Comparisons between analogue and numerical models of thrust wedge development

Analogue and finite element numerical models with frictional and viscous properties are used to model thrust wedge development. Comparison between model types yields valuable information about analogue model evolution, scaling laws and the relative strengths and limitations of the techniques. Both model types show a marked contrast in structural style between ‘frictional-viscous domains’ underlain by a thin viscous layer and purely ‘frictional domains’. Closely spaced thrusts form a narrow and highly asymmetric fold-and-thrust belt in the frictional domain, characterized by in-sequence propagation of forward thrusts. In contrast, the frictional-viscous domain shows a wide and low taper wedge and a thrust belt with a more symmetrical vergence, with both forward and back thrusts. The frictional-viscous domain numerical models show that the viscous layer initially simple shears as deformation propagates along it, while localized deformation resulting in the formation of a pop-up structure occurs in the overlying frictional layers. In both domains, thrust shear zones in the numerical model are generally steeper than the equivalent faults in the analogue model, because the finite element code uses a non-associated plasticity flow law. Nevertheless, the qualitative agreement between analogue and numerical models is encouraging. It shows that the continuum approximation used in numerical models can be used to model frictional materials, such as sand, provided caution is taken to properly scale the experiments, and some of the limitations are taken into account.

Tectonic evolution of the Proterozoic Itremo Group metasediments in central Madagascar

The major geologic units of the Itremo region in central Madagascar include: (1) upper amphibolite to granulite facies (higher grade) Precambrian rocks, mainly para- and orthogneisses, and migmatites; (2) the newly defined Itremo Nappes, a fold-and-thrust belt containing the Proterozoic Itremo Group sediments, metamorphosed at greenschist to lower amphibolite facies (lower grade) conditions: (3) Middle Neoproterozoic and Late Neoproterozoic-Cambrian intrusives. The stratigraphic succession of the Itremo Group in the eastern part of the Itremo region is, from bottom to top: quartzites, metapelites, metacarbonates and metapelites overlain by metacarbonates. During D1 the Itremo Group sediments were detached from their continental substratum, deformed into a fold-and-thrust nappe (Itremo Nappes), and transported on top of higher grade rocks that are intruded by Middle Neoproterozoic (c. 797–780 Ma) granites and gabbros. A second phase of deformation shortening (D2) affected both the Itremo Sedimentary Nappes and structurally underlying higher-grade rocksunits, and formed large-scale N-S-trending F2 folds. S1 axial plane foliations in Itremo Group sediments are truncated by Late Neoproterozoic-Cambrian granites (c. 570–540 Ma). The age of the formation of the Itremo Nappes is not well constrained: they formed in Neoproterozoic times between 780 and 570 Ma.

Age constraints on the tectonic evolution of the Itremo region in Central Madagascar

The Itremo region in Central Madagascar comprises a deformed metasedimentary sequence (Itremo Group) that has undergone greenschist to lower amphibolite facies metamorphism. During a first phase of deformation (D1) Itremo Group sediments were deformed into a fold-and-thrust belt and transported toward the E to NE on top of migmatitic gneisses rocks of Anatananarivo block. A second phase of deformation (D2) affected both the fold-and-thrust belt and structurally underlying units, and formed large-scale N-S trending folds with steeply dipping axial planes. A Late Neoproterozoic Th–U–Pb XRF monazite age (565±17 Ma) dates the emplacement of a granite that truncates first-phase structures in the Itremo Group, and indicates that the fold-and-thrust belt formed prior to ≈565 Ma. Th–U–Pb electron microprobe dating was applied to elongated monazites that lie within the first-phase foliation of Itremo Group metapelites. The detrital cores of zoned monazites reveal two distinct age populations at ∼2000 and 1700 Ma, the latter age giving a maximum depositional age for the Itremo Group. Statistical analysis of ages determined from the rims of zoned monazites and from unzoned monazites indicates three Late Proterozoic–Early Paleozoic monazite growth events at about 565–540, 500 and 430 Ma. The oldest age population is contemporaneous within error, with the intrusion of the dated granite. The two younger age populations are found both in the Th–U–Pb and Ar–Ar data; together with the perturbation of the Rb–Sr system we interpret both ages as due to alteration related to fluid circulation events, possibly connected to the emplacement of pegmatite fields in Central Madagascar. Syn-D1 tectonic growth of contact metamorphism minerals such as andalusite has been observed locally in metapelites along the margin of Middle Neoproterozoic (≈800 Ma) granites, suggesting that D1 in the Itremo Group is contemporaneous with the intrusion of granites at ≈800 Ma. The N-S trending D2 folds are associated with ≈E-W shortening during the final assembly of Gondwana in Late Neoproterozoic–Early Cambrian times.

Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process

Owing to their pathogenical role and unique ability to exist both as soluble proteins and transmembrane complexes, pore-forming toxins (PFTs) have been a focus of microbiologists and structural biologists for decades. PFTs are generally secreted as water-soluble monomers and subsequently bind the membrane of target cells. Then, they assemble into circular oligomers, which undergo conformational changes that allow membrane insertion leading to pore formation and potentially cell death. Aerolysin, produced by the human pathogen Aeromonas hydrophila, is the founding member of a major PFT family found throughout all kingdoms of life. We report cryo-electron microscopy structures of three conformational intermediates and of the final aerolysin pore, jointly providing insight into the conformational changes that allow pore formation. Moreover, the structures reveal a protein fold consisting of two concentric β-barrels, tightly kept together by hydrophobic interactions. This fold suggests a basis for the prion-like ultrastability of aerolysin pore and its stoichiometry.

Tectono-metamorphic evolution of a nappe stack: A case study of the Swiss Alps

Fold-and-thrust belts are prominent structures that occur at the front of compressional orogens. To unravel the tectonic and metamorphic evolution of such complexes, kinematic investigations, quantitative microstructural analysis and geothermometry (calcite–graphite, calcite–dolomite) were performed on carbonate mylonites from thrust faults of the Helvetic nappe stack in Central Switzerland. Paleo-isotherms of peak temperature conditions and cooling stages (fission track) of the nappe pile were reconstructed in a vertical section and linked with the microstructural and kinematic evolution. Mylonitic microstructures suggest that under metamorphic conditions close to peak temperature, strain was highly localized within thrust faults where deformation temperatures spatially continuously increased in both directions, from N to S within each nappe and from top–down in the nappe stack, covering a temperature range of 180–380 °C. Due to the higher metamorphic conditions, thrusting of the lowermost nappe, the Doldenhorn nappe, was accompanied by a much more pronounced nappe internal ductile deformation of carbonaceous rock types than was the case for the overlying Wildhorn- and Gellihorn nappes. Ongoing thrusting brought the Doldenhorn nappe closer to the surface. The associated cooling resulted in a freezing in of the paleo-isotherms of peak metamorphic conditions. Contemporaneous shearing localized in the basal thrust, initially still in the ductile deformation regime and finally as brittle faulting and cataclasis inducing ultimately an inverse metamorphic zonation. With ongoing exhumation and the formation of the Helvetic antiformal nappe stack, a bending of large-scale tectonic structures (thrusts, folds), peak temperature isotherms and cooling isotherms occurred. While this local bending can directly be attributed to active deformation underneath the section investigated up to times of 2–3 ma, a more homogeneous uplift of the entire region is suggested for the very late and still active exhumation stage.

The tectonometamorphic evolution of the Sesia–Dent Blanche nappes (internal Western Alps): review and synthesis

This study reviews and synthesizes the present knowledge on the Sesia–Dent Blanche nappes, the highest tectonic elements in the Western Alps (Switzerland and Italy), which comprise pieces of pre-Alpine basement and Mesozoic cover. All of the available data are integrated in a crustal-scale kinematic model with the aim to reconstruct the Alpine tectono-metamorphic evolution of the Sesia–Dent Blanche nappes. Although major uncertainties remain in the pre-Alpine geometry, the basement and cover sequences of the Sesia–Dent Blanche nappes are seen as part of a thinned continental crust derived from the Adriatic margin. The earliest stages of the Alpine evolution are interpreted as recording late Cretaceous subduction of the Adria-derived Sesia–Dent Blanche nappes below the South-Alpine domain. During this subduction, several sheets of crustal material were stacked and separated by shear zones that rework remnants of their Mesozoic cover. The recently described Roisan-Cignana Shear Zone of the Dent Blanche Tectonic System represents such a shear zone, indicating that the Sesia–Dent Blanche nappes represent a stack of several individual nappes. During the subsequent subduction of the Piemonte–Liguria Ocean large-scale folding of the nappe stack (including the Roisan-Cignana Shear Zone) took place under greenschist facies conditions, which indicates partial exhumation of the Dent Blanche Tectonic System. The entrance of the Briançonnais micro-continent within the subduction zone led to a drastic change in the deformation pattern of the Alpine belt, with rapid exhumation of the eclogite-facies ophiolite bearing units and thrust propagation towards the foreland. Slab breakoff probably was responsible for allowing partial melting in the mantle and Oligocene intrusions into the most internal parts of the Sesia–Dent Blanche nappes. Finally, indentation of the Adriatic plate into the orogenic wedge resulted in the formation of the Vanzone back-fold, which marks the end of the pervasive ductile deformation within the Sesia–Dent Blanche nappes during the earliest Miocene.

