Isolation of pathogenic bacteria from sputum samples using a 3D-printed cartridge system.

Within this contribution we introduce a 3D-printed cartridge system enabling the convenient and cost-efficient sample preparation from sputum for subsequent PCR based detection schemes. The developed fluidic system operates on pneumatic actuations. The closed system ensures a very low probability for contamination during sample processing, which is crucial when using a highly sensitive detection method such as PCR. The enrichment of the bacterial cells is achieved using different types of amine-functionalized particles. Our particle-based sample preparation approach yields intact and viable bacterial cells. Accordingly, not only PCR-based detection schemes can be employed, but also spectroscopic methods and biochemical tests, which require cultivation steps, are possible. The cartridge design in principle is compatible with magnetic and non-magnetic particle types. We investigated both variants and found that the performance of expanded glass beads is superior over the magnetic particles within the cartridge. Owing to the rather large size of the expanded glass beads, the dimensions of the channels can be enlarged, leading to lower hydrodynamic resistances, which is beneficial when processing viscous samples such as sputum. We verified the performance of our system using both artificial and real sputum samples containing Escherichia coli and Moraxella catarrhalis.

Bax and Bak are the critical complementary effectors of colorectal cancer cell apoptosis by chemopreventive resveratrol.

Resveratrol (RS) exerts a large number of cell-protective and anti-tumor effects, among them the induction of tumor cell apoptosis. Since the bioavailability of ingested RS at distant organs is low and apoptosis induction often requires relatively high RS levels (above 20 micromol/l), this polyphenolic food ingredient might be particularly effective as a chemopreventive in the digestive tract. Previous studies have suggested that chemoprevention by non-steroidal anti-inflammatory drugs (NSAIDs) is blunted by the loss of a single component of the apoptotic machinery - the Bax protein. Here, we report that RS efficiently provokes apoptosis in human colorectal carcinoma cells deficient for Bax, although at a reduced rate compared to the parental cells, through the activation of the mitochondrial death pathway. Knockdown of pro-apoptotic Bak by RNA interference reduced the apoptotic response to a similar extent as Bax deficiency in the parental cells and completely abolished apoptosis in Bax-null cells. Notably, although negative for RS-induced, mitochondria-mediated apoptosis, Bax+Bak double-deficient cells were sensitized by RS to ligand-induced, death receptor-mediated apoptosis. Thus, in contrast to NSAIDs, RS may remain effective as a pro-apoptotic chemopreventive as long as Bax and Bak have not both been inactivated during clonal selection.

Retinoblastoma protein is required for efficient colorectal carcinoma cell apoptosis by histone deacetylase inhibitors in the absence of p21Waf.

Colorectal cancer accounts for approximately 10% of all new cancer cases reported worldwide. High dietary fiber intake has been associated with a reduced risk for this type of neoplasia, and much of this effect is ascribed to the histone acetylase (HDAC) inhibitor n-butyrate produced in the gastrointestinal tract. Natural chemopreventive and several new synthetic HDAC inhibitors exert multiple effects on tumor cells including the induction of differentiation, cell cycle arrest and apoptosis. Since cancer cells undergo mutational changes, it will be important to understand precisely which pathway gains or losses modulate or compromise HDAC inhibitor efficacy. We have recently documented that n-butyrate can provoke apoptosis in human HCT116 colorectal carcinoma cells independently of the p53 tumor suppressor and p21Waf inhibitor. Here, we have developed cell lines on the basis of HCT116 p21-/- cells and HCT116 cells in which the retinoblastoma tumor suppressor protein Rb has been specifically knocked down by antisense expression. The cells were exposed to the DNA-damaging drugs adriamycin (ADR) and etoposide or the HDAC inhibitors n-butyrate and trichostatin A (TSA). While the maximal apoptotic response, observed in the absence of p21Waf, was unaffected by the additional knockdown of Rb when cells were treated with ADR or etoposide, the toxicity of the HDAC inhibitors was significantly reduced. This indicates that hyperphosphorylated Rb itself, dissociated from E2F1 transcription factor, can contribute - directly or indirectly - to tumor cell apoptosis provoked by HDAC inhibitors.

Mitochondrial p53 levels parallel total p53 levels independent of stress response in human colorectal carcinoma and glioblastoma cells.

p53 can eliminate damaged cells through the induction of mitochondria-mediated apoptosis. Recent observations have provided strong evidence that a fraction of total p53 translocates to mitochondria specifically in response to a death stimulus. Unexpectedly, mutant p53, which is expressed at much higher levels than wild type in unstressed cells, is apparently always present at the mitochondria, independent of apoptotic signal. This prompted us to ask whether cell lines with intact p53-dependent apoptosis and cell cycle arrest pathways exist in which the mitochondrial localization of wild-type p53, like that of mutant, is independent of a death stimulus and instead, correlates with the total p53 levels. Here, we document that human HCT116 colorectal carcinoma cells treated with adriamycin or 5-fluorouracil (5FU) can accumulate total p53 to equally high levels, and mitochondrial p53 to proportionate levels, although only 5FU treatment provoked p53-dependent apoptosis. Along the same line, HCT116 derivatives with increased basal p53 levels, and glioblastoma cells with a doxycycline-inducible p53, also revealed proportionate mitochondrial p53 levels, and even unstressed HCT116 cells had some p53 located at the mitochondria. Finally, mitochondrial and total p53 showed distinct post-translational modifications. Thus, cell lines exist in which the mitochondrial p53 levels parallel total levels independent of apoptosis.