ResearchPad - transfection https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Regulation of cell growth and migration by miR-96 and miR-183 in a breast cancer model of epithelial-mesenchymal transition]]> https://www.researchpad.co/article/elastic_article_7836 Breast cancer is the most commonly diagnosed malignancy in women, and has the second highest mortality rate. Over 90% of all cancer-related deaths are due to metastasis, which is the spread of malignant cells from the primary tumor to a secondary site in the body. It is hypothesized that one cause of metastasis involves epithelial-mesenchymal transition (EMT). When epithelial cells undergo EMT and transition into mesenchymal cells, they display increased levels of cell proliferation and invasion, resulting in a more aggressive phenotype. While many factors regulate EMT, microRNAs have been implicated in driving this process. MicroRNAs are short noncoding RNAs that suppress protein production, therefore loss of microRNAs may promote the overexpression of specific target proteins important for EMT. The goal of this study was to investigate the role of miR-96 and miR-183 in EMT in breast cancer. Both miR-96 and miR-183 were found to be downregulated in post-EMT breast cancer cells. When microRNA mimics were transfected into these cells, there was a significant decrease in cell viability and migration, and a shift from a mesenchymal to an epithelial morphology (mesenchymal-epithelial transition or MET). These MET-related changes may be facilitated in part by the regulation of ZEB1 and vimentin, as both of these proteins were downregulated when miR-96 and miR-183 were overexpressed in post-EMT cells. These findings indicate that the loss of miR-96 and miR-183 may help facilitate EMT and contribute to the maintenance of a mesenchymal phenotype. Understanding the role of microRNAs in regulating EMT is significant in order to not only further elucidate the pathways that facilitate metastasis, but also identify potential therapeutic options for preventing or reversing this process.

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<![CDATA[OAZ1 knockdown enhances viability and inhibits ER and LHR transcriptions of granulosa cells in geese]]> https://www.researchpad.co/article/5989db50ab0ee8fa60bdc164

An increasing number of studies suggest that ornithine decarboxylase antizyme 1 (OAZ1), which is regarded as a tumor suppressor gene, regulates follicular development, ovulation, and steroidogenesis. The granulosa cells in the ovary play a critical role in these ovarian functions. However, the action of OAZ1 mediating physiological functions of granulosa cells is obscure. OAZ1 knockdown in granulosa cells of geese was carried out in the current study. The effect of OAZ1 knockdown on polyamine metabolism, cell proliferation, apoptosis, and hormone receptor transcription of primary granulosa cells in geese was measured. The viability of granulosa cells transfected with the shRNA OAZ1 at 48 h was significantly higher than the control (p<0.05). The level of putrescine and spermidine in granulosa cells down-regulating OAZ1 was 7.04- and 2.11- fold higher compared with the control, respectively (p<0.05). The CCND1, SMAD1, and BCL-2 mRNA expression levels in granulosa cells down-regulating OAZ1 were each significantly higher than the control, respectively (p<0.05), whereas the PCNA and CASPASE 3 expression levels were significantly lower than the control (p<0.05). The estradiol concentration, ER and LHR mRNA expression levels were significantly lower in granulosa cells down-regulating OAZ1 compared with the control (p<0.05). Taken together, our results indicated that OAZ1 knockdown elevated the putrescine and spermidine contents and enhanced granulosa cell viability and inhibited ER and LHR transcriptions of granulosa cells in geese.

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<![CDATA[Highly efficient serum-free manipulation of miRNA in human NK cells without loss of viability or phenotypic alterations is accomplished with TransIT-TKO]]> https://www.researchpad.co/article/N4e6e8e95-63ae-420d-a6d7-c2f1aa3d99e6

Natural killer (NK) cells are innate lymphocytes with functions that include target cell killing, inflammation and regulation. NK cells integrate incoming activating and inhibitory signals through an array of germline-encoded receptors to gauge the health of neighbouring cells. The reactive potential of NK cells is influenced by microRNA (miRNA), small non-coding sequences that interfere with mRNA expression. miRNAs are highly conserved between species, and a single miRNA can have hundreds to thousands of targets and influence entire cellular programs. Two miRNA species, miR-155-5p and miR-146a-5p are known to be important in controlling NK cell function, but research to best understand the impacts of miRNA species within NK cells has been bottlenecked by a lack of techniques for altering miRNA concentrations efficiently and without off-target effects. Here, we describe a non-viral and straightforward approach for increasing or decreasing expression of miRNA in primary human NK cells. We achieve >90% transfection efficiency without off-target impacts on NK cell viability, education, phenotype or function. This opens the opportunity to study and manipulate NK cell miRNA profiles and their impacts on NK cellular programs which may influence outcomes of cancer, inflammation and autoimmunity.

