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Combatting infectious disease is a critical global health concern and involves tackling both emerging infectious agents and newly–drug resistant strains of previously curable pathogens. The widespread and inappropriate use of antimicrobial agents has increased the frequency of resistance among human pathogens, including bacteria, fungi, and protozoan parasites, and threatened to undermine the efficacy of all existing antimicrobial drugs. Whereas lethal doses of antimicrobials may select for preexisting resistant microbes, there is increasing interest in uncovering the cellular consequences of sublethal antimicrobial exposure on the development of antimicrobial resistance. There are numerous circumstances under which microbial organisms are exposed to low doses of antimicrobials, including in patients, in livestock animals, and in the environment. Sublethal antimicrobial exposure can trigger DNA damage and genomic instability across the diversity of microbial pathogens, including bacterial and fungal species., Banting Postdoctoral Fellowship

Gold nanobeacons can be used as a powerful tool for cancer theranostics. Here, we proposed a nanomaterial platform based on gold nanobeacons to detect, target and inhibit the expression of a mutant Kras gene in an in vivo murine gastric cancer model. The conjugation of fluorescently-labeled antisense DNA hairpin oligonucleotides to the surface of gold nanoparticles enables using their localized surface plasmon resonance properties to directly track the delivery to the primary gastric tumor and to lung metastatic sites. The fluorescently labeled nanobeacons reports on the interaction with the target as the fluorescent Cy3 signal is quenched by the gold nanoparticle and only emit light following conjugation to the Kras target owing to reorganization and opening of the nanobeacons, thus increasing the distance between the dye and the quencher. The systemic administration of the anti-Kras nanobeacons resulted in approximately 60% tumor size reduction and a 90% reduction in tumor vascularization. More important, the inhibition of the Kras gene expression in gastric tumors prevents the occurrence of metastasis to lung (80% reduction), increasing mice survival in more than 85%. Our developed platform can be easily adjusted to hybridize with any specific target and provide facile diagnosis and treatment for neoplastic diseases., Science Foundation Ireland (Grant 11/PI/08), National Key Basic Research Program of China (973 Program) (2011CB933101), National Key Basic Research Program of China (973 Program) (2015CB931802), National Natural Scientific Fund (China) (81225010), 863 Project of China (2012AA022703), 863 Project of China (2014AA020700), Shanghai Science and Technology Fund (13NM1401500), Marie Curie International Fellowship (FP7-PEOPLE-2013-IOF, Project 626386)

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.

An innovative, microwell-based platform for single-cell RNA sequencing (RNA-seq) combines cost efficiency, scalability and parallelizability, and will enable many new avenues of biological inquiry.

The enormous genetic diversity and mutability of HIV has prevented effective control of this virus by natural immune responses or vaccination. Evolution of the circulating HIV population has thus occurred in response to diverse, ultimately ineffective, immune selection pressures that randomly change from host to host. We show that the interplay between the diversity of human immune responses and the ways that HIV mutates to evade them results in distinct sets of sequences defined by similar collectively coupled mutations. Scaling laws that relate these sets of sequences resemble those observed in linguistics and other branches of inquiry, and dynamics reminiscent of neural networks are observed. Like neural networks that store memories of past stimulation, the circulating HIV population stores memories of host–pathogen combat won by the virus. We describe an exactly solvable model that captures the main qualitative features of the sets of sequences and a simple mechanistic model for the origin of the observed scaling laws. Our results define collective mutational pathways used by HIV to evade human immune responses, which could guide vaccine design., Ragon Institute of MGH, MIT and Harvard

Does the affinity or half-life of peptide-MHC-T cell receptor (TCR) interactions determine T cell activation? In this issue of Immunity, Aleksic et al. (2010) propose a role for the on rate through multiple rebindings to the same TCR.

