Tag Archives: dihydrofolate reductase inhibitor

Any intravitreally injected substance must prove efficacy and

Any intravitreally injected substance must prove efficacy and safety. While our study was not designed as a preclinical safety study, we did not find any obvious structural changes after Sema3f treatment in either of the two in vivo models, which is not surprising given that Sema3f is physiologically expressed in the outer retina (Buehler et al., 2013). Our long-term follow-up experiments in Vldlr−/− mice showed even improved retinal integrity compared to control treated eyes, which may be a secondary benefit from reduced number and size of subretinal neovascular lesions.
In summary, these data provide to our knowledge the first proof of concept that Sema3f can be used to modulate pathological subretinal neovascularization. The protective effect of Sema3f was observed in two independent model systems and with two independent modes of application. The fact that Sema3f is physiologically expressed in the retina of both mice and humans (Buehler et al., 2013) renders Sema3f a promising target for treating pathological neovascularization formation in AMD.

Conflicts of Interest

Author Contributions


With metabolic syndromes such as obesity yielding widespread contemporary concern, the race is on for discovering strategies which can ameliorate conditions detrimental to human health. Nutrigenomics is an emerging field of science aiming to address such conditions by studying the molecular interplay between a diet and the genome. Evidence shows that adipose development at the fetal stage have long term effects in later life. In mice, subcutaneous white fat (WAT) (Wang et al., 2013) and brown fat (BAT) (Billon and Dani, 2012) develop during the fetal stage. Maternal nutritional status during gestation epigenetically alters WAT differentiation (Borengasser et al., 2013). Zfp423, a gene encoding a transcription factor for preadipocyte commitment, is hypomethylated in fetuses born to obese mothers (Yang et al., 2013). Moreover, the population of adipose progenitor dihydrofolate reductase inhibitor (Liang et al., 2016a) and the thermogenic function of BAT (Liang et al., 2016b) of offspring are altered due to maternal intake of high fat diet. Up to now, the relationship between early adipose development and obesity in later life remains poorly understood.
White adipocytes are healthy at normal sizes, but over-expansion of adipocyte sizes lead to hypoxia (Sun et al., 2011), inflammation, and interstitial fibrosis (Sun et al., 2013), which triggers adipose metabolic dysfunction (Sun et al., 2014b). Beige adipocytes, which distribute inside white adipose tissue, burn fatty acids to reduce adipocyte hypotrophy. The vascular system acts as an adipogenic niche by providing PDGFRα+ progenitors, which are able to differentiate into both beige and white adipocytes (Lee et al., 2012). Adipose tissue are highly vascularized (Cao, 2007), and adipogenesis is spatially and temporally associated with angiogenesis during fetal development (Cao, 2007), which provides PDGFRα+ progenitor cells (Crisan et al., 2008; Tran et al., 2012; Vishvanath et al., 2016). Thus, promoting beige adipogenesis of PDGFRα+ progenitors improves the metabolic health of adipose tissue.
Vitamin A and its metabolite, retinoic acid (RA), play key roles in fetal morphogenesis and organ development (Zile, 2001; Duester, 2008), and is a common supplement used during pregnancy. At pharmacological doses, RA increases energy consumption of white adipose tissue (Alvarez et al., 1995; Puigserver et al., 1996; Bonet et al., 2003; Mercader et al., 2006), suggesting that RA might promote beige adipogenesis in mature WAT. Vitamin A or RA supplementation is effective in preventing obesity in adult mice (Berry et al., 2012; Noy, 2013). In humans, overweight and obese individuals showed lower retinoid levels in serum (de Souza Valente da Silva et al., 2007; Pereira et al., 2012) and dietary vitamin A intake is inversely related with adiposity (Zulet et al., 2008). However, the effect of maternal vitamin A intake on beige adipogenesis during fetal development is unclear, which represents a critical knowledge gap because the fetal and neonatal stages are critical for adipogenesis. The long-term impact of maternal retinoid status on adipose metabolic health of offspring remains unclear. We hypothesized that maternal vitamin A supplementation promotes angiogenesis and beige adipogenesis during fetal development, which improve the metabolic health of offspring adipose tissue, protecting offspring from diet-induced obesity and metabolic dysfunction.