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<![CDATA[Generation of targeted homozygosity in the genome of human induced pluripotent stem cells]]> https://www.researchpad.co/article/Nc0b5af8d-f419-410c-9036-89fcaed1eba6

When loss of heterozygosity (LOH) is correlated with loss or gain of a disease phenotype, it is often necessary to identify which gene or genes are involved. Here, we developed a region-specific LOH-inducing system based on mitotic crossover in human induced pluripotent stem cells (hiPSCs). We first tested our system on chromosome 19. To detect homozygous clones generated by LOH, a positive selection cassette was inserted at the AASV1 locus of chromosome 19. LOHs were generated by the combination of allele-specific double-stranded DNA breaks introduced by CRISPR/Cas9 and suppression of Bloom syndrome (BLM) gene expression by the Tet-Off system. The BLM protein inhibitor ML216 exhibited a similar crossover efficiency and distribution of crossover sites. We next applied this system to the short arm of chromosome 6, where human leukocyte antigen (HLA) loci are located. Genotyping and flow cytometric analysis demonstrated that LOHs associated with chromosomal crossover occurred at the expected positions. Although careful examination of HLA-homozygous hiPSCs generated from parental cells is needed for cancer predisposition and effectiveness of differentiation, they may help to mitigate the current shortcoming of hiPSC-based transplantation related to the immunological differences between the donor and host.

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<![CDATA[PDIP38/PolDIP2 controls the DNA damage tolerance pathways by increasing the relative usage of translesion DNA synthesis over template switching]]> https://www.researchpad.co/article/5c897795d5eed0c4847d30a7

Replicative DNA polymerases are frequently stalled at damaged template strands. Stalled replication forks are restored by the DNA damage tolerance (DDT) pathways, error-prone translesion DNA synthesis (TLS) to cope with excessive DNA damage, and error-free template switching (TS) by homologous DNA recombination. PDIP38 (Pol-delta interacting protein of 38 kDa), also called Pol δ-interacting protein 2 (PolDIP2), physically associates with TLS DNA polymerases, polymerase η (Polη), Polλ, and PrimPol, and activates them in vitro. It remains unclear whether PDIP38 promotes TLS in vivo, since no method allows for measuring individual TLS events in mammalian cells. We disrupted the PDIP38 gene, generating PDIP38-/- cells from the chicken DT40 and human TK6 B cell lines. These PDIP38-/- cells did not show a significant sensitivity to either UV or H2O2, a phenotype not seen in any TLS-polymerase-deficient DT40 or TK6 mutants. DT40 provides a unique opportunity of examining individual TLS and TS events by the nucleotide sequence analysis of the immunoglobulin variable (Ig V) gene as the cells continuously diversify Ig V by TLS (non-templated Ig V hypermutation) and TS (Ig gene conversion) during in vitro culture. PDIP38-/- cells showed a shift in Ig V diversification from TLS to TS. We measured the relative usage of TLS and TS in TK6 cells at a chemically synthesized UV damage (CPD) integrated into genomic DNA. The loss of PDIP38 also caused an increase in the relative usage of TS. The number of UV-induced sister chromatid exchanges, TS events associated with crossover, was increased a few times in PDIP38-/- human and chicken cells. Collectively, the loss of PDIP38 consistently causes a shift in DDT from TLS to TS without enhancing cellular sensitivity to DNA damage. We propose that PDIP38 controls the relative usage of TLS and TS increasing usage of TLS without changing the overall capability of DDT.

