Near Infrared Optical Projection Tomography for Assessments of β-cell Mass Distribution in Diabetes Research

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of pancreatic tissue, such as the rat pancreas, and to increase the number of channels (cell types) that may be studied in a single specimen. We further describe the implementation of a number of computational tools that provide: 1/ accurate positioning of a specimen''s (in our case the pancreas) centre of mass (COM) at the axis of rotation (AR)²; 2/ improved algorithms for post-alignment tuning which prevents geometric distortions during the tomographic reconstruction² and 3/ a protocol for intensity equalization to increase signal to noise ratios in OPT-based BCM determinations³. In addition, we describe a sample holder that minimizes the risk for unintentional movements of the specimen during image acquisition. Together, these protocols enable assessments of BCM distribution and other features, to be performed throughout the volume of intact pancreata or other organs (e.g. in studies of islet transplantation), with a resolution down to the level of individual islets of Langerhans.     

Conserved and Variable Features of Gag Structure and Arrangement in Immature Retrovirus Particles

The assembly of retroviruses is driven by oligomerization of the Gag polyprotein. We have used cryo-electron tomography together with subtomogram averaging to describe the three-dimensional structure of in vitro-assembled Gag particles from human immunodeficiency virus, Mason-Pfizer monkey virus, and Rous sarcoma virus. These represent three different retroviral genera: the lentiviruses, betaretroviruses and alpharetroviruses. Comparison of the three structures reveals the features of the supramolecular organization of Gag that are conserved between genera and therefore reflect general principles of Gag-Gag interactions and the features that are specific to certain genera. All three Gag proteins assemble to form approximately spherical hexameric lattices with irregular defects. In all three genera, the N-terminal domain of CA is arranged in hexameric rings around large holes. Where the rings meet, 2-fold densities, assigned to the C-terminal domain of CA, extend between adjacent rings, and link together at the 6-fold symmetry axis with a density, which extends toward the center of the particle into the nucleic acid layer. Although this general arrangement is conserved, differences can be seen throughout the CA and spacer peptide regions. These differences can be related to sequence differences among the genera. We conclude that the arrangement of the structural domains of CA is well conserved across genera, whereas the relationship between CA, the spacer peptide region, and the nucleic acid is more specific to each genus.Retrovirus assembly is driven by the oligomerization of Gag, a multidomain protein, including an N-terminal membrane binding domain (MA), a two-domain structural component (CA), and an RNA binding domain (NC). The Gag proteins of all orthoretroviruses, including the alpha-, beta-, and lentiretroviruses discussed here, share this conserved modular architecture (Fig. ​(Fig.1).1). Despite very weak sequence conservation, the tertiary structures of MA, CA, and NC are conserved among retroviruses. Outside these conserved domains the Gag proteins of different retroviruses exhibit substantial variability. Other domains may be present or absent, and the length and sequence of linker peptides may also vary (12) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Modular architecture of the full-length Gag proteins of HIV, M-PMV, and RSV. White rectangles illustrate Gag polyprotein cleavage products. The extent of the constructs used in the electron microscopic analysis is specified under each protein as a black rectangle. Gray triangles specify cleavage sites. Residue numbers are counted from the beginning of Gag.Oligomerization of Gag in an infected cell leads to the formation of roughly spherical immature virus particles, where Gag is arranged in a radial fashion with the N-terminal MA domain associated with a surrounding lipid bilayer, and the more C-terminal NC pointing toward the center of the particle (15, 44, 46). Subsequent multiple cleavages of Gag by the viral protease lead to a rearrangement of the virus. NC and the RNA condense in the center of the particle, CA assembles into a capsid or shell around the nucleoprotein, and MA remains associated with the viral membrane. This proteolytic maturation is required to generate an infectious virion (2). In contrast to the mature CA lattice, which has been extensively studied (11, 16, 36), the Gag lattice in immature particles is incompletely understood.Gag itself contains all of the necessary determinants for particle assembly. For example, the expression of Gag alone in an insect cell expression system is sufficient to generate viruslike particles (3, 17, 22, 38). Retroviral Gag proteins also can be assembled in vitro in the presence of nucleic acids to form spherical particles (9, 19, 39, 43, 47). The arrangement of Gag within these in vitro-assembled Gag particles is indistinguishable from that found in immature virus particles (6), and the in vitro assembly systems have proved valuable for unraveling the principles of virus assembly (18, 28, 29, 39). Multiple layers of interaction promote the assembly of Gag in vivo, including MA-membrane-MA interactions, CA-CA interactions, and NC-RNA-NC interactions. An extensive body of literature has explored which regions of Gag are required for assembly and which can be replaced or deleted without compromising assembly. MA-membrane-MA interactions contribute but are not essential. NC-RNA-NC interactions appear to function to nonspecifically link Gag molecules together and can be replaced both in vivo and in vitro by other interaction domains such as leucine zippers (4, 13, 20, 32, 48). The C-terminal domain of CA (referred