The results of the fifth blind test of crystal structure prediction, which show important success with more challenging large and flexible molecules, are presented and discussed.
Following on from the success of the previous crystal structure prediction blind tests (CSP1999, CSP2001, CSP2004 and CSP2007), a fifth such collaborative project (CSP2010) was organized at the Cambridge Crystallographic Data Centre. A range of methodologies was used by the participating groups in order to evaluate the ability of the current computational methods to predict the crystal structures of the six organic molecules chosen as targets for this blind test. The first four targets, two rigid molecules, one semi-flexible molecule and a 1:1 salt, matched the criteria for the targets from CSP2007, while the last two targets belonged to two new challenging categories – a larger, much more flexible molecule and a hydrate with more than one polymorph. Each group submitted three predictions for each target it attempted. There was at least one successful prediction for each target, and two groups were able to successfully predict the structure of the large flexible molecule as their first place submission. The results show that while not as many groups successfully predicted the structures of the three smallest molecules as in CSP2007, there is now evidence that methodologies such as dispersion-corrected density functional theory (DFT-D) are able to reliably do so. The results also highlight the many challenges posed by more complex systems and show that there are still issues to be overcome.
prediction; blind test; polymorph; crystal structure prediction
The results of the sixth blind test of organic crystal structure prediction methods are presented and discussed, highlighting progress for salts, hydrates and bulky flexible molecules, as well as on-going challenges.
The sixth blind test of organic crystal structure prediction (CSP) methods has been held, with five target systems: a small nearly rigid molecule, a polymorphic former drug candidate, a chloride salt hydrate, a co-crystal and a bulky flexible molecule. This blind test has seen substantial growth in the number of participants, with the broad range of prediction methods giving a unique insight into the state of the art in the field. Significant progress has been seen in treating flexible molecules, usage of hierarchical approaches to ranking structures, the application of density-functional approximations, and the establishment of new workflows and ‘best practices’ for performing CSP calculations. All of the targets, apart from a single potentially disordered Z′ = 2 polymorph of the drug candidate, were predicted by at least one submission. Despite many remaining challenges, it is clear that CSP methods are becoming more applicable to a wider range of real systems, including salts, hydrates and larger flexible molecules. The results also highlight the potential for CSP calculations to complement and augment experimental studies of organic solid forms.
crystal structure prediction; polymorphism; lattice energies; Cambridge Structural Database
Plasmodium vivax is the most widespread species of Plasmodium, causing up to 50% of the malaria cases occurring outside sub-Saharan Africa. An effective vaccine is essential for successful control and potential eradication. A well-characterized vaccine candidate is the circumsporozoite protein (CSP). Preclinical and clinical trials have shown that both antibodies and cellular immune responses have been correlated with protection induced by immunization with CSP. On the basis of our reported approach of developing chimeric Plasmodium yoelii proteins to enhance protective efficacy, we designed PvRMC-CSP, a recombinant chimeric protein based on the P. vivax CSP (PvCSP). In this engineered protein, regions of the PvCSP predicted to contain human T cell epitopes were genetically fused to an immunodominant B cell epitope derived from the N-terminal region I and to repeat sequences representing the two types of PvCSP repeats. The chimeric protein was expressed in soluble form with high yield. As the immune response to PvCSP has been reported to be genetically restricted in the murine model, we tested the immunogenicity of PvRMC-CSP in groups of six inbred strains of mice. PvRMC-CSP was able to induce robust antibody responses in all the mouse strains tested. Synthetic peptides representing the allelic forms of the P. vivax CSP were also recognized to a similar extent regardless of the mouse strain. Furthermore, the immunization regimen induced high frequencies of multifunctional CD4+ and CD8+ PvRMC-CSP-specific T cells. The depth and breadth of the immune responses elicited suggest that immunization with PvRMC-CSP can circumvent the genetic restriction of the immune response to P. vivax CSP. Interestingly, PvRMC-CSP was also recognized by naturally acquired antibodies from individuals living in areas where malaria is endemic. These features make PvRMC-CSP a promising vaccine candidate for further development.
This article describes an important improvement in the CrystalPredictor II code: adaptive Local Approximate Models (LAMs). This improvement allows the most efficient use of computational effort to cover a flexible molecule’s conformational space, and is illustrated with a crystal structure prediction (CSP) investigation into the sixth blind test molecule 26.
The global search stage of crystal structure prediction (CSP) methods requires a fine balance between accuracy and computational cost, particularly for the study of large flexible molecules. A major improvement in the accuracy and cost of the intramolecular energy function used in the CrystalPredictor II [Habgood et al. (2015 ▸). J. Chem. Theory Comput.
11, 1957–1969] program is presented, where the most efficient use of computational effort is ensured via the use of adaptive local approximate model (LAM) placement. The entire search space of the relevant molecule’s conformations is initially evaluated using a coarse, low accuracy grid. Additional LAM points are then placed at appropriate points determined via an automated process, aiming to minimize the computational effort expended in high-energy regions whilst maximizing the accuracy in low-energy regions. As the size, complexity and flexibility of molecules increase, the reduction in computational cost becomes marked. This improvement is illustrated with energy calculations for benzoic acid and the ROY molecule, and a CSP study of molecule (XXVI) from the sixth blind test [Reilly et al. (2016 ▸). Acta Cryst. B72, 439–459], which is challenging due to its size and flexibility. Its known experimental form is successfully predicted as the global minimum. The computational cost of the study is tractable without the need to make unphysical simplifying assumptions.
crystal structure prediction; solid-state science; local apprioximate model
Generating sets of trial structures
that sample the configurational
space of crystal packing possibilities is an essential step in the
process of ab initio crystal structure prediction
(CSP). One effective methodology for performing such a search relies
on low-discrepancy, quasi-random sampling, and our implementation
of such a search for molecular crystals is described in this paper.
