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We construct and investigate paper-based microfluidic devices, which model long-term fluid harvesting, transport, sensing, and analysis in new wearables for sweat analysis. Such devices can continuously wick fluid mimicking sweat and dispose of it on evaporation pads. We characterize and analyze how the action of capillarity and evaporation can cooperatively be used to transport and process sweat mimics containing dissolved salts and model analytes. The results point out that non-invasive osmotic extraction combined with paper microfluidics and evaporative disposal can enable sweat collection and monitoring for durations longer than 10 days. We model the fluid flow in the new capillary-evaporative devices and identify the parameters enabling their long-term operation. We show that the transport rates are sufficiently large to handle natural sweat rates, while we envision that such handling can be interfaced with osmotic harvesting of sweat, a concept that we demonstrated recently. Finally, we illustrate that the salt film deposited at the evaporation pad would eventually lead to cessation of the process but at the same time will preserve a record of analytes that may be used for long-term biomarker monitoring in sweat. These principles can be implemented in future platforms for wearable skin-interfacing assays or electronic biomarker monitors., (Copyright © 2020 Author(s).)

As treatment of the early, inflammatory phase of sepsis improves, post-sepsis immunosuppression and secondary infection have increased in importance. How early inflammation drives immunosuppression remains unclear. Although IFN-γ typically helps microbial clearance, we found that increased plasma IFN-γ in early clinical sepsis was associated with the later development of secondary Candida infection. Consistent with this observation, we found that exogenous IFN-γ suppressed macrophage phagocytosis of zymosan in vivo, and antibody blockade of IFN-γ after endotoxemia improved survival of secondary candidemia. Transcriptomic analysis of innate lymphocytes during endotoxemia suggested that NKT cells drove IFN-γ production by NK cells via mTORC1. Activation of invariant NKT (iNKT) cells with glycolipid antigen drove immunosuppression. Deletion of iNKT cells in Cd1d-/- mice or inhibition of mTOR by rapamycin reduced immunosuppression and susceptibility to secondary Candida infection. Thus, although rapamycin is typically an immunosuppressive medication, in the context of sepsis, rapamycin has the opposite effect. These results implicated an NKT cell/mTOR/IFN-γ axis in immunosuppression following endotoxemia or sepsis. In summary, in vivo iNKT cells activated mTORC1 in NK cells to produce IFN-γ, which worsened macrophage phagocytosis, clearance of secondary Candida infection, and mortality.

Introduction: Cranioplasty (CP) is a multifaceted procedure in a heterogenous patient population, with a high risk for complication. However, no previous large-scale studies have compared outcomes in primary (ie, first attempt) CP versus revision CP (ie, following previous attempts). The authors, therefore, analyzed long-term outcomes of 506 consecutive primary and revision CPs, performed by a single surgeon., Methods: All CPs performed between 2012 and 2019 were analyzed under IRB protocol approval. Surgeries were categorized as either primary (no previous CP; n = 279) or revision CP (at least one previous CP; n = 227). Complications were defined as either major or minor. Subgroup analyses investigated whether or not CP complication risk directly correlated with the number of previous neuro-cranial surgeries and/or CP attempts., Results: The primary CP group experienced a major complication rate of 9% (26/279). In comparison, the revision CP group demonstrated a major complication rate of 32% (73/227). For the revision CP group, the rate of major complications rose with each additional surgery, from 4% (1 prior surgery) to 17% (2 prior surgeries) to 39% (3-4 prior surgeries) to 47% (≥5 prior surgeries)., Conclusion: In a review of 506 consecutive cases, patients undergoing revision CP had a 3-fold increase in incidence of major complications, as compared to those undergoing primary CP. These results provide critical insight into overall CP risk stratification and may guide preoperative risk-benefit discussions. Furthermore, these findings may support a center-of-excellence care model, particularly for those patients with a history of previous neuro-cranial surgeries and/or CP attempts.

Left hand dominance is a minority trait historically regarded as disadvantageous for surgeons. Contemporary scientific literature and folklore have shed new light on left handedness as a "boutique trait" and possible marker of gifted and exceptional individuals. Our subjective impression that left handedness is prevalent in the unique field of plastic surgery raised questions regarding the scope and possible causality of this phenomenon., Methods: One hundred eleven medical doctors in our medical center filled out a 13-item questionnaire regarding hand dominance, medical speciality, and various creative outlets or hobbies., Results: Sixty-four percent of the participating plastic surgeons were left handed (significantly higher than the approximate 12% of the general population; P = 0.007). Many of the left-handed doctors admitted to practicing musical instruments and various arts, crafts, and other hobbies., Conclusions: Plastic surgery is a unique profession requiring astute minds capable of creative and "outside-the-box" thinking; traits we have learned in recent decades may be particularly keen in left-handed individuals, perhaps suggest a causal relationship to the conglomeration of a majority of left-handed plastic surgeons., (Copyright © 2020 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of The American Society of Plastic Surgeons.)

Full-thickness large scalp defects with underlying exposed calvarium pose a significant reconstructive challenge. Traditional reconstructive techniques are usually not an option in patients with irradiated scalp with thin skin and reduced laxity.Dermal substitutes-based reconstruction techniques have been described in recent years. A common approach is the staged methodology, with the initial application of skin substitute followed by a split-thickness skin graft few weeks later; however, this method involves a prolonged period of local wound management prior to skin grafting and is often associated with complications that interfere with wound healing.This report describes a single-stage triple-layer technique for the reconstruction of a large scalp defect with exposed bone in a patient with a history of radiation treatment, using 3 turnover pericranial flaps in conjunction with a Matriderm dermal substitute and split-thickness skin graft. This immediate multilayered reconstruction provides a long-lasting structural and aesthetic outcome, with minimal donor site morbidity and reduced complications.

