Regulation of Multidrug Resistance Proteins by Genistein in a Hepatocarcinoma Cell Line: Impact on Sorafenib Cytotoxicity

Hepatocellular carcinoma (HCC) is the fifth most frequent cancer worldwide. Sorafenib is the only drug available that improves the overall survival of HCC patients. P-glycoprotein (P-gp), Multidrug resistance-associated proteins 2 and 3 (MRP2 and 3) and Breast cancer resistance protein (BCRP) are efflux pumps that play a key role in cancer chemoresistance. Their modulation by dietary compounds may affect the intracellular accumulation and therapeutic efficacy of drugs that are substrates of these transporters. Genistein (GNT) is a phytoestrogen abundant in soybean that exerts its genomic effects through Estrogen-Receptors and Pregnane-X-Receptor (PXR), which are involved in the regulation of the above-mentioned transporters. We evaluated the effect of GNT on the expression and activity of P-gp, MRP2, MRP3 and BCRP in HCC-derived HepG2 cells. GNT (at 1.0 and 10 μM) increased P-gp and MRP2 protein expression and activity, correlating well with an increased resistance to sorafenib cytotoxicity as detected by the methylthiazole tetrazolium (MTT) assay. GNT induced P-gp and MRP2 mRNA expression at 10 but not at 1.0 μM concentration suggesting a different pattern of regulation depending on the concentration. Induction of both transporters by 1.0 μM GNT was prevented by cycloheximide, suggesting translational regulation. Downregulation of expression of the miR-379 by GNT could be associated with translational regulation of MRP2. Silencing of PXR abolished P-gp induction by GNT (at 1.0 and 10 μM) and MRP2 induction by GNT (only at 10 μM), suggesting partial mediation of GNT effects by PXR. Taken together, the data suggest the possibility of nutrient-drug interactions leading to enhanced chemoresistance in HCC when GNT is ingested with soy rich diets or dietary supplements.     

An Internally Translated MAVS Variant Exposes Its Amino-terminal TRAF-Binding Motifs to Deregulate Interferon Induction

Activation of pattern recognition receptors and proper regulation of downstream signaling are crucial for host innate immune response. Upon infection, the NF-κB and interferon regulatory factors (IRF) are often simultaneously activated to defeat invading pathogens. Mechanisms concerning differential activation of NF-κB and IRF are not well understood. Here we report that a MAVS variant inhibits interferon (IFN) induction, while enabling NF-κB activation. Employing herpesviral proteins that selectively activate NF-κB signaling, we discovered that a MAVS variant of ~50 kDa, thus designated MAVS50, was produced from internal translation initiation. MAVS50 preferentially interacts with TRAF2 and TRAF6, and activates NF-κB. By contrast, MAVS50 inhibits the IRF activation and suppresses IFN induction. Biochemical analysis showed that MAVS50, exposing a degenerate TRAF-binding motif within its N-terminus, effectively competed with full-length MAVS for recruiting TRAF2 and TRAF6. Ablation of the TRAF-binding motif of MAVS50 impaired its inhibitory effect on IRF activation and IFN induction. These results collectively identify a new means by which signaling events is differentially regulated via exposing key internally embedded interaction motifs, implying a more ubiquitous regulatory role of truncated proteins arose from internal translation and other related mechanisms.     

The Impact of the Carer Support Needs Assessment Tool (CSNAT) in Community Palliative Care Using a Stepped Wedge Cluster Trial

