Utilizing fMRI to Guide TMS Targets: the Reliability and Sensitivity of fMRI Metrics at 3 T and 1.5 T.

US Food and Drug Administration (FDA) cleared a Transcranial Magnetic Stimulation (TMS) system with functional Magnetic Resonance Imaging-guided (fMRI) individualized treatment protocol for major depressive disorder, which employs resting state-fMRI (RS-fMRI) functional connectivity (FC) to pinpoint the target individually to increase the accuracy and effeteness of the stimulation. Furthermore, task activation-guided TMS, as well as the use of RS-fMRI local metrics for targeted the specific abnormal brain regions, are considered a precise scheme for TMS targeting. Since 1.5 T MRI is more available in hospitals, systematic evaluation of the test-retest reliability and sensitivity of fMRI metrics on 1.5 T and 3 T MRI may provide reference for the application of fMRI-guided individualized-precise TMS stimulation. Twenty participants underwent three RS-fMRI scans and one scan of finger-tapping task fMRI with self-initiated (SI) and visual-guided (VG) conditions at both 3 T and 1.5 T. Then the location reliability derived by FC (with three seed regions) and peak activation were assessed by intra-individual distance. The test-retest reliability and sensitivity of five RS-fMRI local metrics were evaluated using intra-class correlation and effect size, separately. The intra-individual distance of peak activation location between 1.5 T and 3 T was 15.8 mm and 19 mm for two conditions, respectively. The intra-individual distance for the FC derived targets at 1.5 T was 9.6-31.2 mm, compared to that of 3 T (7.6-31.1 mm). The test-retest reliability and sensitivity of RS-fMRI local metrics showed similar trends on 1.5 T and 3 T. These findings hasten the application of fMRI-guided individualized TMS treatment in clinical practice.

Toward Coordinate-based Cognition Dictionaries: A BrainMap and Neurosynth Demo.

Characterizing the functional involvement of specific brain regions has long been a central challenge in cognitive neuroscience. Functional magnetic resonance imaging (fMRI) techniques have offered solutions for mapping functional neural networks. The complex nature of structure-function correspondence makes an elaborate task design difficult to fully capture higher-order cognitive function. Other research practices, such as brain-behavior association or between-group comparisons, are thus widely used to explore cognitive correlations with specific brain regions. However, interpreting the results derived from a specific brain region with their underlying cognitive functions has been too general in publications. Here, we use two examples, i.e., a brain-intelligence correlation study and a depression-control comparison meta-study, to demonstrate use of two neuroimaging online databases, BrainMap and Neurosynth. One key utility of the two databases is collecting results from massive cognitive task-based fMRI (tb-fMRI) studies, i.e., coordinates in standard brain space. Just like looking up a "coordinate-based cognition dictionary", researchers can receive a plethora of related tb-fMRI activation information characterized by cognitive domains, specific cognitive functions, cognitive task paradigms, and related publications. Surprisingly, we found that only less than 1% of brain-behavior association or between-group comparison studies have utilized this dictionary approach. We encourage the community to further engage with the existing databases for specific and comprehensive interpretation of neuroimaging as well as guidance of future experimental tb-fMRI design.

Design and validation of an H5 TaqMan real-time one-step reverse transcription-PCR and confirmatory assays for diagnosis and verification of influenza A virus H5 infections in humans.

Increasing diversity among influenza H5N1 viruses has resulted in the need for sensitive and specific diagnostic assays, fully validated for the detection of H5 viruses belonging to all hemagglutinin (HA) clades, particularly the recently circulating H5N1 viruses of clade 2. In this report, the development and validation of a real-time, one-step TaqMan reverse transcription-PCR (RT-PCR) assay specific for the detection of influenza A H5 viruses from clades 1, 1', 2, and 3 is described. The real-time assay for H5 virus was shown to be highly sensitive, detecting H5 virus levels of <1 PFU from each of the HA clades. Specificity of the H5 RT-PCR for influenza A H5 viruses was demonstrated by using influenza A viruses of different subtypes, clinical samples containing influenza A viruses H1N1, H3N2, and H5N1, influenza B viruses, and other respiratory viruses. The usefulness of the inclusion of a distinguishable assay positive control and of confirmatory assays for the laboratory diagnosis and verification of H5 virus infections was demonstrated. A real-time RT-PCR pyrosequencing assay, a restriction enzyme digestion assay, and direct sequencing of the H5 real-time RT-PCR amplicon were validated for the confirmation of H5 detection by the diagnostic real-time assay. The H5 real-time assay was applied to diagnostic testing for suspected cases of influenza A virus H5 infection in the United Kingdom. Influenza A H5 viruses were not detected in the cases analyzed; however, influenza A H3N2 virus was detected in 57% of the suspected cases of H5. The H5 TaqMan real-time RT-PCR and confirmatory assays will be useful tools for the laboratory surveillance and rapid diagnosis of H5 infections in humans.

Precise epitope mapping of malaria parasite inhibitory antibodies by TROSY NMR cross-saturation.

