A Shorter Door-In-Door-Out Time Is Associated with Improved Outcome in Large Vessel Occlusion Stroke.

Introduction: Endovascular thrombectomy (EVT) significantly improves outcomes in large vessel occlusion stroke (LVOS). When a patient with a LVOS arrives at a hospital that does not perform EVT, emergent transfer to an endovascular stroke center (ESC) is required. Our objective was to determine the association between door-in-door-out time (DIDO) and 90-day outcomes in patients undergoing EVT. Methods: We conducted an analysis of the Optimizing Prehospital Stroke Systems of Care-Reacting to Changing Paradigms (OPUS-REACH) registry of 2,400 LVOS patients treated at nine ESCs in the United States. We examined the association between DIDO times and 90-day outcomes as measured by the modified Rankin scale. Results: A total of 435 patients were included in the final analysis. The mean DIDO time for patients with good outcomes was 17 minute shorter than patients with poor outcomes (122 minutes [min] vs 139 min, P = 0.04). Absolute DIDO cutoff times of ≤60 min, ≤90 min, or ≤120 min were not associated with improved functional outcomes (46.4 vs 32.3%, P = 0.12; 38.6 vs 30.6%, P = 0.10; and 36.4 vs 28.9%, P = 0.10, respectively). This held true for patients with hyperacute strokes of less than four-hour onset. Lower baseline National Institutes of Health Stroke Scale (NIHSS) score (11.9 vs 18.2, P = <.001) and younger age (62.5 vs 74.9 years (P < .001) were associated with improved outcomes. On multiple regression analysis, age (odds ratio [OR] 1.71, 95% confidence interval [CI] 1.45-2.02) and baseline NIHSS score (OR 1.67, 95% CI 1.42-1.98) were associated with improved outcomes while DIDO time was not associated with better outcome (OR 1.13, 95% CI 0.99-1.30). Conclusion: Although the DIDO time was shorter for patients with a good outcome, this was non-significant in multiple regression analysis. Receipt of intravenous thrombolysis and time to EVT were not associated with better outcomes, while male gender, lower age, arrival by private vehicle, and lower NIHSS score portended better outcomes. No absolute DIDO-time cutoff or modifiable factor was associated with improved outcomes for LVOS. This study underscores the need to streamline DIDO times but not to set an artificial DIDO time benchmark to meet.

Delay in hospital presentation is the main reason large vessel occlusion stroke patients do not receive intravenous thrombolysi.

Objectives: Intravenous thrombolysis (IVT) and endovascular therapy (EVT) are the mainstays of treatment for large vessel occlusion stroke (LVOS). Prior studies have examined why patients have not received IVT, the most cited reasons being last-known-well (LKW) to hospital arrival of >4.5 hours and minor/resolving stroke symptoms. Given that LVOS patients typically present moderate-to-severe neurologic deficits, these patients should be easier to identify and treat than patients with minor strokes. This investigation explores why IVT was not administered to a cohort of LVOS patients who underwent EVT. Methods: This is an analysis of the Optimizing the Use of Prehospital Stroke Systems of Care (OPUS-REACH) registry, which contains patients from 9 endovascular centers who underwent EVT between 2015 and 2020. The exposure of interest was the receipt of intravenous thrombolysis. Descriptive summary statistics are presented as means and SDs for continuous variables and as frequencies with percentages for categorical variables. Two-sample t tests were used to compare continuous variables and the chi-square test was used to compare categorical variables between those who received IVT and those who did not receive EVT. Results: Two thousand forty-three patients were included and 60% did not receive IVT. The most common reason for withholding IVT was LKW to arrival of >4.5 (57.2%). The second most common contraindication was oral anticoagulation (15.5%). On multivariable analysis, 2 factors were associated with not receiving IVT: increasing age (odds ratio [OR] 0.86; 95% confidence interval [CI] 0.78-0.93) and increasing time from LKW-to hospital arrival (OR 0.45 95% CI 0.46-0.49). Conclusion: Like prior studies, the most frequent reason for exclusion from IVT was a LKW to hospital presentation of >4.5 hours; the second reason was anticoagulation. Efforts must be made to increase awareness of the time-sensitive nature of IVT and evaluate the safety of IVT in patients on oral anticoagulants.