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<![CDATA[miR-7977 inhibits the Hippo-YAP signaling pathway in bone marrow mesenchymal stromal cells]]> https://www.researchpad.co/article/5c8823ead5eed0c4846392f6

We and others have demonstrated that various abnormalities of the bone marrow (BM) mesenchymal stromal cells (MSCs) such as aberrant cytokine expression, abnormal hedgehog signaling, and impaired miRNA biogenesis are observed in patients with acute myeloid leukemia (AML). However, underlying mechanisms to induce the dysfunction of BM MSCs have not yet been clarified. We previously showed that AML cells release abundant exosomal miR-7977, which, in turn, enters BM mesenchymal stromal cells (MSCs). However, the precise function of miR-7977 is not known. In this study, we performed transduction of a miR-7977 mimic into MSCs, compared transcriptomes between control-transduced (n = 3) and miR-7977-transduced MSCs (n = 3), and conducted pathway analysis. The array data revealed that the expression of 0.05% of genes was reduced 2-fold and the expression of 0.01% of genes was increased 2-fold. Interestingly, approximately half of these genes possessed a miR-7977 target site, while the other genes did not, suggesting that miR-7977 regulates the gene expression level directly and indirectly. Gene set enrichment analysis showed that the gene sets of Yes-associated protein 1 (YAP1) _up were significantly enriched (p<0.001, q<0.25), suggesting that miR-7977 modulates the Hippo-YAP signaling pathway. Visualization of pathway and network showed that miR-7977 significantly reduced the expression of Hippo core kinase, STK4. miR-7977 inactivated the Hippo-YAP signaling pathway as proven by GFP-tagged YAP nuclear trans localization and TEAD reporter assay. The miR-7977-transduced MSC cell line, HTS-5, showed elevated saturation density and enhanced entry into the cell cycle. These results suggest that miR-7977 is a critical factor that regulates the Hippo-YAP signaling pathway in BM-MSCs and may be involved in the upregulation of leukemia-supporting stroma growth.

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<![CDATA[Molecular predictors of brain metastasis-related microRNAs in lung adenocarcinoma]]> https://www.researchpad.co/article/5c5df358d5eed0c484581189

Brain metastasis (BM) is a major complication of lung adenocarcinoma (LAD). An investigation of the pathogenic mechanisms of BM, as well as the identification of appropriate molecular markers, is necessary. The aim of this study was to determine the expression patterns of microRNAs (miRNAs) in LAD with BM, and to investigate the biological role of these miRNAs during tumorigenesis. miRNA array profiles were used to identify BM-associated miRNAs. These miRNAs were independently validated in 155 LAD patients. Several in vivo and in vitro assays were performed to verify the effects of miRNAs on BM. We identified six miRNAs differentially expressed in patients with BM as compared to patients with BM. Of these, miR-4270 and miR-423-3p were further investigated. miR-4270 and miR-423-3p directly targeted MMP19 and P21, respectively, to influence cell viability, migration, and colony formation in vitro. miR-4270 downregulation and miR-423-3p upregulation was associated with an increased risk of BM in LAD patients. Thus, our results suggested that miR-4270 and miR-423-3p might play an important role in BM pathogenesis in LAD patients, and that these miRNAs might be useful prognostic and clinical treatment targets.

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<![CDATA[The genetic intractability of Symbiodinium microadriaticum to standard algal transformation methods]]> https://www.researchpad.co/article/5c75ac68d5eed0c484d08712

Modern transformation and genome editing techniques have shown great success across a broad variety of organisms. However, no study of successfully applied genome editing has been reported in a dinoflagellate despite the first genetic transformation of Symbiodinium being published about 20 years ago. Using an array of different available transformation techniques, we attempted to transform Symbiodinium microadriaticum (CCMP2467), a dinoflagellate symbiont of reef-building corals, with the view to performing subsequent CRISPR-Cas9 mediated genome editing. Plasmid vectors designed for nuclear transformation containing the chloramphenicol resistance gene under the control of the CaMV p35S promoter as well as several putative endogenous promoters were used to test a variety of transformation techniques including biolistics, electroporation and agitation with silicon carbide whiskers. Chloroplast-targeted transformation was attempted using an engineered Symbiodinium chloroplast minicircle encoding a modified PsbA protein expected to confer atrazine resistance. We report that we have been unable to confer chloramphenicol or atrazine resistance on Symbiodinium microadriaticum strain CCMP2467.