Herein we restrict ourselves to rigid organic molecules and, by considering
their geometric properties, build trial crystal packings as starting
points for local lattice energy minimization. We also describe a method
to match instances of the same structure, which we use to measure
the convergence of our packing search toward completeness. The use
of these tools is demonstrated for a set of molecules with diverse
molecular characteristics and as representative of areas of application
where CSP has been applied. An important finding is that the lowest
energy crystal structures are typically located early and frequently
during a quasi-random search of phase space. It is usually the complete
sampling of higher energy structures that requires extended sampling.
We show how the procedure can first be refined, through targetting
the volume of the generated crystal structures, and then extended
across a range of space groups to make a full CSP search and locate
experimentally observed and lists of hypothetical polymorphs. As the
described method has also been created to lie at the base of more
involved approaches to CSP, which are being developed within the Global
Lattice Energy Explorer (Glee) software, a few of these extensions
are briefly discussed.
Among the multiple conservative modalities, physiotherapy is a commonly utilized treatment modality in managing chronic non-specific spinal pain. Despite the scientific progresses with regard to pain and motor control neuroscience, treatment of chronic spinal pain (CSP) often tends to stick to a peripheral biomechanical model, without targeting brain mechanisms. With a view to enhance clinical efficacy of existing physiotherapeutic treatments for CSP, the development of clinical strategies targeted at ‘training the brain’ is to be pursued. Promising proof-of-principle results have been reported for the effectiveness of a modern neuroscience approach to CSP when compared to usual care, but confirmation is required in a larger, multi-center trial with appropriate evidence-based control intervention and long-term follow-up.
The aim of this study is to assess the effectiveness of a modern neuroscience approach, compared to usual care evidence-based physiotherapy, for reducing pain and improving functioning in patients with CSP. A secondary objective entails examining the effectiveness of the modern neuroscience approach versus usual care physiotherapy for normalizing brain gray matter in patients with CSP.
The study is a multi-center, triple-blind, two-arm (1:1) randomized clinical trial with 1-year follow-up. 120 CSP patients will be randomly allocated to either the experimental (receiving pain neuroscience education followed by cognition-targeted motor control training) or the control group (receiving usual care physiotherapy), each comprising of 3 months treatment. The main outcome measures are pain (including symptoms and indices of central sensitization) and self-reported disability. Secondary outcome measures include brain gray matter structure, motor control, muscle properties, and psychosocial correlates. Clinical assessment and brain imaging will be performed at baseline, post-treatment and at 1-year follow-up. Web-based questionnaires will be completed at baseline, after the first 3 treatment sessions, post-treatment, and at 6 and 12-months follow-up.
Findings may provide empirical evidence on: (1) the effectiveness of a modern neuroscience approach to CSP for reducing pain and improving functioning, (2) the effectiveness of a modern neuroscience approach for normalizing brain gray matter in CSP patients, and (3) factors associated with therapy success. Hence, this trial might contribute towards refining guidelines for good clinical practice and might be used as a basis for health authorities’ recommendations.
ClinicalTrials.gov Identifier: NCT02098005.
Chronic pain; Low back pain; Neck pain; Education; Exercise; Motor control; Neuroscience; Randomized controlled trial
Induction of potent long lasting effector T cell responses against liver stage malaria antigens strongly correlates with protection from malaria. While Adenovirus serotype 5 (Ad5) based malaria vaccine platforms have the ability to induce potent effector T cell responses against transgenes, high rates of pre-existing Ad5 immunity in malaria endemic regions has prompted study of alternative Ad serotype based malaria vaccines as replacements for Ad5 based malaria vaccines. The research described in this article examines the utility of alternative serotype adenovirus serotype 4 (Ad4) expressing a sporozoite surface protein (circumsporozoite protein (CSP)) (Ad4-CSP) to induce immune responses against CSP. The immunogenicity of Ad4-CSP was also tested in homologous and heterologous prime boost vaccinations in both Ad5 naïve and Ad5 immune backgrounds as compared to use of Ad5-CSP.
In Ad5 naïve animals, use of Ad4-CSP priming vaccinations followed by boosting with Ad5-CSP (Ad4-CSP/Ad5-CSP) maximally increased the numbers of CSP specific cytokine secreting cytotoxic T cells relative to repeated use of Ad5-CSP. The Ad4-CSP/Ad5-CSP regimen also induced equivalent levels of CSP specific cell killing as did homologous prime-boost vaccinations with Ad5-CSP, despite stimulating lower numbers of CSP specific cytotoxic T cells. Priming with Ad4-CSP followed by a homologous boost resulted in significantly less CSP specific humoral responses than any other vaccination regimen tested in Ad naïve animals. In Ad5 immune animals, addition of Ad4-CSP in homologous or heterologous prime boost resulted in inductions of higher CSP specific responses than animals repeatedly vaccinated with Ad5-CSP alone. However, the observed responses were well below those observed in similarly treated Ad naïve mice.
While the Ad4-CSP/Ad5-CSP and Ad5-CSP/Ad5-CSP vaccination regimens resulted in equivalent CSP specific killing in Ad naïve animals, Ad4-CSP/Ad5-CSP achieved this result with a lower percentage of CSP specific CD8+ T cells and a higher number of IFNγ secreting cells, suggesting that the Ad4-CSP/Ad5-CSP vaccination regimen elicits more efficient cytotoxic T cells. In Ad5 immune animals use of Ad4-CSP improved CSP specific immune responses as compared to repeated use of Ad5-CSP, but could not achieve the levels of immunogenicity observed when the same vaccine regimens were used in Ad naïve animals. These data indicate the existence of some level of immunological cross-reactivity between these two adenovirus subgroups. Based on these results, it is suggested that future studies should undertake similarly stringent analyses of alternative Ad serotypes to establish their effectiveness as replacements for Ad5.