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

Cancer progression is an example of a rapid adaptive process where evolving new traits is essential for survival and requires a high mutation rate. Precancerous cells acquire a few key mutations that drive rapid population growth and carcinogenesis. Cancer genomics demonstrates that these few driver mutations occur alongside thousands of random passenger mutations—a natural consequence of cancer’s elevated mutation rate. Some passengers are deleterious to cancer cells, yet have been largely ignored in cancer research. In population genetics, however, the accumulation of mildly deleterious mutations has been shown to cause population meltdown. Here we develop a stochastic population model where beneficial drivers engage in a tug-of-war with frequent mildly deleterious passengers. These passengers present a barrier to cancer progression describable by a critical population size, below which most lesions fail to progress, and a critical mutation rate, above which cancers melt down. We find support for this model in cancer age–incidence and cancer genomics data that also allow us to estimate the fitness advantage of drivers and fitness costs of passengers. We identify two regimes of adaptive evolutionary dynamics and use these regimes to understand successes and failures of different treatment strategies. A tumor’s load of deleterious passengers can explain previously paradoxical treatment outcomes and suggest that it could potentially serve as a biomarker of response to mutagenic therapies. The collective deleterious effect of passengers is currently an unexploited therapeutic target. We discuss how their effects might be exacerbated by current and future therapies., National Cancer Institute (U.S.) (Grant U54CA143874), MIT Department of Physics Pappalardo Program

The restriction-modification systems use epigenetic modification to distinguish between self and nonself DNA. A modification enzyme transfers a methyl group to a base in a specific DNA sequence while its cognate restriction enzyme introduces breaks in DNA lacking this methyl group. So far, all the restriction enzymes hydrolyze phosphodiester bonds linking the monomer units of DNA. We recently reported that a restriction enzyme (R.PabI) of the PabI superfamily with half-pipe fold has DNA glycosylase activity that excises an adenine base in the recognition sequence (5′-GTAC). We now found a second activity in this enzyme: at the resulting apurinic/apyrimidinic (AP) (abasic) site (5′-GT#C, # = AP), its AP lyase activity generates an atypical strand break. Although the lyase activity is weak and lacks sequence specificity, its covalent DNA–R.PabI reaction intermediates can be trapped by NaBH[subscript 4] reduction. The base excision is not coupled with the strand breakage and yet causes restriction because the restriction enzyme action can impair transformation ability of unmethylated DNA even in the absence of strand breaks in vitro. The base excision of R.PabI is inhibited by methylation of the target adenine base. These findings expand our understanding of genetic and epigenetic processes linking those in prokaryotes and eukaryotes.

Classical nonparametric spectral analysis uses sliding windows to capture the dynamic nature of most real-world time series. This universally accepted approach fails to exploit the temporal continuity in the data and is not well-suited for signals with highly structured time–frequency representations. For a time series whose time-varying mean is the superposition of a small number of oscillatory components, we formulate nonparametric batch spectral analysis as a Bayesian estimation problem. We introduce prior distributions on the time–frequency plane that yield maximum a posteriori (MAP) spectral estimates that are continuous in time yet sparse in frequency. Our spectral decomposition procedure, termed spectrotemporal pursuit, can be efficiently computed using an iteratively reweighted least-squares algorithm and scales well with typical data lengths. We show that spectrotemporal pursuit works by applying to the time series a set of data-derived filters. Using a link between Gaussian mixture models, ℓ[subscript 1] minimization, and the expectation–maximization algorithm, we prove that spectrotemporal pursuit converges to the global MAP estimate. We illustrate our technique on simulated and real human EEG data as well as on human neural spiking activity recorded during loss of consciousness induced by the anesthetic propofol. For the EEG data, our technique yields significantly denoised spectral estimates that have significantly higher time and frequency resolution than multitaper spectral estimates. For the neural spiking data, we obtain a new spectral representation of neuronal firing rates. Spectrotemporal pursuit offers a robust spectral decomposition framework that is a principled alternative to existing methods for decomposing time series into a small number of smooth oscillatory components., National Institutes of Health (U.S.) (Transformative Research Award GM 104948), National Institutes of Health (U.S.) (New Innovator Award R01-EB006385)

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)

