Selection of genotypes harbouring mutations in the cytochrome b gene of Theileria annulata is associated with resistance to buparvaquone.

Buparvaquone remains the only effective therapeutic agent for the treatment of tropical theileriosis caused by Theileria annulata. However, an increase in the rate of buparvaquone treatment failures has been observed in recent years, raising the possibility that resistance to this drug is associated with the selection of T. annulata genotypes bearing mutation(s) in the cytochrome b gene (Cyto b). The aim of the present study was: (1) to demonstrate whether there is an association between mutations in the T. annulata Cyto b gene and selection of parasite-infected cells resistant to buparvaquone and (2) to determine the frequency of these mutations in parasites derived from infected cattle in the Aydin region of Türkiye. Susceptibility to buparvaquone was assessed by comparing the proliferative index of schizont-infected cells obtained from cattle with theileriosis before and/or after treatment with various doses of buparvaquone, using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colourimetric assay. The DNA sequence of the parasite Cyto b gene from cell lines identified as resistant or susceptible was determined. A total of six nonsynonymous and six synonymous mutations were identified. Two of the nonsynonymous mutations resulted in the substitutions V135A and P253S which are located at the putative buparvaquone binding regions of cytochrome b. Allele-specific PCR (AS-PCR) analyses detected the V135A and P253S mutations at a frequency of 3.90% and 3.57% respectively in a regional study population and revealed an increase in the frequency of both mutations over the years. The A53P mutation of TaPIN1 of T. annulata, previously suggested as being involved in buparvaquone resistance, was not detected in any of the clonal cell lines examined in the present study. The observed data strongly suggested that the genetic mutations resulting in V135A and P253S detected at the putative binding sites of buparvaquone in cytochrome b play a significant role in conferring, and promoting selection of, T. annulata genotypes resistant to buparvaquone, whereas the role of mutations in TaPIN1 is more equivocal.

Measurement of Adenovirus-Based Vector Heterogeneity.

Adenovirus vectors have become an important class of vaccines with the recent approval of Ebola and COVID-19 products. In-process quality attribute data collected during Adenovirus vector manufacturing has focused on particle concentration and infectivity ratios (based on viral genome: cell-based infectivity), and data suggest only a fraction of viral particles present in the final vaccine product are efficacious. To better understand this product heterogeneity, lab-scale preparations of two Adenovirus viral vectors, (Chimpanzee adenovirus (ChAdOx1) and Human adenovirus Type 5 (Ad5), were studied using transmission electron microscopy (TEM). Different adenovirus morphologies were characterized, and the proportion of empty and full viral particles were quantified. These proportions showed a qualitative correlation with the sample's infectivity values. Liquid chromatography-mass spectrometry (LC-MS) peptide mapping was used to identify key adenovirus proteins involved in viral maturation. Using peptide abundance analysis, a ∼5-fold change in L1 52/55k abundance was observed between low-(empty) and high-density (full) fractions taken from CsCl ultracentrifugation preparations of ChAdOx1 virus. The L1 52/55k viral protein is associated with DNA packaging and is cleaved during viral maturation, so it may be a marker for infective particles. TEM and LC-MS peptide mapping are promising higher-resolution analytical characterization tools to help differentiate between relative proportions of empty, non-infectious, and infectious viral particles as part of Adenovirus vector in-process monitoring, and these results are an encouraging initial step to better differentiate between the different product-related impurities.

Assessing and Promoting Independence in Young Adults with Autism Spectrum Disorder.

OBJECTIVE: Quality of life, independence, and employment outcomes are poor for young adults with autism spectrum disorder (YA-ASD). This study explored the desires and experiences of YA-ASD as they transition into adulthood. METHODS: Fifteen YA-ASD, age 18 to 25 years, were recruited from autism spectrum disorder centers, participant registries, and social media advertising. Interested individuals completed a survey and individual interview. Semistructured interview guides included questions about transition experiences, current independence, and future goals. Interview transcripts were analyzed using thematic analysis. RESULTS: The desire of young adults with autism spectrum disorder for independence was shown within 4 themes. YA-ASD described their vision of adulthood along with their need for improved skills in driving, living independently, and decision-making. CONCLUSION: The findings indicate YA-ASD desire to be independent but lack the specific support services to get there.

