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Progressive kidney diseases are often associated with scarring of the kidney’s filtration unit, a condition called focal segmental glomerulosclerosis (FSGS). This scarring is due to loss of podocytes, cells critical for glomerular filtration, and leads to proteinuria and kidney failure. Inherited forms of FSGS are caused by Rac1-activating mutations, and Rac1 induces TRPC5 ion channel activity and cytoskeletal remodeling in podocytes. Whether TRPC5 activity mediates FSGS onset and progression is unknown. We identified a small molecule, AC1903, that specifically blocks TRPC5 channel activity in glomeruli of proteinuric rats. Chronic administration of AC1903 suppressed severe proteinuria and prevented podocyte loss in a transgenic rat model of FSGS. AC1903 also provided therapeutic benefit in a rat model of hypertensive proteinuric kidney disease. These data indicate that TRPC5 activity drives disease and that TRPC5 inhibitors may be valuable for the treatment of progressive kidney diseases., National Institutes of Health (U.S.) (Grant DK095045), National Institutes of Health (U.S.) (Grant DK099465), National Institutes of Health (U.S.) (Grant DK103658), National Institutes of Health (U.S.) (Grant DK083511), National Institutes of Health (U.S.) (Grant DK093746)

Antibiotic lethality is a complex physiological process, sensitive to external cues. Recent advances using systems approaches have revealed how events downstream of primary target inhibition actively participate in antibiotic death processes. In particular, altered metabolism, translational stress and DNA damage each contribute to antibiotic-induced cell death. Moreover, environmental factors such as oxygen availability, extracellular metabolites, population heterogeneity and multidrug contexts alter antibiotic efficacy by impacting bacterial metabolism and stress responses. Here we review recent studies on antibiotic efficacy and highlight insights gained on the involvement of cellular respiration, redox stress and altered metabolism in antibiotic lethality. We discuss the complexity found in natural environments and highlight knowledge gaps in antibiotic lethality that may be addressed using systems approaches., National Institutes of Health (U.S.) (Grant K99GM118907), National Institutes of Health (U.S.) (Grant U19AI111276), National Science Foundation (U.S.) (Award 1122374), Defense Threat Reduction Agency (DTRA) (Award HDTRA1-15-1-0051)

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.

Metabolically dormant bacteria present a critical challenge to effective antimicrobial therapy because these bacteria are genetically susceptible to antibiotic treatment but phenotypically tolerant. Such tolerance has been attributed to impaired drug uptake, which can be reversed by metabolic stimulation. Here, we evaluate the effects of central carbon metabolite stimulations on aminoglycoside sensitivity in the pathogen Pseudomonas aeruginosa. We identify fumarate as a tobramycin potentiator that activates cellular respiration and generates a proton motive force by stimulating the tricarboxylic acid (TCA) cycle. In contrast, we find that glyoxylate induces phenotypic tolerance by inhibiting cellular respiration with acetyl-coenzyme A diversion through the glyoxylate shunt, despite drug import. Collectively, this work demonstrates that TCA cycle activity is important for both aminoglycoside uptake and downstream lethality and identifies a potential strategy for potentiating aminoglycoside treatment of P. aeruginosa infections. Keyword: aminoglycoside susceptibility; Pseudomonas aeruginosa; TCA cycle; respiration; electron transport chain; fumarate; glyoxylate; biochemical persistence; LC-MS metabolomics, Defense Threat Reduction Agency (DTRA) (Grant HDTRA1-15-1-0051), National Institutes of Health (U.S.) (Grant NIH K99GM118907)

Chromatin moves with subdiffusive and spatially constrained dynamics within the cell nucleus. Here, we use single-locus tracking by time-lapse fluorescence microscopy to uncover information regarding the forces that influence chromatin movement following the induction of a persistent DNA double-strand break (DSB). Using improved time-lapse imaging regimens, we monitor trajectories of tagged DNA loci at a high temporal resolution, which allows us to extract biophysical parameters through robust statistical analysis. Polymer modeling based on these parameters predicts chromatin domain expansion near a DSB and damage extrusion from the domain. Both phenomena are confirmed by live imaging in budding yeast. Calculation of the anomalous exponent of locus movement allows us to differentiate forces imposed on the nucleus through the actin cytoskeleton from those that arise from INO80 remodeler-dependent changes in nucleosome organization. Our analytical approach can be applied to high-density single-locus trajectories obtained in any cell type.

