Abstract
Background
Due to post-cleavage residence of the Cas9-sgRNA complex at its target, Cas9-induced DNA double-strand breaks (DSBs) have to be exposed to engage DSB repair pathways. Target ...interaction of Cas9-sgRNA determines its target binding affinity and modulates its post-cleavage target residence duration and exposure of Cas9-induced DSBs. This exposure, via different mechanisms, may initiate variable DNA damage responses, influencing DSB repair pathway choices and contributing to mutational heterogeneity in genome editing. However, this regulation of DSB repair pathway choices is poorly understood.
Results
In repair of Cas9-induced DSBs, repair pathway choices vary widely at different target sites and classical nonhomologous end joining (c-NHEJ) is not even engaged at some sites. In mouse embryonic stem cells, weakening the target interaction of Cas9-sgRNA promotes bias towards c-NHEJ and increases target dissociation and reduces target residence of Cas9-sgRNAs in vitro. As an important strategy for enhancing homology-directed repair, inactivation of c-NHEJ aggravates off-target activities of Cas9-sgRNA due to its weak interaction with off-target sites. By dislodging Cas9-sgRNA from its cleaved targets, DNA replication alters DSB end configurations and suppresses c-NHEJ in favor of other repair pathways, whereas transcription has little effect on c-NHEJ engagement. Dissociation of Cas9-sgRNA from its cleaved target by DNA replication may generate three-ended DSBs, resulting in palindromic fusion of sister chromatids, a potential source for CRISPR/Cas9-induced on-target chromosomal rearrangements.
Conclusions
Target residence of Cas9-sgRNA modulates DSB repair pathway choices likely through varying dissociation of Cas9-sgRNA from cleaved DNA, thus widening on-target and off-target mutational spectra in CRISPR/Cas9 genome editing.
BackgroundCirculating tumor cells (CTCs) can survive in the circulation and return to primary tumors through a self-seeding process. However, the mechanisms underlying CTCs escape from natural killer ...(NK) cell-mediated immune surveillance remain unclear.MethodSelf-seeded tumor cells were isolated and characterized using a modified contralateral seeding model. A comparison of transcriptional profiles was performed between the parental cells and self-seeded cells. The molecular mechanism of self-seeded tumor cells escaping from NK cell was demonstrated through in vitro experiments and verified in a CTC-mimicking in vivo model. Then, the expression level of key protein mediating CTCs immune escape was detected in 24 paired primary and recurrent tumor samples of patients with oral cancer by the immunohistochemical method.ResultSelf-seeded cells displayed resistance to NK cell-mediated lysis and a higher tumor seeding ability than their parental cells. Elevated expression levels of the CDH2 gene and its protein product, N-cadherin were found in self-seeded cells. NK cells secreted cytokines, and fluid shear stress facilitated N-cadherin release by promoting A disintegrin and metalloprotease 10 (ADAM10) translation or converting the precursor ADAM10 to the mature form. Soluble N-cadherin triggered NK cell functional exhaustion by interacting with the killer cell lectin-like receptor subfamily G member 1 (KLRG1) receptor and therefore protected tumor cells from NK cell killing in the circulation. In vivo experimental results showed that overexpression of N-cadherin promoted tumor self-seeding and facilitated the survival of CTCs. Compared with primary tumors, N-cadherin expression was significantly increased in matched recurrent tumor tissues.ConclusionTogether, our findings illustrate an unknown mechanism by which CTCs evaded NK cell-mediated immune surveillance, and indicate that targeting N-cadherin is an effective strategy to prevent CTCs from homing to primary tumor.
Rheumatoid arthritis (RA) is a prevalent inflammatory autoimmune disease characterised by persistent inflammation and joint damage with elevated levels of reactive oxygen species (ROS). Current ...treatment modalities for RA have significant limitations, including poor bioavailability, severe side effects, and inadequate targeting of inflamed joints. Herein, we synthesised cerium/manganese oxide nanoparticles (NPs) as efficient drug carriers with antioxidant and catalytic-like functions that can eliminate ROS to facilitate the polarization of macrophages phenotype from M1 to M2 and alleviate inflammation. Methotrexate (MTX), a first-line RA medication, was loaded into the NPs, which were further modified with bovine serum albumin (BSA) and integrated into dissolving hyaluronic acid-based microneedles (MNs) for transdermal delivery.
This innovative approach significantly enhanced drug delivery efficiency, reduced RA inflammation, and successfully modulated macrophage polarization toward an anti-inflammatory phenotype.
