Role of EscP (Orf16) in Injectisome Biogenesis and Regulation of Type III Protein Secretion in Enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli employs a type III secretion system (T3SS) to translocate virulence effector proteins directly into enterocyte host cells, leading to diarrheal disease. The T3SS is encoded within the chromosomal locus of enterocyte effacement (LEE). The function of some of the LEE-encoded proteins remains unknown. Here we investigated the role of the Orf16 protein in T3SS biogenesis and function. An orf16 deletion mutant showed translocator and effector protein secretion profiles different from those of wild-type cells. The orf16 null strain produced T3S structures with abnormally long needles and filaments that caused weak hemolysis of red blood cells. Furthermore, the number of fully assembled T3SSs was also reduced in the orf16 mutant, indicating that Orf16, though not essential, is required for efficient T3SS assembly. Analysis of protein secretion revealed that Orf16 is a T3SS-secreted substrate and regulates the secretion of the inner rod component EscI. Both pulldown and yeast two-hybrid assays showed that Orf16 interacts with the C-terminal domain of an inner membrane component of the secretion apparatus, EscU; the inner rod protein EscI; the needle protein EscF; and the multieffector chaperone CesT. These results suggest that Orf16 regulates needle length and, along with EscU, participates in a substrate specificity switch from early substrates to translocators. Taken together, our results suggest that Orf16 acts as a molecular measuring device in a way similar to that of members of the Yersinia YscP and flagellar FliK protein family. Therefore, we propose that this protein be renamed EscP.     

Computed tomography imaging of primary lung cancer in mice using a liposomal-iodinated contrast agent

Purpose

To investigate the utility of a liposomal-iodinated nanoparticle contrast agent and computed tomography (CT) imaging for characterization of primary nodules in genetically engineered mouse models of non-small cell lung cancer.

Methods

Primary lung cancers with mutations in K-ras alone (Kras^LA1) or in combination with p53 (LSL-Kras^G12D;p53^FL/FL) were generated. A liposomal-iodine contrast agent containing 120 mg Iodine/mL was administered systemically at a dose of 16 µl/gm body weight. Longitudinal micro-CT imaging with cardio-respiratory gating was performed pre-contrast and at 0 hr, day 3, and day 7 post-contrast administration. CT-derived nodule sizes were used to assess tumor growth. Signal attenuation was measured in individual nodules to study dynamic enhancement of lung nodules.

Results

A good correlation was seen between volume and diameter-based assessment of nodules (R²>0.8) for both lung cancer models. The LSL-Kras^G12D;p53^FL/FL model showed rapid growth as demonstrated by systemically higher volume changes compared to the lung nodules in Kras^LA1 mice (p<0.05). Early phase imaging using the nanoparticle contrast agent enabled visualization of nodule blood supply. Delayed-phase imaging demonstrated significant differential signal enhancement in the lung nodules of LSL-Kras^G12D;p53^FL/FL mice compared to nodules in Kras^LA1 mice (p<0.05) indicating higher uptake and accumulation of the nanoparticle contrast agent in rapidly growing nodules.

Conclusions

The nanoparticle iodinated contrast agent enabled visualization of blood supply to the nodules during the early-phase imaging. Delayed-phase imaging enabled characterization of slow growing and rapidly growing nodules based on signal enhancement. The use of this agent could facilitate early detection and diagnosis of pulmonary lesions as well as have implications on treatment response and monitoring.     

Oxidative Stress Induced in Sunflower Seedling Roots by Aqueous Dry Olive-Mill Residues

The contamination of soils with dry olive-mill residue can represent a serious problem as being an environmental stressor in plants. It has been demonstrated that inoculation of aqueous extract of olive oil-mill residue (ADOR) with saprobe fungi removes some phenolic compounds. In this paper we studied the effect of ADOR uninoculated or inoculated with saprobe fungi in sunflower seedling roots. The germination and root growth, O₂·^- generation, superoxide dismutase (SOD) and extracellular peroxidases (EC-POXs) activities, and the content of some metabolites involved in the tolerance of stress were tested. The roots germinated in ADOR uninoculated show a decrease in meristem size, resulting in a reduction of the root length and fresh weight, and in the number of layers forming the cortex, but did not alter the dry weight, protein and soluble amino acid content. ADOR caused the decreases in O₂·^- generation and EC-POX′s activities and protein oxidation, but enhanced SOD activity, lipid peroxidation and proline content. Fluorescence imaging showed that ADOR induced O₂·^- and H₂O₂ accumulation in the roots. The increase in SOD and the decrease in EC-POX′s activities might be involved in the enhancement of H₂O₂ content and lipid peroxidation. Control roots treated with ADOR for 10 min show an oxidative burst. Roots germinated in ADOR inoculated with saprobe fungi partially recovered normal levels of ROS, morphological characteristics and antioxidant activities. These results suggested that treatment with ADOR caused a phytotoxic effect during germination inducing an oxidative stress. The inoculation of ADOR with saprobe fungi limited the stress.     