During low arousal states such as drowsiness and sleep, cortical neurons exhibit rhythmic slow wave activity associated with periods of neuronal silence. Slow waves are locally regulated, and local slow wave dynamics are important for memory, cognition, and behaviour. While several brainstem structures for controlling global sleep states have now been well characterized, a mechanism underlying fast and local modulation of cortical slow waves has not been identified. Here, using optogenetics and whole cortex electrophysiology, we show that local tonic activation of thalamic reticular nucleus (TRN) rapidly induces slow wave activity in a spatially restricted region of cortex. These slow waves resemble those seen in sleep, as cortical units undergo periods of silence phase-locked to the slow wave. Furthermore, animals exhibit behavioural changes consistent with a decrease in arousal state during TRN stimulation. We conclude that TRN can induce rapid modulation of local cortical state., National Institutes of Health (U.S.) (TR01 GM104948), Canadian Institutes of Health Research (Fellowship), Harvard University. Society of Fellows (Fellowship)

Rapid eye movement (REM) sleep is an important component of the natural sleep/wake cycle, yet the mechanisms that regulate REM sleep remain incompletely understood. Cholinergic neurons in the mesopontine tegmentum have been implicated in REM sleep regulation, but lesions of this area have had varying effects on REM sleep. Therefore, this study aimed to clarify the role of cholinergic neurons in the pedunculopontine tegmentum (PPT) and laterodorsal tegmentum (LDT) in REM sleep generation. Selective optogenetic activation of cholinergic neurons in the PPT or LDT during non-REM (NREM) sleep increased the number of REM sleep episodes and did not change REM sleep episode duration. Activation of cholinergic neurons in the PPT or LDT during NREM sleep was sufficient to induce REM sleep., National Institutes of Health (U.S.) (Grant DP1-OD003646), National Institutes of Health (U.S.) (Grant TR01-GM104948), National Institutes of Health (U.S.) (Grant T32-HL07901), Massachusetts General Hospital (Executive Committee on Research Fellowship), Massachusetts General Hospital. Dept. of Anesthesia, Critical Care, and Pain Medicine

Single-cell transcriptomics reveals gene expression heterogeneity but suffers from stochastic dropout and characteristic bimodal expression distributions in which expression is either strongly non-zero or non-detectable. We propose a two-part, generalized linear model for such bimodal data that parameterizes both of these features. We argue that the cellular detection rate, the fraction of genes expressed in a cell, should be adjusted for as a source of nuisance variation. Our model provides gene set enrichment analysis tailored to single-cell data. It provides insights into how networks of co-expressed genes evolve across an experimental treatment. MAST is available at https://github.com/RGLab/MAST.

To study the mechanisms of death following a single lethal dose of thoracic radiation, WAG/RijCmcr (Wistar) rats were treated with 15 Gy to the whole thorax and followed until they were morbid or sacrificed for invasive assays at 6 weeks. Lung function was assessed by breathing rate and arterial oxygen saturation. Lung structure was evaluated histologically. Cardiac structure and function were examined by echocardiography. The frequency and characteristics of pleural effusions were determined. Morbidity from 15 Gy radiation occurred in all rats 5 to 8 weeks after exposure, coincident with histological pneumonitis. Increases in breathing frequencies peaked at 6 weeks, when profound arterial hypoxia was also recorded. Echocardiography analysis at 6 weeks showed pulmonary hypertension and severe right ventricular enlargement with impaired left ventricular function and cardiac output. Histologic sections of the heart revealed only rare foci of lymphocytic infiltration. Total lung weight more than doubled. Pleural effusions were present in the majority of the irradiated rats and contained elevated protein, but low lactate dehydrogenase, when compared with serum from the same animal. Pleural effusions had a higher percentage of macrophages and large monocytes than neutrophils and contained mast cells that are rarely present in other pathological states. Lethal irradiation to rat lungs leads to hypoxia with infiltration of immune cells, edema and pleural effusion. These changes may contribute to pulmonary vascular and parenchymal injury that result in secondary changes in heart structure and function. We report that conditions resembling congestive heart failure contribute to death during radiation pneumonitis, which indicates new targets for therapy.

