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1.  The Ratio of Monocytes to Lymphocytes in Peripheral Blood Correlates with Increased Susceptibility to Clinical Malaria in Kenyan Children 
PLoS ONE  2013;8(2):e57320.
Plasmodium falciparum malaria remains a major cause of illness and death in sub-Saharan Africa. Young children bear the brunt of the disease and though older children and adults suffer relatively fewer clinical attacks, they remain susceptible to asymptomatic P. falciparum infection. A better understanding of the host factors associated with immunity to clinical malaria and the ability to sustain asymptomatic P. falciparum infection will aid the development of improved strategies for disease prevention.
Methods and Findings
Here we investigate whether full differential blood counts can predict susceptibility to clinical malaria among Kenyan children sampled at five annual cross-sectional surveys. We find that the ratio of monocytes to lymphocytes, measured in peripheral blood at the time of survey, directly correlates with risk of clinical malaria during follow-up. This association is evident among children with asymptomatic P. falciparum infection at the time the cell counts are measured (Hazard ratio (HR)  =  2.7 (95% CI 1.42, 5.01, P  =  0.002) but not in those without detectable parasitaemia (HR  =  1.0 (95% CI 0.74, 1.42, P  =  0.9).
We propose that the monocyte to lymphocyte ratio, which is easily derived from routine full differential blood counts, reflects an individual's capacity to mount an effective immune response to P. falciparum infection.
PMCID: PMC3577721  PMID: 23437368
3.  Mature induced-pluripotent-stem-cell-derived human podocytes reconstitute kidney glomerular-capillary-wall function on a chip 
An in vitro model of the human kidney glomerulus — the major site of blood filtration — could facilitate drug discovery and illuminate kidney-disease mechanisms. Microfluidic organ-on-a-chip technology has been used to model the human proximal tubule, yet a kidney-glomerulus-on-a-chip has not been possible because of the lack of functional human podocytes — the cells that regulate selective permeability in the glomerulus. Here, we demonstrate an efficient (> 90%) and chemically defined method for directing the differentiation of human induced pluripotent stem (hiPS) cells into podocytes that express markers of the mature phenotype (nephrin+, WT1+, podocin+, Pax2−) and that exhibit primary and secondary foot processes. We also show that the hiPS-cell-derived podocytes produce glomerular basement-membrane collagen and recapitulate the natural tissue/tissue interface of the glomerulus, as well as the differential clearance of albumin and inulin, when co-cultured with human glomerular endothelial cells in an organ-on-a-chip microfluidic device. The glomerulus-on-a-chip also mimics adriamycin-induced albuminuria and podocyte injury. This in vitro model of human glomerular function with mature human podocytes may facilitate drug development and personalized-medicine applications.
PMCID: PMC5639718  PMID: 29038743
4.  A multiple reader scoring system for Nasal Potential Difference parameters 
Nasal Potential Difference (NPD) is a biomarker of CFTR activity used to diagnose CF and monitor experimental therapies. Limited studies have been performed to assess agreement between expert readers of NPD interpretation using a scoring algorithm.
We developed a standardized scoring algorithm for “interpretability” and “confidence” for PD (potential difference) measures, and sought to determine the degree of agreement on NPD parameters between trained readers.
There was excellent agreement for interpretability between NPD readers for CF and fair agreement for normal tracings but slight agreement of interpretability in indeterminate tracings. Amongst interpretable tracings, excellent correlation of mean scores for Ringer’s Baseline PD, Δamiloride, and ΔCl-free + Isoproterenol was observed. There was slight agreement regarding confidence of the interpretable PD tracings, resulting in divergence of the Ringers and Δamiloride, and ΔCl-free + Isoproterenol PDs between “high” and “low” confidence CF tracings.
A multi-reader process with adjudication is important for scoring NPDs for diagnosis and in monitoring of CF clinical trials.
PMCID: PMC5671639  PMID: 28465124
Nasal Potential Difference; CFTR; Clinical trial outcomes
5.  Flexible, high-resolution micro-optical coherence tomography endobronchial probe toward in vivo imaging of cilia 
Optics letters  2017;42(4):867-870.