to here as C-CA) and the stretch of amino acids immediately following this domain (termed the spacer peptide [SP] region) are critical for assembly and sensitive to mutation (1, 22, 27, 30).We set out to understand how the substantial sequence variation among Gag proteins in different retroviruses is manifested in structural differences in the immature Gag lattice. To do this, we studied three retroviruses from different genera: the lentivirus human immunodeficiency virus type 1 (HIV-1), the betaretrovirus Mason-Pfizer monkey virus (M-PMV), and the alpharetrovirus Rous sarcoma virus (RSV). These retroviruses are those for which in vitro assembly was first established and has been most extensively studied (6, 19, 24, 28, 29, 35, 43, 47).The domain structures of the three retroviruses differ most substantially upstream of CA. Both M-PMV and RSV have domains located between MA and CA that are absent in HIV (Fig. ​(Fig.1).1). In M-PMV there are 198 residues forming the pp24 and p12 domains; in RSV there are 84 residues forming the p2a, p2b, and p10 domains. The three retroviruses have different requirements for regions upstream of CA during assembly. The C-terminal 25 residues of p10 are essential for proper immature RSV assembly, both in vitro and in vivo, and these residues are inferred to interact directly with N-CA to stabilize the hexamer by forming contacts between adjacent N-CA domains (35). An equivalent assembly domain has not been described for other retroviruses. Within M-PMV p12 is the so-called internal scaffolding domain that is not essential for assembly in vitro (43) but is required for particle assembly when the precursor is expressed under the control of the M-PMV promoter (41). It is a key domain for the membrane-independent assembly of immature capsids (40).In HIV, five residues upstream of CA must be present for assembly of immature virus-like spherical particles in vitro, although larger upstream extensions, including part of MA, are required for efficient assembly of regular particles, both for HIV and RSV. For HIV, if the entire MA domain is included, in vitro assembly requires the presence of inositol penta- or hexakis phosphate (8). If no sequences upstream of CA are present, the in vitro particles in both of these viruses adopt a mature-type tubular morphology (10, 18). It has been hypothesized that cleavage at the N terminus of N-CA during maturation leads to the N-terminal residues of CA folding back into the N-CA structure to form a β-hairpin. The β-hairpin is important for assembly of the mature CA lattice, whereas its absence is important for immature assembly (23, 42). These requirements explain why, in HIV and RSV, immature Gag lattice-like structures are formed only if regions upstream of CA are present (18). In M-PMV, an immature Gag lattice can be produced when the regions upstream of CA are deleted if this is combined with mutations (such as deleting the initial proline of CA), which prevent β-hairpin formation (43).During maturation, HIV and RSV Gag proteins are cleaved twice between CA and NC to release a small peptide called SP1 or SP. In RSV the most N-terminal of these two cleavages can occur at one of two possible positions such that the released peptide is either 9 or 12 amino acids long (33). In M-PMV only one cleavage occurs between CA and NC, and no short peptide is produced. The region between the final helix of CA and the Zn fingers has been proposed to adopt a helical bundle architecture in HIV and RSV based on bioinformatic prediction, on mutational analysis, and on structural studies (1, 22, 27, 45). In all three viruses, C-CA and the residues immediately downstream are critical for assembly and are sensitive to mutation. C-CA contains the major homology region, a group of residues that are highly conserved across the retroviruses.Cryo-electron tomography (cET) studies of immature virus particles (6, 45) have resolved the electron density of the HIV Gag lattice in three dimensions at low resolution. Using these methods, we have also described the three-dimensional architecture of in vitro-assembled HIV Gag particles (6). In immature viruses and in vitro-assembled particles, Gag is seen to adopt an 8 nm hexameric lattice, as was predicted from previous Fourier analysis of two-dimensional images (7, 46). The hexameric lattice is interrupted by irregularly shaped holes and cracks in the lattice (6, 45). A similar observation has been made using AFM of in vitro-assembled particles of M-PMV Gag (26). These holes and cracks allow an otherwise planar hexameric lattice to form the surface of an approximately spherical particle.The radial positions of the MA, CA, and NC domains had been assigned previously from cryo-electron micrographs (44, 46). Based on these assignments and the shape of the density, the position and relative orientations of CA domains can be modeled into the low-resolution structure of the HIV lattice (6, 45). Density ascribed to the N-terminal domain of CA (N-CA) forms rings around large holes at the 6-fold symmetry positions in the lattice. Below this layer, at the expected radius of the C-CA, are 2-fold densities, interpreted as corresponding to dimers of C-CA. These densities are linked by rodlike densities, which descend into the NC-nucleic acid layer.HIV is the only retrovirus for which the arrangement of Gag in the immature particle has been described in three dimensions. Prior to this work, important open questions were therefore: which features of the arrangement of Gag are conserved between genera and therefore reflect general principles of Gag-Gag interactions, and which features are specific to certain genera? We have applied subtomogram averaging of cryo-electron tomograms to generate reconstructions of in vitro-assembled Gag particles from HIV, M-PMV, and RSV. These allow identification of the general and variable features of the arrangement of Gag and the architecture of immature retroviruses.     

