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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

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

B cells constitute an essential line of defense from pathogenic infections through the generation of class-switched antibody-secreting cells (ASCs) in germinal centers. Although this process is known to be regulated by follicular helper T (TfH) cells, the mechanism by which B cells initially seed germinal center reactions remains elusive. We found that NKT cells, a population of innate-like T lymphocytes, are critical for the induction of B cell immunity upon viral infection. The positioning of NKT cells at the interfollicular areas of lymph nodes facilitates both their direct priming by resident macrophages and the localized delivery of innate signals to antigen-experienced B cells. Indeed, NKT cells secrete an early wave of IL-4 and constitute up to 70% of the total IL-4-producing cells during the initial stages of infection. Importantly, the requirement of this innate immunity arm appears to be evolutionarily conserved because early NKT and IL-4 gene signatures also positively correlate with the levels of neutralizing antibodies in Zika-virus-infected macaques. In conclusion, our data support a model wherein a pre-TfH wave of IL-4 secreted by interfollicular NKT cells triggers the seeding of germinal center cells and serves as an innate link between viral infection and B cell immunity. NKT cells are required for the initial formation of germinal centers and production of effective neutralizing antibody responses against viruses. Keywords: NKT cells; B cells; macrophages; germinal center seeding; IL-4; viral infection; influenza; Zika virus; lymph node; CXCR3

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

In 1985, the serendipitous discovery of fullerene triggered the research of carbon structures into the world of symmetric nanomaterials. Consequently, Robert F. Curl, Harold W. Kroto and Richard E. Smalley were awarded the Noble prize in chemistry for their discovery of the buckminsterfullerene (C[subscript 60] with a cage-like fused-ring structure). Fullerene, as the first symmetric nanostructure in carbon nanomaterials family, opened up new perspectives in nanomaterials field leading to discovery and research on other symmetric carbon nanomaterials like carbon nanotubes and two-dimensional graphene which put fullerenes in the shade, while fullerene as the most symmetrical molecule in the world with incredible properties deserves more attention in nanomaterials studies. Buckyball with its unique structure consisting of sp[superscript 2] carbons which form a high symmetric cage with different sizes (C[subscript 60], C[subscript 70] and so on); however, the most abundant among them is C[subscript 60] which possesses 60 carbon atoms. The combination of unique properties of this molecule extends its applications in divergent areas of science, especially those related to biomedical engineering. This review aims to be a comprehensive review with a broad interest to the biomedical engineering community, being a substantial overview of the most recent advances on fullerenes in biomedical applications that have not been exhaustively and critically reviewed in the past few years.

The cell fate decision between interferon-producing plasmacytoid DC (pDC) and antigen-presenting classical DC (cDC) is controlled by the E protein transcription factor TCF4 (E2-2). We report that TCF4 comprises two transcriptional isoforms, both of which are required for optimal pDC development in vitro. The long Tcf4 isoform is expressed specifically in pDCs, and its deletion in mice impaired pDCs development and led to the expansion of non-canonical CD8⁺ cDCs. The expression of Tcf4 commenced in progenitors and was further upregulated in pDCs, correlating with stage-specific activity of multiple enhancer elements. A conserved enhancer downstream of Tcf4 was required for its upregulation during pDC differentiation, revealing a positive feedback loop. The expression of Tcf4 and the resulting pDC differentiation were selectively sensitive to the inhibition of enhancer-binding BET protein activity. Thus, lineage-specifying function of E proteins is facilitated by lineage-specific isoform expression and by BET-dependent feedback regulation through distal regulatory elements.

Background & Aims Regeneration of the hepatic mass is crucial to liver repair. Proliferation of hepatic parenchyma is intimately dependent on angiogenesis and resident macrophage-derived cytokines. However the role of circulating monocyte interactions in vascular and hepatic regeneration is not well-defined. We investigated the role of these interactions in regeneration in the presence and absence of intact monocyte adhesion. Methods Partial hepatectomy was performed in wild-type mice and those lacking the monocyte adhesion molecule CD11b. Vascular architecture, angiogenesis and macrophage location were analyzed in the whole livers using simultaneous angiography and macrophage staining with fluorescent multiphoton microscopy. Monocyte adhesion molecule expression and sprouting-related pathways were evaluated. Results Resident macrophages (Kupffer cells) did not migrate to interact with vessels whereas infiltrating monocytes were found adjacent to sprouting points. Infiltrated monocytes colocalized with Wnt5a, angiopoietin 1 and Notch-1 in contact points and commensurate with phosphorylation and disruption of VE-cadherin. Mice deficient in CD11b showed a severe reduction in angiogenesis, liver mass regeneration and survival following partial hepatectomy, and developed unstable and leaky vessels that eventually produced an aberrant hepatic vascular network and Kupffer cell distribution. Conclusions Direct vascular interactions of infiltrating monocytes are required for an ordered vascular growth and liver regeneration. These outcomes provide insight into hepatic repair and new strategies for hepatic regeneration., National Institutes of Health (U.S.) (Grant R01 GM 49039)

