Optimization of a Plasmodium falciparum circumsporozoite protein repeat vaccine using the tobacco mosaic virus platform.

Plasmodium falciparum vaccine RTS,S/AS01 is based on the major NPNA repeat and the C-terminal region of the circumsporozoite protein (CSP). RTS,S-induced NPNA-specific antibody titer and avidity have been associated with high-level protection in naïve subjects, but efficacy and longevity in target populations is relatively low. In an effort to improve upon RTS,S, a minimal repeat-only, epitope-focused, protective, malaria vaccine was designed. Repeat antigen copy number and flexibility was optimized using the tobacco mosaic virus (TMV) display platform. Comparing antigenicity of TMV displaying 3 to 20 copies of NPNA revealed that low copy number can reduce the abundance of low-affinity monoclonal antibody (mAb) epitopes while retaining high-affinity mAb epitopes. TMV presentation improved titer and avidity of repeat-specific Abs compared to a nearly full-length protein vaccine (FL-CSP). NPNAx5 antigen displayed as a loop on the TMV particle was found to be most optimal and its efficacy could be further augmented by combination with a human-use adjuvant ALFQ that contains immune-stimulators. These data were confirmed in rhesus macaques where a low dose of TMV-NPNAx5 elicited Abs that persisted at functional levels for up to 11 mo. We show here a complex association between NPNA copy number, flexibility, antigenicity, immunogenicity, and efficacy of CSP-based vaccines. We hypothesize that designing minimal epitope CSP vaccines could confer better and more durable protection against malaria. Preclinical data presented here supports the evaluation of TMV-NPNAx5/ALFQ in human trials.

Synthesis and biological evaluation of novel 8- substituted sampangine derivatives as potent inhibitor of Zn2+-Aß complex mediated toxicity, oxidative stress and inflammation.

A series of 8-substituted sampangine derivatives have been designed, synthesized and tested for their ability to inhibit cholinesterase and penetrate the blood-brain barrier. Their chelating ability toward Zn2+ and other biologically relevant metal ions was also demonstrated by isothermal titration calorimetry. The new derivatives exhibited high acetylcholinesterase inhibitory activity, high blood-brain barrier penetration ability and high chelating selectivity for Zn2+. Moreover, compound 10 with the strongest binding affinity to Zn2+ was selected for further research. Western blotting analysis, transmission electron microscopy, DCFH-DA assay and paralysis experiment indicated that compound 10 suppressed the formation of Zn2+-Aß complexes, alleviated the Zn2+ induced neurotoxicity and inhibited the production of ROS catalyzed by Zn2+ in Aß42 transgenic C. elegans. Furthermore, compound 10 also inhibited the expressions of pro-inflammatory cytokines, such as NO, TNF-α, IL-6 and IL-1ß, induced by Zn2+ + Aß1-42 in BV2 microglial cells. In general, this work provided new insights into the design and development of potent metal-chelating agents for Alzheimer's disease treatment.

Characteristics of Plastic Pollution in the Environment: A Review.

Plastics are ubiquitous in the environment and have become a hot topic in academic circles. Extensive studies have focused on analytical methods, source, abundance, transport, fate, degradation of plastics in the environment and threats to natural surroundings, wildlife or even human health. However, characteristics of plastic pollution, which are critical to understand this emerging problem, remain unknown up to now. Here, this paper reviews the major characteristics of plastic pollution in the environment to enhance present understanding of this issue. These characteristics, including diversity, persistence, global issues, combined pollution and threats to organisms and human health, are critically summarized in this work. Further, "plastic cycle" in the environment, namely, aquatic, atmospheric, and terrestrial system, is also discussed in this review. Finally, we highlight current challenges of plastic pollution posed to the public and also recommend the research trends in future work.

Identification of Plasmodium falciparum proteoforms from liver stage models.

BACKGROUND: Immunization with attenuated malaria sporozoites protects humans from experimental malaria challenge by mosquito bite. Protection in humans is strongly correlated with the production of T cells targeting a heterogeneous population of pre-erythrocyte antigen proteoforms, including liver stage antigens. Currently, few T cell epitopes derived from Plasmodium falciparum, the major aetiologic agent of malaria in humans are known. METHODS: In this study both in vitro and in vivo malaria liver stage models were used to sequence host and pathogen proteoforms. Proteoforms from these diverse models were subjected to mild acid elution (of soluble forms), multi-dimensional fractionation, tandem mass spectrometry, and top-down bioinformatics analysis to identify proteoforms in their intact state. RESULTS: These results identify a group of host and malaria liver stage proteoforms that meet a 5% false discovery rate threshold. CONCLUSIONS: This work provides proof-of-concept for the validity of this mass spectrometry/bioinformatic approach for future studies seeking to reveal malaria liver stage antigens towards vaccine development.