We report the design and fabrication of a flexible, longitudinally scanning high-resolution micro-optical coherence tomography (μOCT) endobronchial probe, optimized for micro-anatomical imaging in airways. The 2.4 mm diameter and flexibility of the probe allows it to be inserted into the instrument channel of a standard bronchoscope, enabling real-time video guidance of probe placement. To generate a depth-of-focus enhancing annular beam, we utilized a new fabrication method, whereby a hollow glass ferrule was angle-polished and gold-coated to produce an elongated annular reflector. We present validation data that verifies the preservation of linear scanning, despite the use of flexible materials. When utilized on excised, cultured mouse trachea, the probe acquired images of comparable quality to those obtained by a benchtop μOCT system.
PMCID: PMC5665567  PMID: 28198885
6.  Interpreting Mobile and Handheld Air Sensor Readings in Relation to Air Quality Standards and Health Effect Reference Values: Tackling the Challenges 
Atmosphere  2017;8(10):182.
The US Environmental Protection Agency (EPA) and other federal agencies face a number of challenges in interpreting and reconciling short-duration (seconds to minutes) readings from mobile and handheld air sensors with the longer duration averages (hours to days) associated with the National Ambient Air Quality Standards (NAAQS) for the criteria pollutants-particulate matter (PM), ozone, carbon monoxide, lead, nitrogen oxides, and sulfur oxides. Similar issues are equally relevant to the hazardous air pollutants (HAPs) where chemical-specific health effect reference values are the best indicators of exposure limits; values which are often based on a lifetime of continuous exposure. A multi-agency, staff-level Air Sensors Health Group (ASHG) was convened in 2013. ASHG represents a multi-institutional collaboration of Federal agencies devoted to discovery and discussion of sensor technologies, interpretation of sensor data, defining the state of sensor-related science across each institution, and provides consultation on how sensors might effectively be used to meet a wide range of research and decision support needs. ASHG focuses on several fronts: improving the understanding of what hand-held sensor technologies may be able to deliver; communicating what hand-held sensor readings can provide to a number of audiences; the challenges of how to integrate data generated by multiple entities using new and unproven technologies; and defining best practices in communicating health-related messages to various audiences. This review summarizes the challenges, successes, and promising tools of those initial ASHG efforts and Federal agency progress on crafting similar products for use with other NAAQS pollutants and the HAPs. NOTE: The opinions expressed are those of the authors and do not necessary represent the opinions of their Federal Agencies or the US Government. Mention of product names does not constitute endorsement.
PMCID: PMC5662140
air pollutants; ambient air; indoor air; citizen science; toxic chemicals
7.  A neuroprotective astrocyte state is induced by neuronal signal EphB1 but fails in ALS models 
Nature Communications  2017;8:1164.
Astrocyte responses to neuronal injury may be beneficial or detrimental to neuronal recovery, but the mechanisms that determine these different responses are poorly understood. Here we show that ephrin type-B receptor 1 (EphB1) is upregulated in injured motor neurons, which in turn can activate astrocytes through ephrin-B1-mediated stimulation of signal transducer and activator of transcription-3 (STAT3). Transcriptional analysis shows that EphB1 induces a protective and anti-inflammatory signature in astrocytes, partially linked to the STAT3 network. This is distinct from the response evoked by interleukin (IL)-6 that is known to induce both pro inflammatory and anti-inflammatory processes. Finally, we demonstrate that the EphB1–ephrin-B1 pathway is disrupted in human stem cell derived astrocyte and mouse models of amyotrophic lateral sclerosis (ALS). Our work identifies an early neuronal help-me signal that activates a neuroprotective astrocytic response, which fails in ALS, and therefore represents an attractive therapeutic target.
Astrocytes can have protective or detrimental effects on neurons during injury, but the molecular mechanisms that determine these different states are unresolved. Here the authors identify a pathway via neuronal EphB1 that induces neuroprotective signalling in astrocytes through ephrin-B1 mediated STAT3 activation, which is impaired in models of amyotrophic lateral sclerosis.
