In Vitro Antimalarial Activity of Trichothecenes against Liver and Blood Stages of Plasmodium Species.

Trichothecenes (TCNs) are a large group of tricyclic sesquiterpenoid mycotoxins that have intriguing structural features and remarkable biological activities. Herein, we focused on three TCNs (anguidine, verrucarin A, and verrucarol) and their ability to target both the blood and liver stages of Plasmodium species, the parasite responsible for malaria. Anguidine and verrucarin A were found to be highly effective against the blood and liver stages of malaria, while verrucarol had no effect at the highest concentration tested. However, these compounds were also found to be cytotoxic and, thus, not selective, making them unsuitable for drug development. Nonetheless, they could be useful as chemical probes for protein synthesis inhibitors due to their direct impact on parasite synthesis processes.

Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis.

Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.

Potential benefits of gene editing for the future of poultry farming.

The chicken is an exemplar of efficient intensive animal agriculture and provides two valuable food products, chicken meat and eggs. Only aquaculture is better, by efficiency, but poultry is still top, by mass of animal protein produced as food in the global context. However this efficiency and intensive production comes with a number of challenges. Though the genetics of selective breeding have led to dramatic improvements in yield, efficiency and product quality, traits that relate to disease and welfare outcomes have not been so tractable. These two issues are major impacts to the industry in terms of production and in terms of public perception. Both transgenic technology and genome editing have clear potential for impact in these two important areas. The reproductive biology of birds requires techniques very specific to birds to achieve heritable (germline) edited traits. These are quite involved and, even though they are now well-defined and reliable, there is room for improvement and advances can be expected in the future. Currently the key targets for this technology are modifying chicken genes involved in virus-receptor interactions and cellular response involved in infection. For the egg industry the technology is being applied to the issue of sex-selection for layer hens (and the removal of males), removal of allergens from egg white and the tailoring of eggs system to enhance the yield of influenza vaccine doses. Regulation and trading of the animals generated, and resulting food products, will significantly impact the value and future development of genome editing for poultry.

Overexpressing ovotransferrin and avian ß-defensin-3 improves antimicrobial capacity of chickens and poultry products.

Zoonotic and foodborne diseases pose a significant burden, decreasing both human and animal health. Modifying chickens to overexpress antimicrobials has the potential to decrease bacterial growth on poultry products and boost chicken innate immunity. Chickens overexpressing either ovotransferrin or avian ß-defensin-3 (AvßD3) were generated using Tol-2 transposons. Transgene expression at the RNA and protein level was seen in egg white, breast muscle, and serum. There were significant differences in the immune cell populations in the blood, bursa, and spleen associated with transgene expression including an increased proportion of CD8+ cells in the blood of ovotransferrin and AvßD3 transgenic birds. Expression of the antimicrobials inhibited the in vitro growth of human and chicken bacterial pathogens and spoilage bacteria. For example, transgene expression significantly reduced growth of aerobic and coliform bacteria in breast muscle and decreased the growth of Salmonella enterica in egg white. Overall these results indicate that overexpression of antimicrobials in the chicken can impact the immune system and increase the antimicrobial capacity of poultry products.

Salmonella enterica subsp. salamae serovar Sofia, a prevalent serovar in Australian broiler chickens, is also capable of transient colonisation in layers.

1. Salmonella enterica subsp. salamae serovar sofia (S. sofia) is a prevalent strain of Salmonella in Australian broilers and has been isolated from broiler chickens, litter, dust, as well as pre- and post-processing carcasses, and retail chicken portions but has never been reported in commercial Australian layers or eggs. 2. To investigate whether a S. sofia isolate from a broiler could colonise layers, one-month-old Hyline brown layers were orally inoculated with S. sofia and colonisation was monitored for 2-4 weeks. 3. Overall, 30-40% of the chickens shed S. sofia from the cloaca between 6 and 14 d post-inoculation which then declined to 10% by d 21. Necropsy at 2 weeks post-inoculation revealed 80% of birds harboured S. sofia in the caecum, whilst, by 4 weeks post-infection, no chickens were colonised with S. sofia in the gastrointestinal tract, liver or spleen. Additionally, no aerosol 'bird to bird' transfer was evident. 4. This study demonstrated that laying hens can be colonised by broiler-derived S. sofia; however, this colonisation was transient, reaching a peak at 14 d post-inoculation, and was completely cleared by 28 d post-inoculation. The transience of colonisation of S. sofia in layers could be a factor explaining why S. sofia has never been detected when screening for Salmonella serotypes found in Australian laying hens or eggs.