Factors associated with door-in-door-out times in large vessel occlusion stroke patients undergoing endovascular therapy.

INTRODUCTION: In the management of large vessel occlusion stroke (LVOS), patients are frequently evaluated first at a non-endovascular stroke center and later transferred to an endovascular stroke center (ESC) for endovascular treatment (EVT). The door-in-door-out time (DIDO) is frequently used as a benchmark for transferring hospitals though there is no universally accepted nor evidenced-based DIDO time. The goal of this study was to identify factors affecting DIDO times in LVOS patients who ultimately underwent EVT. METHODS: The Optimizing Prehospital Use of Stroke Systems of Care-Reacting to Changing Paradigms (OPUS-REACH) registry is comprised of all LVOS patients who underwent EVT at one of nine endovascular centers in the Northeast United States between 2015 and 2020. We queried the registry for all patients who were transferred from a non-ESC to one of the nine ESCs for EVT. Univariate analysis was performed using t-tests to obtain a p value. A priori, we defined a p value of <0.05 as significant. Multiple logistic regression was conducted to determine the association of variables to estimate an odds ratio. RESULTS: 511 patients were included in the final analysis. The mean DIDO times for all patients was 137.8 min. Vascular imaging and treatment at a non-certified stroke center were associated with longer DIDO times by 23 and 14 min, respectively. On multivariate analyses, the acquisition of vascular imaging was associated with 16 additional minutes spent at the non-ESC while presentation to a non-stroke certified hospital was associated with 20 additional minutes spent at the transferring hospital. The administration of intravenous thrombolysis (IVT) was associated with 15 min less spent at the non-ESC. DISCUSSION: Vascular imaging and non-stroke certified stroke centers were associated with longer DIDO times. Non-ESCs should integrate vascular imaging into their workflow as feasible to reduce DIDO times. Further work examining other details regarding the transfer process such as transfer via ground or air, could help further identify opportunities to improve DIDO times.

Porous liquid metal-elastomer composites with high leakage resistance and antimicrobial property for skin-interfaced bioelectronics.

Liquid metal-elastomer composite is a promising soft conductor for skin-interfaced bioelectronics, soft robots, and others due to its large stretchability, ultrasoftness, high electrical conductivity, and mechanical-electrical decoupling. However, it often suffers from deformation-induced leakage, which can smear skin, deteriorate device performance, and cause circuit shorting. Besides, antimicrobial property is desirable in soft conductors to minimize microbial infections. Here, we report phase separation-based synthesis of porous liquid metal-elastomer composites with high leakage resistance and antimicrobial property, together with large stretchability, tissue-like compliance, high and stable electrical conductivity over deformation, high breathability, and magnetic resonance imaging compatibility. The porous structures can minimize leakage through damping effects and lower percolation thresholds to reduce liquid metal usage. In addition, epsilon polylysine is loaded into elastic matrices during phase separation to provide antimicrobial property. The enabled skin-interfaced bioelectronics can monitor cardiac electrical and mechanical activities and offer electrical stimulations in a mechanically imperceptible and electrically stable manner even during motions.

Evolution of endovascular stroke centers and disparities in access to stroke care in four Northeastern states: 2015-2019.

OBJECTIVES: Disparities exist throughout our healthcare system, especially related to access to care. Advanced stroke care for strokes is only available at selected endovascular centers (ESCs) in the United States. Although the number of ESCs increase each year, this does not necessarily reflect increased access to care. Here, we look at the evolution of ESC in four states and disparities in access to advanced stroke care. MATERIALS AND METHODS: This is a descriptive study of access to ESCs in four Northeastern states between 2015-2019. Using data from the United States Census Bureau and spatial analysis, we examined the proportion of the population with drive times of less than 60 minutes stratified by income, race/ethnicity, population density, and insurance. We also calculated the mean drive time for each of these socioeconomic groups from their census tracts to the nearest ESC. RESULTS: Between 2015 and 2019, the number of ESCs increased from 15 to 48. The proportion of patients within a 60-minute drive of an ESC increased from 77% to 88%. However, only 66% of the least densely populated quartile lived within 60 min of an ESC. By income, access to ESCs in the wealthiest quartile was 96.6% compared to 83.7% in the lowest quartile. Hispanics and non-Hispanic Blacks had the largest proportions of populations within 60 minutes of an ESC while Non-Hispanic Whites had the smallest. CONCLUSIONS: This study underscores the need to evaluate the placement of new ESCs to assure that these hospitals decrease disparities and increase access to advanced stroke care.