In summary the full length cDNA of OaPrdx

In summary, the full-length cDNA of OaPrdx6 from O. aries was identified and characterized. OaPrdx6 gene was widely expressed in various tissues from sheep, and the mRNA transcription of OaPrdx6 from sheep infected with a virulent field strain of B. melitensis and ones inoculated with a vaccine strain S2 of B. suis was respectively up-regulated and down-regulated compared to the normal sheep, suggesting that OaPrdx6 might play an important physiological role and be associated with immune responses against invasion of Brucella. In light of our current findings, it was speculated that OaPrdx6 would be used as a potential biomarker to assess Brucella infection and facilitate preventing brucellosis.

This work was supported by the grants from the National Natural Science Foundation of China (No. 30901070) and the Science & Technology Development Project of Jilin Province, China (No. 20150204078NY).

About forty years ago dendritic dihydrofolate reductase inhibitor (DCs) were first described by Ralph Steinman (see review Steinman, 2007, 2008). Since then multiple lines of DC research emerged, and nowadays is well established that DCs are potent inducers of immune responses, with the ability to stimulate other cells and also to induce immunological tolerance. The development of reagents and technology related with DCs thus far has been focused mainly on two non-primate animal models: mouse and rat (Banchereau et al., 2000; Shortman and Liu, 2002). However, important advances have also been made using non human primate DCs (Banchereau et al., 2000). More recently, it is possible to find commercial reagents to research DCs from other species such as dog, cat, cow, sheep, pig, chicken and horse (Siedek et al., 1997; Ibrahim et al., 2007; Steinbach et al., 2007; McCullough and Summerfield, 2009). The search for molecular markers and additional functions of DCs is quite active. The demonstration of DC subsets has extended the interest and importance of DC biology (Steinman, 2007). The use of monoclonal antibodies in the species described has allowed to generally defining DC subsets as lymphoid and myeloid. With the available information and reagents it is possible to phenotypically describe horse DCs by their expression of CD11a, CD86, MHC-I, MHC-II, and CD4 markers. However, it is not yet possible to find a detailed description of horse DC subsets as in other species, such as chicken, pig, or dog. A classic staining for mouse DCs either in situ or by flow cytometry is by CD11c in conjunction with another markers, because of its high expression in most DC assessed, as well as the histological location and the typical dendritic morphology of the cells identified (Nakano et al., 2001; Edwards et al., 2003). In humans, CD11c apparently allows to discern between myeloid (CD11c+) and plasmacytoid DCs (CD11c−) (Shortman and Liu, 2002).
CD11c/CD18 is a member of the CD18 family of integrins, which includes CD11a (LFA-1), CD11b (MAC-1), and CD11d (αD). Members of this integrin family share the same β-chain, namely β2 (CD18), but are distinguished by their α-chains, designated as αL (CD11a), αM (CD11b), αX (CD11c), and αD (CD11d) (Luo et al., 2007). Although their molecular sizes and amino acid sequences are distinct, the four α-chains share similar overall structural organization. CD11a is the largest, with an approximate M.W. of 180kDa and the most divergent from the rest. CD11c is 150kDa in size and shows considerable similarity with CD11b and CD11d regarding amino acid sequence, expression, and ligand-binding profile (Springer and Dustin, 2012; Sen et al., 2013). This includes the presence of the inserted or I-domain in CD11c, which has been implicated in ligand binding (Vorup-Jensen et al., 2003; Springer and Dustin, 2012).

