HIV Protease Inhibitors Sensitize Human Head and Neck Squamous Carcinoma Cells to Radiation by Activating Endoplasmic Reticulum Stress

BackgroundHuman head and neck squamous cell carcinoma (HNSCC) is the sixth most malignant cancer worldwide. Despite significant advances in the delivery of treatment and surgical reconstruction, there is no significant improvement of mortality rates for this disease in the past decades. Radiotherapy is the core component of the clinical combinational therapies for HNSCC. However, the tumor cells have a tendency to develop radiation resistance, which is a major barrier to effective treatment. HIV protease inhibitors (HIV PIs) have been reported with radiosensitizing activities in HNSCC cells, but the underlying cellular/molecular mechanisms remain unclear. Our previous study has shown that HIV PIs induce cell apoptosis via activation of endoplasmic reticulum (ER) stress. The aim of this study was to examine the role of ER stress in HIV PI-induced radiosensitivity in human HNSCC.

Methodology and Principal Findings

HNSCC cell lines, SQ20B and FaDu, and the most commonly used HIV PIs, lopinavir and ritonavir (L/R), were used in this study. Clonogenic assay was used to assess the radiosensitivity. Cell viability, apoptosis and cell cycle were analyzed using Cellometer Vision CBA. The mRNA and protein levels of ER stress-related genes (eIF2α, CHOP, ATF-4, and XBP-1), as well as cell cycle related protein, cyclin D1, were detected by real time RT-PCR and Western blot analysis, respectively. The results demonstrated that L/R dose-dependently sensitized HNSCC cells to irradiation and inhibited cell growth. L/R-induced activation of ER stress was correlated to down-regulation of cyclin D1 expression and cell cycle arrest under G0/G1 phase.

Conclusion and Significance

HIV PIs sensitize HNSCC cells to radiotherapy by activation of ER stress and induction of cell cycle arrest. Our results provided evidence that HIV PIs can be potentially used in combination with radiation in the treatment of HNSCC.     

Short-term adaptation during propagation improves the performance of xylose-fermenting <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in simultaneous saccharification and co-fermentation

Background

Inhibitors that are generated during thermochemical pretreatment and hydrolysis impair the performance of microorganisms during fermentation of lignocellulosic hydrolysates. In omitting costly detoxification steps, the fermentation process relies extensively on the performance of the fermenting microorganism. One attractive option of improving its performance and tolerance to microbial inhibitors is short-term adaptation during propagation. This study determined the influence of short-term adaptation on the performance of recombinant Saccharomyces cerevisiae in simultaneous saccharification and co-fermentation (SSCF). The aim was to understand how short-term adaptation with lignocellulosic hydrolysate affects the cell mass yield of propagated yeast and performance in subsequent fermentation steps. The physiology of propagated yeast was examined with regard to viability, vitality, stress responses, and upregulation of relevant genes to identify any links between the beneficial traits that are promoted during adaptation and overall ethanol yields in co-fermentation.

Results

The presence of inhibitors during propagation significantly improved fermentation but lowered cell mass yield during propagation. Xylose utilization of adapted cultures was enhanced by increasing amounts of hydrolysate in the propagation. Ethanol yields improved by over 30 % with inhibitor concentrations that corresponded to ≥2.5 % water-insoluble solids (WIS) load during the propagation compared with the unadapted culture. Adaptation improved cell viability by >10 % and increased vitality by >20 %. Genes that conferred resistance against inhibitors were upregulated with increasing amounts of inhibitors during the propagation, but the adaptive response was not associated with improved ethanol yields in SSCF. The positive effects in SSCF were observed even with adaptation at inhibitor concentrations that corresponded to 2.5 % WIS. Higher amounts of hydrolysate in the propagation feed further improved the fermentation but increased the variability in fermentation outcomes and resulted in up to 20 % loss of cell mass yield.

Conclusions

Short-term adaptation during propagation improves the tolerance of inhibitor-resistant yeast strains to inhibitors in lignocellulosic hydrolysates and improves their ethanol yield in fermentation and xylose-fermenting capacity. A low amount of hydrolysate (corresponding to 2.5 % WIS) is optimal, whereas higher amounts decrease cell mass yield during propagation.

     

Crack Detection in Fibre Reinforced Plastic Structures Using Embedded Fibre Bragg Grating Sensors: Theory,Model Development and Experimental Validation

In a fibre-reinforced polymer (FRP) structure designed using the emerging damage tolerance and structural health monitoring philosophy, sensors and models that describe crack propagation will enable a structure to operate despite the presence of damage by fully exploiting the material’s mechanical properties. When applying this concept to different structures, sensor systems and damage types, a combination of damage mechanics, monitoring technology, and modelling is required. The primary objective of this article is to demonstrate such a combination. This article is divided in three main topics: the damage mechanism (delamination of FRP), the structural health monitoring technology (fibre Bragg gratings to detect delamination), and the finite element method model of the structure that incorporates these concepts into a final and integrated damage-monitoring concept. A novel method for assessing a crack growth/damage event in fibre-reinforced polymer or structural adhesive-bonded structures using embedded fibre Bragg grating (FBG) sensors is presented by combining conventional measured parameters, such as wavelength shift, with parameters associated with measurement errors, typically ignored by the end-user. Conjointly, a novel model for sensor output prediction (virtual sensor) was developed using this FBG sensor crack monitoring concept and implemented in a finite element method code. The monitoring method was demonstrated and validated using glass fibre double cantilever beam specimens instrumented with an array of FBG sensors embedded in the material and tested using an experimental fracture procedure. The digital image correlation technique was used to validate the model prediction by correlating the specific sensor response caused by the crack with the developed model.     

