A Stem-Cell-Derived Platform Enables Complete Cryptosporidium Development In Vitro and Genetic Tractability.

Despite being a frequent cause of severe diarrheal disease in infants and an opportunistic infection in immunocompromised patients, Cryptosporidium research has lagged due to a lack of facile experimental methods. Here, we describe a platform for complete life cycle development and long-term growth of C. parvum in vitro using "air-liquid interface" (ALI) cultures derived from intestinal epithelial stem cells. Transcriptomic profiling revealed that differentiating epithelial cells grown under ALI conditions undergo profound changes in metabolism and development that enable completion of the parasite life cycle in vitro. ALI cultures support parasite expansion > 100-fold and generate viable oocysts that are transmissible in vitro and to mice, causing infection and animal death. Transgenic parasite lines created using CRISPR/Cas9 were used to complete a genetic cross in vitro, demonstrating Mendelian segregation of chromosomes during meiosis. ALI culture provides an accessible model that will enable innovative studies into Cryptosporidium biology and host interactions.

Monoclonal Antibodies to Intracellular Stages of Cryptosporidium parvum Define Life Cycle Progression In Vitro.

Among the obstacles hindering Cryptosporidium research is the lack of an in vitro culture system that supports complete life development and propagation. This major barrier has led to a shortage of widely available anti-Cryptosporidium antibodies and a lack of markers for staging developmental progression. Previously developed antibodies against Cryptosporidium were raised against extracellular stages or recombinant proteins, leading to antibodies with limited reactivity across the parasite life cycle. Here we sought to create antibodies that recognize novel epitopes that could be used to define intracellular development. We identified a mouse epithelial cell line that supported C. parvum growth, enabling immunization of mice with infected cells to create a bank of monoclonal antibodies (MAbs) against intracellular parasite stages while avoiding the development of host-specific antibodies. From this bank, we identified 12 antibodies with a range of reactivities across the parasite life cycle. Importantly, we identified specific MAbs that can distinguish different life cycle stages, such as trophozoites, merozoites, type I versus II meronts, and macrogamonts. These MAbs provide valuable tools for the Cryptosporidium research community and will facilitate future investigation into parasite biology.IMPORTANCECryptosporidium is a protozoan parasite that causes gastrointestinal disease in humans and animals. Currently, there is a limited array of antibodies available against the parasite, which hinders imaging studies and makes it difficult to visualize the parasite life cycle in different culture systems. In order to alleviate this reagent gap, we created a library of novel antibodies against the intracellular life cycle stages of Cryptosporidium We identified antibodies that recognize specific life cycle stages in distinctive ways, enabling unambiguous description of the parasite life cycle. These MAbs will aid future investigation into Cryptosporidium biology and help illuminate growth differences between various culture platforms.

Effect of oregano essential oil and carvacrol on Cryptosporidium parvum infectivity in HCT-8 cells.

Cryptosporidium parvum is the second leading cause of persistent diarrhea among children in low-resource settings. This study examined the effect of oregano essential oil (OEO) and carvacrol (CV) on inhibition of C. parvum infectivity in vitro. HCT-8 cells were seeded (1×106) in 96-well microtiter plates until confluency. Cell viability and infectivity were assessed by seeding HCT-8 cell monolayers with C. parvum oocysts (1×104) in two modalities: 1) 4h co-culture with bioactive (0-250µg/mL) followed by washing and incubation (48h, 37°C, 5% CO2) in bioactive-free media; and 2) 4h co-culture of C. parvum oocysts followed by washing and treatment with bioactive (0-250µg/mL) during 48-h incubation. Cell viability was tested using Live/Dead™ assay whereas infectivity was measured using C. parvum-specific antibody staining via immunofluorescence detection. Loss of cell viability was observed starting at 125µg/mL and 60µg/mL for OEO and CV, respectively. Neither OEO nor CV modulated the invasion of C. parvum sporozoites in HCT-8 cells. Treatment with bioactive after invasion reduced relative C. parvum infectivity in a dose-dependent manner to 55.6±10.4% and 45.8±4.1% at 60 and 30µg/mL of OEO and CV, respectively. OEO and CV are potential bioactives to counteract C. parvum infection in children.

