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BackgroundFrontotemporal lobar degeneration (FTLD) is a leading cause of dementia in individuals aged

ObjectivesTraditional fat contouring is now regularly performed using numerous office- based less invasive techniques. However, some limitations of these minimally invasive techniques include high cost or limited selectivity with performing localized contouring and reduction of fat. These shortcomings may potentially be addressed by electrochemical lipolysis (ECLL), a novel approach that involves the insertion of electrodes into tissue followed by application of a direct current (DC) electrical potential. This results in the hydrolysis of tissue water creating active species that lead to fat necrosis and apoptosis. ECLL can be accomplished using a simple voltage-driven system (V-ECLL) or a potential-driven feedback cell (P-ECLL) both leading to water electrolysis and the creation of acid and base in situ. The aim of this study is to determine the long-lasting effects of targeted ECLL in a Yucatan pig model.MethodsA 5-year-old Yucatan pig was treated with both V-ECLL and P-ECLL in the subcutaneous fat layer using 80:20 platinum:iridium needle electrodes along an 8 cm length. Dosimetry parameters included 5 V V-ECLL for 5, 10, and 20 minutes, and -1.5 V P-ECLL, -2.5 V P-ECLL, -3.5 V P-ECLL for 5 minutes. The pig was assessed for changes in fat reduction over 3 months with digital photography and ultrasound. After euthanasia, tissue sections were harvested and gross pathology and histology were examined.ResultsV-ECLL and P-ECLL treatments led to visible fat reduction (12.1%-27.7% and 9.4%-40.8%, respectively) and contour changes across several parameters. An increased reduction of the superficial fat layer occurred with increased dosimetry parameters with an average charge transfer of 12.5, 24.3, and 47.5 C transferred for 5 V V-ECLL for 5, 10, and 20 minutes, respectively, and 2.0, 11.5, and 24.0 C for -1.5 V P-ECLL, -2.5 V P-ECLL, -3.5 V P-ECLL for 5 minutes, respectively. These dose-dependent changes were also evidenced by digital photography, gross pathology, ultrasound imaging, and histology.ConclusionsECLL results in selective damage and long-lasting changes to the adipose layer in vivo. These changes are dose-dependent, thus allowing for more precise contouring of target areas. P-ECLL has greater efficiency and control of total charge transfer compared to V-ECLL, suggesting that a low-voltage potentiostat treatment can result in fat apoptosis equivalent to a high-voltage DC system.

ObjectivesThe purpose of this study was to show that optical coherence tomography (OCT) and thermal imaging can be used to monitor changes in the structure and activity of caries lesions over time after treatment with silver diamine fluoride (SDF).MethodsArtificial caries lesions were formed on enamel and dentin bovine blocks. Each block was partitioned into five windows with the central three windows exposed to a demineralization solution to create lesions: one sound window served as a sound control (SC), one sound window was exposed to SDF to serve as a test control (SCT), one lesion window served as a lesion control (LC), one lesion window received one application of SDF (L1), while the other lesion window received two applications of SDF (L2). Each window was scanned using OCT before SDF application, and every week subsequently, for 12 weeks after initial SDF treatment. Changes in the mean intensity and the width of the peak of increased reflectivity due to the lesion and SDF along with the intensity at a depth of 180 µm from the surface representing optical penetration through the lesion were monitored. Changes in the heat lost, ΔQ (temperature integrated over time) of each window during drying with air were also monitored using a thermal imaging camera. Transverse microradiography (TMR), and high-resolution microscopy were also used for the analysis of selected samples.ResultsThe reflectivity and optical penetration of sound and lesion areas of enamel and dentin manifested significant changes in OCT images after SDF application. Thermal imaging showed significant differences in ΔQ indicative of permeability changes in the sound and lesion areas of enamel and dentin after SDF application.

Introduction: Taking an ear impression is a minimally invasive procedure. A review of existing literature suggests that contactless methods of scanning the ear have not been developed. We proposed to establish a correlation between external ear features with the ear canal and with this proof of concept to develop a prototype and an algorithm for capturing and predicting ear canal information. Methods: We developed a novel prototype using structured light imaging to capture external images of the ear. Using a large database of existing ear impression images obtained by traditional methods, correlation analyses were carried out and established. A deep neural network was devised to build a predictive algorithm. Patients undergoing hearing aid evaluation undertook both methods of ear impression-taking. We evaluated their subjective feedback and determined if there was a close enough objective match between the images obtained from the impression techniques. Results: A prototype was developed and deployed for trial, and most participants were comfortable with this novel method of ear impression-taking. Partial matching of the ear canal could be obtained from the images taken, and the predictive algorithm applied for a few sample images was within good standard of error with proof of concept established. Discussion: Further studies are warranted to strengthen the predictive capabilities of the algorithm and determine optimal prototype imaging positions so that sufficient ear canal information can be obtained for three-dimensional printing. Ear impression-taking may then have the potential to be automated, with the possibility of same-day three-dimensional printing of the earmold to provide timely access. [ABSTRACT FROM AUTHOR]

Mechanistic insights into the role of the human microbiome in the predisposition to and treatment of disease are limited by the lack of methods to precisely add or remove microbial strains or genes from complex communities. Here, we demonstrate that engineered bacteriophage M13 can be used to deliver DNA to Escherichia coli within the mouse gastrointestinal (GI) tract. Delivery of a programmable exogenous CRISPR-Cas9 system enables the strain-specific depletion of fluorescently marked isogenic strains during competitive colonization and genomic deletions that encompass the target gene in mice colonized with a single strain. Multiple mechanisms allow E. coli to escape targeting, including loss of the CRISPR array or even the entire CRISPR-Cas9 system. These results provide a robust and experimentally tractable platform for microbiome editing, a foundation for the refinement of this approach to increase targeting efficiency, and a proof of concept for the extension to other phage-bacterial pairs of interest.

Nonalcoholic fatty liver disease is the leading cause of liver disease worldwide and can progress to nonalcoholic steatohepatitis (NASH) through physical inactivity and gut dysbiosis.1 Exercise training reverses gut dysbiosis in non-NASH persons with obesity and in NASH animal models.2,3 Consequently, we conducted a proof-of-concept study investigating the effect of exercise training on gut dysbiosis in NASH patients.

Drug development for Alzheimer disease and other neurodegenerative dementias, including frontotemporal dementia, has experienced a long history of phase 2 and phase 3 clinical trials that failed to show efficacy of investigational drugs. Despite differences in clinical and behavioral characteristics, these disorders have shared pathologies and face common challenges in designing early-phase trials that are predictive of late-stage success. Here, we discuss exploratory clinical trials in neurodegenerative dementias. These are generally phase 1b or phase 2a trials that are designed to assess pharmacologic effects and rely on biomarker outcomes, with shorter treatment durations and fewer patients than traditional phase 2 studies. Exploratory trials can establish go/no-go decision points, support proof of concept and dose selection, and terminate drugs that fail to show target engagement with suitable exposure and acceptable safety profiles. Early failure saves valuable resources including opportunity costs. This is especially important for programs in academia and small biotechnology companies but may be applied to high-risk projects in large pharmaceutical companies to achieve proof of concept more rapidly at lower costs than traditional approaches. Exploratory studies in a staged clinical development program may provide promising data to warrant the substantial resources needed to advance compounds through late-stage development. To optimize the design and application of exploratory trials, the Alzheimer's Drug Discovery Foundation and the Association for Frontotemporal Degeneration convened an advisory panel to provide recommendations on outcome measures and statistical considerations for these types of studies and study designs that can improve efficiency in clinical development.

