CD8 kinetically promotes ligand binding to the T-cell antigen receptor

The mechanism of CD8 cooperation with the TCR in antigen recognition was studied on live T cells. Fluorescence correlation measurements yielded evidence of the presence of two TCR and CD8 subpopulations with different lateral diffusion rate constants. Independently, evidence for two subpopulations was derived from the experimentally observed two distinct association phases of cognate peptide bound to class I MHC (pMHC) tetramers and the T cells. The fast phase rate constant ((1.7 +/- 0.2) x 10(5) M(-1) s(-1)) was independent of examined cell type or MHC-bound peptides' structure. Its value was much faster than that of the association of soluble pMHC and TCR ((7.0 +/- 0.3) x 10(3) M(-1) s(-1)), and close to that of the association of soluble pMHC with CD8 ((1-2) x 10(5) M(-1) s(-1)). The fast binding phase disappeared when CD8-pMHC interaction was blocked by a CD8-specific mAb. The latter rate constant was slowed down approximately 10-fold after cells treatment with methyl-beta-cyclodextrin. These results suggest that the most efficient pMHC-cell association route corresponds to a fast tetramer binding to a colocalized CD8-TCR subpopulation, which apparently resides within membrane rafts: the reaction starts by pMHC association with the CD8. This markedly faster step significantly increases the probability of pMHC-TCR encounters and thereby promotes pMHC association with CD8-proximal TCR. The slow binding phase is assigned to pMHC association with a noncolocalized CD8-TCR subpopulation. Taken together with results of cytotoxicity assays, our data suggest that the colocalized, raft-associated CD8-TCR subpopulation is the one capable of inducing T-cell activation.     

CD8+ cytotoxic T lymphocyte activation by soluble major histocompatibility complex-peptide dimers

CD8+ cytotoxic T lymphocyte (CTL) can recognize and kill target cells that express only a few cognate major histocompatibility complex class I-peptide (pMHC) complexes. To better understand the molecular basis of this sensitive recognition process, we studied dimeric pMHC complexes containing linkers of different lengths. Although dimers containing short (10-30-A) linkers efficiently bound to and triggered intracellular calcium mobilization and phosphorylation in cloned CTL, dimers containing long linkers (> or = 80 A) did not. Based on this and on fluorescence resonance energy transfer experiments, we describe a dimeric binding mode in which two T cell receptors engage in an anti-parallel fashion two pMHC complexes facing each other with their constant domains. This binding mode allows integration of diverse low affinity interactions, which increases the overall binding and, hence, the sensitivity of antigen recognition. In proof of this, we demonstrated that pMHC dimers containing one agonist and one null ligand efficiently activate CTL, corroborating the importance of endogenous pMHC complexes in antigen recognition.     

Relationship between Phylogeny and Pathotype for the Bacterial Blight Pathogen of Rice

Several transposable elements were isolated from the genome of Xanthomonas oryzae pv. oryzae. These elements and an avirulence gene isolated from X. oryzae pv. oryzae were used as hybridization probes for a collection of X. oryzae pv. oryzae strains from the Philippines. Each of the sequences was present in multiple copies in all strains examined and showed distinct patterns of hybridizing bands. Phenograms were derived from the restriction fragment length polymorphism data obtained for each of the individual probes and for pooled data from multiple probes. The phenograms derived from the different probes differed in topology and, on the basis of bootstrap analysis, were not equally robust. For all of the probes, including the avirulence gene, some groups (even some haplotypes) consisted of multiple races. The strains were grouped into four major clusters on the basis of the two probes giving the highest bootstrap values. These groups were inferred to represent phylogenetic lineages. Three of the six races of X. oryzae pv. oryzae appeared in more than one of the lineages, and another was present in two sublineages. For three of the races, strains representing different phenetic groups were inoculated on rice cultivars carrying 10 resistance genes. Two new races were differentiated, corresponding to pathogen lineages identified by DNA typing. On the basis of DNA and pathotypic analyses, together with information on the spatial and temporal distribution of the pathogen types from this and other studies, a general picture of X. oryzae pv. oryzae evolution in the Philippines is presented.     

