Independent Cell Lines Research Articles

Identification of TAZ-Dependent Breast Cancer Vulnerabilities Using a Chemical Genomics Screening Approach.

Breast cancer stem cells (BCSCs) represent a subpopulation of tumor cells that can self-renew and generate tumor heterogeneity. Targeting BCSCs may ameliorate therapy resistance, tumor growth, and metastatic progression. However, the origin and molecular mechanisms underlying their cellular properties are poorly understood. The transcriptional coactivator with PDZ-binding motif (TAZ) promotes mammary stem/progenitor cell (MaSC) expansion and maintenance but also confers stem-like traits to differentiated tumor cells. Here, we describe the rapid generation of experimentally induced BCSCs by TAZ-mediated reprogramming of human mammary epithelial cells, hence allowing for the direct analysis of BCSC phenotypes. Specifically, we establish genetically well-defined TAZ-dependent (TAZDEP) and -independent (TAZIND) cell lines with cancer stem cell (CSC) traits, such as self-renewal, variable resistance to chemotherapeutic agents, and tumor seeding potential. TAZDEP cells were associated with the epithelial to mesenchymal transition, embryonic, and MaSC signature genes. In contrast, TAZIND cells were characterized by a neuroendocrine transdifferentiation transcriptional program associated with Polycomb repressive complex 2 (PRC2). Mechanistically, we identify Cyclin D1 (CCND1) as a critical downstream effector for TAZ-driven tumorigenesis. Overall, our results reveal a critical TAZ-CCND1-CDK4/CDK6 signaling axis, suggesting novel therapeutic approaches to eliminate both BCSCs and therapy-resistant cancer cells.

Revisiting the consequences of deleting the X inactivation center

Mammalian cells equalize X-linked dosages between the male (XY) and female (XX) sexes by silencing one X chromosome in the female sex. This process, known as "X chromosome inactivation" (XCI), requires a master switch within the X inactivation center (Xic). The Xic spans several hundred kilobases in the mouse and includes a number of regulatory noncoding genes that produce functional transcripts. Over three decades, transgenic and deletional analyses have demonstrated both the necessity and sufficiency of the Xic to induce XCI, including the steps of X chromosome counting, choice, and initiation of whole-chromosome silencing. One recent study, however, reported that deleting the noncoding sequences of the Xic surprisingly had no effect for XCI and attributed a sufficiency to drive counting to the coding gene, Rnf12/Rlim Here, we revisit the question by creating independent Xic deletion cell lines. Multiple independent clones carrying heterozygous deletions of the Xic display an inability to up-regulate Xist expression, consistent with a counting defect. This defect is rescued by a second site mutation in Tsix occurring in trans, bypassing the defect in counting. These findings reaffirm the essential nature of noncoding Xic elements for the initiation of XCI.

The chaperone-like protein Cdc48 regulates ubiquitin-proteasome system in plants.

The degradation of misfolded proteins is mainly mediated by the ubiquitin-proteasome system (UPS). UPS can be assisted by the protein Cdc48 but the relationship between UPS and Cdc48 in plants has been poorly investigated. Here, we analysed the regulation of UPS by Cdc48 in tobacco thanks to two independent cell lines overexpressing Cdc48 constitutively and plant leaves overexpressing Cdc48 transiently. In the cell lines, the accumulation of ubiquitinated proteins was affected both quantitatively and qualitatively and the number of proteasomal subunits was modified, while proteolytic activities were unchanged. Similarly, the over-expression of Cdc48 in planta impacted the accumulation of ubiquitinated proteins. A similar process occurred in leaves overexpressing transiently Rpn3, a proteasome subunit. Cdc48 being involved in plant immunity, its regulation of UPS was also investigated in response to cryptogein, an elicitor of immune responses. In the cell lines stably overexpressing Cdc48 and in leaves transiently overexpressing Cdc48 and/or Rpn3, cryptogein triggered a premature cell death while no increase of the proteasomal activity occurred. Overall, this study highlights a role for Cdc48 in ubiquitin homeostasis and confirms its involvement, as well as that of Rpn3, in the processes underlying the hypersensitive response.