This research not only presents a promising drug delivery strategy for RA but also contributes broadly to the field of immune disease treatment by offering an advanced approach for macrophage phenotypic reprogramming.
LINE1 retrotransposons are mobile DNA elements that copy and paste themselves into new sites in the genome. To ensure their evolutionary success, heritable new LINE-1 insertions accumulate in cells ...that can transmit genetic information to the next generation (i.e., germ cells and embryonic stem cells). It is our hypothesis that LINE1 retrotransposons, insertional mutagens that affect expression of genes, may be causal agents of early miscarriage in humans. The cell has evolved various defenses restricting retrotransposition-caused mutation, but these are occasionally relaxed in certain somatic cell types, including those of the early embryo. We predict that reduced suppression of L1s in germ cells or early-stage embryos may lead to excessive genome mutation by retrotransposon insertion, or to the induction of an inflammatory response or apoptosis due to increased expression of L1-derived nucleic acids and proteins, and so disrupt gene function important for embryogenesis. If correct, a novel threat to normal human development is revealed, and reverse transcriptase therapy could be one future strategy for controlling this cause of embryonic damage in patients with recurrent miscarriages.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Osteoarthritis (OA) is a common degenerative joint disease characterized by progressive cartilage degradation and inflammation. In recent years, mesenchymal stem cells (MSCs) derived exosomes ...(MSCs-Exo) have attracted widespread attention for their potential role in modulating OA pathology. However, the unpredictable therapeutic effects of exosomes have been a significant barrier to their extensive clinical application. In this study, we investigated whether fucoidan-pretreated MSC-derived exosomes (F-MSCs-Exo) could better protect chondrocytes in osteoarthritic joints and elucidate its underlying mechanisms. In order to evaluate the role of F-MSCs-Exo in osteoarthritis, both in vitro and in vivo studies were conducted. MiRNA sequencing was employed to analyze MSCs-Exo and F-MSCs-Exo, enabling the identification of differentially expressed genes and the exploration of the underlying mechanisms behind the protective effects of F-MSCs-Exo in osteoarthritis. Compared to MSCs-Exo, F-MSCs-Exo demonstrated superior effectiveness in inhibiting inflammatory responses and extracellular matrix degradation in rat chondrocytes. Moreover, F-MSCs-Exo exhibited enhanced activation of autophagy in chondrocytes. MiRNA sequencing of both MSCs-Exo and F-MSCs-Exo revealed that miR-146b-5p emerged as a promising candidate mediator for the chondroprotective function of F-MSCs-Exo, with TRAF6 identified as its downstream target. In conclusion, our research results demonstrate that miR-146b-5p encapsulated in F-MSCs-Exo effectively inhibits TRAF6 activation, thereby suppressing inflammatory responses and extracellular matrix degradation, while promoting chondrocyte autophagy for the protection of osteoarthritic cartilage cells. Consequently, the development of a therapeutic approach combining fucoidan with MSC-derived exosomes provides a promising strategy for the clinical treatment of osteoarthritis.
In class II transcription activation, the transcription factor normally binds to the promoter near the −35 position and contacts the domain 4 of σ factors (σ4) to activate transcription. However, σ4 ...of σ70 appears to be poorly folded on its own. Here, by fusing σ4 with the RNA polymerase β‐flap‐tip‐helix, we constructed two σ4 chimera proteins, one from σ70 σ470c and another from σS σ4Sc of Klebsiella pneumoniae. The two chimera proteins well folded into a monomeric form with strong binding affinities for −35 element DNA. Determining the crystal structure of σ4Sc in complex with −35 element DNA revealed that σ4Sc adopts a similar structure as σ4 in the Escherichia coli RNA polymerase σS holoenzyme and recognizes −35 element DNA specifically by several conserved residues from the helix‐turn‐helix motif. By using nuclear magnetic resonance (NMR), σ470c was demonstrated to recognize −35 element DNA similar to σ4Sc. Carr‐Purcell‐Meiboom‐Gill relaxation dispersion analyses showed that the N‐terminal helix and the β‐flap‐tip‐helix of σ470c have a concurrent transient α‐helical structure and DNA binding reduced the slow dynamics on σ470c. Finally, only σ470c was shown to interact with the response regulator PmrA and its promoter DNA. The chimera proteins are capable of −35 element DNA recognition and can be used for study with transcription factors or other factors that interact with domain 4 of σ factors.