Sexual dimorphism in the mammalian immune system is manifested as more frequent and severe infectious diseases in males and, on the other hand, higher rates of autoimmune disease in females, yet insights underlying those differences are still lacking. Here we characterize sex differences in the immune system by RNA and ATAC sequence profiling of untreated and interferon-induced immune cell types in male and female mice. We detect very few differentially expressed genes between male and female immune cells except in macrophages from three different tissues. Accordingly, very few genomic regions display differences in accessibility between sexes. Transcriptional sexual dimorphism in macrophages is mediated by genes of innate immune pathways, and increases after interferon stimulation. Thus, the stronger immune response of females may be due to more activated innate immune pathways prior to pathogen invasion.

Background: Ethiopia has one of the lowest rates of facility delivery and is promoting birth preparedness among pregnant women through its community health services to increase the rate of institutional delivery and reduce maternal mortality. Observational studies of birth preparedness in Ethiopia have thus far only reported the marginal effect of birth preparedness when controlling for other factors, such as parity and education., Objectives: In this cross-sectional study, we use propensity score modeling to estimate the average population-level effect of birth preparedness on the likelihood of delivering at a facility., Methods: We conducted secondary analysis of household survey data collected from 215 women with a recent live birth within the catchment areas of 10 semi-urban health centers. A mother was considered well prepared for birth if she reported completing four of the following six actions: identified a skilled provider, identified an institution, saved money, identified transport, prepared clean delivery materials, and prepared food. We performed unadjusted and multivariate logistic regression analyses, with and without propensity score weighting, to assess the relationship between birth preparedness and institutional delivery., Findings: One hundred respondents (47%) delivered in an institution, and over two-thirds (151, 71%) were considered well prepared for birth. Institutional delivery was more common among women who were considered well prepared (57%) versus those who were considered not well prepared (19%). In the model with propensity score weighting, women who were well prepared for birth had 3.83 times higher odds of delivering at a facility (95% CI: 1.41-10.40, p-value = 0.010)., Conclusions: This study contributes to existing evidence supporting the inclusion of antenatal birth preparedness counseling as a part of an antenatal care package for promoting institutional delivery. Important gaps remain in operationalizing the definition of birth preparedness and understanding the pathway from exposure to outcome., Competing Interests: The authors have no competing interests to declare., (© 2019 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See http://creativecommons.org/licenses/by/4.0/.)

[This corrects the article DOI: 10.1371/journal.pone.0205499.].

Single-cell RNA sequencing (scRNA-seq) is an emerging technology for profiling the gene expression of thousands of cells at the single cell resolution. Currently, the labeling of cells in an scRNA-seq dataset is performed by manually characterizing clusters of cells or by fluorescence-activated cell sorting (FACS). Both methods have inherent drawbacks: The first depends on the clustering algorithm used and the knowledge and arbitrary decisions of the annotator, and the second involves an experimental step in addition to the sequencing and cannot be incorporated into the higher throughput scRNA-seq methods. We therefore suggest a different approach for cell labeling, namely, classifying cells from scRNA-seq datasets by using a model transferred from different (previously labeled) datasets. This approach can complement existing methods, and-in some cases-even replace them. Such a transfer-learning framework requires selecting informative features and training a classifier. The specific implementation for the framework that we propose, designated ''CaSTLe-classification of single cells by transfer learning,'' is based on a robust feature engineering workflow and an XGBoost classification model built on these features. Evaluation of CaSTLe against two benchmark feature-selection and classification methods showed that it outperformed the benchmark methods in most cases and yielded satisfactory classification accuracy in a consistent manner. CaSTLe has the additional advantage of being parallelizable and well suited to large datasets. We showed that it was possible to classify cell types using transfer learning, even when the databases contained a very small number of genes, and our study thus indicates the potential applicability of this approach for analysis of scRNA-seq datasets., Competing Interests: The authors have declared that no competing interests exist.

Supplemental Digital Content is available in the text.

Background: One-stage direct-to-implant post-mastectomy breast reconstruction has been gaining popularity over the traditional two-stage/tissue-expander approach., Objectives: To evaluate the outcome of the two post-mastectomy breast reconstruction procedures in terms of patient satisfaction., Methods: Clinical data were collected by file review for patients who underwent mastectomy with immediate breast reconstruction at two tertiary medical centers in 2010-2013. Patients were asked to complete the BREAST-Q instrument, sent to them by post with a self-addressed, stamped, return envelope. Scores were compared by type of reconstruction performed., Results: Of the 92 patients who received the questionnaire, 59 responded: 39 had one-stage breast reconstruction and 20 underwent two-stage reconstruction. The two-stage reconstruction group was significantly older, had more background diseases, and were followed for a longer period. The one-stage reconstruction group had a higher proportion of BRCA mutation carriers. There was no significant between-group difference in postoperative complications. Mean BREAST-Q scores were similar in the two groups for all dimensions except satisfaction with information, which was higher in the patients after one-stage reconstruction. Women with more background diseases had better sexual well-being, and married women had better psychological well-being. Breast satisfaction was lower among patients treated with radiation and higher among patients with bilateral reconstruction; the latter subgroup also had higher physical well-being. Complications did not affect satisfaction., Conclusions: Patients were equally satisfied with the outcome of one- and two-stage breast reconstruction. The choice of technique should be made on a case-by-case basis. Cost analyses are needed to construct a decision-making algorithm.