All-Optical Electrophysiology in hiPSC-Derived Neurons With Synthetic Voltage Sensors.

Voltage imaging and “all-optical electrophysiology” in human induced pluripotent stem cell (hiPSC)-derived neurons have opened unprecedented opportunities for high-throughput phenotyping of activity in neurons possessing unique genetic backgrounds of individual patients. While prior all-optical electrophysiology studies relied on genetically encoded voltage indicators, here, we demonstrate an alternative protocol using a synthetic voltage sensor and genetically encoded optogenetic actuator that generate robust and reproducible results. We demonstrate the functionality of this method by measuring spontaneous and evoked activity in three independent hiPSC-derived neuronal cell lines with distinct genetic backgrounds.

Low Replicative Stress Triggers Cell-Type Specific Inheritable Advanced Replication Timing.

DNA replication timing (RT), reflecting the temporal order of origin activation, is known as a robust and conserved cell-type specific process. Upon low replication stress, the slowing of replication forks induces well-documented RT delays associated to genetic instability, but it can also generate RT advances that are still uncharacterized. In order to characterize these advanced initiation events, we monitored the whole genome RT from six independent human cell lines treated with low doses of aphidicolin. We report that RT advances are cell-type-specific and involve large heterochromatin domains. Importantly, we found that some major late to early RT advances can be inherited by the unstressed next-cellular generation, which is a unique process that correlates with enhanced chromatin accessibility, as well as modified replication origin landscape and gene expression in daughter cells. Collectively, this work highlights how low replication stress may impact cellular identity by RT advances events at a subset of chromosomal domains.

Application of machine learning to large in vitro databases to identify drug-cancer cell interactions: azithromycin and KLK6 mutation status.

Recent advances in machine learning promise to yield novel insights by interrogation of large datasets ranging from gene expression and mutation data to CRISPR knockouts and drug screens. We combined existing and new algorithms with available experimental data to identify potentially clinically relevant relationships to provide a proof of principle for the promise of machine learning in oncological drug discovery. Specifically, we screened cell line data from the Cancer Dependency Map for the effects of azithromycin, which has been shown to kill cancer cells in vitro. Our findings demonstrate a strong relationship between Kallikrein Related Peptidase 6 (KLK6) mutation status and the ability of azithromycin to kill cancer cells in vitro. While the application of azithromycin showed no meaningful average effect in KLK6 wild-type cell lines, statistically significant enhancements of cell death are seen in multiple independent KLK6-mutated cancer cell lines. These findings suggest a potentially valuable clinical strategy in patients with KLK6-mutated malignancies.

Acquisition of Resistance to MTI‐101 Selects for Favorable MET phenotype in Lung Cancer

Lung cancer has a high mutational burden and therefore, individualized therapies often prove to be most effective. The biggest obstacle is the development of resistance to these therapies. MTI-101 is a novel cancer therapeutic drug that has been shown to be effective in treatment of multiple cancers including prostate, multiple myeloma and lung cancer. Further analysis of MTI-101's effect on lung cancer could give insight to its mechanism of action. Two independent isogenic cell lines were created to analyze what leads to MTI-101 resistance. By comparing the Wild type and drug resistant lines derived from the same parent, a better understanding can be gained as to genotypic and phenotypic selection of MTI-101. Resistance to the drug illustrated the development of a reverse EMT phenotype. MTI-101 selects for cells with increased RNA and protein levels of E-cadherin, decreased vimentin, along with decreased migration and invasion capacity. It was also found that MTI-101 shows synergistic effects when combined with Osimertinib in EGFR driven cell lines. In addition, the cells MTI-101 selects for are more sensitive to Osimertinib as a single agent than the respective wild type. Therefore, this is a novel finding that MTI-101 selects for a less fit and less aggressive phenotype that proves more sensitive to current FDA approved drugs for lung cancer.

Examining FOLFIRINOX resistance in pancreatic cancer through creating resistant cell culture models.

