A Quantitative Proteome Map of the Human Body.

Determining protein levels in each tissue and how they compare with RNA levels is important for understanding human biology and disease as well as regulatory processes that control protein levels. We quantified the relative protein levels from over 12,000 genes across 32 normal human tissues. Tissue-specific or tissue-enriched proteins were identified and compared to transcriptome data. Many ubiquitous transcripts are found to encode tissue-specific proteins. Discordance of RNA and protein enrichment revealed potential sites of synthesis and action of secreted proteins. The tissue-specific distribution of proteins also provides an in-depth view of complex biological events that require the interplay of multiple tissues. Most importantly, our study demonstrated that protein tissue-enrichment information can explain phenotypes of genetic diseases, which cannot be obtained by transcript information alone. Overall, our results demonstrate how understanding protein levels can provide insights into regulation, secretome, metabolism, and human diseases.

Infection-induced plasmablasts are a nutrient sink that impairs humoral immunity to malaria.

Plasmodium parasite-specific antibodies are critical for protection against malaria, yet the development of long-lived and effective humoral immunity against Plasmodium takes many years and multiple rounds of infection and cure. Here, we report that the rapid development of short-lived plasmablasts during experimental malaria unexpectedly hindered parasite control by impeding germinal center responses. Metabolic hyperactivity of plasmablasts resulted in nutrient deprivation of the germinal center reaction, limiting the generation of memory B cell and long-lived plasma cell responses. Therapeutic administration of a single amino acid to experimentally infected mice was sufficient to overcome the metabolic constraints imposed by plasmablasts and enhanced parasite clearance and the formation of protective humoral immune memory responses. Thus, our studies not only challenge the current model describing the role and function of blood-stage Plasmodium-induced plasmablasts but they also reveal new targets and strategies to improve anti-Plasmodium humoral immunity.

Complement in malaria immunity and vaccines.

Developing efficacious vaccines for human malaria caused by Plasmodium falciparum is a major global health priority, although this has proven to be immensely challenging over the decades. One major hindrance is the incomplete understanding of specific immune responses that confer protection against disease and/or infection. While antibodies to play a crucial role in malaria immunity, the functional mechanisms of these antibodies remain unclear as most research has primarily focused on the direct inhibitory or neutralizing activity of antibodies. Recently, there is a growing body of evidence that antibodies can also mediate effector functions through activating the complement system against multiple developmental stages of the parasite life cycle. These antibody-complement interactions can have detrimental consequences to parasite function and viability, and have been significantly associated with protection against clinical malaria in naturally acquired immunity, and emerging findings suggest these mechanisms could contribute to vaccine-induced immunity. In order to develop highly efficacious vaccines, strategies are needed that prioritize the induction of antibodies with enhanced functional activity, including the ability to activate complement. Here we review the role of complement in acquired immunity to malaria, and provide insights into how this knowledge could be used to harness complement in malaria vaccine development.

The Plasmodium falciparum parasitophorous vacuole protein P113 interacts with the parasite protein export machinery and maintains normal vacuole architecture.

Infection with Plasmodium falciparum parasites results in approximately 627,000 deaths from malaria annually. Key to the parasite's success is their ability to invade and subsequently grow within human erythrocytes. Parasite proteins involved in parasite invasion and proliferation are therefore intrinsically of great interest, as targeting these proteins could provide novel means of therapeutic intervention. One such protein is P113 which has been reported to be both an invasion protein and an intracellular protein located within the parasitophorous vacuole (PV). The PV is delimited by a membrane (PVM) across which a plethora of parasite-specific proteins are exported via the Plasmodium Translocon of Exported proteins (PTEX) into the erythrocyte to enact various immune evasion functions. To better understand the role of P113 we isolated its binding partners from in vitro cultures of P. falciparum. We detected interactions with the protein export machinery (PTEX and exported protein-interacting complex) and a variety of proteins that either transit through the PV or reside on the parasite plasma membrane. Genetic knockdown or partial deletion of P113 did not significantly reduce parasite growth or protein export but did disrupt the morphology of the PVM, suggesting that P113 may play a role in maintaining normal PVM architecture.

Anti-C1s humanized monoclonal antibody SAR445088: A classical pathway complement inhibitor specific for the active form of C1s.

