Rapid Operationalization of COVID-19 Immunization Clinics With Medical and Physician Assistant Students Serving as Vaccinators.

Introduction: In anticipation of institutional and community-wide COVID-19 immunization clinics, an educational program for the administration of COVID-19 vaccines was developed, collaborating with the Virginia Department of Health to train doctor of medicine and physician assistant students to serve as vaccinators. Faculty and students also worked with state legislatures to propose and enact a bill that would enable such students to vaccinate patients. Methods: Between January 2021 and August 2022, 263 student volunteers completed 3,685 person-hours, administering 48,279 doses. On the basis of community need, the majority of vaccines were administered at mass vaccination clinics in Chesapeake (47%) and Norfolk (22%) in Virginia. One year after the first COVID-19 immunization clinic, the authors surveyed students who assisted with clinics, utilizing a Likert scale and free-text responses to elicit feedback about the training and volunteer experience. Results: Volunteers ranked the vaccination clinics among the top third of established volunteer experiences offered at Eastern Virginia Medical School, and 75% of respondents believed that this training should be a permanent part of their program curriculum. Conclusions: This paper presents a strategy for preparing students to serve as vaccinators during public health emergencies and show how other institutions of medical education can prepare for and engage student participation in vaccination campaigns and emergent health initiatives.

Course and Treatment of a Rare Neurological Sequelae After COVID-19: Miller Fisher Syndrome.

Reports of COVID-19 infection detailing its symptoms and outcomes point to its effects systemically, including that of the nervous system, such as the rare Miller Fisher syndrome (MFS). In this report, we identified a 43-year-old Caribbean man who arrived in the USA with ataxia and ascending bilateral lower extremity weakness after COVID-19 infection. Before arrival, the patient was diagnosed with Guillain-Barré syndrome (GBS). He was treated with IV methylprednisolone and a round of IV immunoglobulin (IVIG); however, he showed a minimal response. Upon admission to our ED, he had severe tachypnea and flaccid symmetrical quadriparesis combined with areflexia. Moreover, he had begun to exhibit signs of multiple cranial nerve palsies, including ophthalmoplegia and facial diplegia. Additionally, his laboratory cerebrospinal fluid (CSF) analysis was grossly normal. Therefore, he was diagnosed with MFS. Furthermore, he developed acute depression and exhibited signs of mania. The patient was treated with IV methylprednisolone and the second round of a five-day course of IVIG, resulting in marked clinical improvement. This case highlights the need for a multidisciplinary care approach in patients with MFS. It also points to the possible benefit of multiple IVIG rounds in MFS patients who do not improve after the first course.

Structure and Computational Electrophysiology of Ac-LS3, a Synthetic Ion Channel.

Computer simulations are reported on Ac-LS3, a synthetic ion channel, containing 21 residues with a Leu-Ser-Ser-Leu-Leu-Ser-Leu heptad repeat, which forms ions channels upon application of voltage. A hexameric, coiled-coil bundle initially positioned perpendicular to the membrane settled into a stable, tilted structure after 1.5 µs, most likely to improve contacts between the non-polar exterior of the channel and the hydrophobic core of the membrane. Once tilted, the bundle remained in this state during subsequent simulations of nearly 10 µs at voltages ranging from 200 to -100 mV. In contrast, attempts to identify a stable pentameric structure failed, thus supporting the hypothesis that the channel is a hexamer. Results at 100 mV were used to reconstruct the free energy profiles for K+ and Cl- in the channel. This was done by way of several methods in which results of molecular dynamics (MD) simulations were combined with the electrodiffusion model. Two of them developed recently do not require knowledge of the diffusivity. Instead, they utilize one-sided density profiles and committor probabilities. The consistency between different methods is very good, supporting the utility of the newly developed methods for reconstructing free energies of ions in channels. The flux of K+, which accounts for most of the current through the channel, calculated directly from MD matches well the total measured current. However, the current of Cl- is somewhat overestimated, possibly due to a slightly unbalanced force field involving chloride. The current-voltage dependence was also reconstructed by way of a recently developed, efficient method that requires simulations only at a single voltage, yielding good agreement with the experiment. Taken together, the results demonstrate that computational electrophysiology has become a reliable tool for studying how channels mediate ion transport through membranes.

