Effects of storage and the presence of a beef microflora on the thermal resistance of Salmonella Typhimurium DT104 in beef and broth systems

AIMS: To investigate the effects of storage and the presence of a beef microflora on the thermal resistance of Salmonella serotype Typhimurium DT104 on beef surfaces and in a broth system during subsequent heat treatments after extended low-temperature storage (4 degrees C for 14 days) or mild temperature abuse (10 degrees C for 7 days). METHODS AND RESULTS: Surviving Salm. Typhimurium DT104 cells were estimated after heating in a water bath (55 degrees C) by plating beef and broth samples on tryptone soya agar and overlaying with xylose-lysine-deoxycholate agar. In beef and broth systems, D(55) values for Salm. Typhimurium DT104 stored at 4 degrees C or 10 degrees C in the presence or absence of a beef microflora were significantly lower (P < 0.01) than the D values for this organism heat-treated immediately after inoculation. In beef systems, the D(55) values were significantly lower (P < 0.05) in the presence of a beef microflora than the D(55) values obtained in 'pure' culture under all temperature/storage combinations. However, in broth systems, there was no significant difference between the D(55) values obtained in 'pure' culture and the D(55) values obtained from systems containing beef microflora. CONCLUSIONS: Storage of Salm. Typhimurium DT104 significantly reduced the thermal resistance of the pathogen in beef and broth systems. In the presence of high numbers of a Gram-negative beef microflora, the heat sensitivity of the pathogen was further increased on beef surfaces but not in broth. SIGNIFICANCE AND IMPACT OF THE STUDY: Studies investigating the survival of Salm. Typhimurium DT104 in different food systems will help define safe food preservation processes and will aid in the elimination this pathogen from the food production environments.     

Developing the ethics of implementation research in health

Implementation research (IR) is growing in recognition as an important generator of practical knowledge that can be translated into health policy. With its aim to answer questions about how to improve access to interventions that have been shown to work but have not reached many of the people who could benefit from them, IR involves a range of particular ethical considerations that have not yet been comprehensively covered in international guidelines on health research ethics. The fundamental ethical principles governing clinical research apply equally in IR, but the application of these principles may differ depending on the IR question, context, and the nature of the proposed intervention. IR questions cover a broad range of topics that focus on improving health system functioning and improving equitable and just access to effective health care interventions. As such, IR designs are flexible and often innovative, and ethical principles cannot simply be extrapolated from their applications in clinical research. Meaningful engagement with all stakeholders including communities and research participants is a fundamental ethical requirement that cuts across all study phases of IR and links most ethical concerns. Careful modification of the informed consent process may be required in IR to permit study of a needed intervention. The risks associated with IR may be difficult to anticipate and may be very context-specific. The benefits of IR may not accrue to the same groups who participate in the research, therefore justifying the risks versus benefits of IR may be ethically challenging. The expectation that knowledge generated through IR should be rapidly translated into health policy and practice necessitates up-front commitments from decision-makers to sustainability and scalability of effective interventions. Greater awareness of the particular ethical implications of the features of IR is urgently needed to facilitate optimal ethical conduct of IR and uniform ethical review.     

Downregulation of the CCK-B receptor in pancreatic cancer cells blocks proliferation and promotes apoptosis

Gastrin stimulates the growth of pancreatic cancer cells through the activation of the cholecystokinin-B receptor (CCK-BR), which has been found to be overexpressed in pancreatic cancer. In this study, we proposed that the CCK-BR drives growth of pancreatic cancer; hence, interruption of CCK-BR activity could potentially be an ideal target for cancer therapeutics. The effect of CCK-BR downregulation in the human pancreatic adenocarcinoma cells was examined by utilizing specific CCK-BR-targeted RNA interference reagents. The CCK-BR receptor expression was both transiently and stably downregulated by transfection with selective CCK-BR small-interfering RNA or short-hairpin RNA, respectively, and the effects on cell growth and apoptosis were assessed. CCK-BR downregulation resulted in reduced cancer cell proliferation, decreased DNA synthesis, and cell cycle arrest as demonstrated by an inhibition of G(1) to S phase progression. Furthermore, CCK-BR downregulation increased caspase-3 activity, TUNEL-positive cells, and decreased X-linked inhibitor of apoptosis protein expression, suggesting apoptotic activity. Pancreatic cancer cell mobility was decreased when the CCK-BR was downregulated, as assessed by a migration assay. These results show the importance of the CCK-BR in regulation of growth and apoptosis in pancreatic cancer. Strategies to decrease the CCK-BR expression and activity may be beneficial for the development of new methods to improve the treatment for patients with pancreatic cancer.     