The objective of this study was to characterize the complement-inhibiting activity of SAR445088, a novel monoclonal antibody specific for the active form of C1s. Wieslab® and hemolytic assays were used to demonstrate that SAR445088 is a potent, selective inhibitor of the classical pathway of complement. Specificity for the active form of C1s was confirmed in a ligand binding assay. Finally, TNT010 (a precursor to SAR445088) was assessed in vitro for its ability to inhibit complement activation associated with cold agglutinin disease (CAD). TNT010 inhibited C3b/iC3b deposition on human red blood cells incubated with CAD patient serum and decreased their subsequent phagocytosis by THP-1 cells. In summary, this study identifies SAR445088 as a potential therapeutic for the treatment of classical pathway-driven diseases and supports its continued assessment in clinical trials.

RobNorm: model-based robust normalization method for labeled quantitative mass spectrometry proteomics data.

MOTIVATION: Data normalization is an important step in processing proteomics data generated in mass spectrometry experiments, which aims to reduce sample-level variation and facilitate comparisons of samples. Previously published methods for normalization primarily depend on the assumption that the distribution of protein expression is similar across all samples. However, this assumption fails when the protein expression data is generated from heterogenous samples, such as from various tissue types. This led us to develop a novel data-driven method for improved normalization to correct the systematic bias meanwhile maintaining underlying biological heterogeneity. RESULTS: To robustly correct the systematic bias, we used the density-power-weight method to down-weigh outliers and extended the one-dimensional robust fitting method described in the previous work to our structured data. We then constructed a robustness criterion and developed a new normalization algorithm, called RobNorm.In simulation studies and analysis of real data from the genotype-tissue expression project, we compared and evaluated the performance of RobNorm against other normalization methods. We found that the RobNorm approach exhibits the greatest reduction in systematic bias while maintaining across-tissue variation, especially for datasets from highly heterogeneous samples. AVAILABILITYAND IMPLEMENTATION: https://github.com/mwgrassgreen/RobNorm. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Development of reconstructed intestinal micronucleus cytome (RICyt) assay in 3D human gut model for genotoxicity assessment of orally ingested substances.

The micronucleus (MN) assay is widely used as part of a battery of tests applied to evaluate the genotoxic potential of chemicals, including new food additives and novel food ingredients. Micronucleus assays typically utilise homogenous in vitro cell lines which poorly recapitulate the physiology, biochemistry and genomic events in the gut, the site of first contact for ingested materials. Here we have adapted and validated the MN endpoint assay protocol for use with complex 3D reconstructed intestinal microtissues; we have named this new protocol the reconstructed intestine micronucleus cytome (RICyt) assay. Our data suggest the commercial 3D microtissues replicate the physiological, biochemical and genomic responses of native human small intestine to exogenous compounds. Tissues were shown to maintain log-phase proliferation throughout the period of exposure and expressed low background MN. Analysis using the RICyt assay protocol revealed the presence of diverse cell types and nuclear anomalies (cytome) in addition to MN, indicating evidence for comprehensive DNA damage and mode(s) of cell death reported by the assay. The assay correctly identified and discriminated direct-acting clastogen, aneugen and clastogen requiring exogenous metabolic activation, and a non-genotoxic chemical. We are confident that the genotoxic response in the 3D microtissues more closely resembles the native tissues due to the inherent tissue architecture, surface area, barrier effects and tissue matrix interactions. This proof-of-concept study highlights the RICyt MN cytome assay in 3D reconstructed intestinal microtissues is a promising tool for applications in predictive toxicology.

Multifunctional Antibodies Are Induced by the RTS,S Malaria Vaccine and Associated With Protection in a Phase 1/2a Trial.

