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1.  Towards crystal structure prediction of complex organic compounds – a report on the fifth blind test 
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.
PMCID: PMC3222142  PMID: 22101543
2.  Efficacy of a modern neuroscience approach versus usual care evidence-based physiotherapy on pain, disability and brain characteristics in chronic spinal pain patients: protocol of a randomized clinical trial 
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.
Trial registration Identifier: NCT02098005.
PMCID: PMC4028010  PMID: 24885889
Chronic pain; Low back pain; Neck pain; Education; Exercise; Motor control; Neuroscience; Randomized controlled trial
3.  Immunogenicity when utilizing adenovirus serotype 4 and 5 vaccines expressing circumsporozoite protein in naïve and Adenovirus (Ad5) immune mice 
Malaria Journal  2012;11:209.
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.
PMCID: PMC3472263  PMID: 22720732
Serotype 5; Serotype 4; Adenovirus; Malaria; Circumsporozoite protein; Vaccine; Heterologous; Homologous; Prime; Boost
4.  Structure and Function of Cold Shock Proteins in Archaea▿  
Journal of Bacteriology  2007;189(15):5738-5748.
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.
PMCID: PMC1951829  PMID: 17545280
5.  BcL-xL Conformational Changes upon Fragment Binding Revealed by NMR 
PLoS ONE  2013;8(5):e64400.
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.
PMCID: PMC3662666  PMID: 23717610
6.  Cavum Septum Pellucidum in Schizophrenia: Clinical and Neuropsychological Correlates 
Psychiatry research  2007;154(2):147-155.
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.
PMCID: PMC1858669  PMID: 17291728
Schizophrenia; MRI; Cavum Septum Pellucidum; Neuropsychology
7.  CspA, the major cold shock protein of Escherichia coli, negatively regulates its own gene expression. 
Journal of Bacteriology  1997;179(22):7081-7088.
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.
PMCID: PMC179650  PMID: 9371456
8.  Ultrastructural Characterization of Olfactory Sensilla and Immunolocalization of Odorant Binding and Chemosensory Proteins from an Ectoparasitoid Scleroderma guani (Hymenoptera: Bethylidae) 
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.
PMCID: PMC3149280  PMID: 21814481
Scleroderma guani; OBP; CSP; tertiary structure; sensilla; immunolocalization
9.  Sulfated and sulfonated polysaccharide as chiral stationary phases for capillary electrochromatography and capillary electrochromatography–mass spectrometry 
Journal of chromatography. A  2008;1216(5):857-872.
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.
PMCID: PMC2752677  PMID: 19108837
Sulfonated polysaccharide; Chiral stationary phase loading; Pore size; Normal-phase CEC–MS; Reversed-phase CEC–MS; Polar organic phase CEC–MS
10.  Pathogenic Yersinia Species Carry a Novel, Cold-Inducible Major Cold Shock Protein Tandem Gene Duplication Producing both Bicistronic and Monocistronic mRNA 
Journal of Bacteriology  1999;181(20):6449-6455.
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.
PMCID: PMC103781  PMID: 10515936
11.  Adenovirus 5-Vectored P. falciparum Vaccine Expressing CSP and AMA1. Part A: Safety and Immunogenicity in Seronegative Adults 
PLoS ONE  2011;6(10):e24586.
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.
Methodology/Principal Findings
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.
Trial Registration NCT00392015
PMCID: PMC3189181  PMID: 22003383
12.  Localization of Cold Shock Proteins to Cytosolic Spaces Surrounding Nucleoids in Bacillus subtilis Depends on Active Transcription 
Journal of Bacteriology  2001;183(21):6435-6443.
