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Data Management Outreach to Junior Faculty Members: A Case Study

Fri, 08/21/2015 - 2:06pm

New tenure track faculty members are generally in positions as leaders of a research laboratory or group for the first time. In addition to building up the infrastructure of a research lab (whether space, equipment, funding, or personnel), the new faculty member is also setting the research process and expectations for the first time as well. This article highlights outreach to new faculty members assisting those individuals with developing a data management protocol that effectively supports the laboratory researchers to make quality data available internally to and externally from a research laboratory. Using a self-assessment tool and reflective conversation, junior faculty were offered insight and advice into creating a data management protocol for use in their research laboratory.

Chromosome Conformation Capture (3C) in Budding Yeast

Mon, 08/17/2015 - 3:36pm

Chromosome conformation capture (3C) is a method for studying chromosomal organization that takes advantage of formaldehyde cross-linking to measure the spatial association of two pieces of chromatin. The 3C method begins with whole-cell formaldehyde fixation of chromatin. After cell lysis, solubilized chromatin is digested with a type II restriction endonuclease, and cross-linked DNA fragments are ligated together. Cross-links are reversed by degradation with proteinase K, and chimeric DNA molecules are purified by standard phenol:chloroform extraction. The resulting 3C library represents chromatin fragments that may be separated by large genomic distances or located on different chromosomes, but are close enough in three-dimensional space for cross-linking. Locus-specific oligonucleotide primers are used to detect interactions of interest in the 3C library using end-point polymerase chain reaction (PCR).

Randomized ligation control for chromosome conformation capture

Mon, 08/17/2015 - 3:36pm

In experiments using chromosome conformation capture followed by PCR (3C-PCR) or chromosome conformation capture carbon copy (5C), it is critical to control for intrinsic biases in the restriction fragments of interest and the probes or primers used for detection. Characteristics such as GC%, annealing temperature, efficiency of 3C primers or 5C probes, and length of restriction fragment can cause variations in primer or probe performance and fragment ligation efficiency. Bias can be measured empirically by production of a random control library, as described here, to be used with the 3C library of interest.

Chromosome Conformation Capture Carbon Copy (5C) in Budding Yeast

Mon, 08/17/2015 - 3:36pm

Chromosome conformation capture carbon copy (5C) is a high-throughput method for detecting ligation products of interest in a chromosome conformation capture (3C) library. 5C uses ligation-mediated amplification (LMA) to generate carbon copies of 3C ligation product junctions using single-stranded oligonucleotide probes. This procedure produces a 5C library of short DNA molecules which represent the interactions between the corresponding restriction fragments. The 5C library can be amplified using universal primers containing the Illumina paired-end adaptor sequences for subsequent high-throughput sequencing.

Hi-C in Budding Yeast

Mon, 08/17/2015 - 3:36pm

Hi-C enables simultaneous detection of interaction frequencies between all possible pairs of restriction fragments in the genome. The Hi-C method is based on chromosome conformation capture (3C), which uses formaldehyde cross-linking to fix chromatin regions that interact in three-dimensional space, irrespective of their genomic locations. In the Hi-C protocol described here, cross-linked chromatin is digested with HindIII and the ends are filled in with a nucleotide mix containing biotinylated dCTP. These fragments are ligated together, and the resulting chimeric molecules are purified and sheared to reduce length. Finally, biotinylated ligation junctions are pulled down with streptavidin-coated beads, linked to high-throughput sequencing adaptors, and amplified via polymerase chain reaction (PCR). The resolution of the Hi-C data set will depend on the depth of sequencing and choice of restriction enzyme. When sufficient sequence reads are obtained, information on chromatin interactions and chromosome conformation can be derived at single restriction fragment resolution for complete genomes.

Measuring Chromatin Structure in Budding Yeast

Mon, 08/17/2015 - 3:36pm

Chromosome conformation capture (3C) has revolutionized the ways in which the conformation of chromatin and its relationship to other molecular functions can be studied. 3C-based techniques are used to determine the spatial arrangement of chromosomes in organisms ranging from bacteria to humans. In particular, they can be applied to the study of chromosome folding and organization in model organisms with small genomes and for which powerful genetic tools exist, such as budding yeast. Studies in yeast allow the mechanisms that establish or maintain chromatin structure to be analyzed at very high resolution with relatively low cost, and further our understanding of these fundamental processes in higher eukaryotes as well. Here we provide an overview of chromatin structure and introduce methods for performing 3C, with a focus on studies in budding yeast. Variations of the basic 3C approach (e.g., 3C-PCR, 5C, and Hi-C) can be used according to the scope and goals of a given experiment.