Serotype 5; Serotype 4; Adenovirus; Malaria; Circumsporozoite protein; Vaccine; Heterologous; Homologous; Prime; Boost
Malaria, caused by Plasmodium falciparum, continues to have a devastating impact on global health, emphasizing the great need for a malaria vaccine. The circumsporozoite protein (CSP) is an attractive target for a malaria vaccine, and forms a major component of RTS,S, the most clinically advanced malaria vaccine. The clinical efficacy of RTS,S has been moderate, yet has demonstrated the viability of a CSP-based malaria vaccine. In this study, a vaccine comprised of the full-length CSP antigen presented on a virus-like particle (VLP) is produced using a split-intein conjugation system (SpyTag/SpyCatcher) and the immunogenicity is tested in mice.
Full-length 3d7 CSP protein was genetically fused at the C-terminus to SpyCatcher. The CSP-SpyCatcher antigen was then covalently attached (via the SpyTag/SpyCatcher interaction) to Acinetobacter phage AP205 VLPs which were modified to display one SpyTag per VLP subunit. To evaluate the VLP-display effect, the immunogenicity of the VLP vaccine was tested in mice and compared to a control vaccine containing AP205 VLPs plus unconjugated CSP.
Full-length CSP was conjugated at high density (an average of 112 CSP molecules per VLP) to AP205 SpyTag-VLPs. Vaccination of mice with the CSP Spy-VLP vaccine resulted in significantly increased antibody titres over a course of 7 months as compared to the control group (2.6-fold higher at 7 months after immunization). Furthermore, the CSP Spy-VLP vaccine appears to stimulate production of IgG2a antibodies, which has been linked with a more efficient clearing of intracellular parasite infection.
This study demonstrates that the high-density display of CSP on SpyTag-VLPs, significantly increases the level and quality of the vaccine-induced humoral response, compared to a control vaccine consisting of soluble CSP plus AP205 VLPs. The SpyTag-VLP platform utilized in this study constitutes a versatile and rapid method to develop highly immunogenic vaccines. It might serve as a generic tool for the cost-effective development of effective VLP-vaccines, e.g., against malaria.
Virus-like particle; VLP; Pre-erythrocytic; Malaria vaccine; Circumsporozoite protein; CSP; Spycatcher; Spytag; Bacterial superglue; Split-intein
Increased frequency of cavum septum pellucidum (CSP) has been inconsistently observed in schizophrenia, and little is known about its functional implications. We investigated whether patients with schizophrenia were more likely than healthy controls to have CSP, and among patients assessed the relationship between CSP, psychiatric symptoms, and selected neuropsychological functions. Seventy-seven patients with diagnoses of DSM-IV schizophrenia spectrum disorders and 55 healthy controls were studied and completed a 1.5 T MRI scan. Two raters, blind to group membership, determined the presence, length and grade of the CSP. A subset of participants also underwent neuropsychological testing. A CSP of at least 1 mm in length was present in 68.8% of patients and 76.4% of controls, and the groups did not differ significantly with respect to presence or absence, length, overall size, or percent with an abnormally large CSP (≥ 6 mm). Patients with an abnormally large CSP demonstrated poorer performance on measures of verbal learning and memory than patients with smaller CSP. Among patients, CSP length was significantly correlated with negative symptoms, verbal learning, and sentence comprehension. Among patients with abnormally large CSP, CSP length was correlated with reaction time on two conditions of a Continuous Performance Test. CSP, while prevalent, was not more frequent in our sample of patients with schizophrenia, and had few associations with symptom severity or neuropsychological deficits.
Schizophrenia; MRI; Cavum Septum Pellucidum; Neuropsychology
Circumsporozoite protein (CSP) of Plasmodium falciparum is a promising malaria vaccine target. RTS,S, the most advanced malaria vaccine candidate consists of the central NANP repeat and carboxy-terminal region of CSP displayed on a hepatitis B virus-like particle (VLP). To build upon the success of RTS,S, we produced a near full-length Plasmodium falciparum CSP that also includes the conserved amino-terminal region of CSP. We recently showed that this soluble CSP, combined with a synthetic Toll-like-receptor-4 (TLR4) agonist in stable oil-in-water emulsion (GLA/SE), induces a potent and protective immune response in mice against transgenic parasite challenge. Here we have investigated whether the immunogenicity of soluble CSP could be further augmented by presentation on a VLP. Bacteriophage Qβ VLPs can be readily produced in E.coli, they have a diameter of 25 nm and contain packaged E. coli RNA which serves as a built in adjuvant through the activation of TLR7/8. CSP was chemically conjugated to Qβ and the CSP-Qβ vaccine immunogenicity and efficacy were compared to adjuvanted soluble CSP in the C57Bl/6 mouse model. When formulated with adjuvants lacking a TLR4 agonist (Alum, SE and Montanide) the Qβ-CSP induced higher anti-NANP repeat titers, higher levels of cytophilic IgG2b/c antibodies and a trend towards higher protection against transgenic parasite challenge as compared to soluble CSP formulated in the same adjuvant. The VLP and soluble CSP immunogenicity difference was most pronounced at low antigen dose, and within the CSP molecule, the titers against the NANP repeats were preferentially enhanced by Qβ presentation. While a TLR4 agonist enhanced the immunogenicity of soluble CSP to levels comparable to the VLP vaccine, the TLR4 agonist did not further improve the immunogenicity of the Qβ-CSP vaccine. The data presented here pave the way for further improvement in the Qβ conjugation chemistry and evaluation of both the Qβ-CSP and soluble CSP vaccines in the non-human primate model.
