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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)

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)

Summary: Metabolism is an emerging stem cell hallmark tied to cell fate, pluripotency, and self-renewal, yet systems-level understanding of stem cell metabolism has been limited by the lack of genome-scale network models. Here, we develop a systems approach to integrate time-course metabolomics data with a computational model of metabolism to analyze the metabolic state of naive and primed murine pluripotent stem cells. Using this approach, we find that one-carbon metabolism involving phosphoglycerate dehydrogenase, folate synthesis, and nucleotide synthesis is a key pathway that differs between the two states, resulting in differential sensitivity to anti-folates. The model also predicts that the pluripotency factor Lin28 regulates this one-carbon metabolic pathway, which we validate using metabolomics data from Lin28-deficient cells. Moreover, we identify and validate metabolic reactions related to S-adenosyl-methionine production that can differentially impact histone methylation in naive and primed cells. Our network-based approach provides a framework for characterizing metabolic changes influencing pluripotency and cell fate. : Chandrasekaran et al. use computational modeling, metabolomics, and metabolic inhibitors to discover metabolic differences between various pluripotent stem cell states and infer their impact on stem cell fate decisions. Keywords: systems biology, stem cell biology, metabolism, genome-scale modeling, pluripotency, histone methylation, naive (ground) state, primed state, cell fate, metabolic network

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)

Synthetic biology aims to develop engineering-driven approaches to the programming of cellular functions that could yield transformative technologies. Synthetic gene circuits that combine DNA, protein, and RNA components have demonstrated a range of functions such as bistability, oscillation, feedback, and logic capabilities. However, it remains challenging to scale up these circuits owing to the limited number of designable, orthogonal, high-performance parts, the empirical and often tedious composition rules, and the requirements for substantial resources for encoding and operation. Here, we report a strategy for constructing RNA-only nanodevices to evaluate complex logic in living cells. Our ‘ribocomputing’ systems are composed of de-novo-designed parts and operate through predictable and designable base-pairing rules, allowing the effective in silico design of computing devices with prescribed configurations and functions in complex cellular environments. These devices operate at the post-transcriptional level and use an extended RNA transcript to co-localize all circuit sensing, computation, signal transduction, and output elements in the same self-assembled molecular complex, which reduces diffusion-mediated signal losses, lowers metabolic cost, and improves circuit reliability. We demonstrate that ribocomputing devices in Escherichia coli can evaluate two-input logic with a dynamic range up to 900-fold and scale them to four-input AND, six-input OR, and a complex 12-input expression (A1 AND A2 AND NOT A1*) OR (B1 AND B2 AND NOT B2*) OR (C1 AND C2) OR (D1 AND D2) OR (E1 AND E2). Successful operation of ribocomputing devices based on programmable RNA interactions suggests that systems employing the same design principles could be implemented in other host organisms or in extracellular settings., National Institutes of Health (U.S.) (Grant 1DP2OD007292), National Institutes of Health (U.S.) (Grant 1R01EB018659), United States. Office of Naval Research (Award N000141110914), United States. Office of Naval Research (Grant N000141010827), United States. Office of Naval Research (Grant N000141310593), United States. Office of Naval Research (Grant N000141410610), United States. Office of Naval Research (Grant N000141612410), National Science Foundation (U.S.) (Grant CCF1054898), National Science Foundation (U.S.) (Grant CCF1317291), National Science Foundation (U.S.) (Grant CCF1162459), United States. Defense Advanced Research Projects Agency (Grant HR001112C0061), Defense Threat Reduction Agency (DTRA) (Grant HDTRA1-15-1-0040)

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)

