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2.  Tiered Human Integrated Sequence Search Databases for Shotgun Proteomics 
Journal of proteome research  2016;15(11):4091-4100.
The results of analysis of shotgun proteomics mass spectrometry data can be greatly affected by the selection of the reference protein sequence database against which the spectra are matched. For many species there are multiple sources from which somewhat different sequence sets can be obtained. This can lead to confusion about which database is best in which circumstances – a problem especially acute in human sample analysis. All sequence databases are genome-based, with sequences for the predicted gene and their protein translation products compiled. Our goal is to create a set of primary sequence databases that comprise the union of sequences from many of the different available sources and make the result easily available to the community. We have compiled a set of four sequence databases of varying sizes, from a small database consisting of only the ~20,000 primary isoforms plus contaminants to a very large database that includes almost all non-redundant protein sequences from several sources. This set of tiered, increasingly complete human protein sequence databases suitable for mass spectrometry proteomics sequence database searching is called the Tiered Human Integrated Search Proteome set. In order to evaluate the utility of these databases, we have analyzed two different data sets, one from the HeLa cell line and the other from normal human liver tissue, with each of the four tiers of database complexity. The result is that approximately 0.8%, 1.1%, and 1.5% additional peptides can be identified for Tiers 2, 3, and 4, respectively, as compared with the Tier 1 database, at substantially increasing computational cost. This increase in computational cost may be worth bearing if the identification of sequence variants or the discovery of sequences that are not present in the reviewed knowledge base entries is an important goal of the study. We find that it is useful to search a data set against a simpler database, and then check the uniqueness of the discovered peptides against a more complex database. We have set up an automated system that downloads all the source databases on the first of each month and automatically generates a new set of search databases and makes them available for download at http://www.peptideatlas.org/thisp/.
doi:10.1021/acs.jproteome.6b00445
PMCID: PMC5096980  PMID: 27577934
shotgun mass spectrometry; search databases; human
3.  Role of B-scan ocular ultrasound as an adjuvant for the clinical assessment of eyeball diseases: a pictorial essay 
Journal of Ultrasound  2014;18(3):265-277.
We report our experience in B-mode ocular ultrasonography, focusing on its contribution when the clinical examination proves to be difficult, mainly due to the existence of intraocular opacities of the ocular fundus or diagnostic doubts. We revise the ocular ultrasound technique, its indications and contraindications, comparing to the other imaging techniques. In our experience ultrasonography revealed pathological findings which confirmed the clinical suspicion in most of cases or provide additional information. With understanding of the indications for ultrasonography and proper examination technique, one can gather a vast amount of information not possible with clinical examination alone.
doi:10.1007/s40477-014-0153-y
PMCID: PMC4529413  PMID: 26261467
Ophthalmic ultrasonography; Cataract; Hyphema; Hypopyon; Hemovitreous; Vogt–Koyanagi–Harada
4.  Modeling the Pro-inflammatory Tumor Microenvironment in Acute Lymphoblastic Leukemia Predicts a Breakdown of Hematopoietic-Mesenchymal Communication Networks 
Lineage fate decisions of hematopoietic cells depend on intrinsic factors and extrinsic signals provided by the bone marrow microenvironment, where they reside. Abnormalities in composition and function of hematopoietic niches have been proposed as key contributors of acute lymphoblastic leukemia (ALL) progression. Our previous experimental findings strongly suggest that pro-inflammatory cues contribute to mesenchymal niche abnormalities that result in maintenance of ALL precursor cells at the expense of normal hematopoiesis. Here, we propose a molecular regulatory network interconnecting the major communication pathways between hematopoietic stem and progenitor cells (HSPCs) and mesenchymal stromal cells (MSCs) within the BM. Dynamical analysis of the network as a Boolean model reveals two stationary states that can be interpreted as the intercellular contact status. Furthermore, simulations describe the molecular patterns observed during experimental proliferation and activation. Importantly, our model predicts instability in the CXCR4/CXCL12 and VLA4/VCAM1 interactions following microenvironmental perturbation due by temporal signaling from Toll like receptors (TLRs) ligation. Therefore, aberrant expression of NF-κB induced by intrinsic or extrinsic factors may contribute to create a tumor microenvironment where a negative feedback loop inhibiting CXCR4/CXCL12 and VLA4/VCAM1 cellular communication axes allows for the maintenance of malignant cells.
doi:10.3389/fphys.2016.00349
PMCID: PMC4990565  PMID: 27594840
cancer systems biology; acute lymphoblastic leukemia; tumor microenvironment; CXCL12; pro-inflammatory bone marrow; early hematopoiesis; network modeling; dynamical systems
5.  The State of the Human Proteome in 2014/2015 as viewed through PeptideAtlas: enhancing accuracy and coverage through the AtlasProphet 
Journal of proteome research  2015;14(9):3461-3473.
