Pulmonary and systemic endotoxin tolerance in preterm fetal sheep exposed to chorioamnionitis

In a model of human chorioamnionitis, fetal sheep exposed to a single injection, but not repeated injections, of intra-amniotic endotoxin develop lung injury responses. We hypothesized that repeated exposure to intra-amniotic endotoxin induces endotoxin tolerance. Fetal sheep were given intra-amniotic injections of saline (control) or Escherichia coli LPS O55:B5 (10 mg) either 2 days (2-day group, single exposure), 7 days (7-day group, single exposure), or 2 plus 7 days (2- and 7-day repeat exposure) before preterm delivery at 124 days gestation (term=150 days). Endotoxin responses were assessed in vivo in the lung and liver, and in vitro in monocytes from the blood and the lung. Compared with the single 2-day LPS exposure group, the (2 plus 7 days) repeat LPS-exposed lambs had: 1) decreased lung neutrophil and monocyte inducible NO synthase (NOSII) expression, and 2) decreased lung cytokine and liver serum amyloid A3 mRNA expression. In the lung, serum amyloid A3 mRNA expression decreased in the airway epithelial cells but not in the lung inflammatory cells. Unlike the single 7-day LPS exposure group, peripheral blood and lung monocytes from the repeat-LPS group did not increase IL-6 secretion or hydrogen peroxide production in response to in vitro LPS. Compared with controls, TLR4 expression did not change but IL-1R-associated kinase M expression increased in the monocytes from repeat LPS-exposed lambs. These results are consistent with the novel finding of endotoxin tolerance in preterm fetal lungs exposed to intra-amniotic LPS. The findings have implications for preterm infants exposed to chorioamnionitis for both responses to lung injury and postnatal nosocomial infections.     

Identification of Tannerella forsythia antigens specifically expressed in patients with periodontal disease

Molecular pathogenesis of Tannerella forsythia, a putative periodontal pathogen, has not yet been adequately elucidated due to limited information on its virulence factors. Here, identification of in vivo expressed antigens of T. forsythia is reported using in vivo-induced antigen technology (IVIAT). Among 13 000 recombinant clones screened, 16 positive clones were identified that reacted reproducibly with sera obtained from patients with periodontal disease. DNA sequences from 12 of these in vivo-induced genes were determined. IVIAT-identified protein antigens of T. forsythia include: BspA, a well-defined virulence factor of T. forsythia; enzymes involved in housekeeping functions (tRNA synthetases, glycine hydroxymethyltransferase, and glucoside glucohydrolase); enzymes implicated in tissue destruction (dipeptidyl peptidase IV); a DNA mismatch repair protein; and putative outer membrane proteins of unknown function. The in vivo gene expression of these IVIAT-identified antigens was confirmed by a quantitative real-time PCR analysis. This is, to the best of the authors' knowledge, the first report using IVIAT in T. forsythia. It is anticipated that detailed analysis of the in vivo-induced genes identified by IVIAT in this study will lead to a better understanding of the molecular mechanisms mediating periodontal infection by T. forsythia.     

Wild plant seed storage at Neolithic Çatalhöyük East,Turkey

Full analysis of eight seed samples collected in the 1960's excavations at Neolithic Çatalhöyük East, Turkey, is presented. Detailed investigation of the composition and context of the samples suggests that the Neolithic population collected, processed and stored seeds from Capsella sp. and Descurainia sp. (wild crucifers) for food use. In addition seeds of Vicia/Lathyrus sp. (wild vetch), Helianthemum spp. and Taeniatherum caput-medusae mixed with Eremopyrum type (grasses) were also found, some of which may have been used for food or other purposes. The analysis demonstrates that wild seed exploitation was a regular part of subsistence practice alongside the economic staple of crop production, and again demonstrates how diverse plant use practices were at the site.     

Biodiversity associated with restored small-scale mussel habitats has restoration decision implications

The global loss of marine ecosystem engineers has caused an unprecedented decline in biodiversity. Although wild shellfish habitats have been shown to support biodiverse ecosystems, little is known about how biodiversity is altered by restored shellfish habitats, particularly mussels. To explore the biodiversity response to restored mussel habitats we deposited mussels on the seafloor in 1.5?×?1.5 m plots across a gradient of benthic environments. To understand a holistic community response, this study looks at the response of three faunal classifications over 1 year: infauna, epifauna, and pelagic fauna, compared with adjacent control plots (no mussels). The restored mussel habitats recorded 42 times more demersal fish than control areas, while macroalgae and mobile benthic invertebrates had over a twofold increase in abundance. Overall, the addition of mussels to the seafloor resulted in a general reduction of infaunal abundance and biodiversity, but an increase in epifaunal and pelagic faunal abundances, specifically from those species that benefit from benthic habitat complexity and an increase in food availability. From a management perspective, we highlight location-specific differences to consider for future restoration efforts, including environmental conditions and potential observed factors such as nearby sources of species, particularly predators, and relevant demersal fish ranges. Ultimately, measuring biodiversity responses in small-scale studies will serve as a valuable guide for larger scale restoration efforts and this study recommends considerations to enhance biodiversity outcomes in restored mussel habitats.

