Whole-Genome Characterization and Genotyping of Global WU Polyomavirus Strains

Exploration of the genetic diversity of WU polyomavirus (WUV) has been limited in terms of the specimen numbers and particularly the sizes of the genomic fragments analyzed. Using whole-genome sequencing of 48 WUV strains collected in four continents over a 5-year period and 16 publicly available whole-genome sequences, we identified three main WUV clades and five subtypes, provisionally termed Ia, Ib, Ic, II, IIIa, and IIIb. Overall nucleotide variation was low (0 to 1.2%). The discriminatory power of the previous VP2 fragment typing method was found to be limited, and a new, larger genotyping region within the VP2/1 interface was proposed.In 2007, two new human polyomaviruses isolated from respiratory samples of pediatric patients suffering from respiratory disease were discovered, with one being KI polyomavirus (KIV) (2) and the other being WU polyomavirus (WUV) (8).WU polyomavirus shares most of the genomic characteristics of other polyomaviruses, with a noncoding control region (NCCR) separating the early and late coding regions on opposite strands. However, unlike for JCV and BKV, but similar to what was observed for KIV, a late-region-residing agnoprotein gene has not been identified in WUV (8).Despite being frequently detected in respiratory samples of ill patients, no distinct disease associations have so far been conclusively identified for WUV (1, 2, 4, 8, 10, 27). There have been some suggestions that sequence variation plays a role in disease severity and pathogenesis in other polyomaviruses (6, 24). Unfortunately, due to the early nature of research into WUV, there has been a dearth of available complete genomic sequences.In this study, we set out to investigate a large sample set of whole WUV genomes from diverse geographical, temporal, and clinical origins. Incorporating existing WUV genomes with this data set allowed us to investigate global WUV genomic diversity, to characterize the WUV genome, and to propose a new robust typing scheme.     

High-efficiency derivation of human embryonic stem cell lines following pre-implantation genetic diagnosis

Pre-implantation genetic diagnosis allows the characterisation of embryos that carry a gene responsible for a severe monogenic disease and to transfer to the mother’s uterus only the unaffected one(s). The genetically affected embryos can be used to establish human embryonic stem cell (hESC) lines. We are currently establishing a cell bank of ESC lines carrying specific disease-causing mutant genes. These cell lines are available to the scientific community. For this purpose, we have designed a technique that requires only minimal manipulation of the embryos. At the blastocyst stage, we just removed the zona pellucida before seeding the embryo as a whole on a layer of feeder cells. This approach gave a good success rate (>20%), whatever the quality of the embryos, and allowed us to derive 11 new hESC lines, representing seven different pathologies. Full phenotypic validation of the cell lines according to ISCI guidelines confirmed their pluripotent nature, as they were positive for hESC markers and able to differentiate in vitro in all three germ layers derivatives. Nine out of 11 stem cell lines had normal karyotypes. Our results indicate that inner cell mass isolation is not mandatory for hESC derivation and that minimal manipulation of embryos can lead to high success rate.     

Viscoelastic behaviour aids extrusion from and reabsorption of the liquid phase into the digesta plug: creep rheometry of hindgut digesta in the common brushtail possum Trichosurus vulpecula

Temporal variation in the rheometric properties of the proximal and distal colonic digesta of an arboreal marsupial folivore, the common brushtail possum, was examined to assess flow behaviour during peristalsis, segmentation and other aspects of intestinal motility. The time-dependent rheometric characteristics on application of a constant shear stress within the physiological range showed an initial elastic and subsequent viscoelastic phase, which fitted Burger’s model of creep compliance. Similarly, the time-dependent rheometric characteristics on recovery from shear stress fitted with a generalised two-component Maxwell model of elastic and viscoelastic components for creep recovery. Differences in the relative magnitudes of the viscoelastic components during recovery from those during shear indicated that the physical properties of the digesta plug changed with sustained shear stress, a phenomenon, which is likely to result from extrusion of the liquid phase from the solid elements of the digesta plug. There was significant viscoelastic recovery during the initial 4 s following cessation of stress, which would allow for prompt concomitant reabsorption of the liquid phase into the digesta plug. This supports a hypothesis of alternate extrusion and reabsorption of the liquid phase of the digesta plug. This would promote both nutrient absorption across the intestinal wall (from liquid extrusion) and enzyme permeation and digestion (from liquid absorption into the plug). However, the presence of a slower component of viscoelastic recovery indicates that liquid phase reabsorption into the digesta plug is incomplete if the interval before a subsequent contraction is less than 150 s, in which case unreabsorbed liquid may be driven either orally or aborally. This would at least partly account for differences in retention times of liquid and solid phase digesta markers reported for the gastrointestinal tracts of numerous vertebrate species.     