Environmental fate and behavior of silver nanoparticles in natural estuarine systems.

Silver nanoparticles (AgNPs) are widely used in many consumer products, whereas their environmental behaviors in natural aquatic systems remain unknown, especially in natural brackish media. Therefore, it is urgent to investigate the environmental fate of AgNPs in natural brackish waters. Here, we investigated the stability of citrate-coated AgNPs in natural brackish water collected from 6 different sites with distinct salinities in the Xinglinwan Reservoir, located in Xiamen City, southeast China. The obtained results showed that AgNP colloids remained stable in low-salinity waters, which was mainly determined by the effects of dissolved organic matter (DOM) promoting the stability of the nanoparticles. However, the environmental fate of AgNPs in high-salinity waters was dominated by the salinity or ionic strength, especially the free ion concentrations of Cl-, SO42-, or S2-, resulting in rapid sedimentation and dissolution. In addition, both DOM and salinity contributed to the environmental behavior of AgNPs in moderate-salinity waters, ultimately resulting in either colloidal stability or sedimentation. Overall, these results may reveal that AgNPs remain relatively stable for a long period in low-salinity natural waters, and that the stability might gradually decrease as AgNPs are transferred from freshwaters through brackish waters and eventually end up in seawater along the bay. Our findings also further indicate that the toxicity and potential risks of AgNPs may present more serious threats to the environment and organisms in natural freshwaters than in natural estuarine systems or seawater.

Comparison of immunogenicity and safety outcomes of a malaria vaccine FMP013/ALFQ in rhesus macaques (Macaca mulatta) of Indian and Chinese origin.

BACKGROUND: Indian-origin rhesus (InR) are preferred for research, but strict export restrictions continue to limit their use. Chinese-origin rhesus (ChR), although easier to procure, are genetically distinct from InR and differ in their immune response to infectious agents, such as the Simian Immunodeficiency Virus. The most advanced malaria vaccine, RTS,S (GlaxoSmithKline), is based on the circumsporozoite protein (CSP) of Plasmodium falciparum. The efficacy of RTS,S vaccine in the field remains low and short-lived; efforts are underway to improve CSP-based vaccines. Rhesus models can accelerate preclinical down-selection of the next generation of malaria vaccines. This study was used to determine if the safety and immunogenicity outcomes following vaccination with a CSP vaccine would differ in the InR and ChR models, given the genetic differences between the two sub-populations of rhesus. METHODS: The FMP013 vaccine, was composed of nearly full-length soluble P. falciparum CSP produced in Escherichia coli and was adjuvanted with the Army liposomal formulation (ALFQ). Three doses of the vaccine were administered in InR and ChR (n = 6) at 1-month intervals and the antibody and T cell responses were assessed. RESULTS: Local and systemic toxicity profile of FMP013 vaccine in InR and ChR were similar and they revealed that the FMP013 vaccine was safe and caused only mild and transient inflammatory adverse reactions. Following the first 2 vaccines, there was a slower acquisition of antibodies to the CSP repeat region in ChR. However after the 3rd vaccination the titers in the two models were comparable. The ChR group repeat-specific antibodies had higher avidity and ChR group showed higher inhibition of liver stage development activity compared to InR. There was no difference in T-cell responses to the FMP013 vaccine between the two models. CONCLUSIONS: A difference in the quality of serological responses was detected between the two sub-populations of rhesus. However, both models confirmed that FMP013/ALFQ vaccine was safe, highly immunogenic, elicited functional antibodies and T-cell responses. Overall, the data suggests that rhesus of Indian and Chinese origins can be interchangeably used to compare the safety and immunogenicity of next-generation of malaria vaccines and adjuvants.

A Near-infrared Persistent Luminescence Imaging Technique for Tracking Nanoparticles in Zebrafish (Danio rerio).