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)

Drug-eluting stents are accepted as mainstream endovascular therapy, yet concerns for their safety may be under-appreciated. While failure from restenosis has dropped to below 5%, the risk of stent thrombosis and associated mortality remain relatively high. Further optimization of drug release is required to minimize thrombosis risk while maintaining therapeutic dose. The complex three-dimensional geometry of deployed stents together with the combination of diffusive and advective drug transport render an intuitive understanding of the situation exceedingly difficult. In situations such as this, computational modeling has proven essential, helping define the limits of efficacy, determine the mode and mechanism of drug release, and identify alternatives to avoid toxicity. A particularly challenging conformation is encountered in coronary arteries with overlapping stents. To study hemodynamics and drug deposition in such vessels we combined high-resolution, multi-scale ex vivo computed tomography with a flow and mass transfer computational model. This approach ensures high geometric fidelity and precise, simultaneous calculation of blood flow velocity, shear stress and drug distribution. Our calculations show that drug uptake by the arterial tissue is dependent both on the patterns of flow disruption near the wall, as well as on the relative positioning of drug-eluting struts. Overlapping stent struts lead to localized peaks of drug concentration that may increase the risk of thrombosis. Such peaks could be avoided by anisotropic stent structure or asymmetric drug release designed to yield homogeneous drug distribution along the coronary artery and, at the least, suggest that these issues need to remain in the forefront of consideration in clinical practice., National Institutes of Health (U.S.) (Grant R01 GM 49039)

Hematopoietic stem cells (HSCs) sustain long-term reconstitution of hematopoiesis in transplantation recipients, yet their role in the endogenous steady-state hematopoiesis remains unclear. In particular, recent studies suggested that HSCs provide a relatively minor contribution to immune cell development in adults. We directed transgene expression in a fraction of HSCs that maintained reconstituting activity during serial transplantations. Inducible genetic labeling showed that transgene-expressing HSCs gave rise to other phenotypic HSCs, confirming their top position in the differentiation hierarchy. The labeled HSCs rapidly contributed to committed progenitors of all lineages and to mature myeloid cells and lymphocytes, but not to B-1a cells or tissue macrophages. Importantly, labeled HSCs gave rise to more than two-thirds of all myeloid cells and platelets in adult mice, and this contribution could be accelerated by an induced interferon response. Thus, classically defined HSCs maintain immune cell development in the steady state and during systemic cytokine responses.

The precise neural mechanisms underlying transitions between consciousness and anesthetic-induced unconsciousness remain unclear. Here, we studied intracortical neuronal dynamics leading to propofol-induced unconsciousness by recording single-neuron activity and local field potentials directly in the functionally interconnecting somatosensory (S1) and frontal ventral premotor (PMv) network during a gradual behavioral transition from full alertness to loss of consciousness (LOC) and on through a deeper anesthetic level. Macaque monkeys were trained for a behavioral task designed to determine the trial-by-trial alertness and neuronal response to tactile and auditory stimulation. We show that disruption of coherent beta oscillations between S1 and PMv preceded, but did not coincide with, the LOC. LOC appeared to correspond to pronounced but brief gamma-/high-beta-band oscillations (lasting ∼3 min) in PMv, followed by a gamma peak in S1. We also demonstrate that the slow oscillations appeared after LOC in S1 and then in PMv after a delay, together suggesting that neuronal dynamics are very different across S1 versus PMv during LOC. Finally, neurons in both S1 and PMv transition from responding to bimodal (tactile and auditory) stimulation before LOC to only tactile modality during unconsciousness, consistent with an inhibition of multisensory integration in this network. Our results show that propofol-induced LOC is accompanied by spatiotemporally distinct oscillatory neuronal dynamics across the somatosensory and premotor network and suggest that a transitional state from wakefulness to unconsciousness is not a continuous process, but rather a series of discrete neural changes., Foundation for Anesthesia Education and Research (Grant 5T32GM007592), Harvard Medical School. Eleanor and Miles Shore 50th Anniversary Fellowship Program for Scholars in Medicine

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)

