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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.  Crystallization and X-ray structure of cold-shock protein E from Salmonella typhimurium  
The crystal structure of cold-shock protein E from S. typhimurium (StCspE) has been determined at 1.1 Å resolution.
In prokaryotic organisms, cold shock triggers the production of a small highly conserved family of cold-shock proteins (CSPs). CSPs have been well studied structurally and functionally in Escherichia coli and Bacillus subtilis, but Salmonella typhimurium CSPs remain relatively uncharacterized. In S. typhi­­murium, six homologous CSPs have been identified: StCspA–E and StCspH. The crystal structure of cold-shock protein E from S. typhimurium (StCspE) has been determined at 1.1 Å resolution and has an R factor of 0.203 after refinement. The three-dimensional structure is similar to those of previously determined CSPs and is composed of five antiparallel β-strands forming a classic OB fold/five-stranded β-barrel. This first structure of a CSP from S. typhimurium provides new insight into the cold-shock response of this bacterium.
PMCID: PMC2802871  PMID: 20054119
cold-shock proteins; Salmonella typhimurium
3.  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
4.  Crystal Structure Prediction (CSP) of Flexible Molecules using Parallel Genetic Algorithms with a Standard Force Field 
Journal of computational chemistry  2009;30(13):1973-1985.
This paper describes the application of our distributed computing framework for crystal structure prediction (CSP), Modified Genetic Algorithms for Crystal and Cluster Prediction (MGAC) to predict the crystal structure of flexible molecules using the General Amber Force Field (GAFF) and the CHARMM program. The MGAC distributed computing framework which includes a series of tightly integrated computer programs for generating the molecule’s force field, sampling crystal structures using a distributed parallel genetic algorithm, local energy minimization of the structures followed by the classifying, sorting and archiving of the most relevant structures. Our results indicate that the method can consistently find the experimentally known crystal structures of flexible molecules, but the number of missing structures and poor ranking observed in some crystals show the need for further improvement of the potential.
PMCID: PMC2720422  PMID: 19130496
5.  9-Ethynyl-9H-carbazole 
The title compound, C14H9N, is the second crystallographically characterized example of an ynamine with an H atom in the C-terminal position. There are two independent mol­ecules (A and B) in the asymmetric unit. The structures of both mol­ecules are essentially planar (r.m.s. deviation = 0.0312 and 0.0152 Å). The N—Csp bond lengths are 1.353 (4) and 1.350 (4) Å, and those of the acetyl­ene bonds are 1.189 (4) and 1.190 (4) Å. The Csp—H bond lengths are 0.95 (5) and 0.97 (4) Å. These geometries are consistent with those of the previously reported ynamine characterized by crystallography. In the crystal, the mol­ecules stack along the c axis, forming two kinds of columnar structures. The acetyl­ene C atoms of mol­ecule A have a short contact [3.341 (4) Å and 3.396 (4) Å] with an adjacent mol­ecule A at the C—C bond of the fused part, which originates in π–π stacking inter­action; no remarkable spatial contact is recognized within the stacking of mol­ecule B.
PMCID: PMC3297888  PMID: 22412691
6.  1,6-Bis(diphenyl­arsino)hexa­ne 
The title diphenyl­arsino compound, C30H32As2 or Ph2As(CH2)6AsPh2, lies about a crystallographic inversion centre located at the mid-point of the central Csp 3—Csp 3 bond of the methyl­ene chain. The two benzene rings bonded to As are inclined to one another at a dihedral angle of 75.98 (8)°. In the crystal structure, weak inter­molecular C—H⋯π inter­actions stack the mol­ecules down the b axis.
PMCID: PMC2970425  PMID: 21578027
7.  Recent Advances in Ligand-Based Drug Design: Relevance and Utility of the Conformationally Sampled Pharmacophore Approach 
In the absence of three-dimensional (3D) structures of potential drug targets, ligand-based drug design is one of the popular approaches for drug discovery and lead optimization. 3D structure-activity relationships (3D QSAR) and pharmacophore modeling are the most important and widely used tools in ligand-based drug design that can provide crucial insights into the nature of the interactions between drug target and ligand molecule and provide predictive models suitable for lead compound optimization. This review article will briefly discuss the features and potential application of recent advances in ligand-based drug design, with emphasis on a detailed description of a novel 3D QSAR method based on the conformationally sample pharmacophore (CSP) approach (denoted CSP-SAR). In addition, data from a published study is used to compare the CSP-SAR approach to the Catalyst method, emphasizing the utility of the CSP approach for ligand-based model development.