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<![CDATA[ARR22 overexpression can suppress plant Two-Component Regulatory Systems]]> https://www.researchpad.co/article/5c6b2616d5eed0c4842892c3

In plants, several developmental processes are co-coordinated by cytokinins via phosphorylation dependent processes of the Two-Component System (TCS). An outstanding challenge is to track phosphorelay flow from cytokinin perception to its molecular outputs, of which gene activation plays a major role. To address this issue, a kinetic-based reporter system was expounded to track TCS phosphorelay activity in vivo that can distinguish between basal and cytokinin dependent effects of overexpressed TCS members. The TCS phosphorelay can be positively activated by cytokinin and inhibited by pharmaceuticals or naturally interfering components. In this case we took advantage of the phosphohistidine-phosphatase Arabidopsis Response Regulator (ARR) 22 and investigated its phosphocompetition with other TCS members in regulating promoters of ARR5 and WUS in Arabidopsis thaliana cell culture protoplasts. In congruency with the proposed function of ARR22, overexpression of ARR22 blocked the activation of all B-type ARRs in this study in a TCS dependent manner. Furthermore, this effect could not be mimicked by A-type response regulator overexpression or compensated by AHP overexpression. Compared to other reporter assays, ours mimicked effects previously observed only in transgenic plants for all of the TCS proteins studied, suggesting that it is possible to expose phosphocompetition. Thus, our approach can be used to investigate gene signaling networks involving the TCS by leveraging ARR22 as a TCS inhibitor along with B-type ARR overexpression.

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<![CDATA[Deregulation of LRSAM1 expression impairs the levels of TSG101, UBE2N, VPS28, MDM2 and EGFR]]> https://www.researchpad.co/article/5c648d54d5eed0c484c825ab

CMT is the most common hereditary neuromuscular disorder of the peripheral nervous system with a prevalence of 1/2500 individuals and it is caused by mutations in more than 80 genes. LRSAM1, a RING finger ubiquitin ligase also known as TSG101-associated ligase (TAL), has been associated with Charcot-Marie-Tooth disease type 2P (CMT2P) and to date eight causative mutations have been identified. Little is currently known on the pathogenetic mechanisms that lead to the disease. We investigated the effect of LRSAM1 deregulation on possible LRSAM1 interacting molecules in cell based models. Possible LRSAM1 interacting molecules were identified using protein-protein interaction databases and literature data. Expression analysis of these molecules was performed in both CMT2P patient and control lymphoblastoid cell lines as well as in LRSAM1 and TSG101 downregulated SH-SY5Y cells.TSG101, UBE2N, VPS28, EGFR and MDM2 levels were significantly decreased in the CMT2P patient lymphoblastoid cell line as well as in LRSAM1 downregulated cells. TSG101 downregulation had a significant effect only on the expression of VPS28 and MDM2 and it did not affect the levels of LRSAM1. This study confirms that LRSAM1 is a regulator of TSG101 expression. Furthermore, deregulation of LRSAM1 significantly affects the levels of UBE2N, VPS28, EGFR and MDM2.

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<![CDATA[Role of BRCA1-associated protein (BRAP) variant in childhood pulmonary arterial hypertension]]> https://www.researchpad.co/article/5c5ca2b0d5eed0c48441e8b1

