Shear and hydrostatic stress regulate fetal heart valve remodeling through YAP-mediated mechanotransduction.

Clinically serious congenital heart valve defects arise from improper growth and remodeling of endocardial cushions into leaflets. Genetic mutations have been extensively studied but explain less than 20% of cases. Mechanical forces generated by beating hearts drive valve development, but how these forces collectively determine valve growth and remodeling remains incompletely understood. Here, we decouple the influence of those forces on valve size and shape, and study the role of YAP pathway in determining the size and shape. The low oscillatory shear stress promotes YAP nuclear translocation in valvular endothelial cells (VEC), while the high unidirectional shear stress restricts YAP in cytoplasm. The hydrostatic compressive stress activated YAP in valvular interstitial cells (VIC), whereas the tensile stress deactivated YAP. YAP activation by small molecules promoted VIC proliferation and increased valve size. Whereas YAP inhibition enhanced the expression of cell-cell adhesions in VEC and affected valve shape. Finally, left atrial ligation was performed in chick embryonic hearts to manipulate the shear and hydrostatic stress in vivo. The restricted flow in the left ventricle induced a globular and hypoplastic left atrioventricular (AV) valves with an inhibited YAP expression. By contrast, the right AV valves with sustained YAP expression grew and elongated normally. This study establishes a simple yet elegant mechanobiological system by which transduction of local stresses regulates valve growth and remodeling. This system guides leaflets to grow into proper sizes and shapes with the ventricular development, without the need of a genetically prescribed timing mechanism.

Effects of ultrasound on the immunoreactivity of amandin, an allergen in apricot kernels during debitterizing.

In this paper, an investigation was conducted on the effects of ultrasound time, power and temperatures on the immunoreactivity of the allergenic amandin in apricot kernels by western blotting analysis during the ultrasonically accelerated debitterizing. And its influencing mechanism on the structure of amandin was also analyzed by SDS-PAGE, circular dichroism spectrum, extrinsic fluorescence spectrum, surface hydrophobicity and zeta potential determination, respectively. The results indicate that ultrasound could significantly reduce the immunoreactivity of amandin during ultrasonically accelerated debitterizing, and the optimal ultrasound condition was 60 min, 300 W, 55 °C and 59 kHz and decreased the immunoreactivity to 15.61%, which might be attributed to the changes of the protein subunits, secondary and tertiary structure, and molecular aggregation state induced by ultrasound. In a word, ultrasound could not only accelerate debitterizing, but also significantly decrease the immunoreactivity of apricot kernels, which proved the feasibility of ultrasound in practical processing of apricot kernels.

Soft, strong, tough, and durable protein-based fiber hydrogels.

Load-bearing soft tissues normally show J-shaped stress-strain behaviors with high compliance at low strains yet high strength at high strains. They have high water content but are still tough and durable. By contrast, naturally derived hydrogels are weak and brittle. Although hydrogels prepared from synthetic polymers can be strong and tough, they do not have the desired bioactivity for emerging biomedical applications. Here, we present a thermomechanical approach to replicate the combinational properties of soft tissues in protein-based photocrosslinkable hydrogels. As a demonstration, we create a gelatin methacryloyl fiber hydrogel with soft tissue-like mechanical properties, such as low Young's modulus (0.1 to 0.3 MPa), high strength (1.1 ± 0.2 MPa), high toughness (9,100 ± 2,200 J/m3), and high fatigue resistance (2,300 ± 500 J/m2). This hydrogel also resembles the biochemical and architectural properties of native extracellular matrix, which enables a fast formation of 3D interconnected cell meshwork inside hydrogels. The fiber architecture also regulates cellular mechanoresponse and supports cell remodeling inside hydrogels. The integration of tissue-like mechanical properties and bioactivity is highly desirable for the next-generation biomaterials and could advance emerging fields such as tissue engineering and regenerative medicine.

CRISPR-typing PCR (ctPCR), a new Cas9-based DNA detection method.

