The Role of the st313-td Gene in Virulence of Salmonella Typhimurium ST313

Multidrug-resistant Salmonella enterica serovar Typhimurium ST313 has emerged in sub-Saharan Africa causing severe infections in humans. Therefore, it has been speculated that this specific sequence type, ST313, carries factors associated with increased pathogenicity. We assessed the role in virulence of a gene with a yet unknown function, st313-td, detected in ST313 through comparative genomics. Additionally, the structure of the genomic island ST313-GI, harbouring the gene was determined. The gene st313-td was cloned into wild type S. Typhimurium 4/74 (4/74-C) as well as knocked out in S. Typhimurium ST313 02–03/002 (Δst313-td) followed by complementation (02-03/002-C). Δst313-td was less virulent in mice following i.p. challenge than the wild type and this phenotype could be partly complemented in trans, indicating that st313-td plays a role during systemic infection. The gene st313-td was shown not to affect invasion of cultured epithelial cells, while the absence of the gene significantly affects uptake and intracellular survival within macrophages. The gene st313-td was proven to be strongly associated to invasiveness, harboured by 92.5% of S. Typhimurium blood isolates (n = 82) and 100% of S. Dublin strains (n = 50) analysed. On the contrary, S. Typhimurium isolates of animal and food origin (n = 82) did not carry st313-td. Six human, non-blood isolates of S. Typhimurium from Belarus, China and Nepal harboured the gene and belonged to sequence types ST398 and ST19. Our data showed a global presence of the st313-td gene and in other sequence types than ST313. The gene st313-td was shown to be expressed during logarithmic phase of growth in 14 selected Salmonella strains carrying the gene. This study reveals that st313-td plays a role in S. Typhimurium ST313 pathogenesis and adds another chapter to understanding of the virulence of S. Typhimurium and in particular of the emerging sequence type ST313.     

Demonstration of the Effect of Generic Anatomical Divisions versus Clinical Protocols on Computed Tomography Dose Estimates and Risk Burden

Objective

Choosing to undertake a CT scan relies on balancing risk versus benefit, however risks associated with CT scanning have generally been limited to broad anatomical locations, which do not provided adequate information to evaluate risk against benefit. Our study aimed to determine differences in radiation dose and risk estimates associated with modern CT scanning examinations when computed for clinical protocols compared with those using anatomical area.

Methods

Technical data were extracted from a tertiary hospital Picture Archiving Communication System for random samples of 20–40 CT examinations per adult clinical CT protocol. Organ and whole body radiation dose were calculated using ImPACT Monte Carlo simulation software and cancer incidence and mortality estimated using BEIR VII age and gender specific lifetime attributable risk weights.

Results

Thirty four unique CT protocols were identified by our study. When grouped according to anatomic area the radiation dose varied substantially, particularly for abdominal protocols. The total estimated number of incident cancers and cancer related deaths using the mean dose of anatomical area were 86 and 69 respectively. Using more specific protocol doses the estimates rose to 214 and 138 incident cancers and cancer related deaths, at least doubling the burden estimated.

Conclusions

Modern CT scanning produces a greater diversity of effective doses than much of the literature describes; where a lack of focus on actual scanning protocols has produced estimates that do not reflect the range and complexity of modern CT practice. To allow clinicians, patients and policy makers to make informed risk versus benefit decisions the individual and population level risks associated with modern CT practices are essential.     

