CRISPR effectors, which comprise a CRISPR-Cas protein and a guide (g)RNA derived from the bacterial immune system, are widely used for target-specific genome editing. When the gRNA recognizes genomic ...loci with sequences that are similar to the target, deleterious mutations can occur. Off-target mutations with a frequency below 0.5% remain mostly undetected by current genome-wide off-target detection techniques. Here we report a method to effectively detect extremely small amounts of mutated DNA based on predicted off-target-specific amplification. In this study, we used various genome editors to induce intracellular genome mutations, and the CRISPR amplification method detected off-target mutations at a significantly higher rate (1.6~984 fold increase) than an existing targeted amplicon sequencing method. In the near future, CRISPR amplification in combination with genome-wide off-target detection methods will allow detection of genome editor-induced off-target mutations with high sensitivity and in a non-biased manner.
Research on a replacement of amorphous silicon for a thin-film transistor (TFT) and large area electronics has been driven by costly vacuum processed indium-gallium-zinc oxide (IGZO). Even though ...widely studied, the performances still require improvement, and a wide number of other materials have been tested. While indium-zinc oxide, IGZO, indium-zinc-tin oxide (ZTO), and ZTO have been widely investigated, gallium-doped indium oxide (IGO) has not been under highlight. Here, we report the use of simple and cost effective spin-coated IGO TFT using spin-coated AlO x gate dielectric. We achieved high mobility over 50 cm 2 /Vs and high stability. The thin films are studied by transmission electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, Raman spectra, atomic force microscopy, and field-effect measurements. Analyses reveal the strong dependence between crystallinity, mobility, and stability. All TFTs show excellent operation, with champion characteristics for the 10% Ga-doped InO x , revealing a mobility of 52.6 cm 2 /Vs, ON/OFF ratios of 10 8 , and V TH variation of <;0.1 V during 1 h of stress measurement.
Antimicrobial peptides (AMPs) are promising therapeutic agents for treating antibiotic-resistant bacterial infections. Previous studies showed that magainin 2 (isolated from African clawed fogs
) has ...antimicrobial activity against gram-positive and gram-negative bacteria. The present study was conducted to investigate the antibacterial activity of magainin 2 against
. Magainin 2 showed excellent antibacterial activity against
strains and high stability at physiological salt concentrations. This peptide was not cytotoxic towards HaCaT cells and showed no hemolytic activity. Biofilm inhibition and elimination were significantly induced in all
strains exposed to magainin 2. We confirmed the mechanism of magainin 2 on the bacterial outer and inner membranes. Collectively, these results suggest that magainin 2 is an effective antimicrobial and antibiofilm agent against
strains.
Coupling is the process that links bone resorption to bone formation in a temporally and spatially coordinated manner within the remodeling cycle. Several lines of evidence point to the critical ...roles of osteoclast-derived coupling factors in the regulation of osteoblast performance. Here, we used a fractionated secretomic approach and identified the axon-guidance molecule SLIT3 as a clastokine that stimulated osteoblast migration and proliferation by activating β-catenin. SLIT3 also inhibited bone resorption by suppressing osteoclast differentiation in an autocrine manner. Mice deficient in Slit3 or its receptor, Robo1, exhibited osteopenic phenotypes due to a decrease in bone formation and increase in bone resorption. Mice lacking Slit3 specifically in osteoclasts had low bone mass, whereas mice with either neuron-specific Slit3 deletion or osteoblast-specific Slit3 deletion had normal bone mass, thereby indicating the importance of SLIT3 as a local determinant of bone metabolism. In postmenopausal women, higher circulating SLIT3 levels were associated with increased bone mass. Notably, injection of a truncated recombinant SLIT3 markedly rescued bone loss after an ovariectomy. Thus, these results indicate that SLIT3 plays an osteoprotective role by synchronously stimulating bone formation and inhibiting bone resorption, making it a potential therapeutic target for metabolic bone diseases.
