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Rapid diagnostic tests (point-of-care devices) are critical components of informed patient care and public health monitoring (surveillance applications). We propose that among the many rapid diagnostics platforms that have been tested or are in development, lateral flow immunoassays and synthetic biology–based diagnostics (including CRISPR-based diagnostics) represent the best overall options given their ease of use, scalability for manufacturing, sensitivity, and specificity. This review describes the identification of lateral flow immunoassay monoclonal antibody pairs that detect and distinguish between closely related pathogens and that are used in combination with functionalized multicolored nanoparticles and computational methods to deconvolute data. We also highlight the promise of synthetic biology–based diagnostic tests, which use synthetic genetic circuits that activate upon recognition of a pathogen-associated nucleic acid sequence, and discuss how the combined or parallel use of lateral flow immunoassays and synthetic biology tools may represent the future of scalable rapid diagnostics.

Understanding and reshaping cellular behaviors with synthetic gene networks requires the ability to sense and respond to changes in the intracellular environment. Intracellular proteins are involved in almost all cellular processes, and thus can provide important information about changes in cellular conditions such as infections, mutations, or disease states. Here we report the design of a modular platform for intrabody-based protein sensing-Actuation devices with transcriptional output triggered by detection of intracellular proteins in mammalian cells. We demonstrate reporter activation response (fluorescence, apoptotic gene) to proteins involved in hepatitis C virus (HCV) infection, human immunodeficiency virus (HIV) infection, and Huntington's disease, and show sensor-based interference with HIV-1 downregulation of HLA-I in infected T cells. Our method provides a means to link varying cellular conditions with robust control of cellular behavior for scientific and therapeutic applications., United States. Defense Advanced Research Projects Agency (Grant W911NF-11-2-0054), National Institutes of Health (U.S.) (Grant P50 GM098792)

We describe a method that enables the multiplex screening of a pool of many different donor cell lines. Our method accurately predicts each donor proportion from the pool without requiring the use of unique DNA barcodes as markers of donor identity. Instead, we take advantage of common single nucleotide polymorphisms, whole-genome sequencing, and an algorithm to calculate the proportions from the sequencing data. By testing using simulated and real data, we showed that our method robustly predicts the individual proportions from a mixed-pool of numerous donors, thus enabling the multiplexed testing of diverse donor cells en masse., National Human Genome Research Institute (U.S.) (Grant RM1HG008525), Robert Wood Johnson Foundation (Grant 74178)

Background: Despite the increasing recognition that microbial communities within the human body are linked to health, we have an incomplete understanding of the environmental and molecular interactions that shape the composition of these communities. Although host genetic factors play a role in these interactions, these factors have remained relatively unexplored given the requirement for large population-based cohorts in which both genotyping and microbiome characterization have been performed. Methods: We performed whole-genome sequencing of 298 donors from the Human Microbiome Project (HMP) healthy cohort study to accompany existing deep characterization of their microbiomes at various body sites. This analysis yielded an average sequencing depth of 32x, with which we identified 27 million (M) single nucleotide variants and 2.3 M insertions-deletions. Results: Taxonomic composition and functional potential of the microbiome covaried significantly with genetic principal components in the gastrointestinal tract and oral communities, but not in the nares or vaginal microbiota. Example associations included validation of known associations between FUT2 secretor status, as well as a variant conferring hypolactasia near the LCT gene, with Bifidobacterium longum abundance in stool. The associations of microbial features with both high-level genetic attributes and single variants were specific to particular body sites, highlighting the opportunity to find unique genetic mechanisms controlling microbiome properties in the microbial communities from multiple body sites. Conclusions: This study adds deep sequencing of host genomes to the body-wide microbiome sequences already extant from the HMP healthy cohort, creating a unique, versatile, and well-controlled reference for future studies seeking to identify host genetic modulators of the microbiome. Keywords: Human Microbiome Project, Microbiome and human genetics, Human genome sequence, Microbiome metagenome sequence, Association studies, National Institutes of Health (U.S.) (Grant U54HG003067 ), National Institutes of Health (U.S.) (Grant U54DE023798), United States. Army Research Office (Grant W911NF-11-1-0429)

Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas13a (previously known as C2c2) exhibits a "collateral effect" of promiscuous ribonuclease activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications., United States. Air Force Office of Scientific Research (Grant FA9550-14-1-0060), Defense Threat Reduction Agency (DTRA) (Grant HDTRA1-14-1-0006), National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706), National Institutes of Health (U.S.) (Grant 1R01-MH110049)

Cisplatin and its platinum analogs, carboplatin and oxaliplatin, are some of the most widely used cancer chemotherapeutics. Although cisplatin and carboplatin are used primarily in germ cell, breast and lung malignancies, oxaliplatin is instead used almost exclusively to treat colorectal and other gastrointestinal cancers. Here we utilize a unique, multi-platform genetic approach to study the mechanism of action of these clinically established platinum anti-cancer agents, as well as more recently developed cisplatin analogs. We show that oxaliplatin, unlike cisplatin and carboplatin, does not kill cells through the DNA-damage response. Rather, oxaliplatin kills cells by inducing ribosome biogenesis stress. This difference in drug mechanism explains the distinct clinical implementation of oxaliplatin relative to cisplatin, and it might enable mechanistically informed selection of distinct platinum drugs for distinct malignancies. These data highlight the functional diversity of core components of front-line cancer therapy and the potential benefits of applying a mechanism-based rationale to the use of our current arsenal of anti-cancer drugs.

Opioids are the preferred medications for the treatment of pain in the intensive care unit. While undertreatment leads to unrelieved pain and poor clinical outcomes, excessive use of opioids puts patients at risk of experiencing multiple adverse effects. In this work, we present a sequential decision making framework for opioid dosing based on deep reinforcement learning. It provides real-time clinically interpretable dosing recommendations, personalized according to each patient's evolving pain and physiological condition. We focus on morphine, one of the most commonly prescribed opioids. To train and evaluate the model, we used retrospective data from the publicly available MIMIC-3 database. Our results demonstrate that reinforcement learning may be used to aid decision making in the intensive care setting by providing personalized pain management interventions., 2019 41st Annual International Conference of the IEEE Engineering in Medicine & Biology Society (EMBC)

Xenophagy is a form of selective autophagy that involves the targeting and elimination of intracellular pathogens through several recognition, recruitment, and ubiquitination events. E3 ubiquitin ligases control substrate selectivity in the ubiquitination cascade; however, systematic approaches to map the role of E3 ligases in antibacterial autophagy have been lacking. We screened more than 600 putative human E3 ligases, identifying E3 ligases that are required for adaptor protein recruitment and LC3-bacteria colocalization, critical steps in antibacterial autophagy. An unbiased informatics approach pinpointed RNF166 as a key gene that interacts with the autophagy network and controls the recruitment of ubiquitin as well as the autophagy adaptors p62 and NDP52 to bacteria. Mechanistic studies demonstrated that RNF166 catalyzes K29- and K33-linked polyubiquitination of p62 at residues K91 and K189. Thus, our study expands the catalog of E3 ligases that mediate antibacterial autophagy and identifies a critical role for RNF166 in this process., Leona M. and Harry B. Helmsley Charitable Trust (2014PG-IBD016), National Institutes of Health (U.S.) (R01DK097485), National Institutes of Health (U.S.) (U19AI109725), National Institutes of Health (U.S.) (P30DK043351)