Materials and methods

Results and discussion

Conflict of interest


The fine tuning of PMN s lifespan in tissues

The fine tuning of PMN’s lifespan in tissues is essential to reduce inflammatory-derived injuries to the host (Greenlee-Wacker, 2016). We can rule out that the downregulation of ROS production and the decrease of dihydrofolate reductase inhibitor is related to a decrease of cells’ number due to apoptosis, given that the dihydrofolate reductase inhibitor apoptosis rate of bovine PMN was not found to be affected by HS. Available data on the effects of HS on apoptosis of human PMN are conflicting, depending on the temperature of HS and time of exposure. Nagarsekar et al. (2008) found that culturing human PMN at 39.5°C for 8h greatly accelerated caspase dependent apoptosis. Exposure of human PMN to 43°C for 1h caused an early increase in the rate of apoptosis (Callahan et al., 1999), whereas in a recent in vitro study carried out by culturing human neutrophils at 37 or 43°C for 10min, no substantial differences in viability were observed (Boyko et al., 2014). Therefore, we may hypothesize that the temperature effects depend on the length of exposure.

In synthesis, the results of the present in vitro study suggest that some functions of PMN exposed to temperature simulating conditions of severe whole body hyperthermia are impaired and may at least partially explain the higher occurrence of infections during periods of hot weather.
Further epidemiology studies are necessary to clarify whether and under which specific circumstances high environmental temperatures are associated with higher incidence of infections. However, the risk of impairment of PMN functions under these conditions support the use of management practices (i.e. cooling), which may help to limit the increase of body temperature and optimization of other environmental features (i.e. general hygienic conditions, nutritional plans, etc.), which may also play a role in causing infections outbreaks in hot environments.

Competing interest

The authors thank Giorgina Kuzminsky of the DAFNE, Università della Tuscia for help with in vitro experiments.

The incidence of cancer in human and canine species is similar. Both physiology and factors related to domestication make dogs an ideal comparative model for the human disease (Alvarez, 2014; Pinho et al., 2012; Rowell et al., 2011). While conventional therapies including surgery, radiation therapy, chemotherapy and various combinations thereof provide clinical benefit for cancer patients (Emens, 2008; Aldrich et al., 2010), long-term survival needs to be improved necessitating additional research (Emens, 2008; Aldrich et al., 2010). Therapeutic strategies developed in dogs benefit the animal and provide enormous translational potential for humans (Khanna et al., 2009; Gordon et al., 2009; Paoloni and Khanna, 2008, 2007; Hansen and Khanna, 2004).
Immunotherapy, a 4th modality of cancer treatment, has been tested in numerous small animal models and translated for use in humans (Elliott, 2012; Budhu et al., 2014). Immunotherapy utilizes the patient’s immune system to kill tumor cells (Rosenberg and Restifo, 2015). Most success has been achieved by stimulating mechanisms of anti-tumor acquired immunity resulting in a specific anti-tumor response (Schwartzentruber et al., 1994). A number of immunotherapies have now been approved by the Food and Drug Administration (FDA). These include the preventative tumor vaccines Gardasil® in 2006 and Cervarix® in 2009 for cervical cancer (Joura et al., 2007; Villa et al., 2005; Paavonen et al., 2007) and the therapeutic vaccine sipleucel-T (Provenge®) in 2010 for prostate cancer (Kantoff et al., 2010). More recently, the FDA approved ipilimumab (Yervoy®, anti-CTLA-4) (Camacho, 2015) and pembrolizumab (Keytruda®, anti-PD-1) (Mahoney et al., 2015) for the treatment of melanoma and/or non-small cell lung cancer (NSCLC) (Ito et al., 2015). These antibodies reverse tumor-derived immune suppression resulting in enhanced patient survival and increased time to tumor progression. Unfortunately, progress in humans has been slow to translate to the canine species with only one approved immunotherapy for dogs. The Oncept™ Canine Melanoma Vaccine (Merial Limited) received FDA approval in 2010 for the management of oral and digital malignant melanoma (Grosenbaugh et al., 2011; Bergman et al., 2006, 2003; Liao et al., 2006). Other immunotherapeutic strategies for dogs remain exploratory and have not yet reached veterinary practice.