Pancreatic ductal adenocarcinoma (PDAC) is extremely aggressive, often characterized by its early stage metastasis, resistance to cytotoxic chemotherapy, and extremely poor prognosis. FOLFIRINOX is the current standard of care for patients who can tolerate its side effects, and is a cocktail of four anti-cancer drugs: 5-fluorouricil, leucovorin, oxaliplatin, and irinotecan. This investigation focuses on chemotherapy resistance to FOLFIRINOX in multiple PDAC cell lines. Using dosages based on ratios of each drug given clinically, GI50 assays with a cocktail of all 4 anti-cancer drugs in FOLFIRINOX were conducted in four independent PDAC cell lines. Initial measurements for relative sensitivity based on the concentration of drug cocktail needed to inhibit the growth of 50% of the population, the PANC 03.27 cell line was found to be the most sensitive to FOLFIRINOX with a GI50 value of 0.71 mM, while PANC 05.04 was found to be the most resistant with a GI50 value of 1.50 mM. These two cell lines were chosen to then be cultured in FOLFIRINOX in order to create a model of chemotherapy resistance. Both PDAC lines were treated with their GI10 dosage for 3 days and then allowed to grow until confluent. This treatment process was repeated multiple times with the goal of building and maintaining resistance to FOLFIRINOX. Currently, both cell lines have undergone three FOLFIRINOX treatments, and GI50 assays are being run to identify their change in resistance. After resistance is achieved, RNA sequencing will be performed on both treated (resistant) and untreated cells. Changes in gene expression accompanying resistance may lend insight into the underlining mechanism of resistance in FOLFIRINOX experienced by many patients with pancreatic cancer.

The anti‐inflammatory and anti‐oxidative potential of synbiotics in two independent cell lines

This study was performed to help the researchers who are investigating or screening functional Lactobacillus strains with some kinds of prebiotics such as oligosaccharide, polysaccharide and fibre throughout serial in vitro study. We selected some Lactobacillus strains with prebiotics to be treated on two cell lines that we hypothesised them as gut and brain when the host consumes synergistic synbiotics. Selected synergistic synbiotics candidate significantly protected each cell against different stressors. The results may be used to develop supplementary functional dairy product as diet therapy or as medication to protect gut and brain who suffer from brain‐related disease.

The role of protein hydrolysates in prolonging viability and enhancing antibody production of CHO cells.

Four independent mAb-producing CHO cell lines were grown in media supplemented with one of seven protein hydrolysates of animal and plant origin. This generated a 7x4 matrix of replicate cultures which was analysed for viable cell density and mAb productivity. In all cultures, a consistent growth rate was shown in batch culture up to 4 to 5 days. Differences between cultures appeared in the decline phase which was followed up to 7 days beyond the start of the cultures. There was a marginal but significant overall increase(x1.1) in the integral viable cell density (IVCD) in the presence of hydrolysate but a more substantial increase in the cell-specific mAb (qMab) productivity (x1.5). There were individual differences between hydrolysates in terms of enhancement of mAb productivity, the highest being a 166% increase of mAb titre (to 117 mg/L) in batch cultures of CHO-EG2 supplemented with UPcotton hydrolysate. The effect of one of the most active hydrolysates (HP7504) on antibody glycosylation was investigated. This showed no change in the predominant seven glycans produced but a significant increase in the galactosylation and sialylation of some but not all the antibodies. Overall, the animal hydrolysate, Primatone and two cotton-derived hydrolysates provided the most substantial benefit for enhanced productivity. The cotton-based hydrolysates can be viewed as valuable supplements for animal-derived component-free (ADCF) media and as a source for the investigation of chemically defined bioactive components. KEY POINTS: • Protein hydrolysates enhanced both IVCD & qMab; the effect on qMab being consistently greater. • Cotton-based hydrolysates showed high bioactivity and potential for use in serum-free media. • Enhanced galactosylation and sialylation was shown for some of the Mabs tested.