Young Pigs Consuming Lysozyme Transgenic Goat Milk Are Protected from Clinical Symptoms of Enterotoxigenic Escherichia coli Infection.

Background: Diarrheal diseases in infancy and childhood are responsible for substantial morbidity and mortality in developing nations. Lysozyme, an antimicrobial component of human milk, is thought to play a role in establishing a healthy intestinal microbiota and immune system. Consumption of breast milk has been shown to prevent intestinal infections and is a recommended treatment for infants with diarrhea.Objective: This study aimed to examine the ability of lysozyme-rich goat milk to prevent intestinal infection.Methods: Six-week-old Hampshire-Yorkshire pigs were assigned to treatment groups balanced for weight, sex, and litter and were fed milk from nontransgenic control goats (GM group) or human lysozyme transgenic goats (hLZM group) for 2 wk before they were challenged with porcine-specific enterotoxigenic Escherichia coli (ETEC). Fecal consistency, complete blood counts, intestinal histology, and microbial populations were evaluated.Results: Pigs in the hLZM group had less severe diarrhea than did GM pigs at 24 and 48 h after ETEC infection (P = 0.01 and 0.05, respectively), indicating a less severe clinical disease state. Relative to baseline, postmilk hLZM pigs had 19.9% and 137% enrichment in fecal Bacteroidetes (P = 0.028) and Paraprevotellaceae (P = 0.003), respectively, and a 93.8% reduction in Enterobacteriaceae (P = 0.007), whereas GM pigs had a 60.9% decrease in Lactobacillales (P = 0.003) and an 83.3% enrichment in Burkholderiales (P = 0.010). After ETEC infection, hLZM pigs tended to have lower amounts (68.7% less) of fecal Enterobacteriaceae than did GM pigs (P = 0.058). There were 83.1% fewer bacteria translocated into the mesenteric lymph nodes of hLZM pigs than into those of GM pigs (P = 0.039), and hLZM pigs had 34% lower mucin 1 and 61% higher tumor necrosis factor-α expression in the ileum than did GM pigs (P = 0.046 and 0.034, respectively).Conclusion: Results of this study indicate that human lysozyme milk consumption before and during ETEC infection has a positive effect on clinical disease, intestinal mucosa, and gut microbiota in young pigs.

Generation of gene edited birds in one generation using sperm transfection assisted gene editing (STAGE).

Generating transgenic and gene edited mammals involves in vitro manipulation of oocytes or single cell embryos. Due to the comparative inaccessibility of avian oocytes and single cell embryos, novel protocols have been developed to produce transgenic and gene edited birds. While these protocols are relatively efficient, they involve two generation intervals before reaching complete somatic and germline expressing transgenic or gene edited birds. Most of this work has been done with chickens, and many protocols require in vitro culturing of primordial germ cells (PGCs). However, for many other bird species no methodology for long term culture of PGCs exists. Developing methodologies to produce germline transgenic or gene edited birds in the first generation would save significant amounts of time and resource. Furthermore, developing protocols that can be readily adapted to a wide variety of avian species would open up new research opportunities. Here we report a method using sperm as a delivery mechanism for gene editing vectors which we call sperm transfection assisted gene editing (STAGE). We have successfully used this method to generate GFP knockout embryos and chickens, as well as generate embryos with mutations in the doublesex and mab-3 related transcription factor 1 (DMRT1) gene using the CRISPR/Cas9 system. The efficiency of the method varies from as low as 0% to as high as 26% with multiple factors such as CRISPR guide efficiency and mRNA stability likely impacting the outcome. This straightforward methodology could simplify gene editing in many bird species including those for which no methodology currently exists.

