Visualization of cyclooxygenase-2 using a 2,3-diarylsubstituted indole-based inhibitor and confocal laser induced cryofluorescence microscopy at 20 K in melanoma cells in vitro

This study aimed at visualization of cyclooxygenase-2 (COX-2) protein expression in melanoma cells by confocal laser induced cryofluorescence microscopy using 4-(3-(4-methoxyphenyl)-1H-indol-2-yl)benzene-sulfonamide (C1) representative for a novel class of autofluorescent 2,3-diarylsubstituted indole-based selective COX-2 inhibitors.COX-2 expression was measured in human melanoma cell lines A2058 and MelJuso by immunocytochemistry and immunoblotting. Cellular uptake experiments using varying C1 concentrations down to 0.1 nM (with/without molar excess of celecoxib as control) were performed at 37 °C. Cryofluorescence microscopy was conducted at 20 K.COX-2 protein expression was successfully visualized by C1 in A2058 cells. COX-2-negative MelJuso cells showed no specific accumulation of C1. Control experiments using celecoxib and, additionally, implemented fluorescence spectroscopy confirmed specificity of both cellular uptake and intracellular association of C1.Cryofluorescence microscopy in combination with spectroscopy allowed for visualization of COX-2 protein expression in melanoma cells in vitro using a selective COX-2 inhibitor at very low concentrations.     

West Africa - A Safe Haven for Frogs? A Sub-Continental Assessment of the Chytrid Fungus (Batrachochytrium dendrobatidis)

A putative driver of global amphibian decline is the panzootic chytrid fungus Batrachochytrium dendrobatidis (Bd). While Bd has been documented across continental Africa, its distribution in West Africa remains ambiguous. We tested 793 West African amphibians (one caecilian and 61 anuran species) for the presence of Bd. The samples originated from seven West African countries - Bénin, Burkina Faso, Côte d''Ivoire, Ghana, Guinea, Liberia, Sierra Leone - and were collected from a variety of habitats, ranging from lowland rainforests to montane forests, montane grasslands to humid and dry lowland savannahs. The species investigated comprised various life-history strategies, but we focused particularly on aquatic and riparian species. We used diagnostic PCR to screen 656 specimen swabs and histology to analyse 137 specimen toe tips. All samples tested negative for Bd, including a widespread habitat generalist Hoplobatrachus occipitalis which is intensively traded on the West African food market and thus could be a potential dispersal agent for Bd. Continental fine-grained (30 arc seconds) environmental niche models suggest that Bd should have a broad distribution across West Africa that includes most of the regions and habitats that we surveyed. The surprising apparent absence of Bd in West Africa indicates that the Dahomey Gap may have acted as a natural barrier. Herein we highlight the importance of this Bd-free region of the African continent - especially for the long-term conservation of several threatened species depending on fast flowing forest streams (Conraua alleni (“Vulnerable”) and Petropedetes natator (“Near Threatened”)) as well as the “Critically Endangered” viviparous toad endemic to the montane grasslands of Mount Nimba (Nimbaphrynoides occidentalis).     

Elm defence against herbivores and pathogens: morphological,chemical and molecular regulation aspects

Elms (Ulmus spp.) have long been appreciated for their environmental tolerance, landscape and ornamental value, and the quality of their wood. Although elm trees are extremely hardy against abiotic stresses such as wind and pollution, they are susceptible to attacks of biotic stressors. Over 100 phytopathogens and invertebrate pests are associated with elms: fungi, bacteria and insects like beetles and moths, and to a lesser extent aphids, mites, viruses and nematodes. While the biology of the pathogen and insect vector of the Dutch elm disease has been intensively studied, less attention has been paid so far to the defence mechanisms of elms to other biotic stressors. This review highlights knowledge of direct and indirect elm defences against biotic stressors focusing on morphological, chemical and gene regulation aspects. First, we report how morphological defence mechanisms via barrier formation and vessel occlusion prevent colonisation and spread of wood- and bark-inhabiting fungi and bacteria. Second, we outline how secondary metabolites such as terpenoids (volatile terpenoids, mansonones and triterpenoids) and phenolics (lignans, coumarins, flavonoids) in leaves and bark are involved in constitutive and induced chemical defence mechanisms of elms. Third, we address knowledge on how the molecular regulation of elm defence is orchestrated through the interaction of a huge variety of stress- and defence-related genes. We conclude by pointing to the gaps of knowledge on the chemical and molecular mechanisms of elm defence against pest insects and diseases. An in-depth understanding of defence mechanisms of elms will support the development of sustainable integrated management of pests and diseases attacking elms.     

Interaction exists between matriptase inhibitors and intestinal epithelial cells

The type II trypsin-like transmembrane serine protease matriptase, is mainly expressed in epithelial cells and one of the key regulators in the formation and maintenance of epithelial barrier integrity. Therefore, we have studied the inhibition of matriptase in a non-transformed porcine intestinal IPEC-J2 cell monolayer cultured on polyester membrane inserts by the non-selective 4-(2-aminoethyl)-benzosulphonylfluoride (AEBSF) and four more selective 3-amidinophenylalanine-derived matriptase inhibitors. It was found that suppression of matriptase activity by MI-432 and MI-460 led to decreased transepithelial electrical resistance (TER) of the cell monolayer and to an enhanced transport of fluorescently labelled dextran, a marker for paracellular transport between apical and basolateral compartments. To this date this is the first report in which the inhibition of matriptase activity by synthetic inhibitors has been correlated to a reduced barrier integrity of a non-cancerous IPEC-J2 epithelial cell monolayer in order to describe interaction between matriptase activity and intestinal epithelium in vitro.     