Periprostatic adipose tissue promotes prostate cancer resistance to docetaxel by paracrine IGF‐1 upregulation of TUBB2B beta‐tubulin isoform

Growing evidence supports the pivotal role played by periprostatic adipose tissue (PPAT) in prostate cancer (PCa) microenvironment. We investigated whether PPAT can affect response to Docetaxel (DCTX) and the mechanisms associated. Conditioned medium was collected from the in vitro differentiated adipocytes isolated from PPAT which was isolated from PCa patients, during radical prostatectomy. Drug efficacy was studied by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide citotoxicity assay. Culture with CM of human PPAT (AdipoCM) promotes DCTX resistance in two different human prostate cancer cell lines (DU145 and PC3) and upregulated the expression of BCL‐xL, BCL‐2, and TUBB2B. AG1024, a well‐known IGF‐1 receptor inhibitor, counteracts the decreased response to DCTX observed in presence of AdipoCM and decreased TUBB2B expression, suggesting that a paracrine secretion of IGF‐1 by PPAT affect DCTX response of PCa cell. Collectively, our study showed that factors secreted by PPAT elicits DCTX resistance through antiapoptotic proteins and TUBB2B upregulation in androgen independent PCa cell lines. These findings reveal the potential of novel therapeutic strategies targeting adipocyte‐released factors and IGF‐1 axis to overcome DCTX resistance in patients with PCa.

In silico analysis of altered expression of long non-coding RNA in SARS-CoV-2 infected cells and their possible regulation by STAT1, STAT3 and interferon regulatory factors

Altered expression of long noncoding RNA (lncRNA), longer than 200 nucleotides without potential for coding protein, has been observed in diverse human diseases including viral diseases. It is largely unknown whether lncRNA would deregulate in SARS-CoV-2 infection, causing ongoing pandemic COVID-19. To identify, if lncRNA was deregulated in SARS-CoV-2 infected cells, we analyzed in silico the data in GSE147507. It was revealed that expression of 20 lncRNA like MALAT1, NEAT1 was increased and 4 lncRNA like PART1, TP53TG1 was decreased in at least two independent cell lines infected with SARS-CoV-2. Expression of NEAT1 was also increased in lungs tissue of COVID-19 patients. The deregulated lncRNA could interact with more than 2800 genes/proteins and 422 microRNAs as revealed from the database that catalogs experimentally determined interactions. Analysis with the interacting gene/protein partners of deregulated lncRNAs revealed that these genes/proteins were associated with many pathways related to viral infection, inflammation and immune functions. To find out whether these lncRNAs could be regulated by STATs and interferon regulatory factors (IRFs), we used ChIPBase v2.0 that catalogs experimentally determined binding from ChIP-seq data. It was revealed that any one of the transcription factors IRF1, IRF4, STAT1, STAT3 and STAT5A had experimentally determined binding at regions within -5kb to +1kb of the deregulated lncRNAs in at least 2 independent cell lines/conditions. Our analysis revealed that several lncRNAs could be regulated by IRF1, IRF4 STAT1 and STAT3 in response to SARS-CoV-2 infection and lncRNAs might be involved in antiviral response. However, these in silico observations are necessary to be validated experimentally.

Modulated Electro-Hyperthermia Facilitates NK-Cell Infiltration and Growth Arrest of Human A2058 Melanoma in a Xenograft Model.

Modulated electro-hyperthermia (mEHT), induced by 13.56 MHz radiofrequency, has been demonstrated both in preclinical and clinical studies to efficiently induce tumor damage and complement other treatment modalities. Here, we used a mouse xenograft model of human melanoma (A2058) to test mEHT (~42°C) both alone and combined with NK-cell immunotherapy. A single 30 min shot of mEHT resulted in significant tumor damage due to induced stress, marked by high hsp70 expression followed by significant upregulation of cleaved/activated caspase-3 and p53. When mEHT was combined with either primary human NK cells or the IL-2 independent NK-92MI cell line injected subcutaneously, the accumulation of NK cells was observed at the mEHT pretreated melanoma nodules but not at the untreated controls. mEHT induced the upregulation of the chemoattractant CXCL11 and increased the expression of the matrix metalloproteinase MMP2 which could account for the NK-cell attraction into the treated melanoma. In conclusion, mEHT monotherapy of melanoma xenograft tumors induced irreversible heat and cell stress leading to caspase dependent apoptosis to be driven by p53. mEHT could support the intratumoral attraction of distantly injected NK-cells, contributed by CXCL11 and MMP2 upregulation, resulting in an additive tumor destruction and growth inhibition. Therefore, mEHT may offer itself as a good partner for immunotherapy.

