RNA-guided CRISPR-Cas12a endonucleases are promising tools for genome engineering. Here we demonstrate that LbCas12a variants derived from Lachnospiraceae bacterium show a broad PAM preference, recognizing certain non-canonical PAMs with high efficiency. Furthermore, we engineered LbABE8e to carry G532R and/or K595R mutations, altering its original PAM specificities; these variants exhibited superior base editing activity in human cells compared with wild-type LbABE8e at sites with non-canonical PAMs. Based on this finding, we utilized the most effective LbCas12a and LbABE8e variants to demonstrate multiplexed and mutant-allele-specific gene editing in oncogenes, made possible by the variant's recognition of non-canonical PAMs. Importantly, LbCas12a-G532R/K595R and LbABE8e-G532R/K595R with optimized crRNA arrays targeted to triple oncogenic mutations inhibited colon cancer cell proliferation. Taken together, these results demonstrate the potential of engineered LbCas12a and LbABE8e as tools for targeting sites with alternative PAMs for genome engineering and therapeutic editing in cancer cells.
Face-centered cubic (FCC) materials containing twins such as twin-induced plasticity (TWIP) steels and nano-crystalline copper and nickel have exhibited an attractive combination of properties such as strength and ductility. Recently, there has been thus a significant interest in the deformation behavior of FCC metals involving twins. Traditionally, the coherent twin boundary (CTB) is regarded as a strong barrier to dislocation penetration unless dislocations run through the boundary or transfer with easy cross-slip. Although it is well established that slip is strongly affected by twin boundaries, the detailed aspects of dislocation-twin boundary interactions are not yet fully understood.
We here present the detailed reactions between dislocations and CTB and the resultant formation of dislocation networks in several FCC metals using atomistic simulations. It is found that dislocation networks are mainly composed of sessile Frank dislocations and partially of sessile stair-rod and Hirth dislocations and glissile twinning dislocations. The density and type of dislocations in the networks were found to be dependent on the materials' factors such as generalized stacking fault energy and also external factors like loading axis. The present work could provide insight to understand the source of the huge work-hardening rate and high stability of twin boundaries exhibited in TWIP steels.
The toxic side effects of therapies against breast cancer can affect the quality of life of patients, necessitating the use of naturally-derived therapeutics. Here, we investigated the effects of Dendropanax morbiferus H. Lév. leaf (DPL) extract on breast cancer cells in vitro and in vivo to assess its anticancer potential.MDA-MB-231 breast cancer cells were treated with DPL, and the in vitro effect of DPL on the cells was evaluated through an MTT assay, DAPI staining, annexin V/propidium iodide double staining, and western blotting. The in vivo effects of DPL were measured through the MDA-MB-231 tumor xenograft mouse model. A TUNEL assay and immunohistochemistry were used to determine the extent of apoptosis and p-p38 expression in tumor tissues, respectively.DPL treatment significantly suppressed cell viability in a concentration-dependent manner. Furthermore, DPL treatment resulted in increased apoptotic body formation, apoptosis rate, cleaved poly (ADP-ribose) polymerase and B-cell lymphoma 2 (Bcl-2)-associated X protein levels, phosphorylation of mitogen-activated protein kinase (MAPK) pathway proteins, and decreased Bcl-2 levels. In addition, the antitumor effect in vivo was confirmed through the xenograft model, where decreased tumor volume and weight following DPL administration were observed. Further, apoptosis and increased p-p38 levels in tumor tissues were observed, and no pathological abnormalities were found in the liver or kidney.DPL inhibits proliferation through MAPK-mediated apoptosis in breast cancer cells and tumors, suggesting the potential of DPL as a natural therapeutic agent for breast cancer.
Immunosuppressants are used to prevent rejection in transplant patients. Many of these medications commonly cause gastrointestinal (GI) symptoms. We present a 38-year-old kidney and pancreas transplant recipient who had severe ulceration throughout his GI tract leading to perforations of his stomach and cecum, despite early discontinuation of mycophenolate mofetil-the most likely culprit medication. The ongoing injury observed despite holding mycophenolate suggests a possible compounding effect of tacrolimus and everolimus. Both these agents are underrepresented causes of GI injury. This perfect storm of agents may have accounted for the severity and extensive presentation observed in our patient.
The anaphase-promoting complex (APC) regulates cell division in eukaryotes by targeting specific proteins for destruction. APC substrates generally contain one or more short degron sequences that help mediate their recognition and poly-ubiquitination by the APC. The most common and well characterized degrons are the destruction box (D box) and the KEN box. The budding yeast Acm1 protein, an inhibitor of Cdh1-activated APC (APC(Cdh1)) also contains several conserved D and KEN boxes, and here we report that two of these located in the central region of Acm1 constitute a pseudosubstrate sequence required for APC(Cdh1) inhibition. Acm1 interacted with and inhibited substrate binding to the WD40 repeat domain of Cdh1. Combined mutation of the central D and KEN boxes strongly reduced both binding to the Cdh1 WD40 domain and APC(Cdh1) inhibition. Despite this, the double mutant, but not wild-type Acm1, was poly-ubiquitinated by APC(Cdh1) in vitro. Thus, unlike substrates in which D and KEN boxes promote ubiquitination, these same elements in the central region of Acm1 prevent ubiquitination. We propose that this unique property of the Acm1 degron sequences results from an unusually high affinity interaction with the substrate receptor site on the WD40 domain of Cdh1 that may serve both to promote APC inhibition and protect Acm1 from destruction.
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