Metallothionein – Immunohistochemical Cancer Biomarker: A Meta-Analysis

Metallothionein (MT) has been extensively investigated as a molecular marker of various types of cancer. In spite of the fact that numerous reviews have been published in this field, no meta-analytical approach has been performed. Therefore, results of to-date immunohistochemistry-based studies were summarized using meta-analysis in this review.Web of science, PubMed, Embase and CENTRAL databases were searched (up to April 30, 2013) and the eligibility of individual studies and heterogeneity among the studies was assessed. Random and fixed effects model meta-analysis was employed depending on the heterogeneity, and publication bias was evaluated using funnel plots and Egger''s tests.A total of 77 studies were included with 8,015 tissue samples (4,631 cases and 3,384 controls). A significantly positive association between MT staining and tumors (vs. healthy tissues) was observed in head and neck (odds ratio, OR 9.95; 95% CI 5.82–17.03) and ovarian tumors (OR 7.83; 1.09–56.29), and a negative association was ascertained in liver tumors (OR 0.10; 0.03–0.30). No significant associations were identified in breast, colorectal, prostate, thyroid, stomach, bladder, kidney, gallbladder, and uterine cancers and in melanoma. While no associations were identified between MT and tumor staging, a positive association was identified with the tumor grade (OR 1.58; 1.08–2.30). In particular, strong associations were observed in breast, ovarian, uterine and prostate cancers. Borderline significant association of metastatic status and MT staining were determined (OR 1.59; 1.03–2.46), particularly in esophageal cancer. Additionally, a significant association between the patient prognosis and MT staining was also demonstrated (hazard ratio 2.04; 1.47–2.81). However, a high degree of inconsistence was observed in several tumor types, including colorectal, kidney and prostate cancer.Despite the ambiguity in some tumor types, conclusive results are provided in the tumors of head and neck, ovary and liver and in relation to the tumor grade and patient survival.     

Identification of Novel in Vivo Phosphorylation Sites of the Human Proapoptotic Protein BAD: PORE-FORMING ACTIVITY OF BAD IS REGULATED BY PHOSPHORYLATION*