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)

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)

Cells, the basic units of biological structure and function, vary broadly in type and state. Single-cell genomics can characterize cell identity and function, but limitations of ease and scale have prevented its broad application. Here we describe Drop-seq, a strategy for quickly profiling thousands of individual cells by separating them into nanoliter-sized aqueous droplets, associating a different barcode with each cell’s RNAs, and sequencing them all together. Drop-seq analyzes mRNA transcripts from thousands of individual cells simultaneously while remembering transcripts’ cell of origin. We analyzed transcriptomes from 44,808 mouse retinal cells and identified 39 transcriptionally distinct cell populations, creating a molecular atlas of gene expression for known retinal cell classes and novel candidate cell subtypes. Drop-seq will accelerate biological discovery by enabling routine transcriptional profiling at single-cell resolution., National Human Genome Research Institute (U.S.) (P50 HG006193)

Early detection and accurate estimation of the extent of coarctation of the aorta (COA) is critical to long-term outcome. Peak-to-peak trans-coarctation pressure gradient (PKdP) higher than 20 mmHg is an indication for interventional/surgical repair. Patients with COA have reduced proximal and distal aortic compliances. A comprehensive study investigating the effects of variations of proximal COA and systemic compliances on PKdP, and consequently on the COA severity evaluation has never been done. This study evaluates the effect of aortic compliance on diagnostic accuracy of PKdP. Lumped parameter modeling and in vitro experiments were performed for COA severities of 50%, 75% and 90% by area. Modeling and in vitro results were validated against retrospective clinical data of PKdP, measured in 54 patients with COA. Modeling and in vitro. PKdP increases with reduced proximal COA compliance (+36%, +38% and +53% for COA severities of 50%, 75% and 90%, respectively; p<0.05), but decreases with reduced systemic compliance (−62%, −41% and −36% for COA severities of 50%, 75% and 90%, respectively; p<0.01). Clinical study. PKdP has a modest correlation with COA severity (R=0.29). The main determinants of PKdP are COA severity, stroke volume index and systemic compliance. Systemic compliance was found to be as influential as COA severity in PKdP determination (R=0.30 vs. R =0.34). In conclusion, PKdP is highly influenced by both stroke volume index and arterial compliance. Low values of PKdP cannot be used to exclude the severe COA presence since COA severity may be masked by reduced systemic compliance and/or low flow conditions.

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)

In 1953, Joseph Osborn examined the physiologic effects of hypothermia and defined typical associated changes in the electrocardiogram (ECG), now known as J waves of Osborn. There is a subtlety, however: Osborn's J waves were absent in hypothermic animals whose pH was maintained via mechanical ventilation. Osborn wrote: “We regard this as evidence that the ECG changes … may not be associated with the low temperature directly, but rather may be more closely associated with faulty elimination of CO2under hypothermic conditions” (1). This principle is illustrated in a 64-year-old man who presented hypothermic to 92°F and profoundly acidemic (pH 7.03) after cardiac arrest. Striking J waves are evident on initial ECG (A). Controlled cooling was initiated; hypothermia was maintained to preserve brain function. Intubation and resuscitation restored bicarbonate, carbon dioxide concentrations, and pH. At pH 7.33, although body temperature was identical at 92°F, the J waves had resolved (B).

Summary The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets with a protospacer adjacent mot if (PAM) and complementarity to the guide RNA. Recently, we harnessed Staphylococcus aureus Cas9 (SaCas9), which is significantly smaller than Streptococcus pyogenes Cas9 (SpCas9), to facilitate efficient in vivo genome editing. Here, we report the crystal structures of SaCas9 in complex with a single guide RNA (sgRNA) and its double-stranded DNA targets, containing the 5′-TTGAAT-3′ PAM and the 5′-TTGGGT-3′ PAM, at 2.6 and 2.7 Å resolutions, respectively. The structures revealed the mechanism of the relaxed recognition of the 5′-NNGRRT-3′ PAM by SaCas9. A structural comparison of SaCas9 with SpCas9 highlighted both structural conservation and divergence, explaining their distinct PAM specificities and orthologous sgRNA recognition. Finally, we applied the structural information about this minimal Cas9 to rationally design compact transcriptional activators and inducible nucleases, to further expand the CRISPR-Cas9 genome editing toolbox., National Institute of General Medical Sciences (U.S.) (Grant T32GM007753), National Institutes of Health (U.S.) (Award 1DP1-MH100706)

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)