The Human PeptideAtlas is a compendium of the highest quality peptide identifications from over 1000 shotgun mass spectrometry proteomics experiments collected from many different labs, all reanalyzed through a uniform processing pipeline. The latest 2015-03 build contains substantially more input data than past releases, is mapped to a recent version of our merged reference proteome, and uses improved informatics processing and the development of the AtlasProphet to provide the highest quality results. Within the set of ~20,000 neXtProt primary entries, 14,070 (70%) are confidently detected in the latest build, 5% are ambiguous, 9% are redundant, leaving the total percentage of proteins for which there are no mapping detections at just 16% (3166), all derived from over 133 million peptide-spectrum matches identifying more than 1 million distinct peptides using AtlasProphet to characterize and classify the protein matches. Improved handling for detection and presentation of single amino-acid variants (SAAVs) reveals the detection of 5,326 uniquely mapping SAAVs across 2,794 proteins. With such a large amount of data, the control of false positives is a challenge. We present the methodology and results for maintaining rigorous quality, along with a discussion of the implications of the remaining sources of errors in the build. We check our uncertainty estimates against a set of olfactory receptor proteins not expected to be present in the set. We show how the use of synthetic reference spectra can provide confirmatory evidence for claims of detection of proteins with weak evidence.
doi:10.1021/acs.jproteome.5b00500
PMCID: PMC4755269  PMID: 26139527
shotgun proteomics; tandem mass spectrometry; repositories; PeptideAtlas; Human Proteome Project; observed proteome
6.  reSpect: Software for Identification of High and Low Abundance Ion Species in Chimeric Tandem Mass Spectra 
Most shotgun proteomics data analysis workflows are based on the assumption that each fragment ion spectrum is explained by a single species of peptide ion isolated by the mass spectrometer; however, in reality mass spectrometers often isolate more than one peptide ion within the window of isolation that contributes to additional peptide fragment peaks in many spectra. We present a new tool called reSpect, implemented in the Trans-Proteomic Pipeline (TPP), that enables an iterative workflow whereby fragment ion peaks explained by a peptide ion identified in one round of sequence searching or spectral library search are attenuated based on the confidence of the identification, and then the altered spectrum is subjected to further rounds of searching. The reSpect tool is not implemented as a search engine, but rather as a post search engine processing step where only fragment ion intensities are altered. This enables the application of any search engine combination in the following iterations. Thus, reSpect is compatible with all other protein sequence database search engines as well as peptide spectral library search engines that are supported by the TPP. We show that while some datasets are highly amenable to chimeric spectrum identification and lead to additional peptide identification boosts of over 30% with as many as four different peptide ions identified per spectrum, datasets with narrow precursor ion selection only benefit from such processing at the level of a few percent. We demonstrate a technique that facilitates the determination of the degree to which a dataset would benefit from chimeric spectrum analysis. The reSpect tool is free and open source, provided within the TPP and available at the TPP website.
doi:10.1007/s13361-015-1252-5
PMCID: PMC4750398  PMID: 26419769
7.  A Network Model to Describe the Terminal Differentiation of B Cells 
PLoS Computational Biology  2016;12(1):e1004696.
Terminal differentiation of B cells is an essential process for the humoral immune response in vertebrates and is achieved by the concerted action of several transcription factors in response to antigen recognition and extracellular signals provided by T-helper cells. While there is a wealth of experimental data regarding the molecular and cellular signals involved in this process, there is no general consensus regarding the structure and dynamical properties of the underlying regulatory network controlling this process. We developed a dynamical model of the regulatory network controlling terminal differentiation of B cells. The structure of the network was inferred from experimental data available in the literature, and its dynamical behavior was analyzed by modeling the network both as a discrete and a continuous dynamical systems. The steady states of these models are consistent with the patterns of activation reported for the Naive, GC, Mem, and PC cell types. Moreover, the models are able to describe the patterns of differentiation from the precursor Naive to any of the GC, Mem, or PC cell types in response to a specific set of extracellular signals. We simulated all possible single loss- and gain-of-function mutants, corroborating the importance of Pax5, Bcl6, Bach2, Irf4, and Blimp1 as key regulators of B cell differentiation process. The model is able to represent the directional nature of terminal B cell differentiation and qualitatively describes key differentiation events from a precursor cell to terminally differentiated B cells.