Differential CircRNA Expression Signatures May Serve as Potential Novel Biomarkers in Prostate Cancer.

Circular RNAs (circRNAs), a recently discovered non-coding RNA, have a number of functions including the regulation of miRNA expression. They have been detected in a number of malignancies including prostate cancer (PCa). The differential expression pattern of circRNAs associated with PCa and androgen receptor (AR) status was investigated in this study. circRNA profiling was performed using a high throughout microarray assay on a panel of prostate cell lines, which consisted of normal, benign, and malignant cells (n = 9). circRNAs were more commonly significantly up-regulated (p < 0.05) than downregulated in malignant cell lines (n = 3,409) vs. benign cell lines (n = 2,949). In a grouped analysis based on AR status, there were 2,127 down-regulated circRNAs in androgen independent cell lines compared to 2,236 in androgen dependent cell lines, thus identifying a potential circRNA signature reflective of androgen dependency. Through a bioinformatics approach, the parental genes associated with the top 10 differentially expressed circRNAs were identified such as hsa_circ_0064644, whose predicted parental gene target is RBMS3, and hsa_circ_0060539, whose predicted gene target is SDC4. Furthermore, we identified three circRNAs associated with the parental gene Caprin1 (hsa_circ_0021652, hsa_circ_0000288, and hsa_circ_0021647). Other studies have shown the importance of Caprin1 in PCa cell survival and drug resistance. Given the modified circRNA expression signatures identified here, these hypothesis generating results suggest that circRNAs may serve as potential putative diagnostic and predictive markers in PCa. However, further validation studies are required to assess the true potential of these markers in the clinical setting.

Facilitating Drug Discovery in Breast Cancer by Virtually Screening Patients Using In Vitro Drug Response Modeling.

Simple SummaryWhile patient datasets such as The Cancer Genome Atlas (TCGA) often contain a plethora of “-omics” data, the corresponding drug response information are limited and not suited for novel drug discovery. By integrating in vitro high throughput drug screening data and patient tumor molecular information, we created a virtual drug screening pipeline that enables drug discovery with simultaneous biomarker identification for a patient population. Using triple-negative breast cancer (TNBC) as our population of interest, we demonstrated the pipeline from lead identification, to biomarker discovery, to in vitro and in vivo validation of the compound AZD-1775.(1) Background: Drug imputation methods often aim to translate in vitro drug response to in vivo drug efficacy predictions. While commonly used in retrospective analyses, our aim is to investigate the use of drug prediction methods for the generation of novel drug discovery hypotheses. Triple-negative breast cancer (TNBC) is a severe clinical challenge in need of new therapies. (2) Methods: We used an established machine learning approach to build models of drug response based on cell line transcriptome data, which we then applied to patient tumor data to obtain predicted sensitivity scores for hundreds of drugs in over 1000 breast cancer patients. We then examined the relationships between predicted drug response and patient clinical features. (3) Results: Our analysis recapitulated several suspected vulnerabilities in TNBC and identified a number of compounds-of-interest. AZD-1775, a Wee1 inhibitor, was predicted to have preferential activity in TNBC (p < 2.2 × 10−16) and its efficacy was highly associated with TP53 mutations (p = 1.2 × 10−46). We validated these findings using independent cell line screening data and pathway analysis. Additionally, co-administration of AZD-1775 with standard-of-care paclitaxel was able to inhibit tumor growth (p < 0.05) and increase survival (p < 0.01) in a xenograft mouse model of TNBC. (4) Conclusions: Overall, this study provides a framework to turn any cancer transcriptomic dataset into a dataset for drug discovery. Using this framework, one can quickly generate meaningful drug discovery hypotheses for a cancer population of interest.

