Protein ligand and nanotopography separately drive the phenotype of mouse embryonic stem cells.

Biochemical and biomechanical signals regulate stem cell function in the niche environments in vivo. Current in vitro culture of mouse embryonic stem cells (mESC) uses laminin (LN-511) to provide mimetic biochemical signaling (LN-521 for human systems) to maintain stemness. Alternative approaches propose topographical cues to provide biomechanical cues, however combined biochemical and topographic cues may better mimic the in vivo environment, but are largely unexplored for in vitro stem cell expansion. In this study, we directly compare in vitro signals from LN-511 and/or topographic cues to maintain stemness, using systematically-varied submicron pillar patterns or flat surfaces with or without preadsorbed LN-511. The adhesion of cells, colony formation, expression of the pluripotency marker,octamer-binding transcription factor 4 (Oct4), and transcriptome profiling were characterized. We observed that either biochemical or topographic signals could maintain stemness of mESCs in feeder-free conditions, indicated by high-level Oct4 and gene profiling by RNAseq. The combination of LN-511 with nanotopography reduced colony growth, while maintaining stemness markers, shifted the cellular phenotype indicating that the integration of biochemical and topographic signals is antagonistic. Overall, significantly faster (up to 2.5 times) colony growth was observed at nanotopographies without LN-511, suggesting for improved ESC expansion.

Effect of histone deacetylase 8 gene deletion on breast cancer cellular mechanism in vitro and in vivo study.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most aggressive type of cancer without any approved targeted therapy. Epigenetic processes have a pivotal role in cancer cell progression and while histone deacetylase 8 (HDAC8) has been proven as a potential oncogene in breast cancer, its underlying molecular mechanism is not known. Therefore, the present study, aimed to evaluate the underlying mechanism of the HDAC8 carcinogenesis in breast cancer progression. METHODS: The potential role of HDAC8 in cancer cell processes such as apoptosis, invasion, migration, angiogenesis, and cancer stem cells (CSCs) markers were evaluated by using flow cytometry Annexin V-FITC/propidium iodide (PI), reverse transcription-polymerase chain reaction (RT-qPCR), Matrigel-coated transwell plates and wound healing assay on both cell lines. The impact of HDAC8 on tumor development was also studied using a breast cancer xenograft model. RESULTS: HDAC8 expression was significantly downregulated in the cell lines, post-transfection with KO-vector. Downregulation of HDAC8 dramatically decreased cell migration, angiogenesis, and invasion while inducing apoptosis in MDAMB-468 and MDA-MB-231 cell lines. HDAC8 knocked out TNBC cell lines had lower levels of cancer stemness markers, such as prominin-1 (CD133), CD44, BMI1, and Aldehyde dehydrogenase 1 (ALDH1). Additionally, the knockout of HDAC8 inhibited tumor growth in a breast cancer xenograft model. CONCLUSION: The findings show that knocking out HDAC8 retains several anticancer actions in BC cells, such as inducing apoptosis, reducing migration, invasion, angiogenesis and removing CSCs markers.

miR-589-5p Inhibits Cell Proliferation by Targeting Histone Deacetylase 3 in Triple Negative Breast Cancer.

BACKGROUND: Histone deacetylase 3 (HDAC3) is a potential oncogene that is significantly up-regulated in patients with breast cancer. MicroRNAs (miRs) are a group of small non-coding and regulatory RNAs which have recently been proposed as promising molecules for breast cancer target therapy. In the current study, we investigated the impact of miR-589-5p/ HDAC3 axis on cancer cell development in triple negative breast cancer (TNBC) cells. METHODS: In-silico analysis determined that miR-589-5p potentially targets HDAC3. We evaluated the HDAC3 and mir-589-5p expression levels in clinical samples and breast cancer cell lines including MDA-MB-231, MDA-MB-468, MCF-7 and MCF-10A. HDAC3 was knocked out to investigate its role on cancer cell progression. Anti-cancerous role of the miR-589-5p was assessed using an expression vector. We evaluated possible alteration in the cell cycle progression, cell viability and cell proliferation, after transient transfection. RESULTS: HDAC3 was over-expressed in TNBC clinical samples and breast cancer cell lines compared to non-cancerous controls while miR-589-5p was down regulated in cancer cells. Suppression of HDAC3 decreased the cell viability, cell proliferation and colony formation. Similar results were observed after over-expression of the miR-589-5p. Dual-Luciferase reporter assay confirmed the direct targeting of HDAC3 by miR-589-5p. CONCLUSION: Our results showed that miR-589-5p mediates its anti-proliferative effects on breast cancer cells via targeting HDAC3. These findings suggest that the miR-589-5p/ HDAC3 axis could be considered as a possible therapeutic strategy in TNBC.