Models of immunity to malaria indicate the importance of CD8+ T cell responses for targeting intrahepatic stages and antibodies for targeting sporozoite and blood stages. We designed a multistage adenovirus 5 (Ad5)-vectored Plasmodium falciparum malaria vaccine, aiming to induce both types of responses in humans, that was tested for safety and immunogenicity in a Phase 1 dose escalation trial in Ad5-seronegative volunteers.
The NMRC-M3V-Ad-PfCA vaccine combines two adenovectors encoding circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1). Group 1 (n = 6) healthy volunteers received one intramuscular injection of 2×10∧10 particle units (1×10∧10 each construct) and Group 2 (n = 6) a five-fold higher dose. Transient, mild to moderate adverse events were more pronounced with the higher dose. ELISpot responses to CSP and AMA1 peaked at 1 month, were higher in the low dose (geomean CSP = 422, AMA1 = 862 spot forming cells/million) than in the high dose (CSP = 154, p = 0.049, AMA1 = 423, p = 0.045) group and were still positive at 12 months in a number of volunteers. ELISpot depletion assays identified dependence on CD4+ or on both CD4+ and CD8+ T cells, with few responses dependent only on CD8+ T cells. Intracellular cytokine staining detected stronger CD8+ than CD4+ T cell IFN-γ responses (CSP p = 0.0001, AMA1 p = 0.003), but similar frequencies of multifunctional CD4+ and CD8+ T cells secreting two or more of IFN-γ, TNF-α or IL-2. Median fluorescence intensities were 7–10 fold higher in triple than single secreting cells. Antibody responses were low but trended higher in the high dose group and did not inhibit growth of cultured P. falciparum blood stage parasites.
As found in other trials, adenovectored vaccines appeared safe and well-tolerated at doses up to 1×10∧11 particle units. This is the first demonstration in humans of a malaria vaccine eliciting strong CD8+ T cell IFN-γ responses.
Archaea are abundant and drive critical microbial processes in the Earth's cold biosphere. Despite this, not enough is known about the molecular mechanisms of cold adaptation and no biochemical studies have been performed on stenopsychrophilic archaea (e.g., Methanogenium frigidum). This study examined the structural and functional properties of cold shock proteins (Csps) from archaea, including biochemical analysis of the Csp from M. frigidum. csp genes are present in most bacteria and some eucarya but absent from most archaeal genome sequences, most notably, those of all archaeal thermophiles and hyperthermophiles. In bacteria, Csps are small, nucleic acid binding proteins involved in a variety of cellular processes, such as transcription. In this study, archaeal Csp function was assessed by examining the ability of csp genes from psychrophilic and mesophilic Euryarchaeota and Crenarchaeota to complement a cold-sensitive growth defect in Escherichia coli. In addition, an archaeal gene with a cold shock domain (CSD) fold but little sequence identity to Csps was also examined. Genes encoding Csps or a CSD structural analog from three psychrophilic archaea rescued the E. coli growth defect. The three proteins were predicted to have a higher content of solvent-exposed basic residues than the noncomplementing proteins, and the basic residues were located on the nucleic acid binding surface, similar to their arrangement in E. coli CspA. The M. frigidum Csp was purified and found to be a single-domain protein that folds by a reversible two-state mechanism and to exhibit a low conformational stability typical of cold-adapted proteins. Moreover, M. frigidum Csp was characterized as binding E. coli single-stranded RNA, consistent with its ability to complement function in E. coli. The studies show that some Csp and CSD fold proteins have retained sufficient similarity throughout evolution in the Archaea to be able to function effectively in the Bacteria and that the function of the archaeal proteins relates to cold adaptation. The initial biochemical analysis of M. frigidum Csp has developed a platform for further characterization and demonstrates the potential for expanding molecular studies of proteins from this important archaeal stenopsychrophile.
Protein-protein interactions represent difficult but increasingly important targets for the design of therapeutic compounds able to interfere with biological processes. Recently, fragment-based strategies have been proposed as attractive approaches for the elaboration of protein-protein surface inhibitors from fragment-like molecules. One major challenge in targeting protein-protein interactions is related to the structural adaptation of the protein surface upon molecular recognition. Methods capable of identifying subtle conformational changes of proteins upon fragment binding are therefore required at the early steps of the drug design process. In this report we present a fast NMR method able to probe subtle conformational changes upon fragment binding. The approach relies on the comparison of experimental fragment-induced Chemical Shift Perturbation (CSP) of amine protons to CSP simulated for a set of docked fragment poses, considering the ring-current effect from fragment binding. We illustrate the method by the retrospective analysis of the complex between the anti-apoptotic Bcl-xL protein and the fragment 4′-fluoro-[1,1′-biphenyl]-4-carboxylic acid that was previously shown to bind one of the Bcl-xL hot spots. The CSP-based approach shows that the protein undergoes a subtle conformational rearrangement upon interaction, for residues located in helices 2, 3 and the very beginning of 5. Our observations are corroborated by residual dipolar coupling measurements performed on the free and fragment-bound forms of the Bcl-xL protein. These NMR-based results are in total agreement with previous molecular dynamic calculations that evidenced a high flexibility of Bcl-xL around the binding site. Here we show that CSP of protein amine protons are useful and reliable structural probes. Therefore, we propose to use CSP simulation to assess protein conformational changes upon ligand binding in the fragment-based drug design approach.
