Summary 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.

Acid phosphatases (Acp) of intracellular pathogens have recently been implicated as virulence factors that enhance intracellular survival through suppression of the respiratory burst. We describe here the identification, purification, characterization, and sequencing of a novel burst-inhibiting acid phosphatase from the facultative intracellular bacterium, Francisella tularensis. Similar to other the burst-inhibiting Acps, F. tularensis Acp (AcpA) is tartrate-resistant and has broad substrate specificity. The AcpA enzyme is unique, however, in that it is easily released from the bacterial cell in soluble form, is a basic enzyme, suppresses the respiratory burst of not only fMet-Leu-Phe but also phorbol 12-myristate 13-acetate-stimulated neutrophils and does not fit into any of the three currently recognized classes of acid phosphatase. We also report the complete nucleotide sequence of the gene acpA, encoding AcpA, and the deduced primary structure of its encoded polypeptide. Comparative sequence analyses of AcpA is discussed. To our knowledge, this is the first report describing the cloning and sequencing of a burst-inhibiting acid phosphatase.

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 À 1 HMO (HMO) or a prebiotic mixture of 9:1 short-chain galactooligosaccharides (3.6 g l À 1 ) and long-chain fructooligosaccharides (0.4 g l À 1 ) (PRE) (n ¼ 19-21 per group) for 15 days. Piglets (n ¼ 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.

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.

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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.

Isoflavones and their related flavonoid compounds exert antiviral properties in vitro and in vivo against a wide range of viruses. Genistein is, by far, the most studied soy isoflavone in this regard, and it has been shown to inhibit the infectivity of enveloped or nonenveloped viruses, as well as single-stranded or double-stranded RNA or DNA viruses. At concentrations ranging from physiological to supraphysiological (3.7-370 muM), flavonoids, including genistein, have been shown to reduce the infectivity of a variety of viruses affecting humans and animals, including adenovirus, herpes simplex virus, human immunodeficiency virus, porcine reproductive and respiratory syndrome virus, and rotavirus. Although the biological properties of the flavonoids are well studied, the mechanisms of action underlying their antiviral properties have not been fully elucidated. Current results suggest a combination of effects on both the virus and the host cell. Isoflavones have been reported to affect virus binding, entry, replication, viral protein translation and formation of certain virus envelope glycoprotein complexes. Isoflavones also affect a variety of host cell signaling processes, including induction of gene transcription factors and secretion of cytokines. The efficacy of isoflavones and related flavonoids in virus infectivity in in vitro bioassays is dependent on the dose, frequency of administration and combination of isoflavones used. Despite promising in vitro results, there is lack of data confirming the in vivo efficacy of soy isoflavones. Thus, investigations using appropriate in vivo virus infectivity models to examine pharmacological and especially physiological doses of flavonoids are warranted.

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 0 -fucosyllactose) and acidic HMO (aHMO, 3 0 -sialyllactose, 3 0 -SL; 6 0 -sialyllactose, 6 0 -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 0 -SL and 6 0 -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 0 -SL/10 % 3 0 -SL/50 % SA) or media with or without the RV OSU strain (1 £ 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.Key words: Human milk oligosaccharides: Rotavirus: Piglets: Infection Rotaviruses (RV) are double-stranded RNA viruses of the family Reoviridae, which are the most common viral agents causing viral gastroenteritis and diarrhoea in infants and young children worldwide. Each year, approximately 1·4 billion episodes of RV gastroenteritis (RVGE) occur in children under 5 years of age in developing countries, and half a million children die (1,2) .Vaccination is the main public health intervention to prevent RV infection. Systematic reviews of vaccine effectiveness and vaccination-impact studies in industrialised countries (USA, Europe and Australia) have demonstrated an effectiveness of 85 -100 % associated with decreased hospitalisations for RVGE (3) . Vaccination-impact studies have demonstrated that the burden of RVGE has been reduced significantly since the introduction of RV vaccination (3) . However, efficacy trials in developing countries in Africa and Asia showed that vaccine efficacy was lower than that observed in other countries, typically 40 -70 % (4) . Although the efficacy of RV vaccines correlates closely with the national per capita income (5) , it is unclear why vaccination is less efficacious in developing countries (6) . This reduced vaccine efficacy coupled with the high cost and barriers to a widespread distribution of RV vaccines (7) suggest that other means for preventing RV should...