Presence of the cannabinoid receptors, CB1 and CB2, in human omental and subcutaneous adipocytes

To investigate the expression of the endocannabinoid 1 and 2 receptors by human adipocyte cells of omental and subcutaneous fat tissue, as well as to determine whether these receptors are functional. The expression of CB1 and CB2 receptors on human adipocytes was analyzed by western blotting, immunohistology and immunocytology. We also investigated intracytoplasmic cyclic AMP level modulation following CB1 and CB2 receptor stimulation by an enzymatic immuno assay. All mature adipocytes, from visceral (epiploon) and subcutaneous fat tissue, express CB1 and CB2 on their plasma membranes. We also demonstrate in this study that adipocyte precursors (pre-adipocytes) express CB1 and CB2 on their plasma membranes and that both receptors are functional. Activation of CB1 increases intracytoplasmic cyclic AMP whilst CB2 activation leads to a cyclic AMP decrease. Here we demonstrate, for the first time, that adipocytes of human adipose tissue (mature adipocytes and pre-adipocytes) express functional plasma membrane CB1 and CB2 receptors. Their physiological role on the adipose tissue is not known. However, their major involvement in the physiology of other tissues leads us to suppose that they could play a significant role in the homeostasis of the energy balance and/or in the regulation of adipose tissue inflammation.     

The H275Y Neuraminidase Mutation of the Pandemic A/H1N1 Influenza Virus Lengthens the Eclipse Phase and Reduces Viral Output of Infected Cells, Potentially Compromising Fitness in Ferrets

The H275Y amino acid substitution of the neuraminidase gene is the most common mutation conferring oseltamivir resistance in the N1 subtype of the influenza virus. Using a mathematical model to analyze a set of in vitro experiments that allow for the full characterization of the viral replication cycle, we show that the primary effects of the H275Y substitution on the pandemic H1N1 (H1N1pdm09) strain are to lengthen the mean eclipse phase of infected cells (from 6.6 to 9.1 h) and decrease (by 7-fold) the viral burst size, i.e., the total number of virions produced per cell. We also find, however, that the infectious-unit-to-particle ratio of the H275Y mutant strain is 12-fold higher than that of the oseltamivir-susceptible strain (0.19 versus 0.016 per RNA copy). A parallel analysis of the H275Y mutation in the prior seasonal A/Brisbane/59/2007 background shows similar changes in the infection kinetic parameters, but in this background, the H275Y mutation also allows the mutant to infect cells five times more rapidly. Competitive mixed-strain infections in vitro, where the susceptible and resistant H1N1pdm09 strains must compete for cells, are characterized by higher viral production by the susceptible strain but suggest equivalent fractions of infected cells in the culture. In ferrets, however, the mutant strain appears to suffer a delay in its infection of the respiratory tract that allows the susceptible strain to dominate mixed-strain infections.     

In vitro and in vivo antiangiogenic properties of the serpin protease nexin-1

The serpin protease nexin-1 (PN-1) is expressed by vascular cells and secreted by platelets upon activation, and it is known to interact with several modulators of angiogenesis, such as proteases, matrix proteins, and glycosaminoglycans. We therefore investigated the impact of PN-1 on endothelial cell angiogenic responses in vitro and ex vivo and in vivo in PN-1-deficient mice. We found that PN-1 is antiangiogenic in vitro: it inhibited vascular endothelial growth factor (VEGF)-induced endothelial cell responses, including proliferation, migration, and capillary tube formation, and decreased cell spreading on vitronectin. These effects do not require the antiprotease activity of PN-1 but involve PN-1 binding to glycosaminoglycans. In addition, our results indicated that PN-1 does not act by blocking VEGF binding to its heparan sulfate proteoglycan coreceptors. The results obtained in vitro were supported ex vivo in PN-1-deficient mice, where the microvascular network sprouting from aortic rings was significantly enhanced. Moreover, in vivo, neovessel formation was promoted in the Matrigel plug assay in PN-1-deficient mice compared to wild-type mice, and these effects were reversed by the addition of recombinant PN-1. Taken together, our results demonstrate that PN-1 has direct antiangiogenic properties and is a yet-unrecognized player in the angiogenic balance.     

