It is widely assumed that cognitive functions decline with age and that these decrements are associated with age-related changes in patterns of functional activity. However, these functional changes may be due to age-related increased responsiveness to task demands and not to other cognitive processes on which neural and behavioural responses rely, since many ageing studies use task paradigms that may not be orthogonal to the cognitive function being investigated. Here we test this hypothesis in adults aged 20-86 years by combining measures of language comprehension, functional connectivity and neural integrity to identify functional networks activated in two language experiments with varying task demands. In one, participants listened to spoken sentences without performing an overt task (the natural listening condition) while in the other they performed a task in response to the same sentences. Using task-based ICA of fMRI, we identified a left-lateralised frontotemporal network associated with syntactic analysis, which remained consistently activated regardless of task demands. In contrast, in the task condition only a separate set of components showed task-specific activity in Opercular, Frontoparietal, and bilateral PFC. Only the PFC showed age-related increases in activation which, furthermore, was strongly mediated by grey matter health. These results suggest that, contrary to prevailing views, age-related changes in cognitive activation may be due in part to differential responses to task-related processes.
Activation of frontal and parietal brain regions is associated with attentional control during visual search. We used fMRI to characterize age-related differences in frontoparietal activation in a highly efficient feature search task, detection of a shape singleton. On half of the trials, a salient distractor (a color singleton) was present in the display. The hypothesis was that frontoparietal activation mediated the relation between age and attentional capture by the salient distractor. Participants were healthy, community-dwelling individuals, 21 younger adults (19-29years of age) and 21 older adults (60-87years of age). Top-down attention, in the form of target predictability, was associated with an improvement in search performance that was comparable for younger and older adults. The increase in search reaction time (RT) associated with the salient distractor (attentional capture), standardized to correct for generalized age-related slowing, was greater for older adults than for younger adults. On trials with a color singleton distractor, search RT increased as a function of increasing activation in frontal regions, for both age groups combined, suggesting increased task difficulty. Mediational analyses disconfirmed the hypothesized model, in which frontal activation mediated the age-related increase in attentional capture, but supported an alternative model in which age was a mediator of the relation between frontal activation and capture.
We show that in conventional, competition-based bioluminescence resonance energy transfer (BRET) assays of membrane protein stoichiometry, the presence of competitors can alter tagged-protein density and artifactually reduce energy transfer efficiency. A well-characterized monomeric type I membrane protein, CD86, and two G protein-coupled receptors ?2AR and mCannR2, all of which behave as dimers in these conventional assays, exhibit monomeric behavior in an improved competition-based type-3 BRET assay designed to circumvent such artifacts.
Voluntary episodic memories require an intentional memory search, whereas involuntary episodic memories come to mind spontaneously without conscious effort. Cognitive neuroscience has largely focused on voluntary memory, leaving the neural mechanisms of involuntary memory largely unknown. We hypothesized that, because the main difference between voluntary and involuntary memory is the controlled retrieval processes required by the former, there would be greater frontal activity for voluntary than involuntary memories. Conversely, we predicted that other components of the episodic retrieval network would be similarly engaged in the two types of memory. During encoding, all participants heard sounds, half paired with pictures of complex scenes and half presented alone. During retrieval, paired and unpaired sounds were presented, panned to the left or to the right. Participants in the involuntary group were instructed to indicate the spatial location of the sound, whereas participants in the voluntary group were asked to additionally recall the pictures that had been paired with the sounds. All participants reported the incidence of their memories in a postscan session. Consistent with our predictions, voluntary memories elicited greater activity in dorsal frontal regions than involuntary memories, whereas other components of the retrieval network, including medial-temporal, ventral occipitotemporal, and ventral parietal regions were similarly engaged by both types of memories. These results clarify the distinct role of dorsal frontal and ventral occipitotemporal regions in predicting strategic retrieval and recalled information, respectively, and suggest that, although there are neural differences in retrieval, involuntary memories share neural components with established voluntary memory systems.
The reliable neuroimaging finding that older adults often show greater activity (over-recruitment) than younger adults is typically attributed to compensation. Yet, the neural mechanisms of over-recruitment in older adults (OAs) are largely unknown. Rodent electrophysiology studies have shown that as number of afferent fibers within a circuit decreases with age, the fibers that remain show higher synaptic field potentials (less wiring, more firing). Extrapolating to system-level measures in humans, we proposed and tested the hypothesis that greater activity in OAs compensates for impaired white-matter connectivity. Using a neuropsychological test battery, we measured individual differences in executive functions associated with the prefrontal cortex (PFC) and memory functions associated with the medial temporal lobes (MTLs). Using event-related functional magnetic resonance imaging, we compared activity for successful versus unsuccessful trials during a source memory task. Finally, we measured white-matter integrity using diffusion tensor imaging. The study yielded 3 main findings. First, low-executive OAs showed greater success-related activity in the PFC, whereas low-memory OAs showed greater success-related activity in the MTLs. Second, low-executive OAs displayed white-matter deficits in the PFC, whereas low-memory OAs displayed white-matter deficits in the MTLs. Finally, in both prefrontal and MTL regions, white-matter decline and success-related activations occurred in close proximity and were negatively correlated. This finding supports the less-wiring-more-firing hypothesis, which provides a testable account of compensatory over-recruitment in OAs.