Structural studies of tri-functional human GART

Human purine de novo synthesis pathway contains several multi-functional enzymes, one of which, tri-functional GART, contains three enzymatic activities in a single polypeptide chain. We have solved structures of two domains bearing separate catalytic functions: glycinamide ribonucleotide synthetase and aminoimidazole ribonucleotide synthetase. Structures are compared with those of homologous enzymes from prokaryotes and analyzed in terms of the catalytic mechanism. We also report small angle X-ray scattering models for the full-length protein. These models are consistent with the enzyme forming a dimer through the middle domain. The protein has an approximate seesaw geometry where terminal enzyme units display high mobility owing to flexible linker segments. This resilient seesaw shape may facilitate internal substrate/product transfer or forwarding to other enzymes in the pathway.     

EEG applications for sport and performance

One approach to understanding processes that underlie skilled performing has been to study electrical brain activity using electroencephalography (EEG). A notorious problem with EEG is that genuine cerebral data is often contaminated by artifacts of non-cerebral origin. Unfortunately, such artifacts tend to be exacerbated when the subject is in motion, meaning that obtaining reliable data during exercise is inherently problematic. These problems may explain the limited number of studies using EEG as a methodological tool in the sports sciences. This paper discusses how empirical studies have generally tackled the problem of movement artifact by adopting alternative paradigms which avoid recording during actual physical exertion. Moreover, the specific challenges that motion presents to obtaining reliable EEG data are discussed along with practical and computational techniques to confront these challenges. Finally, as EEG recording in sports is often underpinned by a desire to optimise performance, a brief review of EEG-biofeedback and peak performance studies is also presented. A knowledge of practical aspects of EEG recording along with the advent of new technology and increasingly sophisticated processing models offer a promising approach to minimising, if perhaps not entirely circumventing, the problem of obtaining reliable EEG data during motion.     