     

Characterization of dental pulp stem/stromal cells of Huntington monkey tooth germs

Background

Activation by extracellular ligands of G protein-coupled (GPCRs) and tyrosine kinase receptors (RTKs), results in the generation of second messengers that in turn control specific cell functions. Further, modulation/amplification or inhibition of the initial signalling events, depend on the recruitment onto the plasma membrane of soluble protein effectors. High throughput methodologies to monitor quantitatively second messenger production, have been developed over the last years and are largely used to screen chemical libraries for drug development. On the contrary, no such high throughput methods are yet available for the other aspect of GPCRs regulation, i.e. protein translocation to the plasma membrane, despite the enormous interest of this phenomenon for the modulation of receptor downstream functions. Indeed, to date, the experimental procedures available are either inadequate or complex and expensive.

Results

Here we describe the development of a novel conceptual approach to the study of cytosolic proteins translocation to the inner surface of the plasma membrane. The basis of the technique consists in: i) generating chimeras between the protein of interests and the calcium (Ca²⁺)-sensitive, luminescent photo-protein, aequorin and ii) taking advantage of the large Ca²⁺ concentration [Ca²⁺] difference between bulk cytosolic and the sub-plasma membrane rim.

Conclusion

This approach, that keeps unaffected the translocation properties of the signalling protein, can in principle be applied to any protein that, upon activation, moves from the cytosol to the plasma membrane. Thus, not only the modulation of GPCRs and RTKs can be investigated in this way, but that of all other proteins that can be recruited to the plasma membrane also independently of receptor activation. Moreover, its automated version, which can provide information about the kinetics and concentration-dependence of the process, is also applicable to high throughput screening of drugs affecting the translocation process.     

Structural insights into the design of nonpeptidic isothiazolidinone-containing inhibitors of protein-tyrosine phosphatase 1B

Structural analyses of the protein-tyrosine phosphatase 1B (PTP1B) active site and inhibitor complexes have aided in optimization of a peptide inhibitor containing the novel (S)-isothiazolidinone (IZD) phosphonate mimetic. Potency and permeability were simultaneously improved by replacing the polar peptidic backbone of the inhibitor with nonpeptidic moieties. The C-terminal primary amide was replaced with a benzimidazole ring, which hydrogen bonds to the carboxylate of Asp(48), and the N terminus of the peptide was replaced with an aryl sulfonamide, which hydrogen bonds to Asp(48) and the backbone NH of Arg(47) via a water molecule. Although both substituents retain the favorable hydrogen bonding network of the peptide scaffold, their aryl rings interact weakly with the protein. The aryl ring of benzimidazole is partially solvent exposed and only participates in van der Waals interactions with Phe(182) of the flap. The aryl ring of aryl sulfonamide adopts an unexpected conformation and only participates in intramolecular pi-stacking interactions with the benzimidazole ring. These results explain the flat SAR for substitutions on both rings and the reason why unsubstituted moieties were selected as candidates. Finally, substituents ortho to the IZD heterocycle on the aryl ring of the IZD-phenyl moiety bind in a small narrow site adjacent to the primary phosphate binding pocket. The crystal structure of an o-chloro derivative reveals that chlorine interacts extensively with residues in the small site. The structural insights that have led to the discovery of potent benzimidazole aryl sulfonamide o-substituted derivatives are discussed in detail.     

Aerobic fitness enhances relational memory in preadolescent children: The FITKids randomized control trial

It is widely accepted that aerobic exercise enhances hippocampal plasticity. Often, this plasticity co-occurs with gains in hippocampal-dependent memory. Cross-sectional work investigating this relationship in preadolescent children has found behavioral differences in higher versus lower aerobically fit participants for tasks measuring relational memory, which is known to be critically tied to hippocampal structure and function. The present study tested whether similar differences would arise in a clinical intervention setting where a group of preadolescent children were randomly assigned to a 9-month after school aerobic exercise intervention versus a wait-list control group. Performance measures included eye-movements as a measure of memory, based on recent work linking eye-movement indices of relational memory to the hippocampus. Results indicated that only children in the intervention increased their aerobic fitness. Compared to the control group, those who entered the aerobic exercise program displayed eye-movement patterns indicative of superior memory for face-scene relations, with no differences observed in memory for individual faces. The results of this intervention study provide clear support for the proposed linkage among the hippocampus, relational memory, and aerobic fitness, as well as illustrating the sensitivity of eye-movement measures as a means of assessing memory. ? 2012 Wiley Periodicals, Inc.