BACKGROUND: RTS,S is the leading malaria vaccine candidate but only confers partial efficacy against malaria in children. RTS,S is based on the major Plasmodium falciparum sporozoite surface antigen, circumsporozoite protein (CSP). The induction of anti-CSP antibodies is important for protection; however, it is unclear how these protective antibodies function. METHODS: We quantified the induction of functional anti-CSP antibody responses in healthy malaria-naive adults (N = 45) vaccinated with RTS,S/AS01. This included the ability to mediate effector functions via the fragment crystallizable (Fc) region, such as interacting with human complement proteins and Fcγ-receptors (FcγRs) that are expressed on immune cells, which promote various immunological functions. RESULTS: Our major findings were (1) RTS,S-induced antibodies mediated Fc-dependent effector functions, (2) functional antibodies were generally highest after the second vaccine dose, (3) functional antibodies targeted multiple regions of CSP, (4) participants with higher levels of functional antibodies had a reduced probability of developing parasitemia following homologous challenge (P < .05), and (5) nonprotected subjects had higher levels of anti-CSP IgM. CONCLUSIONS: Our data suggest a role for Fc-dependent antibody effector functions in RTS,S-induced immunity. Enhancing the induction of these functional activities may be a strategy to improve the protective efficacy of RTS,S or other malaria vaccines. CLINICAL TRIALS REGISTRATION: NCT00075049.

Reduced risk of placental parasitemia associated with complement fixation on Plasmodium falciparum by antibodies among pregnant women.

BACKGROUND: The pathogenesis of malaria in pregnancy (MiP) involves accumulation of P. falciparum-infected red blood cells (pRBCs) in the placenta, contributing to poor pregnancy outcomes. Parasite accumulation is primarily mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1). Magnitude of IgG to pRBCs has been associated with reduced risk of MiP in some studies, but associations have been inconsistent. Further, antibody effector mechanisms are poorly understood, and the role of antibody complement interactions is unknown. METHODS: Studying a longitudinal cohort of pregnant women (n=302) from a malaria-endemic province in Papua New Guinea (PNG), we measured the ability of antibodies to fix and activate complement using placental binding pRBCs and PfEMP1 recombinant domains. We determined antibody-mediated complement inhibition of pRBC binding to the placental receptor, chondroitin sulfate A (CSA), and associations with protection against placental parasitemia. RESULTS: Some women acquired antibodies that effectively promoted complement fixation on placental-binding pRBCs. Complement fixation correlated with IgG1 and IgG3 antibodies, which dominated the response. There was, however, limited evidence for membrane attack complex activity or pRBC lysis or killing. Importantly, a higher magnitude of complement fixing antibodies was prospectively associated with reduced odds of placental infection at delivery. Using genetically modified P. falciparum and recombinant PfEMP1 domains, we found that complement-fixing antibodies primarily targeted a specific variant of PfEMP1 (known as VAR2CSA). Furthermore, complement enhanced the ability of antibodies to inhibit pRBC binding to CSA, which was primarily mediated by complement C1q protein. CONCLUSIONS: These findings provide new insights into mechanisms mediating immunity to MiP and reveal potential new strategies for developing malaria vaccines that harness antibody-complement interactions.

Safety, infectivity and immunogenicity of a genetically attenuated blood-stage malaria vaccine.

BACKGROUND: There is a clear need for novel approaches to malaria vaccine development. We aimed to develop a genetically attenuated blood-stage vaccine and test its safety, infectivity, and immunogenicity in healthy volunteers. Our approach was to target the gene encoding the knob-associated histidine-rich protein (KAHRP), which is responsible for the assembly of knob structures at the infected erythrocyte surface. Knobs are required for correct display of the polymorphic adhesion ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1), a key virulence determinant encoded by a repertoire of var genes. METHODS: The gene encoding KAHRP was deleted from P. falciparum 3D7 and a master cell bank was produced in accordance with Good Manufacturing Practice. Eight malaria naïve males were intravenously inoculated (day 0) with 1800 (2 subjects), 1.8 × 105 (2 subjects), or 3 × 106 viable parasites (4 subjects). Parasitemia was measured using qPCR; immunogenicity was determined using standard assays. Parasites were rescued into culture for in vitro analyses (genome sequencing, cytoadhesion assays, scanning electron microscopy, var gene expression). RESULTS: None of the subjects who were administered with 1800 or 1.8 × 105 parasites developed parasitemia; 3/4 subjects administered 3× 106 parasites developed significant parasitemia, first detected on days 13, 18, and 22. One of these three subjects developed symptoms of malaria simultaneously with influenza B (day 17; 14,022 parasites/mL); one subject developed mild symptoms on day 28 (19,956 parasites/mL); and one subject remained asymptomatic up to day 35 (5046 parasites/mL). Parasitemia rapidly cleared with artemether/lumefantrine. Parasitemia induced a parasite-specific antibody and cell-mediated immune response. Parasites cultured ex vivo exhibited genotypic and phenotypic properties similar to inoculated parasites, although the var gene expression profile changed during growth in vivo. CONCLUSIONS: This study represents the first clinical investigation of a genetically attenuated blood-stage human malaria vaccine. A P. falciparum 3D7 kahrp- strain was tested in vivo and found to be immunogenic but can lead to patent parasitemia at high doses. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (number: ACTRN12617000824369 ; date: 06 June 2017).