The three-dimensional structures of two odorant binding proteins (OBPs) and one chemosensory protein (CSP) from a polyphagous ectoparasitoid Scleroderma guani (Hymenoptera: Bethylidae) were resolved bioinformatically. The results show that both SguaOBP1 and OBP2 are classic OBPs, whereas SguaCSP1 belongs to non-classic CSPs which are considered as the “Plus-C” CSP in this report. The structural differences between the two OBPs and between OBP and CSP are thoroughly described, and the structural and functional significance of the divergent C-terminal regions (e.g., the prolonged C-terminal region in SguaOBP2 and the additional pair of cysteines in SguaCSP1) are discussed. The immunoblot analyses with antisera raised against recombinant SguaOBP1, OBP2, and CSP1, respectively, indicate that two SguaOBPs are specific to antennae, whereas SguaCSP1, which are more abundant than OBPs and detected in both male and female wasps, expresses ubiquitously across different tissues.
We also describe the ultrastructure of the antennal sensilla types in S. guani and compare them to 19 species of parasitic Hymenoptera. There are 11 types of sensilla in the flagellum and pedicel segments of antennae in both male and female wasps. Seven of them, including sensilla placodea (SP), long sensilla basiconica (LSB), sensilla coeloconica (SC), two types of double-walled wall pore sensilla (DWPS-I and DWPS-II), and two types of sensilla trichodea (ST-I and ST-II), are multiporous chemosensilla. The ultralsturctures of these sensilla are morphologically characterized. In comparison to monophagous specialists, the highly polyphagous generalist ectoparasitoids such as S. guani possess more diverse sensilla types which are likely related to their broad host ranges and complex life styles. Our immunocytochemistry study demonstrated that each of the seven sensilla immunoreacts with at least one antiserum against SguaOBP1, OBP2, and CSP1, respectively. Anti-OBP2 is specifically labeled in DWPS-II, whereas the anti-OBP1 shows a broad spectrum of immunoactivity toward four different sensilla (LSB, SP, ST-I and ST-II). On the other hand, anti-CSP1 is immunoactive toward SP, DWPS-I and SC. Interestingly, a cross co-localization pattern between SguaOBP1 and CSP1 is documented for the first time. Given that the numbers of OBPs and CSPs in many insect species greatly outnumber their antennal sensilla types, it is germane to suggest such phenomenon could be the rule rather than the exception.
Scleroderma guani; OBP; CSP; tertiary structure; sensilla; immunolocalization
When the gene for CspA, the major cold shock protein of Escherichia coli, was disrupted by a novel positive/negative selection method, the deltacspA cells did not show any discernible growth defect at either 37 or 15 degrees C. By two-dimensional gel electrophoresis, total protein synthesis was analyzed after temperature downshift in the deltacspA strain. The production of the CspA homologs CspB and CspG increased, and the duration of their expression was prolonged, suggesting that both CspB and CspG compensate for the function of CspA in the absence of CspA during cold shock adaptation. Interestingly, the production of the 159-base 5'-untranslated region (5'-UTR) of cspA from the chromosomal cspA::cat gene, detected by primer extension, failed to be repressed after cold shock. When an independent system to produce CspA was added to the deltacspA strain, the 5'-UTR production for the cspA::cat gene was significantly reduced compared to that of the deltacspA strain. By examining the expression of translationally fused cspA and cspB genes to lacZ in the deltacspA strain, it was found that cspA is more strongly regulated by CspA than cspB is. We showed that the increased expression of the 5'-UTR of the cspA mRNA in the deltacspA strain occurred mainly at the level of transcription and, to a certain extent, at the level of mRNA stabilization. The mRNA stabilization in the deltacspA strain was observed for other mRNAs, supporting the notion that CspA functions as an mRNA chaperone to destabilize secondary structures in mRNAs.
The applications of polysaccharide phenyl carbamate derivatives as chiral stationary phases (CSPs) for capillary electrochromatography (CEC) are often hindered by longer retention times, especially using a normal-phase (NP) eluent due to very low electroosmotic flow (EOF). Therefore, in this study, we propose an approach for the aforementioned problems by introducing two new types of negatively charged sulfate and sulfonated groups for polysaccharide CSPs. These CSPs were utilized to pack CEC columns for enantioseparation with a NP eluent. Compared to conventional cellulose tris(3,5-dimethylphenyl carbamate) or CDMPC CSPs, the sulfated CDMPC CSP (sulfur content 4.25%, w/w) shortened the analysis time up to 50% but with a significant loss of enantiomeric resolution (~60%). On the other hand, the sulfonated CDMPC CSP (sulfur content 1.76%, w/w) not only provided fast throughput but also maintained excellent resolving power. In addition, its synthesis is much more straightforward than the sulfated one. Furthermore, we studied several stationary phase parameters (CSP loading and silica gel pore size) and mobile phase parameters (including type of mobile phase and its composition) to evaluate the throughput and enantioselectivity. Using the optimized conditions, a chiral pool containing 66 analytes was screened to evaluate the enantioselectivity under three different mobile phase modes (i.e., NP, polar organic phase (POP) and reversed-phase (RP) eluents). Among these mobile phase modes, the RP mode showed the highest success rate, whereas some degree of complementary enantioselectivity was observed with NP and POP. Finally, the feasibility of applying this CSP for CEC–MS enantioseparation using internal tapered column was evaluated with NP, POP and RP eluents. In particular, the NP-CEC–MS provided significantly enhanced sensitivity when methanol was replaced with isopropanol in the sheath liquid. Using aminog-lutethimide as model chiral analyte, all three modes of CEC–MS demonstrated excellent durability as well as excellent reproducibility of retention time and enantioselectivity.