The recent Zika virus outbreak highlights the need for low-cost diagnostics that can be rapidly developed for distribution and use in pandemic regions. Here, we report a pipeline for the rapid design, assembly, and validation of cell-free, paper-based sensors for the detection of the Zika virus RNA genome. By linking isothermal RNA amplification to toehold switch RNA sensors, we detect clinically relevant concentrations of Zika virus sequences and demonstrate specificity against closely related Dengue virus sequences. When coupled with a novel CRISPR/Cas9-based module, our sensors can discriminate between viral strains with single-base resolution. We successfully demonstrate a simple, field-ready sample-processing workflow and detect Zika virus from the plasma of a viremic macaque. Our freeze-dried biomolecular platform resolves important practical limitations to the deployment of molecular diagnostics in the field and demonstrates how synthetic biology can be used to develop diagnostic tools for confronting global health crises., Defense Threat Reduction Agency (DTRA) (HDTRA1-14-1-0006), United States. National Institutes of Health (NIH AI100190)

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)

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)

Purpose To investigate ultrahigh-speed, swept-source optical coherence tomography (SSOCT) angiography for visualizing vascular changes in eyes with nonexudative age-related macular degeneration (AMD) with geographic atrophy (GA). Design Observational, prospective, cross-sectional study. Participants A total of 63 eyes from 32 normal subjects and 12 eyes from 7 patients with nonexudative AMD with GA. Methods A 1050-nm, 400-kHz A-scan rate SSOCT system was used to perform volumetric optical coherence tomography angiography (OCTA) of the retinal and choriocapillaris (CC) vasculatures in normal subjects and patients with nonexudative AMD with GA. Optical coherence tomography angiography using variable interscan time analysis (VISTA) was performed to assess CC alteration and differentiate varying degrees of CC flow impairment. Main Outcome Measures Qualitative comparison of retinal and CC vasculatures in normal subjects versus those in patients with a clinical diagnosis of nonexudative AMD with GA. Results In all 12 eyes with GA, OCTA showed pronounced CC flow impairment within the region of GA. In 10 of the 12 eyes with GA, OCTA with VISTA showed milder CC flow impairment extending beyond the margin of GA. Of the 5 eyes exhibiting foveal-sparing GA, OCTA showed CC flow within the region of foveal sparing in 4 of the eyes. Conclusions The ability of ultrahigh-speed, swept-source OCTA to noninvasively visualize alterations in the retinal and CC vasculatures makes it a promising tool for assessing nonexudative AMD with GA. Optical coherence tomography angiography using VISTA can distinguish varying degrees of CC alteration and flow impairment and may be useful for elucidating disease pathogenesis, progression, and response to therapy., National Institutes of Health (U.S.) ( R01-EY011289-27), National Institutes of Health (U.S.) ( R44-EY022864-01), National Institutes of Health (U.S.) (R44- EY022864-02), National Institutes of Health (U.S.) (R01-CA075289-16), United States. Air Force Office of Scientific Research (FA9550-10-1-0551), United States. Air Force Office of Scientific Research (FA9550-12-1-0499)

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.

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.

Microbiota-modulating interventions are an emerging strategy to promote gastrointestinal homeostasis. Yet, their use in the detection, prevention, and treatment of acute infections remains underexplored. We report the basis of a probiotic-based strategy to promote colonization resistance and point-of-need diagnosis of cholera, an acute diarrheal disease caused by the pathogen Vibrio cholerae. Oral administration of Lactococcus lactis, a common dietary fermentative bacterium, reduced intestinal V. cholerae burden and improved survival in infected infant mice through the production of lactic acid. Furthermore, we engineered an L. lactis strain that specifically detects quorum-sensing signals of V. cholerae in the gut and triggers expression of an enzymatic reporter that is readily detected in fecal samples. We postulate that preventive dietary interventions with fermented foods containing natural and engineered L. lactis strains may hinder cholera progression and improve disease surveillance in populations at risk of cholera outbreaks.