In addition to an innate immune system that battles pathogens in a nonspecific fashion, higher organisms, such as humans, possess an adaptive immune system to combat diverse (and evolving) microbial pathogens. Remarkably, the adaptive immune system mounts pathogen-specific responses, which can be recalled upon reinfection with the same pathogen. It is difficult to see how the adaptive immune system can be preprogrammed to respond specifically to a vast and unknown set of pathogens. Although major advances have been made in understanding pertinent molecular and cellular phenomena, the precise principles that govern many aspects of an immune response are largely unknown. We discuss complementary approaches from statistical mechanics and cell biology that can shed light on how key components of the adaptive immune system, T cells, develop to enable pathogen-specific responses against many diverse pathogens. The mechanistic understanding that emerges has implications for how host genetics may influence the development of T cells with differing responses to the human immunodeficiency virus (HIV) infection., Ragon Institute of MGH, MIT and Harvard, National Institutes of Health (U.S.) (Grant 1PO1-AI071195-01), National Science Foundation (U.S.) (Grant DMR-12-06323), National Institutes of Health (U.S.) (Director's Pioneer Award)

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. We generated model xenograft tumors in mice by subcutaneous injection of a human ovarian cancer cell line, OVCAR-8 (NCI DCTD Tumor Repository, Frederick, MD). We transformed attenuated strains of S. typhimurium bacteria (ELH430:SL1344 phoPQ- 1) with a constitutively expressed luciferase (luxCDABE) plasmid for visualization2. These strains specifically colonize tumors while remaining essentially non-virulent to the mouse1. Once measurable tumors were established, bacteria were injected intravenously via the tail vein with varying dosage. Tumor-localized, bacterial gene expression was monitored in real time over the course of 60 hours using an in vivo imaging system (IVIS). At each time point, tumors were excised, homogenized, and plated to quantitate bacterial colonies for correlation with gene expression data. Together, this data yields a quantitative measure of the in vivo growth and gene expression dynamics of bacteria growing inside tumors., David H. Koch Institute for Integrative Cancer Research at MIT (Misrock Postdoctoral fellowship), Howard Hughes Medical Institute (Investigator), United States. Dept. of Defense (National Defense Science and Engineering Graduate Fellowships (NDSEG))

Chromosomes must be highly compacted and organized within cells, but how this is achieved in vivo remains poorly understood. We report the use of chromosome conformation capture coupled with deep sequencing (Hi-C) to map the structure of bacterial chromosomes. Analysis of Hi-C data and polymer modeling indicates that the Caulobacter crescentus chromosome consists of multiple, largely independent spatial domains that are probably composed of supercoiled plectonemes arrayed into a bottle brush–like fiber. These domains are stable throughout the cell cycle and are reestablished concomitantly with DNA replication. We provide evidence that domain boundaries are established by highly expressed genes and the formation of plectoneme-free regions, whereas the histone-like protein HU and SMC (structural maintenance of chromosomes) promote short-range compaction and the colinearity of chromosomal arms, respectively. Collectively, our results reveal general principles for the organization and structure of chromosomes in vivo., National Institutes of Health (U.S.) (Grant R01GM082899), National Institutes of Health (U.S.) (Grant U54CA143874)

Although brain-machine interfaces (BMIs) have focused largely on performing single-targeted movements, many natural tasks involve planning a complete sequence of such movements before execution. For these tasks, a BMI that can concurrently decode the full planned sequence before its execution may also consider the higher-level goal of the task to reformulate and perform it more effectively. Using population-wide modeling, we discovered two distinct subpopulations of neurons in the rhesus monkey premotor cortex that allow two planned targets of a sequential movement to be simultaneously held in working memory without degradation. Such marked stability occurred because each subpopulation encoded either only currently held or only newly added target information irrespective of the exact sequence. On the basis of these findings, we developed a BMI that concurrently decodes a full motor sequence in advance of movement and can then accurately execute it as desired., National Institutes of Health (U.S.) (DP1 OD003646)

