Vascular Smooth Muscle Cell Stiffness and Adhesion to Collagen I Modified by Vasoactive Agonists

In vascular smooth muscle cells (VSMCs) integrin-mediated adhesion to extracellular matrix (ECM) proteins play important roles in sustaining vascular tone and resistance. The main goal of this study was to determine whether VSMCs adhesion to type I collagen (COL-I) was altered in parallel with the changes in the VSMCs contractile state induced by vasoconstrictors and vasodilators. VSMCs were isolated from rat cremaster skeletal muscle arterioles and maintained in primary culture without passage. Cell adhesion and cell E-modulus were assessed using atomic force microscopy (AFM) by repetitive nano-indentation of the AFM probe on the cell surface at 0.1 Hz sampling frequency and 3200 nm Z-piezo travelling distance (approach and retraction). AFM probes were tipped with a 5 μm diameter microbead functionalized with COL-I (1mg\ml). Results showed that the vasoconstrictor angiotensin II (ANG-II; 10−6 ) significantly increased (p<0.05) VSMC E-modulus and adhesion probability to COL-I by approximately 35% and 33%, respectively. In contrast, the vasodilator adenosine (ADO; 10−4 ) significantly decreased (p<0.05) VSMC E-modulus and adhesion probability by approximately −33% and −17%, respectively. Similarly, the NO donor (PANOate, 10−6 M), a potent vasodilator, also significantly decreased (p<0.05) the VSMC E-modulus and COL-I adhesion probability by −38% and −35%, respectively. These observations support the hypothesis that integrin-mediated VSMC adhesion to the ECM protein COL-I is dynamically regulated in parallel with VSMC contractile activation. These data suggest that the signal transduction pathways modulating VSMC contractile activation and relaxation, in addition to ECM adhesion, interact during regulation of contractile state

Week 48 resistance analyses of the once-daily, single-tablet regimen darunavir/cobicistat/emtricitabine/tenofovir alafenamide (D/C/F/TAF) in adults living with HIV-1 from the Phase III Randomized AMBER and EMERALD Trials

Darunavir/cobicistat/emtricitabine/tenofovir alafenamide (D/C/F/TAF) 800/150/200/10 mg is being investigated in two Phase III trials, AMBER (NCT02431247; treatment-naive adults) and EMERALD (NCT02269917; treatment-experienced, virologically suppressed adults). Week 48 AMBER and EMERALD resistance analyses are presented. Postbaseline samples for genotyping/phenotyping were analyzed from protocol-defined virologic failures (PDVFs) with viral load (VL) >= 400 copies/mL at failure/later time points. Post hoc analyses were deep sequencing in AMBER, and HIV-1 proviral DNA from baseline samples (VL = 3 thymidine analog-associated mutations (24% not fully susceptible to tenofovir) detected at screening. All achieved VL <50 copies/mL at week 48 or prior discontinuation. D/C/F/TAF has a high genetic barrier to resistance; no darunavir, primary PI, or tenofovir RAMs were observed through 48 weeks in AMBER and EMERALD. Only one postbaseline M184I/V RAM was observed in HIV-1 of an AMBER participant. In EMERALD, baseline archived RAMs to darunavir, emtricitabine, and tenofovir in participants with prior VF did not preclude virologic response

The frequency of osteolytic bone metastasis is determined by conditions of the soil, not the number of seeds; evidence from in vivo models of breast and prostate cancer

Background While both preclinical and clinical studies suggest that the frequency of growing skeletal metastases is elevated in individuals with higher bone turnover, it is unclear whether this is a result of increased numbers of tumour cells arriving in active sites or of higher numbers of tumour cells being induced to divide by the bone micro-environment. Here we have investigated how the differences in bone turnover affect seeding of tumour cells and/or development of overt osteolytic bone metastasis using in vivo models of hormone-independent breast and prostate cancer. Methods Cohorts of 6 (young) and 16 (mature)-week old BALB/c nude mice were culled 1, 7 and 21 days after received intracardiac injection of luciferase expressing human prostate (PC3) or breast cancer (MDA-MB-231) cell lines labelled with a fluorescent cell membrane dye (Vybrant DiD). The presence of growing bone metastases was determined by bioluminescence using an in vivo imaging system (IVIS) and followed by anatomical confirmation of tumour metastatic sites post mortem, while the presence of individual fluorescently labelled tumour cells was evaluated using two-photon microscopy ex vivo. The bone remodelling activities were compared between young and mature naïve mice (both male and female) using micro-CT analysis, ELISA and bone histomorphometry. Results Both prostate and breast cancer cells generated higher numbers of overt skeletal lesions in young mice (~80%) than in mature mice (~20%). Although mature mice presented with fewer overt bone metastases, the number of tumour cells arriving/colonizing in the tibias was comparable between young and mature animals. Young naïve mice had lower bone volume but higher bone formation and resorption activities compared to mature animals. Conclusions Our studies suggest that higher frequencies of growing osteolytic skeletal metastases in these models are linked to increased bone turnover and not to the initial number of tumour cells entering the bone microenvironment

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Continuous real-time recordings of adhesion probability of VSMC to COL-I coated AFM probe following ADO or NO administration.