Mapping Nucleosome Resolution Chromosome Folding in Yeast by Micro-C

Mon, 08/17/2015 - 3:36pm

We describe a Hi-C-based method, Micro-C, in which micrococcal nuclease is used instead of restriction enzymes to fragment chromatin, enabling nucleosome resolution chromosome folding maps. Analysis of Micro-C maps for budding yeast reveals abundant self-associating domains similar to those reported in other species, but not previously observed in yeast. These structures, far shorter than topologically associating domains in mammals, typically encompass one to five genes in yeast. Strong boundaries between self-associating domains occur at promoters of highly transcribed genes and regions of rapid histone turnover that are typically bound by the RSC chromatin-remodeling complex. Investigation of chromosome folding in mutants confirms roles for RSC, "gene looping" factor Ssu72, Mediator, H3K56 acetyltransferase Rtt109, and the N-terminal tail of H4 in folding of the yeast genome. This approach provides detailed structural maps of a eukaryotic genome, and our findings provide insights into the machinery underlying chromosome compaction.

Condensin-driven remodelling of X chromosome topology during dosage compensation

Mon, 08/17/2015 - 3:36pm

The three-dimensional organization of a genome plays a critical role in regulating gene expression, yet little is known about the machinery and mechanisms that determine higher-order chromosome structure. Here we perform genome-wide chromosome conformation capture analysis, fluorescent in situ hybridization (FISH), and RNA-seq to obtain comprehensive three-dimensional (3D) maps of the Caenorhabditis elegans genome and to dissect X chromosome dosage compensation, which balances gene expression between XX hermaphrodites and XO males. The dosage compensation complex (DCC), a condensin complex, binds to both hermaphrodite X chromosomes via sequence-specific recruitment elements on X (rex sites) to reduce chromosome-wide gene expression by half. Most DCC condensin subunits also act in other condensin complexes to control the compaction and resolution of all mitotic and meiotic chromosomes. By comparing chromosome structure in wild-type and DCC-defective embryos, we show that the DCC remodels hermaphrodite X chromosomes into a sex-specific spatial conformation distinct from autosomes. Dosage-compensated X chromosomes consist of self-interacting domains ( approximately 1 Mb) resembling mammalian topologically associating domains (TADs). TADs on X chromosomes have stronger boundaries and more regular spacing than on autosomes. Many TAD boundaries on X chromosomes coincide with the highest-affinity rex sites and become diminished or lost in DCC-defective mutants, thereby converting the topology of X to a conformation resembling autosomes. rex sites engage in DCC-dependent long-range interactions, with the most frequent interactions occurring between rex sites at DCC-dependent TAD boundaries. These results imply that the DCC reshapes the topology of X chromosomes by forming new TAD boundaries and reinforcing weak boundaries through interactions between its highest-affinity binding sites. As this model predicts, deletion of an endogenous rex site at a DCC-dependent TAD boundary using CRISPR/Cas9 greatly diminished the boundary. Thus, the DCC imposes a distinct higher-order structure onto X chromosomes while regulating gene expression chromosome-wide.

The yeast genome undergoes significant topological reorganization in quiescence

Mon, 08/17/2015 - 3:36pm

We have examined the three-dimensional organization of the yeast genome during quiescence by a chromosome capture technique as a means of understanding how genome organization changes during development. For exponentially growing cells we observe high levels of inter-centromeric interaction but otherwise a predominance of intrachromosomal interactions over interchromosomal interactions, consistent with aggregation of centromeres at the spindle pole body and compartmentalization of individual chromosomes within the nucleoplasm. Three major changes occur in the organization of the quiescent cell genome. First, intrachromosomal associations increase at longer distances in quiescence as compared to growing cells. This suggests that chromosomes undergo condensation in quiescence, which we confirmed by microscopy by measurement of the intrachromosomal distances between two sites on one chromosome. This compaction in quiescence requires the condensin complex. Second, inter-centromeric interactions decrease, consistent with prior data indicating that centromeres disperse along an array of microtubules during quiescence. Third, inter-telomeric interactions significantly increase in quiescence, an observation also confirmed by direct measurement. Thus, survival during quiescence is associated with substantial topological reorganization of the genome.