Infectious Aerosol Capture Mask as Environmental Control to Reduce Spread of Respiratory Viral Particles.

Negative pressure isolation of COVID-19 patients is critical to limiting the nosocomial transmission of SARS-CoV-2; however, airborne isolation rooms are limited. Alternatives to traditional isolation procedures are needed. The evaluation of an Infectious Aerosol Capture Mask (IACM) that is designed to augment the respiratory isolation of COVID-19 patients is described. Efficacy in capturing exhaled breath aerosols was evaluated using laboratory experimentation, computational fluid dynamics (CFD) and measurements of exhaled breath from COVID-19 patients and their surroundings. Laboratory aerosol experiments indicated that the mask captured at least 99% of particles. Simulations of breathing and speaking showed that all particles between 0.1 and 20 µm were captured either on the surface of the mask or in the filter. During coughing, no more than 13% of the smallest particles escaped the mask, while the remaining particles collected on the surfaces or filter. The total exhaled virus concentrations of COVID-positive patients showed a range from undetectable to 1.1 × 106 RNA copies/h of SARS-CoV-2, and no SARS-CoV-2 aerosol was detected in the samples collected that were adjacent to the patient when the mask was being worn. These data indicate that the IACM is useful for containing the exhaled aerosol of infected individuals and can be used to quantify the viral aerosol production rates during respiratory activities.

Nanofiber capsules for minimally invasive sampling of biological specimens from gastrointestinal tract.

Accurate and rapid point-of-care tissue and microbiome sampling is critical for early detection of cancers and infectious diseases and often result in effective early intervention and prevention of disease spread. In particular, the low prevalence of Barrett's and gastric premalignancy in the Western world makes population-based endoscopic screening unfeasible and cost-ineffective. Herein, we report a method that may be useful for prescreening the general population in a minimally invasive way using a swallowable, re-expandable, ultra-absorbable, and retrievable nanofiber cuboid and sphere produced by electrospinning, gas-foaming, coating, and crosslinking. The water absorption capacity of the cuboid- and sphere-shaped nanofiber objects is shown ∼6000% and ∼2000% of their dry mass. In contrast, unexpanded semicircular and square nanofiber membranes showed <500% of their dry mass. Moreover, the swallowable sphere and cuboid were able to collect and release more bacteria, viruses, and cells/tissues from solutions as compared with unexpanded scaffolds. In addition to that, an expanded sphere shows higher cell collection capacity from the esophagus inner wall as compared with the unexpanded nanofiber membrane. Taken together, the nanofiber capsules developed in this study could provide a minimally invasive method of collecting biological samples from the duodenal, gastric, esophagus, and oropharyngeal sites, potentially leading to timely and accurate diagnosis of many diseases. STATEMENT OF SIGNIFICANCE: Recently, minimally invasive technologies have gained much attention in tissue engineering and disease diagnosis. In this study, we engineered a swallowable and retrievable electrospun nanofiber capsule serving as collection device to collect specimens from internal organs in a minimally invasive manner. The sample collection device could be an alternative endoscopy to collect the samples from internal organs like jejunum, stomach, esophagus, and oropharynx without any sedation. The newly engineered nanofiber capsule could be used to collect, bacteria, virus, fluids, and cells from the abovementioned internal organs. In addition, the biocompatible and biodegradable nanofiber capsule on a string could exhibit a great sample collection capacity for the primary screening of Barret Esophagus, acid reflux, SARS-COVID-19, Helicobacter pylori, and gastric cancer.

Optimizing Prehospital Stroke Systems of Care-Reacting to Changing Paradigms (OPUS-REACH): a pragmatic registry of large vessel occlusion stroke patients to create evidence-based stroke systems of care and eliminate disparities in access to stroke care.

