RNA Virus Population Diversity,an Optimum for Maximal Fitness and Virulence

The ability of an RNA virus to exist as a population of genetically distinct variants permits the virus to overcome events during infections that would otherwise limit virus multiplication or drive the population to extinction. Viral genetic diversity is created by the ribonucleotide misincorporation frequency of the viral RNA-dependent RNA polymerase (RdRp). We have identified a poliovirus (PV) RdRp derivative (H273R) possessing a mutator phenotype. GMP misincorporation efficiency for H273R RdRp in vitro was increased by 2–3-fold that manifested in a 2–3-fold increase in the diversity of the H273R PV population in cells. Circular sequencing analysis indicated that some mutations were RdRp-independent. Consistent with the population genetics theory, H273R PV was driven to extinction more easily than WT in cell culture. Furthermore, we observed a substantial reduction in H273R PV virulence, measured as the ability to cause paralysis in the cPVR mouse model. Reduced virulence correlated with the inability of H273R PV to sustain replication in tissues/organs in which WT persists. Despite the attenuated phenotype, H273R PV was capable of replicating in mice to levels sufficient to induce a protective immune response, even when the infecting dose used was insufficient to elicit any visual signs of infection. We conclude that optimal RdRp fidelity is a virulence determinant that can be targeted for viral attenuation or antiviral therapies, and we suggest that the RdRp may not be the only source of mutations in a RNA virus genome.     

Apolipoprotein M modulates erythrocyte efflux and tubular reabsorption of sphingosine-1-phosphate

Sphingosine-1-phosphate (S1P) mediates several cytoprotective functions of HDL. apoM acts as a S1P binding protein in HDL. Erythrocytes are the major source of S1P in plasma. After glomerular filtration, apoM is endocytosed in the proximal renal tubules. Human or murine HDL elicited time- and dose-dependent S1P efflux from erythrocytes. Compared with HDL of wild-type (wt) mice, S1P efflux was enhanced in the presence of HDL from apoM transgenic mice, but not diminished in the presence of HDL from apoM knockout (Apom^−/−) mice. Artificially reconstituted and apoM-free HDL also effectively induced S1P efflux from erythrocytes. S1P and apoM were not measurable in the urine of wt mice. Apom^−/− mice excreted significant amounts of S1P. apoM was detected in the urine of mice with defective tubular endocytosis because of knockout of the LDL receptor-related protein, chloride-proton exchanger ClC-5 (Clcn5^−/−), or the cysteine transporter cystinosin. Urinary levels of S1P were significantly elevated in Clcn5^−/− mice. In contrast to Apom^−/− mice, these mice showed normal plasma concentrations for apoM and S1P. In conclusion, HDL facilitates S1P efflux from erythrocytes by both apoM-dependent and apoM-independent mechanisms. Moreover, apoM facilitates tubular reabsorption of S1P from the urine, however, with no impact on S1P plasma concentrations.     

Hippocampal proteoglycans brevican and versican are linked to spatial memory of Sprague–Dawley rats in the morris water maze

Proteoglycans (PGs) are major constituents of the extracellular matrix and have recently been proposed to contribute to synaptic plasticity. Hippocampal PGs have not yet been studied or linked to memory. The aim of the study, therefore, was to isolate and characterize rat hippocampal PGs and determine their possible role in spatial memory. PGs were extracted from rat hippocampi by anion‐exchange chromatography and analyzed by nano LC‐MS/MS. Twenty male Sprague–Dawley rats were tested in the morris water maze. PGs agrin, amyloid beta A4 protein, brevican, glypican‐1, neurocan, phosphacan, syndecan‐4, and versican were identified in the hippocampi. Brevican and versican levels in the membrane fraction were higher in the trained group, correlating with the time spent in the target quadrant. α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate receptor GluR1 was co‐precipitated with brevican and versican. Levels for a receptor complex containing GluR1 was higher in trained while GluR2 and GluR3‐containing complex levels were higher in yoked rats. The findings provide information about the PGs present in the rat hippocampus, demonstrating that versican and brevican are linked to memory retrieval in the morris water maze and that PGs interact with α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate receptor GluR1, which is linked to memory retrieval.

     

The Laminin Response in Inflammatory Bowel Disease: Protection or Malignancy?

Laminins (LM), basement membrane molecules and mediators of epithelial-stromal communication, are crucial in tissue homeostasis. Inflammatory Bowel Diseases (IBD) are multifactorial pathologies where the microenvironment and in particular LM play an important yet poorly understood role in tissue maintenance, and in cancer progression which represents an inherent risk of IBD. Here we showed first that in human IBD colonic samples and in murine colitis the LMα1 and LMα5 chains are specifically and ectopically overexpressed with a concomitant nuclear p53 accumulation. Linked to this observation, we provided a mechanism showing that p53 induces LMα1 expression at the promoter level by ChIP analysis and this was confirmed by knockdown in cell transfection experiments. To mimic the human disease, we induced colitis and colitis-associated cancer by chemical treatment (DSS) combined or not with a carcinogen (AOM) in transgenic mice overexpressing LMα1 or LMα5 specifically in the intestine. We demonstrated that high LMα1 or LMα5 expression decreased susceptibility towards experimentally DSS-induced colon inflammation as assessed by histological scoring and decrease of pro-inflammatory cytokines. Yet in a pro-oncogenic context, we showed that LM would favor tumorigenesis as revealed by enhanced tumor lesion formation in both LM transgenic mice. Altogether, our results showed that nuclear p53 and associated overexpression of LMα1 and LMα5 protect tissue from inflammation. But in a mutation setting, the same LM molecules favor progression of IBD into colitis-associated cancer. Our transgenic mice represent attractive new models to acquire knowledge about the paradoxical effect of LM that mediate either tissue reparation or cancer according to the microenvironment. In the early phases of IBD, reinforcing basement membrane stability/organization could be a promising therapeutic approach.     