PMCID: PMC2975775  PMID: 20807187
CoMFA; computer-aided drug design; CoMSIA; CSP; drug discovery; lead optimization; pharmacophore
8.  Enhanced Levels of Cold Shock Proteins in Listeria monocytogenes LO28 upon Exposure to Low Temperature and High Hydrostatic Pressure 
Listeria monocytogenes is a psychrotrophic food-borne pathogen that is problematic for the food industry because of its ubiquitous distribution in nature and its ability to grow at low temperatures and in the presence of high salt concentrations. Here we demonstrate that the process of adaptation to low temperature after cold shock includes elevated levels of cold shock proteins (CSPs) and that the levels of CSPs are also elevated after treatment with high hydrostatic pressure (HHP). Two-dimensional gel electrophoresis combined with Western blotting performed with anti-CspB of Bacillus subtilis was used to identify four 7-kDa proteins, designated Csp1, Csp2, Csp3, and Csp4. In addition, Southern blotting revealed four chromosomal DNA fragments that reacted with a csp probe, which also indicated that a CSP family is present in L. monocytogenes LO28. After a cold shock in which the temperature was decreased from 37°C to 10°C the levels of Csp1 and Csp3 increased 10- and 3.5-fold, respectively, but the levels of Csp2 and Csp4 were not elevated. Pressurization of L. monocytogenes LO28 cells resulted in 3.5- and 2-fold increases in the levels of Csp1 and Csp2, respectively. Strikingly, the level of survival after pressurization of cold-shocked cells was 100-fold higher than that of cells growing exponentially at 37°C. These findings imply that cold-shocked cells are protected from HHP treatment, which may affect the efficiency of combined preservation techniques.
PMCID: PMC126669  PMID: 11823178
9.  1,7-Dimethyl­penta­cyclo­[,6.03,10.05,9]undecane-8,11-dione 
The structure of the title compound, C13H14O2, a penta­cyclo­undecane cage derivative, exhibits unusual Csp 3—Csp 3 single-bond lengths ranging from 1.505 (3) to 1.607 (2) Å and strained bond angles as small as 88.7 (1)° and as large as 121.0 (2)°. In this meso compound, an inter­nal non-crystallographic mirror plane exists, bis­ecting the mol­ecule. In the crystal, weak C—H⋯O hydrogen bonds link the mol­ecules into an infinite spiral about a twofold screw axis along the [100] direction.
PMCID: PMC3007444  PMID: 21588235
10.  1,2,3,4,5,6-Hexa-O-acetyl-scyllo-inositol 
The title mol­ecule, C18H24O12, has crystallographic 2/m symmetry with two acetate group located on a mirror plane. The H—Csp 3—O—Csp 2 torsion angles characterizing orientation of the acetyl groups with respect to the cyclo­hexane ring are 0.0, 23.9 and −23.9°. The cyclo­hexane ring is in a chair conformation with all substituents in equatorial positions. In the crystal, mol­ecules are connected through C—H⋯O hydrogen bonds into a chain extending along the c axis.
PMCID: PMC3415030  PMID: 22905017
11.  A Hydrophobic Patch in the Competence-Stimulating Peptide, a Pneumococcal Competence Pheromone, Is Essential for Specificity and Biological Activity 
Journal of Bacteriology  2006;188(5):1744-1749.
Induction of competence for natural genetic transformation in Streptococcus pneumoniae depends on pheromone-mediated cell-cell communication and a signaling pathway consisting of the competence-stimulating peptide (CSP), its membrane-embedded histidine kinase receptor ComD, and the cognate response regulator ComE. Extensive screening of pneumococcal isolates has revealed that two major CSP variants, CSP1 and CSP2, are found in members of this species. Even though the primary structures of CSP1 and CSP2 are about 50% identical, they are highly specific for their respective receptors, ComD1 and ComD2. In the present work, we have investigated the structural basis of this specificity by determining the three-dimensional structure of CSP1 from nuclear magnetic resonance data and comparing the agonist activity of a number of CSP1/CSP2 hybrid peptides toward the ComD1 and ComD2 receptors. Our results show that upon exposure to membrane-mimicking environments, the 17-amino-acid CSP1 pheromone adopts an amphiphilic α-helical configuration stretching from residue 6 to residue 12. Furthermore, the pattern of agonist activity displayed by the various hybrid peptides revealed that hydrophobic amino acids, some of which are situated on the nonpolar side of the α-helix, strongly contribute to CSP specificity. Together, these data indicate that the identified α-helix is an important structural feature of CSP1 which is essential for effective receptor recognition under natural conditions.