Dietary Human Milk Oligosaccharides but Not Prebiotic Oligosaccharides Increase Circulating Natural Killer Cell and Mesenteric Lymph Node Memory T Cell Populations in Noninfected and Rotavirus-Infected Neonatal Piglets.

Background: Human milk oligosaccharides (HMOs) have antimicrobial and immunomodulatory actions. It has previously been reported that these oligosaccharides contribute to the reduced duration of rotavirus-induced diarrhea in pigs.Objective: We measured the effects of HMOs and prebiotic oligosaccharides on immune cell populations from noninfected and rotavirus-infected pigs. We hypothesized that dietary HMOs would modulate systemic and gastrointestinal immunity.Methods: Colostrum-deprived newborn pigs were fed formula, formula with 4 g HMOs/L (2'-fucosyllactose, lacto-N-neotetraose, 6'-sialyllactose, 3'-sialyllactose, and free sialic acid), or formula with 3.6 g short-chain galactooligosaccharides/L and 0.4 g long-chain fructooligosaccharides/L. On day 10, half of the pigs were infected with the porcine rotavirus strain OSU. Peripheral blood mononuclear cell (PBMC), mesenteric lymph node (MLN), and ileal Peyer's patch immune cell populations were assessed with the use of flow cytometry 5 d postinfection. Interferon-γ (IFN-γ)-producing cells were assessed with the use of Enzyme-Linked ImmunoSpot assay.Results: Infection changed immune cell populations with more systemic natural killer (NK) cells, memory effector T cells, and major histocompatibility complex II+ cells in infected than noninfected pigs (P < 0.06). Regardless of infection status, HMO-fed pigs had nearly twice as many PBMC NK cells, 36% more MLN effector memory T cells, and 5 times more PBMC basophils than formula-fed pigs (P < 0.04). These populations were intermediate in pigs fed prebiotics. PBMCs from HMO-fed noninfected pigs had twice as many IFN-γ-producing cells as did those from formula-fed noninfected pigs (P = 0.017). The PBMCs and MLNs of formula-fed noninfected pigs had 3 times more plasmacytoid dendritic cells (pDCs) than those of HMO-fed noninfected and formula-fed infected pigs (P < 0.04). In the MLNs, the formula-fed noninfected pigs had more macrophages, pDCs, and mature DCs (P < 0.04) but fewer immature DCs than HMO-fed noninfected pigs (P = 0.022).Conclusions: Dietary HMOs were more effective than prebiotics in altering systemic and gastrointestinal immune cells in pigs. These altered immune cell populations may mediate the effects of dietary HMOs on rotavirus infection susceptibility.

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 mechanically-induced colon cancer cell population shows increased metastatic potential.