Condensin promotes the juxtaposition of DNA flanking its loading site in Bacillus subtilis

Mon, 08/17/2015 - 3:36pm

SMC condensin complexes play a central role in compacting and resolving replicated chromosomes in virtually all organisms, yet how they accomplish this remains elusive. In Bacillus subtilis, condensin is loaded at centromeric parS sites, where it encircles DNA and individualizes newly replicated origins. Using chromosome conformation capture and cytological assays, we show that condensin recruitment to origin-proximal parS sites is required for the juxtaposition of the two chromosome arms. Recruitment to ectopic parS sites promotes alignment of large tracks of DNA flanking these sites. Importantly, insertion of parS sites on opposing arms indicates that these "zip-up" interactions only occur between adjacent DNA segments. Collectively, our data suggest that condensin resolves replicated origins by promoting the juxtaposition of DNA flanking parS sites, drawing sister origins in on themselves and away from each other. These results are consistent with a model in which condensin encircles the DNA flanking its loading site and then slides down, tethering the two arms together. Lengthwise condensation via loop extrusion could provide a generalizable mechanism by which condensin complexes act dynamically to individualize origins in B. subtilis and, when loaded along eukaryotic chromosomes, resolve them during mitosis.

Studying Cellular Signal Transduction with OMIC Technologies

Mon, 08/17/2015 - 3:36pm

In the gulf between genotype and phenotype exists proteins and, in particular, protein signal transduction systems. These systems use a relatively limited parts list to respond to a much longer list of extracellular, environmental, and/or mechanical cues with rapidity and specificity. Most signaling networks function in a highly nonlinear and often contextual manner. Furthermore, these processes occur dynamically across space and time. Because of these complexities, systems and "OMIC" approaches are essential for the study of signal transduction. One challenge in using OMIC-scale approaches to study signaling is that the "signal" can take different forms in different situations. Signals are encoded in diverse ways such as protein-protein interactions, enzyme activities, localizations, or post-translational modifications to proteins. Furthermore, in some cases signals may be encoded only in the dynamics, duration, or rates of change of these features. Accordingly, systems-level analyses of signaling may need to integrate multiple experimental and/or computational approaches. As the field has progressed, the non-triviality of integrating experimental and computational analyses has become apparent. Successful use of OMIC methods to study signaling will require the "right" experiments and the "right" modeling approaches, and it is critical to consider both in the design phase of the project. In this review, we discuss common OMIC and modeling approaches for studying signaling, emphasizing the philosophical and practical considerations for effectively merging these two types of approaches to maximize the probability of obtaining reliable and novel insights into signaling biology.

Flipping Biological Switches: Solving for Optimal Control: A Dissertation

Mon, 08/17/2015 - 2:57pm

Switches play an important regulatory role at all levels of biology, from molecular switches triggering signaling cascades to cellular switches regulating cell maturation and apoptosis. Medical therapies are often designed to toggle a system from one state to another, achieving a specified health outcome. For instance, small doses of subpathologic viruses activate the immune system’s production of antibodies. Electrical stimulation revert cardiac arrhythmias back to normal sinus rhythm. In all of these examples, a major challenge is finding the optimal stimulus waveform necessary to cause the switch to flip. This thesis develops, validates, and applies a novel model-independent stochastic algorithm, the Extrema Distortion Algorithm (EDA), towards finding the optimal stimulus. We validate the EDA’s performance for the Hodgkin-Huxley model (an empirically validated ionic model of neuronal excitability), the FitzHugh-Nagumo model (an abstract model applied to a wide range of biological systems that that exhibit an oscillatory state and a quiescent state), and the genetic toggle switch (a model of bistable gene expression). We show that the EDA is able to not only find the optimal solution, but also in some cases excel beyond the traditional analytic approaches. Finally, we have computed novel optimal stimulus waveforms for aborting epileptic seizures using the EDA in cellular and network models of epilepsy. This work represents a first step in developing a new class of adaptive algorithms and devices that flip biological switches, revealing basic mechanistic insights and therapeutic applications for a broad range of disorders.