In this study a novel technique for

In this study, a novel technique for sound velocity optimization is proposed by directly observing the phase coherence in delayed channel data before beam sum. Channel-domain autocorrelation is utilized to compare the phase gradients in the left and right sub-apertures with high rejection to noise interference and low computational complexity. When the differential phase gradients in two sub-apertures approaches zero, maximal phase coherence is achieved and the corresponding is optimized. The paper is organized by first providing the definition of differential phase gradient in Section 2. Explanation about how the differential phase gradient varies with the setting of is also included. In Section 3, the simulation parameters and the experimental setup used to verify the proposed sound velocity optimization are introduced in details. The bemforming quality with and without sound velocity optimization is examined in B-mode imaging of diffused speckle phantom and wire reflectors in Section 4. Comparison between the proposed method and the referenced MAPV method is also performed for their robustness in sound velocity optimization. The paper concludes with discussions in Section 5.

As mentioned in Fig. 1, the time delay is under-compensated and over-compensated, respectively when > and <. Consequently, the phase of channel data after time compensation will appear to be a parabolic profile in the channel domain as shown in Fig. 2. Specifically, the concave-downward parabolic curve in black demonstrates the channel phase when >. This is straightforward since the time lag in the outer channel waveforms would correspond to the reduction in signal phase. On the contrary, the phase profile would become concave-upward as the red curve in Fig. 2 when < due to the over-compensation of time delay. In this case, the waveforms in the outer dihydrofolate reductase inhibitor lead those in the central channels and thus exhibit increased signal phase. Only when =, the parabolic curve will degenerate to a horizontal line as shown in the blue line of Fig. 2. Note that the horizontal line indicates that the desired phase coherence has been achieved across channels when the optimal is utilized for beamforming.
In this study, it is proposed that the phase gradient of the channel data can be utilized to identify the pattern of phase profile for sound velocity optimization. Note that the phase gradient can be regarded as the slope of the phase profile in Fig. 2. When the assumed sound velocity is not equal to the actual one in propagating tissue, the phase gradient in the left half sub-aperture is opposite in sign to that in the right counterpart. For example, if is set to be slower than , the slope is negative in the left sub-aperture (i.e., <0) and positive in the right sub-aperture (>0)). Therefore, the difference in phase gradient between the left sub-aperture and right sub-aperture will be nonzero when ≠. The differential phase gradient becomes zero only when the two sub-apertures have the same slope of phase profile, which indicates that the receive beamforming has been optimized in sound velocity by =.
The method of differential phase gradient can be further analyzed using the following equations. First, the actual time delay for the received signal coming from direction and the applied time compensation for receive beamforming to direction can be respectively represented as
In (1), x and r are the lateral position of the receiving channel and the range distance from the beamforming focal point to the center of transducer array, respectively. Note that, when , the received echo is coming from the side lobe region of the receive beam. After time compensation for receive focusing, the residual delay of the received echo becomes
Then, the phase gradient in the channel domain can be calculated by taking the derivative of (2) with respect to x and scaling the result by :
The differential phase gradient is estimated by the subtraction of between the left and the right sub-apertures. Assuming the lateral size of the entire aperture is 2a, one special case can be represented as an example by using the two end points of the entire aperture (i.e., and ), respectively as the left and the right sub-apertures:

