Using Sensors and Generators of H2O2 to Elucidate the Toxicity Mechanism of Piperlongumine and Phenethyl Isothiocyanate

Aims: Chemotherapeutics target vital functions that ensure survival of cancer cells, including their increased reliance on defense mechanisms against oxidative stress compared to normal cells. Many chemotherapeutics exploit this vulnerability to oxidative stress by elevating the levels of intracellular reactive oxygen species (ROS). A quantitative understanding of the oxidants generated and how they induce toxicity will be important for effective implementation and design of future chemotherapeutics. Molecular tools that facilitate measurement and manipulation of individual chemical species within the context of the larger intracellular redox network present a means to develop this understanding. In this work, we demonstrate the use of such tools to elucidate the roles of H[subscript 2]O[subscript 2] and glutathione (GSH) in the toxicity mechanism of two ROS-based chemotherapeutics, piperlongumine and phenethyl isothiocyanate. Results: Depletion of GSH as a result of treatment with these compounds is not an important part of the toxicity mechanisms of these drugs and does not lead to an increase in the intracellular H[subscript 2]O[subscript 2] level. Measuring peroxiredoxin-2 (Prx-2) oxidation as evidence of increased H[subscript 2]O[subscript 2], only piperlongumine treatment shows elevation and it is GSH independent. Using a combination of a sensor (HyPer) along with a generator (D-amino acid oxidase) to monitor and mimic the drug-induced H[subscript 2O[subscript 2] production, it is determined that H[subscript 2]O[subscript 2] produced during piperlongumine treatment acts synergistically with the compound to cause enhanced cysteine oxidation and subsequent toxicity. The importance of H[subscript 2]O[subscript 2] elevation in the mechanism of piperlongumine promotes a hypothesis of why certain cells, such as A549, are more resistant to the drug than others. Innovation and Conclusion: The approach described herein sheds new light on the previously proposed mechanism of these two ROS-based chemotherapeutics and advocates for the use of both sensors and generators of specific oxidants to isolate their effects. Antioxid. Redox Signal. 24, 924–938.National Science Foundation (U.S.). Graduate Research Fellowship ProgramBurroughs Wellcome Fund (Career Award at the Scientific Interface

Association of temperament and acute stress responsiveness with productivity, feed efficiency, and methane emissions in beef cattle: an observational study

The aim of this study was to assess individual differences in temperament and stress response and quantify their impact on feed efficiency, performance, and methane (CH(4)) emissions in beef cattle. Eighty-four steers (castrated males) (Charolais or Luing) were used. Temperament was assessed using two standardized tests: restlessness when restrained [crush score (CS)] and flight speed (FS) on release from restraint. Over a 56-day period individual animal dry matter intake (DMI) and weekly body weight was measured. Ultrasound fat depth was measured at the end of 56 days. Average daily gain (ADG), feed conversion ratio (FCR), and residual feed intake (RFI) were calculated. After the 56-day test period, animals were transported in groups of six/week to respiration chamber facilities. Blood samples were taken before and 0, 3, 6, and 9 h after transport. Plasma cortisol, creatine kinase (CK), glucose, and free fatty acids (FFA) were determined to assess physiological stress response. Subsequently, CH(4) emissions were measured over a 3-day period in individual respiration chambers. CS (1.7 ± 0.09) and FS (1.6 ± 0.60 m/s) were repeatable (0.63 and 0.51, respectively) and correlated (r = 0.36, P < 0.001). Plasma cortisol, CK, and FFA concentrations increased after transport (P = 0.038, P = 0.006, and P < 0.001, respectively). Temperament (CS) and CK concentration were correlated (r = 0.29; P = 0.015). The extreme group analysis reveals that excitable animals (FS; P = 0.032) and higher stress response (cortisol, P = 0.007; FFA, P = 0.007; and CK, P = 0.003) were associated with lower DMI. ADG was lower in more temperamental animals (CS, P = 0.097, and FS, P = 0.030). Fat depth was greater in steers showing calmer CS (P = 0.026) and lower plasma CK (P = 0.058). Temperament did not show any relationship with RFI or CH(4) emissions. However, steers with higher cortisol showed improved feed efficiency (lower FCR and RFI) (P < 0.05) and greater CH(4) emissions (P = 0.017). In conclusion, agitated temperament and higher stress responsiveness is detrimental to productivity. A greater stress response is associated with a reduction in feed intake that may both increase the efficiency of consumed feed and the ratio of CH(4) emissions/unit of feed. Therefore, temperament and stress response should be considered when designing strategies to improve efficiency and mitigate CH(4) emissions in beef cattle