Recently, Chromosome Conformation Capture (3C) based experiments have highlighted the importance of computational models for the study of chromosome organization. In this review, we propose that current computational models can be grouped into roughly four classes, with two classes of data-driven models: consensus structures and data-driven ensembles, and two classes of de novo models: structural ensembles and mechanistic ensembles. Finally, we highlight specific questions mechanistic ensembles can address., National Institutes of Health (U.S.) (Grant R01HG003143), National Institutes of Health (U.S.) (Grant R01 GM114190)

Physical and physiological comfort, at work and during leisure time, is important to human health and motivation. A growing number of jobs require workers to sit. Most clothes, except those intended for wheelchair users, were designed for walking or the standing position. Clothing designs should be user-oriented and meet users’ needs. Garment design should conform to body position and posture, not just shape and size. In this paper we present the ergometric impact of a new type of trousers designed to adapt to changes in position. Concentrations of compression forces, temperature and pressure were documented in an exploratory pilot study and contrasted to traditional designs. The new trousers showed significant decreases in compression force concentration, especially in and around the knees and waist. Most participants identified comfort as an important factor when purchasing a pair of trousers and that, for working purposes, they would prefer these special trousers rather than traditional designs.

Effective immunity requires a complex network of cellular and humoral components that interact with each other and are influenced by different environmental and host factors. We used a systems biology approach to comprehensively assess the impact of environmental and genetic factors on immune cell populations in peripheral blood, including associations with immunoglobulin concentrations, from ∼500 healthy volunteers from the Human Functional Genomics Project. Genetic heritability estimation showed that variations in T cell numbers are more strongly driven by genetic factors, while B cell counts are more environmentally influenced. Quantitative trait loci (QTL) mapping identified eight independent genomic loci associated with leukocyte count variation, including four associations with T and B cell subtypes. The QTLs identified were enriched among genome-wide association study (GWAS) SNPs reported to increase susceptibility to immune-mediated diseases. Our systems approach provides insights into cellular and humoral immune trait variability in humans.

Second language learning becomes increasingly difficult with age, but some adults learn more successfully than others. We examined whether inter-subject variability in the microstructure of white matter pathways, as measured by diffusion tensor imaging (DTI), would predict native English speakers' outcomes in learning Mandarin Chinese. Twenty-one adults were scanned before participating in an intensive 4-week Mandarin course. At the end of the Mandarin course, participants completed a final exam that assessed their skills in both spoken and written Mandarin. Individual participants' white-matter tracts were reconstructed from their native DTI data and related to final-exam performance. Superior language learning was correlated with DTI measures in the right hemisphere, but not in the left hemisphere. In particular, greater initial fractional anisotropy (FA) in both the right superior longitudinal fasciculus (parietal bundle) and the right inferior longitudinal fasciculus was associated with more successful Mandarin learning. The relation between white-matter structure in the right hemisphere of native English speakers and successful initial language learning may reflect the tonal and visuo-spatial properties, respectively, of spoken and written Mandarin Chinese.

Synthetic biology uses living cells as molecular foundries for the biosynthesis of drugs, therapeutic proteins, and other commodities. However, the need for specialized equipment and refrigeration for production and distribution poses a challenge for the delivery of these technologies to the field and to low-resource areas. Here, we present a portable platform that provides the means for on-site, on-demand manufacturing of therapeutics and biomolecules. This flexible system is based on reaction pellets composed of freeze-dried, cell-free transcription and translation machinery, which can be easily hydrated and utilized for biosynthesis through the addition of DNA encoding the desired output. We demonstrate this approach with the manufacture and functional validation of antimicrobial peptides and vaccines and present combinatorial methods for the production of antibody conjugates and small molecules. This synthetic biology platform resolves important practical limitations in the production and distribution of therapeutics and molecular tools, both to the developed and developing world., Defense Threat Reduction Agency (DTRA) (Grant HDTRA1-14-1-0006)

Transcatheter ablation of renal autonomic nerves is a viable option for the treatment of resistant arterial hypertension; however, structured pre-clinical evaluation with standardization of analytical procedures remains a clear gap in this field. Here we discuss the topics relevant to the pre-clinical model for the evaluation of renal denervation (RDN) devices and report methodologies and criteria toward standardization of the safety and efficacy assessment, including histopathological evaluations of the renal artery, periarterial nerves, and associated periadventitial tissues. The pre-clinical swine renal artery model can be used effectively to assess both the safety and efficacy of RDN technologies. Assessment of the efficacy of RDN modalities primarily focuses on the determination of the depth of penetration of treatment-related injury (e.g., necrosis) of the periarterial tissues and its relationship (i.e., location and distance) and the effect on the associated renal nerves and the correlation thereof with proxy biomarkers including renal norepinephrine concentrations and nerve-specific immunohistochemical stains (e.g., tyrosine hydroxylase). The safety evaluation of RDN technologies involves assessing for adverse effects on tissues local to the site of treatment (i.e., on the arterial wall) as well as tissues at a distance (e.g., soft tissue, veins, arterial branches, skeletal muscle, adrenal gland, ureters). Increasing experience will help to create a standardized means of examining all arterial beds subject to ablative energy and in doing so enable us to proceed to optimize the development and assessment of these emerging technologies.