Advances in genetic engineering of the avian genome: "Realising the promise".

This review provides an historic perspective of the key steps from those reported at the 1st Transgenic Animal Research Conference in 1997 through to the very latest developments in avian transgenesis. Eighteen years later, on the occasion of the 10th conference in this series, we have seen breakthrough advances in the use of viral vectors and transposons to transform the germline via the direct manipulation of the chicken embryo, through to the establishment of PGC cultures allowing in vitro modification, expansion into populations to analyse the genetic modifications and then injection of these cells into embryos to create germline chimeras. We have now reached an unprecedented time in the history of chicken transgenic research where we have the technology to introduce precise, targeted modifications into the chicken genome, ranging from; new transgenes that provide improved phenotypes such as increased resilience to economically important diseases; the targeted disruption of immunoglobulin genes and replacement with human sequences to generate transgenic chickens that express "humanised" antibodies for biopharming; and the deletion of specific nucleotides to generate targeted gene knockout chickens for functional genomics. The impact of these advances is set to be realised through applications in chickens, and other bird species as models in scientific research, for novel biotechnology and to protect and improve agricultural productivity.

Strategies to enable the adoption of animal biotechnology to sustainably improve global food safety and security.

The ability to generate transgenic animals has existed for over 30 years, and from those early days many predicted that the technology would have beneficial applications in agriculture. Numerous transgenic agricultural animals now exist, however to date only one product from a transgenic animal has been approved for the food chain, due in part to cumbersome regulations. Recently, new techniques such as precision breeding have emerged, which enables the introduction of desired traits without the use of transgenes. The rapidly growing human population, environmental degradation, and concerns related to zoonotic and pandemic diseases have increased pressure on the animal agriculture sector to provide a safe, secure and sustainable food supply. There is a clear need to adopt transgenic technologies as well as new methods such as gene editing and precision breeding to meet these challenges and the rising demand for animal products. To achieve this goal, cooperation, education, and communication between multiple stakeholders-including scientists, industry, farmers, governments, trade organizations, NGOs and the public-is necessary. This report is the culmination of concepts first discussed at an OECD sponsored conference and aims to identify the main barriers to the adoption of animal biotechnology, tactics for navigating those barriers, strategies to improve public perception and trust, as well as industry engagement, and actions for governments and trade organizations including the OECD to harmonize regulations and trade agreements. Specifically, the report focuses on animal biotechnologies that are intended to improve breeding and genetics and currently are not routinely used in commercial animal agriculture. We put forward recommendations on how scientists, regulators, and trade organizations can work together to ensure that the potential benefits of animal biotechnology can be realized to meet the future needs of agriculture to feed the world.

Production of human lactoferrin and lysozyme in the milk of transgenic dairy animals: past, present, and future.

Genetic engineering, which was first developed in the 1980s, allows for specific additions to animals' genomes that are not possible through conventional breeding. Using genetic engineering to improve agricultural animals was first suggested when the technology was in the early stages of development by Palmiter et al. (Nature 300:611-615, 1982). One of the first agricultural applications identified was generating transgenic dairy animals that could produce altered or novel proteins in their milk. Human milk contains high levels of antimicrobial proteins that are found in low concentrations in the milk of ruminants, including the antimicrobial proteins lactoferrin and lysozyme. Lactoferrin and lysozyme are both part of the innate immune system and are secreted in tears, mucus, and throughout the gastrointestinal (GI) tract. Due to their antimicrobial properties and abundance in human milk, multiple lines of transgenic dairy animals that produce either human lactoferrin or human lysozyme have been developed. The focus of this review is to catalogue the different lines of genetically engineered dairy animals that produce either recombinant lactoferrin or lysozyme that have been generated over the years as well as compare the wealth of research that has been done on the in vitro and in vivo effects of the milk they produce. While recent advances including the development of CRISPRs and TALENs have removed many of the technical barriers to predictable and efficient genetic engineering in agricultural species, there are still many political and regulatory hurdles before genetic engineering can be used in agriculture. It is important to consider the substantial amount of work that has been done thus far on well established lines of genetically engineered animals evaluating both the animals themselves and the products they yield to identify the most effective path forward for future research and acceptance of this technology.