IL-39 Reduces Proliferation and Promotes Apoptosis of Bladder Cancer by Altering the Activity of Cyclin E and Fas.

BACKGROUND/AIM: This study was designed to investigate the effect of IL-39 on T24 bladder cancer (BC) cell line survival and growth. MATERIALS AND METHODS: In order to assess the direct effect of IL-39 on survival, proliferation, and apoptosis of T24 BC cells, we utilized a clonogenic survival assay, a cell proliferation assay, and caspase-3 activity kits. Potential proliferative and apoptotic molecular mechanisms were evaluated by RT-PCR. RESULTS: Treatment of T24 BC cells with IL-39 resulted in a significant reduction in the percentage of colonies. The anti-tumor effect of IL-39 on T24 bladder cancer cells correlated strongly with a decrease in cyclin E, in combination with an increase in the mRNA levels of Fas. CONCLUSION: IL-39 impedes the growth and survival of T24 BC cells by inhibiting growth and promoting apoptosis. This ability to modulate gene transcription in neoplastic cells shows promise and warrants further research in immunotherapy.

Understanding spatial and temporal variability of N leaching reduction by winter cover crops under climate change.

Winter cover crops are sown in between main spring crops (e.g. cash and forage crops) to provide a range of benefits, including the reduction of nitrogen (N) leaching losses to groundwater. However, the extent by which winter cover crops will remain effective under future climate change is unclear. We assess variability and uncertainty of climate change effects on the reduction of N leaching by winter oat cover crops. Field data were collected to quantify ranges of cover crop above-ground biomass (7 to 10 t DM/ha) and N uptake (70 to 180 kg N/ha) under contrasting initial soil conditions. The data were also used to evaluate the APSIM-NextGen model (R2 from 62 to 96% and RMSEr from 7 to 50%), which was then applied to simulate cover crop and fallow conditions across four key agricultural locations in New Zealand, under baseline and future climate scenarios. Cover crops reduced N leaching risks for all location/scenario combinations but with large variability in space and time (e.g. 21 to 47% of fallow) depending on the climate change scenario. For instance, end-of-century estimates for northern (warmer) locations mostly showed non-significant effects of climate change on cover crop effectiveness and N leaching. In contrast for southern (colder) locations, there was a systematic increase in N leaching risks with climate change intensity despite a concomitant, but less than proportional, increase in cover crop effectiveness (up to ~5% of baseline) due to higher winter yields and N uptake. This implies that climate change may not only modify the geography of N leaching hotspots, but also the extent by which cover crops can locally reduce pollution risks, in some cases requiring complementary adaptive measures. The patchy- and threshold-nature of leaching events indicates that fine spatio-temporal resolutions are better suited to evaluate cover crop effectiveness under climate change.

Development and Initial Testing of a Health-Related Independence Measure for Autistic Young Adults as Reported by Caregivers.