The 2019 novel coronavirus infectious disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created an unsustainable need for molecular diagnostic testing. Molecular approaches such as reverse transcription (RT) polymerase chain reaction (PCR) offers highly sensitive and specific means to detect SARS-CoV-2 RNA, however, despite it being the accepted "gold standard", molecular platforms often require a tradeoff between speed versus throughput. Matrix assisted laser desorption ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS) has been proposed as a potential solution for COVID-19 testing and finding a balance between analytical performance, speed, and throughput, without relying on impacted supply chains. Combined with machine learning (ML), this MALDI-TOF-MS approach could overcome logistical barriers encountered by current testing paradigms. We evaluated the analytical performance of an ML-enhanced MALDI-TOF-MS method for screening COVID-19. Residual nasal swab samples from adult volunteers were used for testing and compared against RT-PCR. Two optimized ML models were identified, exhibiting accuracy of 98.3%, positive percent agreement (PPA) of 100%, negative percent agreement (NPA) of 96%, and accuracy of 96.6%, PPA of 98.5%, and NPA of 94% respectively. Machine learning enhanced MALDI-TOF-MS for COVID-19 testing exhibited performance comparable to existing commercial SARS-CoV-2 tests.

BackgroundMitochondrial dysfunction results in an imbalance between energy supply and demand in a failing heart. An innovative therapy that targets the intracellular bioenergetics directly through mitochondria transfer may be necessary.ObjectivesThe purpose of this study was to establish a preclinical proof-of-concept that extracellular vesicle (EV)-mediated transfer of autologous mitochondria and their related energy source enhance cardiac function through restoration of myocardial bioenergetics.MethodsHuman-induced pluripotent stem cell-derived cardiomyocytes (iCMs) were employed. iCM-conditioned medium was ultracentrifuged to collect mitochondria-rich EVs (M-EVs). Therapeutic effects of M-EVs were investigated using in vivo murine myocardial infarction (MI) model.ResultsElectron microscopy revealed healthy-shaped mitochondria inside M-EVs. Confocal microscopy showed that M-EV-derived mitochondria were transferred into the recipient iCMs and fused with their endogenous mitochondrial networks. Treatment with 1.0 × 108/ml M-EVs significantly restored the intracellular adenosine triphosphate production and improved contractile profiles of hypoxia-injured iCMs as early as 3 h after treatment. In contrast, isolated mitochondria that contained 300× more mitochondrial proteins than 1.0 × 108/ml M-EVs showed no effect after 24 h. M-EVs contained mitochondrial biogenesis-related messenger ribonucleic acids, including proliferator-activated receptor γ coactivator-1α, which on transfer activated mitochondrial biogenesis in the recipient iCMs at 24 h after treatment. Finally, intramyocardial injection of 1.0 × 108 M-EVs demonstrated significantly improved post-MI cardiac function through restoration of bioenergetics and mitochondrial biogenesis.ConclusionsM-EVs facilitated immediate transfer of their mitochondrial and nonmitochondrial cargos, contributing to improved intracellular energetics in vitro. Intramyocardial injection of M-EVs enhanced post-MI cardiac function in vivo. This therapy can be developed as a novel, precision therapeutic for mitochondria-related diseases including heart failure.

BackgroundWith increasing intention to treat HIV as early as possible, evidence to confirm the safety and therapeutic drug concentrations of a nevirapine-based antiretroviral regimen in the early neonatal period is needed. This study aims to establish dosing of nevirapine for very early treatment of HIV-exposed neonates at high risk of HIV acquisition.MethodsIMPAACT P1115 is a multinational phase 1/2 proof-of-concept study in which presumptive treatment for in-utero HIV infection is initiated within 48 h of birth in HIV-exposed neonates at high risk of HIV acquisition. Participants were neonates who were at least 34 weeks gestational age at birth and enrolled within 48 h of birth, born to women with presumed or confirmed HIV infection who had not received antiretrovirals during this pregnancy. The regimen consisted of two nucleoside reverse transcriptase inhibitors plus nevirapine dosed at 6 mg/kg twice daily for term neonates (≥37 weeks gestational age) or 4 mg/kg twice daily for 1 week and 6 mg/kg twice daily thereafter for preterm neonates (34 to

Artificial antigen-presenting cells (aAPCs) are synthetic versions of naturally occurring antigen-presenting cells (APCs) that, similar to natural APCs, promote efficient T effector cell responses in vitro. This report describes a method to produce acellular tolerogenic aAPCs made of biodegradable poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) and encapsulating IL-2 and TGF-β for a paracrine release to T cells. We document that these aAPCs can induce both human CD4+ and CD8+ T cells to become FoxP3+ T regulatory cells (Tregs). The aAPC NP-expanded human Tregs are functional in vitro and can modulate systemic autoimmunity in vivo in humanized NSG mice. These findings establish a proof-of-concept to use PLGA NPs as aAPCs for the induction of human Tregs in vitro and in vivo, highlighting the immunotherapeutic potential of this targeted approach to repair IL-2 and/or TGF-β defects documented in certain autoimmune diseases such as systemic lupus erythematosus.

Our understanding of the beads-on-a-string arrangement of nucleosomes has been built largely on high-resolution sequence-agnostic imaging methods and sequence-resolved bulk biochemical techniques. To bridge the divide between these approaches, we present the single-molecule adenine methylated oligonucleosome sequencing assay (SAMOSA). SAMOSA is a high-throughput single-molecule sequencing method that combines adenine methyltransferase footprinting and single-molecule real-time DNA sequencing to natively and nondestructively measure nucleosome positions on individual chromatin fibres. SAMOSA data allows unbiased classification of single-molecular 'states' of nucleosome occupancy on individual chromatin fibres. We leverage this to estimate nucleosome regularity and spacing on single chromatin fibres genome-wide, at predicted transcription factor binding motifs, and across human epigenomic domains. Our analyses suggest that chromatin is comprised of both regular and irregular single-molecular oligonucleosome patterns that differ subtly in their relative abundance across epigenomic domains. This irregularity is particularly striking in constitutive heterochromatin, which has typically been viewed as a conformationally static entity. Our proof-of-concept study provides a powerful new methodology for studying nucleosome organization at a previously intractable resolution and offers up new avenues for modeling and visualizing higher order chromatin structure.

