Prediction of apparent trabecular bone stiffness through fourth-order fabric tensors

The apparent stiffness tensor is an important mechanical parameter for characterizing trabecular bone. Previous studies have modeled this parameter as a function of mechanical properties of the tissue, bone density, and a second-order fabric tensor, which encodes both anisotropy and orientation of trabecular bone. Although these models yield strong correlations between observed and predicted stiffness tensors, there is still space for reducing accuracy errors. In this paper, we propose a model that uses fourth-order instead of second-order fabric tensors. First, the totally symmetric part of the stiffness tensor is assumed proportional to the fourth-order fabric tensor in the logarithmic scale. Second, the asymmetric part of the stiffness tensor is derived from relationships among components of the harmonic tensor decomposition of the stiffness tensor. The mean intercept length (MIL), generalized MIL (GMIL), and fourth-order global structure tensor were computed from images acquired through microcomputed tomography of 264 specimens of the femur. The predicted tensors were compared to the stiffness tensors computed by using the micro-finite element method (\(\upmu \)FE), which was considered as the gold standard, yielding strong correlations (\(R^2\) above 0.962). The GMIL tensor yielded the best results among the tested fabric tensors. The Frobenius error, geodesic error, and the error of the norm were reduced by applying the proposed model by 3.75, 0.07, and 3.16 %, respectively, compared to the model by Zysset and Curnier (Mech Mater 21(4):243–250, 1995) with the second-order MIL tensor. From the results, fourth-order fabric tensors are a good alternative to the more expensive \(\upmu \)FE stiffness predictions.     

Experimental evolution

Experimental evolution is the study of evolutionary processes occurring in experimental populations in response to conditions imposed by the experimenter. This research approach is increasingly used to study adaptation, estimate evolutionary parameters, and test diverse evolutionary hypotheses. Long applied in vaccine development, experimental evolution also finds new applications in biotechnology. Recent technological developments provide a path towards detailed understanding of the genomic and molecular basis of experimental evolutionary change, while new findings raise new questions that can be addressed with this approach. However, experimental evolution has important limitations, and the interpretation of results is subject to caveats resulting from small population sizes, limited timescales, the simplified nature of laboratory environments, and, in some cases, the potential to misinterpret the selective forces and other processes at work.     

Human Rhinovirus 14 Enters Rhabdomyosarcoma Cells Expressing ICAM-1 by a Clathrin-, Caveolin-, and Flotillin-Independent Pathway