ASK1 is a novel molecular target for preventing aminoglycoside-induced hair cell death.

Aminoglycoside antibiotics are lifesaving medicines, crucial for the treatment of chronic or drug resistant infections. However, aminoglycosides are toxic to the sensory hair cells in the inner ear. As a result, aminoglycoside-treated individuals can develop permanent hearing loss and vestibular impairment. There is considerable evidence that reactive oxygen species (ROS) production and the subsequent phosphorylation of c-Jun N-terminal kinase (JNK) and P38 mitogen-activated protein kinase (P38) drives apoptosis in aminoglycoside-treated hair cells. However, treatment strategies that directly inhibit ROS, JNK, or P38 are limited by the importance of these molecules for normal cellular function. Alternatively, the upstream regulator apoptosis signal-regulating kinase 1 (ASK1/MAP3K5) is a key mediator of ROS-induced JNK and P38 activation under pathologic but not homeostatic conditions. We investigated ASK1 as a mediator of drug-induced hair cell death using cochlear explants from Ask1 knockout mice, demonstrating that Ask1 deficiency attenuates neomycin-induced hair cell death. We then evaluated pharmacological inhibition of ASK1 with GS-444217 as a potential otoprotective therapy. GS-444217 significantly attenuated hair cell death in neomycin-treated explants but did not impact aminoglycoside efficacy against P. aeruginosa in the broth dilution test. Overall, we provide significant pre-clinical evidence that ASK1 inhibition represents a novel strategy for preventing aminoglycoside ototoxicity. KEY MESSAGES: ASK1 is an upstream, redox-sensitive regulator of P38 and JNK, which are known mediators of hair cell death. Ask1 knockout does not affect hair cell development in vivo, but significantly reduces aminoglycoside-induced hair cell death in vitro. A small-molecule inhibitor of ASK1 attenuates neomycin-induced hair cell death, and does not impact antibiotic efficacy in vitro. ASK1 may be a novel molecular target for preventing aminoglycoside-induced hearing loss.

Electrophysiological Properties from Computations at a Single Voltage: Testing Theory with Stochastic Simulations.

We use stochastic simulations to investigate the performance of two recently developed methods for calculating the free energy profiles of ion channels and their electrophysiological properties, such as current-voltage dependence and reversal potential, from molecular dynamics simulations at a single applied voltage. These methods require neither knowledge of the diffusivity nor simulations at multiple voltages, which greatly reduces the computational effort required to probe the electrophysiological properties of ion channels. They can be used to determine the free energy profiles from either forward or backward one-sided properties of ions in the channel, such as ion fluxes, density profiles, committor probabilities, or from their two-sided combination. By generating large sets of stochastic trajectories, which are individually designed to mimic the molecular dynamics crossing statistics of models of channels of trichotoxin, p7 from hepatitis C and a bacterial homolog of the pentameric ligand-gated ion channel, GLIC, we find that the free energy profiles obtained from stochastic simulations corresponding to molecular dynamics simulations of even a modest length are burdened with statistical errors of only 0.3 kcal/mol. Even with many crossing events, applying two-sided formulas substantially reduces statistical errors compared to one-sided formulas. With a properly chosen reference voltage, the current-voltage curves can be reproduced with good accuracy from simulations at a single voltage in a range extending for over 200 mV. If possible, the reference voltages should be chosen not simply to drive a large current in one direction, but to observe crossing events in both directions.

Computational Electrophysiology from a Single Molecular Dynamics Simulation and the Electrodiffusion Model.