Although mutations in several genes have been reported in pulmonary arterial hypertension (PAH), most of PAH cases do not carry these mutations. This study aimed to identify a novel cause of PAH. To determine the disease-causing variants, direct sequencing and multiplex ligation-dependent probe amplification were performed to analyze 18 families with multiple affected family members with PAH. In one of the 18 families with PAH, no disease-causing variants were found in any of BMPR2, ACVRL1, ENG, SMAD1/4/8, BMPR1B, NOTCH3, CAV1, or KCNK3. In this family, a female proband and her paternal aunt developed PAH in their childhood. Whole-exome next-generation sequencing was performed in the 2 PAH patients and the proband’s healthy mother, and a BRCA1-associated protein (BRAP) gene variant, p.Arg554Leu, was identified in the 2 family members with PAH, but not in the proband’s mother without PAH. Functional analyses were performed using human pulmonary arterial smooth muscle cells (hPASMCs). Knockdown of BRAP via small interfering RNA in hPASMCs induced p53 signaling pathway activation and decreased cell proliferation. Overexpression of either wild-type BRAP or p.Arg554Leu-BRAP cDNA constructs caused cell death confounding these studies, however we observed higher levels of p53 signaling inactivation and hPASMC proliferation in cells expressing p.Arg554Leu-BRAP compared to wild-type BRAP. In addition, p.Arg554Leu-BRAP induced decreased apoptosis of hPASMCs compared with wild-type BRAP. In conclusion, we have identified a novel variant of BRAP in a Japanese family with PAH and our results suggest it could have a gain-of-function. This study sheds light on new mechanism of PAH pathogenesis.

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<![CDATA[Utilization of proliferable extracellular amastigotes for transient gene expression, drug sensitivity assay, and CRISPR/Cas9-mediated gene knockout in Trypanosoma cruzi]]> https://www.researchpad.co/article/5c466535d5eed0c484517ffa

Trypanosoma cruzi has three distinct life cycle stages; epimastigote, trypomastigote, and amastigote. Amastigote is the replication stage in host mammalian cells, hence this stage of parasite has clinical significance in drug development research. Presence of extracellular amastigotes (EA) and their infection capability have been known for some decades. Here, we demonstrate that EA can be utilized as an axenic culture to aid in stage-specific study of T. cruzi. Amastigote-like property of axenic amastigote can be sustained in LIT medium at 37°C at least for 1 week, judging from their morphology, amastigote-specific UTR-regulated GFP expression, and stage-specific expression of selected endogenous genes. Inhibitory effect of benznidazole and nifurtimox on axenic amastigotes was comparable to that on intracellular amastigotes. Exogenous nucleic acids can be transfected into EA via conventional electroporation, and selective marker could be utilized for enrichment of transfectants. We also demonstrate that CRISPR/Cas9-mediated gene knockout can be performed in EA. Essentiality of the target gene can be evaluated by the growth capability of the knockout EA, either by continuation of axenic culturing or by host infection and following replication as intracellular amastigotes. By taking advantage of the accessibility and sturdiness of EA, we can potentially expand our experimental freedom in studying amastigote stage of T. cruzi.

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<![CDATA[Ligand-activated PPARδ inhibits angiotensin II-stimulated hypertrophy of vascular smooth muscle cells by targeting ROS]]> https://www.researchpad.co/article/5c3e505ed5eed0c484d80f2b

We investigated the effect of peroxisome proliferator-activated receptor δ (PPARδ) on angiotensin II (Ang II)-triggered hypertrophy of vascular smooth muscle cells (VSMCs). Activation of PPARδ by GW501516, a specific ligand of PPARδ, significantly inhibited Ang II-stimulated protein synthesis in a concentration-dependent manner, as determined by [3H]-leucine incorporation. GW501516-activated PPARδ also suppressed Ang II-induced generation of reactive oxygen species (ROS) in VSMCs. Transfection of small interfering RNA (siRNA) against PPARδ significantly reversed the effects of GW501516 on [3H]-leucine incorporation and ROS generation, indicating that PPARδ is involved in these effects. By contrast, these GW501516-mediated actions were potentiated in VSMCs transfected with siRNA against NADPH oxidase (NOX) 1 or 4, suggesting that ligand-activated PPARδ elicits these effects by modulating NOX-mediated ROS generation. The phosphatidylinositol 3-kinase inhibitor LY294002 also inhibited Ang II-stimulated [3H]-leucine incorporation and ROS generation by preventing membrane translocation of Rac1. These observations suggest that PPARδ is an endogenous modulator of Ang II-triggered hypertrophy of VSMCs, and is thus a potential target to treat vascular diseases associated with hypertrophic changes of VSMCs.