Soft and stretchable materials play an important role in the emerging fields of soft robotics, human-machine interfaces, and stretchable electronics. Hydrogels are compelling materials because they are soft, chemically tunable, biocompatible, and ionically conductive. Hydrogels have been used as components of skin mountable sensors, such as electrocardiogram (ECG) electrodes, and show promise in emerging devices as stretchable, transparent electrodes. Ultimately, these types of devices interface the hydrogel with rigid metallic electrodes to connect with electronic circuitry. Here, we show it is possible to interface hydrogel with liquid metal (eutectic gallium indium, EGaIn) electrodes to create completely soft and deformable electrodes that provide low resistance traces through the gel without altering its mechanical properties. As a case study, we created and tested electrodes for ECG monitoring. ECG electrodes require low impedance at biomedically relevant frequencies (1-50 Hz). Potentiostatic electrochemical impedance spectroscopy measurements show that capacitive effects at the hydrogel-EGaIn interface dominate the impedance at these low frequencies, yet can be reduced by interfacing the metal with acidic or basic hydrogels that remove the native oxide skin from the metal. Increasing the ionic strength of the hydrogel also helps in lowering the impedance of the metal-hydrogel electrodes. The resulting devices have signal-to-noise ratios that exceed commercial ECG electrodes. The softness of these hydrogels can be modified without compromising the electrical properties to create truly soft electrodes. Interfacing liquid metal conductors with hydrogels represents a potential strategy of creating soft electrodes for various bioelectronic applications, e-skins, and next-generation soft robotics.

γδ T cells are situated at barrier sites and guard the body from infection and damage. However, little is known about their roles outside of host defense in nonbarrier tissues. Here, we characterize a highly enriched tissue-resident population of γδ T cells in adipose tissue that regulate age-dependent regulatory T cell (T reg ) expansion and control core body temperature in response to environmental fluctuations. Mechanistically, innate PLZF + γδ T cells produced tumor necrosis factor and interleukin (IL) 17 A and determined PDGFRα + and Pdpn + stromal-cell production of IL-33 in adipose tissue. Mice lacking γδ T cells or IL-17A exhibited decreases in both ST2 + T reg cells and IL-33 abundance in visceral adipose tissue. Remarkably, these mice also lacked the ability to regulate core body temperature at thermoneutrality and after cold challenge. Together, these findings uncover important physiological roles for resident γδ T cells in adipose tissue immune homeostasis and body-temperature control.

It is well established that inducible transcription is essential for the consolidation of salient experiences into long-term memory. However, whether inducible transcription relays information about the identity and affective attributes of the experience being encoded, has not been explored. To this end, we analyzed transcription induced by a variety of rewarding and aversive experiences, across multiple brain regions. Our results describe the existence of robust transcriptional signatures uniquely representing distinct experiences, enabling near-perfect decoding of recent experiences. Furthermore, experiences with shared attributes display commonalities in their transcriptional signatures, exemplified in the representation of valence, habituation and reinforcement. This study introduces the concept of a neural transcriptional code, which represents the encoding of experiences in the mouse brain. This code is comprised of distinct transcriptional signatures that correlate to attributes of the experiences that are being committed to long-term memory., Competing Interests: DM, BI, EI, BG, HT, LI, DH, NB, CC, IA, TS, BG, AZ, AC No competing interests declared, (© 2018, Mukherjee et al.)

Recent advances in single-cell RNA-sequencing (scRNA-seq) technology increase the understanding of immune differentiation and activation processes, as well as the heterogeneity of immune cell types. Although the number of available immune-related scRNA-seq datasets increases rapidly, their large size and various formats render them hard for the wider immunology community to use, and read-level data are practically inaccessible to the non-computational immunologist. To facilitate datasets reuse, we created the JingleBells repository for immune-related scRNA-seq datasets ready for analysis and visualization of reads at the single-cell level (http://jinglebells.bgu.ac.il/). To this end, we collected the raw data of publicly available immune-related scRNA-seq datasets, aligned the reads to the relevant genome, and saved aligned reads in a uniform format, annotated for cell of origin. We also added scripts and a step-by-step tutorial for visualizing each dataset at the single-cell level, through the commonly used Integrated Genome Viewer (www.broadinstitute.org/igv/). The uniform scRNA-seq format used in JingleBells can facilitate reuse of scRNA-seq data by computational biologists. It also enables immunologists who are interested in a specific gene to visualize the reads aligned to this gene to estimate cell-specific preferences for splicing, mutation load, or alleles. Thus JingleBells is a resource that will extend the usefulness of scRNA-seq datasets outside the programming aficionado realm., (Copyright © 2017 by The American Association of Immunologists, Inc.)

This paper describes a technique that utilizes the osmotic properties of hydrogels to passively draw fluid through a membrane and pass it along to a microfluidic network for sensing purposes. This technique may enable non-invasive collection and manipulation of sweat for biosensing. To demonstrate the concept, thin hydrogel discs equilibrated in saline or glycerol were integrated with a microfluidic device. The hydrogel interfaces with a water-permeable membrane. The high concentration of solute in the hydrogel creates an osmotic pressure difference across the membrane, driving fluid flow through the membrane and into the device. The release of solute from the hydrogel autonomously pumps the fluid into an adjacent microfluidic channel. The flowrate of liquid drawn through the membrane is controlled by the osmotic pressure of the hydrogel and its interfacial contact area with the membrane. The flowrate gradually decreases over time as the continuous influx of withdrawn fluid dilutes the concentrated solute in the hydrogel. Initial testing has shown the device can pump accurate levels of glucose across the membrane and through a microchannel to a reservoir with a glucose sensor. Sensors and electrodes can be implemented in future microfluidic devices operating on these principles to test for other bioanalytes in sweat.