BACKGROUND: Metastasis accounts for the majority of deaths from cancer. Although tumor microenvironment has been shown to have a significant impact on the initiation and/or promotion of metastasis, the mechanism remains elusive. We previously reported that HCT-8 colon cancer cells underwent a phenotypic transition from an adhesive epithelial type (E-cell) to a rounded dissociated type (R-cell) via soft substrate culture, which resembled the initiation of metastasis. The objective of current study was to investigate the molecular and metabolic mechanisms of the E-R transition. METHODS: Global gene expressions of HCT-8 E and R cells were measured by RNA Sequencing (RNA-seq); and the results were further confirmed by real-time PCR. Reactive oxygen species (ROS), anoikis resistance, enzyme activity of aldehyde dehydrogenase 3 family, member A1 (ALDH3A1), and in vitro invasion assay were tested on both E and R cells. The deformability of HCT-8 E and R cells was measured by atomic force microscopy (AFM). To study the in vivo invasiveness of two cell types, athymic nude mice were intra-splenically injected with HCT-8 E or R cells and sacrificed after 9 weeks. Incidences of tumor development and metastasis were histologically evaluated and analyzed with Fisher's exact test. RESULTS: Besides HCT-8, E-R transition on soft substrates was also seen in three other cancer cell lines (HCT116, SW480 colon and DU145 prostate cancer). The expression of some genes, such as ALDH3A1, TNS4, CLDN2, and AKR1B10, which are known to play important roles in cancer cell migration, invasion, proliferation and apoptosis, were increased in HCT-8 R cells. R cells also showed higher ALDH3A1 enzyme activity, higher ROS, higher anoikis resistance, and higher softness than E cells. More importantly, in vitro assay and in vivo animal models revealed that HCT-8 R cells were more invasive than E cells. CONCLUSIONS: Our comprehensive comparison of HCT-8 E and R cells revealed differences of molecular, phenotypical, and mechanical signatures between the two cell types. To our knowledge, this is the first study that explores the molecular mechanism of E-R transition, which may greatly increase our understanding of the mechanisms of cancer mechanical microenvironment and initiation of cancer metastasis.

Human milk oligosaccharides shorten rotavirus-induced diarrhea and modulate piglet mucosal immunity and colonic microbiota.

The impact of human milk oligosaccharides (HMO) on mucosal immunity, gut microbiota and response to rotavirus (RV) infection was investigated in the piglet model. Newborn piglets were fed with formula alone (FF) or formula supplemented with 4 g l(-1) HMO (HMO) or a prebiotic mixture of 9:1 short-chain galactooligosaccharides (3.6 g l(-1)) and long-chain fructooligosaccharides (0.4 g l(-1)) (PRE) (n=19-21 per group) for 15 days. Piglets (n=7-8) in each dietary group were orally infected with porcine rotavirus (RV) OSU strain on d10, and stool consistency was assessed daily. Blood, small intestine and colonic contents were collected at day 15. Serum RV-specific antibody concentrations, intestinal histomorphology, RV non-structural protein-4 (NSP4) and cytokine mRNA expression were assessed. Colonic content pH, dry matter (DM) and short-chain fatty acid concentrations were measured. Ascending colonic microbiota was analyzed by 16S rRNA gene v1-3 region pyrosequencing. HMO- and PRE-fed groups had shorter duration of diarrhea than FF piglets. Infection changed intestinal histomorphology, increased serum RV-specific antibody response and intestinal RV NSP4 expression, and modulated ileal cytokine expression. HMO enhanced T helper type 1 (interferon-gamma) and anti-inflammatory (interleukin-10) cytokines in the ileum, while prebiotics promoted RV-specific immunoglobulin M response to the infection. RV infection and HMO supplementation altered intraluminal environment and gut microbiota. HMO increased pH and lowered DM of colonic contents and enhanced the abundance of unclassified Lachnospiraceae, which contains numerous butyrate-producing bacteria. In conclusion, HMO and prebiotics did not prevent the onset of RV infection but reduced the duration of RV-induced diarrhea in piglets, in part, by modulating colonic microbiota and immune response to RV infection.

Role of collector alternating charged patches on transport of Cryptosporidium parvum oocysts in a patchwise charged heterogeneous micromodel.