Finding the components of cellular circuits and determining their functions systematically remains a major challenge in mammalian cells. Here, we introduced genome-wide pooled CRISPR-Cas9 libraries into dendritic cells (DCs) to identify genes that control the induction of tumor necrosis factor (Tnf) by bacterial lipopolysaccharide (LPS), a key process in the host response to pathogens, mediated by the Tlr4 pathway. We found many of the known regulators of Tlr4 signaling, as well as dozens of previously unknown candidates that we validated. By measuring protein markers and mRNA profiles in DCs that are deficient in known or candidate genes, we classified the genes into three functional modules with distinct effects on the canonical responses to LPS and highlighted functions for the PAF complex and oligosaccharyltransferase (OST) complex. Our findings uncover new facets of innate immune circuits in primary cells and provide a genetic approach for dissection of mammalian cell circuits., Broad Institute, National Institutes of Health (U.S.) (NIMH: 5DP1-MH100706), National Institutes of Health (U.S.) (NIDDK: 5R01-DK097768), National Science Foundation (U.S.) (Waterman Award), W. M. Keck Foundation, New York Stem Cell Foundation, Damon Runyon Cancer Research Foundation, Searle Scholars Program, Vallee Foundation, Robert Metcalfe, Massachusetts Institute of Technology. Simons Center for the Social Brain, National Human Genome Research Institute (U.S.) (K99- HG008171), National Science Foundation (U.S.). Graduate Research Fellowship Program (grant number 1122374), Human Frontier Science Program (Strasbourg, France)

Combining electroencephalogram (EEG) recording and functional magnetic resonance imaging (fMRI) offers the potential for imaging brain activity with high spatial and temporal resolution. This potential remains limited by the significant ballistocardiogram (BCG) artifacts induced in the EEG by cardiac pulsation-related head movement within the magnetic field. We model the BCG artifact using a harmonic basis, pose the artifact removal problem as a local harmonic regression analysis, and develop an efficient maximum likelihood algorithm to estimate and remove BCG artifacts. Our analysis paradigm accounts for time-frequency overlap between the BCG artifacts and neurophysiologic EEG signals, and tracks the spatiotemporal variations in both the artifact and the signal. We evaluate performance on: simulated oscillatory and evoked responses constructed with realistic artifacts; actual anesthesia-induced oscillatory recordings; and actual visual evoked potential recordings. In each case, the local harmonic regression analysis effectively removes the BCG artifacts, and recovers the neurophysiologic EEG signals. We further show that our algorithm outperforms commonly used reference-based and component analysis techniques, particularly in low SNR conditions, the presence of significant time-frequency overlap between the artifact and the signal, and/or large spatiotemporal variations in the BCG. Because our algorithm does not require reference signals and has low computational complexity, it offers a practical tool for removing BCG artifacts from EEG data recorded in combination with fMRI., National Institutes of Health (U.S.) (Award DP1-OD003646), National Institutes of Health (U.S.) (Award TR01-GM104948), National Institutes of Health (U.S.) (Grant R44NS071988), National Institute of Neurological Diseases and Stroke (U.S.) (Grant Grant R44NS071988)

