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.
Purpose
To investigate the surgical outcomes of arthroscopic pull-out repair for medial meniscus root tear (MMRT) combined with the marrow stimulation procedures (MSP) for accompanying high-grade ...cartilage lesions.
Methods
Patients who underwent arthroscopic pull-out repair for MMRT between 2010 and 2019 were retrospectively reviewed. Patients who had at least 3 years of follow-up were included and classified into two groups according to whether MSP (microfracture or microdrilling) were performed on cartilage lesions in the medial tibiofemoral joint (group 1, patients with International Cartilage Repair Society ICRS grade 0–3a lesions and did not undergo MSP; group 2, patients with ICRS grade 3b–3d lesions and underwent MSP). Comparative analyses, including non-inferiority trials, were conducted between groups for subjective and objective outcomes. In addition, group 2 was further divided into two subgroups according to cartilage lesion size and compared with group 1 (group S, ≤ 2.0 cm
2
; group L, > 2.0 cm
2
).
Results
A total of 94 patients were included (group 1, 68 patients; group 2, 26 patients). There were no significant differences in clinical scores at postoperative 3 years and final follow-up between groups 1 and 2, but group 2 failed to satisfy the non-inferiority criteria compared to group 1 overall. In objective outcomes, group 2 did not meet the non-inferiority criteria for the rate of osteoarthritis progression compared to group 1, and it also showed a significantly higher proportion of high-grade osteoarthritis at final follow-up (
P
= 0.044) and a higher degree of osteoarthritis progression than group 1 (
P
= 0.03 for pre- to postoperative 3 years, and
P
= 0.006 for pre- to final follow-up). In additional evaluations comparing the subgroups of group 2 and group 1, group S showed relatively favourable results compared to group L in objective outcomes at final follow-up.
Conclusion
Patients who underwent arthroscopic pull-out repair for MMRT combined with MSP for accompanying high-grade cartilage lesions showed suboptimal outcomes compared to those with no or low-grade lesions at mid-term follow-up. High-grade cartilage lesions ≤ 2.0 cm
2
may be candidates for the surgical repair of MMRT if MSP are performed, but those with larger lesions may require alternative treatment strategies.
Level of evidence
III.
With the advance in user-friendly and powerful video editing tools, anyone can easily manipulate videos without leaving prominent visual traces. Frame-rate up-conversion (FRUC), a representative ...temporal-domain operation, increases the motion continuity of videos with a lower frame-rate and is used by malicious counterfeiters in video tampering such as generating fake frame-rate video without improving the quality or mixing temporally spliced videos. FRUC is based on frame interpolation schemes and subtle artifacts that remain in interpolated frames are often difficult to distinguish. Hence, detecting such forgery traces is a critical issue in video forensics. This paper proposes a frame-rate conversion detection network (FCDNet) that learns forensic features caused by FRUC in an end-to-end fashion. The proposed network uses a stack of consecutive frames as the input and effectively learns interpolation artifacts using network blocks to learn spatiotemporal features. Moreover, it can cover the following three types of frame interpolation schemes: nearest neighbor interpolation, bilinear interpolation, and motion-compensated interpolation. In contrast to existing methods that exploit all frames to verify integrity, the proposed approach achieves a high detection speed because it observes only six frames to test its authenticity. Extensive experiments were conducted with conventional forensic methods and neural networks for video forensics to validate our research. The proposed work achieved an outstanding performance in terms of detecting the interpolated artifacts of FRUC. The experimental results also demonstrate that our model is robust against an unseen dataset, unlearned frame-rate, and unlearned quality factor. Furthermore, FCDNet can precisely localize the tampered region applied to manipulation along the time-domain through temporal localization.
Understanding the dynamic behavior of water transport in a compressed gas diffusion layer (GDL) is essential for enhancing water management strategies and designing improved gas channels in polymer ...electrolyte membrane fuel cells. The effects of rib structure and compression are studied using the multiphase lattice Boltzmann method. The water invasion patterns and saturation profiles determine the water behavior characteristics under different compression conditions. The results indicate that the rib structure has a critical influence on water transport in GDL and the breakthrough point. The water distribution and saturation profile results indicate that the region under the rib has a higher resistance force as the compression ratio increases. This phenomenon contributes to the appearance of the water flow path under the channel. Regarding the rib edge effect, the morphological considering of the initiate breakup of carbon fiber more dominantly affects the water distribution in a lower compression ratio.