Author Summary
Generation of antibody-producing cells through terminal B cell differentiation represents a good model to study the formation of multiple effector cells from a progenitor cell type. This process is controlled by the action of several molecules that maintain cell type specific programs in response to cytokines, antigen recognition and the direct contact with T helper cells, forming a complex regulatory network. While there is a large body of experimental data regarding some of the key molecules involved in this process and there have been several efforts to reconstruct the underlying regulatory network, a general consensus about the structure and dynamical behavior of this network is lacking. Moreover, it is not well understood how this network controls the establishment of specific B cell expression patterns and how it responds to specific external signals. We present a model of the regulatory network controlling terminal B cell differentiation and analyze its dynamical behavior under normal and mutant conditions. The model recovers the patterns of differentiation of B cells and describes a large set of gain- and loss-of-function mutants. This model provides an unified framework to generate qualitative descriptions to interpret the role of intra- and extracellular regulators of B cell differentiation.
doi:10.1371/journal.pcbi.1004696
PMCID: PMC4720151  PMID: 26751566
8.  A Boolean network model of human gonadal sex determination 
Background
Gonadal sex determination (GSD) in humans is a complex biological process that takes place in early stages of embryonic development when the bipotential gonadal primordium (BGP) differentiates towards testes or ovaries. This decision is directed by one of two distinct pathways embedded in a GSD network activated in a population of coelomic epithelial cells, the Sertoli progenitor cells (SPC) and the granulosa progenitor cells (GPC). In males, the pathway is activated when the Sex-Determining Region Y (SRY) gene starts to be expressed, whereas in females the WNT4/ β-catenin pathway promotes the differentiation of the GPCs towards ovaries. The interactions and dynamics of the elements that constitute the GSD network are poorly understood, thus our group is interested in inferring the general architecture of this network as well as modeling the dynamic behavior of a set of genes associated to this process under wild-type and mutant conditions.
Methods
We reconstructed the regulatory network of GSD with a set of genes directly associated with the process of differentiation from SPC and GPC towards Sertoli and granulosa cells, respectively. These genes are experimentally well-characterized and the effects of their deficiency have been clinically reported. We modeled this GSD network as a synchronous Boolean network model (BNM) and characterized its attractors under wild-type and mutant conditions.
Results
Three attractors with a clear biological meaning were found; one of them corresponding to the currently known gene expression pattern of Sertoli cells, the second correlating to the granulosa cells and, the third resembling a disgenetic gonad.
Conclusions
The BNM of GSD that we present summarizes the experimental data on the pathways for Sertoli and granulosa establishment and sheds light on the overall behavior of a population of cells that differentiate within the developing gonad. With this model we propose a set of regulatory interactions needed to activate either the SRY or the WNT4/ β-catenin pathway as well as their downstream targets, which are critical for further sex differentiation. In addition, we observed a pattern of altered regulatory interactions and their dynamics that lead to some disorders of sex development (DSD).
Electronic supplementary material
The online version of this article (doi:10.1186/s12976-015-0023-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s12976-015-0023-0
PMCID: PMC4647291  PMID: 26573569
Sex determination; Gonadal sex determination; Boolean model; Gene regulatory network
9.  Trans-Proteomic Pipeline, a standardized data processing pipeline for large-scale reproducible proteomics informatics 
Democratization of genomics technologies has enabled the rapid determination of genotypes. More recently the democratization of comprehensive proteomics technologies is enabling the determination of the cellular phenotype and the molecular events that define its dynamic state. Core proteomic technologies include mass spectrometry to define protein sequence, protein:protein interactions, and protein post-translational modifications. Key enabling technologies for proteomics are bioinformatic pipelines to identify, quantitate, and summarize these events. The Trans-Proteomics Pipeline (TPP) is a robust open-source standardized data processing pipeline for large-scale reproducible quantitative mass spectrometry proteomics. It supports all major operating systems and instrument vendors via open data formats. Here we provide a review of the overall proteomics workflow supported by the TPP, its major tools, and how it can be used in its various modes from desktop to cloud computing. We describe new features for the TPP, including data visualization functionality. We conclude by describing some common perils that affect the analysis of tandem mass spectrometry datasets, as well as some major upcoming features.
doi:10.1002/prca.201400164
PMCID: PMC4506239  PMID: 25631240
bioinformatics; mass spectrometry
10.  Fanconi anemia cells with unrepaired DNA damage activate components of the checkpoint recovery process 
Background
The FA/BRCA pathway repairs DNA interstrand crosslinks. Mutations in this pathway cause Fanconi anemia (FA), a chromosome instability syndrome with bone marrow failure and cancer predisposition. Upon DNA damage, normal and FA cells inhibit the cell cycle progression, until the G2/M checkpoint is turned off by the checkpoint recovery, which becomes activated when the DNA damage has been repaired. Interestingly, highly damaged FA cells seem to override the G2/M checkpoint. In this study we explored with a Boolean network model and key experiments whether checkpoint recovery activation occurs in FA cells with extensive unrepaired DNA damage.