Reliable, closed-chest methods for creating large animal models of acute myocardial hypoperfusion are limited. We demonstrated the feasibility and efficacy of using magnetic resonance (MR)-compatible 3D-printed coronary implants for establishing swine models of myocardial hypoperfusion. We designed, manufactured, and percutaneously deployed implants in 13 swine to selectively create focal coronary stenosis. To test the efficacy of the implants to cause hypoperfusion or ischemia in the perfused territory, we evaluated regional wall motion, myocardial perfusion, and infarction using MR imaging. The overall swine survival rate was 85% (11 of 13). The implant retrieval rate was 92% (12 of 13). Fluoroscopic angiography confirmed focal stenosis. Cine and perfusion MRI showed regional wall motion abnormalities and inducible ischemia, respectively. Late gadolinium enhancement and histopathology showed no myocardial infarction. Our minimally invasive technique has promising applications for validation of new diagnostic methods in cardiac MR. Graphical abstract Our new minimally invasive, percutaneous method for creating swine models of acute focal coronary stenosis can be used for magnetic resonance imaging studies of myocardial ischemia. Comparable to existing methods in its efficacy and reliability, this rapid prototyping technique will allow researchers to more easily conduct translational cardiac imaging studies of coronary artery disease in large animal models.

We give an approximate solution to the difficult inverse problem of inferring the topology of an unknown network from given time-dependent signals at the nodes. For example, we measure signals from individual neurons in the brain, and infer how they are inter-connected. We use Maximum Caliber as an inference principle. The combinatorial challenge of high-dimensional data is handled using two different approximations to the pairwise couplings. We show two proofs of principle: in a nonlinear genetic toggle switch circuit, and in a toy neural network.

BackgroundAlthough acute neurologic impairment might be transient, other long-term effects can be observed with mild traumatic brain injury. However, when pediatric patients with mild traumatic brain injury present for medical care, conventional imaging with CT and MR imaging often does not reveal abnormalities.ObjectiveTo determine whether edge density imaging can separate pediatric mild traumatic brain injury from typically developing controls.Materials and methodsSubjects were recruited as part of the "Therapeutic Resources for Attention Improvement using Neuroimaging in Traumatic Brain Injury" (TRAIN-TBI) study. We included 24 adolescents (χ=14.1 years of age, σ=1.6 years, range 10-16 years), 14 with mild traumatic brain injury (TBI) and 10 typically developing controls. Neurocognitive assessments included the pediatric version of the California Verbal Learning Test (CVLT) and the Attention Network Task (ANT). Diffusion MR imaging was acquired on a 3-tesla (T) scanner. Edge density images were computed utilizing fiber tractography. Principal component analysis (PCA) and support vector machines (SVM) were used in an exploratory analysis to separate mild TBI and control groups. The diagnostic accuracy of edge density imaging, neurocognitive tests, and fractional anisotropy (FA) from diffusion tensor imaging (DTI) was computed with two-sample t-tests and receiver operating characteristic (ROC) metrics.ResultsSupport vector machine-principal component analysis of edge density imaging maps identified three white matter regions distinguishing pediatric mild TBI from controls. The bilateral tapetum, sagittal stratum, and callosal splenium identified mild TBI subjects with sensitivity of 79% and specificity of 100%. Accuracy from the area under the ROC curve (AUC) was 94%. Neurocognitive testing provided an AUC of 61% (CVLT) and 71% (ANT). Fractional anisotropy yielded an AUC of 48%.ConclusionIn this proof-of-concept study, we show that edge density imaging is a new form of connectome mapping that provides better diagnostic delineation between pediatric mild TBI and healthy controls than DTI or neurocognitive assessments of memory or attention.

Coma and disordered consciousness are common manifestations of acute neurological conditions and are among the most pervasive and challenging aspects of treatment in neurocritical care. Gaps exist in patient assessment, outcome prognostication, and treatment directed specifically at improving consciousness and cognitive recovery. In 2019, the Neurocritical Care Society (NCS) launched the Curing Coma Campaign in order to address the "grand challenge" of improving the management of patients with coma and decreased consciousness. One of the first steps was to bring together a Scientific Advisory Council including coma scientists, neurointensivists, neurorehabilitationists, and implementation experts in order to address the current scientific landscape and begin to develop a framework on how to move forward. This manuscript describes the proceedings of the first Curing Coma Campaign Scientific Advisory Council meeting which occurred in conjunction with the NCS Annual Meeting in October 2019 in Vancouver. Specifically, three major pillars were identified which should be considered: endotyping of coma and disorders of consciousness, biomarkers, and proof-of-concept clinical trials. Each is summarized with regard to current approach, benefits to the patient, family, and clinicians, and next steps. Integration of these three pillars will be essential to the success of the Curing Coma Campaign as will expanding the "curing coma community" to ensure broad participation of clinicians, scientists, and patient advocates with the goal of identifying and implementing treatments to fundamentally improve the outcome of patients.

Autonomic nervous system involvement precedes the motor features of Parkinson's disease (PD). Our goal was to develop a proof-of-concept model for identifying subjects at high risk of developing PD by analysis of cardiac electrical activity. We used standard 10-s electrocardiogram (ECG) recordings of 60 subjects from the Honolulu Asia Aging Study including 10 with prevalent PD, 25 with prodromal PD, and 25 controls who never developed PD. Various methods were implemented to extract features from ECGs including simple heart rate variability (HRV) metrics, commonly used signal processing methods, and a Probabilistic Symbolic Pattern Recognition (PSPR) method. Extracted features were analyzed via stepwise logistic regression to distinguish between prodromal cases and controls. Stepwise logistic regression selected four features from PSPR as predictors of PD. The final regression model built on the entire dataset provided an area under receiver operating characteristics curve (AUC) with 95% confidence interval of 0.90 [0.80, 0.99]. The five-fold cross-validation process produced an average AUC of 0.835 [0.831, 0.839]. We conclude that cardiac electrical activity provides important information about the likelihood of future PD not captured by classical HRV metrics. Machine learning applied to ECGs may help identify subjects at high risk of having prodromal PD.

The urea cycle enzyme carbamoyl phosphate synthetase 1 (CPS1) catalyzes the initial step of the urea cycle; bi-allelic mutations typically present with hyperammonemia, vomiting, ataxia, lethargy progressing into coma, and death due to brain edema if ineffectively treated. The enzyme deficiency is particularly difficult to treat; early recognition is essential to minimize injury to the brain. Even under optimal conditions, therapeutic interventions are of limited scope and efficacy, with most patients developing long-term neurologic sequelae. One significant encumberment to gene therapeutic development is the size of the CPS1 cDNA, which, at 4.5 kb, nears the packaging capacity of adeno-associated virus (AAV). Herein we developed a split AAV (sAAV)-based approach, packaging the large transgene and its regulatory cassette into two separate vectors, thereby delivering therapeutic CPS1 by a dual vector system with testing in a murine model of the disorder. Cps1-deficient mice treated with sAAVs survive long-term with markedly improved ammonia levels, diminished dysregulation of circulating amino acids, and increased hepatic CPS1 expression and activity. In response to acute ammonia challenging, sAAV-treated female mice rapidly incorporated nitrogen into urea. This study demonstrates the first proof-of-principle that sAAV-mediated therapy is a viable, potentially clinically translatable approach to CPS1 deficiency, a devastating urea cycle disorder.