BACKGROUND: Large vessel occlusion (LVO) strokes are best treated with rapid endovascular therapy (EVT). There are two routes that LVO stroke patients can take to EVT therapy when transported by EMS: primary transport (ambulance transports directly to an endovascular stroke center (ESC) or secondary transport (EMS transports to a non-ESC then transfers for EVT). There is no clear evidence which path to care results in better functional outcomes for LVO stroke patients. To find this answer, an analysis of a large, real-world population of LVO stroke patients must be performed. METHODS: A pragmatic registry of LVO stroke patients from nine health systems across the United States. The nine health systems span urban and rural populations as well as the spectrum of socioeconomic statuses. We will use univariate and multivariate analysis to explore the relationships between type of EMS transport, socioeconomic factors, and LVO stroke outcomes. We will use geographic information systems and spatial analysis to examine the complex movements of patients in time and space. To detect an 8% difference between groups, with a 3:1 patient ratio of primary to secondary transports, 95% confidence and 80% power, we will need approximately 1600 patients. The primary outcome is the patients with modified Rankin Scale (mRS) ≤ 2 at 90 days. Subgroup analyses include patients who receive intravenous thrombolysis and duration of stroke systems. Secondary analyses include socioeconomic factors associated with poor outcomes after LVO stroke. DISCUSSION: Using the data obtained from the OPUS-REACH registry, we will develop evidence based algorithms for prehospital transport of LVO stroke patients. Unlike prior research, the OPUS-REACH registry contains patient-level data spanning from EMS dispatch to ninety day functional outcomes. We expect that we will find modifiable factors and socioeconomic disparities associated with poor outcomes in LVO stroke. OPUS-REACH with its breadth of locations, detailed patient records, and multidisciplinary researchers will design the optimal prehospital stroke system of care for LVO stroke patients.

The size and culturability of patient-generated SARS-CoV-2 aerosol.

BACKGROUND: Aerosol transmission of COVID-19 is the subject of ongoing policy debate. Characterizing aerosol produced by people with COVID-19 is critical to understanding the role of aerosols in transmission. OBJECTIVE: We investigated the presence of virus in size-fractioned aerosols from six COVID-19 patients admitted into mixed acuity wards in April of 2020. METHODS: Size-fractionated aerosol samples and aerosol size distributions were collected from COVID-19 positive patients. Aerosol samples were analyzed for viral RNA, positive samples were cultured in Vero E6 cells. Serial RT-PCR of cells indicated samples where viral replication was likely occurring. Viral presence was also investigated by western blot and transmission electron microscopy (TEM). RESULTS: SARS-CoV-2 RNA was detected by rRT-PCR in all samples. Three samples confidently indicated the presence of viral replication, all of which were from collected sub-micron aerosol. Western blot indicated the presence of viral proteins in all but one of these samples, and intact virions were observed by TEM in one sample. SIGNIFICANCE: Observations of viral replication in the culture of submicron aerosol samples provides additional evidence that airborne transmission of COVID-19 is possible. These results support the use of efficient respiratory protection in both healthcare and by the public to limit transmission.

Variability in biopsy quality informs translational research applications in hepatocellular carcinoma.

In the era of precision medicine, biopsies are playing an increasingly central role in cancer research and treatment paradigms; however, patient outcomes and analyses of biopsy quality, as well as impact on downstream clinical and research applications, remain underreported. Herein, we report biopsy safety and quality outcomes for percutaneous core biopsies of hepatocellular carcinoma (HCC) performed as part of a prospective clinical trial. Patients with a clinical diagnosis of HCC were enrolled in a prospective cohort study for the genetic, proteomic, and metabolomic profiling of HCC at two academic medical centers from April 2016 to July 2020. Under image guidance, 18G core biopsies were obtained using coaxial technique at the time of locoregional therapy. The primary outcome was biopsy quality, defined as tumor fraction in the core biopsy. 56 HCC lesions from 50 patients underwent 60 biopsy events with a median of 8 core biopsies per procedure (interquartile range, IQR, 7-10). Malignancy was identified in 45/56 (80.4%, 4 without pathology) biopsy events, including HCC (40/56, 71.4%) and cholangiocarcinoma (CCA) or combined HCC-CCA (5/56, 8.9%). Biopsy quality was highly variable with a median of 40% tumor in each biopsy core (IQR 10-75). Only 43/56 (76.8%) and 23/56 (41.1%) samples met quality thresholds for genomic or metabolomic/proteomic profiling, respectively, requiring expansion of the clinical trial. Overall and major complication rates were 5/60 (8.3%) and 3/60 (5.0%), respectively. Despite uniform biopsy protocol, biopsy quality varied widely with up to 59% of samples to be inadequate for intended purpose. This finding has important consequences for clinical trial design and highlights the need for quality control prior to applications in which the presence of benign cell types may substantially alter findings.

