In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express.     

Use of Genetically Engineered Microorganisms (GEMs) for the Bioremediation of Contaminants

ABSTRACT

This paper presents a critical review of the literature on the application of genetically engineered microorganisms (GEMs) in bioremediation. The important aspects of using GEMs in bioremediation, such as development of novel strains with desirable properties through pathway construction and the modification of enzyme specificity and affinity, are discussed in detail. Particular attention is given to the genetic engineering of bacteria using bacterial hemoglobin (VHb) for the treatment of aromatic organic compounds under hypoxic conditions. The application of VHb technology may advance treatment of contaminated sites, where oxygen availability limits the growth of aerobic bioremediating bacteria, as well as the functioning of oxygenases required for mineralization of many organic pollutants. Despite the many advantages of GEMs, there are still concerns that their introduction into polluted sites to enhance bioremediation may have adverse environmental effects, such as gene transfer. The extent of horizontal gene transfer from GEMs in the environment, compared to that of native organisms including benefits regarding bacterial bioremediation that may occur as a result of such transfer, is discussed. Recent advances in tracking methods and containment strategies for GEMs, including several biological systems that have been developed to detect the fate of GEMs in the environment, are also summarized in this review. Critical research questions pertaining to the development and implementation of GEMs for enhanced bioremediation have been identified and posed for possible future research.     

Two different docetaxel resistant MCF-7 sublines exhibited different gene expression pattern

The objective of the present study was to investigate gene expression pattern of two docetaxel resistant MCF-7 breast carcinoma sublines step wisely selected in 30 and 120 nM docetaxel. Cell proliferation assay was performed in order to demonstrate development of docetaxel resistance. cDNA microarray analysis was performed using Affymetrix^® Human Genome U133 Plus 2.0 Arrays in duplicate experiments. Quantitative and semi-quantitative gene expression analysis was also performed to confirm gene expression analysis for selected genes. XTT results demonstrated that 30 (MCF-7/30nM DOC) and 120 nM (MCF-7/120nM DOC) docetaxel selected cells were 13- and 47-fold resistant, respectively. cDNA microarray analysis demonstrated that expression profiles of MCF-7 and MCF-7/30nM DOC were more similar to each other where expression profile of MCF-7/120nM DOC was different as examined by line graphs and scatter plots. 2,837 and 4,036 genes were significantly altered in 30 and 120 nM docetaxel resistant sublines, respectively. Among these, 849 genes were altered in common in two docetaxel resistant sublines. Antiapoptotic gene expression (e.g., Bcl-2 and APRIL) were noticeably altered in MCF-7/30nM DOC. However, docetaxel resistance in MCF-7/120nM DOC were more complicated with the involvement of ECM related gene expression, cytokine and growth factor signaling, ROS metabolism and EMT related gene expression together with higher level of MDR1 expression. Expression profiles in 30 and 120 nM docetaxel resistant sublines changed gradually with increasing resistance index. Drug resistance development seems to be step wise event in MCF-7 cells.     

Investigation of extracellular medium osmolality depending on zinc application and incubation time on A549 cancer cells

Changes in the osmolality of the extracellular medium (ECM) affect cell volume and cellular processes such as cell migration and proliferation. Not only may high concentrations of zinc (Zn) lead to cell death by apoptosis, but Zn is also a physiological suppressor of apoptosis. The aim of our study was to examine whether Zn and regulation of extracellular osmolality had an effect on the lung cancer cell line (A549) and how to be changed in ECM according to elements and osmolality depending on incubation time and Zn application. Our study consisted of four groups: cell-free medium, ECM of cancer cell after 24 h incubation (24hECM), ECM of cancer cell after 48 h incubation (48hECM), and ECM of cancer cell after 48 h incubation with ZnCl₂ (48hECM?+?Zn). ECM osmolality was measured by using osmometer, and the levels of chromium (Cr), iron (Fe), and magnesium (Mg) elements were analyzed using ICP-OES device for all groups. According to the result of the analysis, a statistically significant difference was found when osmolality and element values of ECM of 24hECM and 48hECM groups were compared with the values of the 48hECM?+?Zn group. It was observed that there was a decrease in the levels of Cr, Fe, and Mg with Zn application and incubation period in ECM. The regulation of ECM osmolality is a promising method due to biophysical effects on cancer cells. In our study, we speculated that the understanding of the effects of Zn and osmolality with the relationship between ECM and cancer cell might lead to the discovery of biophysical approaches as a novel therapeutic strategy.

     

Detection of SV40 DNA sequences in malignant mesothelioma specimens from the United States, but not from Turkey

The incidence of malignant mesothelioma (MM) shows a strong epidemiological association with exposure to asbestos fibers. Recently, simian virus 40 (SV40) DNA sequences have been reported in MM tumor specimens from the United States and several European countries, and the SV40 tumor virus has been implicated as a potential co-factor in the etiology of this disease. However, several large studies from the US, Finland, and Turkey did not detect SV40 sequences in MM samples. To address this discrepancy, MM specimens from Turkey and the US were analyzed in the same laboratory under identical conditions to detect the presence of SV40 DNA. We detected SV40 sequences in 4 of 11 specimens from the United States, but in none of the 9 Turkish samples examined. These findings suggest that geographical differences exist with regard to the involvement of SV40 in human tumors.     