Using immunofluorescence microscopy and a fusion of a cold shock protein (CSP), CspB, to green fluorescent protein (GFP), we showed that in growing cells Bacillus subtilis CSPs specifically localize to cytosolic regions surrounding the nucleoid. The subcellular localization of CSPs is influenced by the structure of the nucleoid. Decondensed chromosomes in smc mutant cells reduced the sizes of the regions in which CSPs localized, while cold shock-induced chromosome compaction was accompanied by an expansion of the space in which CSPs were present. As a control, histone-like protein HBsu localized to the nucleoids, while β-galactosidase and GFP were detectable throughout the cell. After inhibition of translation, CspB-GFP was still present around the nucleoids in a manner similar to that in cold-shocked cells. However, in stationary-phase cells and after inhibition of transcription, CspB was distributed throughout the cell, indicating that specific localization of CspB depends on active transcription and is not due to simple exclusion from the nucleoid. Furthermore, we observed that nucleoids are more condensed and frequently abnormal in cspB cspC and cspB cspD double-mutant cells. This suggests that the function of CSPs affects chromosome structure, probably through coupling of transcription to translation, which is thought to decondense nucleoids. In addition, we found that cspB cspD and cspB cspC double mutants are defective in sporulation, with a block at or before stage 0. Interestingly, CspB and CspC are depleted from the forespore compartment but not from the mother cell. In toto, our findings suggest that CSPs localize to zones of newly synthesized RNA, coupling transcription with initiation of translation.
PMCID: PMC100140  PMID: 11591689
13.  Serological responses to a soluble recombinant chimeric Plasmodium vivax circumsporozoite protein in VK210 and VK247 population 
Malaria Journal  2013;12:323.
Circumsporozoite protein (CSP) is essential for sporozoite formation and sporozoite invasion into human hepatocyte. Previously, a recombinant P. vivax CSP based on chimeric repeats (rPvCSP-c) representing two major alleles VK210 and VK247 within central region has been designed. Naturally acquired humoral immune responses study show that antigenicity of rPvCSP-c was much higher than that of native strain. However, the serologic reactivity of rPvCSP-c was still unclear in detail.
In present study, recognition of rPvCSP-c in vivax malaria typed VK210 and VK247 alleles was assessed. VK210 typed and VK247 typed sera from adult residents reacted specifically with rPvCSP-c using protein array and immunoblot assay. Additionally, anti-rPvCSP-c serum recognized the fixed VK210 and VK247 sporozoites by immunofluorescence assay. Furthermore, statistic analysis was performed for correlational detection.
The rPvCSP-c reacted with both VK210 typed and VK247 typed P. vivax infected patient sera and anti-rPvCSP-c immune serum also reacted with VK210 and VK247 sporozoite parasites of P. vivax specifically. There was a positive correlation between increased antibody level, age of patients and also associated with pvcsp repeat number, although the level of responses did vary considerably in their reactivity to the rPvCSP-c from negative to very high level within each age group.
These data confirmed the serologic reactivity of the novel rPvCSP-c in exposed both VK210 and VK247 populations. These results strongly suggested that this recombinant CSP was biologically active and potently immunogenic across major strains and raised the prospect that this protein could be used as serologic marker.
PMCID: PMC3847697  PMID: 24034268
14.  Identification of minimal human MHC-restricted CD8+ T-cell epitopes within the Plasmodium falciparum circumsporozoite protein (CSP) 
Malaria Journal  2013;12:185.
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.
PMCID: PMC3683343  PMID: 23738590
Malaria; Vaccine; Circumsporozoite protein; ELISpot; Flow cytometry; NetMHC; Epitope mapping; Class I restriction; Localization
15.  Identification of a nonameric H-2Kk-restricted CD8+ cytotoxic T lymphocyte epitope on the Plasmodium falciparum circumsporozoite protein. 
Infection and Immunity  1995;63(5):1955-1959.
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.
PMCID: PMC173249  PMID: 7537251
16.  Cold Shock Genes cspA and cspB from Caulobacter crescentus Are Posttranscriptionally Regulated and Important for Cold Adaptation 
Journal of Bacteriology  2012;194(23):6507-6517.