BAD is a proapoptotic member of the Bcl-2 protein family that is regulated by phosphorylation in response to survival factors. Although much attention has been devoted to the identification of phosphorylation sites in murine BAD, little data are available with respect to phosphorylation of human BAD protein. Using mass spectrometry, we identified here besides the established phosphorylation sites at serines 75, 99, and 118 several novel in vivo phosphorylation sites within human BAD (serines 25, 32/34, 97, and 124). Furthermore, we investigated the quantitative contribution of BAD targeting kinases in phosphorylating serine residues 75, 99, and 118. Our results indicate that RAF kinases represent, besides protein kinase A, PAK, and Akt/protein kinase B, in vivo BAD-phosphorylating kinases. RAF-induced phosphorylation of BAD was reduced to control levels using the RAF inhibitor BAY 43-9006. This phosphorylation was not prevented by MEK inhibitors. Consistently, expression of constitutively active RAF suppressed apoptosis induced by BAD and the inhibition of colony formation caused by BAD could be prevented by RAF. In addition, using the surface plasmon resonance technique, we analyzed the direct consequences of BAD phosphorylation by RAF with respect to association with 14-3-3 and Bcl-2/Bcl-X_L proteins. Phosphorylation of BAD by active RAF promotes 14-3-3 protein association, in which the phosphoserine 99 represented the major binding site. Finally, we show here that BAD forms channels in planar bilayer membranes in vitro. This pore-forming capacity was dependent on phosphorylation status and interaction with 14-3-3 proteins. Collectively, our findings provide new insights into the regulation of BAD function by phosphorylation.Apoptosis is a genetically programmed, morphologically distinct form of cell death that can be triggered by a variety of physiological and pathological stimuli (1–3). This form of cellular suicide is widely observed in nature and is not only essential for embryogenesis, immune responses, and tissue homeostasis but is also involved in diseases such as tumor development and progression. Bcl-2 family proteins play a pivotal role in controlling programmed cell death. The major function of these proteins is to directly modulate outer mitochondrial membrane permeability and thereby regulate the release of apoptogenic factors from the intermembrane space into the cytoplasm (for a recent review, see Ref. 4). On the basis of various structural and functional characteristics, the Bcl-2 family of proteins is divided into three subfamilies, including proteins that either inhibit (e.g. Bcl-2, Bcl-X_L, or Bcl-w) or promote programmed cell death (e.g. Bax, Bak, or Bok) (5, 6). A second subclass of proapoptotic Bcl-2 family members, the BH3²-only proteins, comprises BAD, Bik, Bmf, Hrk, Noxa, truncated Bid, Bim, and Puma (4). BH3-only proteins share sequence homology only at the BH3 domain. The amphipathic helix formed by the BH3 domain (and neighboring residues) associates with a hydrophobic groove of the antiapoptotic Bcl-2 family members (7, 8). Originally, truncated Bid has been reported to interact with Bax and Bak (9), suggesting that some BH3-only proteins promote apoptosis via at least two different mechanisms: inactivating Bcl-2-like proteins by direct binding and/or by inducing modification of Bax-like molecules. BAD (Bcl-2-associated death promoter, Bcl-2 antagonist of cell death) was described to promote apoptosis by forming heterodimers with the prosurvival proteins Bcl-2 and Bcl-X_L, thus preventing them from binding with Bax (10). More recently, two major models have been suggested for how BH3-only proteins may induce apoptosis. In the direct model, all BH3-only proteins promote cell death by directly binding and inactivating their specific anti-death Bcl-2 protein partner (11, 12). In this model, the relative killing potency of different BH3-only proteins is based on their affinities for antiapoptotic proteins. Thus, the activation of Bax/Bak would be mediated through their release from antiapoptotic counterparts. Contrary to this model, Kim et al. (13) provided support for an alternative hierarchy model, in which BH3-only proteins are divided into two distinct subsets. According to this model, the inactivator BH3-only proteins, like BAD, Noxa, and some others, respond directly to survival factors, resulting in phosphorylation, 14-3-3 binding, and suppression of the proapoptotic function. In the absence of growth factors, these proteins engage specifically their preferred antiapoptotic Bcl-2 proteins. The targeted Bcl-2 proteins then release the other subset of BH3-only proteins designated the activators (truncated Bid, Bim, and Puma) that in turn bind to and activate Bax and Bak.Non-phosphorylated BAD associated with Bcl-2/Bcl-X_L is found at the outer mitochondrial