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Improvements have been made to the safety and efficacy of bumped kinase inhibitors, and they are advancing toward human and animal use for treatment of cryptosporidiosis. As the understanding of bumped kinase inhibitor pharmacodynamics for cryptosporidiosis therapy has increased, it has become clear that better compounds for efficacy do not necessarily require substantial systemic exposure. We now have a bumped kinase inhibitor with reduced systemic exposure, acceptable safety parameters, and efficacy in both the mouse and newborn calf models of cryptosporidiosis. Potential cardiotoxicity is the limiting safety parameter to monitor for this bumped kinase inhibitor. This compound is a promising pre-clinical lead for cryptosporidiosis therapy in animals and humans., (Copyright © 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Cryptosporidium parvum calcium-dependent protein kinase 1 ( Cp CDPK1) is a promising target for drug development against cryptosporidiosis. We report a series of low-nanomolar Cp CDPK1 5-aminopyrazole-4-carboxamide (AC) scaffold inhibitors that also potently inhibit C. parvum growth in vitro Correlation between anti- Cp CDPK1 and C. parvum growth inhibition, as previously reported for pyrazolopyrimidines, was not apparent. Nonetheless, lead AC compounds exhibited a substantial reduction of parasite burden in the neonatal mouse cryptosporidiosis model when dosed at 25 mg/kg., (Copyright © 2017 American Society for Microbiology.)

There is a substantial need for novel therapeutics to combat the widespread impact caused by Crytosporidium infection. However, there is a lack of knowledge as to which drug pharmacokinetic (PK) characteristics are key to generate an in vivo response, specifically whether systemic drug exposure is crucial for in vivo efficacy. To identify which PK properties are correlated with in vivo efficacy, we generated physiologically based PK models to simulate systemic and gastrointestinal drug concentrations for a series of bumped kinase inhibitors (BKIs) that have nearly identical in vitro potency against Cryptosporidium but display divergent PK properties. When BKI concentrations were used to predict in vivo efficacy with a neonatal model of Cryptosporidium infection, these concentrations in the large intestine were the sole predictors of the observed in vivo efficacy. The significance of large intestinal BKI exposure for predicting in vivo efficacy was further supported with an adult mouse model of Cryptosporidium infection. This study suggests that drug exposure in the large intestine is essential for generating a superior in vivo response, and that physiologically based PK models can assist in the prioritization of leading preclinical drug candidates for in vivo testing., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.)

Bumped kinase inhibitors (BKIs) of Cryptosporidium parvum calcium-dependent protein kinase 1 (CpCDPK1) are leading candidates for treatment of cryptosporidiosis-associated diarrhea. Potential cardiotoxicity related to anti-human ether-à-go-go potassium channel (hERG) activity of the first-generation anti-Cryptosporidium BKIs triggered further testing for efficacy. A luminescence assay adapted for high-throughput screening was used to measure inhibitory activities of BKIs against C. parvum in vitro. Furthermore, neonatal and interferon γ knockout mouse models of C. parvum infection identified BKIs with in vivo activity. Additional iterative experiments for optimum dosing and selecting BKIs with minimum levels of hERG activity and frequencies of other safety liabilities included those that investigated mammalian cell cytotoxicity, C. parvum proliferation inhibition in vitro, anti-human Src inhibition, hERG activity, in vivo pharmacokinetic data, and efficacy in other mouse models. Findings of this study suggest that fecal concentrations greater than parasite inhibitory concentrations correlate best with effective therapy in the mouse model of cryptosporidiosis, but a more refined model for efficacy is needed., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Cryptosporidiosis, caused by the apicomplexan parasite Cryptosporidium parvum, is a diarrheal disease that has produced a large global burden in mortality and morbidity in humans and livestock. There are currently no consistently effective parasite-specific pharmaceuticals available for this disease. Bumped kinase inhibitors (BKIs) specific for parasite calcium-dependent protein kinases (CDPKs) have been shown to reduce infection in several parasites having medical and veterinary importance, including Toxoplasma gondii, Plasmodium falciparum, and C. parvum In the present study, BKIs were screened for efficacy against C. parvum infection in the neonatal mouse model. Three BKIs were then selected for safety and clinical efficacy evaluation in the calf model for cryptosporidiosis. Significant BKI treatment effects were observed for virtually all clinical and parasitological scoring parameters, including diarrhea severity, oocyst shedding, and overall health. These results provide proof of concept for BKIs as therapeutic drug leads in an animal model for human cryptosporidiosis., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Wood decomposition releases almost as much CO2 to the atmosphere as does fossil-fuel combustion, so the factors regulating wood decomposition can affect global carbon cycling. We used metabarcoding to estimate the fungal species diversities of naturally colonized decomposing wood in subtropical China and, for the first time, compared them to concurrent measures of CO2 emissions. Wood hosting more diverse fungal communities emitted less CO2, with Shannon diversity explaining 26 to 44% of emissions variation. Community analysis supports a 'pure diversity' effect of fungi on decomposition rates and thus suggests that interference competition is an underlying mechanism. Our findings extend the results of published experiments using low-diversity, laboratory-inoculated wood to a high-diversity, natural system. We hypothesize that high levels of saprotrophic fungal biodiversity could be providing globally important ecosystem services by maintaining dead-wood habitats and by slowing the atmospheric contribution of CO2 from the world's stock of decomposing wood. However, large-scale surveys and controlled experimental tests in natural settings will be needed to test this hypothesis., Competing Interests: DWY is co-founder of a UK company, NatureMetrics, which provides DNA metabarcoding services to the private and public sector. All other authors declare no conflicts of interest.

