A significant modification to the additive all-atom CHARMM lipid force field (FF) is developed and applied to phospholipid bilayers with both choline and ethanolamine containing head groups and with ...both saturated and unsaturated aliphatic chains. Motivated by the current CHARMM lipid FF (C27 and C27r) systematically yielding values of the surface area per lipid that are smaller than experimental estimates and gel-like structures of bilayers well above the gel transition temperature, selected torsional, Lennard-Jones and partial atomic charge parameters were modified by targeting both quantum mechanical (QM) and experimental data. QM calculations ranging from high-level ab initio calculations on small molecules to semiempirical QM studies on a 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) bilayer in combination with experimental thermodynamic data were used as target data for parameter optimization. These changes were tested with simulations of pure bilayers at high hydration of the following six lipids: DPPC, 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1,2-dilauroyl-sn-phosphatidylcholine (DLPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2-dioleoyl-sn-phosphatidylcholine (DOPC), and 1-palmitoyl-2-oleoyl-sn-phosphatidylethanolamine (POPE); simulations of a low hydration DOPC bilayer were also performed. Agreement with experimental surface area is on average within 2%, and the density profiles agree well with neutron and X-ray diffraction experiments. NMR deuterium order parameters (S(CD)) are well predicted with the new FF, including proper splitting of the S(CD) for the aliphatic carbon adjacent to the carbonyl for DPPC, POPE, and POPC bilayers. The area compressibility modulus and frequency dependence of (13)C NMR relaxation rates of DPPC and the water distribution of low hydration DOPC bilayers also agree well with experiment. Accordingly, the presented lipid FF, referred to as C36, allows for molecular dynamics simulations to be run in the tensionless ensemble (NPT), and is anticipated to be of utility for simulations of pure lipid systems as well as heterogeneous systems including membrane proteins.
No vaccine has yet proven effective against the blood-stages of Plasmodium falciparum, which cause the symptoms and severe manifestations of malaria. We recently found that PfRH5, a P. ...falciparum-specific protein expressed in merozoites, is efficiently targeted by broadly-neutralizing, vaccine-induced antibodies. Here we show that antibodies against PfRH5 efficiently inhibit the in vitro growth of short-term-adapted parasite isolates from Cambodia, and that the EC(50) values of antigen-specific antibodies against PfRH5 are lower than those against PfAMA1. Since antibody responses elicited by multiple antigens are speculated to improve the efficacy of blood-stage vaccines, we conducted detailed assessments of parasite growth inhibition by antibodies against PfRH5 in combination with antibodies against seven other merozoite antigens. We found that antibodies against PfRH5 act synergistically with antibodies against certain other merozoite antigens, most notably with antibodies against other erythrocyte-binding antigens such as PfRH4, to inhibit the growth of a homologous P. falciparum clone. A combination of antibodies against PfRH4 and basigin, the erythrocyte receptor for PfRH5, also potently inhibited parasite growth. This methodology provides the first quantitative evidence that polyclonal vaccine-induced antibodies can act synergistically against P. falciparum antigens and should help to guide the rational development of future multi-antigen vaccines.
A spectrophotometric approach for quantification of dissolved manganese (DMn) with 1-(2-pyridylazo)-2-naphthol (PAN) has been adapted for in situ application in coastal and estuarine waters. The ...analyser uses a submersible microfluidic lab-on-chip device, with low power (~ 1.5 W) and reagent consumption (63 µL per sample). Laboratory characterization showed an absorption coefficient of 40,838 ± 1127 L⋅mol
⋅cm
and a detection limit of 27 nM, determined for a 34.6 mm long optical detection cell. Laboratory tests showed that long-term stability of the PAN reagent was achieved by addition of 4% v/v of a non-ionic surfactant (Triton-X100). To suppress iron (Fe) interferences with the PAN reagent, the Fe(III) masking agents deferoxamine mesylate (DFO-B) or disodium 4,5-dihydroxy-1,3-benzenedisulfonate (Tiron) were added and their Fe masking efficiencies were investigated. The analyser was tested during a deployment over several weeks in Kiel Fjord (Germany), with successful acquisition of 215 in situ data points. The time series was in good agreement with DMn concentrations determined from discretely collected samples analysed via inductively coupled plasma mass spectrometry (ICP-MS), exhibiting a mean accuracy of 87% over the full deployment duration (with an accuracy of > 99% for certain periods) and clear correlations to key hydrographic parameters.