Family caregiving towards the end-of-life entails considerable emotional, social, financial and physical costs for caregivers. Evidence suggests that good support can improve caregiver psychological outcomes. The primary aim of this study was to investigate the impact of using the carer support needs assessment tool (CSNAT), as an intervention to identify and address support needs in end of life home care, on family caregiver outcomes. A stepped wedge design was used to trial the CSNAT intervention in three bases of Silver Chain Hospice Care in Western Australia, 2012-14. The intervention consisted of at least two visits from nurses (2-3 weeks apart) to identify, review and address caregivers’ needs. The outcome measures for the intervention and control groups were caregiver strain and distress as measured by the Family Appraisal of Caregiving Questionnaire (FACQ-PC), caregiver mental and physical health as measured by SF-12v2, and caregiver workload as measured by extent of caregiver assistance with activities of daily living, at baseline and follow up. Total recruitment was 620. There was 45% attrition for each group between baseline and follow-up mainly due to patient deaths resulting in 322 caregivers completing the study (233 in the intervention group and 89 in the control group). At follow-up, the intervention group showed significant reduction in caregiver strain relative to controls, p=0.018, d=0.348 (95% CI 0.25 to 0.41). Priority support needs identified by caregivers included knowing what to expect in the future, having time for yourself in the day and dealing with your feelings and worries. Despite the challenges at the clinician, organisational and trial levels, the CSNAT intervention led to an improvement in caregiver strain. Effective implementation of an evidence-informed and caregiver-led tool represents a necessary step towards helping palliative care providers better assess and address caregiver needs, ensuring adequate family caregiver support and reduction in caregiver strain.     

Proteome analysis of <Emphasis Type="Italic">Aspergillus ochraceus</Emphasis>

Genome sequencing for many important fungi has begun during recent years; however, there is still some deficiency in proteome profiling of aspergilli. To obtain a comprehensive overview of proteins and their expression, a proteomic approach based on 2D gel electrophoresis and MALDI-TOF/TOF mass spectrometry was used to investigate A. ochraceus. The cell walls of fungi are exceptionally resistant to destruction, therefore two lysis protocols were tested: (1) lysis via manual grinding using liquid nitrogen, and (2) mechanical lysis via rapid agitation with glass beads using MagNalyser. Mechanical grinding with mortar and pestle using liquid nitrogen was found to be a more efficient extraction method for our purpose, resulting in extracts with higher protein content and a clear band pattern in SDS-PAGE. Two-dimensional electrophoresis gave a complex spot pattern comprising proteins of a broad range of isoelectric points and molecular masses. The most abundant spots were subjected to mass spectrometric analysis. We could identify 31 spots representing 26 proteins, most of them involved in metabolic processes and response to stress. Seventeen spots were identified by de novo sequencing due to a lack of DNA and protein database sequences of A. ochraceus. The proteins identified in our study have been reported for the first time in A. ochraceus and this represents the first proteomic approach with identification of major proteins, when the fungus was grown under submerged culture.     

Changes in cellular levels of inositol polyphosphates during apoptosis

Accumulations of higher inositol polyphosphates, diphosphoinositol polyphosphates or pyrophosphates, have been implicated to mediate cellular apoptosis. Whether cellular levels of lower inositol phosphates (lower than inositol hexakisphosphates) change during apoptosis is not known, although these inositol phosphates are known to play crucial roles in a number of cellular signaling processes including calcium mobilization. Therefore, in this study, we have examined changes in cellular levels of inositol phosphates following metabolic labeling of these compounds by [³H]myo-inositol and induction of apoptosis. The levels of inositol mono- and bis-phosphates were increased, whereas the levels of inositol tris- and tetrakis-phosphates decreased significantly with an increasing rate of apoptosis induced by etoposide in a dose-dependent manner. NaF treatment, which increased the rate of apoptosis in a time- and dose-dependent manner, also increased the levels of inositol mono- and bis-phosphates and drastically reduced the levels of inositol tris- and tetrakis-phosphates. Prior treatment with antimycin A, a strategy used to reverse the NaF-induced accumulations of higher InsPs, partially reduced the effects of NaF on apoptosis as well as the levels of lower InsPs. Taken together, our results suggest that cellular levels of lower InsPs are altered during apoptosis.     