Prediction of labour onset in women who present with symptoms of preterm labour using cervical length.

BACKGROUND: Diagnosis of preterm labour is difficult because initial symptoms and signs are often mild and may occur in continuing pregnancies. This study aims to investigate the utility of measuring cervical length, using transvaginal ultrasound, in women presenting to the delivery suite with symptoms of preterm labour. METHODS: This was a prospective cohort study performed in KK Women's and Children's Hospital, Singapore from September 2017 to July 2018. Women with singleton pregnancies, presenting with symptoms of contraction pain, between 24+ 0 to 36+ 6 weeks gestation, were included. Transvaginal ultrasound cervical length measurements were done at presentation to the labour ward, after four hours and in the following morning. The primary outcome of the study was delivery within 1 week. All statistical analyses were conducted with Microsoft Excel and Statistical Package for the Social Sciences. RESULTS: A total of 95 subjects were included. A one-millimeter increase in the 1st cervical length increases scan-to-delivery time by 0.802 days (p-value 0.003, CI 0.280-1.323). Receiver Operator Characteristic (ROC) curve analysis for prediction of delivery within 1 week showed an Area Under Curve (AUC) of 0.667, optimal cut-off value of 27.5mm (sensitivity 77.8 %, specificity 61.6 %). A one-millimetre increase in the 3rd cervical length increases scan-to-delivery time by 0.770 days (p-value 0.023, CI 0.108-1.432). ROC curve analysis for prediction of delivery within 1 week showed an AUC of 0.915, optimal cut-off value of 25.5mm (sensitivity 100 %, specificity 73.6 %). However, the change in cervical length over a period of 1 day was not significant in predicting delivery within 1 week. CONCLUSIONS: Our results indicate that by using a cervical length cut off of 27.5mm at presentation, we would have predicted 77.8 % of deliveries within 1 week. If we were to repeat the cervical length scan the next day, with the same cut-off of 27.5mm, we would have predicted 100 % of deliveries within 1 week. In our study, measuring the transvaginal ultrasound cervical length is a reliable diagnostic test for delivery within 1 week. However, the results are limited by the small sample size. Further studies should be conducted with a larger sample size.

PTEX is an essential nexus for protein export in malaria parasites.

During the blood stages of malaria, several hundred parasite-encoded proteins are exported beyond the double-membrane barrier that separates the parasite from the host cell cytosol. These proteins have a variety of roles that are essential to virulence or parasite growth. There is keen interest in understanding how proteins are exported and whether common machineries are involved in trafficking the different classes of exported proteins. One potential trafficking machine is a protein complex known as the Plasmodium translocon of exported proteins (PTEX). Although PTEX has been linked to the export of one class of exported proteins, there has been no direct evidence for its role and scope in protein translocation. Here we show, through the generation of two parasite lines defective for essential PTEX components (HSP101 or PTEX150), and analysis of a line lacking the non-essential component TRX2 (ref. 12), greatly reduced trafficking of all classes of exported proteins beyond the double membrane barrier enveloping the parasite. This includes proteins containing the PEXEL motif (RxLxE/Q/D) and PEXEL-negative exported proteins (PNEPs). Moreover, the export of proteins destined for expression on the infected erythrocyte surface, including the major virulence factor PfEMP1 in Plasmodium falciparum, was significantly reduced in PTEX knockdown parasites. PTEX function was also essential for blood-stage growth, because even a modest knockdown of PTEX components had a strong effect on the parasite's capacity to complete the erythrocytic cycle both in vitro and in vivo. Hence, as the only known nexus for protein export in Plasmodium parasites, and an essential enzymic machine, PTEX is a prime drug target.

Treating hoarding disorder with compassion-focused therapy: A pilot study examining treatment feasibility, acceptability, and exploring treatment effects.