Here, we present a new in situ microfluidic phosphate sensor that features an improved "phosphate blue" assay which includes polyvinylpyrrolidone in place of traditional surfactants-improving ...sensitivity and reducing temperature effects. The sensor features greater power economy and analytical performance relative to commercially available alternatives, with a mean power consumption of 1.8 W, a detection limit of 40 nM, a dynamic range of 0.14-10 μM, and an infield accuracy of 4 ± 4.5%. During field testing, the sensor was continuously deployed for 9 weeks in a chalk stream, revealing complex relations between flow rates and phosphate concentration that suggest changing dominance in phosphate sources. A distinct diel phosphorus signal was observed under low flow conditions, highlighting the ability of the sensor to decouple geochemical and biotic effects on phosphate dynamics in fluvial environments. This paper highlights the importance of high resolution in situ sensors in addressing the current gross under-sampling of aquatic environments.
Antigenic diversity has posed a critical barrier to vaccine development against the pathogenic blood-stage infection of the human malaria parasite Plasmodium falciparum. To date, only strain-specific ...protection has been reported by trials of such vaccines in nonhuman primates. We recently showed that P. falciparum reticulocyte binding protein homolog 5 (PfRH5), a merozoite adhesin required for erythrocyte invasion, is highly susceptible to vaccine-inducible strain-transcending parasite-neutralizing antibody. In vivo efficacy of PfRH5-based vaccines has not previously been evaluated. Here, we demonstrate that PfRH5-based vaccines can protect Aotus monkeys against a virulent vaccine-heterologous P. falciparum challenge and show that such protection can be achieved by a human-compatible vaccine formulation. Protection was associated with anti-PfRH5 antibody concentration and in vitro parasite-neutralizing activity, supporting the use of this in vitro assay to predict the in vivo efficacy of future vaccine candidates. These data suggest that PfRH5-based vaccines have potential to achieve strain-transcending efficacy in humans.
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•Vaccines based on the P. falciparum merozoite antigen PfRH5 were tested in Aotus monkeys•PfRH5-based vaccines afforded protection against heterologous strains of P. falciparum•Protection correlated with anti-PfRH5 IgG concentration and in vivo neutralization
Antigenic diversity has hindered development of vaccines against the pathogenic blood-stage of Plasmodium falciparum. Douglas et al. demonstrate that human-compatible PfRH5-based vaccines can protect Aotus monkeys against vaccine-heterologous P. falciparum challenge. Protection correlated with anti-PfRH5 antibody concentration and parasite-neutralizing activity. PfRH5-based vaccines have potential to achieve strain-transcending efficacy in humans.
Estimates of current global rabies mortality range from 26,000 to 59,000 deaths per annum. Although pre-exposure prophylaxis using inactivated rabies virus vaccines (IRVs) is effective, it requires ...two to three doses and is regarded as being too expensive and impractical for inclusion in routine childhood immunization programmes.
Here we report the development of a simian-adenovirus-vectored rabies vaccine intended to enable cost-effective population-wide pre-exposure prophylaxis against rabies. ChAdOx2 RabG uses the chimpanzee adenovirus serotype 68 (AdC68) backbone previously shown to achieve pre-exposure protection against rabies in non-human primates. ChAdOx2 differs from AdC68 in that it contains the human adenovirus serotype 5 (AdHu5) E4 orf6/7 region in place of the AdC68 equivalents, enhancing ease of manufacturing in cell lines which provide AdHu5 E1 proteins in trans. We show that immunogenicity of ChAdOx2 RabG in mice is comparable to that of AdC68 RabG and other adenovirus serotypes expressing rabies virus glycoprotein. High titers of rabies virus neutralizing antibody (VNA) are elicited after a single dose. The relationship between levels of VNA activity and rabies virus glycoprotein monomer-binding antibody differs after immunization with adenovirus-vectored vaccines and IRV vaccines, suggesting routes to further enhancement of the efficacy of the adenovirus-vectored candidates. We also demonstrate that ChAdOx2 RabG can be thermostabilised using a low-cost method suitable for clinical bio-manufacture and ambient-temperature distribution in tropical climates. Finally, we show that a dose-sparing effect can be achieved by formulating ChAdOx2 RabG with a simple chemical adjuvant. This approach could lower the cost of ChAdOx2 RabG and other adenovirus-vectored vaccines.
ChAdOx2 RabG may prove to be a useful tool to reduce the human rabies death toll. We have secured funding for Good Manufacturing Practice- compliant bio-manufacture and Phase I clinical trial of this candidate.