Agonistic encounters are powerful effectors of future behavior, and the ability to learn from this type of social challenge is an essential adaptive trait. We recently identified a conserved transcriptional program defining the response to social challenge across animal species, highly enriched in transcription factor (TF), energy metabolism, and developmental signaling genes. To understand the trajectory of this program and to uncover the most important regulatory influences controlling this response, we integrated gene expression data with the chromatin landscape in the hypothalamus, frontal cortex, and amygdala of socially challenged mice over time. The expression data revealed a complex spatiotemporal patterning of events starting with neural signaling molecules in the frontal cortex and ending in the modulation of developmental factors in the amygdala and hypothalamus, underpinned by a systems-wide shift in expression of energy metabolism-related genes. The transcriptional signals were correlated with significant shifts in chromatin accessibility and a network of challenge-associated TFs. Among these, the conserved metabolic and developmental regulator ESRRA was highlighted for an especially early and important regulatory role. Cell-type deconvolution analysis attributed the differential metabolic and developmental signals in this social context primarily to oligodendrocytes and neurons, respectively, and we show that ESRRA is expressed in both cell types. Localizing ESRRA binding sites in cortical chromatin, we show that this nuclear receptor binds both differentially expressed energy-related and neurodevelopmental TF genes. These data link metabolic and neurodevelopmental signaling to social challenge, and identify key regulatory drivers of this process with unprecedented tissue and temporal resolution.

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.

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)

The growing prevalence of antibiotic resistance calls for new approaches in the development of antimicrobial therapeutics. Likewise, improved diagnostic measures are essential in guiding the application of targeted therapies and preventing the evolution of therapeutic resistance. Discovery platforms are also needed to form new treatment strategies and identify novel antimicrobial agents. By applying engineering principles to molecular biology, synthetic biologists have developed platforms that improve upon, supplement, and will perhaps supplant traditional broad-spectrum antibiotics. Efforts in engineering bacteriophages and synthetic probiotics demonstrate targeted antimicrobial approaches that can be fine-tuned using synthetic biology-derived principles. Further, the development of paper-based, cell-free expression systems holds promise in promoting the clinical translation of molecular biology tools for diagnostic purposes. In this review, we highlight emerging synthetic biology platform technologies that are geared toward the generation of new antimicrobial therapies, diagnostics, and discovery channels., Defense Threat Reduction Agency (DTRA) (Grant HDTRA1-14-1-0006), Defense Threat Reduction Agency (DTRA) (Grant HDTRA1-15-1-0051)

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.

Synthetic biology is increasingly used to develop sophisticated living devices for basic and applied research. Many of these genetic devices are engineered using multi-copy plasmids, but as the field progresses from proof-of-principle demonstrations to practical applications, it is important to develop single-copy synthetic modules that minimize consumption of cellular resources and can be stably maintained as genomic integrants. Here we use empirical design, mathematical modeling, and iterative construction and testing to build single-copy, bistable toggle switches with improved performance and reduced metabolic load that can be stably integrated into the host genome. Deterministic and stochastic models led us to focus on basal transcription to optimize circuit performance and helped to explain the resulting circuit robustness across a large range of component expression levels. The design parameters developed here provide important guidance for future efforts to convert functional multi-copy gene circuits into optimized single-copy circuits for practical, real-world use., Defense Threat Reduction Agency (DTRA) (Grant HDTRA1-14-1-006), United States. Air Force Office of Scientific Research (Grant FA9550-14-1-0060), United States. Defense Advanced Research Projects Agency (Grant N66001-11-C-4203), United States. Office of Naval Research (Grant N000141110725), National Institutes of Health (U.S.) (Grant P50 GM098792)

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)

Cell-laden hydrogels can regenerate lost, damaged or malfunctioning tissues. Clinical success of such hydrogels is strongly dependent on the ability to tune their chemical, physico-mechanical, and biological properties to a specific application. In particular, mimicking the intricate arrangement of cell-interactive ligands of natural tissues is crucial to proper tissue function. Natural extracellular matrix elements represent a unique source for generating such interactions. A plethora of extracellular matrix-based approaches have been explored to augment the regenerative potential of hydrogels. These efforts include the development of matrix-like hydrogels, hydrogels containing matrix-like molecules, hydrogels containing decellularized matrix, hydrogels derived from decellularized matrix, and decellularized tissues as reimplantable matrix hydrogels. Here we review the evolution, strengths and weaknesses of these developments from the perspective of creating tissue regenerating hydrogels., National Science Foundation (U.S.) (NSF Grant DMR1049381), National Science Foundation (U.S.) (NSF Grant Grant Nos. IIP- 1357109), National Science Foundation (U.S.) (NSF Grant CBET1403545), National Institutes of Health (U.S.) (NIH Grant No. AR063745), Pew Charitable Trusts, National Institutes of Health (U.S.) (NIH Grant No. 1R03EB017402-01), Fonds voor Wetenschappelijk Onderzoek--Vlaanderen (FWO) (Grant No. 1208715N), National Science Foundation (U.S.) (Grant No. 1208715N), National Science Foundation (U.S.) (NSF Grant IMMODGEL (602694)), National Institutes of Health (U.S.) (EB012597), National Institutes of Health (U.S.) (NIH grant AR057837), National Institutes of Health (U.S.) (NIH grant DE021468), National Institutes of Health (U.S.) (NIH grant HL099073), National Institutes of Health (U.S.) (NIH grant AI105024)