Sulfonated polysaccharide; Chiral stationary phase loading; Pore size; Normal-phase CEC–MS; Reversed-phase CEC–MS; Polar organic phase CEC–MS
Class I-restricted CD8+ cytotoxic T lymphocytes (CTL) against the circumsporozoite protein (CSP) protect mice against the rodent malaria parasite, Plasmodium yoelii, and vaccines designed to produce protective CTL against the P. falciparum CSP (PfCSP) are under development. Humans and B10.BR (H-2k) mice have been shown to have CD8+ CTL activity against a 23-amino-acid region of the PfCSP (residues 368 to 390 from the PfCSP 7G8 sequence) that is too long to bind directly to class I major histocompatibility complex molecules. To identify within this 23-amino-acid peptide a shorter peptide that binds to an H-2k class I major histocompatibility molecule, a primarily CD8+ (97.8%) T-cell line (PfCSP TCL.1) was produced by immunizing B10.BR mice with recombinant vaccinia virus expressing the PfCSP and stimulating in vitro spleen cells from these immunized mice with L cells transfected with the PfCSP gene (LPF cells). PfCSP TCL.1 lysed LPF cells and L cells pulsed with peptide PfCSP 7G8 368-390. When 15 overlapping nonamer peptides spanning the 368 to 390 sequence were tested, only one peptide, PfCSP 7G8 375-383 (Y E N D I E K K I), which includes an H-2Kk-binding motif, E at amino acid residue 2, and I at residue 9, sensitized targets for lysis by PfCSP TCL.1. Furthermore, a 10(3)- to 10(4)-fold lower concentration of the nonamer than that of the 23-amino-acid peptide was required to sensitize target cells for lysis by PfCSP TCL.1. Presentation by H-2Kk was demonstrated by using 3T3 fibroblast cells transfected with the murine H-2Kk or H-2Dk genes, and only the H-2Kk transfectants were lysed by PfCSP TCL.1 after incubation with peptide PfCSP 7G8 375-383. Binding to H-2Kk was confirmed by competitive inhibition of binding of labelled peptides to affinity-purified Kk molecules. Substitution of the anchor amino acid residue, E, at position 2 with A dramatically reduced binding to Kk and eliminated the capacity of the peptide to sensitize target cells for killing. Variation of non-anchor residues did not markedly reduce binding to Kk but in some cases eliminated the capacity of the peptide to sensitize targets for cytolysis by PfCSP TCL.1, presumably by eliminating T-cell receptor-binding sites. These data suggest that similar studies with human T cells will be required for optimal development of peptide-based vaccines designed to produce protective class I-restricted CD8+ CTL against the PfCSP in humans.
Plasmodium falciparum circumsporozoite protein (CSP) is a leading malaria vaccine candidate antigen, known to elicit protective antibody responses in humans (RTS,S vaccine). Recently, a DNA prime / adenovirus (Ad) vector boost vaccine encoding CSP and a second P. falciparum antigen, apical membrane antigen-1, also elicited sterile protection, but in this case associated with interferon gamma ELISpot and CD8+ T cell but not antibody responses. The finding that CSP delivered by an appropriate vaccine platform likely elicits protective cell-mediated immunity provided a rationale for identifying class I-restricted epitopes within this leading vaccine candidate antigen.
Limited samples of peripheral blood mononuclear cells from clinical trials of the Ad vaccine were used to identify CD8+ T cell epitopes within pools of overlapping 15mer peptides spanning portions of CSP that stimulated recall responses. Computerized algorithms (NetMHC) predicted 17 minimal class I-restricted 9-10mer epitopes within fifteen 15mers positive in ELISpot assay using PBMC from 10 HLA-matched study subjects. Four additional epitopes were subsequently predicted using NetMHC, matched to other study subjects without initial 15mer ELISpot screening. Nine of the putative epitopes were synthesized and tested by ELISpot assay, and six of these nine were further tested for CD8+ T cell responses by ELISpot CD4+ and CD8+ T cell-depletion and flow cytometry assays for evidence of CD8+ T cell dependence.
Each of the nine putative epitopes, all sequence-conserved, recalled responses from HLA-matched CSP-immunized research subjects. Four shorter sequences contained within these sequences were identified using NetMHC predictions and may have contributed to recall responses. Five (9-10mer) epitopes were confirmed to be targets of CD8+ T cell responses using ELISpot depletion and ICS assays. Two 9mers among these nine epitopes were each restricted by two HLA supertypes (A01/B07; A01A24/A24) and one 9mer was restricted by three HLA supertypes (A01A24/A24/B27) indicating that some CSP class I-restricted epitopes, like DR epitopes, may be HLA-promiscuous.
This study identified nine and confirmed five novel class I epitopes restricted by six HLA supertypes, suggesting that an adenovirus-vectored CSP vaccine would be immunogenic and potentially protective in genetically diverse populations.