The glutamine synthetase-based protein expression system is widely used in industry and academia for producing recombinant proteins but relies on the cloning of transfected cells, necessitating substantial investments in time and handling. We streamlined the production of protein-producing cultures of Chinese hamster ovary cells using this system by co-expressing green fluorescent protein from an internal ribosomal entry site and selecting for high green fluorescent protein-expressing cells using fluorescence-activated cell sorting.
Immune responses to persistent viral infections and cancer often fail because of intense regulation of antigen-specific T cells-a process referred to as immune exhaustion. The mechanisms that underlie the induction of exhaustion are not completely understood. To gain novel insights into this process, we simultaneously examined the dynamics of virus-specific CD8(+) and CD4(+) T cells in the living spleen by two-photon microscopy (TPM) during the establishment of an acute or persistent viral infection. We demonstrate that immune exhaustion during viral persistence maps anatomically to the splenic marginal zone/red pulp and is defined by prolonged motility paralysis of virus-specific CD8(+) and CD4(+) T cells. Unexpectedly, therapeutic blockade of PD-1-PD-L1 restored CD8(+) T cell motility within 30 min, despite the presence of high viral loads. This result was supported by planar bilayer data showing that PD-L1 localizes to the central supramolecular activation cluster, decreases antiviral CD8(+) T cell motility, and promotes stable immunological synapse formation. Restoration of T cell motility in vivo was followed by recovery of cell signaling and effector functions, which gave rise to a fatal disease mediated by IFN-?. We conclude that motility paralysis is a manifestation of immune exhaustion induced by PD-1 that prevents antiviral CD8(+) T cells from performing their effector functions and subjects them to prolonged states of negative immune regulation.
PD-1, a receptor expressed by T cells, B cells, and monocytes, is a potent regulator of immune responses and a promising therapeutic target. The structure and interactions of human PD-1 are, however, incompletely characterized. We present the solution nuclear magnetic resonance (NMR)-based structure of the human PD-1 extracellular region and detailed analyses of its interactions with its ligands, PD-L1 and PD-L2. PD-1 has typical immunoglobulin superfamily topology but differs at the edge of the GFCC sheet, which is flexible and completely lacks a C" strand. Changes in PD-1 backbone NMR signals induced by ligand binding suggest that, whereas binding is centered on the GFCC sheet, PD-1 is engaged by its two ligands differently and in ways incompletely explained by crystal structures of mouse PD-1 · ligand complexes. The affinities of these interactions and that of PD-L1 with the costimulatory protein B7-1, measured using surface plasmon resonance, are significantly weaker than expected. The 3-4-fold greater affinity of PD-L2 versus PD-L1 for human PD-1 is principally due to the 3-fold smaller dissociation rate for PD-L2 binding. Isothermal titration calorimetry revealed that the PD-1/PD-L1 interaction is entropically driven, whereas PD-1/PD-L2 binding has a large enthalpic component. Mathematical simulations based on the biophysical data and quantitative expression data suggest an unexpectedly limited contribution of PD-L2 to PD-1 ligation during interactions of activated T cells with antigen-presenting cells. These findings provide a rigorous structural and biophysical framework for interpreting the important functions of PD-1 and reveal that potent inhibitory signaling can be initiated by weakly interacting receptors.
Single-particle tracking (SPT) is widely used to study processes from membrane receptor organization to the dynamics of RNAs in living cells. While single-dye labeling strategies have the benefit of being minimally invasive, this comes at the expense of data quality; typically a data set of short trajectories is obtained and analyzed by means of the mean square displacements (MSD) or the distribution of the particles displacements in a set time interval (jump distance, JD). To evaluate the applicability of both approaches, a quantitative comparison of both methods under typically encountered experimental conditions is necessary. Here we use Monte Carlo simulations to systematically compare the accuracy of diffusion coefficients (D-values) obtained for three cases: one population of diffusing species, two populations with different D-values, and a population switching between two D-values. For the first case we find that the MSD gives more or equally accurate results than the JD analysis (relative errors of D-values <6%). If two diffusing species are present or a particle undergoes a motion change, the JD analysis successfully distinguishes both species (relative error <5%). Finally we apply the JD analysis to investigate the motion of endogenous LPS receptors in live macrophages before and after treatment with methyl-?-cyclodextrin and latrunculin B.