Declining Trends and Widening Disparities in Overweight and Obesity Prevalence Among Massachusetts Public School Districts, 2009-2014

Mon, 08/17/2015 - 10:44am

OBJECTIVES: We evaluated the overall and sociodemographic disparities in trends in prevalence of childhood overweight and obesity in Massachusetts public school districts between 2009 and 2014.

METHODS: In 2009, Massachusetts mandated annual screening of body mass index for students in grades 1, 4, 7, and 10. This was part of the statewide Mass in Motion prevention programs. We assessed trends in the prevalence of overweight and obesity between 2009 and 2014 by district, gender, grade, and district income.

RESULTS: From 2009 to 2014, prevalence decreased 3.0 percentage points (from 34.3% to 31.3%) statewide. The 2014 district-level rates ranged from 13.9% to 54.5% (median = 31.2%). When stratified by grade, the decreasing trends were significant only for grades 1 and 4. Although rates of districts with a median household income greater than $37 000 improved notably, rates of the poorest remain unchanged and were approximately 40%.

CONCLUSIONS: Although overall prevalence began to decrease, the geographic and socioeconomic disparities in childhood obesity are widening and remain a public health challenge in Massachusetts. Special efforts should be made to address the needs of socioeconomically disadvantaged districts and to narrow the disparities in childhood obesity. (Am J Public Health. Published online ahead of print August 13, 2015: e1-e7. doi:10.2105/AJPH.2015.302807).

Emerging preclinical animal models for FSHD

Mon, 08/17/2015 - 10:26am

Facioscapulohumeral dystrophy (FSHD) is a unique and complex genetic disease that is not entirely solved. Recent advances in the field have led to a consensus genetic premise for the disorder, enabling researchers to now pursue the design of preclinical models. In this review we explore all available FSHD models (DUX4-dependent and -independent) for their utility in therapeutic discovery and potential to yield novel disease insights. Owing to the complex nature of FSHD, there is currently no single model that accurately recapitulates the genetic and pathophysiological spectrum of the disorder. Existing models emphasize only specific aspects of the disease, highlighting the need for more collaborative research and novel paradigms to advance the translational research space of FSHD.

The Recombination Enhancer Modulates the Conformation of Chr. III in Budding Yeast: A Dissertation

Fri, 08/14/2015 - 4:25pm

A hierarchy of different chromosome conformations plays a role in many biological systems. These conformations contribute to the regulation of gene expression, cellular development, chromosome transmission, and defects can lead to human disease. The highest functional level of this hierarchy is the partitioning of the genome into compartments of active and inactive chromatin domains (1’s -10’s Mb). These compartments are further partitioned into Topologically Associating Domains (TADs) that spatially cluster co-regulated genes (100’s kb – 1’s Mb). The final level that has been observed is long range loops formed between regulatory elements and promoters (10’s kb – 100’s Mb). At all of these levels, mechanisms that establish these conformations remain poorly understood. To gain new insights into processes that determine chromosome folding I used the mating type switching system in budding yeast to study the chromosome conformation at length scales analogous to looping interaction. I specifically examined the role in chromosome conformation in the mating type switching system. Budding yeast cells can have two sexes: MATa and MATα. The mating types are determined by allele-specific expression of the MAT locus on chromosome III. The MATa allele encodes for transcription factors responsible for the MATa mating type and the MATα allele encodes transcription factors responsible for the MATα mating type. Yeast cells can switch their mating type by a process that repairs a break at MAT using one of two silent loci, HML or HMR, as a donor to convert the allele at the MAT locus. When MATa cells switch they prefer to use HML, which contains the MATα allele, located at the end of the left arm. MATα cells prefer to use HMR, which contains the MATa allele, located on the end of the right arm of chromosome III. The sequences of the HM loci are not important for donor preference. Instead the cell chooses the donor on the left arm in MATa cells and chooses the donor on the right arm in MATα cells. This lack of sequence specificity has led to the hypothesis that the conformation of the chromosome may play a role in donor preference. I found that the conformation of chromosome III is, indeed, different between the two mating types. In MATa cells the chromosomes displays a more crumpled conformation in which the left arm of the chromosome interacts with a large region of the right arm which includes the centromere and the MAT locus. In MATα cells, on the other hand, the left arm of the chromosomes displays a more extend conformation. I found that the Recombination Enhancer (RE), which enhances recombination along the left arm of the chromosome in MATa cells, is responsible for these mating type-specific conformations. Deleting the RE affects the conformation of the chromosomes in both MATa and MATα cells. The left portion of the RE, which is essential for donor preference during the switching reaction in MATa cells, does not contribute to the conformation in MATa. This region does have a minor effect on the conformation in MATα cells. However, I found that the right portion of the RE is responsible for the conformation of chromosome III in both mating types prior to initiation of switching. This work demonstrates that chromosome conformation is determined by specific cis regulatory elements that drive cell-type specific chromosome conformation.