Susceptibility to Crohn’s disease is mediated by <Emphasis Type="Italic">KIR</Emphasis>2DL2/<Emphasis Type="Italic">KIR</Emphasis>2DL3 heterozygosity and the HLA-C ligand

In the present study, we investigated the relationship between the KIR loci and the genes encoding their HLA ligands and genetic susceptibility to Crohn’s disease (CD). Analyses of the interactions between KIR3DL1, KIR2DL1, KIR2DL2, and KIR2DL3 with their respective HLA ligands indicate that there is a protective effect for KIR2DL2 in the absence of its HLA ligand C1. Given that KIR2DL2 and KIR2DL3 segregate as alleles, we compared their genotypic distributions to expectations under Hardy–Weinberg Equilibrium (HWE) with regard to the HLA ligand C1 status. While all the genotypic distributions conform to expectations under HWE in controls, in C2 ligand homozygous cases there is significant deviation from HWE, with a reduction of KIR2DL2, KIR2DL3 heterozygotes. KIR2DL2, KIR2DL3 heterozygosity is the only genotypic combination that confers protection from CD. In addition to the protective effect (OR = 0.44, CI = 0.22–0.87; p = 0.018) observed in C2 ligand homozygotes, the KIR2DL2, KIR2DL3 genotype is predisposing (OR = 1.34, CI = 1.03–4.53; p = 0.031) in the presence of C1 ligand. A test for trend of HLA class I C ligand group genotypes with KIR2DL2, KIR2DL3 heterozygosity in cases and controls indicates that C1, C2 ligand group heterozygotes have an intermediate effect on predisposition. These results show for the first time that disease susceptibility may be related to heterozygosity at a specific KIR locus, and that HLA ligand genotype influences the relative effect of the KIR genotype.     

IBD-Associated TL1A Gene (TNFSF15) Haplotypes Determine Increased Expression of TL1A Protein

Background

The recently identified member of the TNF superfamily TL1A (TNFSF15) increases IFN-γ production by T cells in peripheral and mucosal CCR9+ T cells. TL1A and its receptor DR3 are up-regulated during chronic intestinal inflammation in ulcerative colitis and Crohn''s disease (CD). TL1A gene haplotypes increase CD susceptibility in Japanese, European, and US cohorts.

Methodology and Principal Findings

Here we report that the presence of TL1A gene haplotype B increases risk in Jewish CD patients with antibody titers for the E. coli outer membrane porin C (OmpC+) (Haplotype B frequency in Jewish CD patients: 24.9% for OmpC negative and 41.9% for OmpC positive patients, respectively, P≤0.001). CD14+ monocytes isolated from Jewish OmpC+ patients homozygous for TL1A gene haplotype B express higher levels of TL1A in response to FcγR stimulation, a known inducing pathway of TL1A, as measured by ELISA. Furthermore, the membrane expression of TL1A is increased on peripheral monocytes from Jewish but not non-Jewish CD patients with the risk haplotype.

Conclusions and Significance

These findings suggest that TL1A gene variation exacerbates induction of TL1A in response to FcγR stimulation in Jewish CD patients and this may lead to chronic intestinal inflammation via overwhelming T cell responses. Thus, TL1A may provide an important target for therapeutic intervention in this subgroup of IBD patients.     

Sub‐picomolar relaxin signalling by a pre‐assembled RXFP1, AKAP79, AC2, β‐arrestin 2, PDE4D3 complex

Biochemical studies suggest that G‐protein‐coupled receptors (GPCRs) achieve exquisite signalling specificity by forming selective complexes, termed signalosomes. Here, using cAMP biosensors in single cells, we uncover a pre‐assembled, constitutively active GPCR signalosome, that couples the relaxin receptor, relaxin family peptide receptor 1 (RXFP1), to cAMP following receptor stimulation with sub‐picomolar concentrations of peptide. The physiological effects of relaxin, a pleiotropic hormone with therapeutic potential in cancer metastasis and heart failure, are generally attributed to local production of the peptide, that occur in response to sub‐micromolar concentrations. The highly sensitive signalosome identified here provides a regulatory mechanism for the extremely low levels of relaxin that circulate. The signalosome includes requisite Gα_s, Gβγ and adenylyl cyclase 2 (AC2); AC2 is functionally coupled to RXFP1 through AKAP79 binding to helix 8 of the receptor; activation of AC2 is tonically opposed by protein kinase A (PKA)‐activated PDE4D3, scaffolded through a β‐arrestin 2 interaction with Ser⁷⁰⁴ of the receptor C‐terminus. This elaborate, pre‐assembled, ligand‐independent GPCR signalosome represents a new paradigm in GPCR signalling and provides a mechanism for the distal actions of low circulating levels of relaxin.     