PMCID: PMC5660125  PMID: 29079839
8.  Rapid sequential in situ multiplexing with DNA-Exchange-Imaging in Neuronal Cells and Tissues 
Nano letters  2017;17(10):6131-6139.
To decipher the molecular mechanisms of biological function, it is critical to map the molecular composition of individual cells or even more importantly tissue samples in the context of their biological environment in situ. Immunofluorescence (IF) provides specific labeling for molecular profiling. However, conventional IF methods have finite multiplexing capabilities due to spectral overlap of the fluorophores. Various sequential imaging methods have been developed to circumvent this spectral limit, but are not widely adopted due to the common limitation of requiring multi-rounds of slow (typically over 2 hours at room temperature to overnight at 4 °C in practice) immunostaining. We present here a practical and robust method, which we call DNA-Exchange-Imaging (DEI), for rapid in situ spectrally-unlimited multiplexing. This technique overcomes speed restrictions by allowing for single-round immunostaining with DNA-barcoded antibodies, followed by rapid (less than 10 minutes) buffer exchange of fluorophore-bearing DNA imager strands. The programmability of DNA-Exchange-Imaging allows us to apply it to diverse microscopy platforms (with Exchange-Confocal, Exchange-SIM, Exchange-STED, and Exchange-PAINT demonstrated here) at multiple desired resolution scales (from ~300 nm down to sub-20-nm). We optimized and validated the use of DEI in complex biological samples, including primary neuron cultures and tissue sections. These results collectively suggest DNA-Exchange as a versatile, practical platform for rapid, highly multiplexed in situ imaging, potentially enabling new applications ranging from basic science, to drug discovery, and to clinical pathology.
Graphical Abstract
PMCID: PMC5658129  PMID: 28933153
Highly multiplexed imaging; super-resolution imaging; in situ protein detection; multiplexed cell type identification; in situ protein-protein co-localization analysis
9.  Informed walks: whispering hints to gene hunters inside networks’ jungle 
BMC Systems Biology  2017;11:97.
Systemic approaches offer a different point of view on the analysis of several types of molecular associations as well as on the identification of specific gene communities in several cancer types. However, due to lack of sufficient data needed to construct networks based on experimental evidence, statistical gene co-expression networks are widely used instead. Many efforts have been made to exploit the information hidden in these networks. However, these approaches still need to capitalize comprehensively the prior knowledge encrypted into molecular pathway associations and improve their efficiency regarding the discovery of both exclusive subnetworks as candidate biomarkers and conserved subnetworks that may uncover common origins of several cancer types.
In this study we present the development of the Informed Walks model based on random walks that incorporate information from molecular pathways to mine candidate genes and gene-gene links. The proposed model has been applied to TCGA (The Cancer Genome Atlas) datasets from seven different cancer types, exploring the reconstructed co-expression networks of the whole set of genes and driving to highlighted sub-networks for each cancer type. In the sequel, we elucidated the impact of each subnetwork on the indication of underlying exclusive and common molecular mechanisms as well as on the short-listing of drugs that have the potential to suppress the corresponding cancer type through a drug-repurposing pipeline.
We have developed a method of gene subnetwork highlighting based on prior knowledge, capable to give fruitful insights regarding the underlying molecular mechanisms and valuable input to drug-repurposing pipelines for a variety of cancer types.
Electronic supplementary material
The online version of this article (10.1186/s12918-017-0473-6) contains supplementary material, which is available to authorized users.
PMCID: PMC5637247  PMID: 29020948
Random walks; Network inference; Network analysis; Gene subnetworks; Molecular mechanisms; Drug repurposing; Cancer types
10.  A Paper-Based “Pop-up” Electrochemical Device for Analysis of Beta-Hydroxy-butyrate 
Analytical chemistry  2016;88(12):6326-6333.