membrane. Phosphorylation of specific serine residues, Ser-112 and Ser-136 of mouse BAD (mBAD) or the corresponding phosphorylation sites Ser-75 and Ser-99 of human BAD (hBAD), results in association with 14-3-3 proteins and subsequent relocation of BAD (14, 15). Phosphorylation of mBAD at Ser-155 (Ser-118 of hBAD) within its BH3 domain disrupts the association with Bcl-2 or Bcl-X_L, promoting cell survival (16). Therefore, the phosphorylation status of BAD at these serine residues reflects a checkpoint for cell death or survival. Although the C-RAF kinase was the first reported BAD kinase (17), its target sites were not clearly defined. However, there is a growing body of evidence for direct participation of RAF in regulation of apoptosis via BAD (18, 19). In addition, Kebache et al. (20) reported recently that the interaction between adaptor protein Grb10 and C-RAF is essential for BAD-mediated cell survival. On the other hand, numerous reports suggest that PKA (21), Akt/PKB (22), PAK (18, 23, 24), Cdc2 (25), RSK (26, 27), CK2 (28), and PIM kinases (29) are involved in BAD phosphorylation as well. The involvement of c-Jun N-terminal kinase in BAD phosphorylation is controversially discussed. Whereas Donovan et al. (30) reported that c-Jun N-terminal kinase phosphorylates mBAD at serine 128, Zhang et al. (31) claimed that c-Jun N-terminal kinase is not a BAD-serine 128 kinase. On the other hand, it has been shown that c-Jun N-terminal kinase is able to suppress IL-3 withdrawal-induced apoptosis via phosphorylation of mBAD at threonine 201 (32). Thus, taken together, with respect to regulation of mBAD by phosphorylation, five serine phosphorylation sites (at positions 112, 128, 136, 155, and 170) and two threonines (117 and 201) have been identified so far. Intriguingly, only little data are available regarding the role of phosphorylation in regulation of hBAD protein, although significant structural differences between these two BAD proteins exist.During apoptosis, some members of the Bcl-2 family of proteins, such as Bax or Bak, have been shown to induce permeabilization of the outer mitochondrial membrane, allowing proteins in the mitochondrial intermembrane space to escape into the cytosol, where they can initiate caspase activation and cell death (for a review, see Refs. 33 and 34). Despite intensive investigation, the mechanism whereby Bax and Bak induce outer membrane permeability remains controversial (34). Based on crystal structure (35), it became evident that Bcl-X_L has a pronounced similarity to the translocation domain of diphtheria toxin (36), a domain that can form pores in artificial lipid bilayers. This discovery provoked the predominant view that upon commitment to apoptosis, the proapoptotic proteins Bax and Bak also form pores in the outer mitochondrial membrane (37). As expected from the structural considerations, Bcl-X_L was found to form channels in synthetic lipid membranes (38). Since then, other Bcl-2 family members like Bcl-2, Bax, and the BH3-only protein Bid have been reported to have channel-forming ability. These pores can be divided into two different types: proteinaceous channels of defined size and ion specificity (38–42) and large lipidic pores that allow free diffusion of 2-megadalton macromolecules (43, 44). With respect to the BH3-only protein BAD, no pore-forming abilities have been reported so far, although human BAD has been found to possess per se high affinity for negatively charged phospholipids and liposomes, mimicking mitochondrial membranes (14).The RAF kinases (A-, B-, and C-RAF) play a central role in the conserved Ras-RAF-MEK-ERK signaling cascade and mediate cellular responses induced by growth factors (45–47). Direct involvement of C-RAF in inhibition of proapoptotic properties of BAD established a link between signal transduction and apoptosis control (48, 49). However, the early works did not identify the exact RAF phosphorylation sites on BAD (17). Here we demonstrate that hBAD serves as a substrate of RAF isoforms. With respect to hBAD phosphorylation by PKA, Akt/PKB, and PAK1 in vivo, we observed different specificity compared with RAF kinases. hBAD phosphorylation by RAF was accompanied by reduced apoptosis in HEK293 cells (transformed human embryonic kidney cells) and NIH 3T3 cells (a mouse embryonic fibroblast cell line). Furthermore, we show that in vitro phosphorylation of hBAD by RAF at serines 75, 99, and 118 regulates the binding of 14-3-3 proteins and association with Bcl-2 and Bcl-X_L. By use of mass spectrometry, we detected several novel in vivo phosphorylation sites of hBAD in addition to the established phosphorylation sites, serines 75, 99, and 118. Finally, we show here that hBAD forms channels in planar bilayer membranes in vitro. This pore-forming capacity was dependent on phosphorylation status and interaction with 14-3-3 proteins.     

