The high frequency Indian rhesus macaque MHC class I molecule, Mamu-B*01, does not appear to be involved in CD8+ T lymphocyte responses to SIVmac239

Although the SIV-infected Indian rhesus macaque (Macaca mulatta) is the animal model most widely used for studying HIV infection, our current understanding of the functional macaque MHC class I molecules is limited. To date, SIV-derived CD8+ T lymphocyte epitopes from only three high frequency macaque MHC class I molecules have been extensively characterized. In this study, we defined the peptide-binding properties of the high frequency Indian rhesus macaque class I molecule, Mamu-B*01 ( approximately 26%). We first identified a preliminary binding motif by eluting and sequencing endogenously bound Mamu-B*01 ligands. We further characterized the peptide-binding characteristics using panels of single amino acid substitution analogs. Using this detailed motif, 507 peptides derived from SIV(mac)239 were identified and tested for their Mamu-B*01 binding capacity. Surprisingly, only 11 (2.2%) of these motif-containing peptides bound with IC50 values < or =500 nM. We assessed the immunogenicity of these peptides using freshly isolated PBMC from ten Mamu-B*01+ SIV-infected rhesus macaques in IFN-gamma ELISPOT and IFN-gamma/TNF-alpha intracellular cytokine staining assays. Lymphocytes from these SIV-infected macaques responded to none of these peptides. Furthermore, there was no sequence variation indicative of escape in the regions of the virus that encoded these peptides. Additionally, we could not confirm previous reports of SIV-derived Mamu-B*01-restricted epitopes in the Env and Gag proteins. Our results suggest that the high frequency MHC class I molecule, Mamu-B*01, is not involved in SIV-specific CD8+ T lymphocyte responses.     

HLA-A*0201, HLA-A*1101, and HLA-B*0702 transgenic mice recognize numerous poxvirus determinants from a wide variety of viral gene products

In virus models explored in detail in mice, CTL typically focus on a few immunodominant determinants. In this study we use a multipronged approach to understand the diversity of CTL responses to vaccinia virus, a prototypic poxvirus with a genome approximately 20-fold larger than that of the model RNA viruses typically studied in mice. Based on predictive computational algorithms for peptide binding to HLA supertypes, we synthesized a panel of 2889 peptides to begin to create an immunomic map of human CTL responses to poxviruses. Using this panel in conjunction with CTLs from vaccinia virus-infected HLA transgenic mice, we identified 14 HLA-A*0201-, 4 HLA-A*1101-, and 3 HLA-B*0702-restricted CD8(+) T cell determinants distributed over 20 distinct proteins. These peptides were capable of binding one or multiple A2, A3, and B7 supertype molecules with affinities typical of viral determinants. Surprisingly, many of the viral proteins recognized are predicted to be late gene products, in addition to the early intermediate gene products expected. Nearly all of the determinants identified have identical counterparts encoded by modified vaccinia virus Ankara as well as variola virus, the agent of smallpox. These findings have implications for the design of new smallpox vaccines and the understanding of immune responses to large DNA viruses in general.     

Circadian rhythm of glucose uptake in cultures of skeletal muscle cells and adipocytes in Wistar-Kyoto, Wistar, Goto-Kakizaki, and spontaneously hypertensive rats

Hypertension and noninsulin-dependent diabetes mellitus are usually associated with marked glucose intolerance. Hypertensive and even nonhypertensive diabetic individuals display disturbances of the normal circadian blood pressure rhythm. However, little is known about circadian changes of the glucose uptake in muscle and fat cells, the major glucose utilizing tissues. Therefore, we investigated circadian rhythms of glucose uptake in primary muscle and fat cell cultures of hypertensive and type II diabetic rats and their respective control strains. 2-Deoxy-D-(1-3H)glucose uptake was measured over 48 h after synchronization of cells by means of medium change with and without addition of insulin, phloretine, and/or staurosporine. The circadian changes of glucose uptake were assessed by fitting cosine curves to the uptake values. Insulin stimulation of deoxyglucose uptake was only present in control animals, not in hypertensive and diabetic rats. Deoxyglucose uptake displayed a circadian rhythm in control animals, and was markedly disturbed in hypertensive and diabetic animals. Blocking of glucose transporters by phloretine abolished the circadian pattern of deoxyglucose uptake indicating a role of glucose transporters in its generation. Inhibition of kinases by staurosporine inhibited the insulin-stimulated deoxyglucose uptake, but did not dampen the circadian rhythmicity of basal deoxyglucose uptake. The generation of the circadian rhythm of glucose uptake in muscle and fat cell cultures is therefore probably insulin independent and independent of protein kinases. In summary, our results show for the first time: (a) a circadian rhythm of deoxyglucose uptake in glucose utilizing muscle and fat cells in vitro, (b) a disruption of this rhythm in cells of hypertensive and diabetic rats.     