Hematopoietic stem cells (HSCs) are rare cells that generate all the various types of blood and immune cells. High-quality transcriptome data have enabled the identification of significant genes for HSCs. However, most genes are expressed in various forms by alternative splicing (AS), extending transcriptome complexity. Here, we delineate AS to determine which isoforms are expressed in mouse HSCs. Our analysis of microarray and RNA-sequencing data includes differential expression of splicing factors that may regulate AS, and a complete map of splicing isoforms. Multiple types of isoforms for known HSC genes and unannotated splicing that may alter gene function are presented. Transcriptome-wide identification of genes and their respective isoforms in mouse HSCs will open another dimension for adult stem cells., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

We report multi-responsive and double-folding bilayer hydrogel sheet actuators, whose directional bending response is tuned by modulating the solvent quality and temperature and where locally crosslinked regions, induced by ionoprinting, enable the actuators to invert their bending axis. The sheets are made multi-responsive by combining two stimuli responsive gels that incur opposing and complementary swelling and shrinking responses to the same stimulus. The lower critical solution temperature (LCST) can be tuned to specific temperatures depending on the EtOH concentration, enabling the actuators to change direction isothermally. Higher EtOH concentrations cause upper critical solution temperature (UCST) behavior in the poly( N -isopropylacrylamide) (pNIPAAm) gel networks, which can induce an amplifying effect during bilayer bending. External ionoprints reliably and repeatedly invert the gel bilayer bending axis between water and EtOH. Placing the ionoprint at the gel/gel interface can lead to opposite shape conformations, but with no clear trend in the bending behavior. We hypothesize that this is due to the ionoprint passing through the neutral axis of the bilayer during shrinking in hot water. Finally, we demonstrate the ability of the actuators to achieve shapes unique to the specific external conditions towards developing more responsive and adaptive soft actuator devices.

Problem/condition: Aquatic facility-associated illness and injury in the United States include disease outbreaks of infectious or chemical etiology, drowning, and pool chemical-associated health events (e.g., respiratory distress or burns). These conditions affect persons of all ages, particularly young children, and can lead to disability or even death. A total of 650 aquatic facility-associated outbreaks have been reported to CDC for 1978-2012. During 1999-2010, drownings resulted in approximately 4,000 deaths each year in the United States. Drowning is the leading cause of injury deaths in children aged 1-4 years, and approximately half of fatal drownings in this age group occur in swimming pools. During 2003-2012, pool chemical-associated health events resulted in an estimated 3,000-5,000 visits to U.S. emergency departments each year, and approximately half of the patients were aged <18 years. In August 2014, CDC released the Model Aquatic Health Code (MAHC), national guidance that can be adopted voluntarily by state and local jurisdictions to minimize the risk for illness and injury at public aquatic facilities., Reporting Period Covered: 2013., Description of System: The Network for Aquatic Facility Inspection Surveillance (NAFIS) was established by CDC in 2013. NAFIS receives aquatic facility inspection data collected by environmental health practitioners when assessing the operation and maintenance of public aquatic facilities. This report presents inspection data that were reported by 16 public health agencies in five states (Arizona, California, Florida, New York, and Texas) and focuses on 15 MAHC elements deemed critical to minimizing the risk for illness and injury associated with aquatic facilities (e.g., disinfection to prevent transmission of infectious pathogens, safety equipment to rescue distressed bathers, and pool chemical safety). Although these data (the first and most recent that are available) are not nationally representative, 15.7% of the estimated 309,000 U.S. public aquatic venues are located in the 16 reporting jurisdictions., Results: During 2013, environmental health practitioners in the 16 reporting NAFIS jurisdictions conducted 84,187 routine inspections of 48,632 public aquatic venues. Of the 84,187 routine inspection records for individual aquatic venues, 78.5% (66,098) included data on immediate closure; 12.3% (8,118) of routine inspections resulted in immediate closure because of at least one identified violation that represented a serious threat to public health. Disinfectant concentration violations were identified during 11.9% (7,662/64,580) of routine inspections, representing risk for aquatic facility-associated outbreaks of infectious etiology. Safety equipment violations were identified during 12.7% (7,845/61,648) of routine inspections, representing risk for drowning. Pool chemical safety violations were identified during 4.6% (471/10,264) of routine inspections, representing risk for pool chemical-associated health events., Interpretation: Routine inspections frequently resulted in immediate closure and identified violations of inspection items corresponding to 15 MAHC elements critical to protecting public health, highlighting the need to improve operation and maintenance of U.S. public aquatic facilities. These findings also underscore the public health function that code enforcement, conducted by environmental health practitioners, has in preventing illness and injury at public aquatic facilities., Public Health Action: Findings from the routine analyses of aquatic facility inspection data can inform program planning, implementation, and evaluation. At the state and local level, these inspection data can be used to identify aquatic facilities and venues in need of more frequent inspections and to select topics to cover in training for aquatic facility operators. At the national level, these data can be used to evaluate whether the adoption of MAHC elements minimizes the risk for aquatic facility-associated illness and injury. These findings also can be used to prioritize revisions or updates to the MAHC. To optimize the collection and analysis of aquatic facility inspection data and thus application of findings, environmental health practitioners and epidemiologists need to collaborate extensively to identify public aquatic facility code elements deemed critical to protecting public health and determine the best way to assess and document compliance during inspections.

Background: HOX genes are a family of developmental genes that are expressed neither in the developing forebrain nor in the normal brain. Aberrant expression of a HOX-gene dominated stem-cell signature in glioblastoma has been linked with increased resistance to chemo-radiotherapy and sustained proliferation of glioma initiating cells. Here we describe the epigenetic and genetic alterations and their interactions associated with the expression of this signature in glioblastoma., Results: We observe prominent hypermethylation of the HOXA locus 7p15.2 in glioblastoma in contrast to non-tumoral brain. Hypermethylation is associated with a gain of chromosome 7, a hallmark of glioblastoma, and may compensate for tumor-driven enhanced gene dosage as a rescue mechanism by preventing undue gene expression. We identify the CpG island of the HOXA10 alternative promoter that appears to escape hypermethylation in the HOX-high glioblastoma. An additive effect of gene copy gain at 7p15.2 and DNA methylation at key regulatory CpGs in HOXA10 is significantly associated with HOX-signature expression. Additionally, we show concordance between methylation status and presence of active or inactive chromatin marks in glioblastoma-derived spheres that are HOX-high or HOX-low, respectively., Conclusions: Based on these findings, we propose co-evolution and interaction between gene copy gain, associated with a gain of chromosome 7, and additional epigenetic alterations as key mechanisms triggering a coordinated, but inappropriate, HOX transcriptional program in glioblastoma.