The production and consumption of the greenhouse gases (GHGs) methane (CH4), carbon dioxide (CO2) and nitrous oxide (N2O) in soil profile are poorly understood. This work sought to quantify the GHG production and consumption at seven depths (0-30, 30-60, 60-90, 90-150, 150-200, 200-250 and 250-300 cm) in a long-term field experiment with a winter wheat-summer maize rotation system, and four N application rates (0; 200; 400 and 600 kg N ha(-1) year(-1)) in the North China Plain. The gas samples were taken twice a week and analyzed by gas chromatography. GHG production and consumption in soil layers were inferred using Fick's law. Results showed nitrogen application significantly increased N2O fluxes in soil down to 90 cm but did not affect CH4 and CO2 fluxes. Soil moisture played an important role in soil profile GHG fluxes; both CH4 consumption and CO2 fluxes in and from soil tended to decrease with increasing soil water filled pore space (WFPS). The top 0-60 cm of soil was a sink of atmospheric CH4, and a source of both CO2 and N2O, more than 90% of the annual cumulative GHG fluxes originated at depths shallower than 90 cm; the subsoil (>90 cm) was not a major source or sink of GHG, rather it acted as a 'reservoir'. This study provides quantitative evidence for the production and consumption of CH4, CO2 and N2O in the soil profile.

Host defence peptides (HDPs) are expressed throughout the animal and plant kingdoms. They have multifunctional roles in the defence against infectious agents of mammals, possessing both bactericidal and immune-modulatory activities. We have identified a novel family of molecules secreted by helminth parasites (helminth defence molecules; HDMs) that exhibit similar structural and biochemical characteristics to the HDPs. Here, we have analyzed the functional activities of four HDMs derived from Schistosoma mansoni and Fasciola hepatica and compared them to human, mouse, bovine and sheep HDPs. Unlike the mammalian HDPs the helminth-derived HDMs show no antimicrobial activity and are non-cytotoxic to mammalian cells (macrophages and red blood cells). However, both the mammalian- and helminth-derived peptides suppress the activation of macrophages by microbial stimuli and alter the response of B cells to cytokine stimulation. Therefore, we hypothesise that HDMs represent a novel family of HDPs that evolved to regulate the immune responses of their mammalian hosts by retaining potent immune modulatory properties without causing deleterious cytotoxic effects.