The development of nanotechnology has drawn increased attention to the risks of nanoparticles (NPs). In this work, the near-infrared persistent luminescence imaging technique was used to track the biodistribution of NPs in vivo in zebrafish. Zebrafish were used as a vertebrate animal model to show NPs distribution and the effects of exposure. ZnGa2O4:Cr (ZGOC) was chosen as the probe in this work. In continuous exposure experiments, the results showed more particles accumulated in the intestines than in the gills in both groups. In both the gills and abdomen, the NPs contents were greater in the ZGOC-NH2-treated groups than in the ZGOC groups, and the NPs caused damage to the gills and intestines. Removal exposure experiments indicated that ZGOC and ZGOC-NH2 could be excreted from the body. The metabolism, excretion of NPs, the quantification and monitoring of NPs behavior in biological systems should be examined in further studies.

Structures and luminescent sensors of mixed-counterions based salen-type lanthanide coordination polymers.

Reactions of N,N'-bis (salicylidene)-1,2-cyclohexanediamine (H2 L) with mixed lanthanide counterions of LnCl3 ·6H2 O and Ln (NO3 )3 ·6H2 O afford six H2 L lanthanide coordination polymers, e.g. {[Pr(H2 L)2 (NO3 )2 Cl]·2CH2 Cl2 }n (1); {[Ln(H2 L)1.5 (NO3 )3 ]2 ·5CHCl3 ·mCH3 OH}n [Ln = Sm (2), Eu (3), Gd (4), Tb (5) and Yb (6); m = 1 (2-5); m = 0 (6)]. X-ray crystallographic analysis reveals that complex 1 exhibits three-dimensional diamondoid topologic structure and complexes 2-6 are of two-dimensional structure. Luminescent spectra show that complexes 1 and 6 have characteristic near-infrared (NIR) emission of praseodymium (III) and ytterbium (III) ions and complexes 2-5 emit luminescence in the visible region. Complexes 3 and 6 reveal sensitive luminescence responses to formaldehyde.

Effect of the Tibetan Medicine Zuotai on Degranulation and Inflammatory Mediator Release in RBL-2H3 Cells.

Zuotai is a drug containing mercury considered to be the king of Tibetan medicine. The biosafety of Zuotai led people's attention and so far little is known about the toxicity of Zuotai to mast cells. RBL-2H3 cells which used as an alternative model of mast cells were treated with Zuotai, ß-HgS and positive drug Compound 48/80 respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the toxicity of drugs to RBL-2H3 cells. The degranulation of RBL-2H3 cells was studied from ß-hexosaminidase, histamine, interleukin (IL)-4 and tumor necrosis factor-α (TNF-α). The result showed that Zuotai can affect the cytotoxicity and degranulation of RBL-2H3 cells and the results can provide reference for the toxicity evaluations of Tibetan medicine Zuotai.

Comparative validation of the D. melanogaster modENCODE transcriptome annotation.

Accurate gene model annotation of reference genomes is critical for making them useful. The modENCODE project has improved the D. melanogaster genome annotation by using deep and diverse high-throughput data. Since transcriptional activity that has been evolutionarily conserved is likely to have an advantageous function, we have performed large-scale interspecific comparisons to increase confidence in predicted annotations. To support comparative genomics, we filled in divergence gaps in the Drosophila phylogeny by generating draft genomes for eight new species. For comparative transcriptome analysis, we generated mRNA expression profiles on 81 samples from multiple tissues and developmental stages of 15 Drosophila species, and we performed cap analysis of gene expression in D. melanogaster and D. pseudoobscura. We also describe conservation of four distinct core promoter structures composed of combinations of elements at three positions. Overall, each type of genomic feature shows a characteristic divergence rate relative to neutral models, highlighting the value of multispecies alignment in annotating a target genome that should prove useful in the annotation of other high priority genomes, especially human and other mammalian genomes that are rich in noncoding sequences. We report that the vast majority of elements in the annotation are evolutionarily conserved, indicating that the annotation will be an important springboard for functional genetic testing by the Drosophila community.

TWEAK/Fn14 activation induces keratinocyte proliferation under psoriatic inflammation.