PMCID: PMC1426553  PMID: 16484185
12.  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
13.  1,3,7,10,14,17,21,28,31,42,52,55-Dodeca­kis(trifluoro­meth­yl)- 1,3,7,10,14,17,21,28,31,42,52,55-dodeca­hydro­(C60–Ih)[5,6]fullerene 
The title compound, C72F36, is one of four isomers of C60(CF3)12 for which crystal structures have been obtained. The fullerene mol­ecule has an idealized Ih C60 core with the 12 CF3 groups arranged in an asymmetric fashion on two ribbons of edge-sharing C6(CF3)2 hexa­gons, a para–meta–para–para–para–meta–para ribbon and a para–meta–para ribbon, giving an overall pmp 3 mp,pmp structure. There are no cage Csp 3—Csp 3 bonds. The F atoms of two CF3 groups are disordered over two positions; the site occupancy factors are 0.85/0.15 and 0.73/0.27. There are intra­molecular F⋯F contacts between pairs of CF3 groups on the same hexa­gon that range from 2.521 (3) to 2.738 (4) Å.
PMCID: PMC2915227  PMID: 21200724
14.  Specific enzyme-linked immunosorbent assay for detection of bovine antibody to Brucella abortus. 
Journal of Clinical Microbiology  1984;20(2):209-213.
Six soluble antigens prepared from Brucella abortus were compared with a salt-extractable protein (CSP) antigen in an enzyme-linked immunosorbent assay for the detection of antibody to B. abortus in cattle sera. Of seven preparations tested, antigens from B. abortus soluble antigen (prepared from an autoclaved cell suspension) and CSP were stable on frozen storage. Enzyme-linked immunosorbent assay with CSP antigen under optimal conditions was from 100- to 700-fold more sensitive than the standard agglutination, card, Rivanol precipitation-plate agglutination, and the complement fixation tests in detecting immunoglobulin G antibody. From a practical point of view, however, using the most stringent criteria for determining an "upper negative" value, the enzyme-linked immunosorbent assay with CSP was at least 12-fold more sensitive than the standard agglutination test and any of the other serological tests. Furthermore, the enzyme-linked immunosorbent assay with CSP was specific for antibody to B. abortus.
PMCID: PMC271288  PMID: 6436294
15.  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
16.  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
17.  2-(Prop-2-enyloxy)benzamide 
In the title mol­ecule, C10H11NO2, the benzene ring forms dihedral angles of 33.15 (2) and 6.20 (2)° with the mean planes of the amide and propen­oxy groups, respectively. The amide –NH2 group is oriented toward the propen­oxy substituent and forms a weak intra­molecular N—H⋯O hydrogen bond to the propen­oxy O atom. The conformation of the propen­oxy group at the Csp 2—Csp 3 and Csp 3—O bonds is synperiplanar and anti­periplanar, respectively. In the crystal, N—H⋯O hydrogen bonds involving the amide groups generate C(4) and R 2 3(7) motifs that organize the mol­ecules into tapes along the a-axis direction. There are C—H⋯π inter­actions between the propen­oxy –CH2 group and the aromatic system of neighboring mol­ecules within the tape. The mean planes of the aromatic ring and the propen­oxy group belonging to mol­ecules located on opposite sites of the tape form an angle of 83.16 (2)°.
PMCID: PMC3515263  PMID: 23284483
18.  4-(Dimethyl­amino)phenyl ethynyl telluride 
The title compound, C10H11NTe, is the first organyl ethynyl telluride, R—Te—C C—H, to be structurally characterized. In the L-shaped mol­ecule, the aryl moiety, viz. Me2NC6H4Te, is almost perpendicular to the Te—C C—H fragment. The Te—Csp 2 bond [2.115 (3) Å] is significantly longer than the Te—Csp bond [2.041 (4) Å]. The Te—C C group is approximately linear [Te—C—C = 178.5 (4)° and C C = 1.161 (5) Å], while the coordination at the Te atom is angular [C—Te—C = 95.92 (14)°]. In the crystal structure, there are Csp—H⋯N hydrogen bonds which are perpendicular to the CNMe2 group; the N atom displays some degree of pyramidalization. Centrosymmetrically related pairs of mol­ecules are linked by Te⋯π(ar­yl) inter­actions, with Te⋯Cg = 3.683 (4) Å and Csp—Te⋯Cg = 159.1 (2)° (Cg is the centroid of the benzene ring). These inter­actions lead to the formation of zigzag ribbons which run along c and are approximately parallel to (110).
PMCID: PMC2968982  PMID: 21582534
19.  Structural and Functional Analysis of the CspB Protease Required for Clostridium Spore Germination 
PLoS Pathogens  2013;9(2):e1003165.