Background:Because no data were available regarding the effect of preexisting left ventricular filling pressure (LVFP) on clinical outcomes in patients with acute myocardial infarction (AMI), we ...evaluated whether preexisting high LVFP can determine outcomes of subsequent AMI events.Methods and Results:Among 399,613 subjects who underwent echocardiography for various reason from August 2004 to June 2019, 231 had experienced subsequent AMI and were stratified according to preexisting LVFP: low LVFP (E/e’ ≤14) and high LVFP (E/e’ >14). The primary outcome was cardiac death at 30 days and 1 year after AMI. Overall, 19.5% had high LVFP prior to AMI events. Preexisting high LVFP was associated with an increased risk of cardiac death at 30 days (3.8% vs. 11.6%; adjusted hazard ratio (HR) 4.56, 95% confidence interval (CI) 1.20–17.24, P=0.026) and 1 year after AMI (7.9% vs. 35.9%; adjusted HR 4.14, 95% CI 1.79–9.57, P<0.001). Preexisting E/e’ as a continuous value was significantly associated with 1-year risk of cardiac death (adjusted HR 1.08, 95% CI 1.02–1.15, P=0.007). Follow-up echocardiography showed that patients with high LVFP did not show improvement in systolic or diastolic function.Conclusions:Preexisting high LVFP was associated with poor clinical course and 1-year cardiac death after subsequent AMI, as well as no improvement in systolic or diastolic function.
To identify genetic factors influencing quantitative traits of biomedical importance, we conducted a genome-wide association study in 8,842 samples from population-based cohorts recruited in Korea. ...For height and body mass index, most variants detected overlapped those reported in European samples. For the other traits examined, replication of promising GWAS signals in 7,861 independent Korean samples identified six previously unknown loci. For pulse rate, signals reaching genome-wide significance mapped to chromosomes 1q32 (rs12731740, P = 2.9 x 10(-9)) and 6q22 (rs12110693, P = 1.6 x 10(-9)), with the latter approximately 400 kb from the coding sequence of GJA1. For systolic blood pressure, the most compelling association involved chromosome 12q21 and variants near the ATP2B1 gene (rs17249754, P = 1.3 x 10(-7)). For waist-hip ratio, variants on chromosome 12q24 (rs2074356, P = 7.8 x 10(-12)) showed convincing associations, although no regional transcript has strong biological candidacy. Finally, we identified two loci influencing bone mineral density at multiple sites. On chromosome 7q31, rs7776725 (within the FAM3C gene) was associated with bone density at the radius (P = 1.0 x 10(-11)), tibia (P = 1.6 x 10(-6)) and heel (P = 1.9 x 10(-10)). On chromosome 7p14, rs1721400 (mapping close to SFRP4, a frizzled protein gene) showed consistent associations at the same three sites (P = 2.2 x 10(-3), P = 1.4 x 10(-7) and P = 6.0 x 10(-4), respectively). This large-scale GWA analysis of well-characterized Korean population-based samples highlights previously unknown biological pathways.
The CRISPR-Cas9 system is widely used for target-specific genome engineering. CRISPR-Cas12a (Cpf1) is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer ...adjacent motif (PAM) sequences. Cas12a has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and modified the Cas12a (cr)RNA to address the off-target cleavage issue. We developed a CRISPR-Cas12a that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR-Cas12a and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based CRISPR- Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential for therapeutic applications in incurable diseases caused by genetic mutations.
In this work, a simple, high-yield biomineralization process is reported for cobalt oxide nanostructures using Gram-positive bacteria, Bacillus subtilis, as the soft templates. Rod-type cobalt oxide ...is prepared at room temperature through an electrostatic interaction between the functional surface structures of the bacteria and the cobalt ions in an aqueous solution. Additionally, porous Co3O4 hollow rods are formed through a subsequent heat treatment at 300 °C. These rods have a high surface area and exhibited an excellent electrochemical performance for rechargeable Li-ion batteries. This facile, inexpensive, and environmentally benign synthesis for transition metal oxides with unique nanostructures can be used for several practical applications, such as batteries, catalysts, sensors, and supercapacitors.
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