The availability of high-resolution structures of ion channels opens the doors to reliable computations of electrophysiological properties, such as the dependence of ionic currents and selectivities on applied voltage. We develop two theoretical approaches for calculating these properties from molecular dynamics simulations at a single voltage, or even in the absence of voltage, combined with the electrodiffusion model in which ion motion in the channel is represented as one-dimensional diffusion in the potential of mean force exerted by other components of the system and the applied electric field. No knowledge of diffusivity or ion densities at other voltages is needed. Instead, in one approach, one-sided ion fluxes and density profiles are used to determine the free energy profile. In the other approach, committor probabilities for ions transported at the selected voltage are used for this purpose. Both approaches have been validated in an example of a simple ion channel formed by trichotoxin. The potentials of mean force calculated by way of the proposed approaches and obtained from traditional methods are in excellent agreement. Furthermore, the current-voltage dependence agrees very well with results obtained by way of computationally more demanding methods. We also have readily calculated the reversal potential, a computationally challenging electrophysiological property. The key assumptions of the electrodiffusion model, such as the independence of crossing events or the insensitivity of the potential of mean force to applied voltage, have been found to be satisfied. We also show that the voltage changes linearly in the hydrophobic core of the membrane and is constant elsewhere.

Case Rates, Treatment Approaches, and Outcomes in Acute Myocardial Infarction During the Coronavirus Disease 2019 Pandemic.

Importance: The coronavirus disease 2019 (COVID-19) pandemic has changed health care delivery worldwide. Although decreases in hospitalization for acute myocardial infarction (AMI) have been reported during the pandemic, the implication for in-hospital outcomes is not well understood. Objective: To define changes in AMI case rates, patient demographics, cardiovascular comorbidities, treatment approaches, and in-hospital outcomes during the pandemic. Design, Setting, and Participants: This cross-sectional study retrospectively analyzed AMI hospitalizations that occurred between December 30, 2018, and May 16, 2020, in 1 of the 49 hospitals in the Providence St Joseph Health system located in 6 states (Alaska, Washington, Montana, Oregon, California, and Texas). The cohort included patients aged 18 years or older who had a principal discharge diagnosis of AMI (ST-segment elevation myocardial infarction [STEMI] or non-ST-segment elevation myocardial infarction [NSTEMI]). Segmented regression analysis was performed to assess changes in weekly case volumes. Cases were grouped into 1 of 3 periods: before COVID-19 (December 30, 2018, to February 22, 2020), early COVID-19 (February 23, 2020, to March 28, 2020), and later COVID-19 (March 29, 2020, to May 16, 2020). In-hospital mortality was risk-adjusted using an observed to expected (O/E) ratio and covariate-adjusted multivariable model. Exposure: Date of hospitalization. Main Outcomes and Measures: The primary outcome was the weekly rate of AMI (STEMI or NSTEMI) hospitalizations. The secondary outcomes were patient characteristics, treatment approaches, and in-hospital outcomes of this patient population. Results: The cohort included 15 244 AMI hospitalizations (of which 4955 were for STEMI [33%] and 10 289 for NSTEMI [67%]) involving 14 724 patients (mean [SD] age of 68 [13] years and 10 019 men [66%]). Beginning February 23, 2020, AMI-associated hospitalizations decreased at a rate of -19.0 (95% CI, -29.0 to -9.0) cases per week for 5 weeks (early COVID-19 period). Thereafter, AMI-associated hospitalizations increased at a rate of +10.5 (95% CI, +4.6 to +16.5) cases per week (later COVID-19 period). No appreciable differences in patient demographics, cardiovascular comorbidities, and treatment approaches were observed across periods. The O/E mortality ratio for AMI increased during the early period (1.27; 95% CI, 1.07-1.48), which was disproportionately associated with patients with STEMI (1.96; 95% CI, 1.22-2.70). Although the O/E mortality ratio for AMI was not statistically different during the later period (1.23; 95% CI, 0.98-1.47), increases in the O/E mortality ratio were noted for patients with STEMI (2.40; 95% CI, 1.65-3.16) and after risk adjustment (odds ratio, 1.52; 95% CI, 1.02-2.26). Conclusions and Relevance: This cross-sectional study found important changes in AMI hospitalization rates and worse outcomes during the early and later COVID-19 periods. Future studies are needed to identify contributors to the increased mortality rate among patients with STEMI.