This study develops a new method for detecting and typing target DNA based on Cas9 nuclease, which was named as ctPCR, representing Cas9-sgRNA- or CRISPR-typing PCR. The technique can detect and type target DNA easily, rapidly, specifically, and sensitively. This technique detects target DNA in three steps: (1) amplifying target DNA with PCR by using a pair of universal primers (PCR1); (2) treating PCR1 products with a process referred to as CAT, representing Cas9 cutting, A tailing and T adaptor ligation; (3) amplifying the CAT-treated DNA with PCR by using a pair of general-specific primers (gs-primers) (PCR2). This method was verified by detecting HPV16 and HPV18 L1 gene in 13 different high-risk human papillomavirus (HPV) subtypes. This method was also verified by detecting the L1 and E6-E7 genes of two high-risk HPVs (HPV16 and 18) in cervical carcinoma cells and many clinical samples. In this method, PCR1 was performed to determine if the detected DNA sample contained the target DNA (such as virus infection), while PCR2 was performed to discriminate which genotypic target DNA was present in the detected DNA sample (such as virus subtypes). Based on these proof-of-concept experiments, this study provides a new CRISPR/Cas9-based DNA detection and typing method.

Automated Microfluidic Instrument for Label-Free and High-Throughput Cell Separation.

Microfluidic technologies for cell separation were reported frequently in recent years. However, a compact microfluidic instrument enabling thoroughly automated cell separation is still rarely reported until today due to the difficult hybrid between the macrosized fluidic control system and the microsized microfluidic device. In this work, we propose a novel and automated microfluidic instrument to realize size-based separation of cancer cells in a label-free and high-throughput manner. Briefly, the instrument is equipped with a fully integrated microfluidic device and a set of robust fluid-driven and control units, and the instrument functions of precise fluid infusion and high-throughput cell separation are guaranteed by a flow regulatory chip and two cell separation chips which are the key components of the microfluidic device. With optimized control programs, the instrument is successfully applied to automatically sort human breast adenocarcinoma cell line MCF-7 from 5 mL of diluted human blood with a high recovery ratio of ∼85% within a rapid processing time of ∼23 min. We envision that our microfluidic instrument will be potentially useful in many biomedical applications, especially cell separation, enrichment, and concentration for the purpose of cell culture and analysis.

Detection of target DNA with a novel Cas9/sgRNAs-associated reverse PCR (CARP) technique.

This study develops a new method for detecting target DNA based on Cas9 nuclease, which was named as CARP, representing CRISPR- or Cas9/sgRNAs-associated reverse PCR. This technique detects target DNA in three steps: (1) cleaving the detected DNA sample with Cas9 in complex with a pair of sgRNAs specific to target DNA; (2) ligating the cleaved DNA with DNA ligase; (3) amplifying target DNA with PCR. In the ligation step, the Cas9-cut target DNA was ligated into intramolecular circular or intermolecular concatenated linear DNA. In the PCR step, the ligated DNA was amplified with a pair of reverse primers. The technique was verified by detecting HPV16 and HPV18 L1 genes in nine different human papillomavirus (HPV) subtypes. The technique also detected the L1 and E6-E7 genes of two high-risk HPVs, HPV16 and HPV18, in the genomic DNA of two HPV-positive cervical carcinoma cells (HeLa and SiHa), in which no L1 and E6-E7 genes were detected in the HPV-negative cervical carcinoma cell, C-33a. By performing these proof-of-concept experiments, this study provides a new CRISPR-based DNA detection and typing method. Especially, the CARP method developed by this study is ready for the clinical HPV detection, which was supported by the final clinical sample detection. Graphical abstract CRISPR-associated reverse PCR (CARP) can be used to detect and type target DNA in a simple three-step procedure, cutting, ligation, and amplification.

Development of a high-speed vacuum ultraviolet (VUV) imaging system for the Experimental Advanced Superconducting Tokamak.

A high-speed vacuum ultraviolet (VUV) imaging system for edge plasma studies is being developed on the Experimental Advanced Superconducting Tokamak (EAST). Its key optics is composed of an inverse type of Schwarzschild telescope made of a set of Mo/Si multilayer mirrors, a micro-channel plate (MCP) equipped with a P47 phosphor screen and a high-speed camera with CMOS sensors. In order to remove the contribution from low-energy photons, a Zr filter is installed in front of the MCP detector. With this optics, VUV photons with a wavelength of 13.5 nm, which mainly come from the line emission from intrinsic carbon (C vi: n = 4-2 transition) or the Ly-α line emission from injected Li iii on the EAST, can be selectively measured two-dimensionally with both high temporal and spatial resolutions. At present, this system is installed to view the plasma from the low field side in a horizontal port in the EAST. It has been operated routinely during the 2016 EAST experiment campaign, and the first result is shown in this work. To roughly evaluate the system performance, synthetic images are created. And it indicates that this system mainly measures the edge localized emissions by comparing the synthetic images and experimental data.