Relation between the circular and linear form of the Elongator Acetyltransferase Complex Subunit 3 in the progression of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive type of breast cancer (BC) that hardly responds to common treatment. Recent studies show that circ-ELP3 (Elongator Acetyltransferase Complex Subunit 3 or hsa-circ-0001785) is involved in the pathogenesis of several malignancies. The present study aimed to evaluate the possible role of this circRNA in the progression of TNBC cells and the possible relation between the circular and linear forms of the ELP3. We evaluated the circ-ELP3 and its host gene expression level in clinical samples and breast cancer cell lines. Using an expression vector, hsa-circ-0001785 was upregulated to investigate its role on cancer cell progression. After a transient transfection, we evaluated possible alterations in the cell cycle progression, cell viability, and cell proliferation. Quantitative real-time polymerase chain reaction analyses verified that circ-ELP3 and its host gene were significantly upregulated in TNBC tissues and breast cancer cells. Overexpression of circ-ELP3 markedly increases the cell viability and proliferation and also the formation of colonies in transfected cells compared to the controls. Briefly, our results showed that Circ-ELP3 and its host gene were significantly upregulated in TNBC. Circ-ELP3 is involved in TNBC progression and may exert its effects by indirectly regulating of ELP3 expression.

Co-delivery of STAT3 siRNA and methotrexate in breast cancer cells.

Co-delivery of anticancer drugs and biologics can provide synergetic effects and outperform single delivery therapies. Here, a nanoparticle (NP) system for co-delivery of methotrexate (MTX) and STAT3 siRNA has been developed and tested in vitro. Mesoporous silica nanoparticles (MSNs) were functionalized with chitosan (ch) by covalent grafting mediated by aminopropyl triethoxysilane (APTES) via glutaraldehyde as the linker. Co-delivery of MTX and STAT3 siRNA to MCF7 cells was demonstrated in cells by flow cytometric analysis and confocal laser scanning fluorescence microscopy for use in breast cancer treatment. MTX either competitively inhibits the dihydrofolate reductase (DHFR) receptor or suppresses the STAT3 metabolic pathway. STAT3 protein plays an essential role in cell division, proliferation and survival. Reduction of the protein by both MTX and STAT3 siRNA, achieved by chMSNs, significantly decreased the viability of breast cancer cells compared to single treatments alone. Cellular uptake of modified NPs was increased over time when additional free MTX was added implicating the DHFR receptor in uptake. In addition, protein corona compositions coated the NPs outer surface, were different between the NPs with and without drug potentially modulating cellular uptake. This study is the first report on co-delivery of MTX and STAT3 siRNA by chitosan modified MSNs.

Nanopore long-read sequencing of circRNAs.

Circular RNA (circRNA) is a group of highly stable RNA molecules with suggested roles in development and disease. They derive from linear pre-mRNAs when a 5'-splice site splices back to an upstream 3'-splice site in a process termed back-splicing. Most circRNAs are multi-exonic and may contain several thousand nucleotides. The extensive sequence overlap between the linear and circular forms of an RNA means that circRNA identification depends on the detection of back-splice-junction sequence reads that are unique to the circRNA. However, the short-read length obtained using standard next-generation sequencing techniques means that the internal sequence, exon composition and alternative splicing of circRNAs are unknown in many cases. Recently, several labs, including ours, have reported protocols for sequencing of circRNAs using long-read nanopore sequencing and thereby expanded our understanding of circRNA size distribution and internal splicing patterns. Here, we review these protocols and discuss the different approaches taken to study the full length composition of circRNAs.

