Inhibition of Rotavirus Infectivity by a Neoglycolipid Receptor Mimetic

Group A rotaviruses are a major cause of diarrhea in the young of many mammalian species. In rotavirus infected piglets mortality can be as high as 60%. Previous research in this laboratory has identified a porcine intestinal GM3 ganglioside receptor that is required for sialic acid-dependent rotavirus recognition of host cells. In addition, we previously demonstrated exogenously added GM3 can competitively inhibit porcine rotavirus binding and infectivity of host cells in vitro. Sialyllactose, the carbohydrate moiety of GM3, is approximately 3 orders of magnitude less effective than GM3 at inhibiting rotavirus binding to cells. Furthermore, production of therapeutic quantities of GM3 ganglioside for use as an oral carbomimetic in swine is cost prohibitive. In an effort to circumvent these problems, a sialyllactose-containing neoglycolipid was synthesized and evaluated for its ability to inhibit rotavirus binding and infectivity of host cells. Sialyllactose was coupled to dipalmitoylphosphatidylethanolamine (PE) by reductive amination and the product (SLPE) purified by HPLC. Characterization of the product showed a single primulin (lipid) and resorcinol (sialic acid) positive band by thin layer chromatography and quantification of phosphate and sialic acid yielded a 1:1 molar ratio. Mass spectroscopy confirmed a molecular weight coinciding with SLPE. Concentration-dependent binding of rotavirus to SLPE was demonstrated using a thin-layer overlay assay. Using concentrations comparable to GM3, SLPE was also shown to inhibit rotavirus binding to host cells by 80%. Furthermore, SLPE was shown to decrease rotavirus infection of host cells by over 90%. Finally, preliminary results of in vivo animal challenge studies using newborn piglets in their natural environment, demonstrated SLPE afforded complete protection from rotavirus disease. The efficacy of SLPE in inhibiting rotavirus binding and infection in vitro and in vivo, coupled with its relatively low-cost, large-scale production capabilities make SLPE a promising candidate for further exploration as a possible prophylactic or therapeutic nutriceutical for combating rotavirus disease in animals. Most importantly, the results presented here provide proof of concept that the nutriceutical approach of providing natural or synthetic dietary receptor mimetics for protection against gastrointestinal virus infectious disease in all species is plausible

Inhibition of calcium-dependent protein kinase 1 (CDPK1) in vitro by pyrazolopyrimidine derivatives does not correlate with sensitivity of Cryptosporidium parvum growth in cell culture

Cryptosporidiosis is a serious diarrheal disease in immunocompromised patients and malnourished children, and treatment is complicated by a lack of adequate drugs. Recent studies suggest that the natural occurrence of a small gatekeeper residue in serine threonine calcium-dependent protein kinase 1 (CDPK1) of Cryptosporidium parvum might be exploited to target this enzyme and block parasite growth. Here were explored the potency with which a series of pyrazolopyrimidine analogs, which are selective for small gatekeeper kinases, inhibit C. parvum CDPK1 and block C. parvum growth in tissue culture in vitro. Although these compounds potently inhibited kinase activity in vitro, most had no effect on parasite growth. Moreover, among those that were active against parasite growth, there was a very poor correlation with their 50% inhibitory concentrations against the enzyme. Active compounds also had no effect on cell invasion, unlike the situation in Toxoplasma gondii, where these compounds block CDPK1, prevent microneme secretion, and disrupt cell invasion. These findings suggest that CPDK1 is not essential for C. parvum host cell invasion or growth and therefore that it is not the optimal target for therapeutic intervention. Nonetheless, several inhibitors with low micromolar 50% effective concentrations were identified, and these may affect other essential targets in C. parvum that are worthy of further exploration

A Legacy of Low-Impact Logging does not Elevate Prevalence of Potentially Pathogenic Protozoa in Free-Ranging Gorillas and Chimpanzees in the Republic of Congo: Logging and Parasitism in African Apes

Many studies have examined the long-term effects of selective logging on the abundance and diversity of free-ranging primates. Logging is known to reduce the abundance of some primate species through associated hunting and the loss of food trees for frugivores; however, the potential role of pathogens in such primate population declines is largely unexplored. Selective logging results in a suite of alterations in host ecology and forest structure that may alter pathogen dynamics in resident wildlife populations. In addition, environmental pollution with human fecal material may present a risk for wildlife infections with zoonotic protozoa, such as Cryptosporidium and Giardia. To better understand this interplay, we compared patterns of infection with these potentially pathogenic protozoa in sympatric western lowland gorillas (Gorilla gorilla gorilla) and chimpanzees (Pan troglodytes troglodytes) in the undisturbed Goualougo Triangle of Nouabalé-Ndoki National Park and the adjacent previously logged Kabo Concession in northern Republic of Congo. No Cryptosporidium infections were detected in any of the apes examined and prevalence of infection with Giardia was low (3.73% overall) and did not differ between logged and undisturbed forest for chimpanzees or gorillas. These results provide a baseline for prevalence of these protozoa in forest-dwelling African apes and suggest that low-intensity logging may not result in long-term elevated prevalence of potentially pathogenic protozoa