The lactoferrin receptor may mediate the reduction of eosinophils in the duodenum of pigs consuming milk containing recombinant human lactoferrin.

Lactoferrin is part of the immune system and multiple tissues including the gastrointestinal (GI) tract, liver, and lung contain receptors for lactoferrin. Lactoferrin has many functions, including antimicrobial, immunomodulatory, and iron binding. Additionally, lactoferrin inhibits the migration of eosinophils, which are constitutively present in the GI tract, and increase during inflammation. Lactoferrin suppresses eosinophil infiltration into the lungs and eosinophil migration in -vitro. Healthy pigs have a large population of eosinophils in their small intestine and like humans, pigs have small intestinal lactoferrin receptors (LFR); thus, pigs were chosen to investigate the effects of consumption of milk containing recombinant human lactoferrin (rhLF-milk) on small intestinal eosinophils and expression of eosinophilic cytokines. In addition, LFR localization was analyzed in duodenum and circulating eosinophils to determine if the LFR could play a role in lactoferrin's ability to inhibit eosinophil migration. In the duodenum there were significantly fewer eosinophils/unit area in pigs fed rhLF-milk compared to pigs fed control milk (p = 0.025); this was not seen in the ileum (p = 0.669). In the duodenum, no differences were observed in expression of the LFR, or any eosinophil migratory cytokines, and the amount of LFR protein was not different (p = 0.386). Immunohistochemistry (IHC) showed that within the duodenum the LFR localized on the brush border of villi, crypts, and within the lamina propria. Circulating eosinophils also contained LFRs, which may be a mechanism allowing lactoferrin to directly inhibit eosinophil migration.

Consumption of transgenic milk containing the antimicrobials lactoferrin and lysozyme separately and in conjunction by 6-week-old pigs improves intestinal and systemic health.

Lactoferrin and lysozyme are antimicrobial and immunomodulatory proteins produced in high quantities in human milk that aid in gastrointestinal (GI) health and have beneficial effects when supplemented separately and in conjunction in human and animal diets. Ruminants produce low levels of lactoferrin and lysozyme; however, there are genetically engineered cattle and goats that respectively secrete recombinant human lactoferrin (rhLF-milk), and human lysozyme (hLZ-milk) in their milk. Effects of consumption of rhLF-milk, hLZ-milk and a combination of rhLF-and hLZ-milk were tested on young pigs as an animal model for the GI tract of children. Compared with control milk-fed pigs, pigs fed a combination of rhLF and hLZ (rhLF+hLZ) milk had a significantly deeper intestinal crypts and a thinner lamina propria layer. Pigs fed hLZ-milk, rhLF-milk and rhLF+hLZ had significantly reduced mean corpuscular volume (MCV) and red blood cells (RBCs) were significantly increased in pigs fed hLZ-milk and rhLF-milk and tended to be increased in rhLF+hLZ-fed pigs, indicating more mature RBCs. These results support previous research demonstrating that pigs fed milk containing rhLF or hLZ had decreased intestinal inflammation, and suggest that in some parameters the combination of lactoferrin and lysozyme have additive effects, in contrast to the synergistic effects reported when utilising in-vitro models.

Consumption of transgenic cows' milk containing human lactoferrin results in beneficial changes in the gastrointestinal tract and systemic health of young pigs.