RIP-seq reveals LINE-1 ORF1p association with p-body enriched mRNAs

BackgroundLong INterspersed Element-1 (LINE-1) is an autonomous retroelement able to “copy-and-paste” itself into new loci of the host genome through a process called retrotransposition. The LINE-1 bicistronic mRNA codes for two proteins, ORF1p, a nucleic acid chaperone, and ORF2p, a protein with endonuclease and reverse transcriptase activity. Both proteins bind LINE-1 mRNA in cis and are necessary for retrotransposition. While LINE-1 transcription is usually repressed in most healthy somatic cells through a plethora of mechanisms, ORF1p expression has been observed in nearly 50% of tumors, and new LINE-1 insertions have been documented in a similar fraction of tumors, including prostate cancer.ResultsHere, we utilized RNA ImmunoPrecipitation (RIP) and the L1EM analysis software to identify ORF1p bound RNA in prostate cancer cells. We identified LINE-1 loci that were expressed in parental androgen sensitive and androgen independent clonal derivatives. In all androgen independent cells, we found higher levels of LINE-1 RNA, as well as unique expression patterns of LINE-1 loci. Interestingly, we observed that ORF1p bound many non-LINE-1 mRNA in all prostate cancer cell lines evaluated, and polyA RNA, and RNA localized in p-bodies were especially enriched. Furthermore, the expression levels of RNAs identified in our ORF1p RIP correlated with RNAs expressed in LINE-1 positive tumors from The Cancer Genome Atlas (TCGA).ConclusionOur results show a significant remodeling of LINE-1 loci expression in androgen independent cell lines when compared to parental androgen dependent cells. Additionally, we found that ORF1p bound a significant amount of non-LINE-1 mRNA, and that the enriched ORF1p bound mRNAs are also amplified in LINE-1 expressing TCGA prostate tumors, indicating the biological relevance of our findings to prostate cancer.

Distinct roles for the RNA-binding protein Staufen1 in prostate cancer

BackgroundProstate cancer is one of the most common malignant cancers with the second highest global rate of mortality in men. During the early stages of disease progression, tumour growth is local and androgen-dependent. Despite treatment, a large percentage of patients develop androgen-independent prostate cancer, which often results in metastases, a leading cause of mortality in these patients. Our previous work on the RNA-binding protein Staufen1 demonstrated its novel role in cancer biology, and in particular rhabdomyosarcoma tumorigenesis. To build upon this work, we have focused on the role of Staufen1 in other forms of cancer and describe here the novel and differential roles of Staufen1 in prostate cancer.MethodsUsing a cell-based approach, three independent prostate cancer cell lines with different characteristics were used to evaluate the expression of Staufen1 in human prostate cancer relative to control prostate cells. The functional impact of Staufen1 on several key oncogenic features of prostate cancer cells including proliferation, apoptosis, migration and invasion were systematically investigated.ResultsWe show that Staufen1 levels are increased in all human prostate cancer cells examined in comparison to normal prostate epithelial cells. Furthermore, Staufen1 differentially regulates growth, migration, and invasion in the various prostate cancer cells assessed. In LNCaP prostate cancer cells, Staufen1 regulates cell proliferation through mTOR activation. Conversely, Staufen1 regulates migration and invasion of the highly invasive, bone metastatic-derived, PC3 prostate cells via the activation of focal adhesion kinase.ConclusionsCollectively, these results show that Staufen1 has a direct impact in prostate cancer development and further demonstrate that its functions vary amongst the prostate cancer cell types. Accordingly, Staufen1 represents a novel target for the development of much-needed therapeutic strategies for prostate cancer.