CRISPR-Cas9 is a versatile genome editing technology for studying the functions of genetic elements. To broadly enable the application of Cas9 in vivo, we established a Cre-dependent Cas9 knockin mouse. We demonstrated in vivo as well as ex vivo genome editing using adeno-associated virus (AAV)-, lentivirus-, or particle-mediated delivery of guide RNA in neurons, immune cells, and endothelial cells. Using these mice, we simultaneously modeled the dynamics of KRAS, p53, and LKB1, the top three significantly mutated genes in lung adenocarcinoma. Delivery of a single AAV vector in the lung generated loss-of-function mutations in p53 and Lkb1, as well as homology-directed repair-mediated Kras[superscript G12D] mutations, leading to macroscopic tumors of adenocarcinoma pathology. Together, these results suggest that Cas9 mice empower a wide range of biological and disease modeling applications., National Science Foundation (U.S.). Graduate Research Fellowship (Grant 1122374), Damon Runyon Cancer Research Foundation (Fellowship DRG-2117-12), Massachusetts Institute of Technology. Simons Center for the Social Brain (Postdoctoral Fellowship), European Molecular Biology Organization (Fellowship), Foundation for Polish Science (Fellowship), American Society for Engineering Education. National Defense Science and Engineering Graduate Fellowship, National Science Foundation (U.S.). Graduate Research Fellowship, Massachusetts Institute of Technology (Presidential Graduate Fellowship), Human Frontier Science Program (Strasbourg, France) (Postdoctoral Fellowship), National Human Genome Research Institute (U.S.) (CEGS P50 HG006193), Howard Hughes Medical Institute, Klarman Cell Observatory, National Cancer Institute (U.S.) (Center of Cancer Nanotechnology Excellence Grant U54CA151884), National Institutes of Health (U.S.) (Controlled Release Grant EB000244), National Heart, Lung, and Blood Institute (Program of Excellence in Nanotechnology (PEN) Award Contract HHSN268201000045C), Massachusetts Institute of Technology (Poitras Gift 1631119), Stanley Center, Simons Foundation (6927482), Nancy Lurie Marks Family Foundation (6928117), United States. Public Health Service (National Institutes of Health (U.S.) R01-CA133404), David H. Koch Institute for Integrative Cancer Research at MIT (Marie D. and Pierre Casimir-Lambert Fund), MIT Skoltech Initiative, National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051), National Institute of Mental Health (U.S.) (Director’s Pioneer Award DP1-MH100706), National Institute of Neurological Disorders and Stroke (U.S.) (Transformative R01 Grant R01-NS 07312401), National Science Foundation (U.S.) (Waterman Award), W. M. Keck Foundation, Kinship Foundation. Searle Scholars Program, Klingenstein Foundation, Vallee Foundation, Merkin Foundation

Dengue virus currently causes 50–100 million infections annually. Comprehensive knowledge about the evolution of Dengue in response to selection pressure is currently unavailable, but would greatly enhance vaccine design efforts. In the current study, we sequenced 187 new dengue virus serotype 3 (DENV-3) genotype III whole genomes isolated from Asia and the Americas. We analyzed them together with previously-sequenced isolates to gain a more detailed understanding of the evolutionary adaptations existing in this prevalent American serotype. In order to analyze the phylogenetic dynamics of DENV-3 during outbreak periods; we incorporated datasets of 48 and 11 sequences spanning two major outbreaks in Venezuela during 2001 and 2007–2008, respectively. Our phylogenetic analysis of newly sequenced viruses shows that subsets of genomes cluster primarily by geographic location, and secondarily by time of virus isolation. DENV-3 genotype III sequences from Asia are significantly divergent from those from the Americas due to their geographical separation and subsequent speciation. We measured amino acid variation for the E protein by calculating the Shannon entropy at each position between Asian and American genomes. We found a cluster of seven amino acid substitutions having high variability within E protein domain III, which has previously been implicated in serotype-specific neutralization escape mutants. No novel mutations were found in the E protein of sequences isolated during either Venezuelan outbreak. Shannon entropy analysis of the NS5 polymerase mature protein revealed that a G374E mutation, in a region that contributes to interferon resistance in other flaviviruses by interfering with JAK-STAT signaling was present in both the Asian and American sequences from the 2007–2008 Venezuelan outbreak, but was absent in the sequences from the 2001 Venezuelan outbreak. In addition to E, several NS5 amino acid changes were unique to the 2007–2008 epidemic in Venezuela and

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)

In the thymus, high-affinity, self-reactive thymocytes are eliminated from the pool of developing T cells, generating central tolerance. Here, we investigate how developing T cells measure self-antigen affinity. We show that very few CD4 or CD8 coreceptor molecules are coupled with the signal-initiating kinase, Lck. To initiate signaling, an antigen-engaged T cell receptor (TCR) scans multiple coreceptor molecules to find one that is coupled to Lck; this is the first and rate-limiting step in a kinetic proofreading chain of events that eventually leads to TCR triggering and negative selection. MHCII-restricted TCRs require a shorter antigen dwell time (0.2 s) to initiate negative selection compared to MHCI-restricted TCRs (0.9 s) because more CD4 coreceptors are Lck-loaded compared to CD8. We generated a model (Lck come&stay/signal duration) that accurately predicts the observed differences in antigen dwell-time thresholds used by MHCI- and MHCII-restricted thymocytes to initiate negative selection and generate self-tolerance., National Institutes of Health (U.S.) (Grant 1-P01-AI091580)