Lactoferrin is an antimicrobial and immunomodulatory protein that is produced in high quantities in human milk and aids in the gastrointestinal (GI) maturation of infants. Beneficial health effects have been observed when supplementing human and animal diets with lactoferrin. A herd of genetically engineered cattle that secrete recombinant human lactoferrin in their milk (rhLF-milk) have been generated which provide an efficient production system and ideal medium for rhLF consumption. The effects of consumption of rhLF-milk were tested on young pigs as an animal model for the GI tract of children. When comparing rhLF-milk fed pigs to non-transgenic milk fed pigs (control), we observed that rhLF-milk fed pigs had beneficial changes in circulating leukocyte populations. There was a significant decrease in neutrophils (p = 0.0036) and increase in lymphocytes (p = 0.0017), leading to a decreased neutrophil to lymphocyte ratio (NLR) (p = 0.0153), which is an indicator of decreased systemic inflammation. We also observed changes in intestinal villi architecture. In the duodenum, rhLF-milk fed pigs tended to have taller villi (p = 0.0914) with significantly deeper crypts (p < 0.0001). In the ileum, pigs consuming rhLF-milk had villi that were significantly taller (p = 0.0002), with deeper crypts (p < 0.0001), and a thinner lamina propria (p = 0.0056). We observed no differences in cytokine expression between rhLF-milk and control-milk fed pigs, indicating that consumption of rhLF-milk did not change cytokine signaling in the intestines. Overall favorable changes in systemic health and GI villi architecture were observed; indicating that consumption of rhLF-milk has the potential to induce positive changes in the GI tract.

Reasons police respond in schools: An analysis of narrative data from police incident reports.

BACKGROUND: Communities across the U.S. have acted to eliminate or curb police presence in schools. These efforts have primarily focused on School Resource Officers. However, school staff also call upon local police to respond in their buildings, for example by calling 911. The reasons that police are called upon to respond in schools has rarely been studied. OBJECTIVE: The current study aimed to identify the primary reasons that local police were asked to respond to incidents in one urban school district, and the outcomes of those responses. PARTICIPANTS AND SETTING: We analyzed 882 police incident report narratives, selected from a stratified sample of 57 schools in one urban school district during the 2017-18 school year. Incident reports, which were written by officers responding in schools, included narrative descriptions of officer intervention, the events precipitating police involvement, and outcomes of incidents. METHODS: By coding incident report narratives, we identified categories describing the reasons for police response to events in schools and the outcomes of those events. RESULTS: Most incident reports originating from school addresses (n = 730; 82.8 %) involved students. Among those reports, police most frequently responded to instances of sexual physical violence (17.5 %), physical assault (15.8 %), dysregulated behavior (11.2 %), threatening language (10.8 %), and threat of or actual school violence (4.4 %). Incidents involving students most often resulted in: (1) parents/guardians being contacted (57.3 %), (2) schools engaging in disciplinary actions (39.7 %) or school safety actions (27.0 %), and (3) child maltreatment reports being made to Crimes Against Children (a subdivision of the police department focused on child maltreatment; 26.0 %) or to the Department of Children and Families (26.2 %). CONCLUSIONS: Findings indicate that many events leading to police responses in schools are related to maltreatment and behavioral health. These events rarely result in a criminal justice response, and most often result in action by families and schools (e.g., contacting parents/guardians, school disciplinary/safety actions), and filed reports of maltreatment. Additional supports in these areas may have the potential to reduce the perceived need to call upon police and to provide more direct access to services for students.

Mapping of equine cerebellar abiotrophy to ECA2 and identification of a potential causative mutation affecting expression of MUTYH.