Background: Becoming an adult comes with education, work, living, and health-related transitions. Health care transition (HCT) services help adolescents prepare for a smooth transition to adult care, ensure health insurance retention, and promote adolescents' independent management of health care and life needs. Lack of HCT services can result in negative outcomes such as unmet needs, overmedication, and loss of decision-making authority. Autistic young adults (AYA) are half as likely to receive HCT services compared with special needs young adults. Furthermore, there are no HCT readiness measures that address the unique needs of AYA. Methods: This study used a mixed-methods approach to develop and test a holistic caregiver-reported measure of HCT readiness for AYA Health-Related Independence (HRI). The phases used to create and test the HRI measure included: (1) construct and question topic development through qualitative data collection with AYA and caregivers; (2) question development with clinicians and caregivers; and (3) initial question testing utilizing cognitive interviews and pretesting of the instrument with caregivers. Results: Measure constructs were developed based on qualitative findings from AYA (n = 27) and caregivers (n = 39). The researchers identified 12 themes related to HRI from the data. Next, questions were developed for each theme by caregivers (n = 5) and clinicians (n = 25). Finally, questions and the survey format were tested using caregiver feedback in the form of cognitive interviews (n = 15) and pretests (n = 21). The final version of the caregiver-reported HRI measure included 8 constructs and 58 questions. Conclusion: The development of the HRI measure was a comprehensive and iterative process. This article highlights the measurement development process and its potential impact on AYA, caregivers, and clinicians. Lay summary: Why was this study done?: Health care transition services help youth keep their health insurance, transition to an adult doctor smoothly, and promote independence. To date, there is no health care transition intervention for autistic young adults. Few studies have examined how to prepare autistic young adults to manage their health and self-care needs and the transition to an adult model of care. We wanted to fill in these gaps by creating a measure of health care transition readiness for autistic young adults.What was the purpose of this study?: The purpose of the study was to develop the Health-Related Independence measure based on autistic young adult and caregiver input. We define Health-Related Independence as a young adult's ability to manage their health, healthcare, and safety needs. We also wanted to examine the measure to make sure it was easy to read, made sense, and was easy to answer.What did the researchers do?: We used a mixed-methods approach to develop and test the Health-Related Independence measure. There were three parts to the study: (1) we conducted individual interviews with autistic young adults and focus groups with caregivers to understand what topics should be included in the measure, (2) clinicians and caregivers then used those topics to create specific survey questions, (3) we conducted interviews and online pretest of the measure with caregivers.What were the results of the study?: The autistic young adults and caregivers identified twelve topics/themes to include in the Health-Related Independence Measure. Caregiver feedback helped make the measure shorter and easier to understand and complete. The final version of the caregiver-reported HRI measure included 58 questions.What do these findings add to what was already known?: We learned that young adults and caregivers have a broad understanding of health-related independence such as safety and sexuality/relationship knowledge. There weren't any measures to capture these ideas. This study created an important new measure that can be used in healthcare clinics, schools, and at home.What are potential weaknesses in the study?: This study aimed to work with autistic young adults to develop the Health-Related Independence measure, but due to funding and study limitations, we only included young adults in the 1st phase of the study. Caregivers were used as proxy reporters in phases 2 and 3. Not including autistic young adults in phases 2 and 3 was a weakness of the study. Future research should aim to fully incorporate young adults into the research process. Their views should inform the development of the qualitative interview guides and all portions of the study.How will these findings help autistic adults now or in the future?: The Health-Related Independence measure can help caregivers and health care providers identify areas in which the autistic young adults are successful and areas of needed improvement to assist in the successful transition to adult care and adult life. The authors are currently working on a study proposal to validate the Health-Related Independence measure as a self-assessment tool for young adults to take themselves.

Adapting crop rotations to climate change in regional impact modelling assessments.