The Study of Two Strategies for Decreasing Mutant Huntingtin: Degradation by Puromycin Sensitive AminoPeptidase and RNA Interference: A Dissertation

Fri, 08/14/2015 - 4:25pm

Huntington’s disease (HD) is a fatal neurodegenerative disease caused by a CAG repeat expansion in exon 1 of the huntingtin gene, resulting in an expanded polyglutamine (polyQ) repeat in the huntingtin protein. Patients receive symptomatic treatment for motor, emotional, and cognitive impairments; however, there is no treatment to slow the progression of the disease, with death occurring 15-20 years after diagnosis. Mutant huntingtin protein interferes with multiple cellular processes leading to cellular dysfunction and neuronal loss. Due to the complexity of mutant huntingtin toxicity, many approaches to treating each effect are being investigated. Unfortunately, addressing one cause of toxicity might not result in protection from other toxic insults, necessitating a combination of treatments for HD patients. Ideally, single therapy targeting the mutant mRNA or protein could prevent all downstream toxicities caused by mutant huntingtin. In this work, I used animal models to investigate a potential therapeutic target for decreasing mutant huntingtin protein, and I apply bioluminescent imaging to investigate RNA interference to silence mutant huntingtin target sites.

The enzyme puromycin sensitive aminopeptidase (PSA) has the unique property of degrading polyQ peptides and been implicated in the degradation of huntingtin. In this study, we looked for an effect of decreased PSA on the pathology and behavior in a mouse model of Huntington’s disease. To achieve this, we crossed HD mice with mice with one functional PSA allele and one inactivated PSA allele. We found that PSA heterozygous HD mice develop a greater number of pathological inclusion bodies, representing an accumulation of mutant huntingtin in neurons. PSA heterozygous HD mice also exhibit worsened performance on the raised-beam test, a test for balance and coordination indicating that the PSA heterozygosity impairs the function of neurons with mutant huntingtin. In order to test whether increasing PSA expression ameliorates the HD phenotype in mice we created an adeno-associated virus (AAV) expressing the human form of PSA (AAV-hPSA). Unexpectedly, testing of AAV-hPSA in non-HD mice resulted in widespread toxicity at high doses. These findings suggest that overexpression of PSA is toxic to neurons in the conditions tested.

In the second part of my dissertation work, I designed a model for following the silencing of huntingtin sequences in the brain. Firefly luciferase is a bioluminescent enzyme that is extensively used as a reporter molecule to follow biological processes in vivo using bioluminescent imaging (BLI). I created an AAV expressing the luciferase gene containing huntingtin sequences in the 3'-untranslated region (AAV-Luc-Htt). After co-injection of AAV-Luc-Htt with RNA-silencing molecules (RNAi) into the brain, we followed luciferase activity. Using this method, we tested cholesterol-conjugated siRNA, un-conjugated siRNA, and hairpin RNA targeting both luciferase and huntingtin sequences. Despite being able to detect silencing on isolated days, we were unable to detect sustained silencing, which had been reported in similar studies in tissues other than the brain. We observed an interesting finding that co-injection of cholesterol-conjugated siRNA with AAV-Luc-Htt increased luminescence, findings that were verified in cell culture to be independent of serotype, siRNA sequence, and cell type. That cc-siRNA affects the expression of AAV-Luc-Htt reveals an interesting interaction possibly resulting in increased delivery of AAV into cells or an increase in luciferase expression within the cell. My work presents a method to follow gene silencing of huntingtin targets in the brain, which needs further optimization in order to detect sustained silencing.