Chemosensitizing effects of methyl jasmonate on paclitaxel-resistant androgen-dependent and independent prostate cancer cell lines

The taxane-based therapy provides survival benefit in patients with metastatic prostate cancer; however, the average survival is less than 20 months due to the partial taxane-related chemoresistance. Innovative strategies are needed to overcome the chemoresistance for improved patient survival. In this project, paclitaxel-resistance was developed on androgen-independent PC3 and androgen-dependent 22Rv1 and LNCaP human prostate cancer (PCa) cell lines to investigate the efficacy of methyl jasmonate (MeJa), an anti-cancer drug, in overcoming the chemoresistance. The PCa cell lines were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) at 37?C under 5% CO2. The cell lines were exposed to the gradually increasing doses of paclitaxel. Since the resistance on LNCaP could not be achieved, the study was continued with 22Rv1 cell line. It was demonstrated that paclitaxel-resistant cell lines overexpress ABCB1. Resistance levels of cells and MeJa activity in all resistant and parental lines were measured using CellTiter-Glo? luminescent assay. Test results were compared with Student?s t-test or analysis of variance (ANOVA). P?0.05 (two-tailed) was considered to be significant. In conclusion, MeJa showed more cytotoxicity on paclitaxel resistant PC3 (PC3-PtxR) cells than resistant 22Rv1 (22Rv1-PtxR) cells. Detection of cytotoxic effects of MeJa in overcoming paclitaxel resistance may contribute to the development of alternative new compounds for the prevention or chemosensitization of resistance to chemotherapeutics such as paclitaxel.

Targeted Gene Integration into Nuclear Genome of Microalgae Using Cre/loxPRecombination System

Genetically modified microalgae have been expected to be a useful tool for bioenergy and recombinant protein production. However, random integration of transgene in the microalgae nuclear genome is susceptible to gene silencing of heterologous gene expression. Here, we attempted to perform targeted gene integration into a pre-determined nuclear genomic site ofChlamydomonas reinhardtiiusing Cre/loxPrecombination system for stable transgene expression. We constructed an expression vector plasmid encoding reporter genes (zeocin resistant gene and green fluorescent protein gene;Zeo-2A-GFP) and mutatedloxPto generate founder cells. A donor vector encodingIFNα-4and paromomycin resistant genes flanked by corresponding mutatedloxPs was constructed and introduced into founder cells together with a Cre expression vector. The optimal ratio of donor vector to Cre expression vector was determined by counting the number of paromomycin resistant colonies. For the established clones, the targeted integration was confirmed by genomic PCR using various specific primer sets. Target genes in the donor vector could be integrated into the expected genomic site ofC. reinhardtiiusing Cre/loxPsystem. RT-PCR revealed thatIFNα-4was expressed in five independent transgenic cell lines tested. This result suggests that Cre-based cell engineering is a promising approach to generate smart microalgae expressing foreign genes.

Tropism of Coxsackie virus A9 depends on the +1 position of the RGD (arginine- glycine- aspartic acid) motif found at the C’ terminus of its VP1 capsid protein

An understanding of how viruses interact with their receptors is vital as this step is a major determinant of host susceptibility and disease. The enterovirus coxsackievirus A9 (CVA9) is an important pathogen responsible for respiratory infections, myocarditis, infections of the central nervous system, chronic dilated cardiomyopathy and possibly type I diabetes. CVA9 harbours an integrin- recognition motif, RGD (Arg-Gly-Asp), in the capsid protein VP1 and this motif is believed to be primarily responsible for binding to integrins αvβ6 and/or αvβ3 during cell entry. Despite the consistent conservation of RGD-flanking amino acids in multiple RGD-containing picornaviruses, the significance of these amino acids to cell tropism has not been thoroughly investigated. In this study we used 10 CVA9 mutants and a panel of cells to analyse cell tropism. We showed that CVA9 infection proceeds by either an RGD- dependent or an apparently RGD- independent pathway. Differences in the amino acid found at the +1 position of the RGD motif affect the cell tropism of CVA9 when an RGD- dependent pathway is used. Naturally occurring CVA9 isolates have either the sequence RGDM and RGDL and we found that the corresponding viruses in our panel infected cells most efficiently. There was also a strong selection pressure for RGDL in adaptation experiments. However, there was also an unexpected selection of an RGDL variant in an apparently RGD- independent cell line. There was also no simple relationship between infection of cells and expression of integrins αvβ3 and αvβ6. The results obtained have greatly improved our understanding of how CVA9 infects cells. This will be useful in the design of antivirus drugs and also gives a framework for the modification of CVA9 or other RGD containing picornaviruses for specific targeting of cancer cells for oncolytic therapy.