Metabolomics reveals attenuation of the SLC6A20 kidney transporter in nonhuman primate and mouse models of type 2 diabetes mellitus

To enhance understanding of the metabolic indicators of type 2 diabetes mellitus (T2DM) disease pathogenesis and progression, the urinary metabolomes of well characterized rhesus macaques (normal or spontaneously and naturally diabetic) were examined. High-resolution ultra-performance liquid chromatography coupled with the accurate mass determination of time-of-flight mass spectrometry was used to analyze spot urine samples from normal (n = 10) and T2DM (n = 11) male monkeys. The machine-learning algorithm random forests classified urine samples as either from normal or T2DM monkeys. The metabolites important for developing the classifier were further examined for their biological significance. Random forests models had a misclassification error of less than 5%. Metabolites were identified based on accurate masses (<10 ppm) and confirmed by tandem mass spectrometry of authentic compounds. Urinary compounds significantly increased (p < 0.05) in the T2DM when compared with the normal group included glycine betaine (9-fold), citric acid (2.8-fold), kynurenic acid (1.8-fold), glucose (68-fold), and pipecolic acid (6.5-fold). When compared with the conventional definition of T2DM, the metabolites were also useful in defining the T2DM condition, and the urinary elevations in glycine betaine and pipecolic acid (as well as proline) indicated defective re-absorption in the kidney proximal tubules by SLC6A20, a Na(+)-dependent transporter. The mRNA levels of SLC6A20 were significantly reduced in the kidneys of monkeys with T2DM. These observations were validated in the db/db mouse model of T2DM. This study provides convincing evidence of the power of metabolomics for identifying functional changes at many levels in the omics pipeline.

Mutagenesis and computer modeling studies of a GPCR conserved residue W5.43(194) in ligand recognition and signal transduction for CB2 receptor

W5.43(194), a conserved tryptophan residue among G-protein coupled receptors (GPCRs) and cannabinoid receptors (CB), was examined in the present report for its significance in CB2 receptor ligand binding and adenylyl cyclase (AC) activity. Computer modeling postulates that this site in CB2 may be involved in the affinity of WIN55212-2 and SR144528 through aromatic contacts. In the present study, we reported that a CB2 receptor mutant, W5.43(194)Y, which had a tyrosine (Y) substitution for tryptophan (W), retained the binding affinity for CB agonist CP55940, but reduced binding affinity for CB2 agonist WIN55212-2 and inverse agonist SR144528 by 8-fold and 5-fold, respectively; the CB2 W5.43(194)F and W5.43(194)A mutations significantly affect the binding activities of CP55940, WIN55212-2 and SR144528. Furthermore, we found that agonist-mediated inhibition of the forskolin-induced cAMP production was dramatically diminished in the CB2 mutant W5.43(194)Y, whereas W5.43(194)F and W5.43(194)A mutants resulted in complete elimination of downstream signaling, suggesting that W5.43(194) was essential for the full activation of CB2. These results indicate that both aromatic interaction and hydrogen bonding are involved in ligand binding for the residue W5.43(194), and the mutations of this tryptophan site may affect the conformation of the ligand binding pocket and therefore control the active conformation of the wild type CB2 receptor. W5.43(194)Y/F/A mutations also displayed noticeable enhancement of the constitutive activation probably attributed to the receptor conformational changes resulted from the mutations.

GDNF family ligands display distinct action profiles on cultured GABAergic and serotonergic neurons of rat ventral mesencephalon