OBJECTIVES: Hoarding disorder (HD) was recognized as a psychiatric disorder in 2013. Existing literature suggests room for improvement in its treatment. The current pilot study aimed to provide an initial evaluation on the potential of compassion-focused therapy (CFT) as an intervention for HD, with the primary aim being assessing its feasibility and acceptability, and the secondary being evaluating its effects. DESIGN: Both CFT and a second round of the current standard of treatment and cognitive behavioural therapy (CBT) were investigated in the current study as follow-up treatment options for individuals who had completed CBT but were still significantly symptomatic. METHODS: Forty eligible individuals were enrolled (20 in each treatment). Treatment feasibility and acceptability were assessed by quantitative and qualitative measures. To explore treatment effects, HD symptom severity, HD-related dysfunctions, and their underlying mechanisms were assessed pre-treatment and post-treatment. RESULTS: Retention rates were 72% for CFT and 37% for CBT. All participants and 79% of the participants rated CFT and CBT, respectively, as good or excellent. After receiving CFT as a follow-up treatment, HD symptom severity dropped below the cut-off point for clinically significant HD for 77% of the treatment completers, and 62% achieved clinically significant reduction in symptom severity. In contrast, after completing a second course of CBT, 23% had HD symptom severity dropped below the cut-off threshold, and 29% achieved clinically significant symptom reduction. CONCLUSIONS: The current study showed satisfactory feasibility and acceptability of CFT. Moreover, it also found promising effects of CFT in addressing hoarding-related mechanisms that may not have been sufficiently addressed by CBT. The results suggest promising potential of CFT as a treatment for HD. Further investigation on this intervention is needed. PRACTITIONER POINTS: CFT may be a promising treatment option, particularly for those who do not respond well to CBT. Improving emotion regulation and negative self-perception by applying CFT interventions may help relieve hoarding symptoms. Generalization of the findings should be applied with caution given the small convenience sample of the current study. Statistical comparison on treatment effect measures between CFT and CBT as follow-up treatments was not available due to small sample size. Therefore, the comparative conclusions based on this pilot study should be made with caution.

Host immunity to Plasmodium falciparum and the assessment of emerging artemisinin resistance in a multinational cohort.

Artemisinin-resistant falciparum malaria, defined by a slow-clearance phenotype and the presence of kelch13 mutants, has emerged in the Greater Mekong Subregion. Naturally acquired immunity to malaria clears parasites independent of antimalarial drugs. We hypothesized that between- and within-population variations in host immunity influence parasite clearance after artemisinin treatment and the interpretation of emerging artemisinin resistance. Antibodies specific to 12 Plasmodium falciparum sporozoite and blood-stage antigens were determined in 959 patients (from 11 sites in Southeast Asia) participating in a multinational cohort study assessing parasite clearance half-life (PCt1/2) after artesunate treatment and kelch13 mutations. Linear mixed-effects modeling of pooled individual patient data assessed the association between antibody responses and PCt1/2.P. falciparum antibodies were lowest in areas where the prevalence of kelch13 mutations and slow PCt1/2 were highest [Spearman ρ = -0.90 (95% confidence interval, -0.97, -0.65), and Spearman ρ = -0.94 (95% confidence interval, -0.98, -0.77), respectively]. P. falciparum antibodies were associated with faster PCt1/2 (mean difference in PCt1/2 according to seropositivity, -0.16 to -0.65 h, depending on antigen); antibodies have a greater effect on the clearance of kelch13 mutant compared with wild-type parasites (mean difference in PCt1/2 according to seropositivity, -0.22 to -0.61 h faster in kelch13 mutants compared with wild-type parasites). Naturally acquired immunity accelerates the clearance of artemisinin-resistant parasites in patients with falciparum malaria and may confound the current working definition of artemisinin resistance. Immunity may also play an important role in the emergence and transmission potential of artemisinin-resistant parasites.

Antibody Targets on the Surface of Plasmodium falciparum-Infected Erythrocytes That Are Associated With Immunity to Severe Malaria in Young Children.