In an effort to understand the role of interhemispheric transfer in numerical development, we investigated the relationship between childrens developing knowledge of numbers and the integrity of their white matter connections between the cerebral hemispheres (the corpus callosum). We used diffusion tensor imaging (DTI) tractography analyses to test the link between the development of the corpus callosum and performance on symbolic and non-symbolic numerical judgment tasks. We were especially interested in the interhemispheric connections of parietal cortex in 6-year-old children, because regions of parietal cortex have been implicated in the development of numerical skills by several prior studies. Our results revealed significant structural differences between children and adults in the fibers of the corpus callosum connecting the left and right parietal lobes. Importantly, these structural differences were predictive of individual differences among children in performance on numerical judgment tasks: children with poor numerical performance relative to their peers exhibited reduced white matter coherence in the fibers passing through the isthmus of the corpus callosum, which connects the parietal hemispheres.
The T lineage glycoprotein CD6 is generally considered to be a costimulator of T-cell activation. Here, we demonstrate that CD6 significantly reduces early and late T-cell responses upon superantigen stimulation or TCR triggering by Abs. Measuring calcium mobilization in single cells responding to superantigen, we found that human T cells expressing rat CD6 react significantly less well compared with T cells not expressing the exogenous receptor. When the cytoplasmic domain of rat CD6 was removed, calcium responses were recovered, indicating that the inhibitory properties of CD6 are attributable to its cytoplasmic domain. Calcium responses, and also late indicators of T-cell activation such as IL-2 release, were also diminished in TCR-activated Jurkat cells expressing human CD6, compared with CD6-deficient cells or cells expressing a cytoplasmic deletion mutant of human CD6. Similarly, calcium signals triggered by anti-CD3 were enhanced in human T lymphocytes following morpholino-mediated suppression of CD6 expression. Finally, the proliferation of T lymphocytes was increased when the CD6-CD166 interaction was blocked with anti-CD166 Abs, but inhibited when anti-CD6 Abs were used. Our data suggest that CD6 is a signaling attenuator whose expression alone, i.e. in the absence of ligand engagement, is sufficient to restrain signaling in T cells.
Understanding the component stoichiometry of the T cell antigen receptor (TCR) triggering apparatus is essential for building realistic models of signal initiation. Recent studies suggesting that the TCR and other signaling-associated proteins are preclustered on resting T cells relied on measurements of the behavior of membrane proteins at interfaces with functionalized glass surfaces. Using fluorescence recovery after photobleaching, we show that, compared with the apical surface, the mobility of TCRs is significantly reduced at Jurkat T cell/glass interfaces, in a signaling-sensitive manner. Using two biophysical approaches that mitigate these effects, bioluminescence resonance energy transfer and two-color coincidence detection microscopy, we show that, within the uncertainty of the methods, the membrane components of the TCR triggering apparatus, i.e. the TCR complex, MHC molecules, CD4/Lck and CD45, are exclusively monovalent or monomeric in human T cell lines, implying that TCR triggering depends only on the kinetics of TCR/pMHC interactions. These analyses also showed that constraining proteins to two dimensions at the cell surface greatly enhances random interactions versus those between the membrane and the cytoplasm. Simulations of TCR-pMHC complex formation based on these findings suggest how unclustered TCR triggering-associated proteins might nevertheless be capable of generating complex signaling outputs via the differential recruitment of cytosolic effectors to the cell membrane.
Modulating the activities of costimulatory molecules controlling immune responses holds considerable promise for immunotherapy. CTLA4Ig (abatacept), a soluble version of the T cell-expressed membrane receptor CTLA-4, is approved for the treatment of rheumatoid arthritis. Like natural CTLA-4 molecules, CTLA4Ig ligates B7-1 and B7-2 on antigen presenting cells, preventing CD28-mediated costimulation of T cells. However, CTLA4Ig can also prevent ligation of CTLA-4, potentially blocking vital inhibitory signals, thereby augmenting immunity. There have been no quantitative analyses of the likely effects of CTLA4Ig on costimulatory interactions at the immunological synapse. We present a mathematical model, based on rigorous biophysical and expression data, for simulating the effects of abatacept and a mutated derivative, LEA29Y, on the synaptic interactions of CD28 and CTLA-4. The simulations reveal an unexpectedly large window within which CD28, but not CTLA-4, ligation is blocked by CTLA4Ig, perhaps explaining the efficacy of abatacept at the recommended therapeutic dose (10mg/kg) and its relative safety. However, the simulations suggest that the present dosing regimen is close to the maximum theoretically safe dose. The simulations also show that, within the therapeutic window, LEA29Y enhances the interaction of CTLA-4 with the more potent of its two native ligands, B7-1. They also suggest that CTLA-4 ligation by B7-1 could, in principle, be enhanced by further decreasing the off-rate of CTLA4Ig for binding to B7-2. Our findings therefore offer molecular explanations for why LEA29Y might prove to be more effective than abatacept in a clinical setting, and suggest ways in which its therapeutic efficacy could be further optimised.