Patients with autism spectrum disorder (ASD) often require sedation or general anesthesia. ASD is thought to arise from deficits in GABAergic signaling leading to abnormal neurodevelopment. We sought to investigate differences in how ASD patients respond to the GABAergic drug propofol by comparing the propofol-induced electroencephalogram (EEG) of ASD and neurotypical (NT) patients. This investigation was a prospective observational study. Continuous 4-channel frontal EEG was recorded during routine anesthetic care of patients undergoing endoscopic procedures between July 1, 2014 and May 1, 2016. Study patients were defined as those with previously diagnosed ASD by DSM-V criteria, aged 2–30 years old. NT patients were defined as those lacking neurological or psychiatric abnormalities, aged 2–30 years old. The primary outcome was changes in propofol-induced alpha (8–13 Hz) and slow (0.1–1 Hz) oscillation power by age. A post hoc analysis was performed to characterize incidence of burst suppression during propofol anesthesia. The primary risk factor of interest was a prior diagnosis of ASD. Outcomes were compared between ASD and NT patients using Bayesian methods. Compared to NT patients, slow oscillation power was initially higher in ASD patients (17.05 vs. 14.20 dB at 2.33 years), but progressively declined with age (11.56 vs. 13.95 dB at 22.5 years). Frontal alpha power was initially lower in ASD patients (17.65 vs. 18.86 dB at 5.42 years) and continued to decline with age (6.37 vs. 11.89 dB at 22.5 years). The incidence of burst suppression was significantly higher in ASD vs. NT patients (23.0% vs. 12.2%, p < 0.01) despite reduced total propofol dosing in ASD patients. Ultimately, we found that ASD patients respond differently to propofol compared to NT patients. A similar pattern of decreased alpha power and increased sensitivity to burst suppression develops in older NT adults; one interpretation of our data could be that ASD patients undergo a form of accelerated neuronal aging in adolescence. Our results suggest that investigations of the propofol-induced EEG in ASD patients may enable insights into the underlying differences in neural circuitry of ASD and yield safer practices for managing patients with ASD.

Inertial measurement units (IMUs) have been demonstrated to reliably measure human joint angles—an essential quantity in the study of biomechanics. However, most previous literature proposed IMU-based joint angle measurement systems that required manual alignment or prescribed calibration motions. This paper presents a simple, physically-intuitive method for IMU-based measurement of the knee flexion/extension angle in gait without requiring alignment or discrete calibration, based on computationally-efficient and easy-to-implement Principle Component Analysis (PCA). The method is compared against an optical motion capture knee flexion/extension angle modeled through OpenSim. The method is evaluated using both measured and simulated IMU data in an observational study (n = 15) with an absolute root-mean-square-error (RMSE) of 9.24∘ and a zero-mean RMSE of 3.49∘. Variation in error across subjects was found, made emergent by the larger subject population than previous literature considers. Finally, the paper presents an explanatory model of RMSE on IMU mounting location. The observational data suggest that RMSE of the method is a function of thigh IMU perturbation and axis estimation quality. However, the effect size for these parameters is small in comparison to potential gains from improved IMU orientation estimations. Results also highlight the need to set relevant datums from which to interpret joint angles for both truth references and estimated data., National Science Foundation (U.S.) (GRFP), National Science Foundation (U.S.) (IIS-1453141)

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

Background Fibronectin in endothelial basement membranes promotes endothelial inflammatory activation and atherosclerosis but also promotes plaque stability and vascular remodeling. The fibronectin receptor α5 subunit is proinflammatory through binding to and activating phosphodiesterase 4D5, which inhibits anti‐inflammatory cyclic adenosine monophosphate and protein kinase A. Replacing the α5 cytoplasmic domain with that of α2 resulted in smaller atherosclerotic plaques. Here, we further assessed plaque phenotype and compensatory vascular remodeling in this model. Methods and Results α5/2 mice in the hyperlipidemic apolipoprotein E null background had smaller plaques in the aortic root, with reduced endothelial NF ‐κB activation and inflammatory gene expression, reduced leukocyte content, and much lower metalloproteinase expression. However, smooth muscle cell content, fibrous cap thickness, and fibrillar collagen were unchanged, indicating no shift toward vulnerability. In vivo knockdown of phosphodiesterase 4D5 also decreased endothelial inflammatory activation and atherosclerotic plaque size. α5/2 mice showed improved recovery from hindlimb ischemia after femoral artery ligation. Conclusions Blocking the fibronectin‐Integrin α5 pathway reduces atherosclerotic plaque size, maintains plaque stability, and improves compensatory remodeling. This pathway is therefore a potential therapeutic target for treatment of atherosclerosis.

