What doesn't kill you makes you stranger: Dipeptidyl peptidase-4 (CD26) proteolysis differentially modulates the activity of many peptide hormones and cytokines generating novel cryptic bioactive ligands

Dipeptidyl peptidase 4 (DPP4) is an exopeptidase found either on cell surfaces where it is highly regulated in terms of its expression and surface availability (CD26) or in a free/circulating soluble constitutively available and intrinsically active form. It is responsible for proteolytic cleavage of many peptide substrates. In this review we discuss the idea that DPP4-cleaved peptides are not necessarily inactivated, but rather can possess either a modified receptor selectivity, modified bioactivity, new antagonistic activity, or even a novel activity relative to the intact parent ligand. We examine in detail five different major DPP4 substrates: glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), peptide tyrosine-tyrosine (PYY), and neuropeptide Y (NPY), and stromal derived factor 1 (SDF-1 aka CXCL12). We note that discussion of the cleaved forms of these five peptides are underrepresented in the research literature, and are both poorly investigated and poorly understood, representing a serious research literature gap. We believe they are understudied and misinterpreted as inactive due to several factors. This includes lack of accurate and specific quantification methods, sample collection techniques that are inherently inaccurate and inappropriate, and a general perception that DPP4 cleavage inactivates its ligand substrates. Increasing evidence points towards many DPP4-cleaved ligands having their own bioactivity. For example, GLP-1 can work through a different receptor than GLP-1R, DPP4-cleaved GIP can function as a GIP receptor antagonist at high doses, and DPP4-cleaved PYY, NPY, and CXCL12 can have different receptor selectivity, or can bind novel, previously unrecognized receptors to their intact ligands, resulting in altered signaling and functionality. We believe that more rigorous research in this area could lead to a better understanding of DPP4’s role and the biological importance of the generation of novel cryptic ligands. This will also significantly impact our understanding of the clinical effects and side effects of DPP4-inhibitors as a class of anti-diabetic drugs that potentially have an expanding clinical relevance. This will be specifically relevant in targeting DPP4 substrate ligands involved in a variety of other major clinical acute and chronic injury/disease areas including inflammation, immunology, cardiology, stroke, musculoskeletal disease and injury, as well as cancer biology and tissue maintenance in aging

Molecular assays for antimalarial drug resistance surveillance: A target product profile.

Antimalarial drug resistance is a major constraint for malaria control and elimination efforts. Artemisinin-based combination therapy is now the mainstay for malaria treatment. However, delayed parasite clearance following treatment with artemisinin derivatives has now spread in the Greater Mekong Sub region and may emerge or spread to other malaria endemic regions. This spread is of great concern for malaria control programmes, as no alternatives to artemisinin-based combination therapies are expected to be available in the near future. There is a need to strengthen surveillance systems for early detection and response to the antimalarial drug resistance threat. Current surveillance is mainly done through therapeutic efficacy studies; however these studies are complex and both time- and resource-intensive. For multiple common antimalarials, parasite drug resistance has been correlated with specific genetic mutations, and the molecular markers associated with antimalarial drug resistance offer a simple and powerful tool to monitor the emergence and spread of resistant parasites. Different techniques to analyse molecular markers associated with antimalarial drug resistance are available, each with advantages and disadvantages. However, procedures are not adequately harmonized to facilitate comparisons between sites. Here we describe the target product profiles for tests to analyse molecular markers associated with antimalarial drug resistance, discuss how use of current techniques can be standardised, and identify the requirements for an ideal product that would allow malaria endemic countries to provide useful spatial and temporal information on the spread of resistance

Narratives to enhance smoking cessation interventions among African-American smokers, the ACCE project

BACKGROUND: Low-income, African-American smokers are less likely to have resources to aid in quitting smoking. Narrative communication may provide an enhancement to traditional smoking cessation interventions like NRT, medications, or behavioral treatments for this audience. After extensive pilot testing of stories and personal experiences with smoking cessation from African-Americans from a low-income community, we conducted a randomized control trial using stories to augment routine inpatient treatment among African-Americans at an urban Southern hospital (N = 300). RESULTS: Differences in smoking cessation outcomes between the intervention (stories DVD + routine clinical treatment) and control (routine clinical treatment) arms were compared using self-report and carbon monoxide measurement at 6-months. Compared to control, individuals who viewed the intervention stories DVD reported greater intentions to quit. Although continuous quitting marginally favored the intervention, our main result did not reach statistical significance (p = 0.16). CONCLUSION: Narrative communication via storytelling to promote smoking cessation among African-Americans in the South is one method to communicate smoking cessation. Results suggest this may not be sufficient as a stand-alone augmentation of routine clinical treatment for continuous smoking cessation. Smoking cessation efforts need to continually assess different means of communicating to smokers about quitting. CLINICAL TRIALS REGISTRATION: The ClinicalTrials.gov Identifier is NCT00101491. This trial was registered January 10, 2005