Contemporary endovascular stents are the product of an iterative design and development process that leverages evolving concepts in vascular biology and engineering. This article reviews how insights into vascular pathophysiology, materials science, and design mechanics drive stent design and explain modes of stent failure. Current knowledge of pathologic processes is providing a more complete picture of the factors mediating stent failure. Further evolution of endovascular stents includes bioresorbable platforms tailored to treat plaques acutely and to then disappear after lesion pacification. Ongoing refinement of stent technology will continue to require insights from pathology to understand adverse events, refine clinical protocols, and drive innovation. Keywords: Coronary artery disease; Pathology; Bare metal stent; Drug-eluting stent; In-stent restenosis; In-stent thrombosis; Atherosclerosis; Neoatherosclerosis, National Institutes of Health (U.S.) (Grant R01 GM-49039)

Human immunodeficiency virus (HIV) evolves within infected persons to escape being destroyed by the host immune system, thereby preventing effective immune control of infection. Here, we combine methods from evolutionary dynamics and statistical physics to simulate in vivo HIV sequence evolution, predicting the relative rate of escape and the location of escape mutations in response to T-cell-mediated immune pressure in a cohort of 17 persons with acute HIV infection. Predicted and clinically observed times to escape immune responses agree well, and we show that the mutational pathways to escape depend on the viral sequence background due to epistatic interactions. The ability to predict escape pathways and the duration over which control is maintained by specific immune responses open the door to rational design of immunotherapeutic strategies that might enable long-term control of HIV infection. Our approach enables intra-host evolution of a human pathogen to be predicted in a probabilistic framework., National Institute of Allergy and Infectious Diseases (U.S.) (Grant UM1-AI100663)

Purpose To compare visualization of choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD) using an ultrahigh-speed swept-source (SS) optical coherence tomography angiography (OCTA) prototype vs a spectral-domain (SD) OCTA device. Design Comparative analysis of diagnostic instruments. Methods Patients were prospectively recruited to be imaged on SD OCT and SS OCT devices on the same day. The SD OCT device employed is the RTVue Avanti (Optovue, Inc, Fremont, California, USA), which operates at ∼840 nm wavelength and 70 000 A-scans/second. The SS OCT device used is an ultrahigh-speed long-wavelength prototype that operates at ∼1050 nm wavelength and 400 000 A-scans/second. Two observers independently measured the CNV area on OCTA en face images from the 2 devices. The nonparametric Wilcoxon signed rank test was used to compare area measurements and P values of, National Institutes of Health (U.S.) (R01-EY011289-28), National Institutes of Health (U.S.) (R44-EY022864-03), National Institutes of Health (U.S.) (R01-CA075289-17), United States. Air Force Office of Scientific Research (FA9550-10-1-0551), United States. Air Force Office of Scientific Research (FA9550-12-1-0499)

Sequencing of exomes and genomes has revealed abundant genetic variation affecting the coding sequences of human transcription factors (TFs), but the consequences of such variation remain largely unexplored. We developed a computational, structure-based approach to evaluate TF variants for their impact on DNA binding activity and used universal protein-binding microarrays to assay sequence-specific DNA binding activity across 41 reference and 117 variant alleles found in individuals of diverse ancestries and families with Mendelian diseases. We found 77 variants in 28 genes that affect DNA binding affinity or specificity and identified thousands of rare alleles likely to alter the DNA binding activity of human sequence-specific TFs. Our results suggest that most individuals have unique repertoires of TF DNA binding activities, which may contribute to phenotypic variation., National Human Genome Research Institute (U.S.) (Grant R01 HG003985)

Proper expression of genes requires communication with their regulatory elements that can be located elsewhere along the chromosome. The physics of chromatin fibers imposes a range of constraints on such communication. The molecular and biophysical mechanisms by which chromosomal communication is established, or prevented, have become a topic of intense study, and important roles for the spatial organization of chromosomes are being discovered. Here we present a view of the interphase 3D genome characterized by extensive physical compartmentalization and insulation on the one hand and facilitated long-range interactions on the other. We propose the existence of topological machines dedicated to set up and to exploit a 3D genome organization to both promote and censor communication along and between chromosomes., National Human Genome Research Institute (U.S.) (Grant R01 HG003143), National Human Genome Research Institute (U.S.) (Grant U54 HG007010), National Human Genome Research Institute (U.S.) (Grant U01 HG007910), National Cancer Institute (U.S.) (Grant U54 CA193419), National Institutes of Health (U.S.) (Grant U54 DK107980), National Institutes of Health (U.S.) (Grant U01 DA 040588), National Institute of General Medical Sciences (U.S.) (Grant R01 GM 112720), National Institute of Allergy and Infectious Diseases (U.S.) (Grant U01 R01 AI 117839)

Thousands of human diseases could be treated by selectively controlling the expression of specific proteins in vivo. A new series of alkenyl amino alcohol (AAA) ionizable lipid nanoparticles (LNPs) capable of delivering human mRNA with unprecedented levels of in vivo efficacy is demonstrated. This study highlights the importance of utilizing synthesis tools in tandem with biological inspiration to understand and improve nucleic acid delivery in vivo., National Science Foundation (U.S.). Graduate Research Fellowship Program, Shire Pharmaceuticals, Wellcome Trust-MIT Postdoctoral Fellowship

Innate lymphoid cells (ILCs) play a central role in the response to infection by secreting cytokines crucial for immune regulation, tissue homeostasis, and repair. Although dysregulation of these systems is central to pathology, the impact of HIV-1 on ILCs remains unknown. We found that human blood ILCs were severely depleted during acute viremic HIV-1 infection and that ILC numbers did not recover after resolution of peak viremia. ILC numbers were preserved by antiretroviral therapy (ART), but only if initiated during acute infection. Transcriptional profiling during the acute phase revealed upregulation of genes associated with cell death, temporally linked with a strong IFN acute-phase response and evidence of gut barrier breakdown. We found no evidence of tissue redistribution in chronic disease and remaining circulating ILCs were activated but not apoptotic. These data provide a potential mechanistic link between acute HIV-1 infection, lymphoid tissue breakdown, and persistent immune dysfunction. Keywords: innate lymphoid cells; HIV-1 infection

Objectives Switching from maintenance of general anesthesia with an ether anesthetic to maintenance with high-dose (concentration >50% and total gas flow rate >4 liters per minute) nitrous oxide is a common practice used to facilitate emergence from general anesthesia. The transition from the ether anesthetic to nitrous oxide is associated with a switch in the putative mechanisms and sites of anesthetic action. We investigated whether there is an electroencephalogram (EEG) marker of this transition. Methods We retrospectively studied the ether anesthetic to nitrous oxide transition in 19 patients with EEG monitoring receiving general anesthesia using the ether anesthetic sevoflurane combined with oxygen and air. Results Following the transition to nitrous oxide, the alpha (8–12 Hz) oscillations associated with sevoflurane dissipated within 3–12 min (median 6 min) and were replaced by highly coherent large-amplitude slow-delta (0.1–4 Hz) oscillations that persisted for 2–12 min (median 3 min). Conclusions Administration of high-dose nitrous oxide is associated with transient, large amplitude slow-delta oscillations. Significance We postulate that these slow-delta oscillations may result from nitrous oxide-induced blockade of major excitatory inputs (NMDA glutamate projections) from the brainstem (parabrachial nucleus and medial pontine reticular formation) to the thalamus and cortex. This EEG signature of high-dose nitrous oxide may offer new insights into brain states during general anesthesia., National Institutes of Health (U.S.) (Grant DP1-OD003646), National Institutes of Health (U.S.) (Grant TR01-GM104948)