Background Previous studies have demonstrated that microvolt T-wave alternans (MTWA) testing is a robust predictor of ventricular tachyarrhythmias and sudden cardiac death (SCD) in at-risk patients. However, recent studies have suggested that MTWA testing is not as good a predictor of “appropriate” implantable cardioverter-defibrillator (ICD) therapy as it is a predictor of SCD in patients without ICDs. Objective To evaluate the utility of MTWA testing for SCD risk stratification in patients without ICDs. Methods Patient-level data were obtained from 5 prospective studies of MTWA testing in patients with no history of ventricular arrhythmia or SCD. In these studies, ICDs were implanted in only a minority of patients and patients with ICDs were excluded from the analysis. We conducted a pooled analysis and examined the 2-year risk for SCD based on the MTWA test result. Results The pooled cohort included 2883 patients. MTWA testing was positive in 856 (30%), negative in 1627 (56%), and indeterminate in 400 (14%) patients. Among patients with a left ventricular ejection fraction (LVEF) of ≤35%, annual SCD event rates were 4.0%, 0.9%, and 4.6% among groups with MTWA positive, negative, and indeterminate test results. The SCD rate was significantly lower among patients with a negative MTWA test result than in patients with either positive or indeterminate MTWA test results (P <.001 for both comparisons). In patients with an LVEF of >35%, annual SCD event rates were 3.0%, 0.3%, and 0.3% among the groups with MTWA positive, negative, and indeterminate test results. The SCD rate associated with a positive MTWA test result was significantly higher than that associated with either negative (P <.001) or indeterminate MTWA test results (P = .003). Conclusions In patients without ICDs, MTWA testing is a powerful predictor of SCD. Among patients with an LVEF of ≤35%, a negative MTWA test result is associated with a low risk for SCD. Conversely, among patients with an LVEF of >35%, National Institute on Aging (Grant 1R21AG035128), National Institutes of Health (U.S.) (Grant 1RO1HL103961), Center for Integration of Medicine and Innovative Technology

siRNA delivery targeting tumor cells and cancer-associated immune cells has been gaining momentum in the last few years. A combinatorial approach for silencing crucial factors essential for tumor progression in cancer-associated immune cells and in cancer cells simultaneously can effectively shift the tumor microenvironment from pro-oncogenic to anti-tumoral. Gene-therapy using RNAi nanomaterials can help shift this balance; however, fully utilizing the potential of RNAi relies on effective and specific delivery. RNAi nanomaterials can act as a Trojan horse which delivers siRNAs against immunosuppressive factors and reverses the regulatory activity of tumor immune cells residing in the tumor microenvironment. Here we review potential RNAi targets, means to activate and control the immune response, as well as ways to design delivery nanovehicles for successful RNAi immunotherapy.

The double-deficit hypothesis of dyslexia posits that both rapid naming and phonological impairments can cause reading difficulties, and that individuals who have both of these deficits show greater reading impairments compared to those with a single deficit. Despite extensive behavioral research, the brain basis of poor reading with a double-deficit has never been investigated. The goal of the study was to evaluate the double-deficit hypothesis using functional MRI. Activation patterns during a printed word rhyme judgment task in 90 children with a wide range of reading abilities showed dissociation between brain regions that were sensitive to phonological awareness (left inferior frontal and inferior parietal regions) and rapid naming (right cerebellar lobule VI). More specifically, the double-deficit group showed less activation in the fronto-parietal reading network compared to children with only a deficit in phonological awareness, who in turn showed less activation than the typically-reading group. On the other hand, the double-deficit group showed less cerebellar activation compared to children with only a rapid naming deficit, who in turn showed less activation than the typically-reading children. Functional connectivity analyses revealed that bilateral prefrontal regions were key for linking brain regions associated with phonological awareness and rapid naming, with the double-deficit group being the most aberrant in their connectivity. Our study provides the first functional neuroanatomical evidence for the double-deficit hypothesis of developmental dyslexia., William & Flora Hewlett Foundation, Richard King Mellon Foundation, Ellison Medical Foundation, Massachusetts Institute of Technology Class of 1976 Funds for Dyslexia Research, Martin Richmond Memorial Fund

Objectives: The aim of this study was to evaluate the impact of intravascular ultrasound (IVUS) guidance on the final volume of contrast agent used in patients undergoing percutaneous coronary intervention (PCI). Background: To date, few approaches have been described to reduce the final dose of contrast agent in PCIs. We hypothesized that IVUS might serve as an alternative imaging tool to angiography in many steps during PCI, thereby reducing the use of iodine contrast. Methods: A total of 83 patients were randomized to angiography-guided PCI or IVUS-guided PCI; both groups were treated according to a pre-defined meticulous procedural strategy. The primary endpoint was the total volume contrast agent used during PCI. Patients were followed clinically for an average of 4 months. Results: The median total volume of contrast was 64.5 ml (interquartile range [IQR]: 42.8 to 97.0 ml; minimum, 19 ml; maximum, 170 ml) in the angiography-guided group versus 20.0 ml (IQR: 12.5 to 30.0 ml; minimum, 3 ml; maximum, 54 ml) in the IVUS-guided group (p < 0.001). Similarly, the median volume of contrast/creatinine clearance ratio was significantly lower among patients treated with IVUS-guided PCI (1.0 [IQR: 0.6 to 1.9] vs. 0.4 [IQR: 0.2 to 0.6, respectively; p < 0.001). In-hospital and 4-month outcomes were not different between patients randomized to angiography-guided and IVUS-guided PCI. Conclusions: Thoughtful and extensive use of IVUS as the primary imaging tool to guide PCI is safe and markedly reduces the volume of iodine contrast compared with angiography-alone guidance. The use of IVUS should be considered for patients at high risk of contrast-induced acute kidney injury or volume overload undergoing coronary angioplasty., National Institutes of Health (U.S.) (NIH grant R01 GM49039), Conselho Nacional de Pesquisas (Brazil) (Grant), Sociedade Brasileira de Cardiologia (SBC) (Arie Fellowship)