BACKGROUND: Sequestration of Plasmodium falciparum-infected erythrocytes (IEs) in the microvasculature contributes to pathogenesis of severe malaria in children. This mechanism is mediated by antigens expressed on the IE surface. However, knowledge of specific targets and functions of antibodies to IE surface antigens that protect against severe malaria is limited. METHODS: Antibodies to IE surface antigens were examined in a case-control study of young children in Papua New Guinea presenting with severe or uncomplicated malaria (n = 448), using isolates with a virulent phenotype associated with severe malaria, and functional opsonic phagocytosis assays. We used genetically modified isolates and recombinant P. falciparum erythrocyte membrane protein 1 (PfEMP1) domains to quantify PfEMP1 as a target of antibodies associated with disease severity. RESULTS: Antibodies to the IE surface and recombinant PfEMP1 domains were significantly higher in uncomplicated vs severe malaria and were boosted following infection. The use of genetically modified P. falciparum revealed that PfEMP1 was a major target of antibodies and that PfEMP1-specific antibodies were associated with reduced odds of severe malaria. Furthermore, antibodies promoting the opsonic phagocytosis of IEs by monocytes were lower in those with severe malaria. CONCLUSIONS: Findings suggest that PfEMP1 is a dominant target of antibodies associated with reduced risk of severe malaria, and function in part by promoting opsonic phagocytosis.

Human Immunization With a Polymorphic Malaria Vaccine Candidate Induced Antibodies to Conserved Epitopes That Promote Functional Antibodies to Multiple Parasite Strains.

Background: Overcoming antigenic diversity is a key challenge in the development of effective Plasmodium falciparum malaria vaccines. Strategies that promote the generation of antibodies targeting conserved epitopes of vaccine antigens may provide protection against diverse parasites strains. Understanding differences between vaccine-induced and naturally acquired immunity is important to achieving this goal. Methods: We analyzed antibodies generated in a phase 1 human vaccine trial, MSP2-C1, which included 2 allelic forms of MSP2, an abundant vaccine antigen on the merozoite surface. Vaccine-induced responses were assessed for functional activity against multiple parasite strains, and cross-reactivity of antibodies was determined using competition ELISA and epitope mapping approaches. Results: Vaccination induced cytophilic antibody responses with strain-transcending opsonic phagocytosis and complement-fixing function. In contrast to antibodies acquired via natural infection, vaccine-induced antibodies were directed towards conserved epitopes at the C-terminus of MSP2, whereas naturally acquired antibodies mainly targeted polymorphic epitopes. Functional activity of C-terminal-targeted antibodies was confirmed using monoclonal antibodies that promoted opsonic phagocytosis against multiple parasite strains. Conclusion: Vaccination generated markedly different responses to polymorphic antigens than naturally acquired immunity and targeted conserved functional epitopes. Induction of antibodies targeting conserved regions of malaria antigens provides a promising vaccine strategy to overcome antigenic diversity for developing effective malaria vaccines.

Patterns of protective associations differ for antibodies to P. falciparum-infected erythrocytes and merozoites in immunity against malaria in children.

Acquired antibodies play an important role in immunity to P. falciparum malaria and are typically directed towards surface antigens expressed by merozoites and infected erythrocytes (IEs). The importance of specific IE surface antigens as immune targets remains unclear. We evaluated antibodies and protective associations in two cohorts of children in Papua New Guinea. We used genetically-modified P. falciparum to evaluate the importance of PfEMP1 and a P. falciparum isolate with a virulent phenotype. Our findings suggested that PfEMP1 was the dominant target of antibodies to the IE surface, including functional antibodies that promoted opsonic phagocytosis by monocytes. Antibodies were associated with increasing age and concurrent parasitemia, and were higher among children exposed to a higher force-of-infection as determined using molecular detection. Antibodies to IE surface antigens were consistently associated with reduced risk of malaria in both younger and older children. However, protective associations for antibodies to merozoite surface antigens were only observed in older children. This suggests that antibodies to IE surface antigens, particularly PfEMP1, play an earlier role in acquired immunity to malaria, whereas greater exposure is required for protective antibodies to merozoite antigens. These findings have implications for vaccine design and serosurveillance of malaria transmission and immunity.

Establishment of a yeast-based VLP platform for antigen presentation.