Implementation of a COVID-19 cohort area resulted in no surface or air contamination in surrounding areas in one academic emergency department.

INTRODUCTION: As a result of the COVID-19 pandemic and highly contagious nature of SARS-CoV-2, emergency departments (EDs) have been forced to implement new measures and protocols to minimize the spread of the disease within their departments. The primary objective of this study was to determine if the implementation of a designated COVID-19 cohort area (hot zone) within a busy ED mitigated the dissemination of SARS-CoV-2 throughout the rest of the department. METHODS: In an ED of a tertiary academic medical center, with 64,000 annual visits, an eight room pod was designated for known COVID-19 or individuals with high suspicion for infection. There was a single entry and exit for donning and doffing personal protective equipment (PPE). Health care workers (HCW) changed gowns and gloves between patients, but maintained their N-95 mask and face shield, cleaning the shield with a germicidal wipe between patients. Staffing assignments designated nurses and technicians to remain in this area for 4 h, where physicians regularly moved between the hot zone and rest of the ED. Fifteen surface samples and four air samples were taken to evaluate SARS-CoV-2 contamination levels and the effectiveness of infection control practices. Samples were collected outside of patient rooms in 3 primary ED patient care areas, the reception area, the primary nurses station, inside the cohort area, and the PPE donning and doffing areas immediately adjacent. Samples were recovered and analyzed for the presence of the E gene of SARS-CoV-2 using RT-PCR. RESULTS: SARS-CoV-2 was not detected on any surface samples, including in and around the cohort area. All air samples outside the COVID-19 hot zone were negative for SARS-CoV-2, but air samples within the cohort area had a low level of viral contamination. CONCLUSION: A designated COVID-19 cohort area resulted in no air or surface contamination outside of the hot zone, and only minimal air, but no surface contamination, within the hot zone.

Interpretative differences of combined cytogenetic and molecular profiling highlights differences between MRC and ELN classifications of AML.

Acute myeloid leukemia (AML) is typically characterized clinically for prognostic purposes using both cytogenetic and molecular characteristics. However, both cytogenetic and molecular risk stratification schemas are varied and few reports have studied correlations between these schemas. We have performed a single institution retrospective review of cytogenetic and molecular classifications of AMLs seen at Penn Medicine between 2013 and 2018. One-hundred fourty-four cases were characterized according to European Leukemia Net (ELN) or Medical Research Council (MRC) criteria for cytogenetics and results compared to molecular profiling. When we analyzed the most common sequencing study results within the risk groupings, negative sequencing studies and FLT3 mutations were common in favorable AMLs, intermediate AMLs had mutations in FLT3, NPM1, DNMT3A and IDH2, while adverse AMLs had a high prevalence of TP53 mutations. We next grouped the genes on the panel by their proteins' functions and found mutations in signaling pathway genes to be common in favorable AMLs while tumor suppressors were commonly mutated in adverse AMLs. AMLs grouped by the type of chromosomal abnormality present showed that FLT3 mutations were common in AMLs with a trisomy while TP53 mutations were common in AMLs with a monosomy or a deletion. TP53 mutations are especially common in AMLs with a monosomal karyotype and often overlap with 17p loss. Interestingly, although all AMLs with TP53 mutations have a defect in the response to DNA damage, expression of P53 protein before and after irradiation is not consistently predicted by phenotype. Overall, these studies confirm the genetic complexity of AML which does not fall into simple patterns of cooperating mutations.

Ultra-absorptive Nanofiber Swabs for Improved Collection and Test Sensitivity of SARS-CoV-2 and other Biological Specimens.