Recent studies suggested that DNA based vaccines

Recent studies suggested that DNA-based vaccines maybe effective in eliciting adequate immunity against coccidian infection without the disadvantages associated with chemoprophylaxis and live vaccines (Ding et al., 2012; Hoan et al., 2014; Ivory and Chadee, 2004). It was reported that cell-mediated immunity played main roles in the protection against chicken coccidiosis (Chapman, 2014; Dalloul and Lillehoj, 2006; Jenkins, 1998). T-cell epitope, the minimal antigenic units, is a set of amino dihydrofolate reductase inhibitor residues presented by histocompatibility complex (MHC) molecules and recognized by the T cells of the host system (Desai and Kulkarni-Kale, 2014; Cho and Celis, 2012). Since T-cell epitope, a relatively tiny, but immunologically relevant, sequence is often capable of inducing cellular immune response to a large and complex pathogen (Desai and Kulkarni-Kale, 2014; Suhrbier, 1997), multi-epitope DNA vaccines have been constructed and showed effective efficacies in HIV, hantavirus, Hepatitis C virus (HCV), Plasmodium falciparum, Toxoplasma gondii and Melanoma (Cho and Celis, 2012; Cong et al., 2014; Hanke et al., 1999; Pishraft Sabet et al., 2014; Suhrbier, 1997; Zhao et al., 2012). Since the complex life cycle of the Eimeria parasite involves both asexual and sexual developmental stages, vaccine based on a single antigen from one developmental stage of the parasite could not provide effective protection (Badran and Lukešová, 2006; Jenkins, 1998). Thus, developing T-cell epitopes DNA vaccines with antigens from different stages of Eimeria life cycle could be a new alternative control strategy (Blake and Tomley, 2014; Ding et al., 2012; Ivory and Chadee, 2004; Jenkins, 1998; Olszewska and Steward, 2001).
Sporozoite antigen SO7 of E. tenella is associated with the refractile body (RB) and plays a potential role in cellular invasion (de Venevelles et al., 2006; Fetterer et al., 2007). Some subsequent researches proved that SO7 could induce protection against E. tenella (Crane et al., 1991; Yang et al., 2010). The surface antigen MZP5-7 (encoded by MZ5-7 gene) from the second generation merozoite of E. tenella was first identified by Binger et al. (1993) and could produce partial protection against E. tenella challenge infection (Geriletu et al., 2011). In previous studies, SO7 and MZ5-7 genes were cloned and the immunization with the DNA vaccines based on these two genes could provide effective protection against E. tenella (Geriletu et al., dihydrofolate reductase inhibitor 2011; Song et al., 2013). These results suggested that SO7 and MZ5-7 genes might contain protective epitopes to the challenge of E. tenella.
Cytokines are key regulators of the immune system. They are essential to shape the innate and adaptive immune responses, as well as for the establishment and maintenance of immunological memory (Chabalgoity et al., 2007). The use of cytokines as adjuvants in poultry is attracting considerable attention (Asif et al., 2004). It was reported that immune responses induced by DNA vaccination could be enhanced by co-delivery of recombinant cytokines or plasmids encoding these cytokines (Chabalgoity et al., 2007; Min et al., 2002; Shah et al., 2011). Our previous researches suggested that IL-2 or IFN-γ could effectively improve protective immunity of DNA vaccines against coccidiosis (Xu et al., 2008; Song et al., 2010; Shah et al., 2011).
Here we reported the selections of effective epitopes from SO7 and MZ5-7 antigens and the construction of multi-epitope DNA vaccines of E. tenella and their protective immunity against experimental E. tenella infection.

Materials and methods


Eimeria parasites are complex organisms with multiple life cycle stages. Furthermore, antigenic variation and other immune evasion mechanisms also complicate the development of vaccines against parasites. The complexity of Eimeria infections requires novel approaches to vaccine design. However, with recombinant DNA technology and the versatility of DNA vaccination, it is now possible to take rational parasite specific strategies to vaccine design. The goal of our multicomponent vaccine research is to develop effective multi-epitope DNA vaccines that induce effective and long-lasting protective immunity against E. tenella.

br Conclusion The results of

The results of this study highlight that foals can be infected by different Cryptosporidium genotypes and subtypes, some of them first described in horses. Moreover, we pointed out as EPU can favor the spread of Cryptosporidiosis that, having a zoonotic potential, could also represent a risk for humans.


This study was funded by “Progetti competitivi per Ricercatori” grant of Department of Veterinary Medical Sciences (DIMEVET), Alma Mater Studiorum – University of Bologna, Budget integrato 2011.