The environmental and economic sustainability of future cropping systems depends on adaptation to climate change. Adaptation studies commonly rely on agricultural systems models to integrate multiple components of production systems such as crops, weather, soil and farmers' management decisions. Previous adaptation studies have mostly focused on isolated monocultures. However, in many agricultural regions worldwide, multi-crop rotations better represent local production systems. It is unclear how adaptation interventions influence crops grown in sequences. We develop a catchment-scale assessment to investigate the effects of tactical adaptations (choice of genotype and sowing date) on yield and underlying crop-soil factors of rotations. Based on locally surveyed data, a silage-maize followed by catch-crop-wheat rotation was simulated with the APSIM model for the RCP 8.5 emission scenario, two time periods (1985-2004 and 2080-2100) and six climate models across the Kaituna catchment in New Zealand. Results showed that direction and magnitude of climate change impacts, and the response to adaptation, varied spatially and were affected by rotation carryover effects due to agronomical (e.g. timing of sowing and harvesting) and soil (e.g. residual nitrogen, N) aspects. For example, by adapting maize to early-sowing dates under a warmer climate, there was an advance in catch crop establishment which enhanced residual soil N uptake. This dynamics, however, differed with local environment and choice of short- or long-cycle maize genotypes. Adaptation was insufficient to neutralize rotation yield losses in lowlands but consistently enhanced yield gains in highlands, where other constraints limited arable cropping. The positive responses to adaptation were mainly due to increases in solar radiation interception across the entire growth season. These results provide deeper insights on the dynamics of climate change impacts for crop rotation systems. Such knowledge can be used to develop improved regional impact assessments for situations where multi-crop rotations better represent predominant agricultural systems.

Flavonoid composition of orange peel and its association with antioxidant and anti-inflammatory activities.

Dried citrus peel derived from Citrus reticulata, also called "chenpi", possesses a complex mixture of flavonoids and has a history of traditional use to treat a variety of digestive disorders. We compared three sources of conventional chenpi from California (USA), Guangxi, Zhejiang, and two sources of "nchenpi", which contain greater nobiletin content, from Sichuan and Xinhui (China). Xinhui orange peel extract (OPE) had highest content of polymethoxylated flavones, along with greatest capacity to scavenge 2,2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), 2,2-diphenyl-1-pcrylhydrazyl (DPPH), and 2,2'-azobis-2-methyl-propanimidamide, dihydrochloride (AAPH) radicals and nitric oxide (NO). OPE also had higher NO, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) inhibitory activity than an equivalent mixture of flavonoids (P<0.05). In conclusion, nobiletin is a good chemical marker for assessing the anti-inflammatory potential of OPE from different sources. Obtaining "nchenpi" from either Sichuan or Xinhui provided potentially superior health benefits compared to conventional chenpi sources.

Identification and Analysis of Immunodominant Antigens for ELISA-Based Detection of Theileria annulata.

Tropical or Mediterranean theileriosis, caused by the protozoan parasite Theileria annulata, remains an economically important bovine disease in North Africa, Southern Europe, India, the Middle East and Asia. The disease affects mainly exotic cattle and imposes serious constraints upon livestock production and breed improvement programmes. While microscopic and molecular methods exist which are capable of detecting T. annulata during acute infection, the identification of animals in the carrier state is more challenging. Serological tests, which detect antibodies that react against parasite-encoded antigens, should ideally have the potential to identify carrier animals with very high levels of sensitivity and specificity. However, assays developed to date have suffered from a lack of sensitivity and/or specificity and it is, therefore, necessary to identify novel parasite antigens, which can be developed for this purpose. In the present study, genes encoding predicted antigens were bioinformatically identified in the T. annulata genome. These proteins, together with a panel of previously described antigens, were assessed by western blot analysis for immunoreactivity, and this revealed that four novel candidates and five previously described antigens were recognised by immune bovine serum. Using a combination of immunoprecipitation and mass spectrophotometric analysis, an immunodominant protein (encoded by TA15705) was identified as Ta9, a previously defined T cell antigen. Western blotting revealed another of the five proteins in the Ta9 family, TA15710, also to be an immunodominant protein. However, validation by Enzyme-Linked Immunosorbent Assay indicated that due to either allelic polymorphism or differential immune responses of individual hosts, none of the novel candidates can be considered ideal for routine detection of T. annulata-infected/carrier animals.

The emergence of resistance to the benzimidazole anthlemintics in parasitic nematodes of livestock is characterised by multiple independent hard and soft selective sweeps.