Bioinformatics-Based Identification of Expanded Repeats: A Non-reference Intronic Pentamer Expansion in RFC1 Causes CANVAS.

Genomic technologies such as next-generation sequencing (NGS) are revolutionizing molecular diagnostics and clinical medicine. However, these approaches have proven inefficient at identifying pathogenic repeat expansions. Here, we apply a collection of bioinformatics tools that can be utilized to identify either known or novel expanded repeat sequences in NGS data. We performed genetic studies of a cohort of 35 individuals from 22 families with a clinical diagnosis of cerebellar ataxia with neuropathy and bilateral vestibular areflexia syndrome (CANVAS). Analysis of whole-genome sequence (WGS) data with five independent algorithms identified a recessively inherited intronic repeat expansion [(AAGGG)exp] in the gene encoding Replication Factor C1 (RFC1). This motif, not reported in the reference sequence, localized to an Alu element and replaced the reference (AAAAG)11 short tandem repeat. Genetic analyses confirmed the pathogenic expansion in 18 of 22 CANVAS-affected families and identified a core ancestral haplotype, estimated to have arisen in Europe more than twenty-five thousand years ago. WGS of the four RFC1-negative CANVAS-affected families identified plausible variants in three, with genomic re-diagnosis of SCA3, spastic ataxia of the Charlevoix-Saguenay type, and SCA45. This study identified the genetic basis of CANVAS and demonstrated that these improved bioinformatics tools increase the diagnostic utility of WGS to determine the genetic basis of a heterogeneous group of clinically overlapping neurogenetic disorders.

An intronic mutation in Chd7 creates a cryptic splice site, causing aberrant splicing in a mouse model of CHARGE syndrome.

Alternate splicing is a critical regulator of gene expression in eukaryotes, however genetic mutations can cause erroneous splicing and disease. Most recorded splicing disorders are caused by mutations of splice donor/acceptor sites, however intronic mutations can affect splicing. Clinical exome analyses largely ignore intronic sequence, limiting the detection of mutations to within coding regions. We describe 'Trooper', a novel mouse model of CHARGE syndrome harbouring a pathogenic point mutation in Chd7. The mutation is 18 nucleotides upstream of exon 10 and creates a cryptic acceptor site, causing exon skipping and partial intron retention. This mutation, though detectable in exome sequence, was initially dismissed by computational filtering due to its intronic location. The Trooper strain exhibited many of the previously described CHARGE-like anomalies of CHD7 deficient mouse lines; including hearing impairment, vestibular hypoplasia and growth retardation. However, more common features such as facial asymmetry and circling were rarely observed. Recognition of these characteristic features prompted manual reexamination of Chd7 sequence and subsequent validation of the intronic mutation, highlighting the importance of phenotyping alongside exome analyses. The Trooper mouse serves as a valuable model of atypical CHARGE syndrome and reveals a molecular mechanism that may underpin milder clinical presentation of the syndrome.

Flexible Proteins at the Origin of Life.