Pancreatic ductal adenocarcinoma (PDAC) is one of the most devastating and painful cancers. It is often highly resistant to therapy owing to inherent chemoresistance and the desmoplastic response that creates a barrier of fibrous tissue preventing transport of chemotherapeutics into the tumor. The growth of the tumor in pancreatic cancer often leads to invasion of other organs and partial or complete biliary obstruction, inducing intense pain for patients and necessitating tumor resection or repeated stenting. Here, we have developed a delivery device to provide enhanced palliative therapy for pancreatic cancer patients by providing high concentrations of chemotherapeutic compounds locally at the tumor site. This treatment could reduce the need for repeated procedures in advanced PDAC patients to debulk the tumor mass or stent the obstructed bile duct. To facilitate clinical translation, we created the device out of currently approved materials and drugs. We engineered an implantable poly(lactic-co-glycolic)-based biodegradable device that is able to linearly release high doses of chemotherapeutic drugs for up to 60 days. We created five patient-derived PDAC cell lines and tested their sensitivity to approved chemotherapeutic compounds. These in vitro experiments showed that paclitaxel was the most effective single agent across all cell lines. We compared the efficacy of systemic and local paclitaxel therapy on the patient-derived cell lines in an orthotopic xenograft model in mice (PDX). In this model, we found up to a 12-fold increase in suppression of tumor growth by local therapy in comparison to systemic administration and reduce retention into off-target organs. Herein, we highlight the efficacy of a local therapeutic approach to overcome PDAC chemoresistance and reduce the need for repeated interventions and biliary obstruction by preventing local tumor growth. Our results underscore the urgent need for an implantable drug-eluting platform to deliver cytotoxic agents directly within the tumor mass as a novel therapeutic strategy for patients with pancreatic cancer. Keywords: Pancreatic cancer; Chemoresistance; Local delivery; Patient-derived xenograft; Paclitaxel; Poly(lactic-co-glycolic acid), National Institutes of Health (U.S.) (Grant P30-CA14051)

Combined measurement of diverse molecular and anatomical traits that span multiple levels remains a major challenge in biology. Here, we introduce a simple method that enables proteomic imaging for scalable, integrated, high-dimensional phenotyping of both animal tissues and human clinical samples. This method, termed SWITCH, uniformly secures tissue architecture, native biomolecules, and antigenicity across an entire system by synchronizing the tissue preservation reaction. The heat- and chemical-resistant nature of the resulting framework permits multiple rounds (>20) of relabeling. We have performed 22 rounds of labeling of a single tissue with precise co-registration of multiple datasets. Furthermore, SWITCH synchronizes labeling reactions to improve probe penetration depth and uniformity of staining. With SWITCH, we performed combinatorial protein expression profiling of the human cortex and also interrogated the geometric structure of the fiber pathways in mouse brains. Such integrated high-dimensional information may accelerate our understanding of biological systems at multiple levels., Simons Foundation. Postdoctoral Fellowship, Life Sciences Research Foundation, Burroughs Wellcome Fund (Career Award at the Scientific Interface), Searle Scholars Program, Michael J. Fox Foundation for Parkinson's Research, United States. Defense Advanced Research Projects Agency, National Institutes of Health (U.S.) (1-U01-NS090473-01)

Mass and growth rate are highly integrative measures of cell physiology not discernable via genomic measurements. Here, we introduce a microfluidic platform enabling direct measurement of single-cell mass and growth rate upstream of highly multiplexed single-cell profiling such as single-cell RNA sequencing. We resolve transcriptional signatures associated with single-cell mass and growth rate in L1210 and FL5.12 cell lines and activated CD8+ T cells. Further, we demonstrate a framework using these linked measurements to characterize biophysical heterogeneity in a patient-derived glioblastoma cell line with and without drug treatment. Our results highlight the value of coupled phenotypic metrics in guiding single-cell genomics. Keywords: Single-cell RNA-Seq, Mass, Growth, Serial suspended microchannel resonator, Multi-omics, Single cell, T cell activation, Glioblastoma, GBM, Drug response, Microfluidics, Biophysical properties

Broadly neutralizing antibodies (bnAbs) against HIV-1 are an effective means of preventing transmission. To better understand the mechanisms by which HIV-specific bnAbs naturally develop, we investigated blood and lymphoid tissue in pediatric infection, since potent bnAbs develop with greater frequency in children than adults. As in adults, the frequency of circulating effector T-follicular helper cells (TFH) in HIV infected, treatment naïve children correlates with neutralization breadth. However, major differences between children and adults were also observed both in circulation, and in a small number of tonsil samples. In children, TFH cells are significantly more abundant, both in blood and in lymphoid tissue germinal centers, than in adults. Second, HIV-specific TFH cells are more frequent in pediatric than in adult lymphoid tissue and secrete the signature cytokine IL-21, which HIV-infected adults do not. Third, the enrichment of IL-21-secreting HIV-specific TFH in pediatric lymphoid tissue is accompanied by increased TFH regulation via more abundant regulatory follicular T-cells and HIV-specific CXCR5+ CD8 T-cells compared to adults. The relationship between regulation and neutralization breadth is also observed in the pediatric PBMC samples and correlates with neutralization breadth. Matching neutralization data from lymphoid tissue samples is not available. However, the distinction between infected children and adults in the magnitude, quality and regulation of HIV-specific TFH responses is consistent with the superior ability of children to develop high-frequency, potent bnAbs. These findings suggest the possibility that the optimal timing for next generation vaccine strategies designed to induce high-frequency, potent bnAbs to prevent HIV infection in adults would be in childhood.

There is a need for large-scale, longitudinal studies to determine the mechanisms by which the gut microbiome and its interactions with the host affect human health and disease. Current methods for profiling the microbiome typically utilize next-generation sequencing applications that are expensive, slow, and complex. Here, we present a synthetic biology platform for affordable, on-demand, and simple analysis of microbiome samples using RNA toehold switch sensors in paper-based, cell-free reactions. We demonstrate species-specific detection of mRNAs from 10 different bacteria that affect human health and four clinically relevant host biomarkers. We develop a method to quantify mRNA using our toehold sensors and validate our platform on clinical stool samples by comparison to RT-qPCR. We further highlight the potential clinical utility of the platform by showing that it can be used to rapidly and inexpensively detect toxin mRNA in the diagnosis of Clostridium difficile infections., National Institutes of Health (U.S.) (Grant T32-DK007191)

We present a novel and time-efficient method for intracoronary lumen detection, which produces three-dimensional (3-D) coronary arteries using optical coherence tomographic (OCT) images. OCT images are acquired for multiple patients and longitudinal cross-section (LOCS) images are reconstructed using different acquisition angles. The lumen contours for each LOCS image are extracted and translated to 2-D cross-sectional images. Using two angiographic projections, the centerline of the coronary vessel is reconstructed in 3-D, and the detected 2-D contours are transformed to 3-D and placed perpendicular to the centerline. To validate the proposed method, 613 manual annotations from medical experts were used as gold standard. The 2-D detected contours were compared with the annotated contours, and the 3-D reconstructed models produced using the detected contours were compared to the models produced by the annotated contours. Wall shear stress (WSS), as dominant hemodynamics factor, was calculated using computational fluid dynamics and 844 consecutive 2-mm segments of the 3-D models were extracted and compared with each other. High Pearson's correlation coefficients were obtained for the lumen area ( r = 0.98) and local WSS ( r = 0.97) measurements, while no significant bias with good limits of agreement was shown in the Bland–Altman analysis. The overlapping and nonoverlapping areas ratio between experts’ annotations and presented method was 0.92 and 0.14, respectively. The proposed computer-aided lumen extraction and 3-D vessel reconstruction method is fast, accurate, and likely to assist in a number of research and clinical applications.