ObjectivesTo assess the validity of a bench-top model of an optical tympanometry device to diagnose in vitro model of middle ear effusion (MEE).Methods and materialsWe illuminated an in vitro model of ear canal and tympanic membrane with broadband light and relayed remitted light to a spectrometer system. We then used our proprietary algorithm to extract spectral features that, together with our logistic regression classifiers, led us to calculate a set of simplified indices related to different middle ear states. Our model included a glass vial covered with a porcine submucosa (representing the tympanic membrane) and filled with air, water, or milk solution (representing different MEE), and a set of cover-glass slips filled with either blood (representing erythema) or cerumen. By interchanging fluid types and cover-glass slips, we made measurements on combinations corresponding to normal healthy ear and purulent or serous MEE.ResultsEach simulated condition had a distinct spectral profile, which was then employed by our algorithm to discriminate clean and cerumen-covered purulent and serous MEE. Two logistic purulent and serous MEE classifiers correctly classified all in vitro middle ear states with 100% accuracy assessed by leave-one-out and k-fold cross validation.ConclusionsThis proof-of-concept in vitro study addressed an unmet need by introducing a device that easily and accurately can assess middle ear effusion. Future in vivo studies aimed at collecting data from clinical settings are warranted to further elucidate the validity of the technology in diagnosing pediatric acute otitis media.

BackgroundThe repair of osteochondral lesions remains a challenge due to its poor vascularity and limited healing potential. Micronized cartilage matrix (MCM) is dehydrated, decellularized, micronized allogeneic cartilage matrix that contains the components of native articular tissue and is hypothesized to serve as a scaffold for the formation of hyaline-like tissue. Our objective was to demonstrate in vitro that the use of MCM combined with mesenchymal stem cells (MSCs) can lead to the formation of hyaline-like cartilage tissue in a single-stage treatment model.DesignIn group 1 (no wash), 250 µL MCM was reconstituted in 150 µL Dulbecco's phosphate-buffered saline (DPBS) for 5 minutes. Group 2 (saline wash) included 250 µL MCM washed in 20 mL DPBS for 30 minutes, then aspirated to remove all DPBS and reconstituted in 150 µL DPBS. Group 3 (serum wash): 250µL MCM washed in 20 mL DPBS for 30 minutes, then aspirated and reconstituted in 150 µL fetal bovine serum. Each group was then added to 50 µL solution of MSC suspended in DPBS at a concentration of 1.2 × 106 cells/350 µL. After 3 weeks, the defects were extracted and sectioned to perform viability and histologic analyses.ResultsStem cells without rehydration of the MCM showed almost no viability whereas near complete cell viability was seen after rehydration with serum or saline solution, ultimately leading to chondrogenic differentiation and adhesion to the MCM particles.ConclusionWe have shown in this proof-of-concept in vitro study that MCM can serve as a scaffold for the growth of cartilage tissue for the treatment of osteochondral lesions.

Background Delivery of hydrogels to the heart is a promising strategy for mitigating the detrimental impact of myocardial infarction (MI). Challenges associated with the in vivo delivery of currently available hydrogels have limited clinical translation of this technology. Gelatin methacryloyl (GelMA) bioadhesive hydrogel could address many of the limitations of available hydrogels. The goal of this proof-of-concept study was to evaluate the cardioprotective potential of GelMA in a mouse model of MI. Methods and Results The physical properties of GelMA bioadhesive hydrogel were optimized in vitro. Impact of GelMA bioadhesive hydrogel on post-MI recovery was then assessed in vivo. In 20 mice, GelMA bioadhesive hydrogel was applied to the epicardial surface of the heart at the time of experimental MI. An additional 20 mice underwent MI but received no GelMA bioadhesive hydrogel. Survival rates were compared for GelMA-treated and untreated mice. Left ventricular function was assessed 3 weeks after experimental MI with transthoracic echocardiography. Left ventricular scar burden was measured with postmortem morphometric analysis. Survival rates at 3 weeks post-MI were 89% for GelMA-treated mice and 50% for untreated mice (P=0.011). Left ventricular contractile function was better in GelMA-treated than untreated mice (fractional shortening 37% versus 26%, P

BackgroundThe mechanisms of aberrant circulating platelet behavior following injury remain unclear. Platelets retain megakaryocyte immature ribonucleic acid (RNA) splicing and protein synthesis machinery to alter their functions based on physiologic signals. We sought to identify fluctuating platelet-specific RNA transcripts in cell-free plasma (CFP) from traumatic brain injury (TBI) patients as proof-of-concept for using RNA sequencing to improve our understanding of postinjury platelet behavior. We hypothesized that we could identify differential expression of activated platelet-specific spliced RNA transcripts from CFP of patients with isolated severe fatal TBI (fTBI) compared with minimally injured trauma controls (t-controls), filtered by healthy control (h-control) data sets.MethodsHigh-read depth RNA sequencing was applied to CFP from 10 patients with fTBI (Abbreviated Injury Scale [AIS] for head ≥3, AIS for all other categories

The National Institute of Mental Health (NIMH) 'fast-fail' approach seeks to improve too-often-misleading early-phase drug development methods by incorporating biomarker-based proof-of-mechanism (POM) testing in phase 2a. This first comprehensive application of the fast-fail approach evaluated the potential of κ-opioid receptor (KOR) antagonism for treating anhedonia with a POM study determining whether robust target engagement favorably impacts the brain circuitry hypothesized to mediate clinical effects. Here we report the results from a multicenter, 8-week, double-blind, placebo-controlled, randomized trial in patients with anhedonia and a mood or anxiety disorder (selective KOR antagonist (JNJ-67953964, 10 mg; n = 45) and placebo (n = 44)). JNJ-67953964 significantly increased functional magnetic resonance imaging (fMRI) ventral striatum activation during reward anticipation (primary outcome) as compared to placebo (baseline-adjusted mean: JNJ-67953964, 0.72 (s.d. = 0.67); placebo, 0.33 (s.d. = 0.68); F(1,86) = 5.58, P

The Gaussian graphical model (GGM) is an increasingly popular technique used in psychology to characterize relationships among observed variables. These relationships are represented as elements in the precision matrix. Standardizing the precision matrix and reversing the sign yields corresponding partial correlations that imply pairwise dependencies in which the effects of all other variables have been controlled for. The graphical lasso (glasso) has emerged as the default estimation method, which uses ℓ1 -based regularization. The glasso was developed and optimized for high-dimensional settings where the number of variables (p) exceeds the number of observations (n), which is uncommon in psychological applications. Here we propose to go 'back to the basics', wherein the precision matrix is first estimated with non-regularized maximum likelihood and then Fisher Z transformed confidence intervals are used to determine non-zero relationships. We first show the exact correspondence between the confidence level and specificity, which is due to 1 minus specificity denoting the false positive rate (i.e., α). With simulations in low-dimensional settings (p ≪ n), we then demonstrate superior performance compared to the glasso for detecting the non-zero effects. Further, our results indicate that the glasso is inconsistent for the purpose of model selection and does not control the false discovery rate, whereas the proposed method converges on the true model and directly controls error rates. We end by discussing implications for estimating GGMs in psychology.

