Discrimination of viable Acanthamoeba castellani trophozoites and cysts by propidium monoazide real-time polymerase chain reaction

Even though the advent of quantitative polymerase chain reaction (PCR) has improved the detection of pathogen microorganisms in most of areas of microbiology, a serious limitation of this method may arise from the inability to discriminate between viable and nonviable pathogens. To overcome it, the use of real-time PCR and selective nucleic acid intercalating dyes like propidium monoazide (PMA) have been effectively evaluated for different microorganisms. To assess whether PMA pretreatment can inhibit PCR amplification of nonviable amoeba DNA, Acanthamoeba castellani survival was measured using cell culture and real-time PCR with and without PMA pretreatment. Autoclave and contact lens disinfecting solutions were used to inactivate amoebae. After these inactivation treatments, the results indicated that the PMA pretreatment approach is appropriate for differentiating viable A. castellani, both trophozoites and cysts. Therefore, the PMA-PCR approach could be useful as a rapid and sensitive analytical tool for monitoring treatment and disease control, assessing effective disinfection treatments, and for a more reliable understanding of the factors that contribute to the interaction amoeba-pathogenic bacteria.     

Antifouling performance of cross-linked hydrogels: refinement of an attachment model

Poly(ethylene glycol) dimethacrylate (PEGDMA), PEGDMA-co-glycidyl methacrylate (PEGDMA-co-GMA), and PEGDMA-co-hydroxyethyl methacrylate (PEGDMA-co-HEMA) hydrogels were polymerized using ammonium persulfate and ascorbic acid as radical initiators. Surface energies of the hydrogels and a standard, poly(dimethylsiloxane) elastomer (PDMSe), were characterized using captive bubble and sessile drop measurements, respectively (γ = 52 mN/m, γ(0) = 19 mN/m). The chemical composition of the hydrogels was characterized by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. All three hydrogel compositions reduced significantly (p = 0.05) initial attachment of zoospores of the green alga Ulva linza (up to 97%), cells of the diatom Navicula incerta (up to 58%) and the bacterium Cobetia marina (up to 62%), compared to a smooth PDMSe standard. A shear stress (45 Pa), generated in a water channel, eliminated up to 95% of the initially attached cells of Navicula from the smooth hydrogel surfaces relative to smooth PDMSe surfaces. Compared to the PDMSe standard, 79% of the cells of C. marina were removed from all smooth hydrogel compositions when exposed to a 50 Pa wall shear stress. Attachment of spores of the green alga Ulva to microtopographies replicated in PEGDMA-co-HEMA was also evaluated. The Sharklet AF microtopography patterned, PEGDMA-co-HEMA surfaces reduced attachment of spores of Ulva by 97% compared to a smooth PDMSe standard. The attachment densities of spores to engineered microtopographies in PDMSe and PEGDMA-co-HEMA were shown to correlate with a modified attachment model through the inclusion of a surface energy term. Attachment densities of spores of Ulva to engineered topographies replicated in a material other than PDMSe are now correlated with the attachment model (R(2) = 0.80).     

Desmin expression in colorectal cancer stroma correlates with advanced stage disease and marks angiogenic microvessels

Introduction  

Biomarkers that improve stratification of colorectal cancer patients for adjuvant therapy versus resection alone, or that are predictive of response to therapeutic agents, have the potential to greatly improve patient selection for such therapies. The aim was to determine proteins differentially expressed within the malignant epithelial glands and closely associated stromal elements compared to matched normal mucosa, and to characterise the over-expression of one such protein as a potential biomarker.     

Arl2-GTP and Arl3-GTP regulate a GDI-like transport system for farnesylated cargo

Lipidated Rho and Rab GTP-binding proteins are transported between membranes in complex with solubilizing factors called 'guanine nucleotide dissociation inhibitors' (GDIs). Unloading from GDIs using GDI displacement factors (GDFs) has been proposed but remains mechanistically elusive. PDEδ is a putative solubilizing factor for several prenylated Ras-subfamily proteins. Here we report the structure of fully modified farnesylated Rheb-GDP in complex with PDEδ. The structure explains the nucleotide-independent binding of Rheb to PDEδ and the relaxed specificity of PDEδ. We demonstrate that the G proteins Arl2 and Arl3 act in a GTP-dependent manner as allosteric release factors for farnesylated cargo. We thus describe a new transport system for farnesylated G proteins involving a GDI-like molecule and an unequivocal GDF. Considering the importance of PDEδ for proper Ras and Rheb signaling, this study is instrumental in developing a new target for anticancer therapy.     