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)

Antisense RNA-mediated transcriptional regulators are powerful tools for controlling gene expression and creating synthetic gene networks. RNA transcriptional repressors derived from natural mechanisms called attenuators are particularly versatile, though their mechanistic complexity has made them difficult to engineer. Here we identify a new structure–function design principle for attenuators that enables the forward engineering of new RNA transcriptional repressors. Using in-cell SHAPE-Seq to characterize the structures of attenuator variants within Escherichia coli, we show that attenuator hairpins that facilitate interaction with antisense RNAs require interior loops for proper function. Molecular dynamics simulations of these attenuator variants suggest these interior loops impart structural flexibility. We further observe hairpin flexibility in the cellular structures of natural RNA mechanisms that use antisense RNA interactions to repress translation, confirming earlier results from in vitro studies. Finally, we design new transcriptional attenuators in silico using an interior loop as a structural requirement and show that they function as desired in vivo. This work establishes interior loops as an important structural element for designing synthetic RNA gene regulators. We anticipate that the coupling of experimental measurement of cellular RNA structure and function with computational modeling will enable rapid discovery of structure–function design principles for a diverse array of natural and synthetic RNA regulators.

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

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)

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)

Protein-DNA interactions are critical for the successful functioning of all natural systems. The key role in these interactions is played by processes of protein search for specific sites on DNA. Although it has been studied for many years, only recently microscopic aspects of these processes became more clear. In this work, we present a review on current theoretical understanding of the molecular mechanisms of the protein target search. A comprehensive discrete-state stochastic method to explain the dynamics of the protein search phenomena is introduced and explained. Our theoretical approach utilizes a first-passage analysis and it takes into account the most relevant physical-chemical processes. It is able to describe many fascinating features of the protein search, including unusually high effective association rates, high selectivity and specificity, and the robustness in the presence of crowders and sequence heterogeneity., National Science Foundation (U.S.) (Grant CHE-1664218), National Science Foundation (U.S.) (Grant PHY-1427654), Robert A. Welch Foundation (Grant C-1559)

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.

Super-enhancers (SEs) are clusters of enhancers that cooperatively assemble a high density of the transcriptional apparatus to drive robust expression of genes with prominent roles in cell identity. Here we demonstrate that the SE-enriched transcriptional coactivators BRD4 and MED1 form nuclear puncta at SEs that exhibit properties of liquid-like condensates and are disrupted by chemicals that perturb condensates. The intrinsically disordered regions (IDRs) of BRD4 and MED1 can form phase-separated droplets, and MED1-IDR droplets can compartmentalize and concentrate the transcription apparatus from nuclear extracts. These results support the idea that coactivators form phase-separated condensates at SEs that compartmentalize and concentrate the transcription apparatus, suggest a role for coactivator IDRs in this process, and offer insights into mechanisms involved in the control of key cell-identity genes., National Institutes of Health (U.S.) (Grant GM123511), National Institutes of Health (U.S.) (Grant P01-CA042063), National Science Foundation (U.S.) (Grant PHY-1743900), National Cancer Institute (U.S.) (Grant P30-CA14051)