Identification of a Subpopulation of Marrow MSC-Derived Medullary Adipocytes That Express Osteoclast-Regulating Molecules: Marrow Adipocytes Express Osteoclast Mediators

Increased marrow medullary adipogenesis and an associated decrease in bone mineral density, usually observed in elderly individuals, is a common characteristic in senile osteoporosis. In this study we investigated whether cells of the medullary adipocyte lineage have the potential to directly support the formation of osteoclasts, whose activity in bone leads to bone degradation. An in vitro mesenchymal stem cell (MSC)-derived medullary adipocyte lineage culture model was used to study the expression of the important osteoclast mediators RANKL, M-CSF, SDF-1, and OPG. We further assessed whether adipocytes at a specific developmental stage were capable of supporting osteoclast-like cell formation in culture. In vitro MSC-derived medullary adipocytes showed an mRNA and protein expression profile of M-CSF, RANKL, and OPG that was dependent on its developmental/metabolic stage. Furthermore, RANKL expression was observed in MSC-derived adipocytes that were at a distinct lineage stage and these cells were also capable of supporting osteoclast-like cell formation in co-cultures with peripheral blood mononuclear cells. These results suggest a connection between medullary adipocytes and osteoclast formation in vivo and may have major significance in regards to the mechanisms of decreased bone density in senile osteoporosis.     

Getting to Compliance in Forced Exercise in Rodents: A Critical Standard to Evaluate Exercise Impact in Aging-related Disorders and Disease

There is a major increase in the awareness of the positive impact of exercise on improving several disease states with neurobiological basis; these include improving cognitive function and physical performance. As a result, there is an increase in the number of animal studies employing exercise. It is argued that one intrinsic value of forced exercise is that the investigator has control over the factors that can influence the impact of exercise on behavioral outcomes, notably exercise frequency, duration, and intensity of the exercise regimen. However, compliance in forced exercise regimens may be an issue, particularly if potential confounds of employing foot-shock are to be avoided. It is also important to consider that since most cognitive and locomotor impairments strike in the aged individual, determining impact of exercise on these impairments should consider using aged rodents with a highest possible level of compliance to ensure minimal need for test subjects. Here, the pertinent steps and considerations necessary to achieve nearly 100% compliance to treadmill exercise in an aged rodent model will be presented and discussed. Notwithstanding the particular exercise regimen being employed by the investigator, our protocol should be of use to investigators that are particularly interested in the potential impact of forced exercise on aging-related impairments, including aging-related Parkinsonism and Parkinson’s disease.     

Group Mobbing Behaviour and Nest Defence in a Cooperatively Breeding Australian Bird

Cooperatively breeding noisy miners (Manorina melanocephala) are well known in Australia for their persistent and very vocal group mobbing of heterospecifics. Here I investigated the nature of this extraordinary behaviour, in particular its role in nest defence, in a colour banded population of noisy miners in south‐east Queensland, Australia. I focused on two questions. First, did the intensity of mobbing vary according to factors such as the threat to the nest, or the ‘value’ of a clutch? Secondly, what role did group mobbing play in the success of a nest? To answer these questions, I experimentally manipulated the nest defence behaviour by placing one of three stuffed models near active noisy miner nests. The response of noisy miners to intruders was not indiscriminate. However, I found that the number of birds that mobbed a model did not simply reflect the potential threat posed. The response of noisy miners to raptors and other potential nest predators may have reflected their rarity as well as the threat posed. The number of mobbers did not vary with the age or size of a brood. In this study, the fate of nests was independent of the number of mobbers or visitors at nests. Finally, up to 80% of mobbers were never seen to make any other type of contribution to a nest, and many could not be related to the brood that they were ‘defending’. Hence, for some noisy miner ‘helpers’ the benefits that they accrued were probably not wholly dependent on the survival of the broods. I suggest that, in this gregarious species, mobbing behaviour at the nest may be a display of social status or individual quality. This hypothesis warrants further investigation.     

Are tropical fungal endophytes hyperdiverse?

Fungal endophytes are ubiquitous fungi that inhabit healthy plant tissues without causing disease. Endophytes have been found in every plant species examined to date and may be important, but often overlooked, components of fungal biodiversity. In two sites in a lowland, moist tropical forest of central Panama, we quantified endophyte colonization patterns, richness, host preference, and spatial variation in healthy leaves of two co-occurring, understory tree species [ Heisteria concinna (Olacaceae) and Ouratea lucens (Ochnaceae)]. From 83 leaves, all of which were colonized by endophytes, we isolated 418 endophyte morphospecies (estimated 347 genetically distinct taxa), most of which were represented by only a single isolate (59%). Among morphospecies encountered in more than one leaf (nonsingletons), we found evidence of host preference and spatial heterogeneity using both morphospecies frequencies and presence/absence records. Based on these data, we postulate that tropical endophytes themselves may be hyperdiverse and suggest that extrapolative estimates that exclude them will markedly underestimate fungal species diversity.