The role of collector surface charge heterogeneity on transport of Cryptosporidium parvum oocyst and carboxylate microsphere in 2-dimensional micromodels was studied. The cylindrical silica collectors within the micromodels were coated with 0, 10, 20, 50, and 100% Fe(2)O(3) patches. The experimental values of average removal efficiencies (η) of the Fe(2)O(3) patches and on the entire collectors were determined. In the presence of significant (>3500 kT) Derjaguin-Landau-Verwey-Overbeek (DLVO) energy barrier between the microspheres and the silica collectors at pH 5.8 and 8.1, η determined for Fe(2)O(3) patches on the heterogeneous collectors were significantly less (p < 0.05, t test) than those obtained for collectors coated entirely with Fe(2)O(3). However, η calculated for Fe(2)O(3) patches for microspheres at pH 4.4 and for oocysts at pH 5.8 and 8.1, where the DLVO energy barrier was relatively small (ca. 200-360 kT), were significantly greater (p < 0.05, t test) than those for the collectors coated entirely with Fe(2)O(3). The dependence of η for Fe(2)O(3) patches on the DLVO energy barrier indicated the importance of periodic favorable and unfavorable electrostatic interactions between colloids and collectors with alternating Fe(2)O(3) and silica patches. Differences between experimentally determined overall η for charged heterogeneous collectors and those predicted by a patchwise geochemical heterogeneous model were observed. These differences can be explained by the model's lack of consideration for the spatial distribution of charge heterogeneity on the collector surface.

Human milk oligosaccharides inhibit rotavirus infectivity in vitro and in acutely infected piglets.

Human milk (HM) is rich in oligosaccharides (HMO) that exert prebiotic and anti-infective activities. HM feeding reduces the incidence of rotavirus (RV) infection in infants. Herein, the anti-RV activity of oligosaccharides was tested in an established in vitro system for assessing cellular binding and viral infectivity/replication, and also tested in a newly developed, acute RV infection, in situ piglet model. For the in vitro work, crude HMO isolated from pooled HM, neutral HMO (lacto-N-neotetraose, LNnT; 2'-fucosyllactose) and acidic HMO (aHMO, '-sialyllactose, 3'-SL; -sialyllactose, -SL) were tested against the porcine OSU strain and human RV Wa strain. The RV Wa strain was not inhibited by any oligosaccharides. However, the RV OSU strain infectivity was dose-dependently inhibited by sialic acid (SA)-containing HMO. 3'-SL and 6'-SL concordantly inhibited (125)I-radiolabelled RV cellular binding and infectivity/replication. For the in situ study, a midline laparotomy was performed on 21-d-old formula-fed piglets and six 10 cm loops of ileum were isolated in situ. Briefly, 2 mg/ml of LNnT, aHMO mixture (40% 6'-SL/10 % 3'-SL/50 % SA) or media with or without the RV OSU strain (1 x 10(7) focus-forming units)were injected into the loops and maintained for 6 h. The loops treated with HMO treatments þ RV had lower RV replication, as assessed by non-structural protein-4 (NSP4) mRNA expression, than RV-treated loops alone. In conclusion, SA-containing HMO inhibited RV infectivity in vitro; however, both neutral HMO and SA with aHMO decreased NSP4 replication during acute RV infection in situ.

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.

Microbial composition and in vitro fermentation patterns of human milk oligosaccharides and prebiotics differ between formula-fed and sow-reared piglets.

The microbial composition and in vitro fermentation characteristics of human milk oligosaccharides (HMO), lacto-N-neotetraose (LNnT), a 2:1 mixture of polydextrose (PDX) and galactooligosaccharides (GOS), and short-chain fructooligosaccharides (scFOS) by pooled ascending colonic microbiota from 9- and 17-d-old formula-fed (FF) and sow-reared (SR) piglets were assessed. pH change and gas, SCFA, and lactate production were determined after 0, 2, 4, 8, and 12 h of incubation. In most donor groups, the pH change was greater for scFOS fermentation and lower for PDX/GOS than for other substrates. LNnT fermentation produced larger amounts of gas, total SCFA, acetate, and butyrate than did the other substrates, whereas HMO and scFOS produced higher amounts of propionate and lactate, respectively. In general, pH change, total SCFA, acetate, and propionate production were greater in pooled inoculum from FF and 9-d-old piglets, whereas SR-derived inoculum produced higher amounts of butyrate and lactate after 4 h fermentation. Gut microbiota were assessed by 16S ribosomal RNA V3 gene denaturing gradient gel electrophoresis analysis and real-time qPCR. Microbial structures differed among the 4 groups before fermentation, with higher counts of Bifidobacterium in SR piglets and higher counts of Clostridium cluster IV, XIVa, and Bacteroides vulgatus in FF piglets. Lactobacillus counts were higher in 9-d-old piglets than in 17-d-old piglets, regardless of diet. Bifidobacterium, Bacteroides, and clostridial species increased after 8 and 12 h fermentation on most substrates. In summary, piglet diet and age affect gut microbiota, leading to different fermentation patterns. HMO have potential prebiotic effects due to their effects on SCFA production and microbial modulation.