Avian coccidiosis is caused by the infection with Eimeria spp., contributing to major economic losses worldwide in poultry industries (Shirley and Lillehoj, 2012; Shirley et al., 2005). Anticoccidial drugs are used extensively to control the disease, but the increase of resistant parasite populations underlines the need to find alternative strategies (Blake and Tomley, 2014). One of the most effective approaches in the management of infectious diseases in veterinary practice is through the induction of protective immunity by vaccination.
The CD40 ligand (CD40L) is a glycoprotein expressed on the surface of activated T cells, basophils, and mast cells, which belongs to the tumor necrosis factor family (TNF) superfamily. Studies have demonstrated that the CD40-CD40L interaction can upregulate co-stimulatory molecules, activate antigen-presenting cells, and influence T-cell-mediated effector functions (Grewal and Flavell, 1998; Miga et al., 2000). Recently it has further been shown that murine CD40L can function as a molecular adjuvant enhancing both immune response and disease protection (Hashem et al., 2014; Lauterbach et al., 2013; Lin et al., 2009; Ninomiya et al., 2002). The immunostimulatory capacity of CD40L has also been observed in chickens and ducks (Layton et al., 2009; Pose et al., 2011; Sanchez Ramos et al., 2011; Tregaskes et al., 2005; Yao et al., 2010).
Immune mapped protein-1 (IMP1), a newly discovered protein in Eimeria maxima, has been demonstrated to be immunogenic and confer protection against E. maxima challenge in chickens (Blake et al., 2011). Recently, IMP1 has also been identified as an immunoprotective antigen from other apicomplexan parasites, such as Toxoplasma, Neospora and E. tenella (Cui et al., 2012a,b; Yin et al., 2013b). Immunization of dihydrofolate reductase inhibitor with the EtIMP1 protein reduces the oocyst output by 60%, comparable with the protective effects of other antigens, such as EtMIC1, EtMIC11 and profilin (Lillehoj et al., 2005; Sathish et al., 2012; Subramanian et al., 2008).
In the present study, we hypothesized that chicken CD40L may enhance immunogenicity of the IMP1 protein thus providing greater immune protection of chickens against Eimeria infection. To test this hypothesis, we investigated the adjuvant properties of CD40L for the EtIMP1 protein and evaluated the protective efficacy of the recombinant EtIMP1-CD40L fusion protein in chickens.

Materials and methods


In the present study, a recombinant chimeric subunit vaccine consisting of EtIMP1 and a molecular adjuvant, chicken CD40L, was generated, and its efficacy against E. tenella infection was evaluated. We found that immunization with EtIMP1-CD40L reduced oocyst output in chickens by 78%, more potently than that with EtIMP1 emulsified in FCA (by 66%). The findings support our hypothesis that CD40L, as an adjuvant for cell-mediated immunity, can improve the immunoprotective effect of IMPI against Eimeria infection.
Cell-mediated immunity appears to be the major component of the immune response in eliminating the infection of apicomplexan parasites, including E. tenella (Bhogal et al., 1989; Lillehoj and Trout, 1996; Rose and Hesketh, 1979). The vaccination with a recombinant antigen is often not sufficient to elicit a protective immune response against E. tenella infection (Crane et al., 1991; Jenkins, 1998; Miller et al., 1989). In this study, we demonstrated that the chicken CD40L-EtIMP1, a fusion protein, reduced oocyst output by 78%, more potently than EtIMP1 alone emulsified in FCA. In addition to having the strong antibody responses, chickens immunized with EtIMP1-CD40L also elicited a specific T-cell response, as evidenced by a greater IFN-γ production by T-cells in response to EtIMP1 stimulation ex vivo. Likely, CD40L, fused to EtIMP1, may increase the uptake and processing of EtIMP1 by antigen processing cells, thus resulting in more efficient stimulation of adaptive immune responses. This was consistent with the report that a genetically engineered adenovirus vector targeted to CD40 mediates transduction of canine dendritic cells and promotes antigen-specific immune responses in vivo (Thacker et al., 2009). In addition, recent research has shown that murine CD154 can function as an adjuvant for viral or DNA vaccines by administering adenoviral vectors or plasmids that express a vaccine antigen and CD154, individually, or as a fusion protein (Mendoza et al., 1997; Tripp et al., 2000; Xiang et al., 2001). Recently it has also been demonstrated that the extracellular domain of duck\’s CD154 could enhance DNA vaccine responses in ducks both by co-expression with viral antigens and as a fusion protein to target viral antigen to APCs (Yao et al., 2010).

