Relaxed chromatin induced by histone deacetylase inhibitors improves the oligonucleotide-directed gene editing in plant cells

Improving efficiency of oligonucleotide-directed mutagenesis (ODM) is a prerequisite for wide application of this gene-editing approach in plant science and breeding. Here we have tested histone deacetylase inhibitor treatments for induction of relaxed chromatin and for increasing the efficiency of ODM in cultured maize cells. For phenotypic assay we produced transgenic maize cell lines expressing the non-functional Green Fluorescent Protein (mGFP) gene carrying a TAG stop codon. These transgenic cells were bombarded with corrective oligonucleotide as editing reagent to recover GFP expression. Repair of green fluorescent protein function was monitored by confocal fluorescence microscopy and flow cytometry was used for quantification of correction events. Sequencing PCR fragments of the GFP gene from corrected cells indicated a nucleotide exchange in the stop codon (TAG) from T to G nucleotide that resulted in the restoration of GFP function. We show that pretreatment of maize cells with sodium butyrate (5–10 mM) and nicotinamide (1–5 mM) as known inhibitors of histone deacetylases can cause elevated chromatin sensitivity to DNase I that was visualized in agarose gels and confirmed by the reduced presence of intact PCR template for the inserted exogenous mGFP gene. Maize cells with more relaxed chromatin could serve as an improved recipient for targeted nucleotide exchange as indicated by an average of 2.67- to 3.62-fold increase in GFP-positive cells. Our results stimulate further studies on the role of the condition of the recipient cells in ODM and testing the application of chromatin modifying agents in other, programmable nuclease-based genome-editing techniques in higher plants.     

Variations in the treatment of early-stage breast cancer in Quebec between 1988 and 1994

BACKGROUND: The influence of organizational factors on the process and outcomes of the treatment of breast cancer has been extensively investigated. Although the quality of care is presumed to be better in larger centres, evidence is inconsistent. This study was conducted to determine whether therapies for patients with breast cancer varied according to hospital caseload. METHODS: Women newly diagnosed between 1988 and 1994 with early-stage node-negative primary breast cancer were randomly selected from the Quebec tumour registry and the Quebec hospital discharge database. Data were collected from medical charts, and only women having undergone dissection of the axilla were included in the analyses. Logistic regression analysis was used to adjust for case mix and organizational variables. RESULTS: The final sample included 1259 patients with node-negative stage I or II primary breast cancer. The proportion of women who underwent breast-conserving surgery increased significantly with hospital caseload (from 78.0% in hospitals admitting fewer than 25 new cases each year to 88.0% in those admitting 100 patients or more; p for trend < 0.001). This trend remained significant even after statistical adjustment for case mix and organizational factors (p for trend = 0.001). Of the 1039 women who underwent breast-conserving surgery 965 (92.9%) received radiotherapy. Use of systemic adjuvant therapy (tamoxifen or chemotherapy, or both) increased with the number of patients treated in a given centre (from 60.1% to 68.5%), but this trend disappeared after adjustment for case mix and other factors. The proportion of patients receiving systemic adjuvant therapy consistent with published consensus guidelines tended to increase with caseload for those treated in hospitals participating in multicentre clinical trials but decrease with caseload for patients in hospitals not involved in clinical research. INTERPRETATION: The care of patients in Quebec with early-stage breast cancer is characterized by a high prevalence of both breast-conserving surgery and systemic adjuvant therapy. Large centres, especially those actively involved in clinical research, rapidly adopt innovative therapeutic modalities.     

Application of dielectric spectroscopy for monitoring high cell density in monoclonal antibody producing CHO cell cultivations

The application of dielectric spectroscopy was frequently investigated as an on-line cell culture monitoring tool; however, it still requires supportive data and experience in order to become a robust technique. In this study, dielectric spectroscopy was used to predict viable cell density (VCD) at industrially relevant high levels in concentrated fed-batch culture of Chinese hamster ovary cells producing a monoclonal antibody for pharmaceutical purposes. For on-line dielectric spectroscopy measurements, capacitance was scanned within a wide range of frequency values (100–19,490 kHz) in six parallel cell cultivation batches. Prior to detailed mathematical analysis of the collected data, principal component analysis (PCA) was applied to compare dielectric behavior of the cultivations. PCA analysis resulted in detecting measurement disturbances. By using the measured spectroscopic data, partial least squares regression (PLS), Cole–Cole, and linear modeling were applied and compared in order to predict VCD. The Cole–Cole and the PLS model provided reliable prediction over the entire cultivation including both the early and decline phases of cell growth, while the linear model failed to estimate VCD in the later, declining cultivation phase. In regards to the measurement error sensitivity, remarkable differences were shown among PLS, Cole–Cole, and linear modeling. VCD prediction accuracy could be improved in the runs with measurement disturbances by first derivative pre-treatment in PLS and by parameter optimization of the Cole–Cole modeling.     