Following the COVID-19 outbreak, swabs for biological specimen collection were thrust to the forefront of healthcare materials. Swab sample collection and recovery are vital for reducing false negative diagnostic tests, early detection of pathogens, and harvesting DNA from limited biological samples. In this study, we report a new class of nanofiber swabs tipped with hierarchical 3D nanofiber objects produced by expanding electrospun membranes with a solids-of-revolution-inspired gas foaming technique. Nanofiber swabs significantly improve absorption and release of proteins, cells, bacteria, DNA, and viruses from solutions and surfaces. Implementation of nanofiber swabs in SARS-CoV-2 detection reduces the false negative rates at two viral concentrations and identifies SARS-CoV-2 at a 10× lower viral concentration compared to flocked and cotton swabs. The nanofiber swabs show great promise in improving test sensitivity, potentially leading to timely and accurate diagnosis of many diseases.

Leading with inclusion during the COVID-19 pandemic: Stronger together.

Inclusion is the deliberate practice of ensuring that each individual is heard, all personal traits are respected, and all can make meaningful contributions to achieve their full potential. As coronavirus disease 2019 spreads globally and across the United States, we have viewed this pandemic through the lens of equity and inclusion. Here, we discuss how this pandemic has magnified preexisting health and social disparities and will summarize why inclusion is an essential tool to traverse this uncertain terrain and discuss strategies that can be implemented at organizational and individual levels to improve inclusion and address inequities moving forward.

Functional Genetic Screening Enables Theranostic Molecular Imaging in Cancer.

PURPOSE: Targeted therapies for cancer have accelerated the need for functional imaging strategies that inform therapeutic efficacy. This study assesses the potential of functional genetic screening to integrate therapeutic target identification with imaging probe selection through a proof-of-principle characterization of a therapy-probe pair using dynamic nuclear polarization (DNP)-enhanced magnetic resonance spectroscopic imaging (MRSI). EXPERIMENTAL DESIGN: CRISPR-negative selection screens from a public dataset were used to identify the relative dependence of 625 cancer cell lines on 18,333 genes. Follow-up screening was performed in hepatocellular carcinoma with a focused CRISPR library targeting imaging-related genes. Hyperpolarized [1-13C]-pyruvate was injected before and after lactate dehydrogenase inhibitor (LDHi) administration in male Wistar rats with autochthonous hepatocellular carcinoma. MRSI evaluated intratumoral pyruvate metabolism, while T2-weighted segmentations quantified tumor growth. RESULTS: Genetic screening data identified differential metabolic vulnerabilities in 17 unique cancer types that could be imaged with existing probes. Among these, hepatocellular carcinoma required lactate dehydrogenase (LDH) for growth more than the 29 other cancer types in this database. LDH inhibition led to a decrease in lactate generation (P < 0.001) and precipitated dose-dependent growth inhibition (P < 0.01 overall, P < 0.05 for dose dependence). Intratumoral alanine production after inhibition predicted the degree of growth reduction (P < 0.001). CONCLUSIONS: These findings demonstrate that DNP-MRSI of LDH activity using hyperpolarized [1-13C]-pyruvate is a theranostic strategy for hepatocellular carcinoma, enabling quantification of intratumoral LDHi pharmacodynamics and therapeutic efficacy prediction. This work lays the foundation for a novel theranostic platform wherein functional genetic screening informs imaging probe selection to quantify therapeutic efficacy on a cancer-by-cancer basis.

Analysis of In Vitro Cytotoxicity of Carbohydrate-Based Materials Used for Dissolvable Microneedle Arrays.