Almost all modern proteins possess well-defined, relatively rigid scaffolds that provide structural preorganization for desired functions. Such scaffolds require the sufficient length of a polypeptide chain and extensive evolutionary optimization. How ancestral proteins attained functionality, even though they were most likely markedly smaller than their contemporary descendants, remains a major, unresolved question in the origin of life. On the basis of evidence from experiments and computer simulations, we argue that at least some of the earliest water-soluble and membrane proteins were markedly more flexible than their modern counterparts. As an example, we consider a small, evolved in vitro ligase, based on a novel architecture that may be the archetype of primordial enzymes. The protein does not contain a hydrophobic core or conventional elements of the secondary structure characteristic of modern water-soluble proteins, but instead is built of a flexible, catalytic loop supported by a small hydrophilic core containing zinc atoms. It appears that disorder in the polypeptide chain imparts robustness to mutations in the protein core. Simple ion channels, likely the earliest membrane protein assemblies, could also be quite flexible, but still retain their functionality, again in contrast to their modern descendants. This is demonstrated in the example of antiamoebin, which can serve as a useful model of small peptides forming ancestral ion channels. Common features of the earliest, functional protein architectures discussed here include not only their flexibility, but also a low level of evolutionary optimization and heterogeneity in amino acid composition and, possibly, the type of peptide bonds in the protein backbone.

Validity of the Electrodiffusion Model for Calculating Conductance of Simple Ion Channels.

We examine the validity and utility of the electrodiffusion (ED) equation, i.e., the generalized Nernst-Planck equation, to characterize, in combination with molecular dynamics, the electrophysiological behavior of simple ion channels. As models, we consider three systems-two naturally occurring channels formed by α-helical bundles of peptaibols, trichotoxin, and alamethicin, and a synthetic, hexameric channel, formed by a peptide that contains only leucine and serine. All these channels mediate transport of potassium and chloride ions. Starting with equilibrium properties, such as the potential of mean force experienced by an ion traversing the channel and diffusivity, obtained from molecular dynamics simulations, the ED equation can be used to determine the full current-voltage dependence with modest or no additional effort. The potential of mean force can be obtained not only from equilibrium simulations, but also, with comparable accuracy, from nonequilibrium simulations at a single voltage. The main assumptions underlying the ED equation appear to hold well for the channels and voltages studied here. To expand the utility of the ED equation, we examine what are the necessary and sufficient conditions for Ohmic and nonrectifying behavior and relate deviations from this behavior to the shape of the ionic potential of mean force.

Combining molecular dynamics and an electrodiffusion model to calculate ion channel conductance.

Establishing the relation between the structures and functions of protein ion channels, which are protein assemblies that facilitate transmembrane ion transport through water-filled pores, is at the forefront of biological and medical sciences. A reliable way to determine whether our understanding of this relation is satisfactory is to reproduce the measured ionic conductance over a broad range of applied voltages. This can be done in molecular dynamics simulations by way of applying an external electric field to the system and counting the number of ions that traverse the channel per unit time. Since this approach is computationally very expensive we develop a markedly more efficient alternative in which molecular dynamics is combined with an electrodiffusion equation. This alternative approach applies if steady-state ion transport through channels can be described with sufficient accuracy by the one-dimensional diffusion equation in the potential given by the free energy profile and applied voltage. The theory refers only to line densities of ions in the channel and, therefore, avoids ambiguities related to determining the surface area of the channel near its endpoints or other procedures connecting the line and bulk ion densities. We apply the theory to a simple, model system based on the trichotoxin channel. We test the assumptions of the electrodiffusion equation, and determine the precision and consistency of the calculated conductance. We demonstrate that it is possible to calculate current/voltage dependence and accurately reconstruct the underlying (equilibrium) free energy profile, all from molecular dynamics simulations at a single voltage. The approach developed here applies to other channels that satisfy the conditions of the electrodiffusion equation.

Isothermal evaporation of ethanol in a dynamic gas atmosphere.

