On the cytotoxicity of HCR-NTPase in the neuroblastoma cell line SH-SY5Y

Pasdziernik M, Kaltschmidt B, Kaltschmidt C, Klinger C, Kaufmann M. On the cytotoxicity of HCR-NTPase in the neuroblastoma cell line SH-SY5Y. BMC Research Notes. 2009;2(1):102.Background: The human cancer-related nucleoside triphosphatase (HCR-NTPase) is overexpressed in several tumour tissues including neuroblastoma. HCR-NTPase is an enzyme exhibiting a slow in vitro activity in hydrolysing nucleosidetriphosphates. However, its in vivo function is still unknown. To learn more about the physiological role of HCR-NTPase, we both overexpressed and silenced it in the neuroblastoma cell line SH-SY5Y. Findings: No effect was observed when the expression of endogenously expressed HCR-NTPase in the cells was silenced by RNA interference. On the other hand, overexpression of HCR-NTPase led to cytotoxicity of the protein in SH-SY5Y cells. Even if the catalytic essential amino acid glutamate 114 was replaced by alanine (E114A-HCR-NTPase), the protein remained cytotoxic. The results could be confirmed by successfully rescuing the cells via RNA interference. Conclusion: Although expressed in several tumours, at least in SH-SY5Y, HCR-NTPase is not essential for the cells to survive. Increased levels of the protein lead to cytotoxicity due to physical intracellular interactions rather than hydrolysis of nucleosidetriphosphates by its intrinsic residual enzymatic activity

Controlling pH in shake flasks using polymer-based controlled-release discs with pre-determined release kinetics

<p>Abstract</p> <p>Background</p> <p>There are significant differences in the culture conditions between small-scale screenings and large-scale fermentation processes. Production processes are usually conducted in fed-batch cultivation mode with active pH-monitoring and control. In contrast, screening experiments in shake flasks are usually conducted in batch mode without active pH-control, but with high buffer concentrations to prevent excessive pH-drifts. These differences make it difficult to compare results from screening experiments and laboratory and technical scale cultivations and, thus, complicate rational process development. In particular, the pH-value plays an important role in fermentation processes due to the narrow physiological or optimal pH-range of microorganisms. To reduce the differences between the scales and to establish a pH-control in shake flasks, a newly developed easy to use polymer-based controlled-release system is presented in this paper. This system consists of bio-compatible silicone discs embedding the alkaline reagent Na₂CO₃. Since the sodium carbonate is gradually released from the discs in pre-determined kinetics, it will ultimately compensate the decrease in pH caused by the biological activity of microorganisms.</p> <p>Results</p> <p>The controlled-release discs presented here were successfully used to cultivate <it>E. coli </it>K12 and <it>E. coli </it>BL21 pRSET eYFP-IL6 in mineral media with glucose and glycerol as carbon (C) sources, respectively. With glucose as the C-source it was possible to reduce the required buffer concentration in shake flask cultures by 50%. Moreover, with glycerol as the C-source, no buffer was needed at all.</p> <p>Conclusions</p> <p>These novel polymer-based controlled-release discs allowed buffer concentrations in shake flask media to be substantially reduced or omitted, while the pH remains in the physiological range of the microorganisms during the whole cultivation time. Therefore, the controlled-release discs allow a better control of the pH, than merely using high buffer concentrations. The conditions applied here, i.e. with significantly reduced buffer concentrations, enhance the comparability of the culture conditions used in screening experiments and large-scale fermentation processes.</p

