Crystal and molecular structure of two geometrically restricted chemotactic tripeptides, analogues of formyl-methionine-leucine-phenylalanine

The crystal structures of HCO-Met-Leu-Phe-OC(CH3)3, (CH25H39N3O5S), fMLP-OtBu, and HCO-Met psi [CSNH]-Leu-Phe-OCH3, (C22H33N3O4S2), fMS LP-OMe, have been determined by single crystal X-ray diffraction, and their conformational properties investigated by molecular mechanics energy calculations. Crystals of fMLP-OtBu are monoclinic, space group P2(1), a = 12.027(4), b = 9.492(3), c = 12.660(4) A, beta = 101.99(3) degrees, Z = 2; those of fMS LP-OMe are orthorhombic, space group P2(1)2(1)2(1), a = 7.130(1), b = 12.097(2), c = 31.060(5) A, Z = 4. The first compounds fMLP-OtBu is the t-butyl ester of the tripeptide fMLP that represents one of the most potent compounds in inducing the lysozyme release from human neutrophils that reflects the chemotactic activity. From the crystal structure, it is shown that the orientation of the phenylalanine side chain is largely affected by the presence of the bulky group. fMSLP-OMe was shown to be inactive after thionation of the methionine residue in the original tripeptide. Nevertheless, the crystal structure does not reveal any influence of the presence of the thionated peptidic bond on the backbone conformation. The X-ray results have been used to generate parameters for empirical energy calculations. Subsequently, a strategy based on random generation of conformations followed by energy-minimization was applied to investigate the conformational space of thiopeptides, in comparison with normal peptides. From molecular free energy calculations, it is shown that the main influence of the introduction of a thioamide bond on the molecular structure is to prevent the existence of C7(eq) conformations involving the thiomethionine residue. Consequently, a larger number of conformers are found to form intramolecular hydrogen bonds involving the formyl group, reducing its availability to interact with the receptor. For the first time, the theoretical prediction of the existence of C7eq conformations for fMLP is made. The resulting conformers are compared to previously active structures of these chemotactic agents.     

Identification of the Flagellin Glycosylation System in Burkholderia cenocepacia and the Contribution of Glycosylated Flagellin to Evasion of Human Innate Immune Responses

Burkholderia cenocepacia is an opportunistic pathogen threatening patients with cystic fibrosis. Flagella are required for biofilm formation, as well as adhesion to and invasion of epithelial cells. Recognition of flagellin via the Toll-like receptor 5 (TLR5) contributes to exacerbate B. cenocepacia-induced lung epithelial inflammatory responses. In this study, we report that B. cenocepacia flagellin is glycosylated on at least 10 different sites with a single sugar, 4,6-dideoxy-4-(3-hydroxybutanoylamino)-d-glucose. We have identified key genes that are required for flagellin glycosylation, including a predicted glycosyltransferase gene that is linked to the flagellin biosynthesis cluster and a putative acetyltransferase gene located within the O-antigen lipopolysaccharide cluster. Another O-antigen cluster gene, rmlB, which is required for flagellin glycan and O-antigen biosynthesis, was essential for bacterial viability, uncovering a novel target against Burkholderia infections. Using glycosylated and nonglycosylated purified flagellin and a cell reporter system to assess TLR5-mediated responses, we also show that the presence of glycan in flagellin significantly impairs the inflammatory response of epithelial cells. We therefore suggest that flagellin glycosylation reduces recognition of flagellin by host TLR5, providing an evasive strategy to infecting bacteria.     

Intestinal barrier function in response to abundant or depleted mucosal glutathione in Salmonella-infected rats

Background  

Glutathione, the main antioxidant of intestinal epithelial cells, is suggested to play an important role in gut barrier function and prevention of inflammation-related oxidative damage as induced by acute bacterial infection. Most studies on intestinal glutathione focus on oxidative stress reduction without considering functional disease outcome. Our aim was to determine whether depletion or maintenance of intestinal glutathione changes susceptibility of rats to Salmonella infection and associated inflammation.     

Anti-Angiogenic/Vascular Effects of the mTOR Inhibitor Everolimus Are Not Detectable by FDG/FLT-PET