Cold shock proteins (CSPs) are nucleic acid binding chaperones, first described as being induced to solve the problem of mRNA stabilization after temperature downshift. Caulobacter crescentus has four CSPs: CspA and CspB, which are cold induced, and CspC and CspD, which are induced only in stationary phase. In this work we have determined that the synthesis of both CspA and CspB reaches the maximum levels early in the acclimation phase. The deletion of cspA causes a decrease in growth at low temperature, whereas the strain with a deletion of cspB has a very subtle and transient cold-related growth phenotype. The cspA cspB double mutant has a slightly more severe phenotype than that of the cspA mutant, suggesting that although CspA may be more important to cold adaptation than CspB, both proteins have a role in this process. Gene expression analyses were carried out using cspA and cspB regulatory fusions to the lacZ reporter gene and showed that both genes are regulated at the transcriptional and posttranscriptional levels. Deletion mapping of the long 5′-untranslated region (5′-UTR) of each gene identified a common region important for cold induction, probably via translation enhancement. In contrast to what was reported for other bacteria, these cold shock genes have no regulatory regions downstream from ATG that are important for cold induction. This work shows that the importance of CspA and CspB to C. crescentus cold adaptation, mechanisms of regulation, and pattern of expression during the acclimation phase apparently differs in many aspects from what has been described so far for other bacteria.
PMCID: PMC3497529  PMID: 23002229
17.  Insights into the Evolution of the CSP Gene Family through the Integration of Evolutionary Analysis and Comparative Protein Modeling 
PLoS ONE  2013;8(5):e63688.
Insect chemical communication and chemosensory systems rely on proteins coded by several gene families. Here, we have combined protein modeling with evolutionary analysis in order to study the evolution and structure of chemosensory proteins (CSPs) within arthropods and, more specifically, in ants by using the data available from sequenced genomes. Ants and other social insects are especially interesting model systems for the study of chemosensation, as they communicate in a highly complex social context and much of their communication relies on chemicals. Our ant protein models show how this complexity has shaped CSP evolution; the proteins are highly modifiable by their size, surface charge and binding pocket. Based on these findings, we divide ant CSPs into three groups: typical insect CSPs, an ancient 5-helical CSP and hymenopteran CSPs with a small binding pocket, and suggest that these groups likely serve different functions. The hymenopteran CSPs have duplicated repeatedly in individual ant lineages. In these CSPs, positive selection has driven surface charge changes, an observation which has possible implications for the interaction between CSPs and ligands or odorant receptors. Our phylogenetic analysis shows that within the Arthropoda the only highly conserved gene is the ancient 5-helical CSP, which is likely involved in an essential ubiquitous function rather than chemosensation. During insect evolution, the 6-helical CSPs have diverged and perform chemosensory functions among others. Our results contribute to the general knowledge of the structural differences between proteins underlying chemosensation and highlight those protein properties which have been affected by adaptive evolution.
PMCID: PMC3665776  PMID: 23723994
18.  Could linear solvation energy relationships give insights into chiral recognition mechanisms? 2. Characterization of macrocyclic glycopeptide stationary phases 
Journal of chromatography. A  2007;1166(0):70-78.
Five parameter linear solvation energy relationships (LSER) are known to have little or no shape recognition ability. However, it is proposed to use LSER studies to get insights into chiral recognition mechanisms. Since the two enantiomers have exactly the same five A–V solute descriptors being still separated by chiral stationary phases (CSPs), it can be considered that they form two different transient diastereoisomers with the CSP. It is then possible to perform LSER studies on the enantioselectivity factors taken as the two enantiomer retention factor ratios. In a first step, the five a–v system parameters of four CSPs of the macrocyclic glycopeptide types were determined using a set of test solutes with known A–V descriptors, both in the reversed phase and the normal phase modes. In a second step, the A–V descriptors of 18 enantiomeric pairs were tentatively established using five achiral columns with known a–v parameters. This was successful for the five molecular enantiomers only. It was found that the predicted retention factor for the molecular enantiomers separated on a given CSP corresponded either to retention factor of the first experimentally eluted enantiomer or to the second one or to none of them. Using the enantioselectivity factors it was possible to obtain the Δa–Δv parameters corresponding to the difference in CSP properties seen by the two enantiomers. For the five molecular enantiomeric pairs in the reversed phase mode with a teicoplanin CSP, it was found that there was an elevated contribution by the e coefficient that we interpret as a possible interaction between surface charges on the teicoplanin CSP and solute induced dipoles. Steric effects, seen on the v parameter, are second in magnitude followed by H-bond and polar interactions. Only one solute could be studied in the normal phase mode showing a different mechanism with polar and steric major interactions.