Anthelmintic resistance is a major problem for the control of parasitic nematodes of livestock and of growing concern for human parasite control. However, there is little understanding of how resistance arises and spreads or of the "genetic signature" of selection for this group of important pathogens. We have investigated these questions in the system for which anthelmintic resistance is most advanced; benzimidazole resistance in the sheep parasites Haemonchus contortus and Teladorsagia circumcincta. Population genetic analysis with neutral microsatellite markers reveals that T. circumcincta has higher genetic diversity but lower genetic differentiation between farms than H. contortus in the UK. We propose that this is due to epidemiological differences between the two parasites resulting in greater seasonal bottlenecking of H. contortus. There is a remarkably high level of resistance haplotype diversity in both parasites compared with drug resistance studies in other eukaryotic systems. Our analysis suggests a minimum of four independent origins of resistance mutations on just seven farms for H. contortus, and even more for T. circumincta. Both hard and soft selective sweeps have occurred with striking differences between individual farms. The sweeps are generally softer for T. circumcincta than H. contortus, consistent with its higher level of genetic diversity and consequent greater availability of new mutations. We propose a model in which multiple independent resistance mutations recurrently arise and spread by migration to explain the widespread occurrence of resistance in these parasites. Finally, in spite of the complex haplotypic diversity, we show that selection can be detected at the target locus using simple measures of genetic diversity and departures from neutrality. This work has important implications for the application of genome-wide approaches to identify new anthelmintic resistance loci and the likelihood of anthelmintic resistance emerging as selection pressure is increased in human soil-transmitted nematodes by community wide treatment programs.

Probing the interaction between U24 and the SH3 domain of Fyn tyrosine kinase.

The putative membrane protein U24 from HHV-6A shares a seven-residue sequence identity (which includes a PxxP motif) with myelin basic protein (MBP), a protein responsible for the compaction of the myelin sheath in the central nervous system. U24 from HHV-6A also shares a PPxY motif with U24 from the related virus HHV-7, allowing them both to block early endosomal recycling. Recently, MBP has been shown to have protein-protein interactions with a range of proteins, including proteins containing SH3 domains. Given that this interaction is mediated by the proline-rich segment in MBP, and that similar proline-rich segments are found in U24, we investigate here whether U24 also interacts with SH3 domain-containing proteins and what the nature of that interaction might be. The implications of a U24-Fyn tyrosine kinase SH3 domain interaction are discussed in terms of the hypothesis that U24 may function like MBP through molecular mimicry, potentially contributing to the disease state of multiple sclerosis or other demyelinating disorders.

Differential response in downstream processing of CHO cells grown under mild hypothermic conditions.

The manufacture of complex therapeutic proteins using mammalian cells is well established, with several strategies developed to improve productivity. The application of sustained mild hypothermic conditions during culture has been associated with increases in product titer and improved product quality. However, despite associated cell physiological effects, very few studies have investigated the impact on downstream processing (DSP). Characterization of cells grown under mild hypothermic conditions demonstrated that the stationary phase was prolonged by delaying the onset of apoptosis. This enabled cells to maintain viability for extended periods and increase volumetric productivity from 0.74 to 1.02 g L(-1) . However, host cell proteins, measured by ELISA, increased by ∼50%, attributed to the extended time course and higher peak and harvest cell densities. The individual components making up this impurity, as determined by SELDI-TOF MS and 2D-PAGE, were shown to be largely comparable. Under mild hypothermic conditions, cells were less shear sensitive than those maintained at 37°C, enhancing the preliminary primary recovery step. Adaptive changes in membrane fluidity were further investigated by adopting a pronounced temperature shift immediately prior to primary recovery and the improvement observed suggests that such a strategy may be implementable when shear sensitivity is of concern. Early and late apoptotic cells were particularly susceptible to shear, at either temperature, even under the lowest shear rate investigated. These findings demonstrate the importance of considering the impact of cell culture strategies and cell physiology on DSP, by implementing a range of experimental methods for process characterization.

The dynamics of the CHO host cell protein profile during clarification and protein A capture in a platform antibody purification process.