Methods
We performed synchronous/asynchronous simulations of the FA/BRCA pathway Boolean network model. FA-A and normal lymphoblastoid cell lines were used to study checkpoint and checkpoint recovery activation after DNA damage induction. The experimental approach included flow cytometry cell cycle analysis, cell division tracking, chromosome aberration analysis and gene expression analysis through qRT-PCR and western blot.
Results
Computational simulations suggested that in FA mutants checkpoint recovery activity inhibits the checkpoint components despite unrepaired DNA damage, a behavior that we did not observed in wild-type simulations. This result implies that FA cells would eventually reenter the cell cycle after a DNA damage induced G2/M checkpoint arrest, but before the damage has been fixed. We observed that FA-A cells activate the G2/M checkpoint and arrest in G2 phase, but eventually reach mitosis and divide with unrepaired DNA damage, thus resolving the initial checkpoint arrest. Based on our model result we look for ectopic activity of checkpoint recovery components. We found that checkpoint recovery components, such as PLK1, are expressed to a similar extent as normal undamaged cells do, even though FA-A cells harbor highly damaged DNA.
Conclusions
Our results show that FA cells, despite extensive DNA damage, do not loss the capacity to express the transcriptional and protein components of checkpoint recovery that might eventually allow their division with unrepaired DNA damage. This might allow cell survival but increases the genomic instability inherent to FA individuals and promotes cancer.
doi:10.1186/s12976-015-0011-4
PMCID: PMC4575447  PMID: 26385365
DNA damage; Checkpoint recovery; Boolean network model
11.  Normal vs. Malignant hematopoiesis: the complexity of acute leukemia through systems biology 
Frontiers in Genetics  2015;6:290.
doi:10.3389/fgene.2015.00290
PMCID: PMC4566035  PMID: 26442108
acute leukemia; early hematopoiesis; bone marrow; mathematical modeling; regulatory networks; systems biology
12.  A Minimal Regulatory Network of Extrinsic and Intrinsic Factors Recovers Observed Patterns of CD4+ T Cell Differentiation and Plasticity 
PLoS Computational Biology  2015;11(6):e1004324.
CD4+ T cells orchestrate the adaptive immune response in vertebrates. While both experimental and modeling work has been conducted to understand the molecular genetic mechanisms involved in CD4+ T cell responses and fate attainment, the dynamic role of intrinsic (produced by CD4+ T lymphocytes) versus extrinsic (produced by other cells) components remains unclear, and the mechanistic and dynamic understanding of the plastic responses of these cells remains incomplete. In this work, we studied a regulatory network for the core transcription factors involved in CD4+ T cell-fate attainment. We first show that this core is not sufficient to recover common CD4+ T phenotypes. We thus postulate a minimal Boolean regulatory network model derived from a larger and more comprehensive network that is based on experimental data. The minimal network integrates transcriptional regulation, signaling pathways and the micro-environment. This network model recovers reported configurations of most of the characterized cell types (Th0, Th1, Th2, Th17, Tfh, Th9, iTreg, and Foxp3-independent T regulatory cells). This transcriptional-signaling regulatory network is robust and recovers mutant configurations that have been reported experimentally. Additionally, this model recovers many of the plasticity patterns documented for different T CD4+ cell types, as summarized in a cell-fate map. We tested the effects of various micro-environments and transient perturbations on such transitions among CD4+ T cell types. Interestingly, most cell-fate transitions were induced by transient activations, with the opposite behavior associated with transient inhibitions. Finally, we used a novel methodology was used to establish that T-bet, TGF-β and suppressors of cytokine signaling proteins are keys to recovering observed CD4+ T cell plastic responses. In conclusion, the observed CD4+ T cell-types and transition patterns emerge from the feedback between the intrinsic or intracellular regulatory core and the micro-environment. We discuss the broader use of this approach for other plastic systems and possible therapeutic interventions.
Author Summary
CD4+ T cells orchestrate adaptive immune responses in vertebrates. These cells differentiate into several types depending on environmental signals and immunological challenges. Once these cells are committed to a particular fate, they can switch to different cell types, thus exhibiting plasticity that enables the immune system to dynamically adapt to novel challenges. We integrated available experimental data into a large network that was formally reduced to a minimal regulatory module with a sufficient set of components and interactions to recover most CD4+ T cell types and reported plasticity patterns in response to various micro-environments and transient perturbations. We formally demonstrate that transcriptional regulatory interactions are not sufficient to recover CD4+ T cell types and thus propose a minimal network that induces most observed phenotypes. This model is robust and was validated with mutant CD4+ T phenotypes. The model was also used to identify key components for cell differentiation and plasticity under varying immunogenic conditions. The model presented here may be a useful framework to study other plastic systems and guide therapeutic approaches to immune system modulation.
doi:10.1371/journal.pcbi.1004324
PMCID: PMC4475012  PMID: 26090929
13.  Effects of Moderate Amounts of Barley in Late Pregnancy on Growth, Glucose Metabolism and Osteoarticular Status of Pre-Weaning Horses 
PLoS ONE  2015;10(4):e0122596.