Background Single-cell genomic methods now provide unprecedented resolution for characterizing the component cell types and states of tissues such as the epithelial subsets of the gastrointestinal tract. Nevertheless, functional studies of these subsets at scale require faithful in vitro models of identified in vivo biology. While intestinal organoids have been invaluable in providing mechanistic insights in vitro, the extent to which organoid-derived cell types recapitulate their in vivo counterparts remains formally untested, with no systematic approach for improving model fidelity. Results Here, we present a generally applicable framework that utilizes massively parallel single-cell RNA-seq to compare cell types and states found in vivo to those of in vitro models such as organoids. Furthermore, we leverage identified discrepancies to improve model fidelity. Using the Paneth cell (PC), which supports the stem cell niche and produces the largest diversity of antimicrobials in the small intestine, as an exemplar, we uncover fundamental gene expression differences in lineage-defining genes between in vivo PCs and those of the current in vitro organoid model. With this information, we nominate a molecular intervention to rationally improve the physiological fidelity of our in vitro PCs. We then perform transcriptomic, cytometric, morphologic and proteomic characterization, and demonstrate functional (antimicrobial activity, niche support) improvements in PC physiology. Conclusions Our systematic approach provides a simple workflow for identifying the limitations of in vitro models and enhancing their physiological fidelity. Using adult stem cell-derived PCs within intestinal organoids as a model system, we successfully benchmark organoid representation, relative to that in vivo, of a specialized cell type and use this comparison to generate a functionally improved in vitro PC population. We predict that the generation of rationally improved cellular models will facilitate mechanistic exploration of specific disease-associated genes in their respective cell types. Keywords: Single-cell RNA-seq; Chemical biology; Stem cell-derived models; Paneth cell; Intestinal organoid; Intestinal stem cell; Differentiation; Systems biology, National Cancer Institute (U.S.) (Grant P30-CA14051), National Institutes of Health (U.S.) (Grant DE013023), National Institutes of Health (U.S.) (Grant HL095722), National Institutes of Health (U.S.) (Grant 1DP2OD020839), National Institutes of Health (U.S.) (Grant 2U19AI089992), National Institutes of Health (U.S.) (Grant 2R01HL095791), National Institutes of Health (U.S.) (Grant 1U54CA217377), National Institutes of Health (U.S.) (Grant 2P01AI039671), National Institutes of Health (U.S.) (Grant 5U24AI118672), National Institutes of Health (U.S.) (Grant 2RM1HG006193), National Institutes of Health (U.S.) (Grant 1R33CA202820), National Institutes of Health (U.S.) (Grant 1R01HL126554), National Institutes of Health (U.S.) (Grant 1R01DA046277), National Institutes of Health (U.S.) (Grant 1R01AI138546), Bill & Melinda Gates Foundation (Grant OPP1139972), Bill & Melinda Gates Foundation (Grant OPP1137006), Bill & Melinda Gates Foundation (Grant OPP1116944)

We present the case of an HIV-negative patient clinically diagnosed with relapsing-remitting MS who achieved significant disease improvement on Combivir (zidovudine/lamivudine). Within months of treatment, the patient reported complete resolution of previously unremitting fatigue and paresthesiae, with simultaneous improvements in lesion burden detected by MRI. All improvements have been sustained for more than three years. This response may be related to the action of zidovudine as a known inhibitor of EBV lytic DNA replication, suggesting future directions for clinical investigation. Keywords: Multiple sclerosis, Epstein-Barr virus

Although gene transfer to hematopoietic stem cells (HSCs) has shown therapeutic efficacy in recent trials for several individuals with inherited disorders, transduction incompleteness of the HSC population remains a hurdle to yield a cure for all patients with reasonably low integrated vector numbers. In previous attempts at HSC selection, massive loss of transduced HSCs, contamination with non-transduced cells, or lack of applicability to large cell populations has rendered the procedures out of reach for human applications. Here, we fused codon-optimized puromycin N-acetyltransferase to herpes simplex virus thymidine kinase. When expressed from a ubiquitous promoter within a complex lentiviral vector comprising the βAT87Q-globin gene, viral titers and therapeutic gene expression were maintained at effective levels. Complete selection and preservation of transduced HSCs were achieved after brief exposure to puromycin in the presence of MDR1 blocking agents, suggesting the procedure’s suitability for human clinical applications while affording the additional safety of conditional suicide., Recent clinical trials have demonstrated the benefits of hematopoietic gene therapy using lentiviral vectors. In this paper, Payen and colleagues describe a method to maximize the proportion of genetically modified human hematopoietic stem cells while limiting the mean vector copy number.

Upper-extremity exoskeletons have demonstrated potential as augmentative, assistive, and rehabilitative devices. Typical control of upper-extremity exoskeletons have relied on switches, force/torque sensors, and surface electromyography (sEMG), but these systems are usually reactionary, and/or rely on entirely hand-tuned parameters. sEMG-based systems may be able to provide anticipatory control, since they interface directly with muscle signals, but typically require expert placement of sensors on muscle bodies. We present an implementation of an adaptive sEMG-based exoskeleton controller that learns a mapping between muscle activation and the desired system state during interaction with a user, generating a personalized sEMG feature classifier to allow for anticipatory control. This system is robust to novice placement of sEMG sensors, as well as subdermal muscle shifts. We validate this method with 18 subjects using a thumb exoskeleton to complete a book-placement task. This learning-from-demonstration system for exoskeleton control allows for very short training times, as well as the potential for improvement in intent recognition over time, and adaptation to physiological changes in the user, such as those due to fatigue., Jeptha H. and Emily V. Wade Fund

Saccadic eye movements are an inherent component of natural reading, yet their contribution to information processing at subsequent fixation remains elusive. Here we use anatomically-constrained magnetoencephalography (MEG) to examine cortical activity following saccades as healthy human subjects engaged in a one-back word recognition task. This activity was compared with activity following external visual stimulation that mimicked saccades. A combination of procedures was employed to eliminate saccadic ocular artifacts from the MEG signal. Both saccades and saccade-like external visual stimulation produced early-latency responses beginning ∼70 ms after onset in occipital cortex and spreading through the ventral and dorsal visual streams to temporal, parietal and frontal cortices. Robust differential activity following the onset of saccades vs. similar external visual stimulation emerged during 150–350 ms in a left-lateralized cortical network. This network included: (i) left lateral occipitotemporal (LOT) and nearby inferotemporal (IT) cortex; (ii) left posterior Sylvian fissure (PSF) and nearby multimodal cortex; and (iii) medial parietooccipital (PO), posterior cingulate and retrosplenial cortices. Moreover, this left-lateralized network colocalized with word repetition priming effects. Together, results suggest that central saccadic mechanisms influence a left-lateralized language network in occipitotemporal and temporal cortex above and beyond saccadic influences at preceding stages of information processing during visual word recognition., National Institutes of Health (U.S.) (grant 03HD050627 (STemereanca)), National Institutes of Health (U.S.) (DP1OD003646), National Institutes of Health (U.S.) (R01EB006385), National Institutes of Health (U.S.) (R01EB009048), National Institute of Biomedical Imaging and Bioengineering (U.S.) (5P41EB015896), National Institutes of Health (U.S.) (R01HD082527)