Development of a safe and scalable route towards a tau PET tracer precursor

A scalable 5-step synthesis of the diazacarbazole derivative 1 used as tau PET tracer precursor is reported. Key features of this synthesis include a Buchwald-Hartwig amination, a Pd catalyzed CH activation and a Suzuki-Miyaura cross-coupling.     

Bifidobacteria or Fiber Protects against Diet-Induced Microbiota-Mediated Colonic Mucus Deterioration

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Structure and dynamics of the two amphipathic arginine-rich peptides RW9 and RL9 in a lipid environment investigated by solid-state NMR and MD simulations

Cell penetrating peptides (CPPs) are able to cross membranes without using receptors but only little information about the underlying mechanism is available. In this work, we investigate the interaction of the two arginine-rich CPPs RW9 and RL9 with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), and POPC/POPG membranes with varying POPG content using isothermal titration calorimetry (ITC), solid-state nuclear magnetic resonance (NMR) spectroscopy, and molecular dynamics (MD) simulations. Both peptides were derived from the known CPP penetratin and it was shown previously that RW9 is able to penetrate membranes better than RL9. Overall, the results show that both RW9 and RL9 have a relatively small influence on the membrane. They increase the order of the lipids in the headgroup region and reduce order in the acyl chains indicating that they are located in the lipid/water interface. In addition, the flexibility of the membrane is slightly increased by both peptides but RW9 has a larger influence than RL9. The differences observed in the influences on POPC and POPG as well as MD simulations on the mixed POPC/POPG bilayers of 850 ns length each show that both peptides preferentially associate with and enrich the charged PG lipids almost 2fold in an area of 12 Å around the peptides. As expected, we could not observe any membrane crossing on the simulation time scale of 850 ns but observed that some peptides flipped their orientation during binding to the membrane. Interestingly, all observed flips coincided with structural changes in the peptides indicating that structural changes or flexibility might play a role during the binding of arginine-rich CPPs to membranes.     

Unusual Features of Vaccinia Virus Extracellular Virion Form Neutralization Resistance Revealed in Human Antibody Responses to the Smallpox Vaccine

The extracellular virion form (EV) of vaccinia virus (VACV) is essential for viral pathogenesis and is difficult to neutralize with antibodies. Why this is the case and how the smallpox vaccine overcomes this challenge remain incompletely understood. We previously showed that high concentrations of anti-B5 antibodies are insufficient to directly neutralize EV (M. R. Benhnia, et al., J. Virol. 83:1201–1215, 2009). This allowed for at least two possible interpretations: covering the EV surface is insufficient for neutralization, or there are insufficient copies of B5 to allow anti-B5 IgG to cover the whole surface of EV and another viral receptor protein remains active. We endeavored to test these possibilities, focusing on the antibody responses elicited by immunization against smallpox. We tested whether human monoclonal antibodies (MAbs) against the three major EV antigens, B5, A33, and A56, could individually or together neutralize EV. While anti-B5 or anti-A33 (but not anti-A56) MAbs of appropriate isotypes were capable of neutralizing EV in the presence of complement, a mixture of anti-B5, anti-A33, and anti-A56 MAbs was incapable of directly neutralizing EV, even at high concentrations. This remained true when neutralizing the IHD-J strain, which lacks a functional version of the fourth and final known EV surface protein, A34. These immunological data are consistent with the possibility that viral proteins may not be the active component of the EV surface for target cell binding and infectivity. We conclude that the protection afforded by the smallpox vaccine anti-EV response is predominantly mediated not by direct neutralization but by isotype-dependent effector functions, such as complement recruitment for antibodies targeting B5 and A33.     