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

Bactericidal antibiotics alter microbial metabolism as part of their lethality and can damage mitochondria in mammalian cells. In addition, antibiotic susceptibility is sensitive to extracellular metabolites, but it remains unknown whether metabolites present at an infection site can affect either treatment efficacy or immune function. Here, we quantify local metabolic changes in the host microenvironment following antibiotic treatment for a peritoneal Escherichia coli infection. Antibiotic treatment elicits microbiome-independent changes in local metabolites, but not those distal to the infection site, by acting directly on host cells. The metabolites induced during treatment, such as AMP, reduce antibiotic efficacy and enhance phagocytic killing. Moreover, antibiotic treatment impairs immune function by inhibiting respiratory activity in immune cells. Collectively, these results highlight the immunomodulatory potential of antibiotics and reveal the local metabolic microenvironment to be an important determinant of infection resolution. Antibiotic susceptibility is sensitive to metabolites, but how this affects in vivo treatment efficacy remains unexplored. Yang, Bhargava et al. characterize antibiotic-induced changes to the metabolic environment during infection and find that direct actions of antibiotics on host cells induce metabolites that impair drug efficacy and enhance phagocytic activity., United States. Defense Threat Reduction Agency (Grant HDTRA1-15-1-0051), National Institutes of Health (U.S.) (Grant U01AI124316), National Institutes of Health (U.S.) (Grant K99GM118907), Novo Nordisk Foundation (Grant NNF16CC0021858), Paul G. Allen Frontiers Group, Wyss Institute for Biologically Inspired Engineering

Understanding how antibiotics impact bacterial metabolism may provide insight into their mechanisms of action and could lead to enhanced therapeutic methodologies. Here, we profiled the metabolome of Escherichia coli after treatment with three different classes of bactericidal antibiotics (β-lactams, aminoglycosides, quinolones). These treatments induced a similar set of metabolic changes after 30 min that then diverged into more distinct profiles at later time points. The most striking changes corresponded to elevated concentrations of central carbon metabolites, active breakdown of the nucleotide pool, reduced lipid levels, and evidence of an elevated redox state. We examined potential end-target consequences of these metabolic perturbations and found that antibiotic-treated cells exhibited cytotoxic changes indicative of oxidative stress, including higher levels of protein carbonylation, malondialdehyde adducts, nucleotide oxidation, and double-strand DNA breaks. This work shows that bactericidal antibiotics induce a complex set of metabolic changes that are correlated with the buildup of toxic metabolic by-products., National Institutes of Health (U.S.) (Director's Pioneer Award), Howard Hughes Medical Institute, Wyss Institute for Biologically Inspired Engineering, United States. Defense Threat Reduction Agency (Grant HDTRA1-15-1-0051), National Institutes of Health (U.S.) (Grant R01 CA021615)

The widely used electroencephalogram-based indices for depth-of-Anesthesia monitoring assume that the same index value defines the same level of unconsciousness for all anesthetics. In contrast, we show that different anesthetics act at different molecular targets and neural circuits to produce distinct brain states that are readily visible in the electroencephalogram. We present a two-part review to educate anesthesiologists on use of the unprocessed electroencephalogram and its spectrogram to track the brain states of patients receiving anesthesia care. Here in part I, we review the biophysics of the electroencephalogram and the neurophysiology of the electroencephalogram signatures of three intravenous anesthetics: propofol, dexmedetomidine, and ketamine, and four inhaled anesthetics: sevoflurane, isoflurane, desflurane, and nitrous oxide. Later in part II, we discuss patient management using these electroencephalogram signatures. Use of these electroencephalogram signatures suggests a neurophysiologically based paradigm for brain state monitoring of patients receiving anesthesia care., National Institutes of Health (U.S.) (Grant DP1-OD003646), National Institutes of Health (U.S.) (Grant TR01-GM104948)