Osteonecrosis of the femoral head caused by glucocorticoids (GIONFH) is a significant issue resulting from prolonged or excessive clinical glucocorticoid use. Astaxanthin, an orange-red carotenoid ...present in marine organisms, has been the focus of this study to explore its impact and mechanism on osteoblast apoptosis induced by dexamethasone (Dex) and GIONFH.
In this experiment, bioinformatic prediction, molecular docking and dynamics simulation, cytotoxicity assay, osteogenic differentiation, qRT-PCR analysis, terminal uridine nickend labeling (TUNEL) assay, determination of intracellular ROS, mitochondrial function assay, immunofluorescence, GIONFH rat model construction, micro-computed tomography (micro-CT) scans were performed.
Our research demonstrated that a low dose of astaxanthin was non-toxic to healthy osteoblasts and restored the osteogenic function of Dex-treated osteoblasts by reducing oxidative stress, mitochondrial dysfunction, and apoptosis. Furthermore, astaxanthin rescued the dysfunction in poor bone quality, bone metabolism and angiogenesis of GIONFH rats. The mechanism behind this involves astaxanthin counteracting Dex-induced osteogenic damage by activating the Nrf2 pathway.
Astaxanthin shields osteoblasts from glucocorticoid-induced oxidative stress and mitochondrial dysfunction via Nrf2 pathway activation, making it a potential therapeutic agent for GIONFH treatment.
A design method of error-free spur slice cutter is proposed according to urgent demand for the slice cutter in actual production. The structure of the rake face is determined according to ...technological realization of the design, manufacturing, and tool grinding. On that basis, the mathematical model of the rake face is built. Taking the error-free design as the goal, the mathematical model of major flank face is established based on the principle of the gear slicing. The mathematical models of designing angle and working angle are constructed by the equivalent methodology, spatial geometrical theory, and conjugation theory of space surface. The validity of the above study results is verified by using a calculation example. The design method lays a theory foundation for the study of cutter parameter optimization, tool life, and other aspects.
Previously, we identified that both fingers 1 and 2 in the three Cys2His2 zinc-finger domains (TZD) of testis zinc-finger protein specifically bind to its cognate DNA; however, finger 3 is ...non-sequence-specific. To gain insights into the interaction mechanism, here we further investigated the DNA-binding characteristics of TZD bound to non-specific DNAs and its finger segments bound to cognate DNA. TZD in non-specific DNA binding showed smaller chemical shift perturbations, as expected. However, the direction of shift perturbation, change of DNA imino-proton NMR signal, and dynamics on the 15N backbone atom significantly differed between specific and non-specific binding. Using these unique characteristics, we confirmed that the three single-finger segments (TZD1, TZD2 and TZD3) and the two-finger segment (TZD23) non-specifically bind to the cognate DNA. In comparison, the other two-finger segment (TZD12) binding to the cognate DNA features simultaneous non-specific and semi-specific binding, both slowly exchanged in terms of NMR timescale. The process of TZD binding to the cognate DNA is likely stepwise: initially TZD non-specifically binds to DNA, then fingers 1 and 2 insert cooperatively into the major groove of DNA by semi-specific binding, and finally finger 3 non-specifically binds to DNA, which promotes the specific binding on fingers 1 and 2 and stabilizes the formation of a specific TZD-DNA complex.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In Staphylococcus aureus, vancomycin‐resistance‐associated response regulator (VraR) is a part of the VraSR two‐component system, which is responsible for activating a cell wall‐stress stimulon in ...response to an antibiotic that inhibits cell wall formation. Two VraR‐binding sites have been identified: R1 and R2 in the vraSR operon control region. However, the binding of VraR to a promoter DNA enhancing downstream gene expression remains unclear. VraR contains a conserved N‐terminal receiver domain (VraRN) connected to a C‐terminal DNA binding domain (VraRC) with a flexible linker. Here, we present the crystal structure of VraRC alone and in complex with R1‐DNA in 1.87‐ and 2.0‐Å resolution, respectively. VraRC consisting of four α‐helices forms a dimer when interacting with R1‐DNA. In the VraRC–DNA complex structure, Mg2+ ion is bound to Asp194. Biolayer interferometry experiments revealed that the addition of Mg2+ to VraRC enhanced its DNA binding affinity by eightfold. In addition, interpretation of NMR titrations between VraRC with R1‐ and R2‐DNA revealed the essential residues that might play a crucial role in interacting with DNA of the vraSR operon. The structural information could help in designing and screening potential therapeutics/inhibitors to deal with antibiotic‐resistant S. aureus via targeting VraR.
PDB Code(s): 7VE4, 7VE5 and 7VE6
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