BACKGROUND: Chimeric virus-like particles (VLP) allow the display of foreign antigens on their surface and have proved valuable in the development of safe subunit vaccines or drug delivery. However, finding an inexpensive production system and a VLP scaffold that allows stable incorporation of diverse, large foreign antigens are major challenges in this field. RESULTS: In this study, a versatile and cost-effective platform for chimeric VLP development was established. The membrane integral small surface protein (dS) of the duck hepatitis B virus was chosen as VLP scaffold and the industrially applied and safe yeast Hansenula polymorpha (syn. Pichia angusta, Ogataea polymorpha) as the heterologous expression host. Eight different, large molecular weight antigens of up to 412 amino acids derived from four animal-infecting viruses were genetically fused to the dS and recombinant production strains were isolated. In all cases, the fusion protein was well expressed and upon co-production with dS, chimeric VLP containing both proteins could be generated. Purification was accomplished by a downstream process adapted from the production of a recombinant hepatitis B VLP vaccine. Chimeric VLP were up to 95% pure on protein level and contained up to 33% fusion protein. Immunological data supported surface exposure of the foreign antigens on the native VLP. Approximately 40 mg of chimeric VLP per 100 g dry cell weight could be isolated. This is highly comparable to values reported for the optimized production of human hepatitis B VLP. Purified chimeric VLP were shown to be essentially stable for 6 months at 4 °C. CONCLUSIONS: The dS-based VLP scaffold tolerates the incorporation of a variety of large molecular weight foreign protein sequences. It is applicable for the display of highly immunogenic antigens originating from a variety of pathogens. The yeast-based production system allows cost-effective production that is not limited to small-scale fundamental research. Thus, the dS-based VLP platform is highly efficient for antigen presentation and should be considered in the development of future vaccines.

Barriers and facilitators of appropriate vancomycin use: prescribing context is key.

PURPOSE: Audit studies reveal frequent non-compliance with dosing and monitoring guidelines for vancomycin. This study aimed to qualitatively explore the barriers and facilitators of compliance with vancomycin dosing and monitoring guidelines. METHODS: Interviews were conducted with 16 prescribers in a large tertiary teaching hospital in Sydney, Australia. Questions explored knowledge, attitudes, and perceived complexities associated with vancomycin use. Interviews were analysed using thematic analysis. RESULTS: Prescribers reported utilising vancomycin guidelines, citing familiarity with guidelines, a positive perception of guidelines, awareness of poor guideline compliance, and assistance from specialist staff as facilitators of the uptake of guideline recommendations. Barriers existing within the prescribing environment such as the prescribing culture, a lack of time, and poor communication and coordination of therapeutic drug monitoring processes were identified as hindrances to guideline compliance. CONCLUSIONS: The provision of guidelines may not be sufficient in ensuring appropriate prescribing and monitoring of vancomycin when barriers relating to the prescribing environment exist. Developing interventions targeted toward these barriers, such as having dedicated phlebotomists for vancomycin blood sampling, fostering better handover processes, and educating staff on poorly understood aspects of guidelines, is likely to improve the uptake of guideline recommendations for vancomycin and other medications requiring therapeutic drug monitoring.

Prostaglandin D2 Uses Components of ROS Signaling to Enhance Testosterone Production in Keratinocytes.

Elevated levels of prostaglandin D2 (PGD2) have been shown to be present in the bald scalp of androgenic alopecia (AGA) patients and to functionally inhibit hair growth. However, its precise mechanism in AGA has yet to be clearly defined. Although testosterone plays a critical role in the initiation and progression of AGA, the existence of a possible link between PGD2 and testosterone in skin has not been investigated. Here we show that human keratinocytes treated with PGD2 show enhanced capacity to convert the weak androgen, androstenedione, to testosterone. At the same time, treatment with PGD2 induced reactive oxygen species as indicated by generation of the lipid peroxidation product, 4-hydroxynonenal. To determine whether these two events are linked, we used the reactive oxygen species scavenger N-acetyl-cysteine, which blocked the enhanced testosterone production from PGD2-treated keratinocytes. Our study suggests the existence of a possible crosstalk between the PGD2-reactive oxygen species axis and testosterone metabolism in keratinocytes. Thus, we propose that AGA patients might benefit from the use of N-acetyl-cysteine or other antioxidants as a supplement to currently available or emerging AGA therapies such as finasteride, minoxidil, and PGD2 receptor blockers.