Catalogue Search | MBRL
Are you sure you want to remove the book from the shelf?
{{itemTitle}}
1,495
result(s) for
"Guo, Li-Hua"
Sort by:
Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage
Background
Precise genome editing via homology-directed repair (HDR) after double-stranded DNA (dsDNA) cleavage facilitates functional genomic research and holds promise for gene therapy. However, HDR efficiency remains low in some cell types, including some of great research and clinical interest, such as human induced pluripotent stem cells (iPSCs).
Results
Here, we show that a double cut HDR donor, which is flanked by single guide RNA (sgRNA)-PAM sequences and is released after CRISPR/Cas9 cleavage, increases HDR efficiency by twofold to fivefold relative to circular plasmid donors at one genomic locus in 293 T cells and two distinct genomic loci in iPSCs. We find that a 600 bp homology in both arms leads to high-level genome knockin, with 97–100% of the donor insertion events being mediated by HDR. The combined use of CCND1, a cyclin that functions in G1/S transition, and nocodazole, a G2/M phase synchronizer, doubles HDR efficiency to up to 30% in iPSCs.
Conclusions
Taken together, these findings provide guidance for the design of HDR donor vectors and the selection of HDR-enhancing factors for applications in genome research and precision medicine.
Journal Article
Effective control of large deletions after double-strand breaks by homology-directed repair and dsODN insertion
Background
After repairing double-strand breaks (DSBs) caused by CRISPR-Cas9 cleavage, genomic damage, such as large deletions, may have pathogenic consequences.
Results
We show that large deletions are ubiquitous but are dependent on editing sites and cell types. Human primary T cells display more significant deletions than hematopoietic stem and progenitor cells (HSPCs), whereas we observe low levels in induced pluripotent stem cells (iPSCs). We find that the homology-directed repair (HDR) with single-stranded oligodeoxynucleotides (ssODNs) carrying short homology reduces the deletion damage by almost half, while adeno-associated virus (AAV) donors with long homology reduce large deletions by approximately 80%. In the absence of HDR, the insertion of a short double-stranded ODN by NHEJ reduces deletion indexes by about 60%.
Conclusions
Timely bridging of broken ends by HDR and NHEJ vastly decreases the unintended consequences of dsDNA cleavage. These strategies can be harnessed in gene editing applications to attenuate unintended outcomes.
Journal Article
Super-enhancers: a new frontier for epigenetic modifiers in cancer chemoresistance
Although new developments of surgery, chemotherapy, radiotherapy, and immunotherapy treatments for cancer have improved patient survival, the emergence of chemoresistance in cancer has significant impacts on treatment effects. The development of chemoresistance involves several polygenic, progressive mechanisms at the molecular and cellular levels, as well as both genetic and epigenetic heterogeneities. Chemotherapeutics induce epigenetic reprogramming in cancer cells, converting a transient transcriptional state into a stably resistant one. Super-enhancers (SEs) are central to the maintenance of identity of cancer cells and promote SE-driven-oncogenic transcriptions to which cancer cells become highly addicted. This dependence on SE-driven transcription to maintain chemoresistance offers an Achilles’ heel for chemoresistance. Indeed, the inhibition of SE components dampens oncogenic transcription and inhibits tumor growth to ultimately achieve combined sensitization and reverse the effects of drug resistance. No reviews have been published on SE-related mechanisms in the cancer chemoresistance. In this review, we investigated the structure, function, and regulation of chemoresistance-related SEs and their contributions to the chemotherapy via regulation of the formation of cancer stem cells, cellular plasticity, the microenvironment, genes associated with chemoresistance, noncoding RNAs, and tumor immunity. The discovery of these mechanisms may aid in the development of new drugs to improve the sensitivity and specificity of cancer cells to chemotherapy drugs.
Journal Article
Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse
Background
Hemophilia A, a bleeding disorder resulting from
F8
mutations, can only be cured by gene therapy. A promising strategy is CRISPR-Cas9-mediated precise insertion of
F8
in hepatocytes at highly expressed gene loci, such as albumin (
Alb
). Unfortunately, the precise in vivo integration efficiency of a long insert is very low (~ 0.1%).