Transport of Cryptosporidium parvum oocysts in a silicon micromodel.

Effective removal of Cryptosporidium parvum oocysts by granular filtration requires the knowledge of oocyst transport and deposition mechanisms, which can be obtained based on real time microscopic observation of oocyst transport in porous media. Attachment of oocysts to silica surface in a radial stagnation point flow cell and in a micromodel, which has 2-dimensional (2-D) microscopic pore structures consisting of an array of cylindrical collectors, was studied and compared. Real time transport of oocysts in the micromodel was recorded to determine the attached oocyst distributions in transversal and longitudinal directions. In the micromodel, oocysts attached to the forward portion of clean collectors, where the flow velocity was lowest. After initial attachment, oocysts attached onto already attached oocysts. As a result, the collectors ripened and the region available for flow was reduced. Results of attachment and detachment experiments suggest that surface charge heterogeneity allowed for oocyst attachment. In addition to experiments, Lattice-Boltzmann simulations helped understanding the slightly nonuniform flow field and explained differences in the removal efficiency in the transversal direction. However, the hydrodynamic modeling could not explain differences in attachment in the longitudinal direction.

Membrane interaction of Pasteurella multocida toxin involves sphingomyelin.

Pasteurella multocida toxin (PMT) is an AB toxin that causes pleiotropic effects in targeted host cells. The N-terminus of PMT (PMT-N) is considered to harbor the membrane receptor binding and translocation domains responsible for mediating cellular entry and delivery of the C-terminal catalytic domain into the host cytosol. Previous studies have implicated gangliosides as the host receptors for PMT binding. To gain further insight into the binding interactions involved in PMT binding to cell membranes, we explored the role of various membrane components in PMT binding, utilizing four different approaches: (a) TLC-overlay binding experiments with (125) I-labeled PMT, PMT-N or the C-terminus of PMT; (b) pull-down experiments using reconstituted membrane liposomes with full-length PMT; (c) surface plasmon resonance analysis of PMT-N binding to reconstituted membrane liposomes; (d) and surface plasmon resonance analysis of PMT-N binding to HEK-293T cell membranes without or with sphingomyelinase, phospholipase D or trypsin treatment. The results obtained revealed that, in our experimental system, full-length PMT and PMT-N did not bind to gangliosides, including monoasialogangliosides GM(1) , GM(2) or GM(3) , but instead bound to membrane phospholipids, primarily the abundant sphingophospholipid sphingomyelin or phosphatidylcholine with other lipid components. Collectively, these studies demonstrate the importance of sphingomyelin for PMT binding to membranes and suggest the involvement of a protein co-receptor.

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 GM(3) ganglioside receptor that is required for sialic acid-dependent rotavirus recognition of host cells. In addition, we previously demonstrated exogenously added GM(3) can competitively inhibit porcine rotavirus binding and infectivity of host cells in vitro. Sialyllactose, the carbohydrate moiety of GM(3), is approximately 3 orders of magnitude less effective than GM(3) at inhibiting rotavirus binding to cells. Furthermore, production of therapeutic quantities of GM(3) 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 GM(3), 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.

Mechanical force affects expression of an in vitro metastasis-like phenotype in HCT-8 cells.