Intercellular adhesion molecule 1 (ICAM-1) mediates binding and entry of major group human rhinoviruses (HRVs). Whereas the entry pathway of minor group HRVs has been studied in detail and is comparatively well understood, the pathway taken by major group HRVs is largely unknown. Use of immunofluorescence microscopy, colocalization with specific endocytic markers, dominant negative mutants, and pharmacological inhibitors allowed us to demonstrate that the major group virus HRV14 enters rhabdomyosarcoma cells transfected to express human ICAM-1 in a clathrin-, caveolin-, and flotillin-independent manner. Electron microscopy revealed that many virions accumulated in long tubular structures, easily distinguishable from clathrin-coated pits and caveolae. Virus entry was strongly sensitive to the Na⁺/H⁺ ion exchange inhibitor amiloride and moderately sensitive to cytochalasin D. Thus, cellular uptake of HRV14 occurs via a pathway exhibiting some, but not all, characteristics of macropinocytosis and is similar to that recently described for adenovirus 3 entry via α_v integrin/CD46 in HeLa cells.Human rhinoviruses (HRVs), members of the family Picornaviridae that represent a major cause of the common cold, essentially utilize two different receptor types for host cell attachment. The 12 minor group HRVs, exemplified by HRV2, bind low-density lipoprotein receptor (LDLR), LDLR-related protein (LRP) (20), and very-LDLR (VLDLR) (29) and are internalized via the well-characterized clathrin-dependent endocytic pathway (44); however, these ligands, like others, can switch to different entry portals when the clathrin-dependent pathway is blocked (4). Once the virus arrives in endosomal carrier vesicles or late endosomes, uncoating (i.e., the release of the viral RNA genome) is triggered by the acidic pH (35, 39).The 87 major group HRVs, exemplified by HRV14, bind intercellular adhesion molecule-1 (ICAM-1). Following entry, uncoating is triggered by ICAM-1 itself (3), but the low endosomal pH facilitates this process (37). Based on inhibition of infection by the dominant negative (DN) dynamin-2 mutant dyn^K44A, it was proposed that HRV14 also follows a clathrin-dependent pathway in HeLa-H1 cells (9). However, ICAM-1 lacks a clathrin localization signal and even functions as a viral receptor when its cytoplasmic tail is replaced with a glycosylphosphatidylinositol (GPI) anchor (45). Furthermore, dynamin has also been shown to be involved in pathways other than clathrin-mediated endocytosis (CME), such as caveolae- and lipid raft-dependent entry, as a function of ligand and cell type (reviewed in references 30 and 34). Additionally, dynamin might play a role in formation and closure of circular pinocytic ruffles (31). More recently, a specific entry pathway for ICAM-1 ligands into human umbilical vein endothelial cells was identified and termed “cam-mediated endocytosis”; uptake was found to be triggered upon binding of multivalent ligands, such as immunoconjugates and immunobeads, and to occur independently from clathrin and caveolin. Inhibition by amiloride, actin depolymerization, and protein kinase C inhibitors pointed to macropinocytosis (33). So far, it is not known whether these findings are relevant to the entry pathway of HRVs via ICAM-1 as the uptake kinetics was significantly dependent on particle size. For all these reasons, involvement of clathrin in HRV14 uptake is questionable. Accordingly, we explored entry of HRV14 via ICAM-1 and compared the results with the well-characterized clathrin-dependent entry pathway of HRV2 (44). Employing pharmacological compounds, specific DN inhibitors, immunofluorescence, and electron microscopy, we demonstrate that HRV14 enters rhabdomyosarcoma ICAM-1-expressing (RD-ICAM) cells via a pathway independent of clathrin, caveolin, and flotillin.     

Interaction of alpha-and beta-oligoarginine-acids and amides with anionic lipid vesicles: a mechanistic and thermodynamic study

The interaction of alpha- and beta-oligoarginine amides and acids and of alpha-polyarginine with anionic lipid vesicles was studied. The beta-oligoarginines used were beta3-homologues of the alpha-oligoarginines. Lipid bilayers were composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]) containing 5 mol % pyrene-PG (1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-[phospho-rac-1-glycerol]). Kinetic analysis of the binding process onto large unilamellar POPC/POPG (3:7, molar ratio) vesicles (100 nm diameter) shows biphasic time courses for all tested peptides. The first binding step is fast and takes place within approximately 10 s with no disruption of the membrane as indicated by corresponding calcein release measurements. The second binding phase is slow and occurs within the next 30-300 s with substantial membrane disruption. In this context, beta-hexa- and octaarginine amides possess higher second half-times than the beta-hexa- and octaarginine acids of the same chain length. Furthermore beta-octaarginine amide induces a calcein release approximately twice as large as that of the beta-octaarginine acid. Thermodynamic analysis of the binding process, using the complex formation model that assumes that each peptide binds independently to n POPG lipids, reveals apparent binding constants (K(app1)) of approximately 5 x 10(6)-10(8) M(-1) and n-values from 3.7 for beta-hexaarginine acid up to 24.8 for alpha-polyarginine. Although the K(app1)-values are similar, the number of binding sites clearly depends on the chemical nature of the oligoarginine: beta-oligoarginine amides and alpha-oligoarginine acids interact with more lipids than beta-oligoarginine acids of the same length. Calculation of the electrostatic contribution to the total free energy of binding reveals that for all oligoarginines only 25-30% has electrostatic origin. The remaining approximately 70-75% is nonelectrostatic, corresponding to hydrogen bonding and/or hydrophobic interactions. From the obtained data, a mechanism is suggested by which oligoarginines interact with anionic vesicles: (1) initial electrostatic interaction that is fast, nonspecific, and relatively weak; (2) nonelectrostatic interaction that is rate-limiting, stronger, and induces bilayer rigidification as well as release of aqueous contents from the vesicles.     