Glial-cell-line-derived neurotrophic factor (GDNF), neurturin (NRTN), artemin (ARTN) and persephin (PSPN), known as the GDNF family ligands (GFLs), influence the development, survival and differentiation of cultured dopaminergic neurons from ventral mesencephalon (VM). Detailed knowledge about the effects of GFLs on other neuronal populations in the VM is essential for their potential application as therapeutic molecules for Parkinson's disease. Hence, in a comparative study, we investigated the effects of GFLs on cell densities and morphological differentiation of gamma-aminobutyric acid-immunoreactive (GABA-ir) and serotonin-ir (5-HT-ir) neurons in primary cultures of E14 rat VM. We observed that all GFLs [10 ng/ml] significantly increased GABA-ir cell densities (1.6-fold) as well as neurite length/neuron. However, only GDNF significantly increased the number of primary neurites/neuron, and none of the GFLs affected soma size of GABA-ir neurons. In contrast, only NRTN treatment significantly increased 5-HT-ir cells densities at 10 ng/ml (1.3-fold), while an augmentation was seen for GDNF and PSPN at 100 ng/ml (2.4-fold and 1.7-fold, respectively). ARTN had no effect on 5-HT-ir cell densities. Morphological analysis of 5-HT-ir neurons revealed a significant increase of soma size, number of primary neurites/neuron and neurite length/neuron after GDNF exposure, while PSPN only affected soma size, and NRTN and ARTN failed to exert any effect. In conclusion, we identified GFLs as effective neurotrophic factors for VM GABAergic and serotonergic neurons, demonstrating characteristic individual action profiles emphasizing their important and distinct roles during brain development.

Interferon-inducible expression of APOBEC3 editing enzymes in human hepatocytes and inhibition of hepatitis B virus replication

Hypermutations in hepatitis B virus (HBV) DNA by APOBEC3 cytidine deaminases have been detected in vitro and in vivo, and APOBEC3G (A3G) and APOBEC3F (A3F) have been shown to inhibit the replication of HBV in vitro, but the presumably low or even absent hepatic expression of these enzymes has raised the question as to their physiological impact on HBV replication. We show that normal human liver expresses the mRNAs of APOBEC3B (A3B), APOBEC3C (A3C), A3F, and A3G. In primary human hepatocytes, interferon alpha (IFN-alpha) stimulated the expression of these cytidine deaminases up to 14-fold, and the mRNAs of A3G, A3F, and A3B reached expression levels of 10%, 3%, and 3%, respectively, relative to GAPDH mRNA abundance. On transfection, the full-length protein A3B(L) inhibited HBV replication in vitro as efficiently as A3G or A3F, whereas the truncated splice variant A3B(S) and A3C had no effect. A3B(L) and A3B(S) were detected predominantly in the nucleus of uninfected cells; however, in HBV-expressing cells both proteins were found also in the cytoplasm and were associated with HBV viral particles, similarly to A3G and A3F. Moreover, A3G, A3F, and A3B(L), but not A3B(S), induced extensive G-to-A hypermutations in a fraction of the replicated HBV genomes. In conclusion, the editing enzymes A3B(L), A3F, and most markedly A3G, which are expressed in liver and up-regulated by IFN-alpha in hepatocytes, are candidates to contribute to the noncytolytic clearance of HBV.

PEGylation and multimerization of the anti-p185HER-2 single chain Fv fragment 4D5: effects on tumor targeting

A major goal in antibody design for cancer therapy is to tailor the pharmacokinetic properties of the molecule according to specific treatment requirements. Key parameters determining the pharmacokinetics of therapeutic antibodies are target specificity, affinity, stability, and size. Using the p185HER-2 (HER-2)-specific scFv 4D5 as model system, we analyzed how changes in molecular weight and valency independently affect antigen binding and tumor localization. By employing multimerization and PEGylation, four different antibody formats were generated and compared with the scFv 4D5. First, dimeric and tetrameric miniantibodies were constructed by fusion of self-associating, disulfide-linked peptides to the scFv 4D5. Second, we attached a 20-kDa PEG moiety to the monovalent scFv and to the divalent miniantibody at the respective C terminus. In all formats, serum stability and full binding reactivity of the scFv 4D5 were retained. Functional affinity, however, did change. An avidity increase was achieved by multimerization, whereas PEGylation resulted in a 5-fold decreased affinity. Nevertheless, the PEGylated monomer showed an 8.5-fold, and the PEGylated dimer even a 14.5-fold higher tumor accumulation than the corresponding scFv, 48 h post-injection, because of a significantly longer serum half-life. In comparison, the non-PEGylated bivalent and tetravalent miniantibodies showed only a moderate increase in tumor localization compared with the scFv, which correlated with the degree of multimerization. However, these non-PEGylated formats resulted in higher tumor-to-blood ratios. Both multimerization and PEGylation represent thus useful strategies to tailor the pharmacokinetic properties of therapeutic antibodies and their combined use can additively improve tumor targeting.