A two-year study (2009 ~ 2010) was carried out to investigate the dynamics of different carbon (C) forms, and the role of stream export in the C balance of a 23.4-ha headwater catchment in a tropical seasonal rainforest at Xishuangbanna (XSBN), southwest China. The seasonal volumetric weighted mean (VWM) concentrations of total inorganic C (TIC) and dissolved inorganic C (DIC) were higher, and particulate inorganic C (PIC) and organic C (POC) were lower, in the dry season than the rainy season, while the VWM concentrations of total organic C (TOC) and dissolved organic C (DOC) were similar between seasons. With increased monthly stream discharge and stream water temperature (SWT), only TIC and DIC concentrations decreased significantly. The most important C form in stream export was DIC, accounting for 51.8% of the total C (TC) export; DOC, POC, and PIC accounted for 21.8%, 14.9%, and 11.5% of the TC export, respectively. Dynamics of C flux were closely related to stream discharge, with the greatest export during the rainy season. C export in the headwater stream was 47.1 kg C ha(-1) yr(-1), about 2.85% of the annual net ecosystem exchange. This finding indicates that stream export represented a minor contribution to the C balance in this tropical seasonal rainforest.

Cryptosporidium parvum is one of the main causes of diarrhea in neonatal calves resulting in significant morbidity and economic losses for producers worldwide. We have previously demonstrated efficacy of a new class of antimicrobial antibody fusions in a neonatal mouse model for C. parvum infection. Here, we extend efficacy testing of these products to experimental infection in calves, the principal target species. Neonatal calves were challenged with C. parvum oocysts and concomitantly treated with antibody-biocide fusion 4H9-G1-LL37 over the course of four days. This resulted in reduced severity of the disease when compared to control animals. Overall clinical health parameters showed significant improvement in treated animals. Oocyst shedding was reduced in treated when compared to control animals. Control of oocyst shedding is a prerequisite for breaking the cycle of re-infection on dairy farms. Antibody-biocide fusion products thus have the potential to reduce the impact of the infection in both individual animals and in the herd., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The apicomplexan parasite Cryptosporidium parvum is an important cause of diarrhea in humans and cattle, and it can persistently infect immunocompromised hosts. No consistently effective parasite-specific pharmaceuticals or immunotherapies for control of cryptosporidiosis are presently available. The innate immune system represents the first line of host defense against a range of infectious agents, including parasitic protozoa. Several types of antimicrobial peptides and proteins, collectively referred to herein as biocides, constitute a major effector component of this system. In the present study, we evaluated lactoferrin, lactoferrin hydrolysate, 5 cationic peptides (lactoferricin B, cathelicidin LL37, indolicidin, β-defensin 1, β-defensin 2), lysozyme, and 2 phospholipases (phospholipase A2, and phosphatidylinositol-specific phospholipase C) for anti-cryptosporidial activity. The biocides were evaluated either alone or in combination with 3E2, a monoclonal antibody (MAb) against C. parvum that inhibits sporozoite attachment and invasion. Sporozoite viability and infectivity were used as indices of anti-cryptosporidial activity in vitro. All biocides except lactoferrin had a significant effect on sporozoite viability and infectivity. Lactoferrin hydrolysate and each of the 5 cationic peptides were highly parasiticidal and strongly reduced sporozoite infectivity. While each phospholipase also had parasiticidal activity, it was significantly less than that of lactoferrin hydrolysate and each of the cationic peptides. However, each phospholipase reduced sporozoite infectivity comparably to that observed with lactoferrin hydrolysate and the cationic peptides. Moreover, when 3 of the cationic peptides (cathelicidin LL37, β-defensin 1, and β-defensin 2) were individually combined with MAb 3E2, a significantly greater reduction of sporozoite infectivity was observed over that by 3E2 alone. In contrast, reduction of sporozoite infectivity by a combination of either phospholipase with MAb 3E2 was no greater than that by 3E2 alone. These collective observations suggest that cationic peptides and phospholipases neutralize C. parvum by mechanisms that are predominantly either parasiticidal or non-parasiticidal, respectively.

At present no completely effective treatments are available for Cryptosporidium parvum infections in humans and livestock. Based on previous data showing the neutralizing potential of a panel of monoclonal antibodies developed against C. parvum, and based on the fact that innate immune peptides and enzymes have anticryptosporidial activity, we engineered several of these antibodies into antibody-biocide fusion proteins. We hypothesized that the combination of high-affinity antibody targeting with innate immune molecule-mediated killing would result in a highly effective new antiprotozoal agent. To test this hypothesis, we expressed antibody-biocide fusion proteins in a mammalian cell culture system and used the resulting products for in vitro and in vivo efficacy experiments. Antibody-biocide fusion proteins efficiently bound to, and destroyed, C. parvum sporozoites in vitro through a membrane-disruptive mechanism. When antibody-biocide fusion proteins were administered orally to neonatal mice in a prophylactic model of cryptosporidiosis, the induction of infection was reduced by as much as 81% in the mucosal epithelium of the gut, as determined on the basis of histopathological scoring of infectious stages. Several versions of antibody fusion proteins that differed in antigen specificity and in the biocide used had strong inhibitory effects on the initiation of infection. The results lay the groundwork for the development of a new class of antimicrobials effective against Cryptosporidium.