Triggering of the T cell receptor initiates a signaling cascade resulting in the activation of the T cell. These signals are integrated alongside those resulting from the triggering of other receptors whose function is to modulate the overall response. CD5 is an immunotyrosine-based inhibition motif-bearing receptor that antagonizes the overt T cell receptor activation response by recruiting inhibitory intracellular mediators such as SHP-1, RasGAP, or Cbl. We now propose that the inhibitory effects of CD5 are also mediated by a parallel pathway that functions at the level of inhibition of Fyn, a kinase generally associated with T cell receptor-mediated activation. After CD5 ligation, phosphorylation of the negative regulatory tyrosine (Tyr(531)) of Fyn increases, and this correlates with a substantial reduction in the kinase activity of Fyn and a profound inhibition of ZAP-70 activation. The effect requires the last 23 amino acids of the cytoplasmic domain of the receptor, strongly implying the involvement of a new CD5-interacting signaling or adaptor protein. Furthermore, we show that upon CD5 ligation there is a profound shift in its distribution from the bulk fluid phase to the lipid raft environment, where it associates with Fyn, Lck, and PAG. We suggest that the relocation of CD5, which we also show is capable of forming homodimers, to the proximity of raft-resident molecules enables CD5 to inhibit membrane proximal signaling by controlling the phosphorylation and activity of Fyn, possibly by interfering with the disassembly of C-terminal Src kinase (Csk)-PAG-Fyn complexes during T cell activation.
Contralateral recruitment remains a controversial phenomenon in both the clinical and normative populations. To investigate the neural correlates of this phenomenon, we explored the tendency for older adults to recruit prefrontal cortex (PFC) regions contralateral to those most active in younger adults. Participants were scanned with diffusion tensor imaging and functional magnetic rresonance imaging during a lateralized word matching task (unilateral vs. bilateral). Cross-hemispheric communication was measured behaviorally as greater accuracy for bilateral than unilateral trials (bilateral processing advantage [BPA]) and at the neural level by functional and structural connectivity between contralateral PFC. Compared with the young, older adults exhibited 1) greater BPAs in the behavioral task, 2) greater compensatory activity in contralateral PFC during the bilateral condition, 3) greater functional connectivity between contralateral PFC during bilateral trials, and 4) a positive correlation between fractional anisotropy in the corpus callosum and both the BPA and the functional connectivity between contralateral PFC, indicating that older adults ability to distribute processing across hemispheres is constrained by white matter integrity. These results clarify how older adults ability to recruit extra regions in response to the demands of aging is mediated by existing structural architecture, and how this architecture engenders corresponding functional changes that allow subjects to meet those task demands.
Glycoproteins present problems for structural analysis since they often have to be glycosylated in order to fold correctly and because their chemical and conformational heterogeneity generally inhibits crystallization. It is shown that the ?-mannosidase I inhibitor kifunensine, which has previously been used for the purpose of glycoprotein crystallization in short-term (3-5?d) cultures, is apparently stable enough to be used to produce highly endoglycosidase H-sensitive glycoprotein in long-term (3-4?week) cultures of stably transfected Chinese hamster ovary (CHO) cells. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based analysis of the extracellular region of the cytotoxic T-lymphocyte antigen 4 (CTLA-4; CD152) homodimer expressed in long-term CHO cell cultures in the presence of kifunensine revealed that the inhibitor restricted CTLA-4 glycan processing to Man9GlcNAc2 and Man5GlcNAc2 structures. Complex-type glycans were undetectable, suggesting that the inhibitor was active for the entire duration of the cultures. Endoglycosidase treatment of the homodimer yielded protein that readily formed orthorhombic crystals with unit-cell parameters a=43.9, b=51.5, c=102.9?Å and space group P2(1)2(1)2(1) that diffracted to Bragg spacings of 1.8?Å. The results indicate that kifunensine will be effective in most, if not all, transient and long-term mammalian cell-based expression systems.
Mutations within MHC class I-restricted epitopes have been studied in relation to T cell-mediated immune escape, but their impact on NK cells via interaction with killer Ig-like receptors (KIRs) during early HIV infection is poorly understood. In two patients acutely infected with HIV-1, we observed the appearance of a mutation within the B*57-restricted TW10 epitope (G9E) that did not facilitate strong escape from T cell recognition. The NK cell receptor KIR3DL1, carried by these patients, is known to recognize HLA-B*5703 and is associated with good control of HIV-1. Therefore, we tested whether the G9E mutation influenced the binding of HLA-B*5703 to soluble KIR3DL1 protein by surface plasmon resonance, and while the wild-type sequence and a second (T3N) variant were recognized, the G9E variant abrogated KIR3DL1 binding. We extended the study to determine the peptide sensitivity of KIR3DL1 interaction with epitopes carrying mutations near the C termini of TW10 and a second HLA-B*57-restricted epitope, IW9. Several amino acid changes interfered with KIR3DL1 binding, the most extreme of which included the G9E mutation commonly selected by HLA-B*57. Our results imply that during HIV-1 infection, some early-emerging variants could affect KIR-HLA interaction, with possible implications for immune recognition.