Immunoregulatory functions of paf-acether. III. Down-regulation of CD4+ T cells high-affinity IL-2 receptor expression

In the present report, we further explored the mechanisms by which 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (paf-acether), a phospholipid mediator of inflammation inhibited PHA-induced CD4+ cell proliferation. Evidence was obtained that CD4+ cells stimulated with either PHA or immobilized OKT3 in the presence of paf at concentrations that block CD4+ cell proliferation, exhibited a marked decrease in high affinity IL-2R expression. Importantly, paf did not prevent the binding of IL-2 to its receptor. Scatchard analysis of the binding data indicated that paf caused more than 50% decrease in the number of IL-2 high affinity sites per cell, whereas the receptor ligand affinity remained essentially constant. Moreover, the down-regulation of high affinity IL-2R was also accompanied by a loss of IL-2-dependent proliferative capacity. Together these data suggest that decreased expression of high affinity IL-2R may contribute to the diminished proliferative activity observed in CD4+ cells stimulated with PHA or immobilized OKT3 in the presence of paf. They further emphasize the potential role of lipid proinflammatory mediators such as paf in the regulation of T cell activation.     

Impact of the H275Y and I223V Mutations in the Neuraminidase of the 2009 Pandemic Influenza Virus In Vitro and Evaluating Experimental Reproducibility

The 2009 pandemic H1N1 (H1N1pdm09) influenza virus is naturally susceptible to neuraminidase (NA) inhibitors, but mutations in the NA protein can cause oseltamivir resistance. The H275Y and I223V amino acid substitutions in the NA of the H1N1pdm09 influenza strain have been separately observed in patients exhibiting oseltamivir-resistance. Here, we apply mathematical modelling techniques to compare the fitness of the wild-type H1N1pdm09 strain relative to each of these two mutants. We find that both the H275Y and I223V mutations in the H1N1pdm09 background significantly lengthen the duration of the eclipse phase (by 2.5 h and 3.6 h, respectively), consistent with these NA mutations delaying the release of viral progeny from newly infected cells. Cells infected by H1N1pdm09 virus carrying the I223V mutation display a disadvantageous, shorter infectious lifespan (17 h shorter) than those infected with the wild-type or MUT-H275Y strains. In terms of compensating traits, the H275Y mutation in the H1N1pdm09 background results in increased virus infectiousness, as we reported previously, whereas the I223V exhibits none, leaving it overall less fit than both its wild-type counterpart and the MUT-H275Y strain. Using computer simulated competition experiments, we determine that in the presence of oseltamivir at doses even below standard therapy, both the MUT-H275Y and MUT-I223V dominate their wild-type counterpart in all aspects, and the MUT-H275Y outcompetes the MUT-I223V. The H275Y mutation should therefore be more commonly observed than the I223V mutation in circulating H1N1pdm09 strains, assuming both mutations have a similar impact or no significant impact on between-host transmission. We also show that mathematical modelling offers a relatively inexpensive and reliable means to quantify inter-experimental variability and assess the reproducibility of results.     

Engineered Fn3 protein has targeted therapeutic effect on mesothelin-expressing cancer cells and increases tumor cell sensitivity to chemotherapy

Mesothelin is a protein expressed at high levels on the cell surface in a variety of cancers, with limited expression in healthy tissues. The presence of mesothelin on tumor tissue correlates with increased invasion and metastasis, and resistance to traditional chemotherapies, through mechanisms that remain poorly understood. Molecules that specifically recognize mesothelin and interrupt its contribution to tumor progression have significant potential for targeted therapy and targeted drug delivery applications. A number of mesothelin-targeting therapies are in preclinical and clinical development, although none are currently approved for routine clinical use. In this work, we report the development of a mesothelin-targeting protein based on the fibronectin type-III non-antibody protein scaffold, which offers opportunities for applications where antibodies have limitations. We engineered protein variants that bind mesothelin with high affinity and selectively initiate apoptosis in tumor cells expressing mesothelin. Interestingly, apoptosis does not occur through a caspase-mediated pathway and does not require downregulation of cell-surface mesothelin, suggesting a currently unknown pathway through which mesothelin contributes to cancer progression. Importantly, simultaneous treatment with mesothelin-binding protein and chemotherapeutic mitomycin C had a greater cytotoxic effect on mesothelin-positive cells compared to either molecule alone, underscoring the potential for combination therapy including biologics targeting mesothelin.