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) has been reported to induce keratinocyte apoptosis in vitro by engaging its sole receptor of fibroblast growth factor-inducible 14 (Fn14). In this study, we explored the role of TWEAK/Fn14 pathway in the growth of psoriatic keratinocytes that is, however, characterized by suppressed apoptotic cell death. Skin tissues from the patients with psoriasis or healthy donors were determined for TWEAK and Fn14 expression, and primary keratinocytes were evaluated under the stimulation of psoriatic proinflammatory cytokines or plus TWEAK. The results showed that both TWEAK and Fn14 were highly expressed in psoriatic skins. Moreover, the stimulation of psoriatic cytokines enhanced Fn14 expression by keratinocytes in vitro, which expressed TNF receptor 2 predominantly and proliferated increasingly with the addition of TWEAK. Furthermore, TWEAK stimulation enhanced the synthesis of survivin, inhibitor of apoptosis protein 2 and cellular FLICE-inhibitory protein in lesional keratinocytes. Therefore, TWEAK/Fn14 interaction prefers to enhance proliferation but not apoptosis of keratinocytes under psoriatic inflammation. The activation of nuclear factor-κB signalling-dependent anti-apoptotic proteins and biased expression of TNF receptors may be responsible for such a novel principle in keratinocytes under psoriatic inflammation.

[Study on drug sensitivity against Comamonas testosteroni by Kirby-Bauer disk diffusion method].

OBJECTIVE: To determine the drug resistance of Comamonas testosteroni (C. testosteroni) by the Kirby-Bauer (K-B) method without Clinical and Laboratory Standards Institute (CLSI) explanation or the minimum inhibitory concentration (MIC) method with the standard CLSI explanation to evaluate the sensitivity of K-B method in detection of C. testosteroni.
 METHODS: K-B method and MIC method was used to determine the sensitivity of C. testosteroni to Piperacillin, Cefepime, Piperacillin/tazobactam, Imipenem, Meropenem, Amikacin, Gentamicin, Tobramycin, Ceftazidime and Ciprofloxacin. The interpretation standard for Pseudomonas aeruginosa was temporary used for the K-B method. The coincident rate was compared between the two methods.
 RESULTS: The complete or partial coincident rate for K-B method and MIC method to detect Piperacillin and Cefepime was 97.4% or 2.6%; the complete coincidence rate to detect Piperacillin/tazobactam, Imipenem and Meropenem was 100%; the complete or partial coincident rate to detect Amikacin, Gentamicin and Tobramycin 94.7% or 5.3%; the complete or partial coincident rate to detect Ceftazidime was 97.4% or 2.6%; the complete or partial coincident rate to detect Ciprofloxacin 86.8% or 10.6%, and the full non-coincidence rate was 2.6%.
 CONCLUSION: The results of drug sensitive test from the two methods are highly consistent. We suggest that the microbiology labs do not report the interpretive results for C. testosteroni with K-B method but report the test results.

Cytoplasmic delivery and selective, multicomponent labeling with oligoarginine-linked protein tags.

Strategies that leverage bio-orthogonal interactions between small molecule ligands and genetically encoded amino acid sequences can be used to attach high-performance fluorophores to proteins in living cells. However, a major limitation of chemical protein labeling is that cells' plasma membranes are impermeable to many useful probes and biolabels. Here, we show that conjugation to nonaarginine, a cell penetrating peptide (CPP), enables passive cytoplasmic delivery of otherwise membrane-impermeant, small molecule protein labels. Heterodimers consisting of a luminescent Tb(3+) complex, Lumi4, linked to benzyl guanine, benzyl cytosine, and trimethoprim were conjugated to the peptide CysArg9 with a reducible disulfide linker. When added to culture medium, the peptide conjugates rapidly (<30 min) enter the cytoplasm and diffuse freely throughout cells. The benzyl guanine, benzyl cytosine, and trimethoprim derivatives bind selectively to fusion proteins tagged with SNAP-Tag, CLIP-Tag, and Escherichia coli dihydrofolate reductase (eDHFR), respectively. Furthermore, eDHFR and SNAP-Tag fusions can be labeled with Lumi4 analogues in the same cell, and this labeling can be detected using two-color, time-gated Förster resonance energy transfer (FRET) microscopy. Finally, we present quantitative data showing that cytoplasmic uptake of nonaarginine-conjugated probes occurs in multiple cell types (MDCK, HeLa, NIH 3T3), most cells in a culture (>75%) are loaded with probe, and the cellular probe concentration can be controlled by varying incubation conditions. CPP-mediated delivery of Lumi4-linked protein labels will greatly increase the utility of lanthanide-based FRET microscopy. Moreover, our results strongly suggest that this approach can be adapted to deliver a wide variety of protein-targeted fluorophores or other functional probes that were previously unavailable for intracellular imaging studies.