RATIONALE:The Notch pathway stabilizes sprouting angiogenesis by favoring stalk cells over tip cells at the vascular front. Because tip and stalk cells have different properties in morphology and ...function, their transcriptional regulation remains to be distinguished. Transcription factor Sox17 is specifically expressed in endothelial cells, but its expression and role at the vascular front remain largely unknown.
OBJECTIVE:To specify the role of Sox17 and its relationship with the Notch pathway in sprouting angiogenesis.
METHODS AND RESULTS:Endothelial-specific Sox17 deletion reduces sprouting angiogenesis in mouse embryonic and postnatal vascular development, whereas Sox17 overexpression increases it. Sox17 promotes endothelial migration by destabilizing endothelial junctions and rearranging cytoskeletal structure and upregulates expression of several genes preferentially expressed in tip cells. Interestingly, Sox17 expression is suppressed in stalk cells in which Notch signaling is relatively high. Notch activation by overexpressing Notch intracellular domain reduces Sox17 expression both in primary endothelial cells and in retinal angiogenesis, whereas Notch inhibition by delta-like ligand 4 (Dll4) blockade increases it. The Notch pathway regulates Sox17 expression mainly at the post-transcriptional level. Furthermore, endothelial Sox17 ablation rescues vascular network from excessive tip cell formation and hyperbranching under Notch inhibition in developmental and tumor angiogenesis.
CONCLUSIONS:Our findings demonstrate that the Notch pathway restricts sprouting angiogenesis by reducing the expression of proangiogenic regulator Sox17.
In this study, the lattice Boltzmann method was used to simulate the three-dimensional intrusion process of liquid water in the gas diffusion layer (GDL) of a polymer electrolyte membrane fuel cell ...(PEMFC). The GDL was reconstructed by the stochastic method and used to investigate fiber orientation's influence on liquid water transport in the GDL of a PEMFC. The fiber orientation can be described by the angle between a single fiber and the in-plane direction; three different samples were simulated for three different fiber orientation ranges. The simulated permeability correlated well with the anisotropic characteristics of reconstructed carbon papers. It was concluded that the fiber orientation had a significant effect on the liquid invasion pattern in the GDL by changing the pore shape and distribution of the GDL. The results indicated that the stochastically reconstructed GDL, taking into account the fiber orientation, better demonstrates the mass transport properties of the GDL.
•The LBM was used to simulate dynamic liquid water transport in a PEMFC.•The reconstructed 3D GDLs were generated by using the stochastic generation method.•Permeability of through- and in-plane was estimated and well represented the anisotropic characteristic of the GDL.•The fiber orientation affected the pore structure and significantly influenced the capillary-driven transport in the GDL.
The epidermal lamellar bodies (LBs) are specialized organelles that contain pro-barrier lipids imparting a fully lamellar internal structure, but also other cargoes such as enzymes (lipid ...metabolizing and proteolytic), enzyme inhibitors, and antimicrobial peptides. Thus, the LB secretory system, by virtue of delivering these cargoes to the stratum corneum (SC) interstices, is essential for forming the various skin barriers located in the SC. Ultrastructural studies have suggested that the morphologic features of LBs reflect the functional status of the SC. Several ichthyotic skin diseases as well as experimental animal models with defective epidermal lipogenesis show only partial lamellar contents or even empty appearing LB, reflecting an abnormal cargo composition. We suggest that LB polymorphism reflects a wide array of barrier adaptations to environmental challenges, rather than just a defective barrier function, based on observations on a) LB morphology in inherited skin disorders of lipid metabolism (Refsum disease, Chanarin-Dorfman syndrome) characterized by deficiency of lamellar lipids and accumulation of toxic metabolites; b) Psoriasis (with a high expression of Psoriasin antimicrobial peptide within lesions) and c) the Pitohui, a toxic bird where diet-derived toxin is eliminated via the LB secretory system that creates a chemical defense system. Morphological features of LBs from these models suggest a hitherto unrecognized function for the LBs in elimination of toxic substances from the body. We also provide preliminary evidence that indicate yet another function for the LBs- as a type of recycling endosomes allowing for uptake of certain topically applied materials by the epidermis.