Spores are the major transmissive form of the nosocomial pathogen Clostridium difficile, a leading cause of healthcare-associated diarrhea worldwide. Successful transmission of C. difficile requires that its hardy, resistant spores germinate into vegetative cells in the gastrointestinal tract. A critical step during this process is the degradation of the spore cortex, a thick layer of peptidoglycan surrounding the spore core. In Clostridium sp., cortex degradation depends on the proteolytic activation of the cortex hydrolase, SleC. Previous studies have implicated Csps as being necessary for SleC cleavage during germination; however, their mechanism of action has remained poorly characterized. In this study, we demonstrate that CspB is a subtilisin-like serine protease whose activity is essential for efficient SleC cleavage and C. difficile spore germination. By solving the first crystal structure of a Csp family member, CspB, to 1.6 Å, we identify key structural domains within CspB. In contrast with all previously solved structures of prokaryotic subtilases, the CspB prodomain remains tightly bound to the wildtype subtilase domain and sterically occludes a catalytically competent active site. The structure, combined with biochemical and genetic analyses, reveals that Csp proteases contain a unique jellyroll domain insertion critical for stabilizing the protease in vitro and in C. difficile. Collectively, our study provides the first molecular insight into CspB activity and function. These studies may inform the development of inhibitors that can prevent clostridial spore germination and thus disease transmission.
Author Summary
Clostridium difficile is the leading cause of health-care associated diarrhea worldwide. C. difficile infections begin when its spores transform into vegetative cells during a process called germination. In Clostridium sp., germination requires that the spore cortex, a thick, protective layer, be removed by the cortex hydrolase SleC. While previous studies have shown that SleC activity depends on a subtilisin-like protease, CspB, the mechanisms regulating CspB function have not been characterized. In this study, we solved the first crystal structure of the Csp family of proteases and identified its key functional regions. We determined that CspB carries a unique jellyroll domain required for stabilizing the protein both in vitro and in C. difficile and a prodomain required for proper folding of the protease. Unlike all other prokaryotic subtilisin-like proteases, the prodomain remains bound to CspB and inhibits its protease activity until the germination signal is sensed. Our study provides new insight into how germination is regulated in C. difficile and may inform the development of inhibitors that can prevent germination and thus C. difficile transmission.
PMCID: PMC3567191  PMID: 23408892
20.  Discovery of Novel Checkpoint Kinase 1 Inhibitors by Virtual Screening Based on Multiple Crystal Structures 
Incorporating receptor flexibility is considered crucial for improvement of docking-based virtual screening. With an abundance of crystallographic structures freely available, docking with multiple crystal structures is believed to be a practical approach to cope with protein flexibility. Here we describe a successful application of the docking of multiple structures to discover novel and potent Chk1 inhibitors. Forty-six Chk1 structures were first compared in single structure docking by predicting the binding mode and recovering known ligands. Combinations of different protein structures were then compared by recovery of known ligands and an optimal ensemble of Chk1 structures were selected. The chosen structures were used in the virtual screening of over 60,000 diverse compounds for Chk1 inhibitors. Six novel compounds ranked at the top of the hits list were tested experimentally and two of these compounds inhibited Chk1 activity–the best with an IC50 value of 9.6 μM. Further study indicated that achieving a better enrichment and identifying more diverse compounds was more likely using multiple structures than using only a single structure even when protein structures were randomly selected. Taking into account conformational energy difference did not help to improve enrichment in the top ranked list.
PMCID: PMC3244973  PMID: 21955044
21.  ATP driven clathrin dependent entry of carbon nanospheres prefer cells with glucose receptors 
Intrinsically fluorescent glucose derived carbon nanospheres (CSP) efficiently enter mammalian cells and also cross the blood brain barrier (BBB). However, the mechanistic details of CSP entry inside mammalian cells and its specificity are not known.
In this report, the biochemical and cellular mechanism of CSP entry into the living cell have been investigated. By employing confocal imaging we show that CSP entry into the mammalian cells is an ATP-dependent clathrin mediated endocytosis process. Zeta potential studies suggest that it has a strong preference for cells which possess high levels of glucose transporters such as the glial cells, thereby enabling it to target individual organs/tissues such as the brain with increased specificity.
The endocytosis of Glucose derived CSP into mammalian cells is an ATP dependent process mediated by clathrin coated pits. CSPs utilize the surface functional groups to target cells containing glucose transporters on its membrane thereby implicating a potential application for specific targeting of the brain or cancer cells.