This paper describes the design and fabrication of a “pop-up” electrochemical paper-based analytical device (pop-up-EPAD) to measure beta-hydroxybutyrate (BHB)—a key biomarker for diabetic ketoacidosis—using a commercial glucometer. Pop-up-EPADs are inspired by pop-up greeting cards and children’s books. They are made from a single sheet of paper folded into a three-dimensional (3D) device that changes shape, and fluidic and electrical connectivity, by simply folding and unfolding the structure. The reconfigurable 3D structure makes it possible to change the fluidic path and to control timing; it also provides mechanical support for the folded and unfolded structures that enables good registration and repeatability on folding. A pop-up-EPAD designed to detect BHB shows performance comparable to commercially available plastic test strips over the clinically relevant range of BHB in blood when used with a commercial glucometer that integrates the ability to measure glucose and BHB (combination BHB/glucometer). With simple modifications of the electrode and fluid path design, the pop-up-EPAD also detects BHB using a simple glucometer—a device that is much more available than combination BHB/glucometers. Strategies that use a “3D pop-up”—that is, large-scale changes a 3D structure and fluidic paths—by folding/unfolding add functionality (e.g., controlled timing, fluidic handling and path programming, control over complex sequences of steps, and alterations in electrical connectivity) to EPADs, and should enable the development of new classes of paper-based diagnostic devices.
TOC image
Pop-up paper-based electrochemical device.
PMCID: PMC5633928  PMID: 27243791
Pop-up structure; Kirigami; point-of-care diagnostics; clinical chemistry; electroanalytical chemistry; paper diagnostics; beta-hydroxybutyrate (BHB); diabetic ketoacidosis (DKA); diabetes
13.  Stress-related changes in personality: A longitudinal study of perceived stress and trait pessimism 
Although research has shown that certain aspects of personality can change over time, the determinants of such change remain unclear. Stress alters neural dynamics and precipitates disorders that shape personality traits involving negative affectivity. In this study, therefore, we assessed the perceived stress and pessimism levels of 332 young, middle-aged, and older adults for five weeks to examine how levels of stress and pessimism change and interrelate over time. The best fitting longitudinal model was a bivariate latent growth curve model, which indicated that stress and pessimism both changed and exhibited significant variability in change over time. Moreover, changes in stress were associated with changes in pessimism. Pessimism thus changes over time, with alterations in stress potentially structuring these changes.
PMCID: PMC4991032  PMID: 27551162
Life stress; Perceived stress; Personality; Pessimism; Change; Affect; Longitudinal; Development; Modeling; Health
14.  Forgiveness, Stress, and Health: a 5-Week Dynamic Parallel Process Study 
Psychological stress is a well-known risk factor for poor health, and recent research has suggested that the emotion-focused coping process of forgiveness may help mitigate these effects. To date, however, no studies have examined how levels of forgiveness, stress, and health fluctuate and interrelate over time.
We addressed this issue by examining how forgiveness, stress, and mental and physical health symptoms change and relate to one another over 5 weeks. We hypothesized that increases in state levels of forgiveness would be associated with decreases in perceptions of stress, which would in turn be related to decreases in mental and physical health symptoms. A reverse effects model was also tested.
We recruited a large, community-based sample of 332 young, middle-aged, and older adults (16–79 years old; Mage = 27.9). Each week for 5 weeks, participants reported on their levels of state forgiveness, perceived stress, and mental and physical health symptoms.
Levels of forgiveness, stress, and mental and physical health symptoms each showed significant change and individual variability in change over time. As hypothesized, increases in forgiveness were associated with decreases in stress, which were in turn related to decreases in mental (but not physical) health symptoms (i.e., forgiveness→ stress→ health). The reverse effects model (i.e., health → stress → forgiveness) provided a relatively poorer fit.
This study is the first to provide prospective, longitudinal evidence showing that greater forgiveness is associated with less stress and, in turn, better mental health. Strategies for cultivating forgiveness may thus have beneficial effects on stress and health.
PMCID: PMC5055412  PMID: 27068160
Forgiveness; Stress; Coping; Individual differences; Trajectories; Symptoms; Risk; Mechanism; Development; Health; Disease
15.  Presurgical thalamocortical connectivity is associated with response to vagus nerve stimulation in children with intractable epilepsy 
NeuroImage : Clinical  2017;16:634-642.