In stud management, broodmares are commonly fed concentrates in late pregnancy. This practice, however, was shown to correlate with an increased incidence of osteochondrosis in foals, which may be related to insulin sensitivity. We hypothesized that supplementation of the mare with barley in the last trimester of pregnancy alters the pre-weaning foal growth, glucose metabolism and osteoarticular status. Here, pregnant multiparous saddlebred mares were fed forage only (group F, n=13) or both forage and cracked barley (group B, n=12) from the 7th month of pregnancy until term, as calculated to cover nutritional needs of broodmares. Diets were given in two daily meals. All mares and foals returned to pasture after parturition. Post-natal growth, glucose metabolism and osteoarticular status were investigated in pre-weaning foals. B mares maintained an optimal body condition score (>3.5), whereas that of F mares decreased and remained low (<2.5) up to 3 months of lactation, with a significantly lower bodyweight (-7%) than B mares throughout the last 2 months of pregnancy. B mares had increased plasma glucose and insulin after the first meal and after the second meal to a lesser extent, which was not observed in F mares. B mares also had increased insulin secretion during an intravenous glucose tolerance test (IVGTT). Plasma NEFA and leptin were only temporarily affected by diet in mares during pregnancy or in early lactation. Neonatal B foals had increased serum osteocalcin and slightly increased glucose increments and clearance after glucose injection, but these effects had vanished at weaning. Body measurements, plasma IGF-1, T4, T3, NEFA and leptin concentrations, insulin secretion during IVGTT, as well as glucose metabolism rate during euglycemic hyperinsulinemic clamps after weaning, did not differ between groups. Radiographic examination of joints indicated increased osteochondrosis relative risk in B foals, but this was not significant. These data demonstrate that B or F maternal nutrition has very few effects on foal growth, endocrinology and glucose homeostasis until weaning, but may induce cartilage lesions.
doi:10.1371/journal.pone.0122596
PMCID: PMC4395399  PMID: 25875166
14.  A model of the regulatory network involved in the control of the cell cycle and cell differentiation in the Caenorhabditis elegans vulva 
BMC Bioinformatics  2015;16(1):81.
Background
There are recent experimental reports on the cross-regulation between molecules involved in the control of the cell cycle and the differentiation of the vulval precursor cells (VPCs) of Caenorhabditis elegans. Such discoveries provide novel clues on how the molecular mechanisms involved in the cell cycle and cell differentiation processes are coordinated during vulval development. Dynamic computational models are helpful to understand the integrated regulatory mechanisms affecting these cellular processes.
Results
Here we propose a simplified model of the regulatory network that includes sufficient molecules involved in the control of both the cell cycle and cell differentiation in the C. elegans vulva to recover their dynamic behavior. We first infer both the topology and the update rules of the cell cycle module from an expected time series. Next, we use a symbolic algorithmic approach to find which interactions must be included in the regulatory network. Finally, we use a continuous-time version of the update rules for the cell cycle module to validate the cyclic behavior of the network, as well as to rule out the presence of potential artifacts due to the synchronous updating of the discrete model. We analyze the dynamical behavior of the model for the wild type and several mutants, finding that most of the results are consistent with published experimental results.
Conclusions
Our model shows that the regulation of Notch signaling by the cell cycle preserves the potential of the VPCs and the three vulval fates to differentiate and de-differentiate, allowing them to remain completely responsive to the concentration of LIN-3 and lateral signal in the extracellular microenvironment.
Electronic supplementary material
The online version of this article (doi:10.1186/s12859-015-0498-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s12859-015-0498-z
PMCID: PMC4367908  PMID: 25884811
C. elegans; Vulva; Fate determination; Cell cycle
15.  Direct application of Padé approximant for solving nonlinear differential equations 
SpringerPlus  2014;3:563.