We have applied NMR cross-saturation with TROSY detection to the problem of precisely mapping conformational epitopes on complete protein antigen molecules. We have investigated complexes of the Fab fragments of two antibodies that have parasite inhibitory activity, bound to the important malaria vaccine candidate antigen, Plasmodium falciparum MSP1(19). The results indicate remarkable overlap between these epitopes for inhibitory antibodies, and will provide a basis for theoretical modeling of the antibody-antigen interface.

Malaria parasite-inhibitory antibody epitopes on Plasmodium falciparum merozoite surface protein-1(19) mapped by TROSY NMR.

Plasmodium falciparum merozoite surface protein 1 (MSP1)(19), the C-terminal fragment of merozoite surface protein 1, is a leading candidate antigen for development of a vaccine against the blood stages of the malaria parasite. Many human and animal studies have indicated the importance of MSP1(19)-specific immune responses. Anti-MSP1(19) antibodies can prevent invasion of red blood cells by P. falciparum parasites in vitro. However, the fine specificity of anti-MSP1(19) antibodies is also important, as only a fraction of monoclonal antibodies (mAbs) have parasite-inhibitory activity in vitro. Human sera from malaria-endemic locations show strong MSP1(19) reactivity, but individual serum samples vary greatly in inhibitory activity. NMR is an excellent method for studying protein-protein interactions, and has been used widely to study binding of peptides representing known epitopes (as well as non-protein antigens) to antibodies and antibody fragments. The recent development of transverse relaxation optimized spectroscopy (TROSY) and related methods has significantly extended the maximum size limit of molecules that can be studied by NMR. TROSY NMR experiments produce high quality spectra of Fab complexes that allow the mapping of epitopes by the chemical shift perturbation technique on a complete, folded protein antigen such as MSP1(19). We studied the complexes of P. falciparum MSP1(19) with Fab fragments from three monoclonal antibodies. Two of these antibodies have parasite-inhibitory activity in vitro, while the third is non-inhibitory. NMR epitope mapping showed a close relationship between binding sites for the two inhibitory antibodies, distinct from the location of the non-inhibitory antibody. Together with a previously published crystal structure of the P. falciparum MSP1(19) complex with the Fab fragment of another non-inhibitory antibody, these results revealed a surface on MSP1(19) where inhibitory antibodies bind. This information will be useful in evaluating the anti-MSP1(19) immune response in natural populations from endemic areas, as well as in vaccine trials. It will also be valuable for optimizing the MSP1(19) antigen by rational vaccine design. This work also shows that TROSY NMR techniques are very effective for mapping conformational epitopes at the level of individual residues on small- to medium-sized proteins, provided that the antigen can be expressed in a system amenable to stable isotope labelling, such as bacteria or yeast.

Inhibitory and neutral antibodies to Plasmodium falciparum MSP119 form ring structures with their antigen.

Blood-stage malaria vaccine candidates include surface proteins of the merozoite. Antibodies to these proteins may either block essential steps during invasion or render the merozoite susceptible to phagocytosis or complement-mediated degradation. Structural information on merozoite surface proteins complexed to antibodies provides crucial information for knowledge-based vaccine design. The major merozoite surface protein MSP1 is an abundant surface molecule in Plasmodium falciparum. Only a subset of antibodies against MSP119 inhibits invasion (inhibitory antibodies), whereas other antibodies binding to MSP119 have no effect on invasion (neutral antibodies). Here we report on the complex of MSP119 with both inhibitory monoclonal antibody 12.10 and neutral monoclonal antibody 2F10. The complexes were established using both whole IgG's and Fab fragments, and analysed by dynamic light scattering, electron microscopy and analytical ultra centrifugation. Specific ring structures were formed in the ternary complex with the two antibodies, providing direct evidence of non-overlapping epitopes on MSP119. Mutational studies also indicated that the epitopes of the inhibitory and neutral antibodies are spatially remote.

Azidodeoxythymidine and didehydrodeoxythymidine as inhibitors and substrates of the human herpesvirus 8 thymidine kinase.

Human herpesvirus 8 (HHV-8), the aetiological agent of Kaposi's sarcoma (KS), encodes many core genes that have been maintained during evolution of the Herpesviridae. Among these is a thymidine kinase (TK) homologue (ORF21), which has 12% homology to the related TK encoded by herpes simplex virus. We show that the HHV-8 TK is a functional deoxythymidine (dT) kinase, with Michaelis constants (K(m)) for dT and ATP of 18.5 and 6.6 microM, respectively. Using homology modelling coupled with site-directed mutagenesis, we identify Gly265, Asp362 and Phe372 as key amino acid residues involved in the catalytic process. The HHV-8 TK is competitively inhibited by azidodeoxythymidine (zidovudine) and didehydrodeoxythymidine (stavudine) and can also accept these anti-retroviral compounds as substrates. These data have implications for our understanding of changes in AIDS-KS incidence following the clinical licensing of these compounds and in the development of new therapies for KS.