Serum Circ-FAF1/Circ-ELP3: A novel potential biomarker for breast cancer diagnosis.

BACKGROUND: Recently, measurement of serum circular RNAs (circRNAs) as a non-invasive tumor marker has been considered more. We designed the present study to investigate the diagnostic efficiency of serum Circ-ELP3 and Circ-FAF1, separately and simultaneously, for diagnosis of patients with breast cancer. METHODS: Seventy-eight female patients diagnosed as primary breast cancer participated in this study. We measured the level of circRNAs in serum specimens of the studied subjects. A receiver operating characteristic (ROC) curve was plotted and the diagnostic efficiency for both circRNAs was determined. RESULTS: Compared to non-cancerous controls, Circ-ELP3 was upregulated in breast cancer patients (p-value = 0.004). On the other hand, serum Circ-FAF1 was seen to be decreased in breast cancer patients than controls (p-value = 0.001). According to ROC curve results, the area under the curve (AUC) for Circ-ELP3 and Circ-FAF1 was 0.733 and 0.787, respectively. Furthermore, the calculated sensitivity and specificity for Circ-ELP3 and Circ-FAF1 were 65, 64% and 77, 74%, respectively. Merging both circRNAs increased the diagnostic efficiency, with a better AUC, sensitivity and specificity values of 0.891, 96 and 62%, respectively. CONCLUSION: Briefly, our results revealed the high diagnostic value for combined circRNAs panel, including Circ-ELP3 and Circ-FAF1 as a non-invasive marker, in detection of breast carcinomas.

Nanopore sequencing of brain-derived full-length circRNAs reveals circRNA-specific exon usage, intron retention and microexons.

Circular RNA (circRNA) is a class of covalently joined non-coding RNAs with functional roles in a wide variety of cellular processes. Their composition shows extensive overlap with exons found in linear mRNAs making it difficult to delineate their composition using short-read RNA sequencing, particularly for long and multi-exonic circRNAs. Here, we use long-read nanopore sequencing of nicked circRNAs (circNick-LRS) and characterize a total of 18,266 and 39,623 circRNAs in human and mouse brain, respectively. We further develop an approach for targeted long-read sequencing of a panel of circRNAs (circPanel-LRS), eliminating the need for prior circRNA enrichment and find >30 circRNA isoforms on average per targeted locus. Our data show that circRNAs exhibit a large number of splicing events such as novel exons, intron retention and microexons that preferentially occur in circRNAs. We propose that altered exon usage in circRNAs may reflect resistance to nonsense-mediated decay in the absence of translation.

From the environment to the cells: An overview on pivotal factors which affect spreading and infection in COVID-19 pandemic.

Several factors ranging from environmental risks to the genetics of the virus and that of the hosts, affect the spread of COVID-19. The impact of physicochemical variables on virus vitality and spread should be taken into account in experimental and clinical studies. Another avenue to explore is the effect of diet and its interaction with the immune system on SARS-CoV-2 infection and mortality rate. Past year have witnessed extensive studies on virus and pathophysiology of the COVID-19 disease and the cellular mechanisms of virus spreading. However, our knowledge has not reached a level where we plan an efficient therapeutic approach to prevent the virus entry to the cells or decreasing the spreading and morbidity in severe cases of disease. The risk of infection directly correlates with the control of virus spreading via droplets and aerosol transmission, as well as patient immune system response. A key goal in virus restriction and transmission rate is to understand the physicochemical structure of aerosol and droplet formation, and the parameters that affect the droplet-borne and airborne in different environmental conditions. The lifetime of droplets on different surfaces is described based on the contact angle. Hereby, we recommend regular use of high-quality face masks in high temperature and low humidity conditions. However, in humid and cold weather conditions, wearing gloves and frequently hand washing, gain a higher priority. Additionally, social distancing rules should be respected in all aforementioned conditions. We will also discuss different routes of SARS-CoV-2 entry into the cells and how multiple genetic factors play a role in the spread of the virus. Given the role of environmental and nutritional factors, we discuss and recommend some strategies to prevent the disease and protect the population against COVID-19. Since an effective vaccine can prevent the transmission of communicable diseases and abolish pandemics, we added a brief review of candidate SARS-CoV-2 vaccines.