Single-molecule magnet of a tetranuclear dysprosium complex disturbed by a salen-type ligand and chloride counterions.

A series of three salen-type lanthanide complexes, e.g., [Dy4(L)2(HL)2Cl2(µ3-OH)2]2Cl2(OH)2·3CH3CH2OH·H2O (1) and [Ln4(L)2(HL)2Cl2(µ3-OH)2]Cl2·5CH3OH·4CH2Cl2 (Ln = Tb(III), 2; Ho(III), 3) have been isolated by the reactions of H2L (H2L = N,N'-bis(3-methoxysalicylidene)cyclohexane-1,2-diamine) with LnCl3·6H2O. X-ray crystallographic analysis reveals that all complexes 1-3 are isostructural, in which four Ln ions and eight O atoms form the distorted defective dicubane {Dy4O8} cores. Magnetic studies indicate that complex 1 exhibits two slow magnetic relaxation processes with effective energy barrier Ueff = 55.71 K under a zero direct-current field, which is attributed to the two coordination geometries of the Dy(III) ions with a salen-type ligand and coordination of a chloride counterion. It represents the highest energy barrier among the salen-type tetranuclear lanthanide single-molecule magnets.

Near-IR luminescence and field-induced single molecule magnet of four salen-type ytterbium complexes.

A series of rigid hexadentate salen-type (H2L) ytterbium complexes, namely, [Yb2L3(CH3OH)]·3CH3CN (1), [Yb2LL'L″(CH3OH)(H2O)2](ClO4)2·CH3OH·H2O (2), [Yb2L(OAc)4(CH3OH)2]·2CH3OH (3), and {[Yb2L(OAc)4]·3H2O}n (4) (H2L = N,N'-bis(2-oxy-3-methoxybenzylidene)-1,2-phenylenediamine, HL' = 2-(2'-hydroxy-3'-methyloxy-phenyl)benzimidazole and HL" = 3-methoxysalicylaldehyde) have been synthesized by reactions of H2L with multifarious Yb(3+) salts. X-ray crystallographic analyses demonstrate that complex 1 is of a triple-decker sandwich-type Yb2L3 structure with a ratio of H2L/Yb = 3:2, 2 and 3 possess the unique Yb2 core with a ratio of H2L/Yb = 2:2 and 1:2, respectively, 4 exhibits one dimensional coordination polymers in which the polymeric structures are formed by acetate (OAc(-)) groups. All complexes 1-4 exhibit near-IR luminescence, which can be rationalized on the basis of the disparate structural effects. The magnetic analysis unveils that all complexes 1-4 are of field-induced single-molecule magnet behavior with the energy barriers (Ueff/kB) of 14.5, 2.0, 9.5, and 2.4 K at 3 kOe direct current fields, respectively.

Epistasis dominates the genetic architecture of Drosophila quantitative traits.

Epistasis-nonlinear genetic interactions between polymorphic loci-is the genetic basis of canalization and speciation, and epistatic interactions can be used to infer genetic networks affecting quantitative traits. However, the role that epistasis plays in the genetic architecture of quantitative traits is controversial. Here, we compared the genetic architecture of three Drosophila life history traits in the sequenced inbred lines of the Drosophila melanogaster Genetic Reference Panel (DGRP) and a large outbred, advanced intercross population derived from 40 DGRP lines (Flyland). We assessed allele frequency changes between pools of individuals at the extremes of the distribution for each trait in the Flyland population by deep DNA sequencing. The genetic architecture of all traits was highly polygenic in both analyses. Surprisingly, none of the SNPs associated with the traits in Flyland replicated in the DGRP and vice versa. However, the majority of these SNPs participated in at least one epistatic interaction in the DGRP. Despite apparent additive effects at largely distinct loci in the two populations, the epistatic interactions perturbed common, biologically plausible, and highly connected genetic networks. Our analysis underscores the importance of epistasis as a principal factor that determines variation for quantitative traits and provides a means to uncover genetic networks affecting these traits. Knowledge of epistatic networks will contribute to our understanding of the genetic basis of evolutionarily and clinically important traits and enhance predictive ability at an individualized level in medicine and agriculture.