Thermal requirement for development of Sancassania rodionovi (Acari: Acaridae) on mushrooms

The free-living mite species Sancassania rodionovi (Zachvatkin) (Acari: Acaridae), is a serious pest of mushrooms in Iran. Studies were conducted to examine the development of this mite in relation to temperature on two mushroom species: Agaricus bisporus Lange (button mushroom) and Pleurotus ostreatus Kummer (oyster mushroom). The developmental time of this acarid mite was studied at eight constant temperatures, ranging from 5 to 40 degrees C, and developmental rates were modeled as a function of temperature. Sancassania rodionovi completed immature development in 17.35 +/- 0.58 and 20.17 +/- 0.88 d at 25 degrees C on button and oyster mushrooms, respectively. When the mite fed on button mushroom, the rate of development increased gradually from 10 to 35 degrees C. Using a linear model, the developmental zero was estimated to be 3.50 degrees C with a thermal constant of 357.14 degree-days. The Logan 10, Briere 1, and Thermodynamic models adequately described the data for this mite and yielded R2 values >0.95; these models provided estimates of optimum temperature for development of 33.244, 32.145, and 32.148 degrees C, respectively. Understanding the influence of temperature on development of S. rodionovi is discussed with respect to pest management in mushroom production.     

Maintaining nitric oxide-induced airway relaxation with superoxide dismutase

BACKGROUND: We have previously shown that the protective effect of inhaled nitric oxide (iNO) against methacholine-induced bronchoconstriction is negated in airways subjected to hyperosmotic stress. In this study, hypothesizing that the impaired efficiency of iNO was caused by release of reactive oxygen radicals, we examined the effect of the radical scavenging enzyme superoxide dismutase (SOD). METHODS: Hemodynamic and respiratory measurements were performed on anesthetized rabbits after (1) inhalation of methacholine (MCh), (2) iNO (80ppm), followed by MCh, (3) inhalation of hypertonic saline (HS), followed by iNO and MCh and (4) pre-treatment with inhalation of SOD, followed by HS, iNO and MCh. We analyzed plasma for a marker of oxidative stress, 8-iso-prostaglandin (PG)F(2alpha) and for a marker of activation of COX-mediated inflammatory cascades, PGF(2alpha) metabolite. RESULTS: Pre-treatment with SOD restored the bronchoprotective response to iNO in hyperosmotic airways. No direct effect was seen by SOD treatment on levels of 8-iso-PGF(2alpha), but this marker of oxidative stress correlated positively with increased bronchoconstriction. Hyperosmotic challenge elevated levels of PGF(2alpha) metabolite, and pre-treatment with SOD protected against this activation of the inflammatory cascade. CONCLUSION: SOD pre-treatment restores the relaxant effects of iNO in hyperosmotically challenged airways by attenuating oxidative stress and activation of COX-mediated inflammatory cascades.     

Time course and attenuation of ischaemia-reperfusion induced oxidative injury by propofol in human renal transplantation

Ischaemia-reperfusion injury resulting from interruption and restoration of blood flow might be related to free radical mediated oxidative stress and inflammation, and subsequently to post-surgery related complications. We studied the impact of renal transplantation on oxidative stress and inflammation by measuring F(2)-isoprostanes and prostaglandin F(2alpha), respectively, during transplantation and post-surgery. Additionally, due to earlier observations, two dissimilar anaesthetic agents (thiopentone and propofol) were compared to determine their antioxidative capacity rather than their anaesthetic properties. Blood samples were collected before, post-intubation, immediately, 30, 60,120, 240 min, and 12 and 24 h after reperfusion. Oxidative stress and inflammatory response were detected by measuring 8-iso-PGF(2alpha) (a major F(2)-isoprostane and a biomarker of oxidative stress) and 15-keto-dihydro-PGF(2alpha) (a major metabolite of PGF(2alpha) and a biomarker of COX-mediated inflammatory response), respectively. Reperfusion of the transplanted graft significantly increased plasma levels of 8-iso-PGF(2alpha). PGF(2alpha) metabolite levels, although elevated, did not reach statistical significance. In addition, significantly lower levels of 8-iso-PGF(2a) were observed in the propofol group compared to the thiopentone group. Together, these findings underline an augmented oxidative stress activity following an inflammatory response after human renal transplantation. Furthermore, propofol a well-known anaesthetic, counteracted oxidative stress by lowering the formation of a major F(2)-isoprostane.