Magnetoencephalography (MEG) is an important non-invasive method for studying activity within the human brain. Source localization methods can be used to estimate spatiotemporal activity from MEG measurements with high temporal resolution, but the spatial resolution of these estimates is poor due to the ill-posed nature of the MEG inverse problem. Recent developments in source localization methodology have emphasized temporal as well as spatial constraints to improve source localization accuracy, but these methods can be computationally intense. Solutions emphasizing spatial sparsity hold tremendous promise, since the underlying neurophysiological processes generating MEG signals are often sparse in nature, whether in the form of focal sources, or distributed sources representing large-scale functional networks. Recent developments in the theory of compressed sensing (CS) provide a rigorous framework to estimate signals with sparse structure. In particular, a class of CS algorithms referred to as greedy pursuit algorithms can provide both high recovery accuracy and low computational complexity. Greedy pursuit algorithms are difficult to apply directly to the MEG inverse problem because of the high-dimensional structure of the MEG source space and the high spatial correlation in MEG measurements. In this paper, we develop a novel greedy pursuit algorithm for sparse MEG source localization that overcomes these fundamental problems. This algorithm, which we refer to as the Subspace Pursuit-based Iterative Greedy Hierarchical (SPIGH) inverse solution, exhibits very low computational complexity while achieving very high localization accuracy. We evaluate the performance of the proposed algorithm using comprehensive simulations, as well as the analysis of human MEG data during spontaneous brain activity and somatosensory stimuli. These studies reveal substantial performance gains provided by the SPIGH algorithm in terms of computational complexity, localization accuracy, National Institutes of Health (U.S.) (New Innovator Award DP2-OD006454), National Institutes of Health (U.S.) (R01-EB006385-01), National Institutes of Health (U.S.) (P41-RR014075-11)

Encounters between immune cells and invading bacteria ultimately determine the course of infection. These interactions are usually measured in populations of cells, masking cell-to-cell variation that may be important for infection outcome. To characterize the gene expression variation that underlies distinct infection outcomes and monitor infection phenotypes, we developed an experimental system that combines single-cell RNA-seq with fluorescent markers. Probing the responses of individual macrophages to invading Salmonella, we find that variation between individual infected host cells is determined by the heterogeneous activity of bacterial factors in individual infecting bacteria. We illustrate how variable PhoPQ activity in the population of invading bacteria drives variable host type I IFN responses by modifying LPS in a subset of bacteria. This work demonstrates a causative link between host and bacterial variability, with cell-to-cell variation between different bacteria being sufficient to drive radically different host immune responses. This co-variation has implications for host-pathogen dynamics in vivo., National Institutes of Health (U.S.) (grant HG002295), National Institutes of Health (U.S.) (grant DK043351), National Institutes of Health (U.S.) (grant NIH U19AI109725), National Institutes of Health (U.S.) (grant NIH F32 HD075541-02), National Human Genome Research Institute (U.S.) ((CEGS) Center of Cell Circuits (P50 HG006193)), Klarman Cell Observatory

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), National Institutes of Health (U.S.) (NIH Grant No. AR063745)

Extensive cellular heterogeneity exists within specific immune-cell subtypes classified as a single lineage, but its molecular underpinnings are rarely characterized at a genomic scale. Here, we use single-cell RNA-seq to investigate the molecular mechanisms governing heterogeneity and pathogenicity of Th17 cells isolated from the central nervous system (CNS) and lymph nodes (LN) at the peak of autoimmune encephalomyelitis (EAE) or differentiated in vitro under either pathogenic or non-pathogenic polarization conditions. Computational analysis relates a spectrum of cellular states in vivo to in vitro differentiated Th17 cells, and unveils genes governing pathogenicity and disease susceptibility. Using knockout mice, we validate four new genes: Gpr65, Plzp, Toso and Cd5l (in a companion paper). Cellular heterogeneity thus informs Th17 function in autoimmunity, and can identify targets for selective suppression of pathogenic Th17 cells while potentially sparing non-pathogenic tissue-protective ones, National Institutes of Health (U.S.) (Grant P50 HG006193), National Cancer Institute (U.S.) (David H. Koch Institute for Integrative Cancer Research at MIT. Grant P30-CA14051), Klarman Cell Observatory