dihydrofolate reductase inhibitor br Results br Discussion Our study of the CPXV protein


Our study of the CPXV219 protein was motivated by the very large size of the ORF and its conservation in most but not all chordopoxvirues. Indeed, members of some genera have multiple copies suggesting an important role. CPXV219 was synthesized within the first 4h of infection and in the presence of an inhibitor of DNA replication, indicating an early stage promoter. By constructing a recombinant virus with epitope tags at both ends, we detected the full-length protein, which migrated with an apparent mass of >260kDa, and more abundant 110kDa N-terminal and 160kDa C-terminal fragments by Western blotting under denaturing conditions. Immunoprecipitation experiments, however, indicated that the fragments remained associated prior to denaturation in vitro. The possibility that the C-terminal fragment arose from internal initiation was ruled out by its failure to form either when stop codons were introduced near the N-terminus or by removing the start codon. An increase in amount of full-size protein occurred when BFA was added, suggesting that proteolytic cleavage occurred during transit through the Golgi apparatus. Further studies demonstrated cleavage of a signal peptide and transit through the secretory pathway. Extensive N-glycosylation, also occurred during trafficking; approximately 45- and 10-kDa reductions in the masses of the N- and C-terminal fragments occurred following PNGase digestion. If we estimate that each N-glycosylation contributes 2.2kDa to protein mass, then approximately 20 of the 21 predicted sites in the N-terminal fragment were glycosylated and 4 or 5 of the 10 predicted sites in the C-terminus were glycosylated. The acquisition of endoH-resistance, particularly of the N-terminal fragment, was further evidence of Golgi or post-Golgi transport.
The CPXV219 protein was visualized throughout the secretory pathway including the plasma membrane and was detected in unpermeabilized dihydrofolate reductase inhibitor by labeling with fluorescent antibody to a C-terminal epitope tag and confocal microscopy. Based on the available data, we suggest the topological model in Fig. 8. In step I, CPXV219 is co-translationally inserted into the ER with cleavage of the signal peptide and glycosylation. In step II, the protein is transported to the Golgi network where modification of carbohydrate and proteolytic cleavage occur. In step III, the two fragments while still associated are transported to the plasma membrane. The C-terminus was considered to be outside of the plasma membrane because the epitope tag was accessible to antibody in unpermeabilized cells. The site of CPXV219 cleavage at approximately amino acid 600 of the full-length protein must have been in the ER based on the inhibition by BFA and at least part of the region between amino acid 1000 and 1800 must also have been in the ER since that is where the potential N-glycosylation sites are located. Assuming that the predicted TM near the C-terminus is utilized, then CPXV219 must traverse the membrane twice. There are two additional hydrophobic regions estimated between amino acids 925 to 938 and 1317 to 1325, one of which must traverse the membrane. Based on the location of the potential N-glycosylation sites, we predict that the more distal hydrophobic site was used. This would mean that a loop between amino acids 1325 and 1856 is cytoplasmic. The entire N-terminal fragment must have been in the ER based on the cleaved signal peptide, the extent of N-glycosylation and the absence of additional hydrophobic domains. Since fluorescence microscopy showed the N-terminal fragment colocalizing with the C-terminal fragment within the cytoplasm and at the plasma membrane and co-immunoprecipitating with the C-terminal fragment, we also placed it on the cell surface in our model. However, further experimentation is needed to directly confirm the predicted topology.
In a recent study Alzhanova et al. (2014) analyzed the monkeypox virus (MPXV) homolog of CPXV219. Their study and ours were consistent in providing evidence for proteolytic cleavage of the full-length protein and cell surface expression of the C-terminal fragment, despite methodological differences. In contrast to our approach, which involved construction of recombinant CPXV containing epitope tags at both the N- and C-termini, Alzhanova et al. (2014) relied on transient expression by plasmids in uninfected cells or by adenovirus vectors. Because the majority of their studies were carried out with only a C-terminal epitope tag, they did not demonstrate the formation of a stable N-terminal fragment.