PMCID: PMC3479219  PMID: 22857258
22.  Automated-Parameterization of the Motor Evoked Potential and Cortical Silent Period Induced by Transcranial Magnetic Stimulation 
To standardize the characterization of motor evoked potential (MEP) and cortical silent period (CSP) recordings elicited with transcranial magnetic stimulation (TMS).
A computer-based, automated-parameterization program (APP) was developed and tested which provides a comprehensive set of electromyography (EMG) magnitude and temporal measures. The APP was tested using MEP, CSP, and isolated CSP (iCSP) TMS stimulus-response data from a healthy adult population (N = 13).
The APP had the highest internal reliability (Cronbach’s alpha = .98) for CSP offset time compared with two prominent automated methods. The immediate post-CSP EMG recovery level was 49% higher than the pre-TMS EMG level. MEP size (peak amplitude, mean amplitude, peak-to-peak amplitude, and area) correlated higher with effective E-field (Eeff) than other intensity measures (r ≈ 0.5 vs. r ≈ 0.3) suggesting that Eeff is better suited for standardizing MEP stimulus-response relationships.
The APP successfully characterized individual and mean epochs containing MEP, CSP, and iCSP responses. The APP provided common signal and temporal measures consistent with previous studies and novel additional parameters. Significance: With the use of the APP modeling method and the Eeff, a standard approach for the analysis and reporting of MEP-CSP complex and iCSP measurements is achievable.
PMCID: PMC2727570  PMID: 19559646
Transcranial magnetic stimulation; Motor evoked potential; Cortical silent period; Automated Parameterization; Effective electrical-field
23.  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
24.  Cold Shock Response and Major Cold Shock Proteins of Vibrio cholerae 
Applied and Environmental Microbiology  2003;69(11):6361-6369.
When exponentially growing Vibrio cholerae cells were shifted from 37°C to various lower temperatures, it was found that the organism could adapt and grow at temperatures down to 15°C, below which the growth was completely arrested. There was no difference between the patterns of the cold shock responses in toxinogenic and nontoxinogenic strains of V. cholerae. Gel electrophoretic analyses of proteins of cold-exposed cells revealed significant induction of two major cold shock proteins (Csps), whose molecular masses were 7.7 kDa (CspAVC) and 7.5 kDa (CspV), and six other Csps, most of which were much larger. We cloned, sequenced, and analyzed the cspV gene encoding the CspV protein of V. cholerae O139 strain SG24. Although CspAVC and CspV have similar kinetics of synthesis and down-regulation, the corresponding genes, cspA and cspV, which are located in the small chromosome, are not located in the same operon. A comparative analysis of the kinetics of synthesis revealed that the CspV protein was synthesized de novo only during cold shock. Although both CspAVC and CspV were stable for several hours in the cold, the CspV protein was degraded rapidly when the culture was shifted back to 37°C, suggesting that this protein is probably necessary for adaptation at lower temperatures. Northern blot analysis confirmed that the cspV gene is cold shock inducible and is regulated tightly at the level of transcription. Interestingly, the cspV gene has a cold shock-inducible promoter which is only 12 nucleotides from the translational start site, and therefore, it appears that no unusually long 5′ untranslated region is present in its mRNA transcript. Thus, this promoter is an exception compared to other promoters of cold shock-inducible genes of different organisms, including Escherichia coli. Our results suggest that V. cholerae may use an alternative pathway for regulation of gene expression during cold shock.
PMCID: PMC262262  PMID: 14602587
25.  Disseminating a cervical cancer screening program through primary physicians in Hong Kong: a qualitative study 
Organized screening programs are more effective and equitable than opportunistic screening, yet governments face challenges to implement evidence-based programs. The objective of this study was to identify reasons for low levels of adoption among primary care physicians of a government sponsored Cervical Screening Program (CSP).
We conducted in-depth interviews with a snowball sample of primary care private and public primary care physicians in Hong Kong. Rogers’ theory of diffusion of innovation was used to understand the factors that influenced the physicians’ practice decisions.
Our study found that Hong Kong physicians made the decision to encourage cervical screening and to participate in the CSP based primarily upon their clinical and business practice needs rather than upon the scientific evidence. The low rates of adoption of the CSP can be attributed to the physicians’ perceptions that the program’s complexity and incompatibility exceeded its relative advantages. Furthermore, women’s knowledge, attitudes and practices, identified as barriers by physicians, were also barriers to physicians adopting the CSP.
In both private and public health care systems, screening programs that rely on physicians must align program incentives with the physicians’ motivators or pursue additional demand creation policies to achieve objectives.
PMCID: PMC3975957  PMID: 24568606
Cervical cancer screening; Dissemination; Physicians; Prevention; Screening programs

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