Inhibition of inducible nitric oxide synthase expression and nitric oxide production in plateau pika (Ochotona curzoniae) at high altitude on Qinghai-Tibet Plateau.

Nitric oxide (NO), a potent vasodilator, plays an important role in preventing hypoxia induced pulmonary hypertension. Endogenous NO is synthesized by nitric oxide synthases (NOSs) from l-arginine. In mammals, three different NOSs have been identified, including neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). Plateau pika (Ochotona curzoniae) is a typical hypoxia tolerant mammal that lives at 3000-5000 m above sea level on the Qinghai-Tibet Plateau. The aim of this study was to investigate whether NOS expression and NO production are regulated by chronic hypoxia in plateau pika. Quantitative real-time PCR and western blot analyses were conducted to quantify relative abundances of iNOS and eNOS transcripts and proteins in the lung tissues of plateau pikas at different altitudes (4550, 3950 and 3200 m). Plasma NO metabolites, nitrite/nitrate (NO(x)⁻) levels were also examined by Ion chromatography to determine the correlation between NO production and altitude level. The results revealed that iNOS transcript levels were significantly lower in animals at high altitudes (decreased by 53% and 57% at altitude of 3950 and 4550 m compared with that at 3200 m). Similar trends in iNOS protein abundances were observed (26% and 41% at 3950 and 4550 m comparing with at 3200 m). There were no significant differences in eNOS mRNA and protein levels in the pika lungs among different altitudes. The plasma NO(x)⁻ levels of the plateau pikas at high altitudes significantly decreased (1.65±0.19 µg/mL at 3200 m to 0.44±0.03 µg/mL at 3950 m and 0.24±0.01 µg/mL at 4550 m). This is the first evidence describing the effects of chronic hypoxia on NOS expression and NO levels in the plateau pika in high altitude adaptation. We conclude that iNOS expression and NO production are suppressed at high altitudes, and the lower NO concentration at high altitudes may serve crucial roles for helping the plateau pika to survive at hypoxic environment.

Towards an optimized inhibition of liver stage development assay (ILSDA) for Plasmodium falciparum.

BACKGROUND: Experimental vaccines targeting Plasmodium falciparum have had some success in recent years. These vaccines use attenuated parasites, recombinant sporozoite proteins, or DNA and virus combinations to induce cell-mediated immune responses and/or antibodies targeting sporozoite surface proteins. To capitalize on the success of these vaccines and understand the mechanisms by which these vaccines function, it is important to develop assays that measure correlates of protection in volunteers. The inhibition of liver stage development assay (ILSDA) tests antibodies for the ability to block sporozoite development in hepatocytes. As such the ILSDA is an excellent candidate assay to identify correlates of humoral protection, particularly against the liver stage of malaria infection. In addition, the ILSDA can be used as a tool to evaluate novel sporozoite antigens for future vaccine development. Historically the ILSDA has suffered from low sporozoite infection rates, absence of standardized reagents, and the subjectivity associated with the traditional primary outcome measures, which depend on microscopy of stained hepatocyte cultures. This study worked to significantly improve sporozoite infection rates in hepatocytes, modify key steps in the assay protocol to reduce experimental variability, and demonstrate the utility of the ILSDA in testing antibodies targeting the circumsporozoite protein. METHODS: Cryopreserved primary human hepatocytes, Plasmodium falciparum sporozoites, and circumsporozoite antibodies were used to optimize the ILSDA. RESULTS: Inoculation of cryopreserved primary human hepatocytes with Plasmodium falciparum sporozoites improved liver stage development in the ILSDA compared to HCO4 cells. In the ILSDA, circumsporozoite antibodies suppressed liver stage development in cryopreserved primary human hepatocytes in a concentration-dependent manner. Antibody-mediated suppression of parasite development in the ILSDA at a 96-hour endpoint was more robust than the 24-hour endpoint. CONCLUSIONS: ILSDA performance is improved by the use of cryopreserved primary human hepatocytes, expediting interactions between sporozoites and hepatocytes, and extending the assay endpoint.

Mechanisms of protective immune responses induced by the Plasmodium falciparum circumsporozoite protein-based, self-assembling protein nanoparticle vaccine.