The notion of complex energy landscape underpins the intriguing dynamical behaviors in many complex systems ranging from polymers, to brain activity, to social networks and glass transitions. The ...spin glass state found in dilute magnetic alloys has been an exceptionally convenient laboratory frame for studying complex dynamics resulting from a hierarchical energy landscape with rugged funnels. Here, we show, by a bulk susceptibility and Monte Carlo simulation study, that densely populated frustrated magnets in a spin jam state exhibit much weaker memory effects than spin glasses, and the characteristic properties can be reproduced by a nonhierarchical landscape with a wide and nearly flat but rough bottom. Our results illustrate that the memory effects can be used to probe different slow dynamics of glassy materials, hence opening a window to explore their distinct energy landscapes.
Abstract Introduction AMP-activated protein kinase (AMPK) has been reported to stimulate differentiation and proliferation of osteoblasts, but the role of AMPK in the physiology of osteoclasts has ...not been investigated. Method Osteoclasts were differentiated from mouse BMMϕs. TRAP-positive multinucleated cells were considered to be osteoclasts using TRAP staining, and resorption area was determined by incubation of cells on dentine discs. Signaling pathways were investigated using Western blotting and RT-PCR. Results RANKL induced phosphorylation/activation of AMPK-α in BMMϕs and stimulated formation of TRAP-positive multinucleated cells. Pharmacological inhibition of AMPK with compound C and siRNA-mediated knockdown of AMPK-α1, the predominant α-subunit isoform in BMMϕs, increased RANKL-induced formation of TRAP-positive multinucleated cells and bone resorption via activation of the downstream signaling elements p38, JNK, NF-κB, Akt, CREB, c-Fos, and NFATc1. STO-609, an inhibitor of CaMKK, completely blocked the RANKL-induced activation of AMPK-α, but KN-93, an inhibitor of CaMK, did not. siRNA-mediated TAK1 knockdown also blocked RANKL-induced activation of AMPK-α. The AMPK activators metformin, (−)-epigallocatechin-3-gallate, berberine, resveratrol, and α-lipoic acid dose-dependently suppressed formation of TRAP-positive multinucleated cells and bone resorption. Conclusion AMPK negatively regulates RANKL, possibly by acting through CaMKK and TAK1. Thus, the development of AMPK activators may be a useful strategy for inhibiting the resorption of bone that is stimulated under RANKL-activated conditions.
Despite the administration of multiple drugs that are highly effective
, tuberculosis (TB) treatment requires prolonged drug administration and is confounded by the emergence of drug-resistant ...strains. To understand the mechanisms that limit antibiotic efficacy, we performed a comprehensive genetic study to identify
genes that alter the rate of bacterial clearance in drug-treated mice. Several functionally distinct bacterial genes were found to alter bacterial clearance, and prominent among these was the
gene that encodes the glycerol-3-kinase enzyme that is necessary for glycerol catabolism. Growth on glycerol generally increased the sensitivity of
to antibiotics
, and
-deficient bacteria persisted during antibiotic treatment
, particularly during exposure to pyrazinamide-containing regimens. Frameshift mutations in a hypervariable homopolymeric region of the
gene were found to be a specific marker of multidrug resistance in clinical
isolates, and these loss-of-function alleles were also enriched in extensively drug-resistant clones. These data indicate that frequently observed variation in the
coding sequence produces a drug-tolerant phenotype that can reduce antibiotic efficacy and may contribute to the evolution of resistance.
TB control is limited in part by the length of antibiotic treatment needed to prevent recurrent disease. To probe mechanisms underlying survival under antibiotic pressure, we performed a genetic screen for
mutants with altered susceptibility to treatment using the mouse model of TB. We identified multiple genes involved in a range of functions which alter sensitivity to antibiotics. In particular, we found glycerol catabolism mutants were less susceptible to treatment and that common variation in a homopolymeric region in the
gene was associated with drug resistance in clinical isolates. These studies indicate that reversible high-frequency variation in carbon metabolic pathways can produce phenotypically drug-tolerant clones and have a role in the development of resistance.
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