Determining the organisation of key molecules on the surface of live cells in two dimensions and how this changes during biological processes, such as signaling, is a major challenge in cell biology and requires methods with nanoscale resolution. Recent advances in fluorescence imaging both at the diffraction limit tracking single molecules and exploiting super resolution imaging have now reached a stage where they can provide fundamentally new insights. Complementary developments in scanning ion conductance microscopy also allow the cell surface to be imaged with nanoscale resolution. The challenge now is to combine the information obtained using these different methods and on different cells to obtain a coherent view of the cell surface. In the future this needs to be driven by interdisciplinary research between physical scientists and biologists.
The inhibitory T-cell surface-expressed receptor, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), which belongs to the class of cell surface proteins phosphorylated by extrinsic tyrosine kinases that also includes antigen receptors, binds the related ligands, B7-1 and B7-2, expressed on antigen-presenting cells. Conformational changes are commonly invoked to explain ligand-induced "triggering" of this class of receptors. Crystal structures of ligand-bound CTLA-4 have been reported, but not the apo form, precluding analysis of the structural changes accompanying ligand binding. The 1.8-? resolution structure of an apo human CTLA-4 homodimer emphasizes the shared evolutionary history of the CTLA-4/CD28 subgroup of the immunoglobulin superfamily and the antigen receptors. The ligand-bound and unbound forms of both CTLA-4 and B7-1 are remarkably similar, in marked contrast to B7-2, whose binding to CTLA-4 has elements of induced fit. Isothermal titration calorimetry reveals that ligand binding by CTLA-4 is enthalpically driven and accompanied by unfavorable entropic changes. The similarity of the thermodynamic parameters determined for the interactions of CTLA-4 with B7-1 and B7-2 suggests that the binding is not highly specific, but the conformational changes observed for B7-2 binding suggest some level of selectivity. The new structure establishes that rigid-body ligand interactions are capable of triggering CTLA-4 phosphorylation by extrinsic kinase(s).
Exactly how ligand binding triggers T cell receptor (TCR) phosphorylation is unclear. It has been proposed that ligand engagement by the TCR somehow activates the Src kinase Lck, which in turn phosphorylates the receptor. Recent data, however, suggest instead that a significant fraction of the Lck in resting T cells is already activated and that the proportion of active Lck does not change during the early stages of T cell activation. We argue that, caveats notwithstanding, these new observations offer support for the kinetic-segregation model of TCR triggering, which involves spatial reorganization of signalling proteins upon ligand binding and requires a fraction of Lck to be active in resting T cells.
Task switching requires executive control processes that undergo age-related decline. Previous neuroimaging studies have identified age-related differences in brain activation associated with global switching effects (dual-task blocks versus single-task blocks), but age-related differences in activation during local switching effects (switch trials versus repeat trials, within blocks) have not been investigated. This experiment used functional magnetic resonance imaging (fMRI), and diffusion tensor imaging (DTI), to examine adult age differences in task switching across adjacent trials (i.e., local task switching). During fMRI scanning, participants performed a cued, word categorization task. From interspersed cue-only trials, switch-related processing associated with the cue was estimated separately from the target. Activation associated with task switching, within a distributed frontoparietal network, differed for cue- and target-related processing. The magnitude of event-related activation for task switching was similar for younger adults (n=20; 18-27years) and older adults (n=20; 60-85years), although activation sustained throughout the on-tasks periods exhibited some age-related decline. Critically, the functional connectivity of switch-related regions, during cue processing, was higher for younger adults than for older adults, whereas functional connectivity during target processing was comparable across the age groups. Further, individual differences in cue-related functional connectivity shared a substantial portion of the age-related variability in the efficiency of target categorization response (drift rate). This age-related difference in functional connectivity, however, was independent of white matter integrity within task-relevant regions. These findings highlight the functional connectivity of frontoparietal activation as a potential source of age-related decline in executive control.
We present the crystal structure of an immunoglobulin light-chain-like domain, CTLA-4, as a strand-swapped dimer displaying cis-trans proline isomerisation and native-like hydrogen bonding. We also show that CTLA-4 can form amyloid-like fibres and amorphous deposits explainable by the same strand swapping. Our results suggest a molecular basis for the pathological aggregation of immunoglobulin domains and why amyloid-like fibres are more often composed of homologous rather than heterologous subunits.