Although chronic vagus nerve stimulation (VNS) is an established treatment for medically-intractable childhood epilepsy, there is considerable heterogeneity in seizure response and little data are available to pre-operatively identify patients who may benefit from treatment. Since the therapeutic effect of VNS may be mediated by afferent projections to the thalamus, we tested the hypothesis that intrinsic thalamocortical connectivity is associated with seizure response following chronic VNS in children with epilepsy. Twenty-one children (ages 5–21 years) with medically-intractable epilepsy underwent resting-state fMRI prior to implantation of VNS. Ten received sedation, while 11 did not. Whole brain connectivity to thalamic regions of interest was performed. Multivariate generalized linear models were used to correlate resting-state data with seizure outcomes, while adjusting for age and sedation status. A supervised support vector machine (SVM) algorithm was used to classify response to chronic VNS on the basis of intrinsic connectivity. Of the 21 subjects, 11 (52%) had 50% or greater improvement in seizure control after VNS. Enhanced connectivity of the thalami to the anterior cingulate cortex (ACC) and left insula was associated with greater VNS efficacy. Within our test cohort, SVM correctly classified response to chronic VNS with 86% accuracy. In an external cohort of 8 children, the predictive model correctly classified the seizure response with 88% accuracy. We find that enhanced intrinsic connectivity within thalamocortical circuitry is associated with seizure response following VNS. These results encourage the study of intrinsic connectivity to inform neural network-based, personalized treatment decisions for children with intractable epilepsy.
•Children demonstrate variable response to vagus nerve stimulation (VNS).•Enhanced thalamocortical connectivity is associated with better response to VNS.•Functional connectivity may pre-operatively identify responders to VNS.•Further study is indicated to inform personalized treatment decisions.
PMCID: PMC5619991
Functional connectivity; Intrinsic connectivity networks; Resting-state fMRI; Low frequency neural oscillations; VNS
16.  Measuring Five Dimensions of Religiosity across Adolescence 
Review of religious research  2017;59(3):367-393.
This paper theorizes and tests a latent variable model of adolescent religiosity in which five dimensions of religiosity are interrelated: religious beliefs, religious exclusivity, external religiosity, private practice, and religious salience. Research often theorizes overlapping and independent influences of single items or dimensions of religiosity on outcomes such as adolescent sexual behavior, but rarely operationalizes the dimensions in a measurement model accounting for their associations with each other and across time. We use longitudinal structural equation modeling (SEM) with latent variables to analyze data from two waves of the National Study of Youth and Religion. We test our hypothesized measurement model as compared to four alternate measurement models and find that our proposed model maintains superior fit. We then discuss the associations between the five dimensions of religiosity we measure and how these change over time. Our findings suggest how future research might better operationalize multiple dimensions of religiosity in studies of the influence of religion in adolescence.
PMCID: PMC5602559  PMID: 28931956
18.  Influence of the Envelope gp120 Phe 43 Cavity on HIV-1 Sensitivity to Antibody-Dependent Cell-Mediated Cytotoxicity Responses 
Journal of Virology  2017;91(7):e02452-16.
HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cellular-mediated cytotoxicity (ADCC). HIV-1 has evolved sophisticated mechanisms to avoid the exposure of Env ADCC epitopes by downregulating CD4 and by limiting the overall amount of Env on the cell surface. In HIV-1, substitution of large residues such as histidine or tryptophan for serine 375 (S375H/W) in the gp120 Phe 43 cavity, where Phe 43 of CD4 contacts gp120, results in the spontaneous sampling of an Env conformation closer to the CD4-bound state. While residue S375 is well conserved in the majority of group M HIV-1 isolates, CRF01_AE strains have a naturally occurring histidine at this position (H375). Interestingly, CRF01_AE is the predominant circulating strain in Thailand, where the RV144 trial took place. In this trial, which resulted in a modest degree of protection, ADCC responses were identified as being part of the correlate of protection. Here we investigate the influence of the Phe 43 cavity on ADCC responses. Filling this cavity with a histidine or tryptophan residue in Env with a natural serine residue at this position (S375H/W) increased the susceptibility of HIV-1-infected cells to ADCC. Conversely, the replacement of His 375 by a serine residue (H375S) within HIV-1 CRF01_AE decreased the efficiency of the ADCC response. Our results raise the intriguing possibility that the presence of His 375 in the circulating strain where the RV144 trial was held contributed to the observed vaccine efficacy.
