The structural characterization of the O-polysaccharide antigen of the lipopolysaccharide of Escherichiacoli serotype O118 and its relation to the O-antigens of Escherichiacoli O151 and Salmonellaenterica O47

Mild acid hydrolysis of the lipopolysaccharide produced by Escherichiacoli O118:H16 standard strain (NRCC 6613) afforded an O-polysaccharide (O-PS) composed of d-galactose, 2-acetamidoylamino-2,6-dideoxy-l-galactose , 2-acetamido-2-deoxy-d-glucose, ribitol, and phosphate (1:1:1:1:1). From DOC-PAGE, sugar and methylation analyses, one- and two-dimensional NMR spectroscopy, capillary electrophoresis-mass spectrometry, hydrolysis, and sequential Smith-type periodate oxidation studies, the O-PS was determined to be an unbranched linear polymer having the structure:[6)-α-d-Galp-(1→3)-α-l-FucpNAm-(1→3)-β-d-GlcpNAc-(1→3)-Rib-ol-5-P-(O→]_nThe structure of the O-PS is consistent with the reported DNA data on the O-antigen gene-cluster of E. coli O118 and interestingly, the O-PS is similar to the structures of the O-antigens of Salmonellaenterica O47 and E. coli O151:H10 reference strain 880-67, as predicted from the results of DNA sequencing of their respective O-antigen gene-clusters.     

Non-linear chromosomal inversion response in prostate after low dose X-radiation exposure

Somatic intrachromosomal recombination can result in inversions and deletions in DNA, which are important mutations in cancer. The pKZ1 chromosomal inversion assay is a sensitive assay for studying the effects of DNA damaging agents using chromosomal inversion as a mutation end-point. We have previously demonstrated that the chromosomal inversion response in pKZ1 spleen after single low doses of X-radiation exposure does not follow the linear no-threshold dose–response model. Here, we optimised a chromosomal inversion screening method to study the effect of low dose X-radiation exposure in pKZ1 prostatic tissue. In the present study, a significant induction in inversions was observed after ultra-low doses of 0.005–0.01 mGy or after a high dose of 1000 mGy, whereas a reduction in inversions to below the sham-treated frequency was observed between 1 and 10 mGy exposure. This is the first report of a reduction to below endogenous frequency for any mutation end-point in prostate. In addition, the doses of radiation studied were at least three orders of magnitude lower than have been reported in other mutation assays in prostate in vivo or in vitro. In sham-treated pKZ1 controls and in pKZ1 mice treated with low doses of 1–10 mGy the number of inversions/gland cross-section rarely exceeded three. Up to 4 and 7 inversions were observed in individual prostatic gland cross-sections after doses ≤0.02 mGy and after 1000 mGy, respectively. The number of inversions identified in individual cross-sections of prostatic glands of untreated mice and all treated mice other than the 1000 mGy treatment group followed a Poisson distribution. The dose–response curves and fold changes observed after all radiation doses studied were similar in spleen and prostate. These results suggest that the pKZ1 assay is measuring a fundamental response to DNA damage after low dose X-radiation exposure which is independent of tissue type.     

Structural elucidation of the O-antigenic polysaccharide from enterohemorrhagic (EHEC) Escherichia coli O48:H21

The structure of the antigenic O-polysaccharide (O-PS) of the lipopolysaccharide (LPS) produced by the enterohemorrhagic strain of Escherichia coli O48:H21 (EHEC) has been elucidated. The O-PS obtained by mild acid hydrolysis of the LPS had [alpha]D +95 (water) and was composed of L-rhamnose (L-Rha), D-galactose (D-Gal), 2-amino-2-deoxy-D-glucose (D-GlcN), 2-amino-2-deoxy-D-galactose (D-GalN), and D-galacturonic acid (D-GalA) (1:1:1:1:1). From the results of methylation analysis, mass spectrometry, 2D NMR, and DOC-PAGE, the O-PS was shown to be a high molecular mass polymer of a repeating pentasaccharide unit having the structure: [structure: see text]. The D-Gal pA non-reducing end groups in the O-PS were partially O-acetylated (approximately 30%) at the O-2 and O-3 positions and the degree of acetylation was variable from batch to batch cell production.     

A cluster of ring stage-specific genes linked to a locus implicated in cytoadherence in Plasmodium falciparum codes for PEXEL-negative and PEXEL-positive proteins exported into the host cell

Blood stages of Plasmodium falciparum export proteins into their erythrocyte host, thereby inducing extensive host cell modifications that become apparent after the first half of the asexual development cycle (ring stage). This is responsible for a major part of parasite virulence. Export of many parasite proteins depends on a sequence motif termed Plasmodium export element (PEXEL) or vacuolar transport signal (VTS). This motif has allowed the prediction of the Plasmodium exportome. Using published genome sequence, we redetermined the boundaries of a previously studied region linked to P. falciparum virulence, reducing the number of candidate genes in this region to 13. Among these, we identified a cluster of four ring stage-specific genes, one of which is known to encode an exported protein. We demonstrate that all four genes code for proteins exported into the host cell, although only two genes contain an obvious PEXEL/VTS motif. We propose that the systematic analysis of ring stage-specific genes will reveal a cohort of exported proteins not present in the currently predicted exportome. Moreover, this provides further evidence that host cell remodeling is a major task of this developmental stage. Biochemical and photobleaching studies using these proteins reveal new properties of the parasite-induced membrane compartments in the host cell. This has important implications for the biogenesis and connectivity of these structures.     