Acceptability and feasibility of peer assisted supervision and support for intervention practitioners: a Q-methodology evaluation

Evidence-based interventions often include quality improvement methods to support fidelity and improve client outcomes. Clinical supervision is promoted as an effective way of developing practitioner confidence and competence in delivery; however, supervision is often inconsistent and embedded in hierarchical line management structures that may limit the opportunity for reflective learning. The Peer Assisted Supervision and Support (PASS) supervision model uses peer relationships to promote the self-regulatory capacity of practitioners to improve intervention delivery. The aim of the present study was to assess the acceptability and feasibility of PASS amongst parenting intervention practitioners. A Q-methodology approach was used to generate data and 30 practitioners volunteered to participate in the study. Data were analyzed and interpreted using standard Q-methodology procedures and by-person factor analysis yielded three factors. There was consensus that PASS was acceptable. Participants shared the view that PASS facilitated an environment of support where negative aspects of interpersonal relationships that might develop in supervision were not evident. Two factors represented the viewpoint that PASS was also a feasible model of supervision. However, the third factor was comprised of practitioners who reported that PASS could be time consuming and difficult to fit into existing work demands. There were differences across the three factors in the extent to which practitioners considered PASS impacted on their intervention delivery. The findings highlight the importance of organizational mechanisms that support practitioner engagement in supervision

Dendritic cell density and activation status in human breast cancer – CD1a, CMRF-44, CMRF-56 and CD-83 expression

Low CD1a-positive putative dendritic cell numbers in human breast cancer has recently been described and may explain the apparent ‘poor immunogenicity’ previously reported in breast cancer. Little attention has been given to dendritic cell activation within the tumour microenvironment, which is another reason why the in-situ immune response may be severely deficient. We have therefore examined CD1a expression as a marker for dendritic cells, together with CMRF-44 and -56 as markers of dendritic cell activation status, in 40 human breast cancers. The results demonstrate few or no CD1a-positive putative dendritic cells and minimal or no expression of the dendritic cell activation markers. Both dendritic cell number and dendritic cell activation appear substantially deficient in human breast cancers, regardless of tumour histological grade

siRNA-Like Double-Stranded RNAs Are Specifically Protected Against Degradation in Human Cell Extract

RNA interference (RNAi) is a set of intracellular pathways in eukaryotes that controls both exogenous and endogenous gene expression. The power of RNAi to knock down (silence) any gene of interest by the introduction of synthetic small-interfering (si)RNAs has afforded powerful insight into biological function through reverse genetic approaches and has borne a new field of gene therapeutics. A number of questions are outstanding concerning the potency of siRNAs, necessitating an understanding of how short double-stranded RNAs are processed by the cell. Recent work suggests unmodified siRNAs are protected in the intracellular environment, although the mechanism of protection still remains unclear. We have developed a set of doubly-fluorophore labeled RNAs (more precisely, RNA/DNA chimeras) to probe in real-time the stability of siRNAs and related molecules by fluorescence resonance energy transfer (FRET). We find that these RNA probes are substrates for relevant cellular degradative processes, including the RNase H1 mediated degradation of an DNA/RNA hybrid and Dicer-mediated cleavage of a 24-nucleotide (per strand) double-stranded RNA. In addition, we find that 21- and 24-nucleotide double-stranded RNAs are relatively protected in human cytosolic cell extract, but less so in blood serum, whereas an 18-nucleotide double-stranded RNA is less protected in both fluids. These results suggest that RNAi effector RNAs are specifically protected in the cellular environment and may provide an explanation for recent results showing that unmodified siRNAs in cells persist intact for extended periods of time