Once a memory has formed, it is thought to undergo a gradual transition within the brain from short- to long-term storage. This putative process, however, also poses a unique problem to the memory system in that the same learned items must also be retrieved across broadly varying time scales. Here, we find that neurons in the ventrolateral prefrontal cortex (VLPFC) of monkeys, an area interconnected with both temporal and frontal associative neocortical regions, signaled the need to alter between retrieval of memories formed at different times. These signals were most closely related to the time interval between initial learning and later retrieval, and did not correlate with task switch demands, novelty, or behavioral response. Consistent with these physiological findings, focal inactivation of the VLPFC led to a marked degradation in retrieval performance. These findings suggest that the VLPFC plays a necessary regulatory role in retrieving memories over different temporal scales., United States. National Institutes of Health (5R01-HD059852), Whitehall Foundation, Neurosurgery Research & Education Foundation, White House Presidential Early Career Award for Scientists and Engineers

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)

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)

A new class of material resistant to bacterial attachment has been discovered that is formed from polyacrylates with hydrocarbon pendant groups. In this study, the relationship between the nature of the hydrocarbon moiety and resistance to bacteria is explored, comparing cyclic, aromatic, and linear chemical groups. A correlation is shown between bacterial attachment and a parameter derived from the partition coefficient and the number of rotatable bonds of the materials' pendant groups. This correlation is applicable to 86% of the hydrocarbon pendant moieties surveyed, quantitatively supporting the previous qualitative observation that bacteria are repelled from poly(meth)acrylates containing a hydrophilic ester group when the pendant group is both rigid and hydrophobic. This insight will help inform and predict the further development of polymers resistant to bacterial attachment., Wellcome Trust (London, England) (Grant 085245), European Metrology Research Programme (IND56)

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.

Although general anesthetics are routinely administered to surgical patients to induce loss of consciousness, the mechanisms underlying anesthetic-induced unconsciousness are not fully understood. In rats, we characterized changes in the extradural EEG and intracranial local field potentials (LFPs) within the prefrontal cortex (PFC), parietal cortex (PC), and central thalamus (CT) in response to progressively higher doses of the inhaled anesthetic sevoflurane. During induction with a low dose of sevoflurane, beta/low gamma (12–40 Hz) power increased in the frontal EEG and PFC, PC and CT LFPs, and PFC–CT and PFC–PFC LFP beta/low gamma coherence increased. Loss of movement (LOM) coincided with an abrupt decrease in beta/low gamma PFC–CT LFP coherence. Following LOM, cortically coherent slow-delta (0.1–4 Hz) oscillations were observed in the frontal EEG and PFC, PC and CT LFPs. At higher doses of sevoflurane sufficient to induce loss of the righting reflex, coherent slow-delta oscillations were dominant in the frontal EEG and PFC, PC and CT LFPs. Dynamics similar to those observed during induction were observed as animals emerged from sevoflurane anesthesia. We conclude that the rat is a useful animal model for sevoflurane-induced EEG oscillations in humans, and that coherent slow-delta oscillations are a correlate of sevoflurane-induced behavioral arrest and loss of righting in rats., National Institutes of Health (U.S.) (Grant PO1-GM118269), National Institutes of Health (U.S.) (Grant TR01-GM104948)

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

Physiologic and environmental factors can modulate antibiotic activity and thus pose a significant challenge to antibiotic treatment. The quinolone class of antibiotics, which targets bacterial topoisomerases, fails to kill bacteria that have grown to high density; however, the mechanistic basis for this persistence is unclear. Here, we show that exhaustion of the metabolic inputs that couple carbon catabolism to oxidative phosphorylation is a primary cause of growth phase-dependent persistence to quinolone antibiotics. Supplementation of stationary-phase cultures with glucose and a suitable terminal electron acceptor to stimulate respiratory metabolism is sufficient to sensitize cells to quinolone killing. Using this approach, we successfully sensitize high-density populations of Escherichia coli, Staphylococcus aureus, and Mycobacterium smegmatis to quinolone antibiotics. Our findings link growth-dependent quinolone persistence to discrete impairments in respiratory metabolism and identify a strategy to kill non-dividing bacteria. Gutierrez et al. show that activation of cellular respiration is sufficient to sensitize antibiotic refractory bacteria at high densities to drugs targeting DNA topoisomerases. This suggests that the nutrient environment and metabolic state are key components of bacterial persistence phenotypes. Keywords: quinolones; drug persistence; antibiotic; oxidative phosphorylation, Defense Threat Reduction Agency (DTRA) (Grant HDTRA1-15-1-0051)

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))