Overland Transport of Rotavirus and the Effect of Soil Type and Vegetation

Soil and vegetation are two critical factors for controlling the overland transport kinetics of pathogens in a natural environment. With livestock operations moving more towards concentrated animal operations, the need to dispose of a very large amount of manure in a localized area is becoming increasingly important. Animal manure contains a substantial amount of microbial pathogens, including rotavirus, which may pose a threat of contamination of water resources. This study examined the kinetics of rotavirus in overland transport, with an overall objective of optimizing the design of best management practices, especially vegetative filter strips. The overland transport of rotavirus was studied using three soil types (Catlin silt-loam, Darwin silty-clay, Alvin fine sandy-loam), spanning the entire spectrum of typical Illinois soil textures. A 20-min rainfall event was produced using a small-scale (1.07 m × 0.66 m) laboratory rainfall simulator over a soil box measuring 0.610 m × 0.305 m. Each soil type was tested for rotavirus transport kinetics with bare surface conditions, as well as with Smooth Brome and Fescue vegetative covers. Surface runoff, near-surface runoff, soil cores, and vegetation were each analyzed for infective rotavirus particles using cell-culture infectivity assays. Results show that vegetation reduces the recovery of infective rotavirus particles in surface runoff by an average of 73%, in addition to delaying the time to peak recovery. The vegetation, in general, appeared to decrease the recovery of infective rotavirus particles in surface runoff by impeding surface flow and increasing the potential for infiltration into the soil profile

1 Investigation of Rotavirus Survival in Different Soil Fractions and Temperature Conditions

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Rotavirus is a leading cause of gastrointestinal illness worldwide. Rotavirus transmission occurs fecal-orally, and becomes a critical water quality issue when soil and water resources are contaminated with feces. Transport of pathogens to surface water sources depends on their survival in the soil, especially considering the fact that large amounts of fecal material are often applied to agricultural lands as fertilizer. In this study, rotavirus survival was investigated in three different soil fractions and at three different temperatures (4˚C, 25˚C and 37˚C). A rotavirus suspension was mixed with whole soil, sand, and clay and allowed to incubate for up to 18 days. Samples were collected daily to investigate virus survival over time, which was quantified using a tissue-culture infectivity assay. Results indicated, in the absence of any soil particles, rotavirus survival was highest at 4˚C, with survival decreasing as temperature increased. These data also indicated whole soil had some protective effect, allowing rotavirus to survive better in soil for the entire range of temperatures and for more than a week at 37˚C. The results also showed that sand fractions were the most effective media for reducing rotavirus recovery at all temperature conditions tested. Although the mechanism responsible for the low recovery from sand is unknown, there is little or no infective rotavirus extracted from sand fractions. This findin

Attenuation of cell mechanosensitivity in colon cancer cells during in vitro metastasis.

Human colon carcinoma (HCT-8) cells show a stable transition from low to high metastatic state when cultured on appropriately soft substrates (21 kPa). Initially epithelial (E) in nature, the HCT-8 cells become rounded (R) after seven days of culture on soft substrate. R cells show a number of metastatic hallmarks [1]. Here, we use gradient stiffness substrates, a bio-MEMS force sensor, and Coulter counter assays to study mechanosensitivity and adhesion of E and R cells. We find that HCT-8 cells lose mechanosensitivity as they undergo E-to-R transition. HCT-8 R cells' stiffness, spread area, proliferation and migration become insensitive to substrate stiffness in contrast to their epithelial counterpart. They are softer, proliferative and migratory on all substrates. R cells show negligible cell-cell homotypic adhesion, as well as non-specific cell-substrate adhesion. Consequently they show the same spread area on all substrates in contrast to E cells. Taken together, these results indicate that R cells acquire autonomy and anchorage independence, and are thus potentially more invasive than E cells. To the best of our knowledge, this is the first report of quantitative data relating changes in cancer cell adhesion and stiffness during the expression of an in vitro metastasis-like phenotype

Surface non-specific adhesion of E cell islands measured using a micro-fabricated bio-MEMS force sensor.

<p>(a) The non-functionalized micro-fabricated Si force sensor with a flat probe and with known force-deflection relation is manipulated by a high-resolution x-y-z Piezo-stage to contact cell islands' lateral convex surface (on x-y plane). (b) Confocal microscopy of cell islands show the height of islands is on the order of 30∼50 μm. The vertical height of bio-MEMS probe is 5∼10 μm. (c) After a 2-minute contact, force sensor is horizontally pulled away at a constant speed of 2.1±0.4 μm/s. While the cell adhesion between the probe and cell surface hinders retraction of the sensor, the sensor beams deform by δ, giving the force F. Note that the probe is not functionalized. The 2-minute contact between the probe and cells prevents the activation of cell integrins and the formation of any cell focal adhesion, which takes >30 minutes to form.</p

Stiffness and morphology of HCT-8 E cells correlate with substrate rigidity.

<p>Using Atomic Force Microscopy, the stiffness of HCT-8 E cells cultured on the gradient substrate is determined. The HCT-8 E cells increase their cell stiffness as the substrates become more rigid. To facilitate the comparison between different cells on same substrate stiffness, five equal-spaced regions across the entire stiffness range are designated: region 1 covers 1–4 kPa, region 2 covers 5–8 kPa, region 3 covers 9–12 kPa, region 4 covers 13–16 kPa, and region 5 covers 17–20 kPa. (a) From region 1 to region 5, the E cell stiffness progressively increases with values 1.42±0.85 kPa to 1.90±0.77 kPa, 2.06±1.39 kPa, 2.15±1.28 kPa, and 3.82±1.98 kPa, respectively. In contrast, on gel substrates with same stiffness gradient, the post-metastatic R cells show almost invariant cell stiffness. (b) Phase-contrast pictures of HCT-8 E cells on gradient PA substrates.</p