IMPORTANCE HIV-1-infected cells presenting Env in the CD4-bound conformation on their surface are preferentially targeted by ADCC mediated by HIV-positive (HIV+) sera. Here we show that the gp120 Phe 43 cavity modulates the propensity of Env to sample this conformation and therefore affects the susceptibility of infected cells to ADCC. CRF01_AE HIV-1 strains have an unusual Phe 43 cavity-filling His 375 residue, which increases the propensity of Env to sample the CD4-bound conformation, thereby increasing susceptibility to ADCC.
PMCID: PMC5355605  PMID: 28100618
HIV-1; Env; gp120; Phe 43 cavity; ADCC; RV144
19.  Measuring lifetime stress exposure and protective factors in life course research on racial inequality and birth outcomes 
Stress (Amsterdam, Netherlands)  2017;20(4):379-385.
There has been a long-standing interest in better understanding how social factors contribute to racial disparities in health, including birth outcomes. A recent emphasis in this context has been on identifying the effects of stress exposure and protective factors experienced over the entire lifetime. Yet despite repeated calls for a life course approach to research on this topic, very few studies have actually assessed how stressors and protective factors occurring over women’s lives relate to birth outcomes. We discuss this issue here by describing how challenges in the measurement of lifetime stress exposure and protective factors have prevented researchers from developing an empirically-based life course perspective on health. First, we summarize prevailing views on racial inequality and birth outcomes; second, we discuss measurement challenges that exist in this context; and finally, we describe both new tools and needed tools for assessing lifetime stress exposure and suggest opportunities for integrating information on stress exposure and psychosocial protective factors. We conclude that more studies are needed that integrate information about lifetime stress exposures and the protective factors that promote resilience against such exposures to inform policy and practice recommendations to reduce racial disparities in birth outcomes.
PMCID: PMC5589186  PMID: 28660838
Stress; social support; measurement; resilience; birth outcomes; health; racial disparities
20.  SCF-KIT signaling induces endothelin-3 synthesis and secretion: Thereby activates and regulates endothelin-B-receptor for generating temporally- and spatially-precise nitric oxide to modulate SCF- and or KIT-expressing cell functions 
PLoS ONE  2017;12(9):e0184154.
We demonstrate that SCF-KIT signaling induces synthesis and secretion of endothelin-3 (ET3) in human umbilical vein endothelial cells and melanoma cells in vitro, gastrointestinal stromal tumors, human sun-exposed skin, and myenteric plexus of human colon post-fasting in vivo. This is the first report of a physiological mechanism of ET3 induction. Integrating our finding with supporting data from literature leads us to discover a previously unreported pathway of nitric oxide (NO) generation derived from physiological endothelial NO synthase (eNOS) or neuronal NOS (nNOS) activation (referred to as the KIT-ET3-NO pathway). It involves: (1) SCF-expressing cells communicate with neighboring KIT-expressing cells directly or indirectly (cleaved soluble SCF). (2) SCF-KIT signaling induces timely local ET3 synthesis and secretion. (3) ET3 binds to ETBR on both sides of intercellular space. (4) ET3-binding-initiated-ETBR activation increases cytosolic Ca2+, activates cell-specific eNOS or nNOS. (5) Temporally- and spatially-precise NO generation. NO diffuses into neighboring cells, thus acts in both SCF- and KIT-expressing cells. (6) NO modulates diverse cell-specific functions by NO/cGMP pathway, controlling transcriptional factors, or other mechanisms. We demonstrate the critical physiological role of the KIT-ET3-NO pathway in fulfilling high demand (exceeding basal level) of endothelium-dependent NO generation for coping with atherosclerosis, pregnancy, and aging. The KIT-ET3-NO pathway most likely also play critical roles in other cell functions that involve dual requirement of SCF-KIT signaling and NO. New strategies (e.g. enhancing the KIT-ET3-NO pathway) to harness the benefit of endogenous eNOS and nNOS activation and precise NO generation for correcting pathophysiology and restoring functions warrant investigation.