ObjectiveTo demonstrate how bladder ultrasound can be useful in completing morcellation during difficult Holmium Laser Enucleation of the Prostate (HoLEP). As HoLEP has emerged as a standard of care for the treatment of benign prostatic hyperplasia, multiple studies have reported the potentially catastrophic complication of bladder injury during morcellation. This video aims to assist any urologist performing HoLEP by providing step-by-step instruction for using ultrasound to complete morcellation safely.MethodsEnucleation is performed using a 26-French continuous flow scope, off-set laser bridge with a laser stabilization catheter, and a 550 µm holmium laser fiber. Once the median and lateral lobes have been enucleated, the outer sheath is removed and the nephroscope is inserted to facilitate morcellation. Under dual inflow irrigation, the Piranha morcellator (Richard Wolf, Knittlingen, Germany) is introduced and set to the manufacturer's recommended settings of 1500 rpm. A 3.5-MHz convex abdominal ultrasound transducer (Hitachi Prosound Alpha 7; Hitachi Aloka Medical America, Wallingford, CT) under B-mode is used to visualize the bladder, predominantly in the sagittal orientation. Morcellation proceeds under simultaneous ultrasound and direct cystoscopic guidance.ResultsThe distended bladder is visualized concurrently with the ultrasound and via the nephroscope as the Piranha engages the adenoma and begins morcellation. Once the adenoma is engaged, the operator then drops their hands to place the morcellator in the center of the bladder. Ultrasound provides real-time feedback as to the location of the morcellator in relation to the adenoma and bladder.ConclusionThis video highlights the use of intraoperative bladder ultrasound as a visual aid to assist during the morcellation portion of HoLEP. This proof of concept demonstrates that ultrasound can be an additional tool to utilize during difficult cases when cystoscopic visualization during morcellation is limited.

Estrogen contributes to females' strong antibody response to microbial vaccines and proneness to autoimmunity, particularly antibody-mediated systemic autoimmunity, in females. We have hypothesized that this is due to estrogen-mediated potentiation of class switch DNA recombination (CSR) and somatic hypermutation (SHM). As we have shown, estrogen boosts AID expression, which is critical for both CSR and SHM, through upregulation of HoxC4, which together with NF-κB critically mediates Aicda (AID gene) promoter activation. We contend here that additional regulation of Aicda expression by estrogen occurs through epigenetic mechanisms. As we have shown, histone deacetylase inhibitors (HDIs) short-chain fatty acid (SCFA) butyrate and propionate as well as the pharmacologic HDI valproic acid upregulate miRNAs that silence AID expression, thereby modulating specific antibody responses in C57BL/6 mice and autoantibody responses in lupus-prone MRL/Fas lpr/lpr mice. Here, using constitutive knockout Esr1 -/- mice and B cells as well as conditional knockout Aicda cre/cre Esr1 flox/flox mice and B cells, we showed that the HDI-mediated downregulation of Aicda expression as well as the maturation of antibody and autoantibody responses is reversed by estrogen and enhanced by deletion of ERα or E2 inhibition. Estrogen's reversion of HDI-mediated inhibition of Aicda and CSR in antibody and autoantibody responses occurred through downregulation of B cell miR-26a, which, as we showed, targets Aicda mRNA 3'UTR. miR-26a was significantly upregulated by HDIs. Accordingly, enforced expression of miR-26a reduced Aicda expression and CSR, while miR-26a-sponges (competitive inhibitors of miR-26a) increased Aicda expression and CSR. Thus, our findings show that estrogen reverses the HDI-mediated downregulation of AID expression and CSR through selective modulation of miR-26a. They also provide mechanistic insights into the immunomodulatory activity of this hormone and a proof-of-principle for using combined ER inhibitor-HDI as a potential therapeutic approach.

Liver metastasis is a recalcitrant disease that usually leads to death of the patient. The present study established a unique patient-derived orthotopic xenograft (PDOX) nude mouse model of a highly aggressive liver metastasis of colon cancer. The aim of the present study was to demonstrate proof-of-concept that candidate drug combinations could significantly inhibit growth and re-metastasis of this recalcitrant tumor. The patient's liver metastasis was initially established subcutaneously in nude mice and the subcutaneous tumor tissue was then orthotopically implanted in the liver of nude mice to establish a PDOX model. Two studies were performed to test different drugs or drug combination, indicating that 5-fluorouracil (5-FU) + irinotecan (IRI) + bevacizumab (BEV) and regorafenib (REG) + selumetinib (SEL) had significantly inhibited liver metastasis growth (p = 0.013 and p = 0.035, respectively), and prevented liver satellite metastasis. This study is proof of concept that a PDOX model of highly aggressive colon-cancer metastasis can identify effective drug combinations and that the model has future clinical potential.

Robotic assistance can help in physically guiding the drilling trajectory during zygomatic implant positioning. A new robot-assisted strategy for a flapless zygomatic implant placement protocol is reported here. In this protocol, a preoperative computed tomography scan is used to plan the surgical path. After surface registration, the ROSA robot (Zimmer Biomet Robotics) guides several steps, which are performed with shared control. The surgeon performs the drilling and tapping, guided by the robotic arm, which is positioned according to the planned trajectory. Placement of the zygomatic implant is done manually. Immediate intraoperative 3D verification is performed by cone beam computed tomography (flat-panel detector, Medtronic O-arm II). Four zygomatic implants were placed in the case patient according to the flapless protocol, with a mean vector error of 1.78 mm (range 0.52–4.70 mm). A screw-retained temporary prosthesis was placed on the same day. No significant complications were observed. The application of this robot-assisted surgical protocol, which guarantees a very high degree of precision, may reduce inaccuracies in the positioning of zygomatic implants that could deviate from the surgeon's plan. This appears to be a potentially safe flapless surgery technique. Drill slipping on the crest or on the maxillary wall is the main source of error in this procedure, emphasizing the usefulness of the assisted surgical guidance with haptic feedback. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of this study was to define and clarify the concept of the family caregivers’ perceived value of caring for older patients. Methods: The hybrid model of Schwartz-Barcott and Kim was used to perform the concept analysis of perceived value of caring. Concept analysis was conducted in three phases: the theoretical phase, fieldwork phase, and final analysis phase. Results: The perceived value of caring was found to have four dimensions and nine attributes. The providing care dimension consisted of two attributes—care behavior and care attitude—and the goals and expectation dimension included three attributes—social norms, family norms, and personal norms. The other two dimensions consisted of two attributes each: a belief in usefulness and a belief in meaning in the building beliefs dimension and life expansion and insight into the future in the suffering sublimation dimension. Finally, perceived value of caring can be defined as the personal belief that behavior or attitude of providing physical, mental, and social services to those in need of care is usefulness and important based on one’s goals and criteria, and with the personal effort of sublimating suffering that arises from care. Conclusion: Based on the study findings, it is suggested that the use of the perceived value of caring should expand in nursing phenomena. [ABSTRACT FROM AUTHOR]