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<![CDATA[MicroRNA-8073: Tumor suppressor and potential therapeutic treatment]]> https://www.researchpad.co/article/5c2e7fd4d5eed0c48451b92d

The comprehensive screening of intracellular and extracellular microRNAs was performed to identify novel tumor suppressors. We found that miR-8073 was present in exosome and predominantly exported from colorectal cancer cells. Treatment with a synthetic miR-8073 mimic resulted in a dramatic decrease in the proliferation of various types of cancer cells, which was not observed in similarly treated normal cells. As little is known about the biological functions of miR-8073, its target mRNAs were analyzed by both mRNA expression and in silico sequence analyses, leading to five probable target candidates (FOXM1, MBD3, CCND1, KLK10, and CASP2) that enhance survival during the regulation of the cell cycle, cell proliferation, and apoptosis. We experimentally confirmed that miR-8073 binds the 3’-UTR of each of these mRNA target candidates and that the introduction of a synthetic miR-8073 mimic into cancer cells reduced levels of protein expression. Finally, the antiproliferative effects of miR-8073 were validated in vivo: the subcutaneous injection of a synthetic miR-8073 mimic suppressed colorectal tumor volume to 43% in tumor-bearing xenografted mice. These results suggest that because miR-8073 binds, and thus reduces the levels of, these oncogenic targets, cancer cells must actively downregulate miR-8073 as a survival mechanism. The introduction of miR-8073 into tumors could thus inhibit tumor growth, indicating its great potential for cancer therapeutics.

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<![CDATA[CDHR3 extracellular domains EC1-3 mediate rhinovirus C interaction with cells and as recombinant derivatives, are inhibitory to virus infection]]> https://www.researchpad.co/article/5c1813ced5eed0c484775dec

Viruses in the rhinovirus C species (RV-C) are more likely to cause severe wheezing illnesses and asthma exacerbations in children than related isolates of the RV-A or RV-B. The RV-C capsid is structurally distinct from other rhinoviruses and does not bind ICAM-1 or LDL receptors. The RV-C receptor is instead, human cadherin-related family member 3 (CDHR3), a protein unique to the airway epithelium. A single nucleotide polymorphism (rs6967330, encoding C529Y) in CDHR3 regulates the display density of CDHR3 on cell surfaces and is among the strongest known genetic correlates for childhood virus-induced asthma susceptibility. CDHR3 immunoprecipitations from transfected or transduced cell lysates were used to characterize the RV-C interaction requirements. The C529 and Y529 variations in extracellular repeat domain 5 (EC5), bound equivalently to virus. Glycosylase treatment followed by mass spectrometry mapped 3 extracellular N-linked modification sites, and further detected surface-dependent, α2–6 sialyation unique to the Y529 format. None of these modifications were required for RV-C recognition, but removal or even dilution of structurally stabilizing calcium ions from the EC junctions irreversibly abrogated virus binding. CDHR3 deletions expressed in HeLa cells or as bacterial recombinant proteins, mapped the amino-terminal EC1 unit as the required virus contact. Derivatives containing the EC1 domain, could not only recapitulate virus:receptor interactions in vitro, but also directly inhibit RV-C infection of susceptible cells for several virus genotypes (C02, C15, C41, and C45). We propose that all RV-C use the same EC1 landing pad, interacting with putative EC3-mediated multimerization formats of CDHR3.

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<![CDATA[Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence the outcome of mutagenic DNA repair]]> https://www.researchpad.co/article/5c1ab83fd5eed0c48402748f

Genome editing occurs in the context of chromatin, which is heterogeneous in structure and function across the genome. Chromatin heterogeneity is thought to affect genome editing efficiency, but this has been challenging to quantify due to the presence of confounding variables. Here, we develop a method that exploits the allele-specific chromatin status of imprinted genes in order to address this problem in cycling mouse embryonic stem cells (mESCs). Because maternal and paternal alleles of imprinted genes have identical DNA sequence and are situated in the same nucleus, allele-specific differences in the frequency and spectrum of mutations induced by CRISPR-Cas9 can be unequivocally attributed to epigenetic mechanisms. We found that heterochromatin can impede mutagenesis, but to a degree that depends on other key experimental parameters. Mutagenesis was impeded by up to 7-fold when Cas9 exposure was brief and when intracellular Cas9 expression was low. In contrast, the outcome of mutagenic DNA repair was unaffected by chromatin state, with similar efficiencies of homology-directed repair (HDR) and deletion spectra on maternal and paternal chromosomes. Combined, our data show that heterochromatin imposes a permeable barrier that influences the kinetics, but not the endpoint, of CRISPR-Cas9 genome editing and suggest that therapeutic applications involving low-level Cas9 exposure will be particularly affected by chromatin status.