Optimization of evaporation and pyrolysis conditions for ethanol are important in carbon nanotube (CNT) synthesis. The activation enthalpy (ΔH(‡)), the activation entropy (ΔS(‡)), and the free energy barrier (ΔG(‡)) to evaporation have been determined by measuring the molar coefficient of evaporation, k(evap), at nine different temperatures (30-70 °C) and four gas flow rates (25-200 mL/min) using nitrogen and argon as carrier gases. At 70 °C in argon, the effect of the gas flow rate on k(evap) and ΔG(‡) is small. However, this is not true at temperatures as low as 30 °C, where the increase of the gas flow rate from 25 to 200 mL/min results in a nearly 6 times increase of k(evap) and decrease of ΔG(‡) by ~5 kJ/mol. Therefore, at 30 °C, the effect of the gas flow rate on the ethanol evaporation rate is attributed to interactions of ethanol with argon molecules. This is supported by simultaneous infrared spectroscopic analysis of the evolved vapors, which demonstrates the presence of different amounts of linear and cyclic hydrogen bonded ethanol aggregates. While the amount of these aggregates at 30 °C depends upon the gas flow rate, no such dependence was observed during evaporation at 70 °C. When the evaporation was carried out in nitrogen, ΔG(‡) was almost independent of the evaporation temperature (30-70 °C) and the gas flow rate (25-200 mL/min). Thus the evaporation of ethanol in a dynamic gas atmosphere at different temperatures may go via different mechanisms depending on the nature of the carrier gas.

Molecular dynamics simulation of the antiamoebin ion channel: linking structure and conductance.

Molecular-dynamics simulations were carried out to ascertain which of the potential multimeric forms of the transmembrane peptaibol channel, antiamoebin, is consistent with its measured conductance. Estimates of the conductance obtained through counting ions that cross the channel and by solving the Nernst-Planck equation yield consistent results, indicating that the motion of ions inside the channel can be satisfactorily described as diffusive. The calculated conductance of octameric channels is markedly higher than the conductance measured in single channel recordings, whereas the tetramer appears to be nonconducting. The conductance of the hexamer was estimated to be 115 ± 34 pS and 74 ± 20 pS, at 150 mV and 75 mV, respectively, in satisfactory agreement with the value of 90 pS measured at 75 mV. On this basis, we propose that the antiamoebin channel consists of six monomers. Its pore is large enough to accommodate K⁺ and Cl⁻ with their first solvation shells intact. The free energy barrier encountered by K⁺ is only 2.2 kcal/mol whereas Cl⁻ encounters a substantially higher barrier of nearly 5 kcal/mol. This difference makes the channel selective for cations. Ion crossing events are shown to be uncorrelated and follow Poisson statistics.

A case of periostitis secondary to voriconazole therapy in a heart transplant recipient.

A 66-year-old man with a history of heart transplant for idiopathic dilated cardiomyopathy presented with progressive bone pain and myalgias. He has been on voriconazole for a pulmonary Aspergillus infection for 9 months. He had an elevated alkaline phosphatase of 280. There is no history of rheumatologic disease. Drug-induced periostitis has recently been reported in patients on long-term voriconazole therapy after lung transplantation for prophylaxis and treatment of Aspergillus infection. This case demonstrates the same phenomenon in a heart transplant patient. This patient's symptoms improved after discontinuation of voriconazole.

Unoccupied electronic structure of ball-milled graphite.

Changes in electronic and vibrational structure of well characterised macrocrystalline graphite milled by a planetary ball-mill are investigated by Raman spectroscopy and Near Edge X-ray Absorption Fine structure (NEXAFS) measurements at the C K-edge. The electronic structure changes at the surface and in the sub-surface of the particles are examined by comparing two-different NEXAFS detection modes: total fluorescence yield (TFY) and partial electron yield (PEY) respectively. When the in-plane crystallite sizes of graphite are decreased to nanosized (from approximately 160 nm to approximately 9 nm), a new spectral structure appears in TFY at 284.1 eV which is not present in the macrocrystalline graphite. This feature is assigned to electronic states associated with zigzag edges. Further the TFY shows a shift of the main graphite pi* band from 285.5 to 285.9 eV, attributed to breaking the conjugation and hence the electron localization effect during milling, The TFY spectra also show strong spectral features at 287.5 and 288.6 eV, which suggest that the local environment of carbon atoms changes from sp(2) to more sp(3) due to physical damage of the graphite sheets and formation of structures other than aromatic hexagons. Complementary Raman spectroscopic measurements demonstrate an up-shift of the graphite G band from 1575 to 1583 cm(-1)en route to nanosize. The changes in TFY NEXAFS and Raman spectra are attributed to modification of the sub-surface electronic structure due to the presence of defects in the graphite crystal produced during milling. The discovery of the strong spectral feature at 284.1 eV in nanographite and the 0.4 eV up-shift of the pi* band may open up possibilities to influence the electronic transport properties of graphite by manipulation of defects during the preparation of the nanographite.