Finally, in this dissertation I continue the study of bioluminescent imaging in the brain. We use mice that have been injected in the brain with AAV-Luciferase (AAV-Luc) to screen 34 luciferase substrate solutions to identify the greatest light-emitting substrate in the brain. We identify two substrates, CycLuc1 and iPr-amide as substrates with enhanced light-emitting properties compared with D-luciferin, the standard, commercially available substrate. CycLuc1 and iPr-amide were tested in transgenic mice expressing luciferase in dopaminergic neurons. These novel substrates produced luminescence unlike the standard substrate, D-luciferin which was undetectable. This demonstrates that CycLuc1 and iPr-amide improve the sensitivity of BLI in low expression models. We then used CycLuc1 to test silencing of luciferase in the brain using AAV-shRNA (AAV-shLuc). We were unable to detect silencing in treated mice, despite a 50% reduction of luciferase mRNA. The results from this experiment identify luciferase substrates that can be used to image transgenic mice expressing luciferase in dopaminergic neurons.

My work contributes new data on the study of PSA as a modifier of Huntington’s disease in a knock-in mouse model of Huntington’s disease. My work also makes contributions to the field of bioluminescent imaging by identifying and testing luciferase substrates in the brain to detect low level of luciferase expression.

En Français S'il Vous Plaît: Translation and Adaptation of the New England Collaborative Data Management Curriculum’s Introductory Module

Fri, 08/14/2015 - 10:11am

The New England Collaborative Data Management Curriculum (NECDMC) is “an instructional tool for teaching data management best practices to undergraduates, graduate students, and researchers in the health sciences” (Lamar Soutter Library 2015a). This article reports on the French translation and adaptation of the first module of the NECDMC as part of the design of a short library instruction workshop.

Characterization of Envelope-Specific Antibody Response Elicited by HIV-1 Vaccines: A Dissertation

Thu, 08/13/2015 - 4:15pm

Despite 30 years of intensive research,an effective human immunodeficiency virus (HIV) vaccine still remains elusive. The desirable immune response capable of providing protection against HIV acquisition is still not clear. The accumulating evidence learned from a recent vaccine efficacy correlate study not only confirmed the importance of antibody responses, but also highlighted potential protective functions of antibodies with a broad repertoire of HIV-1 epitope specificities and a wide range of different antiviral mechanisms. This necessitates a deep understanding of the complexity and diversity of antibody responses elicited by HIV-1 vaccines. My dissertation characterizes antibody response profiles of HIV-1 Env antibodies elicited by several novel immunogens or different immunization regimens, in terms of magnitude, persistence, epitope specificity, binding affinity, and biological function.

First, to overcome the challenge of studying polyclonal sera without established assays, we expanded a novel platform to isolate Env-specific Rabbit mAbs (RmAb) elicited by DNA prime-protein boost immunization. These RmAbs revealed diverse epitope specificity and cross-reactivity against multiple gp120 antigens from more than one subtype, and several had potent and broad neutralizing activities against sensitive Tier 1 viruses. Further, structural analysis of two V3 mAbs demonstrated that a slight shift of the V3 epitope might have a dramatic impact on their neutralization activity. All of these observations provide a useful tool to study the induction of a desired type of antibody by different immunogens or different immunization regimens.

Since heavily glycosylated HIV Env protein is a critical component of an HIV vaccine, we wanted to determine the impact of the HIV Env-associated glycan shield on antibody responses. We were able to produce Env proteins with a selective and homogeneous pattern of N-glycosylation using a glycoengineered yeast cell line. Antigenicity of these novel Env proteins was examined by well-characterized human mAbs. Immunogenicity studies showed that they were immunogenic and elicited gp120- specific antibody responses. More significantly, sera elicited by glycan-modified gp120 protein immunogens revealed better neutralizing activities and increased diversity of epitopes compared to sera elicited by traditional gp120 produced in Chinese Hamster Ovary (CHO) cells.

