-phase half-life of 179.9±64.7minutes. In control animals, the ratios between normal heart and lung were 1.76±0.21, 1.66±0.22, 1.50±0.20 and 1.75±0.31 at 0.5, 1, 2 and 3hours post-injection, respectively. The ratios between normal heart and liver were 0.88±0.13, 0.80±0.13, 0.82±0.19 and 0.88±0.14. In vivo visualization of focal radioactivity uptake in the ischemic heart was attainable as early as 30min post-injection. The in vivo ischemic-to-normal uptake ratios were 3.57±0.74 and 3.69±0.91 at 1 and 3hours post-injection, respectively. Ischemic-to-lung ratios were 4.89±0.85 and 4.93±0.57; and ischemic-to-liver ratios were 2.05±0.30 to 3.23±0.78. The size of (99m)Tc-duramycin positive myocardium was qualitatively larger than the infarct size delineated by the perfusion defect in (99m)Tc-tetrafosmin uptake. This was consistent with findings from tissue analysis and autoradiography. CONCLUSION: (99m)Tc-duramycin was demonstrated, in a large animal model, to have suitable clearance and biodistribution profiles for imaging. The agent has an avid target uptake and a fast background clearance. It is appropriate for imaging myocardial injury induced by ischemia/reperfusion.

Pierre Robin sequence in a patient with ectrodactyly-ectodermal dysplasia-clefting syndrome: a case report and review of the literature.

Ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome is a rare condition first identified in 1970. The majority of cases are reported to be autosomal dominant with variable expression and penetrance. The most common clinical feature is ectodermal dysplasia. Ectrodactyly, cleft lip with or with out cleft palate and urogenital anomalies are also main clinical features of EEC syndrome. Pierre Robin (PR) sequence has the clinical findings of micro-retrognathia, retroposed tongue (glossoptosis), cleft of the secondary palate, and upper airway obstruction. Etiologically PR sequence is heterogeneous. It has been reported that less than 20% of cases are isolated, non-syndromic PR sequence. We present a case of a newborn male who was referred to our craniofacial clinic with EEC syndrome and PR sequence. This case represents a unique clinical association. We found no other reports in the literature of these two clinical entities occurring together. Upper airway obstruction of PR sequence, secondary to glossoptosis and micro-retrognathia, gives this association clinical relevance. In addition, this clinical association may represent a new gene locus associated with EEC syndrome.

Whole-body imaging of high-dose ionizing irradiation-induced tissue injuries using 99mTc-duramycin.

UNLABELLED: High-dose ionizing irradiation can cause extensive injuries in susceptible tissues. A noninvasive imaging technique that detects a surrogate marker of apoptosis may help characterize the dynamics of radiation-induced tissue damage. The goal of this study was to prove the concept of imaging the temporal and spatial distribution of damage in susceptible tissues after high-dose radiation exposure, using (99m)Tc-duramycin as a phosphatidylethanolamine-binding radiopharmaceutical. METHODS: Rats were subjected to 15 Gy of total-body irradiation with x-rays. Planar whole-body (99m)Tc-duramycin scanning (n = 4 per time point) was conducted at 24, 48, and 72 h using a clinical γ-camera. On the basis of findings from planar imaging, preclinical SPECT data were acquired on control rats and on irradiated rats at 6 and 24 h after irradiation (n = 4 per time point). Imaging data were validated by γ-counting and histology, using harvested tissues in parallel groups of animals (n = 4). RESULTS: Prominent focal uptake was detected in the thymus as early as 6 h after irradiation, followed by a gradual decline in (99m)Tc-duramycin binding accompanied by extensive thymic atrophy. Early (6-24 h) radioactivity uptake in the gastrointestinal region was detected. Significant signal was seen in major bones in a slightly delayed fashion, at 24 h, which persisted for at least 2 d. This finding was paralleled by an elevation in signal intensity in the kidneys, spleen, and liver. The imaging results were consistent with ex vivo γ-counting results and histology. Relatively high levels of apoptosis were detected from histology in the thymus, guts, and bones, with the thymus undergoing substantial atrophy. CONCLUSION: As a proof of principle, this study demonstrated a noninvasive imaging technique that allows characterization of the temporal and spatial dynamics of injuries in susceptible tissues during the acute phase after high-dose ionizing irradiation. Such an imaging capability will potentially be useful for global, whole-body, assessment of tissue damage after radiation exposure. These data, in turn, will contribute to our general knowledge of tissue susceptibility to ionizing irradiation, as well as the onset and progression of tissue injuries.