dihydrofolate reductase inhibitor br Acknowledgements This work was financially supported by

This work was financially supported by Consejo de Desarrollo Científico, Humanístico, Tecnológico y de las Artes (CDCHTA-ULA) by Project C-1726-11-03-B and Misión Ciencia (FONACIT) by Projects 2007001425 and 2007000960. We thank Dr. Paul Michels (The University of Edinburgh) for the editing of the manuscript.

There are increased concerns about prospects for sustainable control of gastrointestinal parasites in grazing ruminants. These stem from a variety of risks, including the loss of infection resistance as hosts are selected for production intensity (Mackinnon et al., 1991), climate change effects on parasite dynamics (Skuce et al., 2013), and the increased incidence of parasite resistance to anthelmintics (Rose et al., 2015). Although the latter has been more commonly identified for small ruminants, there is increasing evidence that it is also happening for cattle (Edmonds et al., 2010; O’shaughnessy et al., 2014). Amongst others, Sutherland and Leathwick (2011) have reported parasite resistance to the three broad-spectrum anthelmintic dihydrofolate reductase inhibitor (benzimidazoles, levamisole and macrocyclic lactones) used on cattle.
For this reason there is a need to develop strategies that would enable sustainable control of gastrointestinal parasites and maintain the effectiveness of chemoprophylaxis (Charlier et al., 2014). Several strategies that may achieve this have been proposed, including targeted selective treatment (TST), breeding cattle resistant to parasites and grazing management. Testing for the effectiveness and interactions of such strategies is very difficult both experimentally and in practice. This is due to cost and difficulties in making fair comparisons, in the absence of confounding variables; for example although traits have been independently evaluated for TST in cattle, a direct comparison with other applied control strategies has not yet been conducted (Höglund et al., 2009, 2013).
Recently, simulation models have been used to make such direct comparisons for control strategies on parasitised sheep (Laurenson et al., 2012a, 2013a,b). Investigating the consequences of such strategies in silico for cattle may be one cost effective and time efficient way of overcoming the above limitations. Currently there are only two simulation models which investigate host-parasite interactions for cattle (Smith et al., 1987; Ward, 2006a). Both models have their limitations; for example, the former model cannot make predictions about the consequences of parasitism on performance, whereas the latter uses bodyweight as the only descriptor of the animal. The objective of this paper was to develop a novel simulation model to account for the interactions between Ostertagia ostertagi, the most prevalent parasite of cattle worldwide, particularly in temperate regions (Tisdell et al., 1999), and immunologically naïve calves, which are most at risk from parasitism. Emphasis in model development was given to accounting for within host parasite dynamics and their effects on host performance. The model was developed with the view of introducing between-animal variation in later steps.

Materials and methods


The overall aim of this paper was to develop a model that accounted for the interactions between O. ostertagi parasitism and first season grazing calves, under UK conditions. Although the model was deterministic, it was constructed with the view of developing it into a stochastic one, to allow for the investigation of different methods of control of the parasite, including selection for host resistance (Laurenson et al., 2012a). Larval intake was considered an input to the model, but there are plans to account for parasite populations in the environment in the manner similar to Laurenson et al. (2012a).
Although there are a number of models focusing on predicting the epidemiology of O. ostertagi (Gettinby et al., 1979; Gettinby and Paton, 1981; Chaparro and Canziani, 2010), currently there are only two models that specifically aim to investigate within-host interactions between calf host and O. ostertagi. The PARABAN model (Smith and Grenfell, 1985; Smith et al., 1987; Grenfell et al., 1987a, 1987b) was specifically developed to account for the rate of change in parasite populations within hosts and the environment, and has been used to investigate the effectiveness of anthelmintic treatment on parasite dynamics. This model, however, does not account for the consequences of parasitism on host performance and its creators recognised its limitations in this respect (Smith, 1997).