Renal sympathetic denervation (RDN) is under investigation as a treatment option in patients with resistant hypertension (RH). Determinants of arterial compliance may, however, help to predict the BP response to therapy. Aortic distensibility (AD) is a well-established parameter of aortic stiffness and can reliably be obtained by CMR. This analysis sought to investigate the effects of RDN on AD and to assess the predictive value of pre-treatment AD for BP changes. We analyzed data of 65 patients with RH included in a multicenter trial. RDN was performed in all participants. A standardized CMR protocol was utilized at baseline and at 6-month follow-up. AD was determined as the change in cross-sectional aortic area per unit change in BP. Office BP decreased significantly from 173/92 ± 24/16 mmHg at baseline to 151/85 ± 24/17 mmHg (p

Iron–sulfur (Fe‐S) clusters are essential for mitochondrial metabolism, but their regulation in pulmonary hypertension (PH) remains enigmatic. We demonstrate that alterations of the miR‐210‐ISCU1/2 axis cause Fe‐S deficiencies in vivo and promote PH. In pulmonary vascular cells and particularly endothelium, hypoxic induction of miR‐210 and repression of the miR‐210 targets ISCU1/2 down‐regulated Fe‐S levels. In mouse and human vascular and endothelial tissue affected by PH, miR‐210 was elevated accompanied by decreased ISCU1/2 and Fe‐S integrity. In mice, miR‐210 repressed ISCU1/2 and promoted PH. Mice deficient in miR‐210, via genetic/pharmacologic means or via an endothelial‐specific manner, displayed increased ISCU1/2 and were resistant to Fe‐S‐dependent pathophenotypes and PH. Similar to hypoxia or miR‐210 overexpression, ISCU1/2 knockdown also promoted PH. Finally, cardiopulmonary exercise testing of a woman with homozygous ISCU mutations revealed exercise‐induced pulmonary vascular dysfunction. Thus, driven by acquired (hypoxia) or genetic causes, the miR‐210‐ISCU1/2 regulatory axis is a pathogenic lynchpin causing Fe‐S deficiency and PH. These findings carry broad translational implications for defining the metabolic origins of PH and potentially other metabolic diseases sharing similar underpinnings., National Institutes of Health (U.S.) (U54‐CA151884), National Institutes of Health (U.S.) (R01‐DE016516‐06), National Institutes of Health (U.S.) (EB000244)

As synthetic biology approaches are extended to diverse applications throughout medicine, biotechnology and basic biological research, there is an increasing need to engineer yeast, plant and mammalian cells. Eukaryotic genomes are regulated by the diverse biochemical and biophysical states of chromatin, which brings distinct challenges, as well as opportunities, over applications in bacteria. Recent synthetic approaches, including 'epigenome editing', have allowed the direct and functional dissection of many aspects of physiological chromatin regulation. These studies lay the foundation for biomedical and biotechnological engineering applications that could take advantage of the unique combinatorial and spatiotemporal layers of chromatin regulation to create synthetic systems of unprecedented sophistication., National Institute of General Medical Sciences (U.S.) (Ruth L. Kirschstein Postdoctoral Fellowship ), United States. Defense Advanced Research Projects Agency, National Institutes of Health (U.S.) (R24 Grant), Wyss Institute for Biologically Inspired Engineering, Howard Hughes Medical Institute

Dyslexia is one of the most common learning disabilities, yet its brain basis and core causes are not yet fully understood. Neuroimaging methods, including structural and functional magnetic resonance imaging, diffusion tensor imaging, and electrophysiology, have significantly contributed to knowledge about the neurobiology of dyslexia. Recent studies have discovered brain differences before formal instruction that likely encourage or discourage learning to read effectively, distinguished between brain differences that likely reflect the etiology of dyslexia versus brain differences that are the consequences of variation in reading experience, and identified distinct neural networks associated with specific psychological factors that are associated with dyslexia., Eunice Kennedy Shriver National Institute of Child Health and Human Development (U.S.) (Grant R01 HD067312)

Neuroimaging has greatly enhanced the cognitive neuroscience understanding of the human brain and its variation across individuals (neurodiversity) in both health and disease. Such progress has not yet, however, propelled changes in educational or medical practices that improve people’s lives. We review neuroimaging findings in which initial brain measures (neuromarkers) are correlated with or predict future education, learning, and performance in children and adults; criminality; health-related behaviors; and responses to pharmacological or behavioral treatments. Neuromarkers often provide better predictions (neuroprognosis), alone or in combination with other measures, than traditional behavioral measures. With further advances in study designs and analyses, neuromarkers may offer opportunities to personalize educational and clinical practices that lead to better outcomes for people., Poitras Center for Affective Disorders Research, National Institutes of Health (U.S.) (Grant R01HD067312)

Chronic kidney disease (CKD/uremia) remains vexing because it increases the risk of atherothrombosis and is also associated with bleeding complications on standard antithrombotic/antiplatelet therapies. Although the associations of indolic uremic solutes and vascular wall proteins [such as tissue factor (TF) and aryl hydrocarbon receptor (AHR)] are being defined, the specific mechanisms that drive the thrombotic and bleeding risks are not fully understood. We now present an indolic solute-specific animal model, which focuses on solute-protein interactions and shows that indolic solutes mediate the hyperthrombotic phenotype across all CKD stages in an AHR- and TF-dependent manner. We further demonstrate that AHR regulates TF through STIP1 homology and U-box-containing protein 1 (STUB1). As a ubiquitin ligase, STUB1 dynamically interacts with and degrades TF through ubiquitination in the uremic milieu. TF regulation by STUB1 is supported in humans by an inverse relationship of STUB1 and TF expression and reduced STUB1-TF interaction in uremic vessels. Genetic or pharmacological manipulation of STUB1 in vascular smooth muscle cells inhibited thrombosis in flow loops. STUB1 perturbations reverted the uremic hyperthrombotic phenotype without prolonging the bleeding time, in contrast to heparin, the standard-of-care antithrombotic in CKD patients. Our work refines the thrombosis axis (STUB1 is a mediator of indolic solute-AHR-TF axis) and expands the understanding of the interconnected relationships driving the fragile thrombotic state in CKD. It also establishes a means of minimizing the uremic hyperthrombotic phenotype without altering the hemostatic balance, a long-sought-after combination in CKD patients., National Institutes of Health (U.S.) (Grant R01HL132325), National Institutes of Health (U.S.) (Grant R01CA175382), National Institute of General Medical Sciences (U.S.) (Grant R01GM49039), American Heart Association (Grant 12FTF12080241)

A better understanding of the cell-fate transitions that occur in complex cellular ecosystems in normal development and disease could inform cell engineering efforts and lead to improved therapies. However, a major challenge is to simultaneously identify new cell states, and their transitions, to elucidate the gene expression dynamics governing cell-type diversification. Here, we present CellRouter, a multifaceted single-cell analysis platform that identifies complex cell-state transition trajectories by using flow networks to explore the subpopulation structure of multi-dimensional, single-cell omics data. We demonstrate its versatility by applying CellRouter to single-cell RNA sequencing data sets to reconstruct cell-state transition trajectories during hematopoietic stem and progenitor cell (HSPC) differentiation to the erythroid, myeloid and lymphoid lineages, as well as during re-specification of cell identity by cellular reprogramming of monocytes and B-cells to HSPCs. CellRouter opens previously undescribed paths for in-depth characterization of complex cellular ecosystems and establishment of enhanced cell engineering approaches., National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (Grant R24DK092760), National Institute of Allergy and Infectious Diseases (U.S.) (Grant R37AI039394), National Heart, Lung, and Blood Institute (Grant UO1-HL100001)