Cell cycle deregulation is a cancer hallmark that has stimulated the development of mitotic inhibitors with differing mechanisms of action. Quantitative phase imaging (QPI) is an emerging approach for determining cancer cell sensitivities to chemotherapies in vitro. Cancer cell fates in response to mitotic inhibitors are agent- and dose-dependent. Fates that lead to chromosomal instabilities may result in a survival advantage and drug resistance. Conventional techniques for quantifying cell fates are incompatible with growth inhibition assays that produce binary live/dead results. Therefore, we used QPI to quantify post-mitotic fates of G0/G1 synchronized HeLa cervical adenocarcinoma and M202 melanoma cells during 24 h of escalating-dose exposures to mitotic inhibitors, including microtubule inhibitors paclitaxel and colchicine, and an Aurora kinase A inhibitor, VX-680. QPI determined cell fates by measuring changes in cell biomass, morphology, and mean phase-shift. Cell fates fell into three groups: (1) bipolar division from drug failure; (2) cell death or sustained mitotic arrest; and (3) aberrant endocycling or multipolar division. In this proof-of-concept study, colchicine was most effective in producing desirable outcomes of sustained mitotic arrest or death throughout its dosing range, whereas both paclitaxel and VX-680 yielded dose-dependent multipolar divisions or endocycling, respectively. Furthermore, rapid completion of mitosis associated with bipolar divisions whereas prolonged mitosis associated with multipolar divisions or cell death. Overall, QPI measurement of drug-induced cancer cell fates provides a tool to inform the development of candidate agents by quantifying the dosing ranges over which suboptimal inhibitor choices lead to undesirable, aberrant cancer cell fates.

We assessed whether a bone resorption marker, measured early in the menopause transition (MT), is associated with change in femoral neck size and strength during the MT. Higher levels of bone resorption were associated with slower increases in femoral neck size and faster decreases in femoral neck strength.PurposeComposite indices of the femoral neck's ability to withstand compressive (compression strength index, CSI) and impact (impact strength index, ISI) forces integrate DXA-derived femoral neck width (FNW), bone mineral density (BMD), and body size. During the menopause transition (MT), FNW increases, and CSI and ISI decrease. This proof-of-concept study assessed whether a bone resorption marker, measured early in the MT, is associated with rates of change in FNW, CSI and ISI during the MT.MethodsWe used previously collected bone resorption marker (urine collagen type I N-telopeptide [U-NTX]) and femoral neck strength data from 696 participants from the Study of Women's Health Across the Nation (SWAN), a longitudinal study of the MT in a multi-ethnic cohort of community-dwelling women.ResultsAdjusted for MT stage (pre- vs. early perimenopause), age, body mass index (BMI), bone resorption marker collection time, and study site in multivariable linear regression, bone resorption in pre- and early perimenopause was not associated with transmenopausal decline rate in femoral neck BMD. However, each standard deviation (SD) increase in bone resorption level was associated with 0.2% per year slower increase in FNW (p = 0.03), and 0.3% per year faster declines in CSI (p = 0.02) and ISI (p = 0.01). When restricted to women in early perimenopause, the associations of bone resorption with change in FNW, CSI, and ISI were similar to those in the full sample.ConclusionsMeasuring a bone resorption marker in pre- and early perimenopause may identify women who will experience the greatest loss in bone strength during the MT.

ObjectivesThe use of near infrared transillumination for caries detection is gaining recognition in daily practice. Differentiation between cavitated and non cavitated proximal lesions is recognized as a threshold for restorative treatment. This investigation focused on the use of a near infrared absorbent dye which may enhance the detection of cavitation on near infrared transillumination images.Materials and methods1: Natural teeth with artificial proximal cavitation were images with 3 different dyes to establish that near infrared absorbent dye can act like a contrast medium. 2: Natural teeth with natural cavitated lesions were used to investigate the contrast enhancing effect of indocyanine green (ICG) on near infrared transillumination images. 3: Artificial teeth with artificial cavitations were used to determine the best consistency of ICG as a contrast medium. 4: natural teeth with proximal lesions were used to confirm that ICG can differentiate between cavitated and non cavitated proximal lesions.Results1: ICG enhanced the contrast of cavitations compared to other dyes (ANOVA; p  0.05). 3: ICG in a gel form enhanced the detection of cavitated lesions when compared to liquid ICG (t-test; p

ObjectiveDysfunction in positive affect is a defining symptom of bipolar I disorder (BD), both during and between mood episodes. We hypothesize that helping people with BD learn skills to create balance in their affective experiences by engaging in strategies that increase low activation positive emotion (LAP; e.g., relaxation) could help to improve well-being during periods of symptom remission. We discuss the development and preliminary outcomes of a positive emotion regulation (PER) group treatment for people with BD, designed as a supplement to pharmacological treatment.MethodThe Learning Affective Understanding for a Rich Emotional Life (LAUREL) intervention is a group-based intervention covering 10 empirically supported skills designed to increase LAP. Sixteen people with BD enrolled in the LAUREL intervention and twelve completed baseline and post-intervention assessments.ResultsParticipants who completed the study (n = 12) attended the majority of groups (87.96%) and reported practicing skills, on average, 16 times a week. We were unable to detect significant differences in mania symptoms following engagement in this PER intervention. Finally, participants reported increases in several areas associated with well-being post-intervention, including mindfulness, reappraisal, and self-compassion.ConclusionThis study provides a theoretical framework and preliminary support for a PER intervention for BD.

BackgroundIn rodents, context specificity of Pavlovian extinction is attenuated by manipulations that impair hippocampal function, including systemic administration of scopolamine, a muscarinic-cholinergic receptor antagonist. Context renewal translates into return of fear following exposure therapy to feared situations. We evaluated the effectiveness of scopolamine for attenuating context renewal of phobic fear in humans.MethodsA total of 60 participants (35 female, 22 male, 1 transgender, 2 undeclared) with social anxiety disorder and fear of public speaking were randomized to placebo, 0.5 mg scopolamine, or 0.6 mg scopolamine. They completed seven exposure sessions in an exposure context and subsequently tested in the exposure context (extinction retest) versus a different context (context renewal test), which were counterbalanced. Testing 1 month later occurred in the exposure context (long-term extinction retest). Fear measures included skin conductance and self-reported distress during speeches. Hippocampus-dependent cognitive tasks were completed as well.ResultsScopolamine augmented extinction across exposure sessions on skin conductance response and skin conductance level. Lower skin conductance response at context renewal in scopolamine groups relative to the placebo group was constrained to simple effects and complicated by unexpected outcomes within placebo and on self-reported fear. Scopolamine led to lower skin conductance response at long-term extinction retest. Scopolamine impaired performance on a cognitive task of hippocampal function.ConclusionsNoninvasive and well-tolerated scopolamine impaired hippocampal processes and augmented extinction during exposure. Drug-free effects persisted 1 month later. Findings at context renewal were limited and suggestive only. Further investigation is warranted with varying scopolamine dosages.