Neandertals and the Middle Paleolithic persisted in the Iberian Peninsula south of the Ebro drainage system for several millennia beyond their assimilation/replacement elsewhere in Europe. As only modern humans are associated with the later stages of the Aurignacian, the duration of this persistence pattern can be assessed via the dating of diagnostic occurrences of such stages.
The platelet receptor CLEC-2 binds to the snake venom toxin rhodocytin and the tumor cell surface protein podoplanin. Binding of either of these ligands promotes phosphorylation of a single tyrosine residue in the YXXL motif in the intracellular domain of CLEC-2. Phosphorylation of this tyrosine initiates binding of spleen tyrosine kinase (Syk) and triggers further downstream signaling events and ultimately potent platelet activation and aggregation. However, it is unclear how a single YXXL motif can interact efficiently with Syk, which usually recognizes two tandem YXXL repeats presented as an immunoreceptor tyrosine-based activation motif (ITAM). Using bioluminescence resonance energy transfer, coimmunopreciptitation, recombinant protein expression and analytical gel filtration chromatography, surface plasmon resonance, Western blotting, multiangle light scattering (MALS), and analytical ultracentrifugation, we show that CLEC-2 exists as a non-disulfide-linked homodimer which could allow each Syk molecule to interact with two YXXL motifs, one from each CLEC-2 monomer.
Valuable insights into the history of human populations have been obtained by studying the genetic composition of their domesticated species. Here we address some of the long-standing questions about the origin and subsequent movements of goat pastoralism in Northern Africa. We present the first study combining results from mitochondrial DNA (mtDNA) and Y chromosome loci for the genetic characterization of a domestic goat population. Our analyses indicate a remarkably high diversity of maternal and paternal lineages in a sample of indigenous goats from the northwestern fringe of the African continent. Median-joining networks and a multidimensional scaling of ours and almost 2000 published mtDNA sequences revealed a considerable genetic affinity between goat populations from the Maghreb (Northwest Africa) and the Near East. It has been previously shown that goats have a weak phylogeographic structure compatible with high levels of gene flow, as demonstrated by the worldwide dispersal of the predominant mtDNA haplogroup A. In contrast, our results revealed a strong correlation between genetic and geographical distances in 20 populations from different regions of the world. The distribution of Y chromosome haplotypes in Maghrebi goats indicates a common origin for goat patrilines in both Mediterranean coastal regions. Taken together, these results suggest that the colonization and subsequent dispersal of domestic goats in Northern Africa was influenced by the maritime diffusion throughout the Mediterranean Sea and its coastal regions of pastoralist societies whose economy included goat herding. Finally, we also detected traces of gene flow between goat populations from the Maghreb and the Iberian Peninsula corroborating evidence of past cultural and commercial contacts across the Strait of Gibraltar.
We present an advanced system for on-line position-specific carbon isotope analysis. The main limitation of on-line intramolecular isotope ratio measurements has been that optimal pyrolytic fragments are obtained mostly at temperatures where the analyte has not completely reacted. As a result of undetermined isotopic fractionation, the isotopic signatures of the pyrolysis products are not strictly equal to these of the equivalent moieties in the parent molecule. We designed a pyrolytic unit in which both temperature and reaction time are variable parameters, enabling determination of the enrichment factor of the pyrolysis at optimal temperature by construction of a Rayleigh plot. In the case of methyl tert-butyl ether (MTBE) presented here, a pre-pyrolysis fractionation of MTBE leading to a depletion of 0.9 per thousand was discovered and the enrichment factor of the optimal pyrolysis reaction was determined at -1.7 per thousand. Absolute delta(13)C values of two functional groups of MTBE - the methoxy group and the 2-methylpropane group - could be determined with 95% confidence intervals of 0.4 per thousand and 0.5 per thousand, respectively.
Disruption of Golgi alpha-mannosidase II activity can result in type II congenital dyserythropoietic anemia and induce lupus-like autoimmunity in mice. Here, we isolated a mutant human embryonic kidney (HEK) 293T cell line called Lec36, which displays sensitivity to ricin that lies between the parental HEK 293T cells, in which the secreted and membrane-expressed proteins are dominated by complex-type glycosylation, and 293S Lec1 cells, which produce only oligomannose-type N-linked glycans. Stem cell marker 19A was transiently expressed in the HEK 293T Lec36 cells and in parental HEK 293T cells with and without the potent Golgi alpha-mannosidase II inhibitor, swainsonine. Negative ion nano-electrospray ionization mass spectra of the 19A N-linked glycans from HEK 293T Lec36 and swainsonine-treated HEK 293T cells were qualitatively indistinguishable and, as shown by collision-induced dissociation spectra, were dominated by hybrid-type glycosylation. Nucleotide sequencing revealed mutations in each allele of MAN2A1, the gene encoding Golgi alpha-mannosidase II: a point mutation that mapped to the active site was found in one allele, and an in-frame deletion of 12 nucleotides was found in the other allele. Expression of the wild type but not the mutant MAN2A1 alleles in Lec36 cells restored processing of the 19A reporter glycoprotein to complex-type glycosylation. The Lec36 cell line will be useful for expressing therapeutic glycoproteins with hybrid-type glycans and as a sensitive host for detecting mutations in human MAN2A1 causing type II congenital dyserythropoietic anemia.