Immunological studies of single proteins in a single cell type have been complemented in recent years by larger studies, enabled by emerging high-throughput technologies. This trend has recently been exemplified by the discovery of gene networks controlling regulatory and effector αβ T cell subset development and human hematopoiesis. The Immunological Genome Project (ImmGen) aims to decipher the gene networks underpinning mouse hematopoiesis. The first phase, completed in 2012, profiled the transcriptome of 249 immune cell types. We discuss the utilities of the datasets in high-resolution mapping of the hematopoietic system. The immune transcriptome compendium has revealed unsuspected cell lineage relations and the network reconstruction has identified novel regulatory factors of hematopoiesis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Hematopoietic stem cells (HSCs) maintain blood homeostasis and are the functional units of bone marrow transplantation. To improve the molecular understanding of HSCs and their proximal progenitors, we performed transcriptome analysis within the context of the ImmGen Consortium data set. Gene sets that define steady-state and mobilized HSCs, as well as hematopoietic stem and progenitor cells (HSPCs), were determined. Genes involved in transcriptional regulation, including a group of putative transcriptional repressors, were identified in multipotent progenitors and HSCs. Proximal promoter analyses combined with ImmGen module analysis identified candidate regulators of HSCs. Enforced expression of one predicted regulator, Hlf, in diverse HSPC subsets led to extensive self-renewal activity ex vivo. These analyses reveal unique insights into the mechanisms that control the core properties of HSPCs.

The differentiation of αβT cells from thymic precursors is a complex process essential for adaptive immunity. Here we exploited the breadth of expression data sets from the Immunological Genome Project to analyze how the differentiation of thymic precursors gives rise to mature T cell transcriptomes. We found that early T cell commitment was driven by unexpectedly gradual changes. In contrast, transit through the CD4(+)CD8(+) stage involved a global shutdown of housekeeping genes that is rare among cells of the immune system and correlated tightly with expression of the transcription factor c-Myc. Selection driven by major histocompatibility complex (MHC) molecules promoted a large-scale transcriptional reactivation. We identified distinct signatures that marked cells destined for positive selection versus apoptotic deletion. Differences in the expression of unexpectedly few genes accompanied commitment to the CD4(+) or CD8(+) lineage, a similarity that carried through to peripheral T cells and their activation, demonstrated by mass cytometry phosphoproteomics. The transcripts newly identified as encoding candidate mediators of key transitions help define the 'known unknowns' of thymocyte differentiation.

Much of the knowledge about cell differentiation and function in the immune system has come from studies in mice, but the relevance to human immunology, diseases, and therapy has been challenged, perhaps more from anecdotal than comprehensive evidence. To this end, we compare two large compendia of transcriptional profiles of human and mouse immune cell types. Global transcription profiles are conserved between corresponding cell lineages. The expression patterns of most orthologous genes are conserved, particularly for lineage-specific genes. However, several hundred genes show clearly divergent expression across the examined cell lineages, and among them, 169 genes did so even with highly stringent criteria. Finally, regulatory mechanisms--reflected by regulators' differential expression or enriched cis-elements--are conserved between the species but to a lower degree, suggesting that distinct regulation may underlie some of the conserved transcriptional responses.

Invariant natural killer T cells (iNKT cells) are innate-like T lymphocytes that act as critical regulators of the immune response. To better characterize this population, we profiled gene expression in iNKT cells during ontogeny and in peripheral subsets as part of the Immunological Genome Project. High-resolution comparative transcriptional analyses defined developmental and subset-specific programs of gene expression by iNKT cells. In addition, we found that iNKT cells shared an extensive transcriptional program with NK cells, similar in magnitude to that shared with major histocompatibility complex (MHC)-restricted T cells. Notably, the program shared by NK cells and iNKT cells also operated constitutively in γδ T cells and in adaptive T cells after activation. Together our findings highlight a core effector program regulated distinctly in innate and adaptive lymphocytes.

We assessed gene expression in tissue macrophages from various mouse organs. The diversity in gene expression among different populations of macrophages was considerable. Only a few hundred mRNA transcripts were selectively expressed by macrophages rather than dendritic cells, and many of these were not present in all macrophages. Nonetheless, well-characterized surface markers, including MerTK and FcγR1 (CD64), along with a cluster of previously unidentified transcripts, were distinctly and universally associated with mature tissue macrophages. TCEF3, C/EBP-α, Bach1 and CREG-1 were among the transcriptional regulators predicted to regulate these core macrophage-associated genes. The mRNA encoding other transcription factors, such as Gata6, was associated with single macrophage populations. We further identified how these transcripts and the proteins they encode facilitated distinguishing macrophages from dendritic cells.

Although much progress has been made in the understanding of the ontogeny and function of dendritic cells (DCs), the transcriptional regulation of the lineage commitment and functional specialization of DCs in vivo remains poorly understood. We made a comprehensive comparative analysis of CD8(+), CD103(+), CD11b(+) and plasmacytoid DC subsets, as well as macrophage DC precursors and common DC precursors, across the entire immune system. Here we characterized candidate transcriptional activators involved in the commitment of myeloid progenitor cells to the DC lineage and predicted regulators of DC functional diversity in tissues. We identified a molecular signature that distinguished tissue DCs from macrophages. We also identified a transcriptional program expressed specifically during the steady-state migration of tissue DCs to the draining lymph nodes that may control tolerance to self tissue antigens.

The signaling pathways that commit cells to migration are incompletely understood. We employed human mammary cells and two stimuli: epidermal growth factor (EGF), which induced cellular migration, and serum factors, which stimulated cell growth. In addition to strong activation of ERK by EGF, and AKT by serum, early transcription remarkably differed: while EGF induced early growth response-1 (EGR1), and this was required for migration, serum induced c-Fos and FosB to enhance proliferation. We demonstrate that induction of EGR1 involves ERK-mediated down-regulation of microRNA-191 and phosphorylation of the ETS2 repressor factor (ERF) repressor, which subsequently leaves the nucleus. Unexpectedly, knockdown of ERF inhibited migration, which implies migratory roles for exported ERF molecules. On the other hand, chromatin immunoprecipitation identified a subset of direct EGR1 targets, including EGR1 autostimulation and SERPINB2, whose transcription is essential for EGF-induced cell migration. In summary, EGR1 and the EGF-ERK-ERF axis emerge from our study as major drivers of growth factor-induced mammary cell migration.

