The effect of the elongation of the proximal aorta on the estimation of the aortic wall distensibility

The compliance of the proximal aortic wall is a major determinant of cardiac afterload. Aortic compliance is often estimated based on cross-sectional area changes over the pulse pressure, under the assumption of a negligible longitudinal stretch during the pulse. However, the proximal aorta is subjected to significant axial stretch during cardiac contraction. In the present study, we sought to evaluate the importance of axial stretch on compliance estimation by undertaking both an in silico and an in vivo approach. In the computational analysis, we developed a 3-D finite element model of the proximal aorta and investigated the discrepancy between the actual wall compliance to the value estimated after neglecting the longitudinal stretch of the aorta. A parameter sensitivity analysis was further conducted to show how increased material stiffness and increased aortic root motion might amplify the estimation errors (discrepancies between actual and estimated distensibility ranging from − 20 to − 62%). Axial and circumferential aortic deformation during ventricular contraction was also evaluated in vivo based on MR images of the aorta of 3 healthy young volunteers. The in vivo results were in good qualitative agreement with the computational analysis (underestimation errors ranging from − 26 to − 44%, with increased errors reflecting higher aortic root displacement). Both the in silico and in vivo findings suggest that neglecting the longitudinal strain during contraction might lead to severe underestimation of local aortic compliance, particularly in the case of women who tend to have higher aortic root motion or in subjects with stiff aortas.

     

The ERdj5-Sel1L complex facilitates cholera toxin retrotranslocation

Cholera toxin (CT) traffics from the host cell surface to the endoplasmic reticulum (ER), where the toxin''s catalytic CTA1 subunit retrotranslocates to the cytosol to induce toxicity. In the ER, CT is captured by the E3 ubiquitin ligase Hrd1 via an undefined mechanism to prepare for retrotranslocation. Using loss-of-function and gain-of-function approaches, we demonstrate that the ER-resident factor ERdj5 promotes CTA1 retrotranslocation, in part, via its J domain. This Hsp70 cochaperone regulates binding between CTA and the ER Hsp70 BiP, a chaperone previously implicated in toxin retrotranslocation. Importantly, ERdj5 interacts with the Hrd1 adaptor Sel1L directly through Sel1L''s N-terminal lumenal domain, thereby linking ERdj5 to the Hrd1 complex. Sel1L itself also binds CTA and facilitates toxin retrotranslocation. By contrast, EDEM1 and OS-9, two established Sel1L binding partners, do not play significant roles in CTA1 retrotranslocation. Our results thus identify two ER factors that promote ER-to-cytosol transport of CTA1. They also indicate that ERdj5, by binding to Sel1L, triggers BiP–toxin interaction proximal to the Hrd1 complex. We postulate this scenario enables the Hrd1-associated retrotranslocation machinery to capture the toxin efficiently once the toxin is released from BiP.     

NuMA localization,stability, and function in spindle orientation involve 4.1 and Cdk1 interactions

The epidermis is a multilayered epithelium that requires asymmetric divisions for stratification. A conserved cortical protein complex, including LGN, nuclear mitotic apparatus (NuMA), and dynein/dynactin, plays a key role in establishing proper spindle orientation during asymmetric divisions. The requirements for the cortical recruitment of these proteins, however, remain unclear. In this work, we show that NuMA is required to recruit dynactin to the cell cortex of keratinocytes. NuMA''s cortical recruitment requires LGN; however, LGN interactions are not sufficient for this localization. Using fluorescence recovery after photobleaching, we find that the 4.1-binding domain of NuMA is important for stabilizing its interaction with the cell cortex. This is functionally important, as loss of 4.1/NuMA interaction results in spindle orientation defects, using two distinct assays. Furthermore, we observe an increase in cortical NuMA localization as cells enter anaphase. Inhibition of Cdk1 or mutation of a single residue in NuMA mimics this effect. NuMA''s anaphase localization is independent of LGN and 4.1 interactions, revealing two distinct mechanisms responsible for NuMA cortical recruitment at different stages of mitosis. This work highlights the complexity of NuMA localization and reveals the importance of NuMA cortical stability for productive force generation during spindle orientation.     

New resource for the computation of cartilage biphasic material properties with the interpolant response surface method

Cartilage material properties are important for understanding joint function and diseases, but can be challenging to obtain. Three biphasic material properties (aggregate modulus, Poisson's ratio and permeability) can be determined using an analytical or finite element model combined with optimisation to find the material properties values that best reproduce an experimental creep curve. The purpose of this study was to develop an easy-to-use resource to determine biphasic cartilage material properties. A Cartilage Interpolant Response Surface was generated from interpolation of finite element simulations of creep indentation tests. Creep indentation tests were performed on five sites across a tibial plateau. A least-squares residual search of the Cartilage Interpolant Response Surface resulted in a best-fit curve for each experimental condition with corresponding material properties. These sites provided a representative range of aggregate moduli (0.48–1.58 MPa), Poisson's ratio (0.00–0.05) and permeability (1.7 × 10^? 15–5.4 × 10^? 15 m⁴/N s) values found in human cartilage. The resource is freely available from https://simtk.org/home/va-squish.     

SNP Detection for Cytochrome P450 Alleles by Target-assembled Tandem Oligonucleotide Systems Based on Exciplexes

Abstract

We report the first use of exciplex-based split-probes for detection of the wild type and *3 mutant alleles of human cytochrome P450 2C9. A tandem 8-mer split DNA oligonucleotide probe system was designed that allows detection of the complementary target DNA sequence. This exciplex-based fluorescence detector system operates by means of a contiguous hybridization of two oligonucleotide exciplex split-probes to a complementary target nucleic acid target. Each probe oligonucleotide is chemically modified at one of its termini by a potential exciplex-forming partner, each of which is fluorescently silent at the wavelength of detection. Under conditions that ensure correct three-dimensional assembly, the chemical moieties on suitable photoexcitation form an exciplex that fluoresces with a large Stokes shift (in this case 130 nm). Preliminary proof-of-concept studies used two 8-mer probe oligonucleotides, but in order to give better specificity for genomic applications, probe length was extended to give coverage of 24 bases. Eight pairs of tandem 12-mer oligonucleotide probes spanning the 2C9*3 region were designed and tested to find the best set of probes. Target sequences tested were in the form of (i) synthetic oligonucleotides, (ii) embedded in short PCR products (150 bp), or (iii) inserted into plasmid DNA (～ 3 Kbp). The exciplex system was able to differentiate wild type and human cytochrome P450 2C9 *3 SNP (1075 A→C) alleles, based on fluorescence emission spectra and DNA melting curves, indicating promise for future applications in genetic testing and molecular diagnostics.     

Highly Sensitive Real-Time In Vivo Imaging of an Influenza Reporter Virus Reveals Dynamics of Replication and Spread

The continual public health threat posed by the emergence of novel influenza viruses necessitates the ability to rapidly monitor infection and spread in experimental systems. To analyze real-time infection dynamics, we have created a replication-competent influenza reporter virus suitable for in vivo imaging. The reporter virus encodes the small and bright NanoLuc luciferase whose activity serves as an extremely sensitive readout of viral infection. This virus stably maintains the reporter construct and replicates in culture and in mice with near-native properties. Bioluminescent imaging of the reporter virus permits serial observations of viral load and dissemination in infected animals, even following clearance of a sublethal challenge. We further show that the reporter virus recapitulates known restrictions due to host range and antiviral treatment, suggesting that this technology can be applied to studying emerging influenza viruses and the impact of antiviral interventions on infections in vivo. These results describe a generalizable method to quickly determine the replication and pathogenicity potential of diverse influenza strains in animals.