Abstract
This work presents a direct procedure to apply Padé method to find approximate solutions for nonlinear differential equations. Moreover, we present some cases study showing the strength of the method to generate highly accurate rational approximate solutions compared to other semi-analytical methods. The type of tested nonlinear equations are: a highly nonlinear boundary value problem, a differential-algebraic oscillator problem, and an asymptotic problem. The high accurate handy approximations obtained by the direct application of Padé method shows the high potential if the proposed scheme to approximate a wide variety of problems. What is more, the direct application of the Padé approximant aids to avoid the previous application of an approximative method like Taylor series method, homotopy perturbation method, Adomian Decomposition method, homotopy analysis method, variational iteration method, among others, as tools to obtain a power series solutions to post-treat with the Padé approximant.
AMS Subject Classification
34L30
doi:10.1186/2193-1801-3-563
PMCID: PMC4194307  PMID: 25332863
Padé transform; Nonlinear differential equations
16.  PASSEL: The PeptideAtlas SRM Experiment Library 
Proteomics  2012;12(8):10.1002/pmic.201100515.
Public repositories for proteomics data have accelerated proteomics research by enabling more efficient cross-analyses of datasets, supporting the creation of protein and peptide compendia of experimental results, supporting the development and testing of new software tools, and facilitating the manuscript review process. The repositories available to date have been designed to accommodate either shotgun experiments or generic proteomic data files. Here, we describe a new kind of proteomic data repository for the collection and representation of data from selected reaction monitoring (SRM) measurements. The PeptideAtlas SRM Experiment Library (PASSEL) allows researchers to easily submit proteomic data sets generated by SRM. The raw data are automatically processed in a uniform manner and the results are stored in a database, where they may be downloaded or browsed via a web interface that includes a chromatogram viewer. PASSEL enables cross-analysis of SRM data, supports optimization of SRM data collection, and facilitates the review process of SRM data. Further, PASSEL will help in the assessment of proteotypic peptide performance in a wide array of samples containing the same peptide, as well as across multiple experimental protocols.
doi:10.1002/pmic.201100515
PMCID: PMC3832291  PMID: 22318887
data repository; MRM; software; SRM; targeted proteomics
18.  The Microvesicle Component of HIV-1 Inocula Modulates Dendritic Cell Infection and Maturation and Enhances Adhesion to and Activation of T Lymphocytes 
PLoS Pathogens  2013;9(10):e1003700.
HIV-1 is taken up by immature monocyte derived dendritic cells (iMDDCs) into tetraspanin rich caves from which the virus can either be transferred to T lymphocytes or enter into endosomes resulting in degradation. HIV-1 binding and fusion with the DC membrane results in low level de novo infection that can also be transferred to T lymphocytes at a later stage. We have previously reported that HIV-1 can induce partial maturation of iMDDCs at both stages of trafficking. Here we show that CD45+ microvesicles (MV) which contaminate purified HIV-1 inocula due to similar size and density, affect DC maturation, de novo HIV-1 infection and transfer to T lymphocytes. Comparing iMDDCs infected with CD45-depleted HIV-1BaL or matched non-depleted preparations, the presence of CD45+ MVs was shown to enhance DC maturation and ICAM-1 (CD54) expression, which is involved in DC∶T lymphocyte interactions, while restricting HIV-1 infection of MDDCs. Furthermore, in the DC culture HIV-1 infected (p24+) MDDCs were more mature than bystander cells. Depletion of MVs from the HIV-1 inoculum markedly inhibited DC∶T lymphocyte clustering and the induction of alloproliferation as well as limiting HIV-1 transfer from DCs to T lymphocytes. The effects of MV depletion on these functions were reversed by the re-addition of purified MVs from activated but not non-activated SUPT1.CCR5-CL.30 or primary T cells. Analysis of the protein complement of these MVs and of these HIV-1 inocula before and after MV depletion showed that Heat Shock Proteins (HSPs) and nef were the likely DC maturation candidates. Recombinant HSP90α and β and nef all induced DC maturation and ICAM-1 expression, greater when combined. These results suggest that MVs contaminating HIV-1 released from infected T lymphocytes may be biologically important, especially in enhancing T cell activation, during uptake by DCs in vitro and in vivo, particularly as MVs have been detected in the circulation of HIV-1 infected subjects.
Author Summary
Dendritic cells (DCs) are vital for immune recognition of pathogens as they capture, internalise, degrade and present foreign peptides to T lymphocytes. It is thought that HIV-1 hijacks the DCs functions, such as migration and maturation, to increase contact with the major target cell CD4+ T lymphocytes leading to dissemination throughout the body. Currently there is still some controversy over the ability of HIV-1 to infect and mature DCs, which may be due to differences in the inoculum used. Here we examined the effect of contaminating microvesicles (MVs) identified in HIV-1 preparations on HIV-1 modulation of DC function. We show that when MVs are present with HIV-1, the inoculum induces greater DC maturation and adhesion probably via cellular HSP90α and β and viral nef within the MVs. The functional consequences are reduced de novo replication of HIV-1 but increased clustering with T lymphocytes, resulting in increased T lymphocyte alloproliferation and HIV-1 transfer. As MVs are produced in HIV-1 susceptible cells and would be present in vivo due to HIV-1 induced cell death and hence are physiologically relevant, these results also indicate that MVs present in HIV-1 inocula should be considered when assessing HIV∶DC interactions.
doi:10.1371/journal.ppat.1003700
PMCID: PMC3798598  PMID: 24204260
19.  A network model for the specification of vulval precursor cells and cell fusion control in Caenorhabditis elegans 
Frontiers in Genetics  2013;4:112.