Utility of SPECT/CT as an adjunct to planar whole body I-131 imaging: liver metastasis from papillary thyroid cancer.

One of the major limitations of planar I-131 imaging is its lack of anatomic precision. SPECT/CT offers the benefit of precise anatomic localization that planar imaging lacks. Whether for confirmation of physiologic uptake or true pathology, SPECT/CT has an important role to play in clarifying equivocal findings. We present a case of papillary thyroid cancer metastatic to the liver, a relatively rare scenario. SPECT/CT allowed definitive lesion characterization at the time of the patient's visit to the nuclear medicine department.

Pitfalls of I-131 whole body scan interpretation: bronchogenic cyst and mucinous cystadenoma.

Whole body iodine scans are routinely performed in the nuclear medicine department as part of the management of differentiated thyroid carcinoma. Similarly, radioactive iodine has a well-established role as an adjunct to thyroidectomy in the treatment of these patients. A thorough understanding of the normal, benign, and pathologic biodistribution of iodine is imperative for the nuclear medicine physician. This knowledge leads to the accurate determination of the presence of metastatic or recurrent carcinoma, and may even facilitate the accurate detection of an undiagnosed condition. Above all, correct image interpretation avoids unnecessary therapeutic doses. The authors describe 2 unusual examples of false positive findings in fluid-filled cavities that showcase the variety of nonmalignant entities one may encounter when interpreting metastatic surveys.

Laparoscopic sentinel lymph node mapping for cervix cancer--a detailed evaluation and time analysis.

OBJECTIVE: To provide data from a US center on laparoscopic (LSC) approach to sentinel lymph node (SLN) detection in cervix cancer with detailed time analysis. METHODS: This prospective trial enrolled patients with stage IA2-IIA cervix cancer undergoing primary radical surgery. Tc-99 radiocolloid was injected the morning of surgery, followed by hybrid SPECT/CT lymphoscintigraphy. Blue dye injection occurred just prior to incision. After bilateral LSC SLN detection, all patients received complete LSC pelvic lymphadenectomy. Institutional SLN protocol was followed for frozen section, hematoxylin and eosin, and cytokeratin staining. RESULTS: Between December 2003 and February 2006, 20 enrolled patients received 9 LSC-assisted radical vaginal hysterectomies, 7 radical abdominal hysterectomies, 2 LSC-assisted radical vaginal trachelectomies, and 2 LSC lymphadenectomies alone (secondary to positive lymph nodes). Mean tumor size was 2.5 cm. Nineteen percent of the 64 SLNs were found in unusual sites, including common iliac (11%), presacral (5%) and para-aortic (3%). The negative predictive value was 100%. The combined technique detected SLNs bilaterally in all patients. If blue dye alone was used, this rate would have dropped to 67.5% and was negatively correlated with elapsed surgical time (-0.7; p=0.002). The ability to visualize blue SLNs remained steady for 30 min and was completely gone by 50 min. CONCLUSIONS: Laparoscopic SLN mapping can be newly introduced into gynecologic oncology centers with high detection rates and negative predictive values. The visualization of blue dye in SLNs is transient, and this negative time correlation may explain the previously reported inferior detection rates with this technique. CLINICAL TRIAL REGISTRATION.: ClinicalTrials.gov, http://www.clinicaltrials.gov, NCT 00205010.