Mitosis-specific promoter of the alfalfa cyclin-dependent kinase gene (Medsa;CDKB2;1) is activated by wounding and ethylene in a non-cell division-dependent manner

Cyclin-dependent serine/threonine kinases (CDKs) have pivotal roles in regulating the eukaryotic cell cycle. Plants possess a unique class of CDKs (B-type CDKs) with preferential protein accumulation at G2/M-phases; however, their exact functions are still enigmatic. Here we describe the functional characterization of a 360-bp promoter region of the alfalfa (Medicago sativa) CDKB2;1 gene in transgenic plants and cell lines. It is shown that the activity of the analyzed promoter was characteristic for proliferating meristematic regions in planta and specific for cells in the G2/M-phases in synchronized cell cultures. Immunohistochemical analysis of transgenic root sections further confirmed the correlation of the expression of the CDKB2;1 promoter-linked reporter genes with the accumulation of the correspondent kinase. It was found that, in addition to auxin (2,4-dichlorophenoxyacetic acid) treatment, wounding could also induce both the reporter and endogenous genes in transgenic leaf explants. Furthermore, ethylene, known as a wound-response mediator, had a similar effect. The gene activation in response to wounding or ethephon was faster and occurred without the induction of cell cycle progression in contrast to the control auxin treatment. In silico analysis of this promoter indeed revealed the presence of a set of cis-elements, indicating not only cell cycle- but wound- and ethylene-dependent regulation of this CDK gene. Based on the presented data, we discuss the functional significance of the complex regulation of mitosis-specific CDK genes in plants.     

Germ cells in the crustacean Parhyale hawaiensis depend on Vasa protein for their maintenance but not for their formation

Germ cells are a population of cells that do not differentiate to form somatic tissue but form the egg and sperm that ensure the reproduction of the organism. To understand how germ cells form, holds a key for identifying what sets them apart from all other cells of the organism. There are large differences between embryos regarding where and when germ cells form but the expression of Vasa protein is a common trait of germ cells. We studied the role of vasa during germ cell formation in the crustacean Parhyale hawaiensis. In a striking difference to the posterior specification of the group of germ cells in the arthropod model Drosophila, all germ cells in Parhyale originate from a single germ line progenitor cell of the 8-cell stage. We found vasa RNA ubiquitously distributed from 1-cell to 16-cell stage in Parhyale and localized to the germ cells from 32-cell stage onwards. Localization of vasa RNA to the germ cells is controlled by its 3′UTR and this could be mimicked by fluorescently labeled 3′UTR RNA. Vasa protein was first detectable at the 100-cell stage. MO-mediated inhibition of vasa translation caused germ cells to die after gastrulation. This means that in Parhyale Vasa protein is not required for the initial generation of the clone of germ cells but is required for their subsequent proliferation and maintenance. It also means that the role of vasa changed substantially during an evolutionary switch in the crustaceans by Parhyale from the specification of a group of germ cells to that of a single germ line progenitor. This is the first functional study of vasa in an arthropod beyond Drosophila.     

Debaryomyces psychrosporus sp. nov., a yeast species from a Venezuelan cave

Three yeast strains, which are phenotypically indistinguishable from Debaryomyces hansenii, were recovered from secondary mineral deposits (stalactites and stromatolites) obtained in the Crystal Eyes Cave, Roraima Tepui Mountain, Venezuela. Analyses of the D1/D2 domains of the LSU rRNA gene as well as the concatenated sequences of the nearly entire SSU rRNA gene, the ITS regions and the D1/D2 domains of the LSU rRNA gene confirmed the placement of these strains in the genus Debaryomyces, but relationship with all valid species of D. hansenii complex was distant. Based on the observed considerable sequence divergence the three strains are proposed as a new species, D. psychrosporus sp. nov., with the type strain NCAIM Y.01972(T) (CBS 11845(T), NRRL Y-48723(T)).     

Identification of differentially expressed genes in isogenic highly metastatic and poorly metastatic cell lines of R3230AC rat mammary adenocarcinoma

Tumour metastasis occurs as a result of a cascade of events including alterations in the expression of various genes. The identification of such genes is essential to understanding formation of metastasis. In a previous study, highly metastatic (LN4.D6) and poorly metastatic (CAb.D5) cell lines were obtained from the rat mammary adenocarcinoma cell line R3230AC. Subtractive hybridization was used to identify differentially expressed genes between these two cell lines. We identified eight cDNA clones in CAb.D5 and six cDNA clones in LN4.D6 that were differentially expressed. One of the cDNA clones in each cell line had no homology with known sequences. Expression patterns of these differentially expressed genes were examined in a pair of rat mammary and prostate adenocarcinoma cell lines. Compared with cell lines examined, cDNA FF-10 was only expressed in CAb.D5; however, cDNA RB-8, RE-1, RF-5 were only expressed in the highly metastatic LN4.D6. No correlation was observed between expression patterns of the differentially expressed genes and metastatic potential of these cells. However, differential expression of genes, especially cytokeratins (CK8 and CK5) and collagens (III and IV) between highly metastatic and low metastatic rat mammary adenocarcinoma cell lines might initiate further investigation of these genes in metastatic process.