Expression of the miR-302/367 microRNA cluster is regulated by a conserved long non-coding host-gene.

MicroRNAs are important regulators of cellular functions. MiR-302/367 is a polycistronic miRNA cluster that can induce and maintain pluripotency. Here we investigate the transcriptional control and the processing of the miR-302 host-gene in mice. Our results indicate that the mmu-miR-302 host-gene is alternatively spliced, polyadenylated and exported from the nucleus. The regulatory sequences extend at least 2 kb upstream of the transcription start site and contain several conserved binding sites for both transcriptional activators and repressors. The gene structure and regulatory elements are highly conserved between mouse and human. So far, regulating miR-302 expression is the only known function of the miR-302 host-gene. Even though we here only provide one example, regulation of microRNA transcription might be a so far little recognized function of long non-coding RNA genes.

Evaluation of miR-302 promoter activity in transgenic mice and pluripotent stem cell lines.

Some miRNAs, including the miR-302 cluster, are critical regulators of the stemness state of embryonic stem cells and cell fate patterning. In this study, we evaluated the activity of the miR-302 core promotor in mice and human pluripotent stem cells, somatic tissue derivatives, and generated transgenic mice expressing EGFP under a miR-302 promoter. The expression of EGFP under the control of the miR-302 promotor was examined in the cell lines and somatic tissues of transgenic mice, transgenic blastocysts, and embryonic stem cells derived from transgenic blastocysts. Our results showed that the miR-302 promoter is highly expressed in the mouse and human pluripotent cells, weakly expressed in the somatic tissue derivatives, is highly expressed in both blastocysts and the first passages of transgenic embryonic stem cells, and lowly expressed in the somatic tissues of transgenic mice. It can be concluded that different temporal and spatial gene expression patterns occur during the embryonic and adult stages of cells in mice.

MiR-646 prevents proliferation and progression of human breast cancer cell lines by suppressing HDAC2 expression.

BACKGROUND: Breast cancer is a type of cancer with a high incidence and mortality rate worldwide. Change in epigenetic mechanisms enhances cancer cell progression. Histon deacetylase 2 (HDAC2) was found to act as a potential oncogene in different malignancies. For better understanding the mechanisms related to breast cancer development, we investigated the role of HDAC2 in breast cancer and the inhibitory effect of miR-646 on this oncogene. METHODS: A total of thirty cancerous tissues and 30 adjacent non-cancerous specimens and also three breast cancer cell lines were enrolled in the study. Quantitative reverse transcriptase PCR (qRT-PCR) was employed to detect the HDAC2 and miR-646 expression level in the studied samples. The biological roles of HDAC2 and miR-646 were investigated through manipulating the expression level of HDAC2 or miR-646 in breast cancer cells. Finally, we evaluated whether the HDAC2 is a direct target for miR-646. RESULTS: In this study, we found HDAC2 is significantly upregulated in cancerous specimens and cell lines compared to non-cancerous tissues and normal cell line. On the other hand, miR-646 expression was decreased in clinical specimens and breast cancer cells compared to non-cancerous samples. Knocking out of the HDAC2 and overexpression of miR-646 inhibited breast cancer cell growth but promoted cell death, while untreated groups showed inverse results. Furthermore, we showed that in the breast cancer cells, miR-646 regulates the progression and proliferation by suppressing HDAC2. CONCLUSION: Taken together, our study identified a miR-646/HDAC2 regulatory function in the breast cancer development and introduced a therapeutically target for breast cancer.