Bacteriophage (phage) have attractive advantages as delivery systems compared with mammalian viruses, but have been considered poor vectors because they lack evolved strategies to confront and overcome mammalian cell barriers to infective agents. We reasoned that improved efficacy of delivery might be achieved through structural modification of the viral capsid to avoid pre- and postinternalization barriers to mammalian cell transduction. We generated multifunctional hybrid adeno-associated virus/phage (AAVP) particles to enable simultaneous display of targeting ligands on the phage's minor pIII proteins and also degradation-resistance motifs on the very numerous pVIII coat proteins. This genetic strategy of directed evolution bestows a next-generation of AAVP particles that feature resistance to fibrinogen adsorption or neutralizing antibodies and ability to escape endolysosomal degradation. This results in superior gene transfer efficacy in vitro and also in preclinical mouse models of rodent and human solid tumors. Thus, the unique functions of our next-generation AAVP particles enable improved targeted gene delivery to tumor cells.

Tryptophan, an essential amino acid, is metabolized into a variety of small molecules capable of impacting human physiology, and aberrant tryptophan metabolism has been linked to a number of diseases. There are three principal routes by which tryptophan is degraded, and thus methods for measuring metabolic flux through these pathways can be used to understand the factors that perturb tryptophan metabolism and potentially to measure disease biomarkers. Here, we describe a method utilizing 6-fluorotryptophan as a probe for detecting tryptophan metabolites in ex vivo tissue samples via 19F nuclear magnetic resonance. As a proof of concept, we demonstrate that 6-fluorotryptophan can be used to measure changes in tryptophan metabolism resulting from antibiotic-induced changes in gut microbiota composition. Taken together, we describe a general strategy for monitoring amino acid metabolism using 19F nuclear magnetic resonance that is operationally simple and does not require chromatographic separation of metabolites.

BackgroundLoss of bronchoprotection (LOBP) with a regularly used long-acting β2-adrenergic receptor agonist (LABA) is well documented. LOBP has been attributed to β2-adrenergic receptor (B2AR) downregulation, a process requiring farnesylation, which is inhibited by alendronate.ObjectiveWe sought to determine whether alendronate can reduce LABA-associated LOBP in inhaled corticosteroid (ICS)-treated patients.MethodsWe conducted a randomized, double-blind, placebo-controlled, parallel-design, proof-of-concept trial. Seventy-eight participants with persistent asthma receiving 250 μg of fluticasone twice daily for 2 weeks were randomized to receive alendronate or placebo while initiating salmeterol for 8 weeks. Salmeterol-protected methacholine challenges (SPMChs) and PBMC B2AR numbers (radioligand binding assay) and signaling (cyclic AMP ELISA) were assessed before randomization and after 8 weeks of ICS plus LABA treatment. LOBP was defined as a more than 1 doubling dose reduction in SPMCh PC20 value.ResultsThe mean doubling dose reduction in SPMCh PC20 value was 0.50 and 0.27 with alendronate and placebo, respectively (P = .62). Thirty-eight percent of participants receiving alendronate and 33% receiving placebo had LOBP (P = .81). The after/before ICS plus LABA treatment ratio of B2AR number was 1.0 for alendronate (P = .86) and 0.8 for placebo (P = .15; P = .31 for difference between treatments). The B2AR signaling ratio was 0.89 for alendronate (P = .43) and 1.02 for placebo (P = .84; P = .44 for difference). Changes in lung function and B2AR number and signaling were similar between those who did and did not experience LOBP.ConclusionThis study did not find evidence that alendronate reduces LABA-associated LOBP, which relates to the occurrence of LOBP in only one third of participants. LOBP appears to be less common than presumed in concomitant ICS plus LABA-treated asthmatic patients. B2AR downregulation measured in PBMCs does not appear to reflect LOBP.

Detecting accurate concentrations of gas in environments with dynamically changing relative humidity conditions has been a challenge in gas sensing technology. We report a method to eliminate effects of humidity response in chemical-sensitive field-effect transistors using microheaters. Using a hydrogen gas sensor with Pt/FOTS active material as a test case, we demonstrate that a sensor response of 3844% to a relative humidity change of 50 to 90% at 25 °C can be reduced to a negligible response of 11.6% by utilizing microheaters. We also show the advantage of this technique in maintaining the same sensitivity in changing ambient temperatures and its application to the nitrogen dioxide gas sensors.

An important advance in biosensor research is the extension and application of laboratory-developed methodologies toward clinical diagnostics, though the propensity toward nonspecific binding of materials in clinically relevant matrices, such as human blood serum and plasma, frequently leads to compromised assays. Several surface chemistries have been developed to minimize nonspecific interactions of proteins and other biological components found within blood and serum samples, though these often exhibit substantially variable outcomes. Herein we report a surface chemistry consisting of a charged-matched supported lipid membrane that has been tailored to form over a gold surface functionalized with protein A. Fine tuning of the interfacial charge of this membrane, along with rational selection of a backfilling self-assembled monolayer, allows for high surface coverage with retention of orientation-controlled capture antibody attachment. We demonstrate using surface-plasmon resonance (SPR) that this highly charged lipid membrane is antifouling, allowing for complete removal of nonspecific human serum and plasma components using only a mild buffer rinse, which we attribute to unique steric interactions with the underlying surface. Furthermore, this surface chemistry is successfully applied for specific detection of IgG and cholera toxin in undiluted human biofluids with negligible sacrifice of SPR signal compared to buffered analysis. This novel lipid membrane interface over protein A may open new avenues for direct biosensing of disease markers within clinical samples.

ObjectivesThe purpose of this prospective proof of concept study was to investigate the feasibility of using percutaneous peripheral nerve stimulation of the femoral nerve to treat pain in the immediate postoperative period following ambulatory anterior cruciate ligament reconstruction with a patellar autograft.Materials and methodsPreoperatively, an electrical lead (SPRINT, SPR Therapeutics, Inc., Cleveland, OH, USA) was percutaneously implanted with ultrasound guidance anterior to the femoral nerve caudad to the inguinal crease. Within the recovery room, subjects received 5 min of either stimulation or sham in a randomized, double-masked fashion followed by a 5-min crossover period, and then continuous active stimulation until lead removal postoperative Day 14-28. Statistics were not applied to the data due to the small sample size of this feasibility study.ResultsDuring the initial 5-min treatment period, subjects randomized to stimulation (n = 5) experienced a slight downward trajectory (decrease of 7%) in their pain over the 5 min of treatment, while those receiving sham (n = 5) reported a slight upward trajectory (increase of 4%) until their subsequent 5-min stimulation crossover, during which time they also experienced a slight downward trajectory (decrease of 11% from baseline). A majority of subjects (80%) used a continuous adductor canal nerve block for rescue analgesia (in addition to stimulation) during postoperative Days 1-3, after which the median resting and dynamic pain scores remained equal or less than 1.5 on the numeric rating scale, respectively, and the median daily opioid consumption was less than 1.0 tablet.ConclusionsThis proof of concept study demonstrates that percutaneous femoral nerve stimulation is feasible for ambulatory knee surgery; and suggests that this modality may be effective in providing analgesia and decreasing opioid requirements following anterior cruciate ligament reconstruction. clinicaltrials.gov: NCT02898103.

