Extension of the crRNA enhances Cpf1 gene editing in vitro and in vivo.

Engineering of the Cpf1 crRNA has the potential to enhance its gene editing efficiency and non-viral delivery to cells. Here, we demonstrate that extending the length of its crRNA at the 5 end can enhance the gene editing efficiency of Cpf1 both in cells and in vivo. Extending the 5 end of the crRNA enhances the gene editing efficiency of the Cpf1 RNP to induce non-homologous end-joining and homology-directed repair using electroporation in cells. Additionally, chemical modifications on the extended 5 end of the crRNA result in enhanced serum stability. Also, extending the 5 end of the crRNA by 59 nucleotides increases the delivery efficiency of Cpf1 RNP in cells and in vivo cationic delivery vehicles including polymer nanoparticle. Thus, 5 extension and chemical modification of the Cpf1 crRNA is an effective method for enhancing the gene editing efficiency of Cpf1 and its delivery in vivo

Structural basis for RNA recognition and activation by human innate immune receptor RIG-1

Innate immunity provides the first line of host defense against pathogenic microbial and viral invasion. Activation of innate immune responses relies on the specific recognition of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs). Retinoic acid Inducible Gene- I (RIG-I) is a crucial PPR in the cytoplasm that induces antiviral and inflammatory immune responses against RNA viruses by selectively detecting PAMP RNAs. The RIG-I signaling pathway is highly regulated. Aberrant signaling can lead to apoptosis and altered cell differentiation, which have been implicated in the development of inflammation, autoimmune diseases including type 1 diabetes, and cancer. We have collaborated with Dr. Michael Gale at University of Washington School of Medicine to identify the poly-uridine motif of the Hepatitis C virus (HCV) genome 3′ non-translated region and its replication intermediate as a PAMP substrate of RIG-I (Saito et al., 2008). To this end, I have developed efficient expression and purification methods for human RIG-I, and characterized the protein using biochemical and biophysical methods. Highly purified RIG-I protein was then used to verify HCV PAMP RNA in vitro by gel shift assay and limited proteolysis. RIG-I consists of two N-terminal caspase recruitment domains (CARDs), a central DExD/H box RNA helicase/ATPase domain, and a C- terminal repressor domain (RD). To understand how the RIG-I helicase binds RNA and leads to activation, I have determined the crystal structure of the human RIG-I helicase-RD domain bound to dsRNA and ADP•BeF3 in collaboration with Dr. Smita Patel’s group at UMDNJ. The structure of ternary complex reveals a major contribution from the helicase domain to RNA binding and a synergy between the helicase and RD in recognition of blunt-ended dsRNA (Jiang et al., 2011). Furthermore, I have determined the crystal structures of RIG-I bound to panhandle-like short hairpin RNAs in the presence or absence of 5’-triphosphorylated modification, and chimeric RNA-DNA duplex at high resolution. These recent structures provide further insights into the molecular mechanics of RNA recognition and RIG-I activation upon viral infection.Ph. D.Includes bibliographical referencesIncludes vitaby Fuguo Jian

The structural biology of CRISPR-Cas systems.

Prokaryotic CRISPR-Cas genomic loci encode RNA-mediated adaptive immune systems that bear some functional similarities with eukaryotic RNA interference. Acquired and heritable immunity against bacteriophage and plasmids begins with integration of ∼30 base pair foreign DNA sequences into the host genome. CRISPR-derived transcripts assemble with CRISPR-associated (Cas) proteins to target complementary nucleic acids for degradation. Here we review recent advances in the structural biology of these targeting complexes, with a focus on structural studies of the multisubunit Type I CRISPR RNA-guided surveillance and the Cas9 DNA endonuclease found in Type II CRISPR-Cas systems. These complexes have distinct structures that are each capable of site-specific double-stranded DNA binding and local helix unwinding

Constitutive Model Prediction and Flow Behavior Considering Strain Response in the Thermal Processing for the TA15 Titanium Alloy

To investigate the flow stress, microstructure, and usability of TA15 titanium alloy, isothermal compression was tested at 1073–1223 K and strain rates of 10, 1, 0.1, 0.01, and 0.001 s−1, and strain of 0.9. The impact of strain and temperature on thermal deformation was investigated through the exponent-type Zener–Hollomon equation. Based on the influence of various material constants (including α, n, Q, and lnA) on the TA15 titanium alloy, the strain effect was included in the constitutive equation considering strain compensation, which is presented in this paper. The validity of the proposed constitutive equation was verified through the correlation coefficient (R) and the average absolute relative error (AARE), the values of which were 0.9929% and 6.85%, respectively. Research results demonstrated that the strain-based constitutive equation realizes consistency between the calculated flow stress and the measured stress of TA15 titanium alloy at high temperatures

Corepressors SsnF and RcoA Regulate Development and Aflatoxin B₁ Biosynthesis in <i>Aspergillus flavus</i> NRRL 3357

Aspergillus flavus is a saprophytic fungus that can be found across the entire world. It can produce aflatoxin B1 (AFB1), which threatens human health. CreA, as the central factor in carbon catabolite repression (CCR), regulates carbon catabolism and AFB1 biosynthesis in A. flavus. Additionally, SsnF-RcoA are recognized as the corepressors of CreA in CCR. In this study, ssnF and rcoA not only regulated the expressions of CCR factors and hydrolase genes, but also positively affected mycelia growth, conidia production, sclerotia formation, and osmotic stress response in A. flavus. More importantly, SsnF and RcoA were identified as positive regulators for AFB1 biosynthesis, as they modulate the AF cluster genes and the relevant regulators at a transcriptional level. Additionally, the interactions of SsnF-CreA and RcoA-CreA were strong and moderate, respectively. However, the interaction of SsnF and RcoA was weak. The interaction models of CreA-SsnF, CreA-RcoA, and SsnF-RcoA were also simulated with a docking analysis. All things considered, SsnF and RcoA are not just the critical regulators of the CCR pathway, but the global regulators involving in morphological development and AFB1 biosynthesis in A. flavus

Low-Profile Directional Ultra-Wideband Antenna for See-Through-Wall Imaging Applications

A compact-size planar antenna with ultra-wideband (UWB) bandwidth and directional patterns is presented. The antenna can be fabricated on a printed circuit board (PCB). On one side of the PCB, it has a circular patch, and on the other side it has a slot-embedded ground plane with a fork-shaped feeding stub in the slot. Directional radiation is achieved by using a reflector below the antenna. To reduce the thickness of the antenna, a new low-profile antenna configuration is proposed. Three types of directional UWB antennas are analyzed. The distance between the antenna and the reflector is 12mm (0:16 lambda0,lambda0 is the free space wavelength at the lowest frequency). In order to validate the design, a prototype is also fabricated and measured. Measured results agree well with the simulated ones. The measured results confirm that the proposed antenna features a reflection coe±cient below -10 dB over the UWB range from 4.2 GHz to 8.5 GHz, a maximum gain around 9 dBi, a front-to-back ratio over 17 dB and pulse fidelity higher than 90% in the time domain. Thus it is promising for see-through-wall imaging applications