Flagella from Five Cronobacter Species Induce Pro-Inflammatory Cytokines in Macrophage Derivatives from Human Monocytes

Cronobacter spp. are opportunistic pathogens linked to lie-threatening infections in neonates and contaminated powdered infant formula that has been epidemiologically associated with these cases. Clinical symptoms of Cronobacter include necrotizing enterocolitis, bacteremia, and meningitis. Flagella from C. sakazakii are involved in biofilm formation and its adhesion to epithelial cells. We investigated the role of flagella from C. sakazakii ST1 and ST4, C. malonaticus, C. muytjensii, C. turicensis and C. dublinensis during the activation of cytokines (IL-8, TNF-α, and IL-10) in macrophage derivatives from human monocytes, which has not been extensively studied. The production and identity of flagella from the five Cronobacter species were visualized and recognized with anti-flagella antibodies by immunogold labeling through transmission electron microscopy. Purified flagella were dissociated into monomers in 12% SDS-PAGE Coomassie blue-stained gels showing a band of ∼28 kDa and, in addition, mass spectrometry revealed the presence of several peptides that correspond to flagellin. Flagella (100 ng) induced the release of IL-8 (3314–6025 pg/ml), TNF-α (39–359 pg/ml), and IL-10 (2–96 pg/ml), in macrophage isolates from human monocytes and similar results were obtained when flagella were dissociated into monomers. Inhibition assays using three dilutions of anti-flagella antibodies (1∶10, 1∶100, and 1∶200) suppressed the secretion of IL-8, TNF-α, and IL-10 between 95–100% using 100 ng of protein. A transfection assay using 293-hTLR5 cells showed IL-8 release of 197 pg/ml and suppression in the secretion of IL-8 when anti-hTLR5-IgA antibodies were used at different concentrations. These observations suggest that flagella and flagellin are involved in an inflammatory response dependent on TLR5 recognition, which could contribute to the pathogenesis of the bacteria.     

N-3 Long-Chain Polyunsaturated Fatty Acid Supplementation Significantly Reduces Liver Oxidative Stress in High Fat Induced Steatosis

Omega-3 (n-3) long-chain polyunsaturated fatty acids (n-3 LCPUFA) are associated with several physiological functions, suggesting that their administration may prevent non transmissible chronic diseases. Therefore, we investigate whether dietary n-3 LCPUFA supplementation triggers an antioxidant response preventing liver steatosis in mice fed a high fat diet (HFD) in relation to n-3 LCPUFA levels. Male C57BL/6J mice received (a) control diet (10% fat, 20% protein, 70% carbohydrate), (b) control diet plus n-3 LCPUFA (108 mg/kg/day eicosapentaenoic acid plus 92 mg/kg/day docosahexaenoic acid), (c) HFD (60% fat, 20% protein, 20% carbohydrate), or (d) HFD plus n-3 LCPUFA for 12 weeks. Parameters of liver steatosis, glutathione status, protein carbonylation, and fatty acid analysis were determined, concomitantly with insulin resistance and serum tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 levels. HFD significantly increased total fat and triacylglyceride contents with macrovesicular steatosis, concomitantly with higher fasting serum glucose and insulin levels, HOMA, and serum TNF-α, IL-1β, and IL-6. Reduced and total liver glutathione contents were diminished by HFD, with higher GSSG/GSH ratio and protein carbonylation, n-3 LCPUFA depletion and elevated n-6/n-3 ratio over control values. These changes were either reduced or normalized to control values in animals subjected to HFD and n-3 LCPUFA, with significant increased hepatic total n-3 LCPUFA content and reduced n-6/n-3 ratio being observed after n-3 LCPUFA supplementation alone. So, repletion of liver n-3 LCPUFA levels by n-3 LCPUFA dietary supplementation in HFD obese mice reduces hepatic lipid content, with concomitant antioxidant and anti-inflammatory responses favouring insulin sensitivity.     