Equine Cerebellar Abiotrophy (CA) is a neurological disease found in Arabian horses. CA is characterized by post-natal degeneration of the Purkinje cells of the cerebellum. Signs of CA include ataxia, head tremors, and a lack of balance equilibrium. We have discovered a linkage of the CA phenotype to a microsatellite marker on ECA2 and identified a region of conserved homozygosity spanning approximately 142 kb. Complete sequencing of the four genes in this region identified one SNP found only in Arabian horses, located in exon 4 of TOE1 and approximately 1200 base pairs upstream of MUTYH, adjacent to a possible binding site for the transcription factor GATA2. qPCR analysis of cDNA from the cerebella of affected and unaffected horses suggested that MUTYH expression is down-regulated in affected horses. This SNP may therefore have a function effect on TOE1, or a regulatory effect on MUTYH by negatively affecting the binding affinity of GATA2.

A novel murine model of post-implantation malaria-induced preterm birth.

Despite major advances made in malaria treatment and control over recent decades, the development of new models for studying disease pathogenesis remains a vital part of malaria research efforts. The study of malaria infection during pregnancy is particularly reliant on mouse models, as a means of circumventing many challenges and costs associated with pregnancy studies in endemic human populations. Here, we introduce a novel murine model that will further our understanding of how malaria infection affects pregnancy outcome. When C57BL/6J (B6) mice are infected with Plasmodium chabaudi chabaudi AS on either embryonic day (E) 6.5, 8.5, or 10.5, preterm birth occurs in all animals by E16.5, E17.5, or E18.5 respectively, with no evidence of intrauterine growth restriction. Despite having the same outcome, we found that the time to delivery, placental inflammatory and antioxidant transcript upregulation, and the relationships between parasitemia and transcript expression prior to preterm birth differed based on the embryonic day of infection. On the day before preterm delivery, E6.5 infected mice did not experience significant upregulation of the inflammatory or antioxidant gene transcripts examined; however, peripheral and placental parasitemia correlated positively with Il1ß, Cox1, Cat, and Hmox1 placental transcript abundance. E8.5 infected mice had elevated transcripts for Ifnγ, Tnf, Il10, Cox1, Cox2, Sod1, Sod2, Cat, and Nrf2, while Sod3 was the only transcript that correlated with parasitemia. Finally, E10.5 infected mice had elevated transcripts for Ifnγ only, with a tendency for Tnf transcripts to correlate with peripheral parasitemia. Tumor necrosis factor deficient (TNF-/-) and TNF receptor 1 deficient (TNFR1-/-) mice infected on E8.5 experienced preterm birth at the same time as B6 controls. Further characterization of this model is necessary to discover the mechanism(s) and/or trigger(s) responsible for malaria-driven preterm birth caused by maternal infection during early pregnancy.

Three synthetic biology applications and their paths to impact in Australia: Cane toads, bacteriophages, and biomining microbes.

Synthetic biology [synbio] applications have the potential to assist in addressing significant global health and environmental challenges. Australian research institutes are investing in formative research to develop synbio technologies capable of meeting these challenges. Alongside the laboratory research, investigating the broader social, institutional, and ethical considerations that synbio presents has been a priority. We conducted targeted qualitative research to uncover the barriers and opportunities for a range of multisectoral stakeholders identified as potential end-users of the science under development. The research provides insights into the research implementation environment for three synthetic biology applications: (1) gene editing cane toads (Rhinella marina) to reduce their environmental impact; (2) engineering bacteriophages to combat antimicrobial resistance in humans; and (3) engineering microbes to improve biomining efficiency in the mining industry. In-depth interviews (N = 23) with government, research and civil society representatives revealed key challenges in the impact pathway for each application. The strongest themes uncovered during interviews related to perceived negative public attitudes towards genetic technologies, a lack of investment in critical research infrastructure, unclear regulatory pathways and the presence of a strong social and environmental imperative underpinning technology development. These findings reveal specific entry points for further engagement with the most immediate end-users of synbio. Separate from research on public attitudes to synbio, the cases highlight the various hurdles to achieving research impact, according to experts who will likely use, approve or invest in these applications in the future. The themes uncovered inform avenues for strengthening engagement and research coordination in Australia and elsewhere.

Liver-stage fate determination in Plasmodium vivax parasites: Characterization of schizont growth and hypnozoite fating from patient isolates.