PMCID: PMC5589172  PMID: 28880927
21.  Leiomyoma of External Auditory Canal 
This article reports a case of piloleiomyoma of external auditory canal, which is the 7th case of leiomyoma of the external auditory canal being reported and the 2nd case of leiomyoma arising from arrectores pilorum muscles, all the other five cases were angioleiomyomas, arising from blood vessels. A 52 years old male presented with a mass in the right external auditory canal and decreased hearing of 6 months duration. Tumor excision done by end aural approach. Histopathological examination report was leiomyoma. It is extremely rare for leiomyoma to occur in the external auditory canal because of the non-availability of smooth muscles in the external canal. So it should be considered as a very rare differential diagnosis for any tumor or polyp in the ear canal.
PMCID: PMC4961651  PMID: 27508144
External auditory canal; Tumor; Leiomyoma
22.  Airway Progenitor Clone Formation Is Enhanced by Y-27632–Dependent Changes in the Transcriptome 
The application of conditional reprogramming culture (CRC) methods to nasal airway epithelial cells would allow more wide-spread incorporation of primary airway epithelial culture models into complex lung disease research. In this study, we adapted the CRC method to nasal airway epithelial cells, investigated the growth advantages afforded by this technique over standard culture methods, and determined the cellular and molecular basis of CRC cell culture effects. We found that the CRC method allowed the production of 7.1 × 1010 cells after 4 passages, approximately 379 times more cells than were generated by the standard bronchial epithelial growth media (BEGM) method. These nasal airway epithelial cells expressed normal basal cell markers and could be induced to form a mucociliary epithelium. Progenitor cell frequency was significantly higher using the CRC method in comparison to the standard culture method, and progenitor cell maintenance was dependent on addition of the Rho-kinase inhibitor Y-27632. Whole-transcriptome sequencing analysis demonstrated widespread gene expression changes in Y-27632–treated basal cells. We found that Y-27632 treatment altered expression of genes fundamental to the formation of the basal cell cytoskeleton, cell–cell junctions, and cell–extracellular matrix (ECM) interactions. Importantly, we found that Y-27632 treatment up-regulated expression of unique basal cell intermediate filament and desmosomal genes. Conversely, Y-27632 down-regulated multiple families of protease/antiprotease genes involved in ECM remodeling. We conclude that Y-27632 fundamentally alters cell–cell and cell–ECM interactions, which preserves basal progenitor cells and allows greater cell amplification.
PMCID: PMC5023026  PMID: 27144410
conditionally reprogrammed cells; clone-forming cell frequency; airway stem progenitor; Y-27632
23.  NT-3 promotes proprioceptive axon regeneration when combined with activation of the mTor intrinsic growth pathway but not with reduction of myelin extrinsic inhibitors 
Experimental neurology  2016;283(Pt A):73-84.
Although previous studies have identified several strategies to stimulate regeneration of CNS axons, extensive regeneration and functional recovery have remained a major challenge, particularly for large diameter myelinated axons. Within the CNS, myelin is thought to inhibit axon regeneration, while modulating activity of the mTOR pathway promotes regeneration of injured axons. In this study, we examined NT-3 mediated regeneration of sensory axons through the dorsal root entry zone in a triple knockout of myelin inhibitory proteins or after activation of mTOR using a constitutively active (ca) Rheb in DRG neurons to determine the influence of environmental inhibitory or activation of intrinsic growth pathways could enhance NT-3-mediate regeneration. Loss of myelin inhibitory proteins showed modest enhancement of sensory axon regeneration. In mTOR studies, we found a dramatic age related decrease in the mTOR activation as determined by phosphorylation of the downstream marker S6 ribosomal subunit. Expression of caRheb within adult DRG neurons in vitro increased S6 phosphorylation and doubled the overall length of neurite outgrowth, which was reversed in the presence of rapamycin. In adult female rats, combined expression of caRheb in DRG neurons and NT-3 within the spinal cord increased regeneration of sensory axons almost 3 fold when compared to NT-3 alone. Proprioceptive assessment using a grid runway indicates functionally significant regeneration of large-diameter myelinated sensory afferents. Our results indicate that caRheb-induced increase in mTOR activation enhances neurotrophin-3 induced regeneration of large-diameter myelinated axons.