Aberrant activation of kinases has emerged to be a key event along with tumor progression, maintenance of tumor phenotype and response to anticancer treatments. This study documents the existence of an oncogenic auto-regulatory feedback loop that includes the Polo-like kinase-2 (Snk/Plk2) and mutant p53 proteins. Plk2 protein binds to and phosphorylates mutant p53, thereby potentiating its oncogenic activities. Phosphorylated mutant p53 binds more efficiently to p300 consequently strengthening its own transcriptional activity. Plk2 gene is regulated at a transcriptional level by both wt- and mutant p53 proteins. This leads to growth suppression or enhanced cell proliferation and chemo-resistance, respectively. In turn, the siRNA-mediated knock down of either mutant p53 or Plk2 proteins significantly curtails the growth properties of tumor cells and their chemo-resistance to anticancer treatments. Therefore, this paper identifies a novel tumor network including Plk2 and mutant p53 proteins whose triggering in response to DNA damage might disclose important implications for the treatment of human cancers.

Gene expression-based prediction of genomic copy number aberrations in the chromosomal region 12q13 to 12q15 that is flanked by MDM2 and CDK4 identified Wnt inhibitory factor 1 (WIF1) as a candidate tumor suppressor gene in glioblastoma. WIF1 encodes a secreted Wnt antagonist and was strongly downregulated in most glioblastomas as compared with normal brain, implying deregulation of Wnt signaling, which is associated with cancer. WIF1 silencing was mediated by deletion (7/69, 10%) or epigenetic silencing by promoter hypermethylation (29/110, 26%). Co-amplification of MDM2 and CDK4 that is present in 10% of glioblastomas was associated in most cases with deletion of the whole genomic region enclosed, including the WIF1 locus. This interesting pathogenetic constellation targets the RB and p53 tumor suppressor pathways in tandem, while simultaneously activating oncogenic Wnt signaling. Ectopic expression of WIF1 in glioblastoma cell lines revealed a dose-dependent decrease of Wnt pathway activity. Furthermore, WIF1 expression inhibited cell proliferation in vitro, reduced anchorage-independent growth in soft agar, and completely abolished tumorigenicity in vivo. Interestingly, WIF1 overexpression in glioblastoma cells induced a senescence-like phenotype that was dose dependent. These results provide evidence that WIF1 has tumor suppressing properties. Downregulation of WIF1 in 75% of glioblastomas indicates frequent involvement of aberrant Wnt signaling and, hence, may render glioblastomas sensitive to inhibitors of Wnt signaling, potentially by diverting the tumor cells into a senescence-like state.

Normal cells require continuous exposure to growth factors in order to cross a restriction point and commit to cell-cycle progression. This can be replaced by two short, appropriately spaced pulses of growth factors, where the first pulse primes a process, which is completed by the second pulse, and enables restriction point crossing. Through integration of comprehensive proteomic and transcriptomic analyses of each pulse, we identified three processes that regulate restriction point crossing: (1) The first pulse induces essential metabolic enzymes and activates p53-dependent restraining processes. (2) The second pulse eliminates, via the PI3K/AKT pathway, the suppressive action of p53, as well as (3) sets an ERK-EGR1 threshold mechanism, which digitizes graded external signals into an all-or-none decision obligatory for S phase entry. Together, our findings uncover two gating mechanisms, which ensure that cells ignore fortuitous growth factors and undergo proliferation only in response to consistent mitogenic signals., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Though many individual transcription factors are known to regulate hematopoietic differentiation, major aspects of the global architecture of hematopoiesis remain unknown. Here, we profiled gene expression in 38 distinct purified populations of human hematopoietic cells and used probabilistic models of gene expression and analysis of cis-elements in gene promoters to decipher the general organization of their regulatory circuitry. We identified modules of highly coexpressed genes, some of which are restricted to a single lineage but most of which are expressed at variable levels across multiple lineages. We found densely interconnected cis-regulatory circuits and a large number of transcription factors that are differentially expressed across hematopoietic states. These findings suggest a more complex regulatory system for hematopoiesis than previously assumed., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The genetic profiling of B-cell malignancies is rapidly expanding, providing important information on the tumorigenic potential, response to treatment, and clinical outcome of these diseases. However, the relative contributions of inherent gene expression versus microenvironmental effects are poorly understood. The regulation of gene expression programs by means of adhesive interactions was studied here in ARH-77 human malignant B-cell variants, derived from the same cell line by selective adhesion to a fibronectin matrix. The populations included cells that adhere to fibronectin and are highly tumorigenic (designated "type A" cells) and cells that fail to adhere to fibronectin and fail to develop tumors in vivo ("type F" cells). To identify genes directly affected by cell adhesion to fibronectin, type A cells deprived of an adhesive substrate (designated "AF cells") were also examined. Bioinformatic analyses revealed a remarkable correlation between cell adhesion and both B-cell differentiation state and the expression of multiple myeloma (MM)-associated genes. The highly adherent type A cells expressed higher levels of NFkappaB-regulated genes, many of them associated with MM. Moreover, we found that the transcription of several MM-related proto-oncogenes is stimulated by adhesion to fibronectin. In contrast, type F cells, which display poor adhesive and tumorigenic properties, expressed genes associated with higher levels of B-cell differentiation. Our findings indicate that B-cell differentiation, as manifested by gene expression profiles, is attenuated by cell adhesion to fibronectin, leading to upregulation of specific genes known to be associated with the pathogenesis of MM., ((c) 2010 AACR.)