Cancer deaths are primarily caused by metastases, not by the parent tumor. During metastasis, malignant cells detach from the parent tumor, and spread through the circulatory system to invade new tissues and organs. The physical-chemical mechanisms and parameters within the cellular microenvironment that initiate the onset of metastasis, however, are not understood. Here we show that human colon carcinoma (HCT-8) cells can exhibit a dissociative, metastasis-like phenotype (MLP) in vitro when cultured on substrates with appropriate mechanical stiffness. This rather remarkable phenotype is observed when HCT-8 cells are cultured on gels with intermediate-stiffness (physiologically relevant 21-47 kPa), but not on very soft (1 kPa) and very stiff (3.6 GPa) substrates. The cell-cell adhesion molecule E-Cadherin, a metastasis hallmark, decreases 4.73 ± 1.43 times on cell membranes in concert with disassociation. Both specific and nonspecific cell adhesion decrease once the cells have disassociated. After reculturing the disassociated cells on fresh substrates, they retain the disassociated phenotype regardless of substrate stiffness. Inducing E-Cadherin overexpression in MLP cells only partially reverses the MLP phenotype in a minority population of the dissociated cells. This important experiment reveals that E-Cadherin does not play a significant role in the upstream regulation of the mechanosensing cascade. Our results indicate, during culture on the appropriate mechanical microenvironment, HCT-8 cells undergo a stable cell-state transition with increased in vitro metastasis-like characteristics as compared to parent cells grown on standard, very stiff tissue culture dishes. Nuclear staining reveals that a large nuclear deformation (major/minor axis ratio, 2:5) occurs in HCT-8 cells when cells are cultured on polystyrene substrates, but it is markedly reduced (ratio, 1:3) in cells grown on 21 kPa substrates, suggesting the cells are experiencing different intracellular forces when grown on stiff as compared to soft substrates. Furthermore, MLP can be inhibited by blebbistatin, which inactivates myosin II activity and relaxes intracellular forces. This novel finding suggests that the onset of metastasis may, in part, be linked to the intracellular forces and the mechanical microenvironment of the tumor.

Composition and conformation of Cryptosporidium parvum oocyst wall surface macromolecules and their effect on adhesion kinetics of oocysts on quartz surface.

We characterized the composition and conformation of Cryptosporidium parvum ( C. parvum ) oocyst wall surface macromolecules and studied their effect on interactions between C. parvum oocyst and quartz surface. Proteinase K and mixed glycosidases were used to modify C. parvum oocyst surface macromolecules. The peptides released by proteinase K and carbohydrates hydrolyzed by mixed glycosidases were respectively analyzed with liquid chromatography/nanoelectrospray ionization tandem mass spectrometry (LC-MS/MS) and phenol-sulfuric acid assay to determine the composition of C. parvum oocyst wall surface macromolecules. Surface potential and polarity of the untreated and proteinase treated C. parvum oocysts revealed information about the conformation of oocyst wall surface macromolecules. The results illustrated that C. parvum oocyst wall is covered by a fluffy layer of glycoproteins. Adhesion kinetics of untreated and proteinase K treated C. parvum oocysts on quartz surface were studied in a radial stagnation point flow cell over a wide range of ionic strength to investigate the effect of C. parvum oocyst wall surface macromolecules on oocysts-quartz interactions. The adhesion rate coefficient of proteinase K treated C. parvum oocysts significantly decreased compared to that of untreated oocysts. This observation indicated that the fluffy layer on C. parvum oocysts wall leads to weaker van der Waals interaction and stronger steric repulsion.

Role of divalent cations on deposition of Cryptosporidium parvum oocysts on natural organic matter surfaces.