Results
We report that the use of a double-cut donor leads to a 10- to 20-fold increase in liver editing efficiency, thereby completely reconstituting serum F8 activity in a mouse model of hemophilia A after hydrodynamic injection of Cas9-sgAlb and B domain-deleted (BDD) F8 donor plasmids. We find that the integration of a double-cut donor at the
Alb
locus in mouse liver is mainly through non-homologous end joining (NHEJ)-mediated knock-in. We then target
BDDF8
to multiple sites on introns 11 and 13 and find that NHEJ-mediated insertion of
BDDF8
restores hemostasis. Finally, using 3 AAV8 vectors to deliver genome editing components, including Cas9, sgRNA, and
BDDF8
donor, we observe the same therapeutic effects. A follow-up of 100 mice over 1 year shows no adverse effects.
Conclusions
These findings lay the foundation for curing hemophilia A by NHEJ knock-in of
BDDF8
at
Alb
introns after AAV-mediated delivery of editing components.
Journal Article
KLF5-mediated Eppk1 expression promotes cell proliferation in cervical cancer via the p38 signaling pathway
Background
Epiplakin1 (Eppk1) is part of epidermal growth factor (EGF) signal and takes part in reorganization of cytoskeleton and cell proliferation. However, the role of Eppk1 in cervical cancer (CC) remains unknown.
Methods
To express Eppk1 and KLF5 and their correlation, we used RNA-sequence, RT-qPCR, TCGA database and immunofluorescence staining in vitro and in different pathological cervical tissues. In CC cell lines, we tested adenovirus-mediated over expression or knockdown of KLF5 and siRNA-mediated knockdown of Eppk1 and a suiting assessment of cell proliferation and cell signaling by western blot and CCK8 tests. We studied the mechanism by which KLF5 regulates Eppk1 expression by reporter gene test and chromatin immunoprecipitation test.
Results
Eppk1 expression promoted in CC tissues and cell lines compared with increased KLF5 expression. The results of immunofluorescence staining further showed the increased co-expression of Eppk1 and KLF5 correlated substantially with tumorigenesis in cervical tissues. Overexpression of KLF5 significantly increased Eppk1 expression at transcription and translation levels. Conversely, the knockdown of KLF5 by siRNA against KLF5 decreased Eppk1 expression. Mechanically, KLF5 activated Eppk1 transcription by direct binding to the Eppk1 promoter. Gain- and loss-of-function experiments reported that KLF5 promoted cell proliferation in Hela partly dependent on Eppk1 upregulation. Besides, KLF5-mediated activation of p38 signaling significantly decreased after Eppk1 knockdown compared with decline of proliferation, suggesting that Eppk1 lies upstream of p38 signaling affecting cell proliferation. Finally, Eppk1 expression is positively correlated with tumor size in clinicopathological features of CC.
Conclusions
Eppk1 may be an effective therapeutic target for affecting p38 signaling pathway and cell proliferation in cervical cancer.
Journal Article
Fluid Reactive Anomalous Transport with Random Waiting Time Depending on the Preceding Jump Length
Anomalous (or non-Fickian) diffusion has been widely found in fluid reactive transport and the traditional advection–diffusion–reaction equation (ADRE) based on Fickian diffusion is proved to be inadequate to predict this anomalous transport of the reactive particle in flows. To capture the complex coupling effect among advection, diffusion and reaction, and the energy-dependent characteristics of fluid reactive anomalous transport, in the present paper we analyze A→B reaction under anomalous diffusion with waiting time depending on the preceding jump length in linear flows, and derive the corresponding generalized master equations in Fourier–Laplace space for the distribution of A and B particles in continuous time random walks scheme. As examples, the generalized ADREs for the jump length of Gaussian distribution and L e´ vy flight with the probability density function of waiting time being quadratic dependent on the preceding jump length are obtained by applying the derived generalized master equations.
Journal Article
Mediation and moderation analyses: exploring the complex pathways between hope and quality of life among patients with schizophrenia
Background
The underlying mechanism between hope and quality of life is as yet unknown. We aim to examine the potential mediating effect of depression and resilience and the moderated effect of sex in this well-established association.