Effect of prostaglandin E2 and tumor necrosis factor alpha on the VEGF-receptor system expression in cultured porcine luteal cells

The purpose of the present study was to investigate the effects of prostaglandin (PG) E(2) and tumor necrosis factor (TNF) alpha on expression of vascular endothelial growth factor (VEGF) and its receptors, fms-like tyrosine kinase (Flt-1) and fetal liver kinase-1/kinase insert domain-containing receptor (Flk-1/KDR), in cultured porcine luteal cells. Real-time PCR was used for quantification of VEGF and its receptors mRNA, whereas VEGF release by luteal cells was determined by radioimmunoassay (RIA). Only the highest dose of PGE(2) (1 microM) after 6 hr of incubation stimulated VEGF release by luteal cells collected in the mid-luteal phase (P < 0.05). Moreover, PGE(2) (100 nM, 1 microM) significantly stimulated VEGF secretion by luteal cells in the late phase and during pregnancy on Days 10-12 (P < 0.05). Elevated mRNA expression of both VEGF 120 and VEGF 164 isoforms was found in luteal cells cultured with PGE(2). The lack of an effect of PGE(2) on VEGF receptors mRNA expression was observed. TNFalpha was able to significantly stimulate VEGF release from cells obtained in the mid- and late luteal phase or during early pregnancy. All tested doses enhanced mRNA levels of VEGF 120 isoform, but not VEGF 164. Additionally, TNFalpha was able to decrease Flk-1/KDR mRNA expression, whereas Flt-1 mRNA levels were not affected. These results indicated that PGE(2) and TNFalpha influenced VEGF ligand-receptor system expression in porcine luteal cells and may therefore play an important role in regulation of luteal functions during the estrous cycle and pregnancy in pigs.     

Morphological, physiological and behavioural evaluation of a ‘Mice in Space’ housing system

Environmental conditions likely affect physiology and behaviour of mice used for life sciences research on Earth or in Space. Here, we analysed the effects of cage confinement on the weightbearing musculoskeletal system, behaviour and stress of wild-type mice (C57BL/6JRj, 30 g b.wt., total n = 24) housed for 25 days in a prototypical ground-based and fully automated life support habitat device called “Mice in Space” (MIS). Compared with control housing (individually ventilated cages) the MIS mice revealed no significant changes in soleus muscle size and myofiber distribution (type I vs. II) and quality of bone (3-D microarchitecture and mineralisation of calvaria, spine and femur) determined by confocal and micro-computed tomography. Corticosterone metabolism measured non-invasively (faeces) monitored elevated adrenocortical activity at only start of the MIS cage confinement (day 1). Behavioural tests (i.e., grip strength, rotarod, L/D box, elevated plus-maze, open field, aggressiveness) performed subsequently revealed only minor changes in motor performance (MIS vs. controls). The MIS habitat will not, on its own, produce major effects that could confound interpretation of data induced by microgravity exposure during spaceflight. Our results may be even more helpful in developing multidisciplinary protocols with adequate scenarios addressing molecular to systems levels using mice of various genetic phenotypes in many laboratories. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Aminoacyl-tRNA formation in the extreme thermophile Thermus thermophilus

Thermophilic organisms must be capable of accurate translation at temperatures in which the individual components of the translation machinery and also specific amino acids are particularly sensitive. Thermus thermophilus is a good model organism for studies of thermophilic translation because many of the components in this process have undergone structural and biochemical characterization. We have focused on the pathways of aminoacyl-tRNA synthesis for glutamine, asparagine, proline, and cysteine. We show that the T. thermophilus prolyl-tRNA synthetase (ProRS) exhibits cysteinyl-tRNA synthetase (CysRS) activity although the organism also encodes a canonical CysRS. The ProRS requires tRNA for cysteine activation, as is known for the characterized archaeal prolyl-cysteinyl-tRNA synthetase (ProCysRS) enzymes. The heterotrimeric T. thermophilus aspartyl-tRNA(Asn) amidotransferase can form Gln-tRNA in addition to Asn-tRNA: however, a 13-amino-acid C-terminal truncation of the holoenzyme A subunit is deficient in both activities when assayed with homologous substrates. A survey of codon usage in completed prokaryotic genomes identified a higher Glu:Gln ratio in proteins of thermophiles compared to mesophiles.