A strategy to determine HLA class II restriction broadly covering the DR, DP, and DQ allelic variants most commonly expressed in the general population

Classic ways to determine MHC restriction involve inhibition with locus-specific antibodies and antigen presentation assays with panels of cell lines matched or mismatched at the various loci of interest. However, these determinations are often complicated by T cell epitope degeneracy and promiscuity. We describe a selection of 46 HLA DR, DQ, and DP specificities that provide worldwide population (phenotypic) coverage of almost 90 % at each locus, and account for over 66 % of all genes at each locus. This panel afforded coverage of at least four HLA class II alleles in over 95 % of the individuals in four study populations of diverse ethnicity from the USA and South Africa. Next, a panel of single HLA class II-transfected cell lines, corresponding to these 46 allelic variants was assembled, consisting of lines previously developed and 15 novel lines generated for the present study. The novel lines were validated by assessing their HLA class II expression by FACS analysis, the in vitro peptide binding activity of HLA molecules purified from the cell lines, and their antigen presenting capacity to T cell lines of known restriction. We also show that these HLA class II-transfected cell lines can be used to rapidly and unambiguously determine HLA restriction of epitopes recognized by an individual donor in a single experiment. This panel of lines will enable high throughput determination of HLA restriction, enabling better characterization of HLA class II-restricted T cell responses and facilitating the development of HLA tetrameric staining reagents.     

The Development and Application of a Quantitative Peptide Microarray Based Approach to Protein Interaction Domain Specificity Space