Recombinant protein products such as monoclonal antibodies (mAbs) for use in the clinic must be clear of host cell impurities such as host cell protein (HCP), DNA/RNA, and high molecular weight immunogenic aggregates. Despite the need to remove and monitor HCPs, the nature, and fate of these during downstream processing (DSP) remains poorly characterized. We have applied a proteomic approach to investigate the dynamics and fate of HCPs in the supernatant of a mAb producing cell line during early DSP including centrifugation, depth filtration, and protein A capture chromatography. The primary clarification technique selected was shown to influence the HCP profile that entered subsequent downstream steps. MabSelect protein A chromatography removed the majority of contaminating proteins, however using 2D-PAGE we could visualize not only the antibody species in the eluate (heavy and light chain) but also contaminant HCPs. These data showed that the choice of secondary clarification impacts upon the HCP profile post-protein A chromatography as differences arose in both the presence and abundance of specific HCPs when depth filters were compared. A number of intracellularly located HCPs were identified in protein A elution fractions from a Null cell line culture supernatant including the chaperone Bip/GRP78, heat shock proteins, and the enzyme enolase. We demonstrate that the selection of early DSP steps influences the resulting HCP profile and that 2D-PAGE can be used for monitoring and identification of HCPs post-protein A chromatography. This approach could be used to screen cell lines or hosts to select those with reduced HCP profiles, or to identify HCPs that are problematic and difficult to remove so that cell-engineering approaches can be applied to reduced, or eliminate, such HCPs.

Impact of aeration strategy on CHO cell performance during antibody production.

Stirred tank bioreactors using suspension adapted mammalian cells are typically used for the production of complex therapeutic proteins. The hydrodynamic conditions experienced by cells within this environment have been shown to directly impact growth, productivity, and product quality and therefore an improved understanding of the cellular response is critical. Here we investigate the sub-lethal effects of different aeration strategies on Chinese hamster ovary cells during monoclonal antibody production. Two gas delivery systems were employed to study the presence and absence of the air-liquid interface: bubbled direct gas sparging and a non-bubbled diffusive silicone membrane system. Additionally, the effect of higher gas flow rate in the sparged bioreactor was examined. Both aeration systems were run using chemically defined media with and without the shear protectant Pluronic F-68 (PF-68). Cells were unable to grow with direct gas sparging without PF-68; however, when a silicone membrane aeration system was implemented growth was comparable to the sparged bioreactor with PF-68, indicating the necessity of shear protectants in the presence of bubbles. The cultures exposed to increased hydrodynamic stress were shown by flow cytometry to have decreased F-actin intensity within the cytoskeleton and enter apoptosis earlier. This indicates that these conditions elicit a sub-lethal physiological change in cells that would not be detected by the at-line assays which are normally implemented during cell culture. These physiological changes only result in a difference in continuous centrifugation performance under high flow rate conditions. Product quality was more strongly affected by culture age than the hydrodynamic conditions tested.

Host cell protein adsorption characteristics during protein A chromatography.

Protein A chromatography is a critical and 'gold-standard' step in the purification of monoclonal antibody (mAb) products. Its ability to remove >98% of impurities in a single step alleviates the burden on subsequent process steps and facilitates the implementation of platform processes, with a minimal number of chromatographic steps. Here, we have evaluated four commercially available protein A chromatography matrices in terms of their ability to remove host cell proteins (HCPs), a complex group of process related impurities that must be removed to minimal levels. SELDI-TOF MS was used as a screening tool to generate an impurity profile fingerprint for each resin and indicated a number of residual impurities present following protein A chromatography, agreeing with HCP ELISA. Although many of these were observed for all matrices there was a significantly elevated level of impurity binding associated with the resin based on controlled pore glass under standard conditions. Use of null cell line supernatant with and without spiked purified mAb demonstrated the interaction of HCPs to be not only with the resin back-bone but also with the bound mAb. A null cell line column overload and sample enrichment method before 2D-PAGE was then used to determine individual components associated with resin back-bone adsorption. The methods shown allow for a critical analysis of HCP removal during protein A chromatography. Taken together they provide the necessary process understanding to allow process engineers to identify rational approaches for the removal of prominent HCPs.