Malaria; Vaccine; Circumsporozoite protein; ELISpot; Flow cytometry; NetMHC; Epitope mapping; Class I restriction; Localization
This study tests Common Sense Parenting (CSP)®, a widely used parent-training program, in its standard form and in a modified form known as CSP Plus, with low-income 8th graders and their families during the high school transition. The six-session CSP program proximally targets parenting and child emotion regulation skills. CSP Plus adds two sessions that include youth, and the eight-session program further targets skills for avoiding negative peers and activities in high school. Over two cohorts, 321 families were enrolled and randomly assigned to either CSP, CSP Plus, or minimal-contact control conditions. To date, pretest and posttest assessments have been completed, with 93% retention over about a 6-month interval. Here, analyses of preliminary outcomes from pretest to posttest focus on data collected from parents, who represent the primary proximal intervention targets. Intent-to-treat structural equation modeling analyses were conducted. CSP and CSP Plus had statistically significant effects on increased parent-reported child emotion regulation skills. CSP Plus further showed a statistically significant effect on increased parent perceptions of their adolescent being prepared for high school, but only in a model that excluded the CSP condition. Neither program had a significant proximal effect on parenting practices. Emotion regulation, one indicator of self-control, is a robust protective factor against problem behaviors. Intervention effects on this outcome may translate into reduced problems during high school. Moreover, CSP Plus showed some limited signs of added value for preparing families for the high school transition.
parent-training; family intervention; prevention; transition; high school; parenting; emotion regulation
Dinoflagellate transcriptomes contain cold shock domain proteins as the major component of the proteins annotated as transcription factors. We show here that the major family of cold shock domain proteins in the dinoflagellate Lingulodinium do not bind specific sequences, suggesting that transcriptional control is not a predominant mechanism for regulating gene expression in this group of protists.
Roughly two-thirds of the proteins annotated as transcription factors in dinoflagellate transcriptomes are cold shock domain-containing proteins (CSPs), an uncommon condition in eukaryotic organisms. However, no functional analysis has ever been reported for a dinoflagellate CSP, and so it is not known if they do in fact act as transcription factors. We describe here some of the properties of two CSPs from the dinoflagellate Lingulodinium polyedrum, LpCSP1 and LpCSP2, which contain a glycine-rich C-terminal domain and an N-terminal cold shock domain phylogenetically related to those in bacteria. However, neither of the two LpCSPs act like the bacterial CSP, since they do not functionally complement the Escherichia coli quadruple cold shock domain protein mutant BX04, and cold shock does not induce LpCSP1 and LpCSP2 to detectable levels, based on two-dimensional gel electrophoresis. Both CSPs bind to RNA and single-stranded DNA in a nonspecific manner in electrophoretic mobility shift assays, and both proteins also bind double-stranded DNA nonspecifically, albeit more weakly. These CSPs are thus unlikely to act alone as sequence-specific transcription factors.
IMPORTANCE Dinoflagellate transcriptomes contain cold shock domain proteins as the major component of the proteins annotated as transcription factors. We show here that the major family of cold shock domain proteins in the dinoflagellate Lingulodinium do not bind specific sequences, suggesting that transcriptional control is not a predominant mechanism for regulating gene expression in this group of protists.
RNA binding domain; DNA binding domain; cold shock domain; dinoflagellates; cold shock protein; transcription
A new class of cold shock-induced proteins that may be involved in an adaptive process required for cell viability at low temperatures or may function as antifreeze proteins in Escherichia coli and Saccharomyces cerevisiae has been identified. We purified a small Bacillus subtilis cold shock protein (CspB) and determined its amino-terminal sequence. By using mixed degenerate oligonucleotides, the corresponding gene (cspB) was cloned on two overlapping fragments of 5 and 6 kb. The gene encodes an acidic 67-amino-acid protein (pI 4.31) with a predicted molecular mass of 7,365 Da. Nucleotide and deduced amino acid sequence comparisons revealed 61% identity to the major cold shock protein of E. coli and 43% identity to a family of eukaryotic DNA binding proteins. Northern RNA blot and primer extension studies indicated the presence of one cspB transcript that was initiated 119 bp upstream of the initiation codon and was found to be induced severalfold when exponentially growing B. subtilis cell cultures were transferred from 37 degrees C to 10 degrees C. Consistent with this cold shock induction of cspB mRNA, a six- to eightfold induction of a cspB-directed beta-galactosidase synthesis was observed upon downshift in temperature. To investigate the function of CspB, we inactivated the cold shock protein by replacing the cspB gene in the B. subtilis chromosome with a cat-interrupted copy (cspB::cat) by marker replacement recombination. The viability of cells of this mutant strain, GW1, at freezing temperatures was strongly affected. However, the effect of having no CspB in GW1 could be slightly compensated for when cells were preincubated at 10 degrees C before freezing. These results indicate that CspB belongs to a new type of stress-inducible proteins that might be able to protect B. subtilis cells from damage caused by ice crystal formation during freezing.
Small molecules can powerfully benefit society, but the study and optimization of their function is too often impeded by the time-intensive and specialist-dependent process that is typically used to make them. In contrast, general and automated platforms have been developed for peptide, oligonucleotide, and increasingly oligosaccharide synthesis, resulting in on-demand access to these molecules, even for non-specialists. A more generalized and automated approach for making small molecules could similarly help shift the rate limiting step in small molecule science from synthesis to function. Targeting this goal, we have developed a fully automated and increasingly general platform for iterative coupling of boronate building blocks. Analogous to peptide synthesis, the process involves iterative coupling of haloboronic acids protected as the corresponding N-methyliminodiacetic acid (MIDA) boronates. This platform has enabled us and other groups to access many polyene natural products, including the polyene motifs in >75% of all polyene natural products. It further allowed us to derivatize and thereby understand the powerful but also highly toxic antifungal natural product amphotericin B, which has led to the development of less toxic derivatives currently under evaluation as drug candidates. We also discovered a stereocontrolled entry into chiral, non-racemic α-boryl aldehydes, which are versatile intermediates for the synthesis of many Csp3 boronate building blocks that are otherwise difficult to access. We have also expanded the scope of the platform to include Csp3-rich, polycyclic molecules using a linear-to-cyclized strategy, in which Csp3 boronate building blocks are iteratively assembled into linear precursors that are then cyclized into the cyclic frameworks found in many natural products and natural product-like structures. Enabled by the serendipitous discovery of a catch-and-release protocol for generally purifying MIDA boronate intermediates, the platform has been automated. The synthesis of 14 distinct classes of small molecules, including pharmaceuticals, materials, and polycyclic natural products has been achieved using this new synthesis machine. It is anticipated that the scope of small molecules accessible by this platform will continue to expand via further developments in building block synthesis, Csp3 cross-coupling methodologies, and cyclization strategies.