Further, we examined the impact of the delivery order of DNA and protein immunization on antibody responses. We found that DNA prime-protein boost induced a comparable level of Env-specific binding Abs at the peak immunogenicity point to codelivery of DNA. However, antibody responses from DNA prime-protein boost had high avidity and diverse specificities, which improved potency and breadth of neutralizing Abs against Tier 1 viruses. Our data indicate that DNA vaccine priming of the immune system is essential for generation of high-quality antibodies.

Additionally, we determined the relative immunogenicity of gp120 and gp160 Env in the context of DNA prime-protein boost vaccination to induce high-quality antibody responses. Immunized sera from gp120 DNA primed animals, but not those primed with gp160 DNA, presented with distinct antibody repertoire specificities, a high magnitude of CD4 binding site-directed binding capabilities as well as neutralizing activities. We confirmed the importance of using the gp120 Env form at the DNA priming phase, which directly determined the quality of antibody response.

Antiplatelet activity, P2Y(1) and P2Y(1)(2) inhibition, and metabolism in plasma of stereoisomers of diadenosine 5',5'''-P(1) ,P(4)-dithio-P(2),P(3)-chloromethylenetetraphosphate

Thu, 08/13/2015 - 10:43am

BACKGROUND: Diadenosine tetraphosphate (Ap4A), a constituent of platelet dense granules, and its P1,P4-dithio and/or P2,P3-chloromethylene analogs, inhibit adenosine diphosphate (ADP)-induced platelet aggregation. We recently reported that these compounds antagonize both platelet ADP receptors, P2Y1 and P2Y12. The most active of those analogs, diadenosine 5',5''''-P1,P4-dithio-P2,P3-chloromethylenetetraphosphate, (compound 1), exists as a mixture of 4 stereoisomers.

OBJECTIVE: To separate the stereoisomers of compound 1 and determine their effects on platelet aggregation, platelet P2Y1 and P2Y12 receptor antagonism, and their metabolism in human plasma.

METHODS: We separated the 4 diastereomers of compound 1 by preparative reversed-phase chromatography, and studied their effect on ADP-induced platelet aggregation, P2Y1-mediated changes in cytosolic Ca2+, P2Y12-mediated changes in VASP phosphorylation, and metabolism in human plasma.

RESULTS: The inhibition of ADP-induced human platelet aggregation and human platelet P2Y12 receptor, and stability in human plasma strongly depended on the stereo-configuration of the chiral P1- and P4-phosphorothioate groups, the SPSP diastereomer being the most potent inhibitor and completely resistant to degradation in plasma, and the RPRP diastereomer being the least potent inhibitor and with the lowest plasma stability. The inhibitory activity of SPRP diastereomers depended on the configuration of the pseudo-asymmetric carbon of the P2,P3-chloromethylene group, one of the configurations being significantly more active than the other. Their plasma stability did not differ significantly, being intermediate to that of the SPSP and the RPRP diastereomers.

CONCLUSIONS: The presently-described stereoisomers have utility for structural, mechanistic, and drug development studies of dual antagonists of platelet P2Y1 and P2Y12 receptors.

DeTEXT: A Database for Evaluating Text Extraction from Biomedical Literature Figures

Thu, 08/13/2015 - 10:43am

Hundreds of millions of figures are available in biomedical literature, representing important biomedical experimental evidence. Since text is a rich source of information in figures, automatically extracting such text may assist in the task of mining figure information. A high-quality ground truth standard can greatly facilitate the development of an automated system. This article describes DeTEXT: A database for evaluating text extraction from biomedical literature figures. It is the first publicly available, human-annotated, high quality, and large-scale figure-text dataset with 288 full-text articles, 500 biomedical figures, and 9308 text regions. This article describes how figures were selected from open-access full-text biomedical articles and how annotation guidelines and annotation tools were developed. We also discuss the inter-annotator agreement and the reliability of the annotations. We summarize the statistics of the DeTEXT data and make available evaluation protocols for DeTEXT. Finally we lay out challenges we observed in the automated detection and recognition of figure text and discuss research directions in this area. DeTEXT is publicly available for downloading at