AKAP79/150 Interacts with AC8 and Regulates Ca2+-dependent cAMP Synthesis in Pancreatic and Neuronal Systems

Protein kinase A anchoring proteins (AKAPs) provide the backbone for targeted multimolecular signaling complexes that serve to localize the activities of cAMP. Evidence is accumulating of direct associations between AKAPs and specific adenylyl cyclase (AC) isoforms to facilitate the actions of protein kinase A on cAMP production. It happens that some of the AC isoforms (AC1 and AC5/6) that bind specific AKAPs are regulated by submicromolar shifts in intracellular Ca²⁺. However, whether AKAPs play a role in the control of AC activity by Ca²⁺ is unknown. Using a combination of co-immunoprecipitation and high resolution live cell imaging techniques, we reveal an association of the Ca²⁺-stimulable AC8 with AKAP79/150 that limits the sensitivity of AC8 to intracellular Ca²⁺ events. This functional interaction between AKAP79/150 and AC8 was observed in HEK293 cells overexpressing the two signaling molecules. Similar findings were made in pancreatic insulin-secreting cells and cultured hippocampal neurons that endogenously express AKAP79/150 and AC8, which suggests important physiological implications for this protein-protein interaction with respect to Ca²⁺-stimulated cAMP production.     

Self-renewal of acute lymphocytic leukemia cells is limited by the Hedgehog pathway inhibitors cyclopamine and IPI-926

Conserved embryonic signaling pathways such as Hedgehog (Hh), Wingless and Notch have been implicated in the pathogenesis of several malignancies. Recent data suggests that Hh signaling plays a role in normal B-cell development, and we hypothesized that Hh signaling may be important in precursor B-cell acute lymphocytic leukemia (B-ALL). We found that the expression of Hh pathway components was common in human B-ALL cell lines and clinical samples. Moreover, pathway activity could be modulated by Hh ligand or several pathway inhibitors including cyclopamine and the novel SMOOTHENED (SMO) inhibitor IPI-926. The inhibition of pathway activity primarily impacted highly clonogenic B-ALL cells expressing aldehyde dehydrogenase (ALDH) by limiting their self-renewal potential both in vitro and in vivo. These data demonstrate that Hh pathway activation is common in B-ALL and represents a novel therapeutic target regulating self-renewal and persistence of the malignant clone.     

Diatom species–environment relationships and inference models from Isachsen, Ellef Ringnes Island, Canadian High Arctic

Surface sediment diatom assemblages were examined from 26 freshwater sites near Isachsen (78°47N, 103°32W), Ellef Ringnes Island, a region of diverse and atypical water chemistry for high arctic sites. One hundred and sixty eight diatom taxa were identified from these samples, over 50% of which had not previously been recorded in the Canadian High Arctic. Variations in diatom assemblages were related to changes in measured environmental variables using multivariate techniques. Canonical correspondence analysis (CCA) indicated that five variables contributed significantly to explaining patterns of diatom variation (i.e., COND, DIC, Mn, TPF, TPU). The first CCA axis (=0.44) was primarily controlled by conductivity-related variables, while CCA axis 2 (=0.21) was related to particulate concentrations. Diatom-based inference models were generated for the reconstruction of conductivity (RMSEP_jack=0.32, r²_jack=0.76) and pH (RMSEP_jack=0.40, r²_jack=0.69). The strengths of these models indicate that it will be possible to reliably infer past trends in conductivity and pH from diatom assemblages preserved in dated sediment cores from the Isachsen region.     

Cryptic species,cryptic endosymbionts,and geographical variation in chemical defences in the bryozoan Bugula neritina

Molecular markers often offer the only means to discriminate between species and to elucidate the specificity of many community interactions, both of which are key to the understanding of ecological patterns. Western Atlantic populations of the bryozoan Bugula neritina vary in the palatability of their larvae to predators: individuals south of Cape Hatteras produce chemical deterrents to fish predators that are absent in more northern individuals. We use mitochondrial cytochrome oxidase c subunit I (COI) sequences to show that the differences in palatability between populations correlate with the geographical distributions of two cryptic species within B. neritina. Furthermore, these cryptic species differ in their associations with bacteria that may confer chemical resistance to predation. Small subunit rRNA primers specific to a subset of gamma-proteobacteria amplified only the bacterium Endobugula sertula from the southern cryptic species. Endobugula sertula produces a family of chemical compounds (bryostatins) that may deter predators of its animal host. In contrast, the same primers amplified an array of gamma-proteobacteria from the unprotected northern cryptic bryozoan species, but never E. sertula. In combination, these findings suggest that the geographical variation in palatability observed in the larvae of B. neritina is not the result of local adaptation of a single species to regions of differing predation pressure, but rather results from the comparison of cryptic species that differ in the presence or absence of a bacterium that may provide protection against predators. The ability to identify the cryptic Bugula species and their differing relationships with bacterial associates provides an example of the important role molecular techniques may play in addressing ecological questions.