The biological basis for the specificity of host infectivity patterns of Cryptosporidium spp., in particular C. hominis and C. parvum, has yet to be fully elucidated. Comparison of the C. parvum and C. hominis P23 and GP900 predicted amino acid sequences revealed 3 differences in P23 and 4 and 17 differences in GP900 domains 1 and 5, respectively. Using monoclonal antibodies developed against the surface (glyco)proteins P23 and GP900 of the C. parvum Iowa isolate, solubilized glycoprotein from three C. hominis isolates was screened for reactivity using Western immunoblots. One of ten P23 MAbs and three of 21 GP900 MAbs were not reactive with any of the three C. hominis isolates. The non-reactive P23 MAb binds to a peptide epitope, while the non-reactive GP900 MAbs bind to either carbohydrate/carbohydrate-dependent or peptide epitopes of C. parvum. These results demonstrate phenotypic differences between C. hominis and C. parvum within two (glyco)proteins that are involved in parasite gliding motility and attachment/invasion.

The objective of this study was to determine whether changes in the ileal intraepithelial lymphocyte (TEL) phenotype and function occurred prior to development of diarrhea in Cryptosporidium parvum-infected calves. Calves were orally inoculated with 10(8) oocysts and maintained in enteric pathogen-free conditions until their use in experiments. Age-matched uninfected calves were used for comparisons. Ileal IELs were isolated and phenotyped to determine whether changes in lymphocyte population dynamics had occurred by 3 days postinoculation (PI). Ex vivo reverse transcriptase-polymerase chain reaction of messenger ribonucleic acid (mRNA) from IELs from infected calves was compared with controls to determine whether changes in cytokine expression had occurred by 3 days PI. No significant changes in lymphocyte population dynamics were documented, however, IELs isolated from 4 out of 8 infected calves, but not from 8 out of 8 control calves, expressed mRNA for interleukin-10 (IL-10). IL-10 expression by IELs was associated with the expression of a significantly larger (P < 0.001) proportion (0.75) of monoclonal antibody-defined C. parvum epitopes within infected ileal epithelium, as compared with a much smaller proportion (0.30) of epitopes with IL-10 lymphocytes. The results suggest that a temporal association exists between the expression of IL-10 by ileal IELs and the expression of C. parvum antigens in infected calf epithelium prior to development of cryptosporidiosis.

Cryptosporidium parvum is an important cause of diarrhea in humans and calves and can persistently infect immunocompromised hosts. Presently, there are no consistently effective parasite-specific drugs for cryptosporidiosis. We hypothesized that neutralizing monoclonal antibodies (MAbs) targeting the apical complex and surface antigens CSL, GP25-200, and P23 could passively immunize against cryptosporidiosis. We recently reported that a formulation of MAbs 3E2 (anti-CSL), 3H2 (anti-GP25-200), and 1E10 (anti-P23) provided significant additive prophylactic efficacy over that of the individual MAbs in neonatal ICR mice. In the present study, these MAbs were evaluated for therapeutic efficacy against persistent infection in adult gamma interferon-depleted SCID mice. 3E2 demonstrated the most significant and consistent therapeutic effect, reducing intestinal infection in two experiments. In one experiment, 3E2 plus 3H2 and 3E2 plus 3H2 plus 1E10 also significantly reduced infection; however, no significant increase in efficacy over 3E2 alone was apparent. The results indicate that anti-CSL MAb 3E2 has highly significant efficacy in reducing, but not eliminating, persistent C. parvum infection.