HES1 is a novel downstream modifier of the SHH-GLI3 Axis in the development of preaxial polydactyly

Sonic Hedgehog/GLI3 signaling is critical in regulating digit number, such that Gli3-deficiency results in polydactyly and Shh-deficiency leads to digit number reductions. SHH/GLI3 signaling regulates cell cycle factors controlling mesenchymal cell proliferation, while simultaneously regulating Grem1 to coordinate BMP-induced chondrogenesis. SHH/GLI3 signaling also coordinates the expression of additional genes, however their importance in digit formation remain unknown. Utilizing genetic and molecular approaches, we identified HES1 as a downstream modifier of the SHH/GLI signaling axis capable of inducing preaxial polydactyly (PPD), required for Gli3-deficient PPD, and capable of overcoming digit number constraints of Shh-deficiency. Our data indicate that HES1, a direct SHH/GLI signaling target, induces mesenchymal cell proliferation via suppression of Cdkn1b, while inhibiting chondrogenic genes and the anterior autopod boundary regulator, Pax9. These findings establish HES1 as a critical downstream effector of SHH/GLI3 signaling in the development of PPD.     

Targeted mutagenesis on PDGFRα-Fc identifies amino acid modifications that allow efficient inhibition of HCMV infection while abolishing PDGF sequestration

Platelet-derived growth factor receptor alpha (PDGFRα) serves as an entry receptor for the human cytomegalovirus (HCMV), and soluble PDGFRα-Fc can neutralize HCMV at a half-maximal effective concentration (EC50) of about 10 ng/ml. While this indicates a potential for usage as an HCMV entry inhibitor PDGFRα-Fc can also bind the physiological ligands of PDGFRα (PDGFs), which likely interferes with the respective signaling pathways and represents a potential source of side effects. Therefore, we tested the hypothesis that interference with PDGF signaling can be prevented by mutations in PDGFRα-Fc or combinations thereof, without losing the inhibitory potential for HCMV. To this aim, a targeted mutagenesis approach was chosen. The mutations were quantitatively tested in biological assays for interference with PDGF-dependent signaling as well as inhibition of HCMV infection and biochemically for reduced affinity to PDGF-BB, facilitating quantification of PDGFRα-Fc selectivity for HCMV inhibition. Mutation of Ile 139 to Glu and Tyr 206 to Ser strongly reduced the affinity for PDGF-BB and hence interference with PDGF-dependent signaling. Inhibition of HCMV infection was less affected, thus increasing the selectivity by factor 4 and 8, respectively. Surprisingly, the combination of these mutations had an additive effect on binding of PDGF-BB but not on inhibition of HCMV, resulting in a synergistic 260fold increase of selectivity. In addition, a recently reported mutation, Val 242 to Lys, was included in the analysis. PDGFRα-Fc with this mutation was fully effective at blocking HCMV entry and had a drastically reduced affinity for PDGF-BB. Combining Val 242 to Lys with Ile 139 to Glu and/or Tyr 206 to Ser further reduced PDGF ligand binding beyond detection. In conclusion, this targeted mutagenesis approach identified combinations of mutations in PDGFRα-Fc that prevent interference with PDGF-BB but maintain inhibition of HCMV, which qualifies such mutants as candidates for the development of HCMV entry inhibitors.     

Phosphatidylcholine as a constituent in the colonic mucosal barrier—Physiological and clinical relevance

Phosphatidylcholine (PC) is an important constituent of the gastrointestinal tract. PC molecules are not only important in intestinal cell membranes but also receiving increasing attention as protective agents in the gastrointestinal barrier. They are largely responsible for establishing the hydrophobic surface of the colon. Decreased phospholipids in colonic mucus could be linked to the pathogenesis of ulcerative colitis, a chronic inflammatory bowel disease. Clinical studies revealed that therapeutic addition of PC to the colonic mucus of these patients alleviated the inflammatory activity. This positive role is still elusive, however, we hypothesized that luminal PC has two possible functions: first, it is essential for surface hydrophobicity, and second, it is integrated into the plasma membrane of enterocytes and it modulates the signaling state of the mucosa. The membrane structure and lipid composition of cells is a regulatory component of the inflammatory signaling pathways. In this perspective, we will shortly summarize what is known about the localization and protective properties of PC in the colonic mucosa before turning to its evident medical importance. We will discuss how PC contributes to our understanding of the pathogenesis of ulcerative colitis and how reinforcing the luminal phospholipid monolayer can be used as a therapeutic concept in humans.     