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<![CDATA[Pro-osteoporotic miR-320a impairs osteoblast function and induces oxidative stress]]> https://www.researchpad.co/article/5c0841e5d5eed0c484fcb16a

MicroRNAs (miRNAs) are important regulators of many cellular processes, including the differentiation and activity of osteoblasts, and therefore, of bone turnover. MiR-320a is overexpressed in osteoporotic bone tissue but its role in osteoblast function is unknown. In the present study, functional assays were performed with the aim to elucidate the mechanism of miR-320a action in osteoblastic cells. MiR-320a was either overexpressed or inhibited in human primary osteoblasts (hOB) and gene expression changes were evaluated through microarray analysis. In addition, the effect of miR-320a on cell proliferation, viability, and oxidative stress in hOB was evaluated. Finally, matrix mineralization and alkaline phosphatase activity were assessed in order to evaluate osteoblast functionality. Microarray results showed miR-320a regulation of a number of key osteoblast genes and of genes involved in oxidative stress. Regulation of osteoblast differentiation and ossification appeared as the best significant biological processes (PANTHER P value = 3.74E-05; and P value = 3.06E-04, respectively). The other enriched pathway was that of the cellular response to cadmium and zinc ions, mostly by the overexpression of metallothioneins. In hOBs, overexpression of miR-320a increased cell proliferation and oxidative stress levels whereas mineralization capacity was reduced. In conclusion, overexpression of miR-320a increased stress oxidation levels and was associated with reduced osteoblast differentiation and functionality, which could trigger an osteoporotic phenotype.

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<![CDATA[Rad51 recruitment and exclusion of non-homologous end joining during homologous recombination at a Tus/Ter mammalian replication fork barrier]]> https://www.researchpad.co/article/5b60218a463d7e3d6d2261bd

Classical non-homologous end joining (C-NHEJ) and homologous recombination (HR) compete to repair mammalian chromosomal double strand breaks (DSBs). However, C-NHEJ has no impact on HR induced by DNA nicking enzymes. In this case, the replication fork is thought to convert the DNA nick into a one-ended DSB, which lacks a readily available partner for C-NHEJ. Whether C-NHEJ competes with HR at a non-enzymatic mammalian replication fork barrier (RFB) remains unknown. We previously showed that conservative “short tract” gene conversion (STGC) induced by a chromosomal Tus/Ter RFB is a product of bidirectional replication fork stalling. This finding raises the possibility that Tus/Ter-induced STGC proceeds via a two-ended DSB intermediate. If so, Tus/Ter-induced STGC might be subject to competition by C-NHEJ. However, in contrast to the DSB response, where genetic ablation of C-NHEJ stimulates HR, we report here that Tus/Ter-induced HR is unaffected by deletion of either of two C-NHEJ genes, Xrcc4 or Ku70. These results show that Tus/Ter-induced HR does not entail the formation of a two-ended DSB to which C-NHEJ has competitive access. We found no evidence that the alternative end-joining factor, DNA polymerase θ, competes with Tus/Ter-induced HR. We used chromatin-immunoprecipitation to compare Rad51 recruitment to a Tus/Ter RFB and to a neighboring site-specific DSB. Rad51 accumulation at Tus/Ter was more intense and more sustained than at a DSB. In contrast to the DSB response, Rad51 accumulation at Tus/Ter was restricted to within a few hundred base pairs of the RFB. Taken together, these findings suggest that the major DNA structures that bind Rad51 at a Tus/Ter RFB are not conventional DSBs. We propose that Rad51 acts as an “early responder” at stalled forks, binding single stranded daughter strand gaps on the arrested lagging strand, and that Rad51-mediated fork remodeling generates HR intermediates that are incapable of Ku binding and therefore invisible to the C-NHEJ machinery.