PURPOSE: Dissolvable microneedle arrays (MNAs) can be used to realize enhanced transdermal and intradermal drug delivery. Dissolvable MNAs are fabricated from biocompatible and water-soluble base polymers, and the biocargo to be delivered is integrated with the base polymer when forming the MNAs. The base polymer is selected to provide mechanical strength, desired dissolution characteristics, and compatibility with the biocargo. However, to satisfy regulatory requirements and be utilized in clinical applications, cytotoxicity of the base polymers should also be thoroughly characterized. This study systematically investigated the cytotoxicity of several important carbohydrate-based base polymers used for production of MNAs, including carboxymethyl cellulose (CMC), maltodextrin (MD), trehalose (Treh), glucose (Gluc), and hyaluronic acid (HA). METHODS: Each material was evaluated using in vitro cell-culture methods on relevant mouse and human cells, including MPEK-BL6 mouse keratinocytes, NIH-3T3 mouse fibroblasts, HaCaT human keratinocytes, and NHDF human fibroblasts. A common laboratory cell line, human embryonic kidney cells HEK-293, was also used to allow comparisons to various cytotoxicity studies in the literature. Dissolvable MNA materials were evaluated at concentrations ranging from 3 mg/mL to 80 mg/mL. RESULTS: Qualitative and quantitative analyses of cytotoxicity were performed using optical microscopy, confocal fluorescence microscopy, and flow cytometry-based assays for cell morphology, viability, necrosis and apoptosis. Results from different methods consistently demonstrated negligible in vitro cytotoxicity of carboxymethyl cellulose, maltodextrin, trehalose and hyaluronic acid. Glucose was observed to be toxic to cells at concentrations higher than 50 mg/mL. CONCLUSIONS: It is concluded that CMC, MD, Treh, HA, and glucose (at low concentrations) do not pose challenges in terms of cytotoxicity, and thus, are good candidates as MNA materials for creating clinically-relevant and well-tolerated biodissolvable MNAs.

Hyperpolarized Metabolic Imaging Detects Latent Hepatocellular Carcinoma Domains Surviving Locoregional Therapy.

BACKGROUND AND AIMS: Advances in cancer treatment have improved survival; however, local recurrence and metastatic disease-the principal causes of cancer mortality-have limited the ability to achieve durable remissions. Local recurrences arise from latent tumor cells that survive therapy and are often not detectable by conventional clinical imaging techniques. Local recurrence after transarterial embolization (TAE) of hepatocellular carcinoma (HCC) provides a compelling clinical correlate of this phenomenon. In response to TAE-induced ischemia, HCC cells adapt their growth program to effect a latent phenotype that precedes local recurrence. APPROACH AND RESULTS: In this study, we characterized and leveraged the metabolic reprogramming demonstrated by latent HCC cells in response to TAE-induced ischemia to enable their detection in vivo using dynamic nuclear polarization (DNP) magnetic resonance spectroscopic imaging (MRSI) of 13 carbon-labeled substrates. Under TAE-induced ischemia, latent HCC cells demonstrated reduced metabolism and developed a dependence on glycolytic flux to lactate. Despite the hypometabolic state of these cells, DNP-MRSI of 1-13 C-pyruvate and its downstream metabolites, 1-13 C-lactate and 1-13 C-alanine, predicted histological viability. CONCLUSIONS: These studies provide a paradigm for imaging latent, treatment-refractory cancer cells, suggesting that DNP-MRSI provides a technology for this application.

Development of Lentiviral Vectors for HIV-1 Gene Therapy with Vif-Resistant APOBEC3G.

Strategies to control HIV-1 replication without antiviral therapy are needed to achieve a functional cure. To exploit the innate antiviral function of restriction factor cytidine deaminase APOBEC3G (A3G), we developed self-activating lentiviral vectors that efficiently deliver HIV-1 Vif-resistant mutant A3G-D128K to target cells. To circumvent APOBEC3 expression in virus-producing cells, which diminishes virus infectivity, a vector containing two overlapping fragments of A3G-D128K was designed that maintained the gene in an inactive form in the virus-producer cells. However, during transduction of target cells, retroviral recombination between the direct repeats reconstituted an active A3G-D128K in 89%-98% of transduced cells. Lentiviral vectors that expressed A3G-D128K transduced CD34+ hematopoietic stem and progenitor cells with a high efficiency (>30%). A3G-D128K expression in T cell lines CEM, CEMSS, and PM1 potently inhibited spreading infection of several HIV-1 subtypes by C-to-U deamination leading to lethal G-to-A hypermutation and inhibition of reverse transcription. SIVmac239 and HIV-2 were not inhibited, since their Vifs degraded A3G-D128K. A3G-D128K expression in CEM cells potently suppressed HIV-1 replication for >3.5 months without detectable resistant virus, suggesting a high genetic barrier for the emergence of A3G-D128K resistance. Because of this, A3G-D128K expression in HIV-1 target cells is a potential anti-HIV gene therapy approach that could be combined with other therapies for the treatment and functional cure of HIV-1 infection.