Biosynthesis of Circular RNA ciRS-7/CDR1as Is Mediated by Mammalian-wide Interspersed Repeats.

Circular RNAs (circRNAs) are stable non-coding RNAs with a closed circular structure. One of the best studied circRNAs is ciRS-7 (CDR1as), which acts as a regulator of the microRNA miR-7; however, its biosynthetic pathway has remained an enigma. Here we delineate the biosynthetic pathway of ciRS-7. The back-splicing events that form circRNAs are often facilitated by flanking inverted repeats of the primate-specific Alu elements. The ciRS-7 gene lacks these elements, but, instead, we identified a set of flanking inverted elements belonging to the mammalian-wide interspersed repeat (MIR) family. Splicing reporter assays in HEK293 cells demonstrated that these inverted MIRs are required to generate ciRS-7 through back-splicing, and CRISPR/Cas9-mediated deletions confirmed the requirement of the endogenous MIR elements in SH-SY5Y cells. Using bioinformatic searches, we identified several other MIR-dependent circRNAs and confirmed them experimentally. We propose that MIR-mediated RNA circularization is used to generate a subset of mammalian circRNAs.

Association of HDAC8 Expression with Pathological Findings in Triple Negative and Non-Triple Negative Breast Cancer: Implications for Diagnosis.

Background: Previous data have shown the tumorigenicity roles of histone deacetylase 8 (HDAC 8) in breast cancer. More recently, the oncogenic effects of this molecule have been revealed in triple negative breast cancer (TNBC). The present study aimed to determine the diagnostic value of HDAC8 for the differentiation of TNBC from nTNBC tumors. Methods: A total of 50 cancerous and normal adjacent tumor specimens were obtained, and the clinical and pathological findings of studied subjects were recorded. The expression of HDAC8 gene was determined by qRT-PCR. Also, immunohistochemical staining was performed on tissue samples. Results: Our results showed that the expression of HDAC8 in breast cancer tissues was significantly higher than the normal adjacent tissues (p = 0.0011). HDAC8 expression was also observed to be higher in TNBC patients than nTNBC group (p = 0.0013). In addition, in the TNBC group, there was a significant association between the HDAC8 overexpression and tumor characteristics, including tumor size (p = 0.039), lymphatic invasion (p = 0.01), tumor grade (p = 0.02), and perineural invasion (p < 0.05). The cut-off value was fixed at 0.6279 r.u., and the corresponding sensitivity and specificity were found to be 73.91% and 70.37%, respectively. Conclusion: According to the findings, among the other markers, HDAC8 oncogene may be used as a potential tumor marker in the diagnosis of TNBC tumors.

miR-483-3p suppresses the proliferation and progression of human triple negative breast cancer cells by targeting the HDAC8>oncogene.

Triple negative breast cancer (TNBC) is a heterogeneous subclass of breast cancer (BC) distinguished by lack of hormone receptor expression. It is highly aggressive and difficult to treat with traditional chemotherapeutic regimens. Targeted-therapy using microRNAs (miR) has recently been proposed to improve the treatment of TNBC in the early stages. Here, we explore the roles of miR-483-3p/HDAC8 HDAC8 premiR-vector on tumorigenicity in TNBC patients. Clinical TNBC specimens and three BC cell lines were prepared. miR-483-3p and expression levels were measured using quantitative real-time polymerase chain reaction. Cell cycle progression was assessed by a flow-cytometry method. We also investigated cell proliferation by 3-2, 5-diphenyl tetrazolium bromide assay and colony formation assay. We used a to overexpress miR-483-3p, and a HDAC8-KO-vector for knocking out the endogenous production of HDAC8. Our data showed significant downregulation of miR-483-3p expression in TNBC clinical and cell line samples. The HDAC8 was also upregulated in both tissue specimens and BC cell lines. We found that increased levels of endogenous miR-483-3p affects tumorigenecity of MDA-MB-231. Downregulation of HDAC8 using the KO-vector showed the same pattern. Our results revealed that the miR-483-3p suppresses cellular proliferation and progression in TNBC cell lines via targeting HDAC8. Overall, our outcomes demonstrated the role of miR-483-3p as a tumor suppressor in TNBC and showed the possible mechanism via HDAC8. In addition, targeted treatment of TNBC with miR-483-3p might be considered in the future.