We present a low-cost kit based on freeze-dried, cell-free reactions to illustrate synthetic and molecular biology concepts., Hands-on demonstrations greatly enhance the teaching of science, technology, engineering, and mathematics (STEM) concepts and foster engagement and exploration in the sciences. While numerous chemistry and physics classroom demonstrations exist, few biology demonstrations are practical and accessible due to the challenges and concerns of growing living cells in classrooms. We introduce BioBits™ Explorer, a synthetic biology educational kit based on shelf-stable, freeze-dried, cell-free (FD-CF) reactions, which are activated by simply adding water. The FD-CF reactions engage the senses of sight, smell, and touch with outputs that produce fluorescence, fragrances, and hydrogels, respectively. We introduce components that can teach tunable protein expression, enzymatic reactions, biomaterial formation, and biosensors using RNA switches, some of which represent original FD-CF outputs that expand the toolbox of cell-free synthetic biology. The BioBits™ Explorer kit enables hands-on demonstrations of cutting-edge science that are inexpensive and easy to use, circumventing many current barriers for implementing exploratory biology experiments in classrooms.

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

The maintenance of DNA supercoiling is essential for the proper regulation of a plethora of biological processes. As a consequence of this mode of regulation, ahead of the replication fork, DNA replication machinery is prone to introducing supercoiled regions into the DNA double helix. Resolution of DNA supercoiling is essential to maintain DNA replication rates that are amenable to life. This resolution is handled by evolutionarily conserved enzymes known as topoisomerases. The activity of topoisomerases is essential, and therefore constitutes a prime candidate for targeting by antibiotics. In this review, we present hallmark investigations describing the mode of action of quinolones, one of the antibacterial classes targeting the function of topoisomerases in bacteria. By chronologically analyzing data gathered on the mode of action of this imperative antibiotic class, we highlight the necessity to look beyond primary drug-target interactions towards thoroughly understanding the mechanism of quinolones at the level of the cell. Keywords: antibiotics; quinolones; topoisomerases; DNA replication; DNA supercoiling

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.

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.

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

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)

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.

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.

Designer microbial consortia are an emerging frontier in synthetic biology that enable versatile microbiome engineering. However, the utilization of such consortia is hindered by our limited capacity in rapidly creating ecosystems with desired dynamics. Here we present the development of synthetic communities through social interaction engineering that combines modular pathway reconfiguration with model creation. Specifically, we created six two-strain consortia, each possessing a unique mode of interaction, including commensalism, amensalism, neutralism, cooperation, competition and predation. These consortia follow distinct population dynamics with characteristics determined by the underlying interaction modes. We showed that models derived from two-strain consortia can be used to design three- and four-strain ecosystems with predictable behaviors and further extended to provide insights into community dynamics in space. This work sheds light on the organization of interacting microbial species and provides a systematic framework—social interaction programming—to guide the development of synthetic ecosystems for diverse purposes., National Science Foundation (U.S.) (Grant 1553649), National Science Foundation (U.S.) (Grant 1227034), United States. Office of Naval Research (Award (N00014-16-12525), American Cancer Society (Grant 12SDG12090025), Defense Threat Reduction Agency (DTRA) (Grant HDTRA1-14-1-0006)

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))

1AbstractDistribution-based operational taxonomic unit-calling (dbOTU) improves on other approaches by incorporating information about the input sequences’ distribution across samples. Previous implementations of dbOTU presented challenges for users. Here we introduce and evaluate a new implementation of dbOTU that is faster and more user-friendly. We show that this new implementation has theoretical and practical improvements over previous implementations of dbOTU, making the algorithm more accessible to microbial ecology and biomedical researchers.

Predicting and constraining RNA virus evolution require understanding the molecular factors that define the mutational landscape accessible to these pathogens. RNA viruses typically have high mutation rates, resulting in frequent production of protein variants with compromised biophysical properties. Their evolution is necessarily constrained by the consequent challenge to protein folding and function. We hypothesized that host proteostasis mechanisms may be significant determinants of the fitness of viral protein variants, serving as a critical force shaping viral evolution. Here, we test that hypothesis by propagating influenza in host cells displaying chemically-controlled, divergent proteostasis environments. We find that both the nature of selection on the influenza genome and the accessibility of specific mutational trajectories are significantly impacted by host proteostasis. These findings provide new insights into features of host-pathogen interactions that shape viral evolution, and into the potential design of host proteostasis-targeted antiviral therapeutics that are refractory to resistance., National Institutes of Health (U.S.) (Award 1DP2GM119162), National Institutes of Health (U.S.) (Grant P30-ES002109)