The vulva of Caenorhabditis elegans has been long used as an experimental model of cell differentiation and organogenesis. While it is known that the signaling cascades of Wnt, Ras/MAPK, and NOTCH interact to form a molecular network, there is no consensus regarding its precise topology and dynamical properties. We inferred the molecular network, and developed a multivalued synchronous discrete dynamic model to study its behavior. The model reproduces the patterns of activation reported for the following types of cell: vulval precursor, first fate, second fate, second fate with reversed polarity, third fate, and fusion fate. We simulated the fusion of cells, the determination of the first, second, and third fates, as well as the transition from the second to the first fate. We also used the model to simulate all possible single loss- and gain-of-function mutants, as well as some relevant double and triple mutants. Importantly, we associated most of these simulated mutants to multivulva, vulvaless, egg-laying defective, or defective polarity phenotypes. The model shows that it is necessary for RAL-1 to activate NOTCH signaling, since the repression of LIN-45 by RAL-1 would not suffice for a proper second fate determination in an environment lacking DSL ligands. We also found that the model requires the complex formed by LAG-1, LIN-12, and SEL-8 to inhibit the transcription of eff-1 in second fate cells. Our model is the largest reconstruction to date of the molecular network controlling the specification of vulval precursor cells and cell fusion control in C. elegans. According to our model, the process of fate determination in the vulval precursor cells is reversible, at least until either the cells fuse with the ventral hypoderm or divide, and therefore the cell fates must be maintained by the presence of extracellular signals.
doi:10.3389/fgene.2013.00112
PMCID: PMC3682179  PMID: 23785384
Caenorhabditis VPCs; vulval precursor cells; regulatory networks; discrete state network model; Caenorhabditis model
20.  Building Qualitative Models of Plant Regulatory Networks with SQUAD 
doi:10.3389/fpls.2012.00072
PMCID: PMC3355663  PMID: 22639661
21.  TraML—A Standard Format for Exchange of Selected Reaction Monitoring Transition Lists* 
Molecular & Cellular Proteomics : MCP  2011;11(4):R111.015040.
Targeted proteomics via selected reaction monitoring is a powerful mass spectrometric technique affording higher dynamic range, increased specificity and lower limits of detection than other shotgun mass spectrometry methods when applied to proteome analyses. However, it involves selective measurement of predetermined analytes, which requires more preparation in the form of selecting appropriate signatures for the proteins and peptides that are to be targeted. There is a growing number of software programs and resources for selecting optimal transitions and the instrument settings used for the detection and quantification of the targeted peptides, but the exchange of this information is hindered by a lack of a standard format. We have developed a new standardized format, called TraML, for encoding transition lists and associated metadata. In addition to introducing the TraML format, we demonstrate several implementations across the community, and provide semantic validators, extensive documentation, and multiple example instances to demonstrate correctly written documents. Widespread use of TraML will facilitate the exchange of transitions, reduce time spent handling incompatible list formats, increase the reusability of previously optimized transitions, and thus accelerate the widespread adoption of targeted proteomics via selected reaction monitoring.
doi:10.1074/mcp.R111.015040
PMCID: PMC3322582  PMID: 22159873
22.  Formulation and in Vitro, ex Vivo and in Vivo Evaluation of Elastic Liposomes for Transdermal Delivery of Ketorolac Tromethamine 
Pharmaceutics  2011;3(4):954-970.