PMCID: PMC4155902  PMID: 17719593
Linear solvation energy relationship; Chiral stationary phase; Enantioselectivity; Chiral mechanisms; Teicoplanin; Macrocyclic glycopeptide; Reversed phase; Normal phase mode
19.  Characterization of cspB, a Bacillus subtilis inducible cold shock gene affecting cell viability at low temperatures. 
Journal of Bacteriology  1992;174(20):6326-6335.
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.
PMCID: PMC207576  PMID: 1400185
20.  Development and Evaluation of a Contrast Sensitivity Perimetry Test for Patients with Glaucoma 
To design a contrast sensitivity perimetry (CSP) protocol that decreases variability in glaucomatous defects while maintaining good sensitivity to glaucomatous loss.
Twenty patients with glaucoma and 20 control subjects were tested with a CSP protocol implemented on a monitor-based testing station. In the protocol 26 locations were tested over the central visual field with Gabor patches with a peak spatial frequency of 0.4 cyc/deg and a two-dimensional spatial Gaussian envelope, with most of the energy concentrated within a 4° circular region. Threshold was estimated by a staircase method. Patients and 10 age-similar control subjects were also tested on conventional automated perimetry (CAP), with the 24−2 pattern with the SITA Standard testing strategy. The neuroretinal rim area of the patients was measured with a retinal tomograph (Retina Tomograph II [HRT]; Heidelberg Engineering, Heidelberg, Germany). A Bland-Altman analysis of agreement was used to assess test–retest variability, compare depth of defect shown by the two perimetric tests, and investigate the relations between contrast sensitivity and neuroretinal rim area.
Variability showed less dependence on defect depth for CSP than for CAP (z = 9.3, P < 0.001). Defect depth was similar for CAP and CSP when averaged by quadrant (r = 0.26, P > 0.13). The relation between defect depth and rim area was more consistent with CSP than with CAP (z = 9, P < 0.001).
The implementation of CSP was successful in reducing test–retest variability in glaucomatous defects. CSP was in general agreement with CAP in terms of depth of defect and was in better agreement than CAP with HRT-determined rim area.
PMCID: PMC2532064  PMID: 18378580
21.  Nucleic acid melting by Escherichia coli CspE 
Nucleic Acids Research  2005;33(17):5583-5590.
Escherichia coli contains nine members of the CspA family. CspA and some of its homologues play critical role in cold acclimation of cells by acting as RNA chaperones, destabilizing nucleicacid secondary structures. Disruption of nucleic acid melting activity of CspE led to loss of its transcription antitermination activity and consequently its cold acclimation activity. To date, the melting activity of Csp proteins was studied using partially double-stranded model nucleic acids substrates forming stem–loop structures. Here, we studied the mechanism of nucleic acid melting by CspE. We show that CspE melts the stem region in two directions, that CspE-induced melting does not require the continuity of the substrate's loop region, and CspE can efficiently melt model substrates with single-stranded overhangs as short as 4 nt. We further show that preferential binding of CspE at the stem–loop junction site initiates melting; binding of additional CspE molecules that fully cover the single-stranded region of a melting substrate leads to complete melting of the stem.
PMCID: PMC1251665  PMID: 16214801
22.  Evaluation of Dalbavancin as chiral selector for HPLC and comparison with Teicoplanin based chiral stationary phases 
Chirality  2010;22(5):495-513.
Dalbavancin is a new compound of the macrocyclic glycopeptide family. It was covalently linked to 5μm silica particles by using two different binding chemsitries. Approximately two hundred and fifty racemates including (A) heterocyclic compounds; (B) chiral acids; (C) chiral amines; (D) chiral alcohols; (E) chiral sulfoxides and sulfilimines; (F) amino acids and amino acid derivatives; and (G) other chiral compounds were tested on the two new chiral stationary phases (CSP) using three different mobile phases. As dalbavancin is structurally related to teicoplanin, the same set of chiral compounds was screened on two commercially available teicoplanin CSPs for comparison. The dalbavancin CSPs were able to separate some enantiomers that were not separated by the teicoplanin CSPs and also showed improved separations for many racemates. However, there were other compounds only separated or better separated on teicoplanin CSPS. Therefore, the dalbavancin CSPs are complementary to the teicoplanin CSPs.