Coronary artery disease (CAD) is a major determinant of the long-term prognosis among patients with diabetes mellitus (DM). DM is associated with a 2 to 4-fold increased mortality risk from heart disease. Furthermore, in patients with DM there is an increased mortality after MI, and worse overall prognosis with CAD. Near-normal glycemic control for a median of 3.5 to 5 years does not reduce cardiovascular events. Thus, the general goal of HbA1c <7% appears reasonable for the majority of patients. Iatrogenic hypoglycemia is the limiting factor in the glycemic management of diabetes, and is an independent cause of excess morbidity and mortality. Statins are effective in reducing major coronary events, stroke, and the need for coronary revascularization.

Soft tissue adhesives are employed to repair and seal many different organs, which range in both tissue surface chemistry and mechanical challenges during organ function. This complexity motivates the development of tunable adhesive materials with high resistance to uniaxial or multiaxial loads dictated by a specific organ environment. Co-polymeric hydrogels comprising aminated star polyethylene glycol and dextran aldehyde (PEG:dextran) are materials exhibiting physico-chemical properties that can be modified to achieve this organ- and tissue-specific adhesion performance. Here we report that resistance to failure under specific loading conditions, as well as tissue response at the adhesive material–tissue interface, can be modulated through regulation of the number and density of adhesive aldehyde groups. We find that atomic force microscopy (AFM) can characterize the material aldehyde density available for tissue interaction, and in this way enable rapid, informed material choice. Further, the correlation between AFM quantification of nanoscale unbinding forces with macroscale measurements of adhesion strength by uniaxial tension or multiaxial burst pressure allows the design of materials with specific cohesion and adhesion strengths. However, failure strength alone does not predict optimal in vivo reactivity. Thus, we demonstrate that the development of adhesive materials is significantly enabled when experiments are integrated along length scales to consider organ chemistry and mechanical loading states concurrently with adhesive material properties and tissue response., National Science Foundation (U.S.) (Career Award), American Society for Engineering Education. National Defense Science and Engineering Graduate Fellowship, National Institutes of Health (U.S.) (Grant ERE GM 49039)

We propose that complex (nonlinear) fluctuations of hemodynamic variables including systemic blood pressure parameters) during cardiovascular surgery contain information relevant to risk assessment and intraoperative management. Preliminary analysis of a pilot study supports the feasibility and potential merits of performing a larger, prospective study to assess the clinical utility of such new dynamical measures and to evaluate their potential role in enhancing contemporary approaches to risk assessment of major adverse events., National Institutes of Health (U.S.) (Grant R01 EB0001659)

Tumor-propagating cells in acute leukemia maintain a stem/progenitor-like immature phenotype and proliferative capacity. Acute myeloid leukemia (AML) and acute T-lymphoblastic leukemia (T-ALL) originate from different lineages through distinct oncogenic events such as MLL fusions and Notch signaling, respectively. We found that Zfx, a transcription factor that controls hematopoietic stem cell self-renewal, controls the initiation and maintenance of AML caused by MLL-AF9 fusion and of T-ALL caused by Notch1 activation. In both leukemia types, Zfx prevents differentiation and activates gene sets characteristic of immature cells of the respective lineages. In addition, endogenous Zfx contributes to gene induction and transformation by Myc overexpression in myeloid progenitors. Key Zfx target genes include the mitochondrial enzymes Ptpmt1 and Idh2, whose overexpression partially rescues the propagation of Zfx-deficient AML. These results show that distinct leukemia types maintain their undifferentiated phenotype and self-renewal by exploiting a common stem-cell-related genetic regulator.

Activation of Ras proteins underlies functional decisions in diverse cell types. Two molecules, RasGRP and SOS, catalyze Ras activation in lymphocytes. Binding of active Ras to SOS' allosteric pocket markedly increases SOS' activity establishing a positive feedback loop for SOS-mediated Ras activation. Integrating in silico and in vitro studies, we demonstrate that digital signaling in lymphocytes (cells are “on” or “off”) is predicated upon feedback regulation of SOS. SOS' feedback loop leads to hysteresis in the dose-response curve, which can enable a capacity to sustain Ras activation as stimuli are withdrawn and exhibit “memory” of past encounters with antigen. Ras activation via RasGRP alone is analog (graded increase in amplitude with stimulus). We describe how complementary analog (RasGRP) and digital (SOS) pathways act on Ras to efficiently convert analog input to digital output. Numerous predictions regarding the impact of our findings on lymphocyte function and development are noted., National Institutes of Health (U.S.). Pioneer Award, National Institutes of Health (U.S.) (1PO1/AI071195/01)