Regulation of lysophosphatidate signaling by autotaxin and lipid phosphate phosphatases with respect to tumor progression, angiogenesis, metastasis and chemo-resistance

Evidence from clinical, animal and cell culture studies demonstrates that increased autotaxin (ATX) expression is responsible for enhancing tumor progression, cell migration, metastases, angiogenesis and chemo-resistance. These effects depend mainly on the rapid formation of lysophosphatidate (LPA) by ATX. Circulating LPA has a half-life of about 3 min in mice and it is degraded by the ecto-activities of lipid phosphate phosphatases (LPPs). These enzymes also hydrolyze extracellular sphingosine 1-phosphate (S1P), a potent signal for cell division, survival and angiogenesis. Many aggressive tumor cells express high ATX levels and low LPP activities. This favors the formation of locally high LPA and S1P concentrations. Furthermore, LPPs attenuate signaling downstream of the activation of G-protein coupled receptors and receptor tyrosine kinases. Therefore, we propose that the low expression of LPPs in many tumor cells makes them hypersensitive to growth promoting and survival signals that are provided by LPA, S1P, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF). One of the key signaling pathways in this respect appears to be activation of phospholipase D (PLD) and phosphatidate (PA) production. This is required for the transactivations of the EGFR and PDGFR and also for LPA-induced cell migration. PA also increases the activities of ERK, mTOR, myc and sphingosine kinase-1 (SK-1), which provide individual signals for cells division, survival, chemo-resistance and angiogenesis. This review focuses on the balance of signaling by bioactive lipids including LPA, phosphatidylinositol 3,4,5-trisphosphate, PA and S1P versus the action of ceramides. We will discuss how these lipid mediators interact to produce an aggressive neoplastic phenotype.     

Preoperative brush and impression cytology in ocular surface squamous neoplasms

OBJECTIVE: Preoperative cytologic diagnoses of ocular surface squamous neoplasms were evaluated and compared with histologic diagnoses. STUDY DESIGN: Impression cytology (Millipore filter paper) and brush cytology were applied to 32 patients who had conjunctival neoplasms. Papanicolaou-stained cytologic preparations and hematoxylin and eosin-stained histologic sections were examined by light microscopy. RESULTS: The brush technique was used on 27 patients; impression cytology was applied in 5 cases. Cytologic and histologic diagnoses were concordant in 26 cases. Squamous cell carcinoma or carcinoma in situ was diagnosed in 18 and dysplasia in 4 cases. Squamous metaplasia and normal-appearing conjunctival epithelial cells were diagnosed cytologically in four cases; of those histologic diagnoses, one was pterygium and three, conjunctival nevus. Four cases revealed discrepancies between the cytologic and histologic preparations. There was one false positive result, and one case was subconjunctival invasion of basal cell carcinoma of the eyelid. CONCLUSION: Impression and brush cytology are fast, cost-effective, reliable and noninvasive diagnostic tools for ocular surface squamous neoplasms. However, the brush technique has several advantages over impression cytology.     

A Working Memory Deficit among Dyslexic Readers with No Phonological Impairment as Measured Using the N-Back Task: An fNIR Study

Data indicated that dyslexic individuals exhibited difficulties on tasks involving Working Memory (WM). Previous studies have suggested that these deficits stem from impaired processing in the Phonological Loop (PL). The PL impairment was connected to poor phonological processing. However, recent data has pointed to the Central Executive (CE) system as another source of WM deficit in dyslexic readers. This opened a debate whether the WM deficit stems solely from PL or can also be seen as an outcome of poor CE processing. In an attempt to verify this question, the current study compared adult skilled and compensated dyslexic readers with no impairment of phonological skills. The participants’ PL and CE processing were tested by using the fNIR device attached to the frontal lobe and measured the changes in brain oxygen values when performing N-back task. As it was previously suggested, the N = 0 represented PL and N = 1 to 3 represent CE processing. It was hypothesized that dyslexic readers who show non-impaired phonological skills will exhibit deficits mainly in the CE subsystem and to a lesser extent in the PL. Results indicated that the two reading level groups did not differ in their accuracy and reaction times in any of the N-Back conditions. However, the dyslexic readers demonstrated significant lower maximum oxyHb values in the upper left frontal lobe, mainly caused due to a significant lower activity under the N = 1 condition. Significant task effects were found in the medial left hemisphere, and the high medial right hemisphere. In addition, significant correlations between fNIR-features, reading performance and speed of processing were found. The higher oxyHb values, the better reading and speed of processing performance obtained. The results of the current study support the hypothesis that at least for the group of dyslexics with non-impaired PL, WM deficit stems from poor CE activity.