Evidence for the prevention of enthesitis in HLA‐B27/hβ2m transgenic rats treated with a monoclonal antibody against TNF‐α

Transgenic rats with high expression of HLA‐B27 and human β₂‐microglobulin (B27TR) develop a multisystem inflammatory disease resembling human inflammatory bowel disease (IBD) and spondyloarthropaties (SpA). Tumour necrosis factor α (TNF‐α) has a crucial role in sustaining chronic inflammation in the gut and joints. The aim of this work was to evaluate whether TNF‐α blockade could prevent or reduce the inflammation of peripheral joints in B27TR. A first group of 9‐week‐old B27TR received an anti‐TNF‐α monoclonal antibody (mAb) or an isotypic IgG2a,k up to the age of 18 weeks. An untreated group was monitored up to the age of 18 weeks and then randomly assigned to a 9‐week treatment with anti‐TNF‐α mAb or IgG2a,k. Each rat was monitored for clinical IBD and peripheral joint manifestations. After sacrifice the colon and hind paws were examined for macroscopical and microscopical pathological changes. Early TNF‐α blockade prevented, and late treatment improved IBD signs in B27TR. Erythema, oedema, inflammatory infiltrate close to the tendons and enthesis, proliferating chondrocyte‐like cells, signs of new endochondral bone ossification and bone erosion were observed in peripheral joints of four out of six IgG2a,k‐treated B27TR, both at 18 and 27 weeks. Immunopositivity for phosphorylated Smad1/5/8 indicated that the process of joint remodelling was activated in B27TR. Some entheses showed chondroid nodules. Anti‐TNF‐α treatment reduced inflammation and preserved the enthesis organization in most animals. Occasional and transient erythema and oedema were still present in three of six of the late anti‐TNF‐α‐treated animals. Smad1/5/8 signalling was not inhibited by late anti‐TNF‐α treatment. In B27TR, articular involvement follows IBD onset and develops at entheses. Early TNF‐α blockade prevents the onset of IBD and consequently the development of enthesitis in peripheral joints in the B27TR model of human SpA.     

Qualitative detection of the NSAIDs diclofenac and ibuprofen in the hair of Eurasian otters (Lutra lutra) occupying UK waterways with GC�CMS

The pervasiveness of pharmaceuticals such as nonsteroidal anti-inflammatory drugs (NSAIDs) in the aquatic ecosystem through the discharge of wastewater, and their potential to biomagnify within this ecosystem, is now recognised. Residues of diclofenac and ibuprofen are currently being detected in surface waters and aquatic organisms throughout the UK and Europe. However, the levels of these residues in fish and other aquatic organisms, particularly lower trophic level prey species, have not yet been determined. While exposure to diclofenac is known to adversely affect fish, the degree to which other aquatic organisms are exposed and impacted through continuous ingestion of contaminated prey and interaction with the aquatic habitat remains unknown. The extent and effects of exposure to ibuprofen also remain largely unknown. As an exploratory subset of a broader study to investigate the detectability of diclofenac in alternative biological matrices, we analysed hair samples from Eurasian otters (Lutra lutra, n?=?28) for residues of the two NSAIDs using GC?CMS. The otters were collected from six counties in England as part of an ongoing otter health monitoring project at the Wildlife Veterinary Investigation Centre in Chacewater, UK. Diclofenac was qualitatively detected in five hair wash and 15 extract samples, and ibuprofen was determined to be present in at least two of the hair extract samples. Here, we provide preliminary evidence that otters are exposed to both NSAIDs and argue for further studies to identify residue loads in the otters and their prey to fully assess the pervasiveness of these compounds and potential risks of ongoing exposure to them.     

The gift of motherhood: Egg donation in a Barcelona infertility clinic

In this paper we analyse the ways in which egg donors from a private infertility clinic in Barcelona try to render their new experience meaningful. Donors are striving to see their action as a contribution to the creation of a particular kinship bond – motherhood in another woman – by means of the abrogation of a bond that also looks very much like kinship, which links them to the individuals that will be born thanks to their eggs. The specific meaning that egg donation has for each donor varies according to her particular circumstances, but the language constructed in order to convey this meaning emerges from the creative expression of several cultural paradoxes and dichotomies that constitute, in themselves, an original and highly significant cultural grammar.     