Discovery of novel,orally available benzimidazoles as melanin concentrating hormone receptor 1 (MCHR1) antagonists

Melanin concentrating hormone (MCH) is an important mediator of energy homeostasis and plays role in several disorders such as obesity, stress, depression and anxiety. The synthesis and biological evaluation of novel benzimidazole derivatives as MCHR1 antagonists are described. The in vivo proof of principle for weight loss with a lead compound from this series is exemplified.     

Effects of ambient versus reduced UV-B radiation on high arctic Salix arctica assessed by measurements and calculations of chlorophyll a fluorescence parameters from fluorescence transients

A UV-B exclusion-experiment was conducted in the high Arctic Zackenberg, NE Greenland, in which Salix arctica leaves during most of the growing season were fixed perpendicular to the solar zenith angle, thereby receiving maximal solar radiation. Covered with Teflon and Mylar foil, the leaves received approximately 90 and 40% of the ambient UV-B irradiance, respectively. The effects were examined through recordings of chlorophyll a fluorescence transients, determination of biomass and analysis of total carbon and nitrogen content and amount of soluble flavonoids in the leaves. The processing of light was analysed by means of the chlorophyll a fluorescence transient, using the so-called JIP test, as evolved by Reto J. Strasser and his coworkers. Reduction of the UV-B irradiance caused a rise in many of the fluorescence parameters during July, but not in August (late season). Thus increases in the efficiency that an absorbed photon will be trapped by the PSII reaction centre with the resultant reduction of Q_A to Q_A^– (ET₀/ABS = F_V/F_M) and the efficiency that an electron residing on Q_A^– will enter the intersystem electron transport chain (ET₀/TR₀) were observed in reduced UV-B. Moreover, estimated per cross-section of leaf sample, the number of active PSII reaction centres (RC/CS_M) and electron transport rate (ET_M/CS_M) and all performance indexes (PI_ABS, PI_CSo and PI_CSm) were increased in reduced UV-B. The total soluble flavonoid content was highest in ambient UV-B. The treatment effects on fluorescence parameters that were directly measured (e.g. F₀ and F_M) and those that were derived (e.g. quantum efficiencies, parameters per PSII reaction centres and per cross-section of leaf sample) are discussed in relation to one another, in relation to daily and seasonal variation, and from the perspective of evaluating the relative importance of UV-B of donor and acceptor side capacity in Photosystem II. In conclusion, the experimental set-up and non-invasive measurements proved to be a sensitive method to screen for effects of UV-B stress.     

Penicillium brasilianum as an enzyme factory; the essential role of feruloyl esterases for the hydrolysis of the plant cell wall

The production of arabinoxylan-degrading enzymes by the fungus Penicillium brasilianum, grown on different carbon and nitrogen sources as well as different environmental conditions was investigated. Highest feruloyl esterase (225 mU/ml) and alpha-L-arabinofuranosidase (211 mU/ml) activities were obtained when P. brasilianum was grown on sugar beet pulp, whereas maximum xylanase (17 U/ml) activity was found during growth on oat spelt xylan. Yeast extract was the preferable nitrogen source for the production of all the three enzymes. Further optimization of the production of the crude enzyme mixture was examined by experimental design using a D-optimal quadratic model. Investigation of the microbial regulation of enzyme production showed that the presence of free ferulic acid further stimulated the production and pointing to that the fungal regulatory mechanism involved a coordinated production and secretion of feruloyl esterase, xylanase and alpha-L-arabinofuranosidase. Since agroindustrial by-products are a potential source of phenolic acids, crude enzyme mixtures of P. brasilianum were tested for their hydrolysis abilities against eight complex or model substrates. While total release of phenolic acids and pentoses was not observed, the synergistic enhancement of hydrolysis in the presence of feruloyl esterase was clearly demonstrated.     

Characterization of a multifunctional inositol phosphate kinase from rice and barley belonging to the ATP-grasp superfamily

OsIpk and HvIpk, inositol phosphate kinases, were cloned from rice (Oryza sativa L. var. indica, IR64) and barley (Hordeum vulgare) respectively. Sequence alignment showed that they belong to the ATP-grasp family, which includes inositol 1,3,4-trisphosphate 5/6-kinase from humans and Arabidopsis. Residues that are binding sites for ATP and coordinate magnesium in absence or presence of inositol phosphate are conserved and in total 23 residues are invariant among the twelve aligned inositol phosphate kinases. The genes were heterologously expressed in Escherichia coli and kinase activity assays with 17 different isomers of inositol mono-/di-/tri-/tetra-/pentaphosphate as well as phytate were performed. The strongest activity for both kinases was observed with Ins(3,4,5,6)P(4), which candidates as the primary substrate for these kinases in plants. Several species-specific differences between the two recombinant Ipks were observed. Rice OsIpk showed detectable kinase activity towards eight different substrates, whereas barley HvIpk showed kinase activity with all the substrates including inositol mono- and bisphosphates. HvIpk showed 3-kinase activity towards the Ins(1,4,5)P(3) substrate and it also interconverted the two substrates Ins(1,3,4,5)P(4) and Ins(1,3,4,6)P(4) by isomerase activity, which was not observed for the rice homologue. Both OsIpk and HvIpk had no detectable 2-kinase activity. Furthermore, the two Ipks showed phosphatase activity towards several inositol phosphates. Expression analysis by RT-PCR demonstrated that the Ipk gene was equally expressed in different tissues and developmental stages. Taken together, these results show that the Ipk kinase plays a significant role in the inositol phosphate interacting network in plants.