The pulsatile release of cortisol from the adrenal glands is controlled by a hierarchical system that involves corticotropin releasing hormone (CRH) from the hypothalamus, adrenocorticotropin hormone (ACTH) from the pituitary, and cortisol from the adrenal glands. Determining the number, timing, and amplitude of the cortisol secretory events and recovering the infusion and clearance rates from serial measurements of serum cortisol levels is a challenging problem. Despite many years of work on this problem, a complete satisfactory solution has been elusive. We formulate this question as a non-convex optimization problem, and solve it using a coordinate descent algorithm that has a principled combination of (i) compressed sensing for recovering the amplitude and timing of the secretory events, and (ii) generalized cross validation for choosing the regularization parameter. Using only the observed serum cortisol levels, we model cortisol secretion from the adrenal glands using a second-order linear differential equation with pulsatile inputs that represent cortisol pulses released in response to pulses of ACTH. Using our algorithm and the assumption that the number of pulses is between 15 to 22 pulses over 24 hours, we successfully deconvolve both simulated datasets and actual 24-hr serum cortisol datasets sampled every 10 minutes from 10 healthy women. Assuming a one-minute resolution for the secretory events, we obtain physiologically plausible timings and amplitudes of each cortisol secretory event with R[superscript 2] above 0.92. Identification of the amplitude and timing of pulsatile hormone release allows (i) quantifying of normal and abnormal secretion patterns towards the goal of understanding pathological neuroendocrine states, and (ii) potentially designing optimal approaches for treating hormonal disorders., National Science Foundation (U.S.). Graduate Research Fellowship Program, National Institutes of Health (U.S.) (NIH DP1 OD003646), National Science Foundation (U.S.) (0836720), National Science Foundation (U.S.). Office of Emerging Frontiers in Research and Innovation (EFRI-0735956)

The development of molecular detection that allows rapid responses with high sensitivity and selectivity remains challenging. Herein, we demonstrate the strategy of novel bio-nanotechnology to successfully fabricate high-performance dopamine (DA) biosensor using DA Receptor-containing uniform-particle-shaped Nanovesicles-immobilized Carboxylated poly(3,4-ethylenedioxythiophene) (CPEDOT) NTs (DRNCNs). DA molecules are commonly associated with serious diseases, such as Parkinson's and Alzheimer's diseases. For the first time, nanovesicles containing a human DA receptor D1 (hDRD1) were successfully constructed from HEK-293 cells, stably expressing hDRD1. The nanovesicles containing hDRD1 as gate-potential modulator on the conducting polymer (CP) nanomaterial transistors provided high-performance responses to DA molecule owing to their uniform, monodispersive morphologies and outstanding discrimination ability. Specifically, the DRNCNs were integrated into a liquid-ion gated field-effect transistor (FET) system via immobilization and attachment processes, leading to high sensitivity and excellent selectivity toward DA in liquid state. Unprecedentedly, the minimum detectable level (MDL) from the field-induced DA responses was as low as 10 pM in real- time, which is 10 times more sensitive than that of previously reported CP based-DA biosensors. Moreover, the FET-type DRNCN biosensor had a rapid response time (<1 s) and showed excellent selectivity in human serum.

Drug-eluting stents reside in a dynamic fluid environment where the extent to which drugs are distributed within the arterial wall is critically modulated by the blood flowing through the arterial lumen. Yet several factors associated with the pulsatile nature of blood flow and their impact on arterial drug deposition have not been fully investigated. We employed an integrated framework comprising bench-top and computational models to explore the factors governing the time-varying fluid dynamic environment within the vasculature and their effects on arterial drug distribution patterns. A custom-designed bench-top framework comprising a model of a single drug-eluting stent strut and a poly-vinyl alcohol-based hydrogel as a model tissue bed simulated fluid flow and drug transport under fully apposed strut settings. Bench-top experiments revealed a relative independence between drug distribution and the factors governing pulsatile flow and these findings were validated with the in silico model. Interestingly, computational models simulating suboptimal deployment settings revealed a complex interplay between arterial drug distribution, Womersley number and the extent of malapposition. In particular, for a stent strut offset from the wall, total drug deposition was sensitive to changes in the pulsatile flow environment, with this dependence increasing with greater wall displacement. Our results indicate that factors governing pulsatile luminal flow on arterial drug deposition should be carefully considered in conjunction with device deployment settings for better utilization of drug-eluting stent therapy., National Institutes of Health (U.S.) (grant NIH R01 GM-49039)