A survey of the trichostrongylid nematode species present on UK sheep farms and associated anthelmintic control practices.

A survey of sheep farms from across the UK was conducted to establish information on farming practices, the trichostrongylid nematode species present and anthelmintic usage. Questionnaires and faecal samples were returned from 118 farms. First stage larvae (L(1)) were cultured from faecal samples and used for PCR analysis to determine the presence/absence of selected trichostrongylid species. Teladorsagia circumcincta was the only species present on 100% of farms. Haemonchus contortus was found on ∼50% of farms and was widespread throughout the UK. The most common Trichostrongylus spp. was T. vitrinus, found on 95% of farms. Determining the anthelmintic dose rate based on the weight of the heaviest animal in the flock to avoid under dosing was carried out on 58% of farms and was associated with a significantly lower mean epg (p<0.001) in lambs. However, the weight of animals was only estimated (as opposed to animals weighed) on 32% of farms. Macrocyclic lactones (ML) were the most commonly used anthelmintic class for ewes, whilst benzimidazoles (BZ) were the most widely used in lambs. Twenty-two of the surveyed farms had confirmed anthelmintic resistance, of these, 18 had BZ resistance, one had levamisole (LEV) resistance and 3 had resistance to both BZ and LEV. No farms in this survey reported resistance to ML. Location had a significant effect on the incidence of anthelmintic resistance on the farms in this survey (p=0.002). There was evidence of a lower risk of anthelmintic resistance occurring on farms from Scotland compared to those in England (p(f)=0.047) and Wales (p(f)=0.012). Farm type, flock type and open or closed status did not have any significant effect on the incidence of anthelmintic resistance when all other factors were taken into consideration.

Overexpression and purification of U24 from human herpesvirus type-6 in E. coli: unconventional use of oxidizing environments with a maltose binding protein-hexahistine dual tag to enhance membrane protein yield.

BACKGROUND: Obtaining membrane proteins in sufficient quantity for biophysical study and biotechnological applications has been a difficult task. Use of the maltose binding protein/hexahistidine dual tag system with E.coli as an expression host is emerging as a high throughput method to enhance membrane protein yield, solubility, and purity, but fails to be effective for certain proteins. Optimizing the variables in this system to fine-tune for efficiency can ultimately be a daunting task. To identify factors critical to success in this expression system, we have selected to study U24, a novel membrane protein from Human Herpesvirus type-6 with potent immunosuppressive ability and a possible role in the pathogenesis of the disease multiple sclerosis. RESULTS: We expressed full-length U24 as a C-terminal fusion to a maltose binding protein/hexahistidine tag and examined the effects of temperature, growth medium type, cell strain type, oxidizing vs. reducing conditions and periplasmic vs. cytoplasmic expression location. Temperature appeared to have the greatest effect on yield; at 37°C full-length protein was either poorly expressed (periplasm) or degraded (cytoplasm) whereas at 18°C, expression was improved especially in the periplasm of C41(DE3) cells and in the cytoplasm of oxidizing Δtrx/Δgor mutant strains, Origami 2 and SHuffle. Expression of the fusion protein in these strains were estimated to be 3.2, 5.3 and 4.3 times greater, respectively, compared to commonly-used BL21(DE3) cells. We found that U24 is isolated with an intramolecular disulfide bond under these conditions, and we probed whether this disulfide bond was critical to high yield expression of full-length protein. Expression analysis of a C21SC37S cysteine-free mutant U24 demonstrated that this disulfide was not critical for full-length protein expression, but it is more likely that strained metabolic conditions favour factors which promote protein expression. This hypothesis is supported by the fact that use of minimal media could enhance protein production compared to nutrient-rich LB media. CONCLUSIONS: We have found optimal conditions for heterologous expression of U24 from Human Herpesvirus type-6 in E.coli and have demonstrated that milligram quantities of pure protein can be obtained. Strained metabolic conditions such as low temperature, minimal media and an oxidizing environment appeared essential for high-level, full-length protein production and this information may be useful for expressing other membrane proteins of interest.