Expansion on specific substrates regulates the phenotype and differentiation capacity of human articular chondrocytes

In this study, we investigated if monolayer expansion of adult human articular chondrocytes (AHAC) on specific substrates regulates cell phenotype and post-expansion multilineage differentiation ability. AHAC isolated from cartilage biopsies of five donors were expanded on plastic dishes (PL), on dishes coated with collagen type II (COL), or on slides coated with a ceramic material (Osteologic, OS). The phenotype of expanded chondrocytes was assessed by flow cytometry and real-time RT-PCR. Cells were then cultured in previously established conditions promoting differentiation toward the chondrogenic or osteogenic lineage. AHAC differentiation was assessed histologically, biochemically, and by real-time RT-PCR. As compared to PL-expanded AHAC, those expanded on COL did not exhibit major phenotypic changes, whereas OS-expanded cells expressed (i) higher bone sialoprotein (BSP) (22.6-fold) and lower collagen type II (9.3-fold) mRNA levels, and (ii) lower CD26, CD90 and CD140 surface protein levels (1.4-11.1-fold). Following chondrogenic differentiation, COL-expanded AHAC expressed higher mRNA levels of collagen type II (2.3-fold) and formed tissues with higher glycosaminoglycan (GAG) contents (1.7-fold), whereas OS-expanded cells expressed 16.5-fold lower collagen type II and generated pellets with 2.0-fold lower GAG contents. Following osteogenic differentiation, OS-expanded cells expressed higher levels of BSP (3.9-fold) and collagen type I (2.8-fold) mRNA. In summary, AHAC expansion on COL or OS modulated the de-differentiated cell phenotype and improved the cell differentiation capacity respectively toward the chondrogenic or osteogenic lineage. Phenotypic changes induced by AHAC expansion on specific substrates may mimic pathophysiological events occurring at different stages of osteoarthritis and may be relevant for the engineering of osteochondral tissues.

Identification of markers to characterize and sort human articular chondrocytes with enhanced in vitro chondrogenic capacity

OBJECTIVE: To identify markers associated with the chondrogenic capacity of expanded human articular chondrocytes and to use these markers for sorting of more highly chondrogenic subpopulations. METHODS: The chondrogenic capacity of chondrocyte populations derived from different donors (n = 21) or different clonal strains from the same cartilage biopsy specimen (n = 21) was defined based on the glycosaminoglycan (GAG) content of tissues generated using a pellet culture model. Selected cell populations were analyzed by microarray and flow cytometry. In some experiments, cells were sorted using antibodies against molecules found to be associated with differential chondrogenic capacity and again assessed in pellet cultures. RESULTS: Significance Analysis of Microarrays indicated that chondrocytes with low chondrogenic capacity expressed higher levels of insulin-like growth factor 1 and of catabolic genes (e.g., matrix metalloproteinase 2, aggrecanase 2), while chondrocytes with high chondrogenic capacity expressed higher levels of genes involved in cell-cell or cell-matrix interactions (e.g., CD49c, CD49f). Flow cytometry analysis showed that CD44, CD151, and CD49c were expressed at significantly higher levels in chondrocytes with higher chondrogenic capacity. Flow cytometry analysis of clonal chondrocyte strains indicated that CD44 and CD151 could also identify more chondrogenic clones. Chondrocytes sorted for brighter CD49c or CD44 signal expression produced tissues with higher levels of GAG per DNA (up to 1.4-fold) and type II collagen messenger RNA (up to 3.4-fold) than did unsorted cells. CONCLUSION: We identified markers that allow characterization of the capacity of monolayer-expanded chondrocytes to form in vitro cartilaginous tissue and enable enrichment for subpopulations with higher chondrogenic capacity. These markers might be used as a means to predict and possibly improve the outcome of cell-based cartilage repair techniques.

Gene and protein expression of galectin-3 and galectin-9 in experimental pneumococcal meningitis

Inflammation of the subarachnoid and ventricular space contributes to the development of brain damage i.e. cortical necrosis and hippocampal apoptosis in pneumococcal meningitis (PM). Galectin-3 and -9 are known pro-inflammatory mediators and regulators of apoptosis. Here, the gene and protein expression profile for both galectins was assessed in the disease progression of PM. The mRNA of Lgals3 and Lgals9 increased continuously in the cortex and in the hippocampus from 22 h to 44 h after infection. At 44 h after infection, mRNA levels of Lgals9 in the hippocampus were 7-fold and those of Lgals3 were 30-fold higher than in uninfected controls (P<0.01). Galectin-9 protein did not change, but galectin-3 significantly increased in cortex and hippocampus with the duration of PM. Galectin-3 was localized to polymorphonuclear neutrophils, microglia, monocytes and macrophages, suggesting an involvement of galectin-3 in the neuroinflammatory processes leading to brain damage in PM.