BACKGROUND: A lack of defined correlates of immunity for malaria, combined with the inability to induce long-lived sterile immune responses in a human host, demonstrate a need for improved understanding of potentially protective immune mechanisms for enhanced vaccine efficacy. Protective sterile immunity (>90%) against the Plasmodium falciparum circumsporozoite protein (CSP) has been achieved using a transgenically modified Plasmodium berghei sporozoite (Tg-Pb/PfCSP) and a self-assembling protein nanoparticle (SAPN) vaccine presenting CSP epitopes (PfCSP-SAPN). Here, several possible mechanisms involved in the independently protective humoral and cellular responses induced following SAPN immunization are described. METHODS: Inbred mice were vaccinated with PfCSP-SAPN in PBS. Serum antibodies were harvested and effects on P. falciparum sporozoites mobility and integrity were examined using phase contrast microscopy. The functionality of SAPN-induced antibodies on inhibition of sporozoite invasion and growth within primary human hepatocytes was also examined. The internal processing of SAPN by bone marrow-derived dendritic cells (BMDDC), using organelle-specific, fluorescent-tagged antibody or gold-encapsulated SAPN, was observed using confocal or electron microscopy, respectively. RESULTS: The results of this work demonstrate that PfCSP-SAPN induces epitope-specific antibody titers, predominantly of the Th2 isotype IgG1, and that serum antibodies from PfCSP-SAPN-immunized mice appear to target P. falciparum sporozoites via the classical pathway of complement. This results in sporozoite death as indicated by cessation of motility and the circumsporozoite precipitation reaction. Moreover, PfCSP-SAPN-induced antibodies are able to inhibit wild-type P. falciparum sporozoite invasion and growth within cultured primary human hepatocytes. In addition, the observation that PfCSP-SAPN are processed (and presented) to the immune system by dendritic cells in a slow and continuous fashion via transporter associated with antigen processing (TAP) recruitment to the early endosome (EE), and have partially delayed processing through the endoplasmic reticulum, has the potential to induce the long-lived, effector memory CD8+ T-cells as described previously. CONCLUSION: This paper describes the examination of humoral and cellular immune mechanisms induced by PfCSP-SAPN vaccination which result in sterile host protection against a transgenic P. berghei malaria sporozoite expressing the P. falciparum CSP, and which significantly inhibits native P. falciparum sporozoites from invading and developing within cultured human hepatocytes. These results may indicate the type and mode of action of protective antibodies needed to control P. falciparum sporozoites from infecting humans as well as a potential mechanism of induction of protective long-lived effector memory CD8+ T-cells.

Machine Learning Applied to Cholesterol-Lowering Pharmacotherapy: Proof-of-Concept in High-Risk Patients Treated in Primary Care.

Objectives: Machine learning has potential to improve the management of lipid disorders. We explored the utility of machine learning in high-risk patients in primary care receiving cholesterol-lowering medications. Methods: Machine learning algorithms were created based on lipid management guidelines for England [National Institute for Health and Care Excellence (NICE) CG181] to reproduce the guidance with >95% accuracy. Natural language processing and therapy identification algorithms were applied to anonymized electronic records from six South London primary care general practices to extract medication information from free text fields. Results: Among a total of 48,226 adult patients, a subset of 5630 (mean ± standard deviation, age = 67 ± 13 years; male:female = 55:45) with a history of lipid-lowering therapy were identified. Additional major cardiometabolic comorbidities included type 2 diabetes in 13% (n = 724) and hypertension in 32% (n = 1791); all three risk factors were present in a further 28% (n = 1552). Of the 5630 patients, 4290 (76%) and 1349 (24%) were in primary and secondary cardiovascular disease prevention cohorts, respectively. Statin monotherapy was the most common current medication (82%, n = 4632). For patients receiving statin monotherapy, 71% (n = 3269) were on high-intensity therapy aligned with NICE guidance with rates being similar for the primary and secondary prevention cohorts. In the combined cohort, only 46% of patients who had been prescribed lipid-lowering therapy in the previous 12 months achieved the NICE treatment goal of >40% reduction in non-high-density lipoprotein cholesterol from baseline pretreatment levels. Based on the most recent data entry for patients not at goal the neural network recommended either increasing the dose of statin, adding complementary cholesterol-lowering medication, or obtaining an expert lipid opinion. Conclusions: Machine learning can be of value in (a) quantifying suboptimal lipid-lowering prescribing patterns, (b) identifying high-risk patients who could benefit from more intensive therapy, and (c) suggesting evidence-based therapeutic options.