PMCID: PMC4309287  PMID: 19676111
dalbavancin; teicoplanin; chiral stationary phases; HPLC; macrocyclic glycopeptides; racemates; enantiomeric separation
23.  Comparison of patients diagnosed with gonorrhoea through community screening with those self-presenting to the genitourinary medicine clinic 
BMJ Open  2014;4(3):e004862.
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.
PMCID: PMC3963091  PMID: 24633530
Genitourinary Medicine; Socioeconomic Status; Mass screening; Community Health Services; Residence Characteristics; Neisseria Gonorrhoeae
24.  SpdR, a Response Regulator Required for Stationary-Phase Induction of Caulobacter crescentus cspD▿  
Journal of Bacteriology  2010;192(22):5991-6000.
The cold shock protein (CSP) family includes small polypeptides that are induced upon temperature downshift and stationary phase. The genome of the alphaproteobacterium Caulobacter crescentus encodes four CSPs, with two being induced by cold shock and two at the onset of stationary phase. In order to identify the environmental signals and cell factors that are involved in cspD expression at stationary phase, we have analyzed cspD transcription during growth under several nutrient conditions. The results showed that expression of cspD was affected by the medium composition and was inversely proportional to the growth rate. The maximum levels of expression were decreased in a spoT mutant, indicating that ppGpp may be involved in the signalization for carbon starvation induction of cspD. A Tn5 mutant library was screened for mutants with reduced cspD expression, and 10 clones that showed at least a 50% reduction in expression were identified. Among these, a strain with a transposon insertion into a response regulator of a two-component system showed no induction of cspD at stationary phase. This protein (SpdR) was able to acquire a phosphate group from its cognate histidine kinase, and gel mobility shift assay and DNase I footprinting experiments showed that it binds to an inverted repeat sequence of the cspD regulatory region. A mutated SpdR with a substitution of the conserved aspartyl residue that is the probable phosphorylation site is unable to bind to the cspD regulatory region and to complement the spdR mutant phenotype.
PMCID: PMC2976457  PMID: 20833806
25.  Cold Shock Proteins of Lactococcus lactis MG1363 Are Involved in Cryoprotection and in the Production of Cold-Induced Proteins 
Applied and Environmental Microbiology  2001;67(11):5171-5178.
Members of the group of 7-kDa cold-shock proteins (CSPs) are the proteins with the highest level of induction upon cold shock in the lactic acid bacterium Lactococcus lactis MG1363. By using double-crossover recombination, two L. lactis strains were generated in which genes encoding CSPs are disrupted: L. lactis NZ9000ΔAB lacks the tandemly orientated cspA and cspB genes, and NZ9000ΔABE lacks cspA, cspB, and cspE. Both strains showed no differences in growth at normal and at low temperatures compared to that of the wild-type strain, L. lactis NZ9000. Two-dimensional gel electrophoresis showed that upon disruption of the cspAB genes, the production of remaining CspE at low temperature increased, and upon disruption of cspA, cspB, and cspE, the production of CspD at normal growth temperatures increased. Northern blot analysis showed that control is most likely at the transcriptional level. Furthermore, it was established by a proteomics approach that some (non-7-kDa) cold-induced proteins (CIPs) are not cold induced in the csp-lacking strains, among others the histon-like protein HslA and the signal transduction protein LlrC. This supports earlier observations (J. A. Wouters, M. Mailhes, F. M. Rombouts, W. M. De Vos, O. P. Kuipers, and T. Abee, Appl. Environ. Microbiol. 66:3756–3763, 2000). that the CSPs of L. lactis might be directly involved in the production of some CIPs upon low-temperature exposure. Remarkably, the adaptive response to freezing by prior exposure to 10°C was significantly reduced in strain NZ9000ΔABE but not in strain NZ9000ΔAB compared to results with wild-type strain NZ9000, indicating a notable involvement of CspE in cryoprotection.
PMCID: PMC93287  PMID: 11679342

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