Neuronal activity regulates alternative exon usage

Neuronal activity-regulated gene transcription underlies plasticity-dependent changes in the molecular composition and structure of neurons. A large number of genes regulated by different neuronal plasticity inducing pathways have been identified, but altered gene expression levels represent only part of the complexity of the activity-regulated transcriptional program. Alternative splicing, the differential inclusion and exclusion of exonic sequence in mRNA, is an additional mechanism that is thought to define the activity-dependent transcriptome. Here, we present a genome wide microarray-based survey to identify exons with increased expression levels at 1, 4 or 8 h following neuronal activity in the murine hippocampus provoked by generalized seizures. We used two different bioinformatics approaches to identify alternative activity-induced exon usage and to predict alternative splicing, ANOSVA (ANalysis Of Splicing VAriation) which we here adjusted to accommodate data from different time points and FIRMA (Finding Isoforms using Robust Multichip Analysis). RNA sequencing, in situ hybridization and reverse transcription PCR validate selected activity-dependent splicing events of previously described and so far undescribed activity-regulated transcripts, including Homer1a, Homer1d, Ania3, Errfi1, Inhba, Dclk1, Rcan1, Cda, Tpm1 and Krt75. Taken together, our survey significantly adds to the comprehensive understanding of the complex activity-dependent neuronal transcriptomic signature. In addition, we provide data sets that will serve as rich resources for future comparative expression analyses.Projekt DEALPeer Reviewe

Digital afx: digital dressing and affective shifts in Sin City and 300

In Sin City (Robert Rodriguez, 2005) and 300 (Zack Snyder, 2006) extensive post-production work has created stylised colour palettes, manipulated areas of the image, and added or subtracted elements. Framing a discussion around the terms ‘affect’ and ‘emotion’, this paper argues that the digital technologies used in Sin City and 300 modify conventional interactions between representational and aesthetic dimensions. Brian Massumi suggests affective imagery can operate through two modes of engagement. One mode is embedded in a meaning system, linked to a speci?c emotion. The second is understood as an intensi?cation whereby a viewer reacts but that reaction is not yet gathered into an alignment with meaning. The term ‘digital afx’ is used to describe manipulations that produce imagery allowing these two modes of engagement to coexist. Digital afx are present when two competing aesthetic strategies remain equally visible within sequences of images. As a consequence the afx mingle with and shift the content of representation

Thyroid hormones enhance mitochondrial function in human epidermis

Since it is unknown whether thyroid hormones (THs) regulate mitochondrial function in human epidermis, we treated organ-cultured human skin, or isolated cultured human epidermal keratinocytes, with triiodothyronine (100 pmol/L) or thyroxine (100 nmol/L). Both THs significantly increased protein expression of the mitochondrially encoded cytochrome C oxidase I (MTCO1), complex I activity, and the number of perinuclear mitochondria. Triiodothyronine also increased mitochondrial transcription factor A (TFAM) protein expression, and thyroxine stimulated complex II/IV activity. Increased mitochondrial function can correlate with increased reactive oxygen species production, DNA damage, and accelerated tissue aging. However, THs neither raised reactive oxygen species production or matrix metalloproteinase-1, -2 and -9 activity nor decreased sirtuin1 (Sirt1) immunoreactivity. Instead, triiodothyronine increased sirtuin-1, fibrillin-1, proliferator-activated receptor-gamma 1-alpha (PGC1 alpha), collagen I and III transcription, and thyroxine decreased cyclin-dependent kinase inhibitor 2A (p16(ink4)) expression in organ-cultured human skin. Moreover, TH treatment increased intracutaneous fibrillin-rich microfibril and collagen III deposition and decreased mammalian target of rapamycin (mTORC1/2) expression ex vivo. This identifies THs as potent endocrine stimulators of mitochondrial function in human epidermis, which down-regulates rather than enhance the expression of skin aging-related biomarkers ex vivo. Therefore, topically applied THs deserve further exploration as candidate agents for treating skin conditions characterized by reduced mitochondrial function

The evolution of breast prostheses.

Every year approximately 1.5 million prostheses are implanted worldwide for breast augmentation and reconstructive indications. The modern breast implant as we know was released to the open market in 1963. It has gone through intense phases of development which have improved the initially primitive and limited devices to current-day devices, which exhibit a tremendous range of surface textures, sizes, gel consistencies, and anatomical shapes. This article explores the evolution of breast implants providing historical facts and technical details

Late Recognition of SARS in Nosocomial Outbreak, Toronto

Late recognition of severe acute respiratory syndrome (SARS) was associated with no known SARS contact, hospitalization before the nosocomial outbreak was recognized, symptom onset while hospitalized, wards with SARS clusters, and postoperative status. SARS is difficult to recognize in hospitalized patients with a variety of underlying conditions in the absence of epidemiologic links