A Radial Stagnation Point Flow (RSPF) system coupled with a microscope was used to study deposition of Cryptosporidium parvum oocysts on quartz and Suwannee River Natural Organic Matter (SRNOM)-coated surfaces in solutions with different Ca(2+) or Mg(2+) concentrations. Both untreated and proteinase K-treated oocysts were used. Deposition of oocysts on a SRNOM surface in Ca(2+) solution was higher than in Mg(2+) solution, even though the energy barriers calculated from Derjaguin-Landau-Verwey-Overbeek (DLVO) theory for Ca(2+) solution were higher than for Mg(2+) solution. On the other hand, the attachment of oocysts on a quartz surface was the same in both Ca(2+) and Mg(2+) solution and in qualitative agreement with the DLVO energy profiles. Inductive coupled plasma (ICP) was employed to measure the free divalent cation concentration in solutions containing oocysts. ICP data showed more Ca(2+) bound to oocyst surface than Mg(2+). Moreover, proteinase K treatment of oocysts led to a significant decrease in deposition rate due to less binding of Ca(2+) to the surface of the treated oocysts as shown by the ICP data. The deposition and ICP results suggested that inner-sphere complexation of Ca(2+) with carboxylate groups on both SRNOM and oocyst surfaces enhanced deposition of oocysts on a SRNOM surface.

Soy formula and isoflavones and the developing intestine.

Soy protein-based infant formulas (SF) are commonly used to feed infants during their first year of life. SF contains isoflavones, which influence cell proliferation, but their actions in the developing intestine have received little attention. Herein, the impact of soy isoflavones and SF on intestinal development and rotavirus (RV) infectivity is described. The isoflavone genistein has been found to reduce intestinal cell proliferation in vitro and in vivo in piglets without affecting intestinal enzyme activity or nutrient transport. However, isoflavones possess antiviral activity. The mix of isoflavones at the concentrations in SF, or genistin alone, inhibited RV infectivity by 40-60%. Thus, soy isoflavones are bioactive within the neonatal intestine and may reduce the severity of RV infections.

Adsorption of rotavirus and bacteriophage MS2 using glass fiber coated with hematite nanoparticles.

Batch and flow-through experiments were conducted to investigate the removal and inactivation of rotavirus (RV) and bacteriophage MS2 using glass fiber coated with hematite nanoparticles. Batch tests showed a high removal of MS2 (2.49x10(11) plaque forming unit/g) and RV (8.9x10(6) focal forming unit/g) at a low concentration of hematite nanoparticles in solution (0.043g/L and 0.26g/L, respectively). Virus adsorption was, however, decreased in the presence of bicarbonate ions and natural organic matter (NOM) in solution, suggesting a great affinity of iron oxide nanoparticles for these competitors. Adsorption on hematite nanoparticles by MS2 and RV was also tested with aquifer groundwater under saturated flow conditions to mimic environmental conditions with promising results (8x10(8) plaque forming unit/g and 3x10(4) focal forming unit/g, respectively). Desorption of up to 63% of infectious MS2 and only 2% of infectious RV from hematite nanoparticles were achieved when an eluant solution containing beef extract and glycine was used. Transmission electron microscopy (TEM) images showed evidence of electrostatic adsorption of apparently intact MS2 and structurally damaged RV particles to hematite nanoparticles. Results from this research suggest that a cartridge made of glass fiber coated with hematite nanoparticles could be used as a point-of-use device for virus removal for drinking water treatment.

Deposition of Cryptosporidium parvum oocysts on natural organic matter surfaces: microscopic evidence for secondary minimum deposition in a radial stagnation point flow cell.

A radial stagnation point flow (RSPF) system combined with a microscope was used to determine the deposition kinetics of Cryptosporidium parvum oocysts on quartz surfaces and silica surfaces coated with Suwannee River natural organic matter (SRNOM) in solutions with different ionic strengths. Microscopic evidence of C. parvum oocysts entrapped in the secondary minimum energy well was presented to show that among the entrapped C. parvum oocysts some were washed away by the radial flow and some were able to transfer to deep primary minima and become irreversibly deposited. Experimental data were compared with simulation results obtained by the convective-diffusion equation and Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. The experimental results suggested that surface charge heterogeneity led to a higher attachment efficiency at low ionic strength. In addition, the maximum attachment efficiency was less than 1 at high ionic strength due to steric interaction.