MiR-216b-5p inhibits cell proliferation in human breast cancer by down-regulating HDAC8 expression.

AIM: Over-expression of histone deacetylase 8 (HDAC8) has been demonstrated in breast cancer. But the underlying molecular mechanism of HDAC8 on the progression of breast cancer remains unknown. MicroRNAs (miRs) are proposed as important molecules in cancer progression by targeting specific oncogenes or tumor-suppressor genes. Our overall objective was to assess the miR-216b-5p role on HDAC8; and its impacts on breast cancer (BC) progression. MAIN METHODS: We acquired cancerous and noncancerous tissues from Iran Tumor Bank (I.T.B). The MDA-MB-231, MCF-7 and MCF-10A BC cell lines were also purchased. The tissue and cell line expression levels of miR-216b-5p and HDAC8 were determined by quantitative real-time PCR (qPCR). We next measured protein levels of HDAC8 by Western blotting assay. The cell cycle, cell proliferation, and colony formation assay were determined. Finally, we investigated the role of HDAC8 using a knockout vector; and confirmed the targeting of 3' untranslated region (3'-UTR) of HDAC8 through miR-216b-5p using a luciferase reporter assay. KEY FINDINGS: Our results demonstrated a significant decrease in miR-216b-5p, and remarkable increase in HDAC8 levels within human breast cancer tissues and cell lines. The lower levels of miR-216b-5p were negatively correlated with lymph node metastasis and advanced tumor size. The overexpression of miR-216b-5p in BC cell lines inhibited cellular proliferation and progression. HDAC8 was directly down-regulated by miR-216b-5p and knockout of HDAC8 showed the similar effects as miR-216b-5p overexpression. SIGNIFICANCE: Briefly, HDAC8 is an oncogene that accelerate breast cancer proliferation and progression and miR-216b-5p modulates those functions by binding to HDAC8 3'-UTR.

Isolation of cancer stem cells by selection for miR-302 expressing cells.

BACKGROUND: Cancer stem cells are believed to be a major reason for long-term therapy failure because they are multi-drug resistant and able to rest mitotically inactive in the hypoxic center of tumors. Due to their variable number and their often low proliferation rate, cancer stem cells are difficult to purify in decent quantities and to grow in cell culture systems, where they are easily outcompeted by faster growing more 'differentiated', i.e., less stem cell-like tumor cells. METHODS: Here we present a proof of principle study based on the idea to select cancer stem cells by means of the expression of a stem cell-specific gene. A selectable egfp-neo coding sequence was inserted in the last exon of the non-coding murine miR-302 host gene. As a stem cell specific regulatory element, 2.1 kb of the genomic region immediately upstream of the miR-302 host gene transcription start site was used. Stable transgenic CJ7 embryonic stem cells were used to induce teratomas. RESULTS: After three weeks, tumors were removed for analysis and primary cultures were established. Stem cell-like cells were selected from these culture based on G418 selection. When the selection was removed, stem cell morphology and miR-302 expression were rapidly lost, indicating that it was not the original ES cells that had been isolated. CONCLUSIONS: We show the possibility to use drug resistance expressed from a regulatory sequence of a stem cell-specific marker, to isolate and propagate cancer stem cells that otherwise might be hidden in the majority of tumor cells.

Generation of Fam83h knockout mice by CRISPR/Cas9-mediated gene engineering.