Rationale Factors associated with one-year mortality after recovery from critical illness are not well understood. Clinicians generally lack information regarding post-hospital discharge outcomes of patients from the intensive care unit, which may be important when counseling patients and families. Objective We sought to determine which factors among patients who survived for at least 30 days post-ICU admission are associated with one-year mortality. Methods Single-center, longitudinal retrospective cohort study of all ICU patients admitted to a tertiary-care academic medical center from 2001–2012 who survived 30 days from ICU admission. Cox’s proportional hazards model was used to identify the variables that are associated with one-year mortality. The primary outcome was one-year mortality. Results 32,420 patients met the inclusion criteria and were included in the study. Among patients who survived to 30 days, 28,583 (88.2%) survived for greater than one year, whereas 3,837 (11.8%) did not. Variables associated with decreased one-year survival include: increased age, malignancy, number of hospital admissions within the prior year, duration of mechanical ventilation and vasoactive agent use, sepsis, history of congestive heart failure, end-stage renal disease, cirrhosis, chronic obstructive pulmonary disease, and the need for renal replacement therapy. Numerous effect modifications between these factors were found. Conclusion Among survivors of critical illness, a significant number survive less than one year. More research is needed to help clinicians accurately identify those patients who, despite surviving their acute illness, are likely to suffer one-year mortality, and thereby to improve the quality of the decisions and care that impact this outcome., National Institute of Biomedical Imaging and Bioengineering (U.S.) (grant R01EB017205-01A1), A*STAR (Graduate Scholarship), National Institutes of Health (U.S.) (grant T32 HL007287-39), National Heart, Lung, and Blood Institute, Philips Healthcare Nederland

BACKGROUND: Human immunity relies on the coordinated responses of many cellular subsets and functional states. Inter-individual variations in cellular composition and communication could thus potentially alter host protection. Here, we explore this hypothesis by applying single-cell RNA-sequencing to examine viral responses among the dendritic cells (DCs) of three elite controllers (ECs) of HIV-1 infection. RESULTS: To overcome the potentially confounding effects of donor-to-donor variability, we present a generally applicable computational framework for identifying reproducible patterns in gene expression across donors who share a unifying classification. Applying it, we discover a highly functional antiviral DC state in ECs whose fractional abundance after in vitro exposure to HIV-1 correlates with higher CD4+ T cell counts and lower HIV-1 viral loads, and that effectively primes polyfunctional T cell responses in vitro. By integrating information from existing genomic databases into our reproducibility-based analysis, we identify and validate select immunomodulators that increase the fractional abundance of this state in primary peripheral blood mononuclear cells from healthy individuals in vitro. CONCLUSIONS: Overall, our results demonstrate how single-cell approaches can reveal previously unappreciated, yet important, immune behaviors and empower rational frameworks for modulating systems-level immune responses that may prove therapeutically and prophylactically useful. Keywords: HIV-1; dendritic cell; single-cell; RNA-seq; single-cell genomics; elite controller; adjuvant; reproducibility; differential expression, National Institutes of Health (U.S.) (Grant DP2OD020839), National Institutes of Health (U.S.) (Grant 5U24AI118672), National Institutes of Health (U.S.) (Grant 2U19AI089992), National Institutes of Health (U.S.) (Grant 2R01HL095791), National Institutes of Health (U.S.) (Grant 2RM1HG006193)

The discovery of sodium taurocholate cotransporting polypeptide (NTCP) as the hepatitis B virus (HBV) receptor enabled researchers to create hepatoma cell lines susceptible to HBV infection. Infection in current systems, however, is inefficient and virus fails to spread. Infection efficiency is enhanced by treating cells with polyethylene glycol 8000 (PEG) during infection. However, this alone does not promote virus spread. Here we show that maintaining PEG in culture medium increases the rate of infection by at least one order of magnitude, and, most importantly, promotes virus spread. To demonstrate the utility of this system, we show that two interferon-stimulated genes (ISGs), ISG20 and tetherin, restrict HBV spread in NTCP-expressing hepatoma cells. Thus, this protocol can be easily applied to existing cell culture systems to study the complete HBV life cycle, including virus spread.

Major surgeries can result in high rates of adverse postoperative events. Reliable prediction of which patient might be at risk for such events may help guide peri- and postoperative care. We show how archiving and mining of intraoperative hemodynamic data in orthotopic liver transplantation (OLT) can aid in the prediction of postoperative 180-day mortality and acute renal failure (ARF), improving upon predictions that rely on preoperative information only. From 101 patient records, we extracted 15 preoperative features from clinical records and 41 features from intraoperative hemodynamic signals. We used logistic regression with leave-one-out cross-validation to predict outcomes, and incorporated methods to limit potential model instabilities from feature multicollinearity. Using only preoperative features, mortality prediction achieved an area under the receiver operating characteristic curve (AUC) of 0.53 (95% CI: 0.44–0.78). By using intraoperative features, performance improved significantly to 0.82 (95% CI: 0.56–0.91, P = 0.001). Similarly, including intraoperative features (AUC = 0.82; 95% CI: 0.66–0.94) in ARF prediction improved performance over preoperative features (AUC = 0.72; 95% CI: 0.50–0.85), though not significantly (P = 0.32). We conclude that inclusion of intraoperative hemodynamic features significantly improves prediction of postoperative events in OLT. Features strongly associated with occurrence of both outcomes included greater intraoperative central venous pressure and greater transfusion volumes.

Homeostatic programs balance immune protection and self-tolerance. Such mechanisms likely impact autoimmunity and tumor formation, respectively. How homeostasis is maintained and impacts tumor surveillance is unknown. Here, we find that different immune mononuclear phagocytes share a conserved steady-state program during differentiation and entry into healthy tissue. IFNγ is necessary and sufficient to induce this program, revealing a key instructive role. Remarkably, homeostatic and IFNγ-dependent programs enrich across primary human tumors, including melanoma, and stratify survival. Single-cell RNA sequencing (RNA-seq) reveals enrichment of homeostatic modules in monocytes and DCs from human metastatic melanoma. Suppressor-of-cytokine-2 (SOCS2) protein, a conserved program transcript, is expressed by mononuclear phagocytes infiltrating primary melanoma and is induced by IFNγ. SOCS2 limits adaptive anti-tumoral immunity and DC-based priming of T cells in vivo, indicating a critical regulatory role. These findings link immune homeostasis to key determinants of anti-tumoral immunity and escape, revealing co-opting of tissue-specific immune development in the tumor microenvironment. Keywords: dendritic cells; homeostasis; differentiation; IFNγ; tumor microenvironment; melanoma tolerance; immunotherapy; suppressor-of-cytokine-signaling 2 (SOCS2); tissue mononuclear phagocytes