Plasmodium vivax, one species of parasite causing human malaria, forms a dormant liver stage, termed the hypnozoite, which activate weeks, months or years after the primary infection, causing relapse episodes. Relapses significantly contribute to the vivax malaria burden and are only killed with drugs of the 8-aminoquinoline class, which are contraindicated in many vulnerable populations. Development of new therapies targeting hypnozoites is hindered, in part, by the lack of robust methods to continuously culture and characterize this parasite. As a result, the determinants of relapse periodicity and the molecular processes that drive hypnozoite formation, persistence, and activation are largely unknown. While previous reports have described vastly different liver-stage growth metrics attributable to which hepatocyte donor lot is used to initiate culture, a comprehensive assessment of how different P. vivax patient isolates behave in the same lots at the same time is logistically challenging. Using our primary human hepatocyte-based P. vivax liver-stage culture platform, we aimed to simultaneously test the effects of how hepatocyte donor lot and P. vivax patient isolate influence the fate of sporozoites and growth of liver schizonts. We found that, while environmental factors such as hepatocyte donor lot can modulate hypnozoite formation rate, the P. vivax case is also an important determinant of the proportion of hypnozoites observed in culture. In addition, we found schizont growth to be mostly influenced by hepatocyte donor lot. These results suggest that, while host hepatocytes harbor characteristics making them more- or less-supportive of a quiescent versus growing intracellular parasite, sporozoite fating toward hypnozoites is isolate-specific. Future studies involving these host-parasite interactions, including characterization of individual P. vivax strains, should consider the impact of culture conditions on hypnozoite formation, in order to better understand this important part of the parasite's lifecycle.

Genetic approaches for increasing fitness in endangered species.

The global rate of wildlife extinctions is accelerating, and the persistence of many species requires conservation breeding programs. A central paradigm of these programs is to preserve the genetic diversity of the founder populations. However, this may preserve original characteristics that make them vulnerable to extinction. We introduce targeted genetic intervention (TGI) as an alternative approach that promotes traits that enable species to persist in the face of threats by changing the incidence of alleles that impact on fitness. The TGI toolkit includes methods with established efficacy in model organisms and agriculture but are largely untried for conservation, such as synthetic biology and artificial selection. We explore TGI approaches as a species-restoration tool for intractable threats including infectious disease and climate change.

Metabolic, Pharmacokinetic, and Activity Profile of the Liver Stage Antimalarial (RC-12).

The catechol derivative RC-12 (WR 27653) (1) is one of the few non-8-aminoquinolines with good activity against hypnozoites in the gold-standard Plasmodium cynomolgi-rhesus monkey (Macaca mulatta) model, but in a small clinical trial, it had no efficacy against Plasmodium vivax hypnozoites. In an attempt to better understand the pharmacokinetic and pharmacodynamic profile of 1 and to identify potential active metabolites, we now describe the phase I metabolism, rat pharmacokinetics, and in vitro liver-stage activity of 1 and its metabolites. Compound 1 had a distinct metabolic profile in human vs monkey liver microsomes, and the data suggested that the O-desmethyl, combined O-desmethyl/N-desethyl, and N,N-didesethyl metabolites (or a combination thereof) could potentially account for the superior liver stage antimalarial efficacy of 1 in rhesus monkeys vs that seen in humans. Indeed, the rate of metabolism was considerably lower in human liver microsomes in comparison to rhesus monkey microsomes, as was the formation of the combined O-desmethyl/N-desethyl metabolite, which was the only metabolite tested that had any activity against liver-stage P. vivax; however, it was not consistently active against liver-stage P. cynomolgi. As 1 and all but one of its identified Phase I metabolites had no in vitro activity against P. vivax or P. cynomolgi liver-stage malaria parasites, we suggest that there may be additional unidentified active metabolites of 1 or that the exposure of 1 achieved in the reported unsuccessful clinical trial of this drug candidate was insufficient to kill the P. vivax hypnozoites.