Recent work has demonstrated that nonstimulatory endogenous peptides can enhance T cell recognition of antigen, but MHCI- and MHCII-restricted systems have generated very different results. MHCII-restricted TCRs need to interact with the nonstimulatory peptide–MHC (pMHC), showing peptide specificity for activation enhancers or coagonists. In contrast, the MHCI-restricted cells studied to date show no such peptide specificity for coagonists, suggesting that CD8 binding to noncognate MHCI is more important. Here we show how this dichotomy can be resolved by varying CD8 and TCR binding to agonist and coagonists coupled with computer simulations, and we identify two distinct mechanisms by which CD8 influences the peptide specificity of coagonism. Mechanism 1 identifies the requirement of CD8 binding to noncognate ligand and suggests a direct relationship between the magnitude of coagonism and CD8 affinity for coagonist pMHCI. Mechanism 2 describes how the affinity of CD8 for agonist pMHCI changes the requirement for specific coagonist peptides. MHCs that bind CD8 strongly were tolerant of all or most peptides as coagonists, but weaker CD8-binding MHCs required stronger TCR binding to coagonist, limiting the potential coagonist peptides. These findings in MHCI systems also explain peptide-specific coagonism in MHCII-restricted cells, as CD4–MHCII interaction is generally weaker than CD8–MHCI., National Institutes of Health (U.S.). Pioneer Award

We develop an approach to quantitative analysis of carbon dioxide concentration in exhaled breath, recorded as a function of time by capnography. The generated waveform – or capnogram – is currently used in clinical practice to establish the presence of respiration as well as determine respiratory rate and end-tidal CO2 concentration. The capnogram shape also has diagnostic value, but is presently assessed qualitatively, by visual inspection. Prior approaches to quantitatively characterizing the capnogram shape have explored the correlation of various geometric parameters with pulmonary function tests. These studies attempted to characterize the capnogram in normal subjects and patients with cardiopulmonary disease, but no consistent progress was made, and no translation into clinical practice was achieved. We apply automated quantitative analysis to discriminate between chronic obstructive pulmonary disease (COPD) and congestive heart failure (CHF), and between COPD and normal. Capnograms were collected from 30 normal subjects, 56 COPD patients, and 53 CHF patients. We computationally extract four physiologically based capnogram features. Classification on a hold-out test set was performed by an ensemble of classifiers employing quadratic discriminant analysis, designed through cross-validation on a labeled training set. Using 80 exhalations of each capnogram record in the test set, performance analysis with bootstrapping yields areas under the receiver operating characteristic (ROC) curve of 0.89 (95% CI: 0.72-0.96) for COPD/CHF classification, and 0.98 (95% CI: 0.82- 1.0) for COPD/normal classification. This classification performance is obtained with a run-time sufficiently fast for realtime monitoring., National Science Foundation (U.S.) (ASEE NDSEG fellowship)

Enhanced signaling by the small guanosine triphosphatase Ras is common in T cell acute lymphoblastic leukemia/lymphoma (T-ALL), but the underlying mechanisms are unclear. We identified the guanine nucleotide exchange factor RasGRP1 (Rasgrp1 in mice) as a Ras activator that contributes to leukemogenesis. We found increased RasGRP1 expression in many pediatric T-ALL patients, which is not observed in rare early T cell precursor T-ALL patients with KRAS and NRAS mutations, such as K-Ras[superscript G12D]. Leukemia screens in wild-type mice, but not in mice expressing the mutant K-Ras[superscript G12D] that encodes a constitutively active Ras, yielded frequent retroviral insertions that led to increased Rasgrp1 expression. Rasgrp1 and oncogenic K-Ras[superscript G12D] promoted T-ALL through distinct mechanisms. In K-Ras[superscript G12D] T-ALLs, enhanced Ras activation had to be uncoupled from cell cycle arrest to promote cell proliferation. In mouse T-ALL cells with increased Rasgrp1 expression, we found that Rasgrp1 contributed to a previously uncharacterized cytokine receptor–activated Ras pathway that stimulated the proliferation of T-ALL cells in vivo, which was accompanied by dynamic patterns of activation of effector kinases downstream of Ras in individual T-ALLs. Reduction of Rasgrp1 abundance reduced cytokine-stimulated Ras signaling and decreased the proliferation of T-ALL in vivo. The position of RasGRP1 downstream of cytokine receptors as well as the different clinical outcomes that we observed as a function of RasGRP1 abundance make RasGRP1 an attractive future stratification marker for T-ALL., National Institutes of Health (U.S.). Pioneer Award, National Cancer Institute (U.S.). Physical Sciences-Oncology Center (U54CA143874), National Institutes of Health (U.S.). (P01 AI091580)