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<![CDATA[Role of UBIAD1 in Intracellular Cholesterol Metabolism and Vascular Cell Calcification]]> https://www.researchpad.co/article/5989da92ab0ee8fa60ba0674

Vascular calcification is an important risk factor associated with mortality among patients with chronic kidney disease. Intracellular cholesterol metabolism is involved in the process of vascular cell calcification. In this study, we investigated the role of UbiA prenyltransferase domain containing 1 (UBIAD1) in intracellular cholesterol metabolism and vascular cell calcification, and identified its subcellular location. Primary human umbilical vein smooth muscle cells (HUVSMCs) were incubated with either growth medium (1.4 mmol/L Pi) or calcification medium (CM) (3.0 mmol/L Pi). Under treatment with CM, HUVSMCs were further incubated with exogenous cholesterol, or menaquinone-4, a product of UBIAD1. The plasmid and small interfering RNA were transfected in HUVSMCs to alter the expression of UBIAD1. Matrix calcium quantitation, alkaline phosphatase activity, intracellular cholesterol level and menaquinone-4 level were measured. The expression of several genes involved in cholesterol metabolism were analyzed. Using an anti-UBIAD1 antibody, an endoplasmic reticulum marker and a Golgi marker, the subcellular location of UBIAD1 in HUVSMCs was analyzed. CM increased matrix calcium, alkaline phosphatase activity and intracellular cholesterol level, and reduced UBIAD1 expression and menaquinone-4 level. Addition of cholesterol contributed to increased matrix calcification and alkaline phosphatase activity in a dose-dependent manner. Elevated expression of UBIAD1 or menaquinone-4 in HUVSMCs treated with CM significantly reduced intracellular cholesterol level, matrix calcification and alkaline phosphatase activity, but increased menaquinone-4 level. Elevated expression of UBIAD1 or menaquinone-4 reduced the gene expression of sterol regulatory element-binding protein-2, and increased gene expression of ATP binding cassette transporters A1, which are in charge of cholesterol synthesis and efflux. UBIAD1 co-localized with the endoplasmic reticulum marker and the Golgi marker in HUVSMCs. In conclusion, high intracellular cholesterol content contributes to phosphate-induced vascular cell differentiation and calcification. UBIAD1 or menaquinone-4 could decrease vascular cell differentiation and calcification, probably via its potent role of inversely modulating cellular cholesterol.

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<![CDATA[Parkinson-Related LRRK2 Mutation R1628P Enables Cdk5 Phosphorylation of LRRK2 and Upregulates Its Kinase Activity]]> https://www.researchpad.co/article/5989dabcab0ee8fa60baf0d8

Background

Recent studies have linked certain single nucleotide polymorphisms in the leucine-rich repeat kinase 2 (LRRK2) gene with Parkinson’s disease (PD). Among the mutations, LRRK2 c.4883G>C (R1628P) variant was identified to have a significant association with the risk of PD in ethnic Han-Chinese populations. But the molecular pathological mechanisms of R1628P mutation in PD is still unknown.

Principle Findings

Unlike other LRRK2 mutants in the Roc-COR-Kinase domain, the R1628P mutation didn’t alter the LRRK2 kinase activity and promote neuronal death directly. LRRK2 R1628P mutation increased the binding affinity of LRRK2 with Cyclin-dependent kinase 5 (Cdk5). Interestingly, R1628P mutation turned its adjacent amino acid residue S1627 on LRRK2 protein to a novel phosphorylation site of Cdk5, which could be defined as a typical type II (+) phosphorylation-related single nucleotide polymorphism. Importantly, we showed that the phosphorylation of S1627 by Cdk5 could activate the LRRK2 kinase, and neurons ectopically expressing R1628P displayed a higher sensitivity to 1-methyl-4-phenylpyridinium, a bioactive metabolite of environmental toxin MPTP, in a Cdk5-dependent manner.

Conclusion

Our data indicate that Parkinson-related LRRK2 mutation R1628P leads to Cdk5 phosphorylation of LRRK2 at S1627, which would upregulate the kinase activity of LRRK2 and consequently cause neuronal death.

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