Glycoproteins of the scavenger receptor cysteine-rich (SRCR) superfamily contain one or more protein modules homologous to the membrane-distal domain of macrophage scavenger receptor I. These domains can be found in the extracellular regions of membrane proteins and in secreted glycoproteins, from the most primitive species to vertebrates. A systematic, bioinformatics-based search for putative human proteins related to the forty-seven known human group B SRCR domains identified a new family member that we have called Soluble Scavenger with 5 Domains (SSc5D). SSc5D is a new soluble protein whose expression is restricted to monocytes/macrophages and T-lymphocytes, and is particularly enriched in the placenta. The gene encoding SSc5D spans 30kb of genomic DNA, and contains fourteen exons producing a 4.8kb-long mRNA. The mature polypeptide is predicted to consist of 1573 amino acids comprising, towards the N-terminus, five very similar SRCR domains that are highly conserved among non-marsupial mammals, and a large (>250nm), very heavily glycosylated, mucin-like sequence towards the C-terminus. Each of the SRCR domains is encoded by a single exon, and contains eight cysteine residues, as observed for all other group B SRCR domains. A shorter isoform encoded by a weakly expressed, alternatively spliced transcript, which lacks the mucin-like C-terminal region, was also identified. It seems likely that SSc5D has a role at the interface between adaptive and innate immunity, or in placental function.
Aging is associated with significant white matter deterioration and this deterioration is assumed to be at least partly a consequence of myelin degeneration. The present study investigated specific predictions of the myelodegeneration hypothesis using diffusion tensor tractography. This technique has several advantages over other methods of assessing white matter architecture, including the possibility of isolating individual white matter tracts and measuring effects along the whole extent of each tract. The study yielded three main findings. First, age-related white matter deficits increased gradually from posterior to anterior segments within specific fiber tracts traversing frontal and parietal, but not temporal cortex. This pattern inverts the sequence of myelination during childhood and early development observed in previous studies and lends support to a "last-in-first-out" theory of the white matter health across the lifespan. Second, both the effects of aging on white matter and their impact on cognitive performance were stronger for radial diffusivity (RD) than for axial diffusivity (AD). Given that RD has previously been shown to be more sensitive to myelin integrity than AD, this second finding is also consistent with the myelodegeneration hypothesis. Finally, the effects of aging on select white matter tracts were associated with age difference in specific cognitive functions. Specifically, FA in anterior tracts was shown to be primarily associated with executive tasks and FA in posterior tracts mainly associated with visual memory tasks. Furthermore, these correlations were mirrored in RD, but not AD, suggesting that RD is more sensitive to age-related changes in cognition. Taken together, the results help to clarify how age-related white matter decline impairs cognitive performance.
We present a general method called dynamic single-molecule colocalization for quantitating the associations of single cell surface molecules labeled with distinct autofluorescent proteins. The chief advantages of the new quantitative approach are that, in addition to stable interactions, it is capable of measuring nonconstitutive associations, such as those induced by the cytoskeleton, and it is applicable to situations where the number of molecules is small.
Previous research has established that age-related decline occurs in measures of cerebral white matter integrity, but the role of this decline in age-related cognitive changes is not clear. To conclude that white matter integrity has a mediating (causal) contribution, it is necessary to demonstrate that statistical control of the white matter-cognition relation reduces the magnitude of age-cognition relation. In this research, we tested the mediating role of white matter integrity, in the context of a task-switching paradigm involving word categorization. Participants were 20 healthy, community-dwelling older adults (60-85 years), and 20 younger adults (18-27 years). From diffusion tensor imaging tractography, we obtained fractional anisotropy (FA) as an index of white matter integrity in the genu and splenium of the corpus callosum and the superior longitudinal fasciculus (SLF). Mean FA values exhibited age-related decline consistent with a decrease in white matter integrity. From a model of reaction time distributions, we obtained independent estimates of the decisional and nondecisional (perceptual-motor) components of task performance. Age-related decline was evident in both components. Critically, age differences in task performance were mediated by FA in two regions: the central portion of the genu, and splenium-parietal fibers in the right hemisphere. This relation held only for the decisional component and was not evident in the nondecisional component. This result is the first demonstration that the integrity of specific white matter tracts is a mediator of age-related changes in cognitive performance.