MIDA boronates; iterative coupling; automated synthesis; polyenes; Amphotericin B
Inverse PCR was used to amplify major cold shock protein (MCSP) gene families from a diverse range of bacteria, including the psychrotolerant Yersinia enterocolitica, which was found to have two almost identical MCSP coding regions (cspA1 and cspA2) located approximately 300 bp apart. This tandem gene duplication was also found in Y. pestis, Y. pseudotuberculosis, and Y. ruckeri but not in other bacteria. Analysis of the transcriptional regulation of this MCSP gene in Y. enterocolitica, performed by using both reverse transcriptase-PCR and Northern blot assays, showed there to be two cold-inducible mRNA templates arising from this locus: a monocistronic template of approximately 450 bp (cspA1) and a bicistronic template of approximately 900 bp (cspA1/A2). The former may be due to a secondary structure between cspA1 and cspA2 causing either 3′ degradation protection of cspA1 or, more probably, partial termination after cspA1. Primer extension experiments identified a putative transcriptional start site (+1) which is flanked by a cold-box motif and promoter elements (−10 and −35) similar to those found in Escherichia coli cold-inducible MCSP genes. At 30°C, the level of both mRNA molecules was negligible; however, upon a temperature downshift to 10°C, transcription of the bicistronic mRNA was both substantial (300-fold increase) and immediate, with transcription of the monocistronic mRNA being approximately 10-fold less (30-fold increase) and significantly slower. The ratio of bicistronic to monocistronic mRNA changed with time after cold shock and was higher when cells were shocked to a lower temperature. High-resolution, two-dimensional protein gel electrophoresis showed that synthesis of the corresponding proteins, both CspA1 and CspA2, was apparent after only 10 min of cold shock from 30°C to 10°C. The data demonstrate an extraordinary capacity of the psychrotolerant Y. enterocolitica to produce major cold shock proteins upon cold shock.
To compare the clinical, socioeconomic and demographic characteristics of individuals diagnosed with Neisseria gonorrhoeae (NG) in the community using a concomitant nucleic acid amplification test (NAAT, AptimaCombo2) as part of the (community-based) UK Chlamydia Screening Programme (CSP), with those diagnosed in hospital-based genitourinary medicine (GUM) services.
A retrospective case note review of all 643 patients treated for NG at a GUM in north west England (January 2007–April 2009).
All 643 treated for NG (including CSP cases, since all cases were referred to GUM for treatment). Limited data were available for 13 CSP cases who failed to attend GUM.
Primary outcome measure
Whether the case was detected in the community or GUM services. Predictors were demographics (age, gender, postcode for deprivation analysis), sexual history (eg, number of partners) and clinical factors (eg, culture positivity).
131 cases were diagnosed by CSP (13 of whom did not attend GUM). A further four cases were contacts of these. The GUM caseload was thus inflated by 23% (from 521 to 643). Community cases were overwhelmingly female (85% vs 27% in GUM, p<0.001) and younger (87% females were <25 years vs 70% GUM females, p=0.001). Logistic regression analysis restricted to the target age of the CSP (<25 years) revealed that CSP cases, compared with GUM cases, were more likely to reside in deprived areas (adjusted OR=5.6, 95% CI 1.4 to 21.8 and 5.3, CI 1.7 to 16.6 for the most and second most deprived group respectively, compared with the averagely deprived group, p=0.037) and be asymptomatic (adjusted OR=1.9, CI 1.1 to 3.4, p=0.02).
Community screening for NG led to a 79% increase in the number of infections detected in women aged <25 years. Screening is targeted at young people, and tends to disproportionately attract young women, a group under-represented at GUM. Screening also contributed further to case detection in deprived areas.
Genitourinary Medicine; Socioeconomic Status; Mass screening; Community Health Services; Residence Characteristics; Neisseria Gonorrhoeae
The purpose of this paper is to describe a clearly defined manual method for calculating cortical silent period (CSP) length that can be employed successfully and reliably by raters after minimal training in subjects with focal hand dystonia (FHD) and healthy subjects. A secondary purpose was to explore intra-subject variability of the CSP in subjects with FHD vs. healthy subjects.
Two raters previously naïve to CSP identification and one experienced rater independently analyzed 170 CSP measurements collected in six subjects with focal hand dystonia (FHD) and nine healthy subjects. Intraclass correlation coefficient (ICC) was calculated to quantify inter-rater reliability within the two groups of subjects. The relative variability of CSP in each group was calculated by coefficient of variation (CV). Relative variation between raters within repeated measures of individual subjects was also quantified by CV.
Reliability measures were as follows: mean of three raters: all subjects: ICC= 0.976; within healthy subjects: ICC=0.965; in subjects with FHD: ICC= 0.956. The median within-subject variability for the healthy group was CV=7.33% and in subjects with FHD: CV= 11.78%. The median variability of calculating individual subject CSP duration between raters was CV=10.23% in subjects with dystonia and CV=10.46% in healthy subjects.
Manual calculation of CSP results in excellent reliability between raters of varied levels of experience. Healthy subjects display less variability in CSP. Despite greater variability, the CSP in impaired subjects can be reliably calculated across raters.
cortical silent period; cortical excitability; focal hand dystonia; reliability; definition; methodology; transcranial magnetic stimulation