The functions of the "Greeting Ceremony" among male mantled howlers (Alouatta palliata) on Agaltepec Island, Mexico

Nonhuman primates use greeting behaviors as nonaggressive communicatory signals in multiple social contexts. Adult male mantled howlers (Alouatta palliata) perform a ritual greeting that has been associated with bond-strengthening functions. The aim of this study is to explore the greeting patterns of male howlers living on Agaltepec Island, Mexico. Specifically, we analyzed the relationships between greetings and several individual, relational, and contextual variables, such as the expression of affiliation and agonism, dominance rank, age, kinship relationships, spatial organization, activity patterns, and subgrouping patterns. Greetings were more frequent between males with closer dominance ranks. Among those dyads that greeted at least once, dominant males initiated greetings more frequently than less-dominant males. On the other hand, more greetings were observed when one of the participants had recently returned to a subgroup and during locomotion. On the basis of these results, we propose that on Agaltepec greetings are a conflict management mechanism used between males of similar ranks. The fission-fusion social system of this group of howlers allows males with conflicting interests to remain separated, and greetings may reduce tension during fusion events.     

A syntaxin 10-SNARE complex distinguishes two distinct transport routes from endosomes to the trans-Golgi in human cells

Mannose 6-phosphate receptors (MPRs) are transported from endosomes to the Golgi after delivering lysosomal enzymes to the endocytic pathway. This process requires Rab9 guanosine triphosphatase (GTPase) and the putative tether GCC185. We show in human cells that a soluble NSF attachment protein receptor (SNARE) complex comprised of syntaxin 10 (STX10), STX16, Vti1a, and VAMP3 is required for this MPR transport but not for the STX6-dependent transport of TGN46 or cholera toxin from early endosomes to the Golgi. Depletion of STX10 leads to MPR missorting and hypersecretion of hexosaminidase. Mouse and rat cells lack STX10 and, thus, must use a different target membrane SNARE for this process. GCC185 binds directly to STX16 and is competed by Rab6. These data support a model in which the GCC185 tether helps Rab9-bearing transport vesicles deliver their cargo to the trans-Golgi and suggest that Rab GTPases can regulate SNARE–tether interactions. Importantly, our data provide a clear molecular distinction between the transport of MPRs and TGN46 to the trans-Golgi.     

Mexican Arnica Anti–Inflammatory Action: Plant Age is Correlated with the Concentration of Anti–Inflammatory Sesquiterpenes in the Medicinal Plant <Emphasis Type="Italic">Heterotheca inuloides</Emphasis> Cass. (Asteraceae)<Superscript>1</Superscript>

Mexican Arnica Anti–Inflammatory Action: Plant Age Is Correlated with the Concentration of Anti–inflammatory Sesquiterpenes in the Medicinal Plant Heterotheca inuloides Cass. (Asteraceae). Mexican árnica (Heterotheca inuloides Cass.) is a widely used anti–inflammatory medicinal plant in Mexican folk medicine. Although it has been suggested that plant age, fertilization, and harvesting regime influence the concentration of secondary compounds affecting the therapeutic activity of the plant, the effect of these variables on the concentration of the Mexican árnica anti–inflammatory compounds was not known. We quantified anti–inflammatory sesquiterpenes (caryolan–1, 9β–diol, cadalen–15–oic acid, 7–hydroxycadalene, 4–hydroxy–2–isopropyl–4, 7–dimethyl–1[4H] naftalinone, 7–hydroxy–4αH–3, 4–dihydrocadalene, β–caryophyllene, and β–caryophyllene epoxide) in Mexican árnica plants subjected to fertilization and successive harvests of flowering stems, conditions that mimic the cultivation and harvesting for árnica in México. Fertilization and successive harvesting and their interaction had no significant effect on the concentration of anti–inflammatory compounds. However, the concentrations of these compounds were 60% higher in flowering stems from 15–month–old plants than in those from 4– or 8–month–old plants and was independent of the number of harvests and fertilization regime applied.