Analysis of NLRP3 in the development of allergic airway disease in mice

The contribution of NLRP3, a member of the nucleotide-binding domain leucine-rich repeat-containing (NLR) family, to the development of allergic airway disease is currently controversial. In this study, we used multiple allergic asthma models to examine the physiologic role of NLRP3. We found no significant differences in airway eosinophilia, histopathologic condition, mucus production, and airway hyperresponsiveness between wild-type and Nlrp3(-/-) mice in either acute (alum-dependent) or chronic (alum-independent) OVA models. In addition to the OVA model, we did not detect a role for NLRP3 in the development of allergic airway disease induced by either acute or chronic house dust mite Ag exposure. Although we did not observe significant phenotypic differences in any of the models tested, we did note a significant reduction of IL-13 and IL-33 in Nlrp3(-/-) mice compared with wild-type controls in the chronic OVA model without added alum. In all of the allergic airway disease models, the NLRP3 inflammasome-associated cytokines IL-1β and IL-18 in the lung were below the level of detection. In sum, this report surveyed four different allergic asthma models and found a modest and selected role for NLRP3 in the alum-free OVA model. However, this difference did not greatly alter the clinical outcome of the disease. This finding suggests that the role of NLRP3 in allergic asthma must be re-evaluated.     

Characterization of NLRP12 during the development of allergic airway disease in mice

Among the 22 members of the nucleotide binding-domain, leucine rich repeat-containing (NLR) family, less than half have been functionally characterized. Of those that have been well studied, most form caspase-1 activating inflammasomes. NLRP12 is a unique NLR that has been shown to attenuate inflammatory pathways in biochemical assays and mediate the lymph node homing of activated skin dendritic cells in contact hypersensitivity responses. Since the mechanism between these two important observations remains elusive, we further evaluated the contribution of NLRP12 to organ specific adaptive immune responses by focusing on the lung, which, like skin, is exposed to both exogenous and endogenous inflammatory agents. In models of allergic airway inflammation induced by either acute ovalbumin (OVA) exposure or chronic house dust mite (HDM) antigen exposure, Nlrp12(-/-) mice displayed subtle differences in eosinophil and monocyte infiltration into the airways. However, the overall development of allergic airway disease and airway function was not significantly altered by NLRP12 deficiency. Together, the combined data suggest that NLRP12 does not play a vital role in regulating Th2 driven airway inflammation using common model systems that are physiologically relevant to human disease. Thus, the allergic airway inflammation models described here should be appropriate for subsequent studies that seek to decipher the contribution of NLRP12 in mediating the host response to agents associated with asthma exacerbation.     

Therapeutic response to CDK4/6 inhibition in breast cancer defined by ex vivo analyses of human tumors

To model the heterogeneity of breast cancer as observed in the clinic, we employed an ex vivo model of breast tumor tissue. This methodology maintained the histological integrity of the tumor tissue in unselected breast cancers, and importantly, the explants retained key molecular markers that are currently used to guide breast cancer treatment (e.g., ER and Her2 status). The primary tumors displayed the expected wide range of positivity for the proliferation marker Ki67, and a strong positive correlation between the Ki67 indices of the primary and corresponding explanted tumor tissues was observed. Collectively, these findings indicate that multiple facets of tumor pathophysiology are recapitulated in this ex vivo model. To interrogate the potential of this preclinical model to inform determinants of therapeutic response, we investigated the cytostatic response to the CDK4/6 inhibitor, PD-0332991. This inhibitor was highly effective at suppressing proliferation in approximately 85% of cases, irrespective of ER or HER2 status. However, 15% of cases were completely resistant to PD-0332991. Marker analyses in both the primary tumor tissue and the corresponding explant revealed that cases resistant to CDK4/6 inhibition lacked the RB-tumor suppressor. These studies provide important insights into the spectrum of breast tumors that could be treated with CDK4/6 inhibitors, and defines functional determinants of response analogous to those identified through neoadjuvant studies.     

Postthaw survival of rhesus macaque sperm: variation in the response of individual males to different freezing protocols

This study was designed to assess the effects of various cryopreservation protocols on the postthaw survival of rhesus macaque sperm. Multiple ejaculates of five mature males were collected and frozen by each of four different methods. Cryopreservation significantly reduced motility in all samples, regardless of the method, and the response of different ejaculates of the same male to each method proved to be consistent. The freezing method was a significant factor in determining the immediate postthaw motility, although this effect disappeared after 4 hr. Moreover, there was a significant interaction between individual males and freezing method suggesting that developing a single freezing protocol that is universally suitable may be difficult.