<p><b>(A)</b> A representative individual experiment shows the decrease in the adhesion probability after introduction of ADO in the cell bath. <b>(B)</b> Decrease in the group average adhesion probability after addition of ADO in the cell bath (n = 10). <b>(C)</b> Average adhesion probability showed a significant decrease in adhesion probability following addition of ADO. Data were summed over 1800 s and were presented as mean ± SEM (n = 10, *<i>P<0.05</i>). <b>(D)</b> NO donor dramatically reduced adhesion probability as shown in a representative individual example. <b>(E)</b> Average adhesion events decreased after treatment with NO (n = 10). <b>(F)</b> Average adhesion probability shows a significant decrease in adhesion probability after the addition of NO donor to the cell bath. Data were summed over 1800 s and were presented as mean ± SEM (n = 10, *<i>P<0.05</i>).</p

Continuous real-time VSMC E-modulus recordings following ADO or NO.

<p><b>(A)</b> VSMC E-modulus immediately reduced after addition of ADO (10⁻⁴ M) as shown in a representative example. <b>(B)</b> Group average of VSMC E-modulus after addition of ADO (n = 10) in cell bath. <b>(C)</b> Average E-modulus summed across all time points for the group of VSMCs before and after ADO (n = 10, *<i>P<0.05</i>). <b>(D)</b> A representative single cell measurement shows decrease in VSMC E-modulus following addition of NO to the cell bath. <b>(E)</b> Alteration in group average of VSMC E-modulus before and after addition of NO (n = 10). <b>(F)</b> Average E-modulus summed for all time points before and after addition of NO showed a significant decrease in VSMC E-modulus (n = 10, *<i>P<0.05</i>). Data were presented as mean ± SEM.</p

Continuous real-time recordings of E-modulus and adhesion probability of VSMC to COL-I coated AFM probe following stimulation with ANG II.

<p>The adhesion probability is presented as the number of adhesion events per AFM retraction curve. <b>(A)</b> A representative individual example shows the increase in VSMC adhesion probability after stimulation with ANG II. <b>(B)</b> Average adhesion probability before and after addition of ANG II for experimental group (n = 10). <b>(C)</b> Average adhesion probability significantly increased after ANG II treatment. Data were summed over 1800 s and were presented as mean ± SEM (n = 10, *<i>P<0.05</i>). <b>(D)</b> A representative single cell record of VSMC E-modulus shows the immediate increase in cell E-modulus after the addition of ANG II in cell bath (10⁻⁶ M). <b>(E)</b> Alteration in group average of VSMC E-modulus before and after stimulation with ANG II (n = 10). <b>(F)</b> Average E-modulus summed across all time points for the group significantly increased after addition of ANG II. Data were summed over 1800 s and were presented as mean ± SEM (n = 10, *<i>P<0.05</i>.</p

Continuous real-time recordings of E-modulus and adhesion probability of VSMC to COL-I coated AFM probe in sham control experiments.

<p><b>(A)</b> A representative individual example shows no effect on adhesion probability after addition of vehicle buffer. <b>(B)</b> No change in adhesion probability was shown in the group average adhesion probability before and after addition of vehicle buffer (n = 10). <b>(C)</b> Average adhesion probability summed across all time points for the group of VSMCs before and after addition of vehicle buffer (n = 10, <i>P>0.05</i>). <b>(D)</b> A representative single cell measurement shows no changes in E-modulus before and after addition of vehicle buffer in cell bath. <b>(E)</b> Group average VSMC E-modulus did not change before and after addition of vehicle buffer in cell bath (n = 10). <b>(F)</b> No significant difference was shown in the average E-modulus summed for all time points before and after addition of vehicle buffer (n = 10, <i>P>0.05</i>). Data were presented as mean ± SEM.</p

Representative force curve recorded by AFM.

<p><b>(A)</b> An example of an approach curve recorded by AFM (red). The blue diamond in the black square is the estimated contact point with the VSMC where the AFM cantilever is in contact with the cell plasma membrane and begins to bend. The blue line is the Hertz fitting to the approach curve. <b>(B)</b> Representative retraction force curve recorded by AFM for pre-drug period (control). <b>(C)</b> Representative retraction force curves after treatment with NO. <b>(D)</b> Representative retraction force curve after treatment with ANG II. The height between the paired red spots was used to compute the adhesion force and the rupture number was used to evaluate the adhesion probability. The 1.5 fold of the average noise (signal fluctuation) was set as the threshold of adhesion force and only the rupture force higher than threshold was considered as the real unbinding force between cell membrane and AFM cantilever. The rupture indicated by two green spots was considered as noise and omitted in adhesion probability computation (<b>C)</b>. The snaps with low gap height were not the particular characteristics of the NO or ADO treated force curve, they also appeared in control and ANG II treated force curve and were omitted as well.</p