Genetic Modifiers of dFMR1 Encode RNA Granule Components in Drosophila

Mechanisms of neuronal mRNA localization and translation are of considerable biological interest. Spatially regulated mRNA translation contributes to cell-fate decisions and axon guidance during development, as well as to long-term synaptic plasticity in adulthood. The Fragile-X Mental Retardation protein (FMRP/dFMR1) is one of the best-studied neuronal translational control molecules and here we describe the identification and early characterization of proteins likely to function in the dFMR1 pathway. Induction of the dFMR1 in sevenless-expressing cells of the Drosophila eye causes a disorganized (rough) eye through a mechanism that requires residues necessary for dFMR1/FMRP''s translational repressor function. Several mutations in dco, orb2, pAbp, rm62, and smD3 genes dominantly suppress the sev-dfmr1 rough-eye phenotype, suggesting that they are required for dFMR1-mediated processes. The encoded proteins localize to dFMR1-containing neuronal mRNPs in neurites of cultured neurons, and/or have an effect on dendritic branching predicted for bona fide neuronal translational repressors. Genetic mosaic analyses indicate that dco, orb2, rm62, smD3, and dfmr1 are dispensable for translational repression of hid, a microRNA target gene, known to be repressed in wing discs by the bantam miRNA. Thus, the encoded proteins may function as miRNA- and/or mRNA-specific translational regulators in vivo.THE subcellular localization and regulated translation of stored mRNAs contributes to cellular asymmetry and subcellular specialization (Lecuyer et al. 2007; Martin and Ephrussi 2009). In mature neurons, local protein synthesis at active synapses may contribute to synapse-specific plasticity that underlies persistent forms of memory (Casadio et al. 1999; Ashraf et al. 2006; Sutton and Schuman 2006; Richter and Klann 2009). During this process, synaptic activity causes local translation of mRNAs normally stored in translationally repressed synaptic mRNPs (Sutton and Schuman 2006; Richter and Klann 2009). While specific neuronal translational repressors and microRNAs have been implicated in this process, their involvement in local translation that underlies memory, as well as the underlying mechanisms, are generally not well understood (Schratt et al. 2006; Keleman et al. 2007; Kwak et al. 2008; Li et al. 2008; Richter and Klann 2009). Furthermore, it remains possible that there are neuron-specific, mRNA-specific, and stimulus-pattern specific pathways for neuronal translational control (Raab-Graham et al. 2006; Giorgi et al. 2007).The Fragile-X Mental Retardation protein (FMRP) is among the best studied of neuronal translational repressors, in part due to its association with human neurodevelopmental disease (Pieretti et al. 1991; Mazroui et al. 2002; Gao 2008). Consistent with function in synaptic translation required for memory formation, mutations in FMRP are associated with increased synaptic translation, enhanced LTD, increased synapse growth, and also with enhanced long-term memory (Zhang et al. 2001; Huber et al. 2002; Bolduc et al. 2008; Dictenberg et al. 2008).FMRP co-immunoprecipitates with components of the RNAi and miRNA machinery and appears to be required for aspects of miRNA function in neurons (Caudy et al. 2002; Ishizuka et al. 2002; Jin et al. 2004b; Gao 2008). In addition, FMRP associates with neuronal polyribosomes as well as with Staufen-containing ribonucleoprotein (mRNP) granules easily observed in neurites of cultured neurons (Feng et al. 1997; Krichevsky and Kosik 2001; Mazroui et al. 2002; Kanai et al. 2004; Barbee et al. 2006; Bramham and Wells 2007; Bassell and Warren 2008; Dictenberg et al. 2008). FMRP-containing neuronal mRNPs contain not only several ubiquitous translational control molecules, but also CaMKII and Arc mRNAs, whose translation is locally controlled at synapses (Rook et al. 2000; Krichevsky and Kosik 2001; Kanai et al. 2004; Barbee et al. 2006). Thus, FMRP-containing RNA particles are probably translationally repressed and transported along microtubules from the neuronal cell body to synaptic sites in dendrites where local synaptic activity can induce their translation (Kiebler and Bassell 2006; Dictenberg et al. 2008).The functions of FMRP/dFMR1 in mRNA localization as well as miRNA-dependent and independent forms of translational control is likely to require several other regulatory proteins. To identify such proteins, we used a previously designed and validated genetic screen (Wan et al. 2000; Jin et al. 2004a; Zarnescu et al. 2005). The overexpression of dFMR1 in the fly eye causes a “rough-eye” phenotype through a mechanism that requires (a) key residues in dFMR1 that mediate translational repression in vitro; (b) Ago1, a known components of the miRNA pathway; and (c) a DEAD-box helicase called Me31B, which is a highly conserved protein from yeast (Dhh1p) to humans (Rck54/DDX6) functioning in translational repression and present on neuritic mRNPs (Wan et al. 2000; Laggerbauer et al. 2001; Jin et al. 2004a; Coller and Parker 2005; Barbee et al. 2006; Chu and Rana 2006). To identify other Me31B-like translational repressors and neuronal granule components, we screened mutations in 43 candidate proteins for their ability to modify dFMR1 induced rough-eye phenotype. We describe the results of this genetic screen and follow up experiments to address the potential cellular functions of five genes identified as suppressors of sev-dfmr1.     