Protein interaction domain (PID) linear peptide motif interactions direct diverse cellular processes in a specific and coordinated fashion. PID specificity, or the interaction selectivity derived from affinity preferences between possible PID-peptide pairs is the basis of this ability. Here, we develop an integrated experimental and computational cellulose peptide conjugate microarray (CPCMA) based approach for the high throughput analysis of PID specificity that provides unprecedented quantitative resolution and reproducibility. As a test system, we quantify the specificity preferences of four Src Homology 2 domains and 124 physiological phosphopeptides to produce a novel quantitative interactome. The quantitative data set covers a broad affinity range, is highly precise, and agrees well with orthogonal biophysical validation, in vivo interactions, and peptide library trained algorithm predictions. In contrast to preceding approaches, the CPCMAs proved capable of confidently assigning interactions into affinity categories, resolving the subtle affinity contributions of residue correlations, and yielded predictive peptide motif affinity matrices. Unique CPCMA enabled modes of systems level analysis reveal a physiological interactome with expected node degree value decreasing as a function of affinity, resulting in minimal high affinity binding overlap between domains; uncover that Src Homology 2 domains bind ligands with a similar average affinity yet strikingly different levels of promiscuity and binding dynamic range; and parse with unprecedented quantitative resolution contextual factors directing specificity. The CPCMA platform promises broad application within the fields of PID specificity, synthetic biology, specificity focused drug design, and network biology.Protein interaction domains (PIDs)¹ often compete for the same linear motif binding sites across a range of affinities, resulting in many potential interactions that may enable the rapid assembly and disassembly of signaling proteins in response to external and internal cues (1, 2). PID-peptide interactions have small binding interfaces, resulting in moderate affinity interactions mediated primarily by a few amino acid “hot-spots” within motifs specific for a particular PID family (3–5). The power of individual residues to direct interactions, the absence of structural constraint for linear motifs, and the modularity of PIDs has enabled the rapid evolution of these networks resulting in many large multimember PID families in higher eukaryotes (6–9). For these large families dedicated to the recognition of similar ligands, PID specificity—or the interaction selectivity derived from affinity preferences between possible PID-peptide pairs—underpins the effective conveyance of specific cell signals. High throughput interaction mapping efforts are used to decipher how this PID “specificity space” is populated, thereby providing insight into protein function and the principles of network architecture and evolution (10–15). The extent of binding overlap or interaction promiscuity within and between PID families for physiological ligands, the affinity range of overlapping interactions, and the biological relevancy of these interactions are important questions thus far poorly resolved by existing high throughput methods. Here, we develop and apply a quantitative high throughput method capable of addressing these questions.Peptide arrays (16, 17), degenerate libraries (18, 19), and phage display (20) are the most frequently applied high throughput approaches for investigating PID specificity. Phage display and degenerate library approaches sample a large ligand space and can produce consensus selectivity motifs that represent the most preferred residues at every position panned. This selectivity data is used to predict interactions, often via position specific scoring matrices (PSSMs) (21–23). However, neither approach can explicitly measure nonbinding events and only large phage display data sets can resolve a limited subset of high-affinity contextual binding information (24). Nonbinding information and contextual interplay, that is, correlated contributions between ligand positions to binding affinity, play important roles in defining the specificity landscapes for multiple PID families (25–27). Having explicit nonbinding or low-affinity information available helps uncover contextual binding information, and improves the accuracy of interaction priority assignment between multiple competing PIDs. Correspondingly, the availability of nonbinding and contextual information improves interaction prediction performance (28, 29). Peptide arrays using physiological ligands do not have these limitations, yet may under-sample PID specificity space because of smaller library sizes. Newly emerging ultrahigh density peptide arrays avoid this particular limitation and are capable of sampling the entire proteome (30). However, a common limitation for all of these techniques is their dependence on nonquantitative interaction information.A comprehensive understanding of PID specificity space requires the quantitative assessment of pairwise interactions across a broad dynamic range of affinities. Common low-throughput biophysical techniques used to measure protein-peptide interaction affinities require highly pure and often large amounts of interactants along with prior knowledge of their interaction. To facilitate discovery and lessen the stringency of the purity and/or quantity requirements of interacting molecules, multiple quantitative high throughput methodologies have been developed. Thus far, the protein microarray (PMA) (31) and high-throughput fluorescence polarization (HTFP) (11, 32) quantitative approaches have been used to examine PID specificity. Unfortunately, PMAs suffer from poor sensitivity, reproducibility, and measurement discrepancies (32, 33). The alternative HTFP assay is more sensitive than PMAs, but also has poor reproducibility and is biased toward high affinity interactions (32). Further, PMAs and HTFP have minimal K_D sensitivities of 2 and 20 μm, respectively. This boundary limits their scope of application considering the importance of moderate affinity interactions for many PID families (34) and the general importance of these interactions in directing emergent phenomena such as ultrasensitivity and interaction gating (35, 36).The Src Homology 2 (SH2) domain phosphotyrosine interactome is a system of prominent physiological importance that has been the subject of multiple preceding interaction mapping and platform development efforts (17, 19, 31, 32, 37, 38). Tyrosine phosphorylation and its recognition by SH2 domains is a uniquely metazoan adaptation (39), prominently involved in the transduction of growth signals (1, 2, 40). SH2 domain specificity has been leveraged in multiple contexts to rewire signaling pathways (15), and specificity dysregulation is associated with multiple diseases, including cancer (40–42). Despite the importance of directing specific signals, many SH2 domains share sequence preferences, resulting in substantial binding crossover over a broad dynamic range of affinities (27). Yet, the extent of this crossover and the peptide motif characteristics responsible for its direction are poorly resolved. In light of these biological attributes and the availability of preceding information to guide platform development and assess its performance, we chose a significantly novel set of 124 physiological phosphopeptides and four SH2 domains as a pilot interactome for our arrays.Here we develop a cellulose peptide conjugate microarray (CPCMA) based approach that is the first broadly applicable protein interaction mapping approach capable of explicitly capturing binding and nonbinding information, resolving the contextual interplay between amino acids that contribute to binding, and providing a quantitative measure of interaction affinity. We optimize multiple production and incubation parameters for the arrays and develop an experimental and computational pipeline to control for systematic artifacts and robustly estimate affinities, ultimately producing a physiological interactome of 368 measurements between 92 peptides and four domains. In contrast to preceding platforms, we find our approach is highly sensitive, extremely precise, and consistent with multiple forms of orthogonal biophysical and in vivo validation. Leveraging unique modes of analysis enabled by the CPCMAs, we demonstrate that SH2 domains vary considerably in promiscuity and binding dynamic range in a manner aligned with biological function. Consistent with recent evidence that pathway crosstalk is subjected to negative selection (10–12, 43) we demonstrate for the first time that the degree distribution of the interactome is a function of affinity, and therefore little binding overlap is found among the highest affinity interactions, suggesting that such sites may be important points of in vivo coregulation. We highlight a few such interactions and corroborate others using available domain-site specific in vivo interaction information. Next, in order to provide insight into the specificity determinants responsible for this segregation we produce for the first time predictive domain specific binding motif affinity matrices. Lastly, we demonstrate the unique ability of the CPCMAs to resolve contextual contributions to binding, and highlight examples where contextual information contributes to binding specificity.Quantitative analysis of systems-level PID properties is a necessary prerequisite for improved mathematical modeling of signal transduction systems, specificity focused drug design, and engineering advanced synthetic biology circuits. Thus, our quantitative CPCMA based approach should have broad application in future network biology and PID specificity efforts.