Natural and Regenerated Saltmarshes Exhibit Similar Soil and Belowground Organic Carbon Stocks,Root Production and Soil Respiration

Ecosystems - Saltmarshes provide many valuable ecosystem services including storage of a large amount of ‘blue carbon’ within their soils. To date, up to 50% of the world’s...     

Involvement of the Adhesion GPCRs Latrophilins in the Regulation of Insulin Release

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Improvement of femoral bone-cement adhesion in cemented revision hip arthroplasty by application of an amphiphilic bonder in a dynamic femur expulsion testing in vitro ]

Cemented femoral stems have shown decreased longevity compared to cementless implants in hip revision arthroplasty. The aim of this study was to evaluate the effect of an amphiphilic bonder on bone cement stability in a biomechanical femur expulsion test. A simplified hip simulator test setup with idealised femur stem specimens was carried out. The stems were implanted into bovine femurs (group 1: no bonder, n=10; group 2: bonder including glutaraldehyde, n=10; group 3: bonder without glutaraldehyde, n=10). A dynamic loading (maximum load: 800 N; minimum load: 100 N; frequency: 3 Hz; 105 cycles) was performed. Subsequently, the stem specimens were expulsed axially out of their implant beds and maximum load at failure was recorded. The static controls showed a mean maximum load to failure of 4123 N in group 1, 8357.5 N in group 2 and 5830.8 N in group 3. After dynamic loading, the specimens of group 2 reached the highest load to failure (8191.5 N), followed by group 3 (5649.5 N) and group 1 (3462 N), respectively. In group 2, we observed nine periprosthetic fractures at a load of 8400 N without signs of interface loosening. Application of an amphiphilic bonder led to a significant improvement of bonding stability, especially when glutaraldehyde was added to the bonder. This technique might offer an increased longevity of cemented femur revision stems in total hip replacement.     

Structural and functional evolution of the P2Y12-like receptor group

Metabotropic pyrimidine and purine nucleotide receptors (P2Y receptors) belong to the superfamily of G protein-coupled receptors (GPCR). They are distinguishable from adenosine receptors (P1) as they bind adenine and/or uracil nucleotide triphosphates or diphosphates depending on the subtype. Over the past decade, P2Y receptors have been cloned from a variety of tissues and species, and as many as eight functional subtypes have been characterized. Most recently, several members of the P2Y₁₂-like receptor group, which includes the clopidogrel-sensitive ADP receptor P2Y₁₂, have been deorphanized. The P2Y₁₂-like receptor group comprises several structurally related GPCR which, however, display heterogeneous agonist specificity including nucleotides, their derivatives, and lipids. Besides the established function of P2Y₁₂ in platelet activation, expression in macrophages, neuronal and glial cells as well as recent results from functional studies implicate that several members of this group may have specific functions in neurotransmission, inflammation, chemotaxis, and response to tissue injury. This review focuses specifically on the structure-function relation and shortly summarizes some aspects of the physiological relevance of P2Y₁₂-like receptor members.     

Natural killer cells promote early CD8 T cell responses against cytomegalovirus

Understanding the mechanisms that help promote protective immune responses to pathogens is a major challenge in biomedical research and an important goal for the design of innovative therapeutic or vaccination strategies. While natural killer (NK) cells can directly contribute to the control of viral replication, whether, and how, they may help orchestrate global antiviral defense is largely unknown. To address this question, we took advantage of the well-defined molecular interactions involved in the recognition of mouse cytomegalovirus (MCMV) by NK cells. By using congenic or mutant mice and wild-type versus genetically engineered viruses, we examined the consequences on antiviral CD8 T cell responses of specific defects in the ability of the NK cells to control MCMV. This system allowed us to demonstrate, to our knowledge for the first time, that NK cells accelerate CD8 T cell responses against a viral infection in vivo. Moreover, we identify the underlying mechanism as the ability of NK cells to limit IFN-alpha/beta production to levels not immunosuppressive to the host. This is achieved through the early control of cytomegalovirus, which dramatically reduces the activation of plasmacytoid dendritic cells (pDCs) for cytokine production, preserves the conventional dendritic cell (cDC) compartment, and accelerates antiviral CD8 T cell responses. Conversely, exogenous IFN-alpha administration in resistant animals ablates cDCs and delays CD8 T cell activation in the face of NK cell control of viral replication. Collectively, our data demonstrate that the ability of NK cells to respond very early to cytomegalovirus infection critically contributes to balance the intensity of other innate immune responses, which dampens early immunopathology and promotes optimal initiation of antiviral CD8 T cell responses. Thus, the extent to which NK cell responses benefit the host goes beyond their direct antiviral effects and extends to the prevention of innate cytokine shock and to the promotion of adaptive immunity.