Background Recently there has been a large outbreak of Zika virus infections in Colombia, South America. The epidemic began in September 2015 and continued to April 2017, for the total number of Zika cases reported of 107,870. For those confirmed Zika cases, there were nearly 20,000 (18.5%) suspected to be pregnant women, resulting in 157 confirmed cases of microcephaly in newborns reported by their health government agency. There is a clear under-estimation of the total number of cases and in addition no prior publications have been published to demonstrate the clinical aspects of the Zika infection in Colombia. We characterized one Zika presentation to be able to compare and contrast with other cases of Zika infection already reported in the literature. Case presentation In this case report, we demonstrate congenital microcephaly at week 19 of gestation in a 34-year-old mother who showed symptoms compatible with Zika virus infection from Sincelejo, State of Sucre, in the Colombian Caribbean. Zika virus RNA was detected in the placenta using real-time reverse transcriptase polymerase chain reaction (RT-PCR). At week 25, the fetus weigh estimate was 770 g, had a cephalic perimeter of 20.2 cm (5th percentile), ventriculomegaly on the right side and dilatation of the fourth ventricle. At week 32, the microcephaly was confirmed with a cephalic perimeter of 22 cm, dilatation of the posterior atrium to 13 mm, an abnormally small cerebellum (29 mm), and an augmented cisterna magna. At birth (39 weeks by cesarean section), the head circumference was 27.5 cm, and computerized axial tomography (Siemens Corp, 32-slides) confirmed microcephaly with calcifications. Conclusion We report a first case of maternal Zika virus infection associated with fetal microcephaly in Colombia and confirmed similar presentation to those observed previous in Brazil, 2015–2016.

A capability for analyzing complex cellular communication among tissues is important in drug discovery and development, and in vitro technologies for doing so are required for human applications. A prominent instance is communication between the gut and the liver, whereby perturbations of one tissue can influence behavior of the other. Here, we present a study on human gut-liver tissue interactions under normal and inflammatory contexts, via an integrative multi-organ platform comprising human liver (hepatocytes and Kupffer cells), and intestinal (enterocytes, goblet cells, and dendritic cells) models. Our results demonstrated long-term (>2 weeks) maintenance of intestinal (e.g., barrier integrity) and hepatic (e.g., albumin) functions in baseline interaction. Gene expression data comparing liver in interaction with gut, versus isolation, revealed modulation of bile acid metabolism. Intestinal FGF19 secretion and associated inhibition of hepatic CYP7A1 expression provided evidence of physiologically relevant gut-liver crosstalk. Moreover, significant non-linear modulation of cytokine responses was observed under inflammatory gut-liver interaction; for example, production of CXCR3 ligands (CXCL9,10,11) was synergistically enhanced. RNA-seq analysis revealed significant upregulation of IFNα/β/γ signaling during inflammatory gut-liver crosstalk, with these pathways implicated in the synergistic CXCR3 chemokine production. Exacerbated inflammatory response in gut-liver interaction also negatively affected tissue-specific functions (e.g., liver metabolism). These findings illustrate how an integrated multi-tissue platform can generate insights useful for understanding complex pathophysiological processes such as inflammatory organ crosstalk., National Institutes of Health (U.S.) (grant UH3TR00069), United States. Defense Advanced Research Projects Agency (grant Microphysiological Systems Program (W911NF-12-2-00))

All rights reserved. Recent advances in cellular profiling have demonstrated substantial heterogeneity in the behaviour of cells once deemed 'identical', challenging fundamental notions of cell 'type' and 'state'. Not surprisingly, these findings have elicited substantial interest in deeply characterizing the diversity, interrelationships and plasticity among cellular phenotypes. To explore these questions, experimental platforms are needed that can extensively and controllably profile many individual cells. Here, microfluidic structures-whether valve-, droplet- or nanowell-based-have an important role because they can facilitate easy capture and processing of single cells and their components, reducing labour and costs relative to conventional plate-based methods while also improving consistency. In this article, we review the current state-of-the-art methodologies with respect to microfluidics for mammalian single-cell 'omics' and discuss challenges and future opportunities., National Institutes of Health (U.S.) (Award DP2OD020839), National Institutes of Health (U.S.) (Grant U24AI118672), National Institutes of Health (U.S.) (Grant P50HG006193)

Background: Calcific atherosclerosis is a major challenge to intraluminal drug delivery in peripheral artery disease (PAD). Objectives: We evaluated the effects of orbital atherectomy on intraluminal paclitaxel delivery to human peripheral arteries with substantial calcified plaque. Methods: Diagnostic angiography and 3-D rotational imaging of five fresh human lower limbs revealed calcification in all main arteries. The proximal or distal segment of each artery was treated using an orbital atherectomy system (OAS) under simulated blood flow and fluoroscopy. Explanted arterial segments underwent either histomorphometric assessment of effect or tracking of [superscript 14]C-labeled or fluorescent–labeled paclitaxel. Radiolabeled drug quantified bulk delivery and fluorescent label established penetration of drug over finer spatial domain in serial microscopic sections. Results: were interpreted using a mathematical model of binding-diffusion mediated arterial drug distribution. Results Lesion composition affected paclitaxel absorption and distribution in cadaveric human peripheral arteries. Pretreatment imaging calcium scores in control femoropopliteal arterial segments correlated with a log-linear decline in the bulk absorption rate-constant of [superscript 14]C-labeled, declining 5.5-fold per calcified quadrant (p = 0.05, n = 7). Compared to controls, OAS-treated femoropopliteal segments exhibited 180 μm thinner intima (p < 0.001), 45% less plaque calcification, and 2 log orders higher paclitaxel bulk absorption rate-constants. Correspondingly, fluorescent paclitaxel penetrated deeper in OAS-treated femoropopliteal segments compared to controls, due to a 70% increase in diffusivity (p < 0.001). Conclusions These data illustrate that calcified plaque limited intravascular drug delivery, and controlled OAS treatment of calcific plaques resulted in greater drug permeability and improved adjunct drug delivery to diseased arteries.Peripheral artery disease Keywords: Drug coated balloons, Drug eluting stents, Atherectomy, Orbital atherectomy, calcified plaque, Paclitaxel, National Institute of Mental Health (U.S.) (Grant R01 GM-49039)

Radiotherapy is a crucial component of cancer care, employed in the treatment of over 50% of cancer patients. Patients undergoing image-guided radiotherapy or brachytherapy routinely have inert radiotherapy (RT) biomaterials implanted into their tumors. The single function of these RT biomaterials is to ensure geometric accuracy during treatment. Recent studies have proposed that the inert biomaterials could be upgraded to ‘smart’ RT biomaterials, designed to do more than one function. Such smart biomaterials include next generation fiducial markers, brachytherapy spacers, and balloon applicators, designed to respond to stimulus and perform additional desirable functions like controlled delivery of therapy-enhancing payloads directly into the tumor sub-volume, while minimizing normal tissue toxicities. More broadly, smart RT biomaterials may include functionalized nanoparticles that can be activated to boost radiotherapy efficacy. This work reviews the rationale for smart radiotherapy biomaterials, the state-of-the-art in this emerging cross-disciplinary research area, challenges/opportunities for further research and development, and a purview of potential clinical applications. Applications covered include using smart RT biomaterials for boosting cancer therapy with minimal side effects, combining radiotherapy with immunotherapy or chemotherapy, reducing treatment time or healthcare costs, and other incipient applications.