A full understanding of leukocyte responses to external stimuli requires knowledge of the full complement of proteins found on their surfaces. Systematic examination of the mammalian cell surfaces at the protein level is hampered by technical difficulties associated with proteomic analysis of so many membrane proteins and the large amounts of starting material required. The use of transcriptomic analyses avoids challenges associated with protein stability and separation and enables the inclusion of an amplification step; thus allowing the use of cell numbers applicable to the study of sub populations of, for example, primary lymphocytes. Here we present a transcriptomic methodology based on Serial Analysis of Gene Expression (SAGE) to recover an essentially complete and quantitative profile of mRNA species in a particular cell. We discuss how, using bioinformatic tools accessible to standard desktop computers, plasma membrane proteins can be identified in silico, from this list. While we describe the use of this approach to characterise the cell surface protein complement of a resting CD8(+) T-cell clone, it is theoretically applicable to any cell surface, where a suitable pure population of cells is available.
Determining the organization of key molecules on the surface of live cells in two dimensions and how this changes during biological processes, such as signalling, is a major challenge in cell biology and requires methods with nanoscale spatial resolution and high temporal resolution. Here, we review biophysical tools, based on scanning ion conductance microscopy and single-molecule fluorescence and the combination of both of these methods, which have recently been developed to address these issues. We then give examples of how these methods have been be applied to provide new insights into cell membrane organization and function, and discuss some of the issues that will need to be addressed to further exploit these methods in the future.
Flashbacks are a defining feature of posttraumatic stress disorder (PTSD), but there have been few studies of their neural basis. We tested predictions from a dual representation model of PTSD that, compared with ordinary episodic memories of the same traumatic event, flashbacks would be associated with activity in dorsal visual stream and related areas rather than in the medial temporal lobe. Participants with PTSD, with depression but not PTSD, and healthy controls were scanned during a recognition task with personally relevant stimuli. The contrast of flashbacks versus ordinary episodic trauma memories in PTSD was associated with increased activation in sensory and motor areas including the insula, precentral gyrus, supplementary motor area, and mid-occipital cortex. The same contrast was associated with decreased activation in the midbrain, parahippocampal gyrus, and precuneus/posterior cingulate cortex. The results were discussed in terms of theories of PTSD and dual-process models of recognition.
Although there has been much success in identifying genetic variants associated with common diseases using genome-wide association studies (GWAS), it has been difficult to demonstrate which variants are causal and what role they have in disease. Moreover, the modest contribution that these variants make to disease risk has raised questions regarding their medical relevance. Here we have investigated a single nucleotide polymorphism (SNP) in the TNFRSF1A gene, that encodes tumour necrosis factor receptor 1 (TNFR1), which was discovered through GWAS to be associated with multiple sclerosis (MS), but not with other autoimmune conditions such as rheumatoid arthritis, psoriasis and Crohn’s disease. By analysing MS GWAS data in conjunction with the 1000 Genomes Project data we provide genetic evidence that strongly implicates this SNP, rs1800693, as the causal variant in the TNFRSF1A region. We further substantiate this through functional studies showing that the MS risk allele directs expression of a novel, soluble form of TNFR1 that can block TNF. Importantly, TNF-blocking drugs can promote onset or exacerbation of MS, but they have proven highly efficacious in the treatment of autoimmune diseases for which there is no association with rs1800693. This indicates that the clinical experience with these drugs parallels the disease association of rs1800693, and that the MS-associated TNFR1 variant mimics the effect of TNF-blocking drugs. Hence, our study demonstrates that clinical practice can be informed by comparing GWAS across common autoimmune diseases and by investigating the functional consequences of the disease-associated genetic variation.
Native and non-native ligands of the T cell receptor (TCR), including antibodies, have been proposed to induce signaling in T cells via intra- or intersubunit conformational rearrangements within the extracellular regions of TCR complexes. We have investigated whether any signatures can be found for such postulated structural changes during TCR triggering induced by antibodies, using crystallographic and mutagenesis-based approaches. The crystal structure of murine CD3? complexed with the mitogenic anti-CD3? antibody 2C11 enabled the first direct structural comparisons of antibody-liganded and unliganded forms of CD3? from a single species, which revealed that antibody binding does not induce any substantial rearrangements within CD3?. Saturation mutagenesis of surface-exposed CD3? residues, coupled with assays of antibody-induced signaling by the mutated complexes, suggests a new configuration for the complex within which CD3? is highly exposed and reveals that no large new CD3? interfaces are required to form during antibody-induced signaling. The TCR complex therefore appears to be a structure that is capable of initiating intracellular signaling in T cells without substantial structural rearrangements within or between the component subunits. Our findings raise the possibility that signaling by native ligands might also be initiated in the absence of large structural rearrangements in the receptor.
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