Using nanoparticles for therapy and imaging holds tremendous promise for the treatment of major diseases such as cancer. However, their translation into the clinic has been slow because it remains difficult to produce nanoparticles that are consistent 'batch-to-batch', and in sufficient quantities for clinical research. Moreover, platforms for rapid screening of nanoparticles are still lacking. Recent microfluidic technologies can tackle some of these issues, and offer a way to accelerate the clinical translation of nanoparticles. In this Progress Article, we highlight the advances in microfluidic systems that can synthesize libraries of nanoparticles in a well-controlled, reproducible and high-throughput manner. We also discuss the use of microfluidics for rapidly evaluating nanoparticles in vitro under microenvironments that mimic the in vivo conditions. Furthermore, we highlight some systems that can manipulate small organisms, which could be used for evaluating the in vivo toxicity of nanoparticles or for drug screening. We conclude with a critical assessment of the near- and long-term impact of microfluidics in the field of nanomedicine., Prostate Cancer Foundation (Award in Nanotherapeutics), MIT-Harvard Center for Cancer Nanotechnology Excellence (U54-CA151884), National Heart, Lung, and Blood Institute (Programs of Excellence in Nanotechnology (HHSN268201000045C)), National Science Foundation (U.S.) (Graduate Research Fellowship)

Background: An inexpensive prophylactic vaccine offers the best hope to curb the HIV/AIDS epidemic gripping sub-Saharan Africa. Systematic means to guide the design of an effective immunogen for this, and other, infectious diseases are not available. What is required is a method to chart the peaks and valleys of viral fitness as a function of amino acid sequence. An efficacious vaccine would eject the virus from the high fitness peaks, and drive it into the valleys where its compromised fitness impairs its ability to replicate and inflict damage to the host. Methods: Appealing to spin glass models in statistical physics, we present a novel approach to translate viral sequence databases into landscapes of viral fitness. These inferred models furnish a quantitative description of viral replicative capacity as a function of amino acid sequence. We illustrate this approach in the development of landscapes for the proteins of HIV-1 clade B Gag. Results: In comparisons to experimental and clinical data, our inferred landscapes demonstrate excellent agreement with: 1) in vitro replicative fitness measurements, 2) clinically observed high-fitness circulating viral strains, 3) documented HLA associated CTL escape mutations, and 4) intra-host temporal adaptation pathways revealed by deep sequencing. These favorable comparisons support our landscapes as reflections of intrinsic viral fitness. We illustrate the value of such descriptions in the computational design of a CTL Gag immunogen. Conclusion: We present a novel methodology to translate viral sequence data into quantitative landscapes of viral fitness. In an application to HIV-1 Gag, we illustrate excellent agreement of our model predictions with experimental and clinical data, and demonstrate a powerful new approach for HIV immunogen design. We anticipate that this approach may represent a heretofore unprecedented means to synthesize fitness landscapes for diverse pathogens, and may provide the basis for the design of i

Dielectrophoresis (DEP) has emerged as an important tool for the manipulation of bioparticles ranging from the submicron to the tens of microns in size. Here we show the use of phospholipid vesicle electroformation techniques to develop a new class of test particles with specifically engineered electrical propserties to enable identifiable dielectrophoretic responses in microfabricated systems. These electrically addressable vesicles (EAVs) enable the creation of electrically distinct populations of test particles for DEP. EAVs offer control of both their inner aqueous core and outer membrane properties; by encapsulating solutions of different electrolyte strength inside the vesicle and by incorporating functionalized phospholipids containing poly(ethylene glycol) (PEG) brushes attached to their hydrophilic headgroup in the vesicle membrane, we demonstrate control of the vesicles’ electrical polarizabilities. This combined with the ability to encode information about the properties of the vesicle in its fluorescence signature forms the first steps toward the development of EAV populations as metrology tools for any DEP-based microsystem., National Institutes of Health (U.S.) (Grant RR199652), National Institutes of Health (U.S.) (Grant EB005753), Merck/CSBi (Fellowship), Solomon Buchsbaum AT&T Research Fund