Trex1 exonuclease degrades ssDNA to prevent chronic checkpoint activation and autoimmune disease

Trex1 is the major 3' DNA exonuclease in mammalian cells, and mutations in the human TREX1 gene can cause Aicardi-Goutières syndrome, characterized by perturbed immunity. Similarly, Trex1(-/-) mice have an autoinflammatory phenotype; however, the mechanism of Trex1-deficient disease is unknown. We report that Trex1, ordinarily associated with the endoplasmic reticulum (ER), relocalizes to the S phase nucleus after gamma irradiation or hydroxyurea treatment. Notably, Trex1-deficient cells show defective G1/S transition and chronic ATM-dependent checkpoint activation, even in the absence of exogenous stress, correlating with persistent single-stranded DNA molecules produced in S phase, which accumulate in the ER. Our data indicate that Trex1 acts on a single-stranded DNA polynucleotide species generated from processing of aberrant replication intermediates to attenuate DNA damage checkpoint signaling and prevent pathological immune activation.     

Contrasting behavior of higher plant photosystem I and II antenna systems during acclimation

In this work we analyzed the photosynthetic apparatus in Arabidopsis thaliana plants acclimated to different light intensity and temperature conditions. Plants showed the ability to acclimate into different environments and avoid photoinhibition. When grown in high light, plants had a faster activation rate for energy dissipation (qE). This ability was correlated to higher accumulation levels of a specific photosystem II subunit, PsbS. The photosystem II antenna size was also regulated according to light exposure; smaller antenna size was observed in high light-acclimated plants with respect to low light plants. Different antenna polypeptides did not behave similarly, and Lhcb1, Lchb2, and Lhcb6 (CP24) are shown to undergo major levels of regulation, whereas Lhcb4 and Lhcb5 (CP29 and CP26) maintained their stoichiometry with respect to the reaction center in all growth conditions. The effect of acclimation on photosystem I antenna was different; in fact, the stoichiometry of any Lhca antenna proteins with respect to photosystem I core complex was not affected by growth conditions. Despite this stability in antenna stoichiometry, photosystem I light harvesting function was shown to be regulated through different mechanisms like the control of photosystem I to photosystem II ratio and the association or dissociation of Lhcb polypeptides to photosystem I.     

A decreased mitochondrial DNA content is related to insulin resistance in adolescents

The aim of this study was to investigate whether mitochondrial DNA (mtDNA) content is associated with insulin resistance (IR) in a sample of adolescents with features of metabolic syndrome. We further studied the link between polymorphisms in three genes involved in mitochondrial biogenesis and the presence of deleted mtDNA and mtDNA content. Data and blood samples were collected from 175 adolescents out of a cross-sectional, population-based study of 934 high school students. On the basis of the median value of homeostasis model assessment of IR (HOMA-IR) of the whole sample (2.2), the population was divided into two groups: noninsulin resistance (NIR) and IR. mtDNA quantification using nuclear DNA (nDNA) as a reference was carried out using a real-time quantitative PCR method. Genotyping for peroxisome proliferator-activated receptor-gamma (PPAR-gamma) (pro12Ala), PPAR- gamma coactivator-1alpha (PGC-1alpha) (Gly482Ser), and Tfam (rs1937 and rs12247015) polymorphisms was performed by PCR-based restriction fragment length polymorphism. Long-extension PCR was performed to amplify the whole mitochondrial genome. The mtDNA/nDNA ratio was significantly lower in the IR group (median: 9.08, range: 68.94) in comparison with the NIR group (12.24, 71.92) (P<0.03). Besides, the mtDNA/nDNA ratio was inversely correlated with HOMA (R: -0.18, P<0.02), glucose (R: -0.21, P<0.008), and uric acid (R: -0.18, P<0.03). Genotypes for the PPAR- gamma, PGC-1alpha, and Tfam variants were not associated with the mtDNA/nDNA ratio. Long-extension PCR did not show significant levels of mtDNA deletions. In conclusion, our findings indicate that reduced mtDNA content in peripheral leukocytes is associated with IR. This result seems not to be related with the previously mentioned variants in genes involved in the regulation of mitochondrial biogenesis.