Prime-boost immunisation against tropical theileriosis with two parasite surface antigens: evidence for protection and antigen synergy.

Current methods for control of tropical theileriosis in cattle suffer from several disadvantages that could be circumvented by development of an effective sub-unit vaccine. Previous work has utilised two major surface antigens (SPAG-1 and Tams1) and conventional adjuvants to provide partial protection against parasite challenge. In this study we have delivered these antigens using the prime-boost system and analysed whether a combination regime can enhance protection against lethal challenge. Delivery of the boost as recombinant protein or expressed from a recombinant MVA vector was also assessed. The results confirmed that immunisation with Tams1 alone could reduce the severity of several disease parameters compared to non-immunised controls and these effects were more marked when recombinant protein was used for boosting compared to MVA delivery. A similar outcome was obtained by immunisation with SPAG-1 alone. Significantly, delivery of SPAG-1 and Tams1 as a cocktail showed enhanced protection. This was manifest by significant improvement in a large range of clinical and parasitological parameters and, most dramatically, by the survival and recovery of 50% of the immunised animals compared to 0% of the controls. Analysis of the antibody response post-challenge showed that while there was a strong response to Tams1, no response to SPAG-1 was detected. In contrast, lymphoproliferation assays showed a significant enhancement of response at day 7 post-challenge in calves of the SPAG-1 group but a dramatic decrease of the proliferation activity in all three groups receiving Tams1. We conclude that immunisation with a cocktail of SPAG-1 and Tams1 generates a synergistic protective response that significantly improves the efficacy of recombinant vaccination against tropical theileriosis. Potential effector mechanisms that could mediate this response are discussed.

Population diversity and multiplicity of infection in Theileria annulata.

The tick-borne apicomplexan parasite Theileria annulata is endemic in many sub-tropical countries and causes the bovine disease tropical theileriosis. Although the parasite is known to be highly diverse, detailed information is lacking on the genetic structure of natural populations and levels of multiplicity of infection in the cattle host. With the widespread deployment of live attenuated vaccines and the emergence of drug-resistant parasites in the field, it is vital to appreciate the factors which shape genetic diversity of the parasite both within individual hosts and in the wider population. This study addresses these issues and represents an extensive genetic analysis of T. annulata populations in two endemic countries utilising a high-throughput adaptation of a micro- and mini-satellite genotyping system. Parasite material was collected from infected cattle in defined regions of Turkey and Tunisia to allow a variety of analyses to be conducted. All animals (n=305) were found to harbour multiple parasite genotypes and only two isolates shared an identical predominant multi-locus profile. A modelling approach was used to demonstrate that host age, location and vaccination status play a measurable role in determining multiplicity of infection in an individual animal. Age was shown to positively correlate with multiplicity of infection and while positive vaccination status exerted a similar effect, it was shown to be due not simply to the presence of the immunising genotype. Importantly, no direct evidence was found for the immunising genotype spreading or recombining within the local parasite community. Genetic analysis confirmed the tentative conclusion of a previous study that the parasite population appears to be, in general, panmictic. Nevertheless, evidence supporting linkage disequilibrium and a departure from panmixia was uncovered in some localities and a number of explanations for these findings are advanced.

Primary carcinosarcoma of the parotid gland.

Carcinosarcoma is a rare malignant 'mixed' tumour in the head and neck region. We present a case of carcinosarcoma in a long standing parotid lump and share our experience in the management of the disease together with a review of recent English literature on the subject.