Different Patterns of Epstein-Barr Virus Latency in Endemic Burkitt Lymphoma (BL) Lead to Distinct Variants within the BL-Associated Gene Expression Signature

Epstein-Barr virus (EBV) is present in all cases of endemic Burkitt lymphoma (BL) but in few European/North American sporadic BLs. Gene expression arrays of sporadic tumors have defined a consensus BL profile within which tumors are classifiable as “molecular BL” (mBL). Where endemic BLs fall relative to this profile remains unclear, since they not only carry EBV but also display one of two different forms of virus latency. Here, we use early-passage BL cell lines from different tumors, and BL subclones from a single tumor, to compare EBV-negative cells with EBV-positive cells displaying either classical latency I EBV infection (where EBNA1 is the only EBV antigen expressed from the wild-type EBV genome) or Wp-restricted latency (where an EBNA2 gene-deleted virus genome broadens antigen expression to include the EBNA3A, -3B, and -3C proteins and BHRF1). Expression arrays show that both types of endemic BL fall within the mBL classification. However, while EBV-negative and latency I BLs show overlapping profiles, Wp-restricted BLs form a distinct subgroup, characterized by a detectable downregulation of the germinal center (GC)-associated marker Bcl6 and upregulation of genes marking early plasmacytoid differentiation, notably IRF4 and BLIMP1. Importantly, these same changes can be induced in EBV-negative or latency I BL cells by infection with an EBNA2-knockout virus. Thus, we infer that the distinct gene profile of Wp-restricted BLs does not reflect differences in the identity of the tumor progenitor cell per se but differences imposed on a common progenitor by broadened EBV gene expression.     

Assessment of the Protective Effect of Imvamune and Acam2000 Vaccines against Aerosolized Monkeypox Virus in Cynomolgus Macaques

To support the licensure of a new and safer vaccine to protect people against smallpox, a monkeypox model of infection in cynomolgus macaques, which simulates smallpox in humans, was used to evaluate two vaccines, Acam2000 and Imvamune, for protection against disease. Animals vaccinated with a single immunization of Imvamune were not protected completely from severe and/or lethal infection, whereas those receiving either a prime and boost of Imvamune or a single immunization with Acam2000 were protected completely. Additional parameters, including clinical observations, radiographs, viral load in blood, throat swabs, and selected tissues, vaccinia virus-specific antibody responses, immunophenotyping, extracellular cytokine levels, and histopathology were assessed. There was no significant difference (P > 0.05) between the levels of neutralizing antibody in animals vaccinated with a single immunization of Acam2000 (132 U/ml) and the prime-boost Imvamune regime (69 U/ml) prior to challenge with monkeypox virus. After challenge, there was evidence of viral excretion from the throats of 2 of 6 animals in the prime-boost Imvamune group, whereas there was no confirmation of excreted live virus in the Acam2000 group. This evaluation of different human smallpox vaccines in cynomolgus macaques helps to provide information about optimal vaccine strategies in the absence of human challenge studies.     

Extracellular Electron Transfer to Fe(III) Oxides by the Hyperthermophilic Archaeon Geoglobus ahangari via a Direct Contact Mechanism

The microbial reduction of Fe(III) plays an important role in the geochemistry of hydrothermal systems, yet it is poorly understood at the mechanistic level. Here we show that the obligate Fe(III)-reducing archaeon Geoglobus ahangari uses a direct-contact mechanism for the reduction of Fe(III) oxides to magnetite at 85°C. Alleviating the need to directly contact the mineral with the addition of a chelator or the electron shuttle anthraquinone-2,6-disulfonate (AQDS) stimulated Fe(III) reduction. In contrast, entrapment of the oxides within alginate beads to prevent cell contact with the electron acceptor prevented Fe(III) reduction and cell growth unless AQDS was provided. Furthermore, filtered culture supernatant fluids had no effect on Fe(III) reduction, ruling out the secretion of an endogenous mediator too large to permeate the alginate beads. Consistent with a direct contact mechanism, electron micrographs showed cells in intimate association with the Fe(III) mineral particles, which once dissolved revealed abundant curled appendages. The cells also produced several heme-containing proteins. Some of them were detected among proteins sheared from the cell''s outer surface and were required for the reduction of insoluble Fe(III) oxides but not for the reduction of the soluble electron acceptor Fe(III) citrate. The results thus support a mechanism in which the cells directly attach and transfer electrons to the Fe(III) oxides using redox-active proteins exposed on the cell surface. This strategy confers on G. ahangari a competitive advantage for accessing and reducing Fe(III) oxides under the extreme physical and chemical conditions of hot ecosystems.