The protozoan parasite Cryptosporidium parvum is a leading cause of diarrhea in humans and neonatal calves. The absence of approved parasite-specific drugs, vaccines, and immunotherapies for cryptosporidiosis relates in part to limited knowledge on the pathogenesis of zoite attachment and invasion. We recently reported that the C. parvum apical complex glycoprotein CSL contains a zoite ligand for intestinal epithelial cells which is defined by monoclonal antibody (MAb) 3E2. In the present study, the host cell receptor for CSL was characterized. For these studies, a panel of epithelial and mesenchymal cell lines was examined for permissiveness to C. parvum and the ability to bind CSL. Cells of epithelial origin were significantly more permissive and bound significantly greater quantities of CSL than cells of mesenchymal origin. Caco-2 intestinal cells were selected from the epithelial panel for further characterization of the CSL receptor. Immunoelectron microscopy demonstrated that CSL bound initially to the surface of Caco-2 cells and was rapidly internalized. The molecule bound by CSL was identified as an 85-kDa Caco-2 cell surface protein by radioimmunoprecipitation and CSL affinity chromatography. Sporozoite incubation with the isolated 85-kDa protein reduced binding of MAb 3E2. Further, attachment and invasion were significantly inhibited when sporozoites were incubated with the 85-kDa protein prior to inoculation onto Caco-2 cells. These observations indicate that the 85-kDa protein functions as a Caco-2 cell receptor for CSL. CSL also bound specifically to intestinal epithelium from calves, indicating receptor expression in a second important host species. Molecular characterization of the CSL receptor may lead to novel avenues for disrupting ligand-receptor interactions in the pathogenesis of C. parvum infection.

The coccidian parasite Cryptosporidium parvum causes diarrhea in humans, calves, and other mammals. Neither immunization nor parasite-specific pharmaceuticals that are consistently effective against this organism are available. While polyclonal antibodies against whole C. parvum reduce infection, their efficacy and predictability are suboptimal. We hypothesized that passive immunization against cryptosporidiosis could be improved by using neutralizing monoclonal antibodies (MAbs) targeting functionally defined antigens on the infective stages. We previously reported that the apical complex and surface-exposed zoite antigens CSL, GP25-200, and P23 are critical in the infection process and are therefore rational targets. In the present study, a panel of 126 MAbs generated against affinity-purified CSL, GP25-200, and P23 was characterized to identify the most efficacious neutralizing MAb formulation targeting each antigen. To identify neutralizing MAbs, sporozoite infectivity following exposure to individual MAbs was assessed by enzyme-linked immunosorbent assay. Of 126 MAbs evaluated, 47 had neutralizing activity. These were then evaluated individually in oocyst-challenged neonatal mice, and 14 MAbs having highly significant efficacy were identified for further testing in formulations. Epitope specificity assays were performed to determine if candidate MAbs recognized the same or different epitopes. Formulations of two or three neutralizing MAbs, each recognizing distinct epitopes, were then evaluated. A formulation of MAbs 3E2 (anti-CSL [alphaCSL]), 3H2 (alphaGP25-200), and 1E10 (alphaP23) provided highly significant additive efficacy over that of either individual MAbs or combinations of two MAbs and reduced intestinal infection by 86 to 93%. These findings indicate that polyvalent neutralizing MAb formulations targeting epitopes on defined antigens may provide optimal passive immunization against cryptosporidiosis.

We estimated the density of subterranean termites Gnathamitermes tubiformans at 800,000 · ha -1 for a standing crop biomass of 2 kg · ha -1 Predation losses were estimated to be 5,73 kg · ha -1 · yr -1 representing the major release of nutrients from termites to surficial soil layers. Nutrient fluxes from termites to predators amounted to 410g N·ha -1 ·yr -1 , 33 g S · ha -1 · yr -1 and 19 g P · ha -1 · yr -1 . These fluxes account for 8% of the litter N, 1.5% of the litter P and 2.9% of the litter S. The termites fixed an estimated 66 g · ha -1 · yr -1 atmospheric N and returned an estimated 100 g · ha -1 · yr -1 in the surface gallery carton. Since losses of elements from subterannean termites were greater than standing crops, we estimated an annual turnover of N at 3.5 times per year, P of 2.5 times per year, and S of 2.5 per times per year.Since surface foraging, predation and alate flights are pulse regulated by rainfall, nutrient flows through subterranean termites are episodic and releases of nutrients accumulated in termite biomass preceeds or is coincident with productivity "pulses" of some shallow rooted plants. We propose that subterranean termites are important as regulators in desert nutrient cycles.