Cytokines and growth factors cross-link heparan sulfate

The glycosaminoglycan heparan sulfate (HS), present at the surface of most cells and ubiquitous in extracellular matrix, binds many soluble extracellular signalling molecules such as chemokines and growth factors, and regulates their transport and effector functions. It is, however, unknown whether upon binding HS these proteins can affect the long-range structure of HS. To test this idea, we interrogated a supramolecular model system, in which HS chains grafted to streptavidin-functionalized oligoethylene glycol monolayers or supported lipid bilayers mimic the HS-rich pericellular or extracellular matrix, with the biophysical techniques quartz crystal microbalance (QCM-D) and fluorescence recovery after photobleaching (FRAP). We were able to control and characterize the supramolecular presentation of HS chains—their local density, orientation, conformation and lateral mobility—and their interaction with proteins. The chemokine CXCL12α (or SDF-1α) rigidified the HS film, and this effect was due to protein-mediated cross-linking of HS chains. Complementary measurements with CXCL12α mutants and the CXCL12γ isoform provided insight into the molecular mechanism underlying cross-linking. Fibroblast growth factor 2 (FGF-2), which has three HS binding sites, was also found to cross-link HS, but FGF-9, which has just one binding site, did not. Based on these data, we propose that the ability to cross-link HS is a generic feature of many cytokines and growth factors, which depends on the architecture of their HS binding sites. The ability to change matrix organization and physico-chemical properties (e.g. permeability and rigidification) implies that the functions of cytokines and growth factors may not simply be confined to the activation of cognate cellular receptors

Tuberculous Granuloma Induction via Interaction of a Bacterial Secreted Protein with Host Epithelium

Granulomas, organized aggregates of immune cells, are a hallmark of tuberculosis, and have traditionally been thought to restrict mycobacterial growth. However, analysis of Mycobacterium marinum in zebrafish has shown that the early granuloma facilitates mycobacterial growth; uninfected macrophages are recruited to the granuloma where they are productively infected by M. marinum. Here, we identified the molecular mechanism by which mycobacteria induce granulomas: the bacterial secreted protein ESAT-6, which has long been implicated in virulence, induced matrix metalloproteinase-9 (MMP9) in epithelial cells neighboring infected macrophages. MMP9 enhanced recruitment of macrophages, which contributed to nascent granuloma maturation and bacterial growth. Disruption of MMP9 function attenuated granuloma formation and bacterial growth. Thus, interception of epithelial MMP9 production could hold promise as a host-targeting tuberculosis therapy

Immunoblot analysis of the seroreactivity to recombinant Borrelia burgdorferi sensu lato antigens, including VlsE, in the long-term course of treated patients with Erythema migrans

Objective: We evaluated whether immunoblotting is capable of substantiating the posttreatment clinical assessment of patients with erythema migrans ( EM), the hallmark of early Lyme borreliosis. Methods: In 50 patients, seroreactivity to different antigens of Borrelia burgdorferi sensu lato was analyzed by a recombinant immunoblot test (IB) in consecutive serum samples from a minimum follow-up period of 1 year. Antigens in the IgG test were decorin- binding protein A, internal fragment of p41 (p41i), outer surface protein C (OspC), p39, variable major protein-like sequence expressed (VlsE), p58 and p100; those in the IgM test were p41i, OspC and p39. Immune responses were correlated with clinical and treatment-related parameters. Results: Positive IB results were found in 50% before, in 57% directly after therapy and in 44% by the end of the follow-up for the IgG class, and in 36, 43 and 12% for the IgM class. In acute and convalescence phase sera, VlsE was most immunogenic on IgG testing 60 and 70%), and p41i (46 and 57%) and OspC (40 and 57%) for the IgM class. By the end of the follow-up, only the anti-p41i lgM response was significantly decreased to 24%. Conclusions: No correlation was found between IB results and treatment-related parameters. Thus, immunoblotting does not add to the clinical assessment of EM patients after treatment. Copyright (c) 2008 S. Karger AG, Basel

Gender-dependent differences in plasma matrix metalloproteinase-8 elevated in pulmonary tuberculosis.