The MST1R (RON) kinase is overexpressed in >80% of human pancreatic cancers, but its role in pancreatic carcinogenesis is unknown. In this study, we examined the relevance of Mst1r kinase to Kras driven pancreatic carcinogenesis using genetically engineered mouse models. In the setting of mutant Kras, Mst1r overexpression increased acinar-ductal metaplasia (ADM), accelerated the progression of pancreatic intraepithelial neoplasia (PanIN), and resulted in the accumulation of (mannose receptor C type 1) MRC1+, (arginase 1) Arg+ macrophages in the tumor microenvironment. Conversely, absence of a functional Mst1r kinase slowed PanIN initiation, resulted in smaller tumors, prolonged survival and a reduced tumor-associated macrophage content. Mst1r expression was associated with increased production of its ligand Mst1, and in orthotopic models, suppression of Mst1 expression resulted in reduced tumor size, changes in macrophage polarization and enhanced T cell infiltration. This study demonstrates the functional significance of Mst1r during pancreatic cancer initiation and progression. Further, it provides proof of concept that targeting Mst1r can modulate pancreatic cancer growth and the microenvironment. This study provides further rationale for targeting Mst1r as a therapeutic strategy.

Despite current immunosuppressive strategies, long-term lung transplant outcomes remain poor due to rapid allogenic responses. Using a stringent mouse model of allo-airway transplantation, we identify the CCR4-ligand axis as a central node driving secondary lymphoid tissue homing and activation of the allogeneic T cells that prevent long-term allograft survival. CCR4 deficiency on transplant recipient T cells diminishes allograft injury and when combined with CTLA4-Ig leads to an unprecedented long-term lung allograft accommodation. Thus, we identify CCR4-ligand interactions as a central mechanism driving allogeneic transplant rejection and suggest it as a potential target to enhance long-term lung transplant survival.

The transcription factor nuclear factor of activated T cells (NFAT) has a key role in both T cell activation and tolerance and has emerged as an important target of immune modulation. NFAT directs the effector arm of the immune response in the presence of activator protein-1 (AP-1), and T cell anergy/exhaustion in the absence of AP-1. Envisioning a strategy for selective modulation of the immune response, we designed a FRET-based high-throughput screen to identify compounds that disrupt the NFAT:AP-1:DNA complex. We screened ∼202,000 small organic compounds and identified 337 candidate inhibitors. We focus here on one compound, N-(3-acetamidophenyl)-2-[5-(1H-benzimidazol-2-yl)pyridin-2-yl]sulfanylacetamide (Compound 10), which disrupts the NFAT:AP-1 interaction at the composite antigen-receptor response element-2 site without affecting the binding of NFAT or AP-1 alone to DNA. Compound 10 binds to DNA in a sequence-selective manner and inhibits the transcription of the Il2 gene and several other cyclosporin A-sensitive cytokine genes important for the effector immune response. This study provides proof-of-concept that small molecules can inhibit the assembly of specific DNA-protein complexes, and opens a potential new approach to treat human diseases where known transcription factors are deregulated.

We developed an integrated, modular approach to predicting chemical toxicity relying on in vitro assay data, linkage of molecular targets to disease categories, and software for ranking chemical activity and examining structural features (chemotypes). We evaluate our approach in a proof-of-concept exercise to identify and prioritize chemicals of potential carcinogenicity concern. We identified 137 cancer pathway-related assays from a subset of U.S. EPA's ToxCast platforms. We mapped these assays to key characteristics of carcinogens and found they collectively assess 5 of 10 characteristics. We ranked all 1061 chemicals screened in Phases I and II of ToxCast by their activity in the selected cancer pathway-related assays using Toxicological Prioritization Index software. More chemicals used as biologically active agents (eg, pharmaceuticals) ranked in the upper 50% versus lower 50%. Twenty-three chemotypes are enriched in the top 5% (n = 54) of chemicals; these features may be important for their activity in cancer pathway-related assays. The biological coverage of the ToxCast assays related to cancer pathways is limited and short-term assays may not capture the biology of some key characteristics. Metabolism is also minimal in the assays. The ability of our approach to identify chemicals with cancer hazard is limited with the current input data, but we expect that our approach can be applied with future iterations of ToxCast and other data for improved chemical prioritization and characterization. The novel approach and proof-of-concept exercise described here for ranking chemicals for potential carcinogenicity concern is modular, adaptable, and amenable to evolving data streams.

BackgroundEstimation of glomerular filtration rate (GFR) using estimated glomerular filtration rate creatinine (eGFRcr) is central to clinical practice but has limitations. We tested the hypothesis that serum metabolomic profiling can identify novel markers that in combination can provide more accurate GFR estimates.MethodsWe performed a cross-sectional study of 200 African American Study of Kidney Disease and Hypertension (AASK) and 265 Multi-Ethnic Study of Atherosclerosis (MESA) participants with measured GFR (mGFR). Untargeted gas chromatography/dual mass spectrometry- and liquid chromatography/dual mass spectrometry-based quantification was followed by the development of targeted assays for 15 metabolites. On the log scale, GFR was estimated from single- and multiple-metabolite panels and compared with eGFR using the Chronic Kidney Disease Epidemiology equations with creatinine and/or cystatin C using established metrics, including the proportion of errors >30% of mGFR (1-P30), before and after bias correction.ResultsOf untargeted metabolites in the AASK and MESA, 283 of 780 (36%) and 387 of 1447 (27%), respectively, were significantly correlated (P ≤ 0.001) with mGFR. A targeted metabolite panel eGFR developed in the AASK and validated in the MESA was more accurate (1-P30 3.7 and 1.9%, respectively) than eGFRcr [11.2 and 18.5%, respectively (P  0.05) and 9.1% (P

Colloidal drug aggregates have been a nuisance in drug screening, yet, because they inherently comprise drug-rich particles, they may be useful in vivo if issues of stability can be addressed. As the first step toward answering this question, we optimized colloidal drug aggregate formulations using a fluorescence-based assay to study fulvestrant colloidal formation and stability in high (90%) serum conditions in vitro. We show, for the first time, that the critical aggregation concentration of fulvestrant depends on media composition and increases with serum concentration. Excipients, such as polysorbate 80, stabilize fulvestrant colloids in 90% serum in vitro for over 48 h. Using fulvestrant and an investigational pro-drug, pentyloxycarbonyl-( p-aminobenzyl) doxazolidinylcarbamate (PPD), as proof-of-concept colloidal formulations, we demonstrate that the in vivo plasma half-life for stabilized colloids is greater than their respective monomeric forms. These studies demonstrate the potential of turning the nuisance of colloidal drug aggregation into an opportunity for drug-rich formulations.