Biochemical and functional characterization of an L-amino acid oxidase isolated from Bothrops pirajai snake venom

In this work we describe the isolation of a new l-amino acid oxidase (LAAO) referred to as BpirLAAO-I from Bothrops pirajai snake venom, which was highly purified using a combination of molecular exclusion, affinity, and hydrophobic chromatography steps. BpirLAAO-I homodimeric acid glycoprotein (approximate Mr and pI of 130,000 and 4.9, respectively) displays high specificity toward hydrophobic/aromatic amino acids, while deglycosylation does not alter its enzymatic activity. The N-terminal LAAO sequence of its first 49 amino acids presented a high similarity between a amino acid sequence with other LAAOs from: Bothrops spp., Crotalus spp., Calloselasma rhodostoma, Agkistrodon spp., Trimeresurus spp., Pseudechis australis, Oxyuranus scutellatus, and Notechis scutatus. BpirLAAO-I induces time-dependent platelet aggregation, mouse paw edema, cytotoxic activity against Escherichia coli, Pseudomonas aeruginosa, Leishmania sp., and tumor cells, and also a typical fago (M13mp18) DNA fragmentation. Platelet aggregation, leishmanicidal and antitumoral activities were reduced by catalase. Thus, BpirLAAO-I is a multifunctional protein with promising biotechnological and medical applications.     

Neuromedin U receptor 2-deficient mice display differential responses in sensory perception, stress, and feeding

Neuromedin U (NMU) is a highly conserved neuropeptide with a variety of physiological functions mediated by two receptors, peripheral NMUR1 and central nervous system NMUR2. Here we report the generation and phenotypic characterization of mice deficient in the central nervous system receptor NMUR2. We show that behavioral effects, such as suppression of food intake, enhanced pain response, and excessive grooming induced by intracerebroventricular NMU administration were abolished in the NMUR2 knockout (KO) mice, establishing a causal role for NMUR2 in mediating NMU's central effects on these behaviors. In contrast to the NMU peptide-deficient mice, NMUR2 KO mice appeared normal with regard to stress, anxiety, body weight regulation, and food consumption. However, the NMUR2 KO mice showed reduced pain sensitivity in both the hot plate and formalin tests. Furthermore, facilitated excitatory synaptic transmission in spinal dorsal horn neurons, a mechanism by which NMU stimulates pain, did not occur in NMUR2 KO mice. These results provide significant insights into a functional dissection of the differential contribution of peripherally or centrally acting NMU system. They suggest that NMUR2 plays a more significant role in central pain processing than other brain functions including stress/anxiety and regulation of feeding.     

Design and preparation of 2-benzamido-pyrimidines as inhibitors of IKK

The design, synthesis, and the biological evaluation of 2-benzamido-pyrimidines as novel IKK inhibitors are described. By optimization of the lead compound 3, compounds 16 and 24 are identified as good inhibitors of IKK2 with IC(50) values of 40 and 25 nM, respectively. Compound 16 also demonstrated significant in vivo activity in an acute model of cytokine release.     

Staufen- and FMRP-containing neuronal RNPs are structurally and functionally related to somatic P bodies

Local control of mRNA translation modulates neuronal development, synaptic plasticity, and memory formation. A poorly understood aspect of this control is the role and composition of ribonucleoprotein (RNP) particles that mediate transport and translation of neuronal RNAs. Here, we show that staufen- and FMRP-containing RNPs in Drosophila neurons contain proteins also present in somatic "P bodies," including the RNA-degradative enzymes Dcp1p and Xrn1p/Pacman and crucial components of miRNA (argonaute), NMD (Upf1p), and general translational repression (Dhh1p/Me31B) pathways. Drosophila Me31B is shown to participate (1) with an FMRP-associated, P body protein (Scd6p/trailer hitch) in FMRP-driven, argonaute-dependent translational repression in developing eye imaginal discs; (2) in dendritic elaboration of larval sensory neurons; and (3) in bantam miRNA-mediated translational repression in wing imaginal discs. These results argue for a conserved mechanism of translational control critical to neuronal function and open up new experimental avenues for understanding the regulation of mRNA function within neurons.