Models of mammalian regulatory networks controlling gene expression have been inferred from genomic data but have largely not been validated. We present an unbiased strategy to systematically perturb candidate regulators and monitor cellular transcriptional responses. We applied this approach to derive regulatory networks that control the transcriptional response of mouse primary dendritic cells to pathogens. Our approach revealed the regulatory functions of 125 transcription factors, chromatin modifiers, and RNA binding proteins, which enabled the construction of a network model consisting of 24 core regulators and 76 fine-tuners that help to explain how pathogen-sensing pathways achieve specificity. This study establishes a broadly applicable, comprehensive, and unbiased approach to reveal the wiring and functions of a regulatory network controlling a major transcriptional response in primary mammalian cells.

ID4 (inhibitor of DNA binding 4) is a member of a family of proteins that function as dominant-negative regulators of basic helix-loop-helix transcription factors. Growing evidence links ID proteins to cell proliferation, differentiation and tumorigenesis. Here we identify ID4 as a transcriptional target of gain-of-function p53 mutants R175H, R273H and R280K. Depletion of mutant p53 protein severely impairs ID4 expression in proliferating tumor cells. The protein complex mutant p53-E2F1 assembles on specific regions of the ID4 promoter and positively controls ID4 expression. The ID4 protein binds to and stabilizes mRNAs encoding pro-angiogenic factors IL8 and GRO-alpha. This results in the increase of the angiogenic potential of cancer cells expressing mutant p53. These findings highlight the transcriptional axis mutant p53, E2F1 and ID4 as a still undefined molecular mechanism contributing to tumor neo-angiogenesis.

Transcriptional regulatory circuits govern how cis and trans factors transform signals into messenger RNA (mRNA) expression levels. With advances in quantitative and high-throughput technologies that allow measurement of gene expression state in different conditions, data that can be used to build and test models of transcriptional regulation is being generated at a rapid pace. Here, we review experimental and computational methods used to derive detailed quantitative circuit models on a small scale and cruder, genome-wide models on a large scale. We discuss the potential of combining small- and large-scale approaches to understand the working and wiring of transcriptional regulatory circuits.

Many types of tumors exhibit characteristic chromosomal losses or gains, as well as local amplifications and deletions. Within any given tumor type, sample specific amplifications and deletions are also observed. Typically, a region that is aberrant in more tumors, or whose copy number change is stronger, would be considered as a more promising candidate to be biologically relevant to cancer. We sought for an intuitive method to define such aberrations and prioritize them. We define V, the "volume" associated with an aberration, as the product of three factors: (a) fraction of patients with the aberration, (b) the aberration's length and (c) its amplitude. Our algorithm compares the values of V derived from the real data to a null distribution obtained by permutations, and yields the statistical significance (p-value) of the measured value of V. We detected genetic locations that were significantly aberrant, and combine them with chromosomal arm status (gain/loss) to create a succinct fingerprint of the tumor genome. This genomic fingerprint is used to visualize the tumors, highlighting events that are co-occurring or mutually exclusive. We apply the method on three different public array CGH datasets of Medulloblastoma and Neuroblastoma, and demonstrate its ability to detect chromosomal regions that were known to be altered in the tested cancer types, as well as to suggest new genomic locations to be tested. We identified a potential new subtype of Medulloblastoma, which is analogous to Neuroblastoma type 1.

Research on phenotypic markers of vulnerability to bipolar disorder has focused on the identification of personality traits uniquely associated with the illness. To expand knowledge in this area, we compared Cloninger's seven temperament and character dimensions in 85 euthymic/subsyndromal bipolar I inpatients and outpatients and 85 age and sex matched community controls. We also examined associations between Cloninger's personality traits and mood state in the patient group. Bipolar subjects were administered the Structured Clinical Interview for DSM-IV (SCID), Hamilton Rating Scale for Depression, and Clinician-Administered Rating Scale for Mania. Controls received the SCID, a family psychiatric history questionnaire, and urine toxicology screen to confirm healthy status. Both groups competed the 240-item Temperament and Character Inventory (TCI). A multivariate analysis of covariance, accounting for demographic factors, was conducted to compare the groups on the TCI. Bipolar I patients scored higher on harm avoidance, lower on self-directedness, and higher on self-transcendence compared to controls. Harm avoidance and self-directedness were correlated with residual depressive symptoms positively and negatively, respectively; persistence was correlated with residual manic symptoms; and selftranscendence was correlated with residual psychotic symptoms in patients. The results indicate that bipolar I subjects do possess personality traits that are significantly different from non-ill individuals. However, only a prospective, longitudinal study may determine whether these traits mark a vulnerability to the disorder, or represent the scarring effect of affective episodes and chronic subsyndromal symptoms.

A technique that employs two seed signals for the purpose of mitigating stimulated Brillouin scattering (SBS) effects in narrow-linewidth Yb-doped fiber amplifiers is investigated theoretically by constructing a self-consistent model that incorporates the laser gain, SBS, and four-wave mixing (FWM). The model reduces to solving a two-point boundary problem consisting of an 8x8 system of coupled nonlinear differential equations. Optimal operating conditions are determined by examining the interplay between the wavelength separation and power ratio of the two seeds. Two variants of this 'two-tone' amplification are considered. In one case the wavelength separation is precisely twice the Brillouin shift, while the other case considers a greater wavelength separation. For the former case, a two-fold increase in total output power over a broad range of seed power ratios centered about a ratio of approximately 2 is obtained, but with fairly large FWM. For the latter case, this model predicts an approximately 100% increase in output power (at SBS threshold with no signs of FWM) for a 'two-tone' amplifier with seed signals at 1064nm and 1068nm, compared to a conventional fiber amplifier with a single 1068nm seed. More significantly for this case, it is found that at a wavelength separation greater than 10nm, it is possible to appreciably enhance the power output of one of the laser frequencies.