Host proteins are essential for HIV entry and replication and can be important nonviral therapeutic targets. Large-scale RNA interference (RNAi)-based screens have identified nearly a thousand candidate host factors, but there is little agreement among studies and few factors have been validated. Here we demonstrate that a genome-wide CRISPR-based screen identifies host factors in a physiologically relevant cell system. We identify five factors, including the HIV co-receptors CD4 and CCR5, that are required for HIV infection yet are dispensable for cellular proliferation and viability. Tyrosylprotein sulfotransferase 2 (TPST2) and solute carrier family 35 member B2 (SLC35B2) function in a common pathway to sulfate CCR5 on extracellular tyrosine residues, facilitating CCR5 recognition by the HIV envelope. Activated leukocyte cell adhesion molecule (ALCAM) mediates cell aggregation, which is required for cell-to-cell HIV transmission. We validated these pathways in primary human CD4 + T cells through Cas9-mediated knockout and antibody blockade. Our findings indicate that HIV infection and replication rely on a limited set of host-dispensable genes and suggest that these pathways can be studied for therapeutic intervention.

To artificially reprogram cell fate, experimentalists manipulate the gene regulatory networks (GRNs) that maintain a cell's phenotype. In practice, reprogramming is often performed by constant overexpression of specific transcription factors (TFs). This process can be unreliable and inefficient. Here, we address this problem by introducing a new approach to reprogramming based on mathematical analysis. We demonstrate that reprogramming GRNs using constant overexpression may not succeed in general. Instead, we propose an alternative reprogramming strategy: a synthetic genetic feedback controller that dynamically steers the concentration of a GRN's key TFs to any desired value. The controller works by adjusting TF expression based on the discrepancy between desired and actual TF concentrations. Theory predicts that this reprogramming strategy is guaranteed to succeed, and its performance is independent of the GRN's structure and parameters, provided that feedback gain is sufficiently high. As a case study, we apply the controller to a model of induced pluripotency in stem cells. Keywords: feedback control; synthetic biology; cell fate; reprogramming; multistability; gene regulatory network, National Institute of General Medical Sciences (U.S.) (Grant P50 GMO98792)

Objective To demonstrate the incremental benefit of using free text data in addition to vital sign and demographic data to identify patients with suspected infection in the emergency department. Methods This was a retrospective, observational cohort study performed at a tertiary academic teaching hospital. All consecutive ED patient visits between 12/17/08 and 2/17/13 were included. No patients were excluded. The primary outcome measure was infection diagnosed in the emergency department defined as a patient having an infection related ED ICD-9-CM discharge diagnosis. Patients were randomly allocated to train (64%), validate (20%), and test (16%) data sets. After preprocessing the free text using bigram and negation detection, we built four models to predict infection, incrementally adding vital signs, chief complaint, and free text nursing assessment. We used two different methods to represent free text: a bag of words model and a topic model. We then used a support vector machine to build the prediction model. We calculated the area under the receiver operating characteristic curve to compare the discriminatory power of each model. Results A total of 230,936 patient visits were included in the study. Approximately 14% of patients had the primary outcome of diagnosed infection. The area under the ROC curve (AUC) for the vitals model, which used only vital signs and demographic data, was 0.67 for the training data set, 0.67 for the validation data set, and 0.67 (95% CI 0.65–0.69) for the test data set. The AUC for the chief complaint model which also included demographic and vital sign data was 0.84 for the training data set, 0.83 for the validation data set, and 0.83 (95% CI 0.81–0.84) for the test data set. The best performing methods made use of all of the free text. In particular, the AUC for the bag-of-words model was 0.89 for training data set, 0.86 for the validation data set, and 0.86 (95% CI 0.85–0.87) for the test data set. The AUC for the topic model was 0.86 for the training data set, 0.86 for the validation data set, and 0.85 (95% CI 0.84–0.86) for the test data set. Conclusion Compared to previous work that only used structured data such as vital signs and demographic information, utilizing free text drastically improves the discriminatory ability (increase in AUC from 0.67 to 0.86) of identifying infection.

The malaria liver stage is an attractive target for antimalarial development, and preclinical malaria models are essential for testing such candidates. Given ethical concerns and costs associated with non‐human primate models, humanized mouse models containing chimeric human livers offer a valuable alternative as small animal models of liver stage human malaria. The best available human liver chimeric mice rely on cellular transplantation into mice with genetically engineered liver injury, but these systems involve a long and variable humanization process, are expensive, and require the use of breeding-challenged mouse strains which are not widely accessible. We previously incorporated primary human hepatocytes into engineered polyethylene glycol (PEG)-based nanoporous human ectopic artificial livers (HEALs), implanted them in mice without liver injury, and rapidly generated human liver chimeric mice in a reproducible and scalable fashion. By re-designing the PEG scaffold to be macroporous, we demonstrate the facile fabrication of implantable porous HEALs that support liver stage human malaria (P. falciparum) infection in vitro, and also after implantation in mice with normal liver function, 60% of the time. This proof-of-concept study demonstrates the feasibility of applying a tissue engineering strategy towards the development of scalable preclinical models of liver stage malaria infection for future applications., Bill & Melinda Gates Foundation (Global Health Grant initiative (Global Health Grant: OPP1023607)), National Cancer Institute (U.S.) (Grant P30-CA14051), National Institute of Environmental Health Sciences (Core Center Grant P30-ES002109), Singapore. Agency for Science, Technology and Research (National Science Scholarship).

Nanotechnology-based approaches hold substantial potential for improving the care of patients with diabetes. Nanoparticles are being developed as imaging contrast agents to assist in the early diagnosis of type 1 diabetes. Glucose nanosensors are being incorporated in implantable devices that enable more accurate and patient-friendly real-time tracking of blood glucose levels, and are also providing the basis for glucose-responsive nanoparticles that better mimic the body's physiological needs for insulin. Finally, nanotechnology is being used in non-invasive approaches to insulin delivery and to engineer more effective vaccine, cell and gene therapies for type 1 diabetes. Here, we analyse the current state of these approaches and discuss key issues for their translation to clinical practice., Leona M. and Harry B. Helmsley Charitable Trust (Grant 09PG-T1D027), Juvenile Diabetes Research Foundation International (17-2007-1063), Juvenile Diabetes Research Foundation International (3-2013-178), Juvenile Diabetes Research Foundation International (3-2011-310), United States. National Institutes of Health (EB000244), United States. National Institutes of Health (EB000351), United States. National Institutes of Health (DE013023), United States. National Institutes of Health (CA151884)

DNA instability is an important contributor to cancer development. Previously, defects in the chromosome segregation and excessive DNA double strand breaks due to the replication or oxidative stresses were implicated in DNA instability in cancer. Here, we demonstrate that DNA instability can directly result from the oncogene-induced senescence signaling. Expression of the activated form of Her2 oncogene, NeuT, in immortalized breast epithelial cells led to downregulation of the major DNA repair factor histone H2AX and a number of other components of the HR and NHEJ double strand DNA breaks repair pathways. H2AX expression was regulated at the transcriptional level via a senescence pathway involving p21-mediated regulation of CDK and Rb1. The p21-dependent downregulation of H2AX was seen both in cell culture and the MMTV-neu mouse model of Her2-positive breast cancer. Importantly, downregulation of H2AX upon Her2/NeuT expression impaired repair of double strand DNA breaks. This impairment resulted in both increased DNA instability in the form of somatic copy number alterations, and in increased sensitivity to the chemotherapeutic drug doxorubicin. Overall, these findings indicate that the Her2/NeuT oncogene signaling directly potentiates DNA instability and increases sensitivity to DNA damaging treatments, National Institutes of Health (U.S.) (NIH CA081244), National Institutes of Health (U.S.) (NIH R21CA178563), National Institutes of Health (U.S.) (NIH 5U54CA143874)