Family with sequence similarity 83 member H (FAM83H) protein-coding geneplay an essential role in the structural organization, calcification of developing enamel, and keratin cytoskeleton disassembly by recruiting Casein kinase 1 alpha (CSNK1A1) to keratin filaments. In this study, we have applied CRISPR Cas9 nickase (D10A) to knockout (KO) the Fam83h gene in NMRI outbred mice. We generated homozygous Fam83h KO mice ( Fam83h Ko/Ko ) through a premature termination codon, which was validated by Sanger sequencing in F0 generation. Next, we also bred the FAM83H KO for two generations. Reverse-transcription polymerase chain reaction and Western blot analysis approved the Fam83h KO mice. The Fam83h KO mice had evidence of normal morphology at the cervical loops, secretory and maturation stages, and mandibular molars. In comparison with the normal wild-type mice ( Fam83h W/W ), the F2 homozygous KO ( Fam83h Ko/Ko ) had sparse, scruffy coats with small body size and decreased general activity. Also, they had the natural reproductive ability and natural lifespan. In addition, delay in opening the eyes and dry eyes among infant mice were seen. The F1 heterozygous mice looked comparable to the normal wild-type mice ( Fam83h W/W ), which showed autosomal recessive inheritance of these phenotypes. The KO of FAM83H had controversial effects on the development of teeth and the formation of enamel. The phenotype defect in dental development and the enamel formation were seen in three mice among four generations. It can be concluded that null FAM83H in outbred mice not only showed the reported phenotypes in null inbred mouse but also showed normal lifespan and reproductive ability; dental deficiency in three homozygous mice; and the symptoms that were similar to the symptoms of dry eye syndrome and curly coat dog syndrome in all four evaluated KO generations.

A Modified Monomeric Red Fluorescent Protein Reporter for Assessing CRISPR Activity.

Gene editing in human embryonic stem cells (hESCs) has been significantly enhanced by the discovery and development of CRISPR Cas9, a programmable nuclease system that can introduce targeted double-stranded breaks. The system relies on the optimal selection of a sgRNA sequence with low off-targets and high efficiency. We designed an improved monomeric red fluorescent protein reporter, GEmCherry2, for assessing CRISPR Cas9 activity and for optimizing sgRNA. By incorporating an out-of-frame sequence to the N-terminal of the red fluorescent protein mCherry, we created a visual tool for assessing the indel frequency after cutting with CRISPR Cas9. When a sgRNA-Cas9 construct is co-transfected with a corresponding GEmCherry2 construct, single nucleotide indels can move the GEmCherry2 sequence back in-frame and allow quantification and comparison of the efficiency of different sgRNA target sites by measuring red fluorescence. With this GEmCherry2 assay, we compared four target sites in the safe harbor AAVS1 locus and found significant differences in target site activity. We verified the activity using TIDE, which ranked our target sites in a similar order as the GEmCherry2 system. We also identified an AAV short inverted terminal repeat sequence within the Cas9 construct that, upon removal significantly improved transient transfection and expression in hESCs. Moreover, using GEmCherry2, we designed a sgRNA to target SORCS2 in hESCs and successfully introduced indels into the coding sequence of SORCS2.

Anionic peroxidase production by Arnebia euchroma callus.

Arnebia euchroma callus, obtained from the root cell culture of an Iranian native specimen, has gained a doubling time of 63 H after regular subculturing on Linsmaier-Skoog (LS) medium containing sugar (50 g/L), 2,4-dichlorophenoxyacetic acid (10(-6) M), and kinetin (10(-5) M) under darkness at 25°C. Despite the observed somaclonal variations, peroxidase production by the A. euchroma calli has been stable over 4 years under the aforementioned conditions. Isoelectric focusing experiments revealed that the partially purified A. euchroma peroxidases (AePoxs) are mainly anionic with pI values of about 5.5 and 6.6. AePox reaches its optimal activity at 55°C and pH 7.5. Results of the various kinetic studies suggest that AePox belongs to the type III plant peroxidases with no activity for the oxidation of 3-indoleacetic acid, but seems to play a role in the lignin biosynthesis and H(2) O(2) regulation during the proliferation of the A. euchroma cells on LS medium. Comparing the biochemical properties of AePox with horseradish peroxidase and in view of the ease of solid cell culture, the A. euchroma callus could be considered as a source of plant peroxidase for some biotechnological applications.