Purpose: Glioblastomas are notorious for resistance to therapy, which has been attributed to DNA-repair proficiency, a multitude of deregulated molecular pathways, and, more recently, to the particular biologic behavior of tumor stem-like cells. Here, we aimed to identify molecular profiles specific for treatment resistance to the current standard of care of concomitant chemoradiotherapy with the alkylating agent temozolomide., Patients and Methods: Gene expression profiles of 80 glioblastomas were interrogated for associations with resistance to therapy. Patients were treated within clinical trials testing the addition of concomitant and adjuvant temozolomide to radiotherapy., Results: An expression signature dominated by HOX genes, which comprises Prominin-1 (CD133), emerged as a predictor for poor survival in patients treated with concomitant chemoradiotherapy (n = 42; hazard ratio = 2.69; 95% CI, 1.38 to 5.26; P = .004). This association could be validated in an independent data set. Provocatively, the HOX cluster was reminiscent of a "self-renewal" signature (P = .008; Gene Set Enrichment Analysis) recently characterized in a mouse leukemia model. The HOX signature and EGFR expression were independent prognostic factors in multivariate analysis, adjusted for the O-6-methylguanine-DNA methyltransferase (MGMT) methylation status, a known predictive factor for benefit from temozolomide, and age. Better outcome was associated with gene clusters characterizing features of tumor-host interaction including tumor vascularization and cell adhesion, and innate immune response., Conclusion: This study provides first clinical evidence for the implication of a "glioma stem cell" or "self-renewal" phenotype in treatment resistance of glioblastoma. Biologic mechanisms identified here to be relevant for resistance will guide future targeted therapies and respective marker development for individualized treatment and patient selection.

Background: Cell migration is a highly complex process, regulated by multiple genes, signaling pathways and external stimuli. To discover genes or pharmacological agents that can modulate the migratory activity of cells, screening strategies that enable the monitoring of diverse migratory parameters in a large number of samples are necessary., Methodology: In the present study, we describe the development of a quantitative, high-throughput cell migration assay, based on a modified phagokinetic tracks (PKT) procedure, and apply it for identifying novel pro-migratory genes in a cancer-related gene library. In brief, cells are seeded on fibronectin-coated 96-well plates, covered with a monolayer of carboxylated latex beads. Motile cells clear the beads, located along their migratory paths, forming tracks that are visualized using an automated, transmitted-light screening microscope. The tracks are then segmented and characterized by multi-parametric, morphometric analysis, resolving a variety of morphological and kinetic features., Conclusions: In this screen we identified 4 novel genes derived from breast carcinoma related cDNA library, whose over-expression induces major alteration in the migration of the stationary MCF7 cells. This approach can serve for high throughput screening for novel ways to modulate cellular migration in pathological states such as tumor metastasis and invasion.

Cell migration driven by the epidermal growth factor receptor (EGFR) propels morphogenesis and involves reorganization of the actin cytoskeleton. Although de novo transcription precedes migration, transcript identity remains largely unknown. Through their actin-binding domains, tensins link the cytoskeleton to integrin-based adhesion sites. Here we report that EGF downregulates tensin-3 expression, and concomitantly upregulates cten, a tensin family member that lacks the actin-binding domain. Knockdown of cten or tensin-3, respectively, impairs or enhances mammary cell migration. Furthermore, cten displaces tensin-3 from the cytoplasmic tail of integrin beta1, thereby instigating actin fibre disassembly. In invasive breast cancer, cten expression correlates not only with high EGFR and HER2, but also with metastasis to lymph nodes. Moreover, treatment of inflammatory breast cancer patients with an EGFR/HER2 dual-specificity kinase inhibitor significantly downregulated cten expression. In conclusion, a transcriptional tensin-3-cten switch may contribute to the metastasis of mammary cancer.

Signaling pathways invoke interplays between forward signaling and feedback to drive robust cellular response. In this study, we address the dynamics of growth factor signaling through profiling of protein phosphorylation and gene expression, demonstrating the presence of a kinetically defined cluster of delayed early genes that function to attenuate the early events of growth factor signaling. Using epidermal growth factor receptor signaling as the major model system and concentrating on regulation of transcription and mRNA stability, we demonstrate that a number of genes within the delayed early gene cluster function as feedback regulators of immediate early genes. Consistent with their role in negative regulation of cell signaling, genes within this cluster are downregulated in diverse tumor types, in correlation with clinical outcome. More generally, our study proposes a mechanistic description of the cellular response to growth factors by defining architectural motifs that underlie the function of signaling networks.

Primary immune response to pathogens involves the maturation of antigen-presenting dendritic cells (DC). Bacterial lipopolysacharride (LPS) is a potent inducer of DC maturation, whereas the transforming growth factor beta (TGFbeta) attenuates much of this process. Here, we analyzed the global gene expression pattern in LPS-treated bone marrow derived DC during inhibition of their maturation process by TGFbeta. Exposure of DC to LPS induces a pronounced cell response, manifested in altered expression of a large number of genes. Interestingly, TGFbeta did not affect most of the LPS responding genes. Nevertheless, analysis identified a subset of genes that did respond to TGFbeta, among them the two inflammatory cytokines interleukin (IL)-12 and IL-18. Expression of IL-12, the major proinflammatory cytokine secreted by mature DC, was downregulated by TGFbeta, whereas the expression level of the proinflammatory cytokine IL-18, known to potentiate the IL-12 effect, was upregulated. Expression of the peroxisome proliferator-activated receptor gamma (PPARgamma) increased in response to TGFbeta, concomitantly with reduced expression of chemokine receptor 7 (CCR7). This finding supports the possibility that TGFbeta-dependent inhibition of CCR7 expression in DC is mediated by PPARgamma.

A novel, highly accurate, all electronic technique for phase locking arrays of optical fibers is demonstrated. We report the first demonstration of the only electronic phase locking technique that doesn't require a reference beam. The measured phase error is lambda/20. Excellent phase locking has been demonstrated for fiber amplifier arrays.