PMCID: PMC5090983  PMID: 27264357
Rheb; mTOR signaling pathway; neurotrophin-3 (NT-3); axonal regeneration; dorsal root ganglion (DRG)
24.  Potential Value of Impaired Cognition in Stroke Prediction: A U.K. Population‐Based Study 
To determine whether the association between impaired cognition and greater risk of incident stroke is also observed when cognitive impairment is defined using different criteria for mild cognitive impairment (MCI).
Prospective cohort study with 10 years of follow‐up.
Large multicentre study in the United Kingdom.
Individuals (aged 64–105) from the Medical Research Council Cognitive Function and Ageing Study (N = 13,004). From this, a subsample of 2,640 individuals was selected based on age, center, and cognitive ability to undergo a detailed cognitive assessment.
Information on sociodemographic characteristics, health, cognition, and functional ability was collected in an interview. The Geriatric Mental State Automated Geriatric Examination for Computer Assisted Taxonomy and the Cambridge Cognitive Examination were used to determine cognitive status. Stroke incidence was derived from self‐report, informant report, and death certificates. Participants were divided into no, mild, moderate, and severe cognitive impairment according to their baseline Mini‐Mental State Examination (MMSE) score. MCI criteria were used to classify persons into four groups: no cognitive impairment, MCI, severe impairment (i.e. other cognitive impairment no dementia: OCIND) and dementia.
Over 10 years, 703 incident strokes occurred. Lower MMSE score at baseline was associated with greater risk of incident stroke. When cognitive status was determined according to MCI criteria, those with severe impairment (odds ratio (OR) = 1.5, 95% confidence interval (CI) = 1.0–2.2) and dementia (OR = 2.6, 95% CI = 1.6–3.4) had a significantly greater risk of stroke than those with no cognitive impairment.
Criteria for MCI, defined using MMSE scores or clinical criteria, can capture individuals at greater stroke risk. The results highlight the need to focus on stroke risk in individuals even with MCI.
PMCID: PMC5574015  PMID: 28369710
mild cognitive impairment (MCI); stroke; cognitive aging; cohort studies; risk factors in epidemiology
25.  Staged induction of HIV-1 glycan–dependent broadly neutralizing antibodies 
Science translational medicine  2017;9(381):eaai7514.
A preventive HIV-1 vaccine should induce HIV-1–specific broadly neutralizing antibodies (bnAbs). However, bnAbs generally require high levels of somatic hypermutation (SHM) to acquire breadth, and current vaccine strategies have not been successful in inducing bnAbs. Because bnAbs directed against a glycosylated site adjacent to the third variable loop (V3) of the HIV-1 envelope protein require limited SHM, the V3-glycan epitope is an attractive vaccine target. By studying the cooperation among multiple V3-glycan B cell lineages and their coevolution with autologous virus throughout 5 years of infection, we identify key events in the ontogeny of a V3-glycan bnAb. Two autologous neutralizing antibody lineages selected for virus escape mutations and consequently allowed initiation and affinity maturation of a V3-glycan bnAb lineage. The nucleotide substitution required to initiate the bnAb lineage occurred at a low-probability site for activation-induced cytidine deaminase activity. Cooperation of B cell lineages and an improbable mutation critical for bnAb activity defined the necessary events leading to breadth in this V3-glycan bnAb lineage. These findings may, in part, explain why initiation of V3-glycan bnAbs is rare, and suggest an immunization strategy for inducing similar V3-glycan bnAbs.
PMCID: PMC5562350  PMID: 28298420

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