Calreticulin (CRT) is a high-capacity Ca2+ protein whose expression is up-regulated during cellular transformation and is associated with disease progression in multiple types of malignancies. At the ...same time, CRT has been characterized as an important stress-response protein capable of inducing immunogenic cell death (ICD) when translocated to the cell surface. It remains unclear why CRT expression is preserved by malignant cells during the course of transformation despite its immunogenic properties. In this study, we identify a novel, critical function of CRT as a cell survival factor in multiple types of human solid-tissue malignancies. CRT knockdown activates p53, which mediates cell-death response independent of executioner caspase activity and accompanied full-length poly ADP ribose polymerase (PARP) cleavage. Mechanistically, we show that down-regulation of CRT results in mitochondrial Ca2+ overload and induction of mitochondria permeability transition pore (mPTP)-dependent cell death, which can be significantly rescued by the mPTP inhibitor, Cyclosporin A (CsA). The clinical importance of CRT expression was revealed in the analysis of the large cohort of cancer patients (N = 2,058) to demonstrate that high levels of CRT inversely correlates with patient survival. Our study identifies intracellular CRT as an important therapeutic target for tumors whose survival relies on its expression.
Intranasal vaccination may induce protective local and systemic immune responses against respiratory pathogens. A number of intranasal SARS-CoV-2 vaccine candidates have achieved protection in ...pre-clinical challenge models, including ChAdOx1 nCoV-19 (AZD1222, University of Oxford / AstraZeneca).
We performed a single-centre open-label Phase I clinical trial of intranasal vaccination with ChAdOx1 nCoV-19 in healthy adults, using the existing formulation produced for intramuscular administration.
Thirty SARS-CoV-2 vaccine-naïve participants were allocated to receive 5 × 109 viral particles (VP, n=6), 2 × 1010 VP (n=12), or 5 × 1010 VP (n=12). Fourteen received second intranasal doses 28 days later. A further 12 received non-study intramuscular mRNA SARS-CoV-2 vaccination between study days 22 and 46.
To investigate intranasal ChAdOx1 nCoV-19 as a booster, six participants who had previously received two intramuscular doses of ChAdOx1 nCoV-19 and six who had received two intramuscular doses of BNT162b2 (Pfizer / BioNTech) were given a single intranasal dose of 5 × 1010 VP of ChAdOx1 nCoV-19.
Objectives were to assess safety (primary) and mucosal antibody responses (secondary).
Reactogenicity was mild or moderate. Antigen-specific mucosal antibody responses to intranasal vaccination were detectable in a minority of participants, rarely exceeding levels seen after SARS-CoV-2 infection. Systemic responses to intranasal vaccination were typically weaker than after intramuscular vaccination with ChAdOx1 nCoV-19. Antigen-specific mucosal antibody was detectable in participants who received an intramuscular mRNA vaccine after intranasal vaccination. Seven participants developed symptomatic SARS-CoV-2 infection.
This formulation of intranasal ChAdOx1 nCoV-19 showed an acceptable tolerability profile but induced neither a consistent mucosal antibody response nor a strong systemic response.
AstraZeneca.
Central neuromodulators (antidepressants, antipsychotics, and other central nervous system−targeted medications) are increasingly used for treatment of functional gastrointestinal disorders (FGIDs), ...now recognized as disorders of gut−brain interaction. However, the available evidence and guidance for the use of central neuromodulators in these conditions is scanty and incomplete. In this Rome Foundation Working Team report, a multidisciplinary team summarized available research evidence and clinical experience to provide guidance and treatment recommendations.
The working team summarized the literature on the pharmacology of central neuromodulators and their effects on gastrointestinal sensorimotor function and conducted an evidence-based review on their use for treating FGID syndromes. Because of the paucity of data for FGIDs, we included data for non-gastrointestinal painful disorders and specific symptoms of pain, nausea, and vomiting. This information was combined into a final document comprising a synthesis of available evidence and recommendations for clinical use guided by the research and clinical experience of the experts on the committee.
The evidence-based review on neuromodulators in FGID, restricted by the limited available controlled trials, was integrated with open-label studies and case series, along with the experience of experts to create recommendations using a consensus (Delphi) approach. Due to the diversity of conditions and complexity of treatment options, specific recommendations were generated for different FGIDs. However, some general recommendations include: (1) low to modest dosages of tricyclic antidepressants provide the most convincing evidence of benefit for treating chronic gastrointestinal pain and painful FGIDs and serotonin noradrenergic reuptake inhibitors can also be recommended, though further studies are needed; (2) augmentation, that is, adding a second treatment (adding quetiapine, aripiprazole, buspirone α2δ ligand agents) is recommended when a single medication is unsuccessful or produces side effects at higher dosages; (3) treatment should be continued for 6−12 months to potentially prevent relapse; and (4) implementation of successful treatment requires effective communication skills to improve patient acceptance and adherence, and to optimize the patient−provider relationship.
Based on systematic and selectively focused review and the consensus of a multidisciplinary panel, we have provided summary information and guidelines for the use of central neuromodulators in the treatment of chronic gastrointestinal symptoms and FGIDs. Further studies are needed to confirm and refine these recommendations.
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