The objective of the current study was to formulate ketorolac tromethamine-loaded elastic liposomes and evaluate their in vitro drug release and their ex vivo and in vivo transdermal delivery. Ketorolac tromethamine (KT), which is a potent analgesic, was formulated in elastic liposomes using Tween 80 as an edge activator. The elastic vesicles were prepared by film hydration after optimizing the sonication time and number of extrusions. The vesicles exhibited an entrapment efficiency of 73 ± 11%, vesicle size of 127.8 ± 3.4 nm and a zeta potential of −12 mV. In vitro drug release was analyzed from liposomes and an aqueous solution, using Franz diffusion cells and a cellophane dialysis membrane with molecular weight cut-off of 8000 Da. Ex vivo permeation of KT across pig ear skin was studied using a Franz diffusion cell, with phosphate buffer (pH 7.4) at 32 °C as receptor solution. An in vivo drug permeation study was conducted on healthy human volunteers using a tape-stripping technique. The in vitro results showed (i) a delayed release when KT was included in elastic liposomes, compared to an aqueous solution of the drug; (ii) a flux of 0.278 μg/cm2h and a lag time of about 10 h for ex vivo permeation studies, which may indicate that KT remains in the skin (with the possibility of exerting a local effect) before reaching the receptor medium; (iii) a good correlation between the total amount permeated, the penetration distance (both determined by tape stripping) and transepidermal water loss (TEWL) measured during the in vivo permeation studies. Elastic liposomes have the potential to transport the drug through the skin, keep their size and drug charge, and release the drug into deep skin layers. Therefore, elastic liposomes hold promise for the effective topical delivery of KT.
doi:10.3390/pharmaceutics3040954
PMCID: PMC3857066  PMID: 24309316
elastic liposomes; ketorolac tromethamine; skin permeation; tape stripping; TEWL
23.  Neurocognitive Functioning in Preschool-age Children with Type 1 Diabetes Mellitus 
Pediatric diabetes  2010;11(6):424-430.
Neurocognitive functioning may be compromised in children with type 1 diabetes mellitus (T1DM). The factor most consistently implicated in the long-term neurocognitive functioning of children with T1DM is age of onset. The pediatric literature suggests that glycemic extremes may have an effect on the neurocognitive functioning of children, but findings are mixed. The purpose of this study was to compare the neurocognitive functioning of young children with T1DM diagnosed before six years of age and healthy children (i.e., without chronic illness). Additionally, in the children with T1DM, we examined the relationship between their neurocognitive functioning and glycemic control. Sixty eight (36 with T1DM and 32 without chronic illness) preschool-age children (M age = 4.4yrs) were recruited and administered a battery of instruments to measure cognitive, language, and fine motor skills. Children with T1DM performed similarly to the healthy controls and both groups' skills fell in the average range. Among children with diabetes, poor glycemic control (higher HbA1c) was related to lower general cognitive abilities (r = -.44, p < .04), slower fine motor speed (r = -.64, p < .02), and lower receptive language scores (r = -.39, p < .04). Such findings indicate that young children with T1DM already demonstrate some negative neurocognitive effects in association with chronic hyperglycemia.
doi:10.1111/j.1399-5448.2009.00618.x
PMCID: PMC2921563  PMID: 20456084
children; type 1 diabetes; neurocognitive
24.  Trans-Proteomic Pipeline supports and improves analysis of electron transfer dissociation datasets 
Proteomics  2010;10(6):1190-1195.
Electron transfer dissociation (ETD) is an alternative fragmentation technique to collision induced dissociation (CID) that has recently become commercially available. ETD has several advantages over CID. It is less prone to fragmenting amino acid side chains, especially those that are modified, thus yielding fragment ion spectra with more uniform peak intensities. Further, precursor ions of longer peptides and higher charge states can be fragmented and identified. However, analysis of ETD spectra has a few important differences that require the optimization of the software packages used for the analysis of CID data, or the development of specialized tools. We have adapted the Trans-Proteomic Pipeline (TPP) to process ETD data. Specifically, we have added support for fragment ion spectra from high charge precursors, compatibility with charge-state estimation algorithms, provisions for the use of the Lys-C protease, capabilities for ETD spectrum library building, and updates to the data formats to differentiate CID and ETD spectra. We show the results of processing datasets from several different types of ETD instruments and demonstrate that application of the ETD-enhanced TPP can increase the number of spectrum identifications at a fixed false discovery rate by as much as 100% over native output from a single sequence search engine.
doi:10.1002/pmic.200900567
PMCID: PMC3018683  PMID: 20082347
shotgun proteomics; electron-transfer dissociation; bioinformatics
25.  A Guided Tour of the Trans-Proteomic Pipeline 
Proteomics  2010;10(6):1150-1159.
The Trans-Proteomic Pipeline (TPP) is a suite of software tools for the analysis of tandem mass spectrometry datasets. The tools encompass most of the steps in a proteomic data analysis workflow in a single, integrated software system. Specifically, the TPP supports all steps from spectrometer output file conversion to protein-level statistical validation, including quantification by stable isotope ratios. We describe here the full workflow of the TPP and the tools therein, along with an example on a sample dataset, demonstrating that the set up and use of the tools is straightforward and well supported and does not require specialized informatics resources or knowledge.
doi:10.1002/pmic.200900375
PMCID: PMC3017125  PMID: 20101611

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