Noninvasive functional imaging of tumors can provide valuable early-response biomarkers, in particular, for targeted chemotherapy. Using various experimental tumor models, we have investigated the ability of positron emission tomography (PET) measurements of 2-deoxy-2-[¹⁸F]fluoro-glucose (FDG) and 3′-deoxy-3′-[¹⁸F]fluorothymidine (FLT) to detect response to the allosteric mammalian target of rapamycin (mTOR) inhibitor everolimus. Tumor models were declared sensitive (murine melanoma B16/BL6 and human lung H596) or relatively insensitive (human colon HCT116 and cervical KB31), according to the IC₅₀ values (concentration inhibiting cell growth by 50%) for inhibition of proliferation in vitro (<10 nM and >1 µM, respectively). Everolimus strongly inhibited growth of the sensitive models in vivo but also significantly inhibited growth of the insensitive models, an effect attributable to its known anti-angiogenic/vascular properties. However, although tumor FDG and FLT uptake was significantly reduced in the sensitive models, it was not affected in the insensitive models, suggesting that endothelial-directed effects could not be detected by these PET tracers. Consistent with this hypothesis, in a well-vascularized orthotopic rat mammary tumor model, other antiangiogenic agents also failed to affect FDG uptake, despite inhibiting tumor growth. In contrast, the cytotoxic patupilone, a microtubule stabilizer, blocked tumor growth, and markedly reduced FDG uptake. These results suggest that FDG/FLT-PET may not be a suitable method for early markers of response to antiangiogenic agents and mTOR inhibitors in which anti-angiogenic/vascular effects predominate because the method could provide false-negative responses. These conclusions warrant clinical testing.     

Influence of inverse dynamics methods on the calculation of inter-segmental moments in vertical jumping and weightlifting

Background  

A vast number of biomechanical studies have employed inverse dynamics methods to calculate inter-segmental moments during movement. Although all inverse dynamics methods are rooted in classical mechanics and thus theoretically the same, there exist a number of distinct computational methods. Recent research has demonstrated a key influence of the dynamics computation of the inverse dynamics method on the calculated moments, despite the theoretical equivalence of the methods. The purpose of this study was therefore to explore the influence of the choice of inverse dynamics on the calculation of inter-segmental moments.     

Ensemble attribute profile clustering: discovering and characterizing groups of genes with similar patterns of biological features

Background  

Ensemble attribute profile clustering is a novel, text-based strategy for analyzing a user-defined list of genes and/or proteins. The strategy exploits annotation data present in gene-centered corpora and utilizes ideas from statistical information retrieval to discover and characterize properties shared by subsets of the list. The practical utility of this method is demonstrated by employing it in a retrospective study of two non-overlapping sets of genes defined by a published investigation as markers for normal human breast luminal epithelial cells and myoepithelial cells.     

Insulin-like growth factor binding protein-6 (IGFBP-6) interacts with DNA-end binding protein Ku80 to regulate cell fate

Insulin-like growth factor binding protein-6 (IGFBP-6) is a growth inhibitory protein that regulates the availability of insulin-like growth factors (IGFs). We recently reported that IGFBP-6 exerts intracellular actions via its translocation to the nucleus. We now show that IGFBP-6 co-purifies by tandem-affinity with nuclear proteins involved in DNA stability and repair such as Ku80, Ku70, histone H2B and importin-α. Furthermore, this report shows that IGFBP-6 and Ku80 interact specifically using two active binding sites for Ku80 in IGFBP-6. One of the binding sites [196RKR199], as part of the NLS-sequence in IGFBP-6 also binds importin-α which may selectively compete with Ku80 regulating its trafficking to the nucleus. Moreover, IGFBP-6 co-localized with Ku80 based on a cell cycle pattern. Overexpression of IGFBP-6 increased the nuclear Ku80 in mitotic cells and reduced it post-mitosis. It is known that if highly expressed IGFBP-6 induces apoptosis and in our model, the down-regulation of Ku80 by specific siRNAs enhanced the apoptotic effect caused by the IGFBP-6 overexpression. This study demonstrates that IGFBP-6 alters cell survival by potentially regulating the availability of Ku80 for the DNA-repair process. This action represents a novel mechanism by which growth inhibitory proteins such as IGFBP-6 regulate cell fate.     

Birth Origin Differentially Affects Depressive-Like Behaviours: Are Captive-Born Cynomolgus Monkeys More Vulnerable to Depression than Their Wild-Born Counterparts?

Background

Adverse early-life experience might lead to the expression of abnormal behaviours in animals and the predisposition to psychiatric disorder (e.g. major depressive disorder) in Humans. Common breeding processes employ weaning and housing conditions different from what happens in the wild.

Methods

The present study, therefore, investigated whether birth origin impacts the possible existence of spontaneous atypical/abnormal behaviours displayed by 40 captive-born and 40 wild-born socially-housed cynomolgus macaques in farming conditions using an unbiased ethological scan-sampling analysis followed by multifactorial correspondence and hierarchical clustering analyses.

Results

We identified 10 distinct profiles (groups A to J) that significantly differed on several behaviours, body postures, body orientations, distances between individuals and locations in the cage. Data suggest that 4 captive-born and 1 wild-born animals (groups G and J) present depressive-like symptoms, unnatural early life events thereby increasing the risk of developing pathological symptoms. General differences were also highlighted between the captive- and wild-born populations, implying the expression of differential coping mechanisms in response to the same captive environment.

Conclusions

Birth origin thus impacts the development of atypical ethologically-defined behavioural profiles, reminiscent of certain depressive-like symptoms. The use of unbiased behavioural observations might allow the identification of animal models of human mental/behavioural disorders and their most appropriate control groups.     

Collection,Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.