Methods
Two hundred seven patients diagnosed with schizophrenia were administered a questionnaire battery that measured hope, depression, resilience and QOL. A multiple mediation model was used to examine the mediating effect of resilience and depression on the association between hope and QOL. A subgroup analysis was performed and a moderated mediation model was examined to find and test the moderated effect of sex on the mediation model. We used Mplus to perform moderation and mediation analyses so that the mediators and moderator could function together in the same model.
Result
Sex was the moderator on the direct path between hope and QOL. The relationship between hope and QOL was mediated by resilience and depression in both sexes. When compared with female patients, the effect of hope on QOL was completely mediated by resilience and depression in males. In female patients, the model was partially mediated, and the direct effect of hope on QOL was significantly negatively correlated with the level of hope.
Conclusion
We present a conceptual model containing the mediated effects of resilience and depression and the moderated effect of sex between hope and QOL, which we believe facilitates the understanding of these associations. This model should be useful in the formulation of strategies to improve QOL.
Journal Article
Effects of cyclic carboxylate nucleating agents on nucleus density and crystallization behavior of isotactic polypropylene
Isothermal crystallization behavior of isotactic polypropylene (iPP) nucleated with three cyclic carboxylate nucleating agents, namely, sodium bicyclic[2,2,1] heptane dicarboxylate (HPN-68), sodium benzoate (Be-Na), and sodium salt of hexahydrophthalic acid (HHPA-Na), was investigated by using differential scanning calorimetry. The classical Avarmi method was used to evaluate the isothermal crystallization behavior of iPP. The nucleus density was obtained by the calculation model of Lamberti based on the Avrami method of crystallization kinetics from calorimetric crystallization curves recorded under isothermal conditions. This method was able to measure nucleus density and growth rate of nucleated iPP, for which the nucleation phenomenon was substantially heterogeneous. The addition of nucleating agents shortened the crystallization halftime (t1/2) and increased the crystallization rate of iPP under isothermal crystallization, eventually increasing the nucleus density of iPP nucleated with nucleating agents for finishing crystallization under higher crystallization temperature, which was larger than that of virgin iPP. The results of polarized optical microscopy also showed that the nucleus density and crystallization rate were greatly increased with addition of nucleating agents. In these three nucleating agents, HPN-68 had the best nucleation effect, and iPP nucleated with HPN-68 had shorter crystallization halftime and higher nucleus density than iPP nucleated with Be-Na and HHPA-Na.
Journal Article
CD226 deletion improves post-infarction healing via modulating macrophage polarization in mice
Macrophages are essential for wound repair after myocardial infarction (MI). CD226, a member of immunoglobulin superfamily, is expressed on inflammatory monocytes, however, the role of CD226 in infarct healing and the effect of CD226 on macrophage remain unknown.
Wild type and CD226 knockout (CD226 KO) mice were subjected to permanent coronary ligation. CD226 expression, cardiac function and ventricular remodeling were evaluated. Profile of macrophages, myofibroblasts, angiogenesis and monocytes mobilization were determined.
CD226 expression increased in the infarcted heart, with a peak on day 7 after MI. CD226 KO attenuated infarct expansion and improved infarct healing after MI. CD226 deletion resulted in increased F4/80
CD206
M2 macrophages and diminished Mac-3
iNOS
M1 macrophages accumulation in the infarcted heart, as well as enrichment of α-smooth muscle actin positive myofibroblasts and Ki67
CD31
endothelial cells, leading to increased reparative collagen deposition and angiogenesis. Furthermore, CD226 deletion restrained inflammatory monocytes mobilization, as revealed by enhanced retention of Ly6C
monocytes in the spleen associated with a decrease of Ly6C
monocytes in the peripheral blood, whereas local proliferation of macrophage in the ischemic heart was not affected by CD226 deficiency.
studies using bone marrow-derived macrophages showed that CD226 deletion potentiated M2 polarization and suppressed M1 polarization.
: CD226 expression is dramatically increased in the infarcted heart, and CD226 deletion improves post-infarction healing and cardiac function by favoring macrophage polarization towards reparative phenotype. Thus, inhibition of CD226 may represent a novel therapeutic approach to improve wound healing and cardiac function after MI.
Journal Article