Biological control of rice bacterial blight by plant-associated bacteria producing 2,4-diacetylphloroglucinol

Certain plant-associated strains of fluorescent Pseudomonas spp. are known to produce the antimicrobial antibiotic 2,4-diacetylphloroglucinol (DAPG). It has antibacterial, antifungal, antiviral, and antihelminthic properties and has played a significant role in the biological control of tobacco, wheat, and sugar beet diseases. It has never been reported from India and has not been implicated in the biological suppression of a major disease of the rice crop. Here, we report that a subpopulation of 27 strains of plant-associated Pseudomonas fluorescens screened in a batch of 278 strains of fluorescent pseudomonads produced DAPG. The DAPG production was detected by a PCR-based screening method that used primers Phl2a and Phl2b and amplified a 745-bp fragment characteristic of DAPG. HPLC, 1H NMR, and IR analyses provided further evidence for its production. We report also that this compound inhibited the growth of the devastating rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae in laboratory assays and suppressed rice bacterial blight up to 59%-64% in net-house and field experiments. Tn5 mutants defective in DAPG production (Phl-) of P. fluorescens PTB 9 were much less effective in their suppression of rice bacterial blight.     

A structural model for duck hepatitis B virus core protein derived by extensive mutagenesis

Duck hepatitis B virus (DHBV) shares many fundamental features with human HBV. However, the DHBV core protein (DHBc), forming the nucleocapsid shell, is much larger than that of HBV (HBc) and, in contrast to HBc, there is little direct information on its structure. Here we applied an efficient expression system for recombinant DHBc particles to the biochemical analysis of a large panel of mutant DHBc proteins. By combining these data with primary sequence alignments, secondary structure prediction, and three-dimensional modeling, we propose a model for the fold of DHBc. Its major features are a HBc-like two-domain structure with an assembly domain comprising the first about 185 amino acids and a C-terminal nucleic acid binding domain (CTD), connected by a morphogenic linker region that is longer than in HBc and extends into the CTD. The assembly domain shares with HBc a framework of four major α-helices but is decorated at its tip with an extra element that contains at least one helix and that is made up only in part by the previously predicted insertion sequence. All subelements are interconnected, such that structural changes at one site are transmitted to others, resulting in an unexpected variability of particle morphologies. Key features of the model are independently supported by the accompanying epitope mapping study. These data should be valuable for functional studies on the impact of core protein structure on virus replication, and some of the mutant proteins may be particularly suitable for higher-resolution structural investigations.     

Food Security Monitoring via Mobile Data Collection and Remote Sensing: Results from the Central African Republic

The Central African Republic is one of the world’s most vulnerable countries, suffering from chronic poverty, violent conflicts and weak disaster resilience. In collaboration with Doctors without Borders/Médecins Sans Frontières (MSF), this study presents a novel approach to collect information about socio-economic vulnerabilities related to malnutrition, access to resources and coping capacities. The first technical test was carried out in the North of the country (sub-prefecture Kabo) in May 2015. All activities were aimed at the investigation of technical feasibility, not at operational data collection, which requires a random sampling strategy. At the core of the study is an open-source Android application named SATIDA COLLECT that facilitates rapid and simple data collection. All assessments were carried out by local MSF staff after they had been trained for one day. Once a mobile network is available, all assessments can easily be uploaded to a database for further processing and trend analysis via MSF in-house software. On one hand, regularly updated food security assessments can complement traditional large-scale surveys, whose completion can take up to eight months. Ideally, this leads to a gain in time for disaster logistics. On the other hand, recording the location of every assessment via the smart phones’ GPS receiver helps to analyze and display the coupling between drought risk and impacts over many years. Although the current situation in the Central African Republic is mostly related to violent conflict it is necessary to consider information about drought risk, because climatic shocks can further disrupt the already vulnerable system. SATIDA COLLECT can easily be adapted to local conditions or other applications, such as the evaluation of vaccination campaigns. Most importantly, it facilitates the standardized collection of information without pen and paper, as well as straightforward sharing of collected data with the MSF headquarters or other aid organizations.