Tuberculosis (TB) remains a global health pandemic and greater understanding of underlying pathogenesis is required to develop novel therapeutic and diagnostic approaches. Matrix metalloproteinases (MMPs) are emerging as key effectors of tissue destruction in TB but have not been comprehensively studied in plasma, nor have gender differences been investigated. We measured the plasma concentrations of MMPs in a carefully characterised, prospectively recruited clinical cohort of 380 individuals. The collagenases, MMP-1 and MMP-8, were elevated in plasma of patients with pulmonary TB relative to healthy controls, and MMP-7 (matrilysin) and MMP-9 (gelatinase B) were also increased. MMP-8 was TB-specific (p<0.001), not being elevated in symptomatic controls (symptoms suspicious of TB but active disease excluded). Plasma MMP-8 concentrations inversely correlated with body mass index. Plasma MMP-8 concentration was 1.51-fold higher in males than females with TB (p<0.05) and this difference was not due to greater disease severity in men. Gender-specific analysis of MMPs demonstrated consistent increase in MMP-1 and -8 in TB, but MMP-8 was a better discriminator for TB in men. Plasma collagenases are elevated in pulmonary TB and differ between men and women. Gender must be considered in investigation of TB immunopathology and development of novel diagnostic markers

Specialized dynamical properties of promiscuous residues revealed by simulated conformational ensembles

The ability to interact with different partners is one of the most important features in proteins. Proteins that bind a large number of partners (hubs) have been often associated with intrinsic disorder. However, many examples exist of hubs with an ordered structure, and evidence of a general mechanism promoting promiscuity in ordered proteins is still elusive. An intriguing hypothesis is that promiscuous binding sites have specific dynamical properties, distinct from the rest of the interface and pre-existing in the protein isolated state. Here, we present the first comprehensive study of the intrinsic dynamics of promiscuous residues in a large protein data set. Different computational methods, from coarse-grained elastic models to geometry-based sampling methods and to full-atom Molecular Dynamics simulations, were used to generate conformational ensembles for the isolated proteins. The flexibility and dynamic correlations of interface residues with a different degree of binding promiscuity were calculated and compared considering side chain and backbone motions, the latter both on a local and on a global scale. The study revealed that (a) promiscuous residues tend to be more flexible than nonpromiscuous ones, (b) this additional flexibility has a higher degree of organization, and (c) evolutionary conservation and binding promiscuity have opposite effects on intrinsic dynamics. Findings on simulated ensembles were also validated on ensembles of experimental structures extracted from the Protein Data Bank (PDB). Additionally, the low occurrence of single nucleotide polymorphisms observed for promiscuous residues indicated a tendency to preserve binding diversity at these positions. A case study on two ubiquitin-like proteins exemplifies how binding promiscuity in evolutionary related proteins can be modulated by the fine-tuning of the interface dynamics. The interplay between promiscuity and flexibility highlighted here can inspire new directions in protein-protein interaction prediction and design methods. © 2013 American Chemical Society

A High-Throughput Screen for Tuberculosis Progression

One-third of the world population is infected with Mycobacterium tuberculosis and multi-drug resistant strains are rapidly evolving. The noticeable absence of a whole organism high-throughput screening system for studying the progression of tuberculosis is fast becoming the bottleneck in tuberculosis research. We successfully developed such a system using the zebrafish Mycobacterium marinum infection model, which is a well-characterized model for tuberculosis progression with biomedical significance, mimicking hallmarks of human tuberculosis pathology. Importantly, we demonstrate the suitability of our system to directly study M. tuberculosis, showing for the first time that the human pathogen can propagate in this vertebrate model, resulting in similar early disease symptoms to those observed upon M. marinum infection. Our system is capable of screening for disease progression via robotic yolk injection of early embryos and visual flow screening of late-stage larvae. We also show that this system can reliably recapitulate the standard caudal vein injection method with a throughput level of 2,000 embryos per hour. We additionally demonstrate the possibility of studying signal transduction leading to disease progression using reverse genetics at high-throughput levels. Importantly, we use reference compounds to validate our system in the testing of molecules that prevent tuberculosis progression, making it highly suited for investigating novel anti-tuberculosis compounds in vivo