Signal recognition particle receptor-ß (SR-ß) coordinates cotranslational N-glycosylation.

Proteins destined for the secretory compartment of the cell are cotranslationally translocated into the endoplasmic reticulum. The majority of these proteins are N-glycosylated, a co- and posttranslational modification that ensures proper protein folding, stability, solubility, and cellular localization. Here, we show that the [Formula: see text] subunit of the signal recognition particle receptor (SR) is required for assembly of the N-glycosylation-competent translocon. We report that guanine analog chemical probes identified by high-throughput screening or mutation of the SR-[Formula: see text] guanosine triphosphate binding site cause an N-glycosylation-deficient phenotype. Neither method alters the association of SR-[Formula: see text] with SR-[Formula: see text], but both approaches reduce the association of SR-[Formula: see text] with the oligosaccharyltransferase complex. These experiments demonstrate that SR-[Formula: see text] has a previously unrecognized function coordinating endoplasmic reticulum translation with N-glycosylation.

Consumers' Acceptability and Perception of Edible Insects as an Emerging Protein Source.

In recent years in Western Europe, studies on entomophagy have drawn the attention of many researchers interested in identifying parameters that could improve the acceptability of insect consumption in order to introduce insects as a sustainable source of protein into the future diet. Analysing the factors involved in consumer acceptability in the Mediterranean area could help to improve their future acceptance. A cross-sectional study was conducted using an ad-hoc questionnaire in which 1034 consumers participated. The questionnaire responses allowed us to study the areas relevant to acceptance: neophobia, social norms, familiarity, experiences of consumption and knowledge of benefits. Only 13.15% of participants had tried insects. Disgust, lack of custom and food safety were the main reasons for avoiding insect consumption. Consequently, preparations with an appetising appearance need to be offered, with flours being the most accepted format. The 40-59-year-old age group was the one most willing to consume them. To introduce edible insects as food in the future, it is important to inform people about their health, environmental and economic benefits because that could increase their willingness to include them in their diet.

Edible Insect Consumption for Human and Planetary Health: A Systematic Review.

This systematic review aimed to examine the health outcomes and environmental impact of edible insect consumption. Following PRISMA-P guidelines, PubMed, Medline ProQuest, and Cochrane Library databases were searched until February 2021. Twenty-five articles met inclusion criteria: twelve animal and six human studies (randomized, non-randomized, and crossover control trials), and seven studies on sustainability outcomes. In animal studies, a supplement (in powdered form) of 0.5 g/kg of glycosaminoglycans significantly reduced abdominal and epididymal fat weight (5-40% and 5-24%, respectively), blood glucose (10-22%), and total cholesterol levels (9-10%), and a supplement of 5 mg/kg chitin/chitosan reduced body weight (1-4%) and abdominal fat accumulation (4%) versus control diets. In other animal studies, doses up to 7-15% of edible insect inclusion level significantly improved the live weight (9-33%), reduced levels of triglycerides (44%), cholesterol (14%), and blood glucose (8%), and increased microbiota diversity (2%) versus control diet. In human studies, doses up to 7% of edible insect inclusion level produced a significant improvement in gut health (6%) and reduction in systemic inflammation (2%) versus control diets and a significant increase in blood concentrations of essential and branched-chain amino acids and slowing of digestion (40%) versus whey treatment. Environmental indicators (land use, water footprint, and greenhouse gas emissions) were 40-60% lower for the feed and food of edible insects than for traditional animal livestock. More research is warranted on the edible insect dose responsible for health effects and on environmental indicators of edible insects for human nutrition. This research demonstrates how edible insects can be an alternative protein source not only to improve human and animal nutrition but also to exert positive effects on planetary health.

Which diet has the lower water footprint in Mediterranean countries?

The Mediterranean region is increasingly water scarce, with the food system being the largest driver of water use. We calculate the water resources related to food consumption in nine major Mediterranean countries, by means of the water footprint (WF), for the existing situation (period 2011-2013) as well as the Mediterranean and EAT-Lancet diets. We account for different food intake requirements according to gender and six age groups. These nine countries - Spain, France, Italy, Greece, Turkey, Egypt, Tunisia, Algeria and Morocco - represent 88% of the population of all countries bordering the Mediterranean. As first major observation, we find that the EAT-Lancet diet, a scientifically optimised diet for both nutrition and certain environmental indicators, requires less water resources than the Mediterranean diet, a culturally accepted diet within the region. In terms of water resources use, adherence to the former is thus more beneficial than adherence to the latter. As second major observation, we find that the EAT-Lancet diet reduces the current WF for all nations consistently, within the range -17% to -48%, whereas the Mediterranean diet reduces the WF of the European countries, Turkey, Egypt and Morocco within the range of -4% to -35%. For the Maghreb countries Tunisia and Algeria, the Mediterranean diet WF is slightly higher compared to the current WF and the proportions of food product groups differ. Such dietary shifts would be important parts of the solution to obtain the sustainable use of water resources in Mediterranean countries.

STING enhances cell death through regulation of reactive oxygen species and DNA damage.

Resistance to DNA-damaging agents is a significant cause of treatment failure and poor outcomes in oncology. To identify unrecognized regulators of cell survival we performed a whole-genome CRISPR-Cas9 screen using treatment with ionizing radiation as a selective pressure, and identified STING (stimulator of interferon genes) as an intrinsic regulator of tumor cell survival. We show that STING regulates a transcriptional program that controls the generation of reactive oxygen species (ROS), and that STING loss alters ROS homeostasis to reduce DNA damage and to cause therapeutic resistance. In agreement with these data, analysis of tumors from head and neck squamous cell carcinoma patient specimens show that low STING expression is associated with worse outcomes. We also demonstrate that pharmacologic activation of STING enhances the effects of ionizing radiation in vivo, providing a rationale for therapeutic combinations of STING agonists and DNA-damaging agents. These results highlight a role for STING that is beyond its canonical function in cyclic dinucleotide and DNA damage sensing, and identify STING as a regulator of cellular ROS homeostasis and tumor cell susceptibility to reactive oxygen dependent, DNA damaging agents.

Neuregulin Signaling Is a Mechanism of Therapeutic Resistance in Head and Neck Squamous Cell Carcinoma.

EGFR signaling confers resistance to radiotherapy and is a validated target in head and neck squamous cell carcinoma (HNSCC). The inhibition of EGFR in combination with radiotherapy improves local control and overall survival in these patients; however, therapeutic resistance limits the efficacy of this approach. We therefore sought to identify cellular mechanisms that cause resistance to EGFR inhibition and radiotherapy in HNSCC. Though clonal isolation of carcinoma cells exposed to increasing concentrations of cetuximab, we found that resistant cells upregulate prosurvival ErbB3 and AKT signaling. Using EFM-19 cells and confirmatory analysis of protein levels, we demonstrate that cetuximab resistance is characterized by enhanced neuregulin expression identifying a novel adaptive mechanism of therapeutic resistance. Inhibition of this autocrine loop with CDX-3379 (an ErbB3 specific antibody) was sufficient to block ErbB3/AKT signaling in cetuximab resistant cells. The combination of CDX-3379 and cetuximab reduced proliferation and survival after radiotherapy in several HNSCC cell lines. These in vitro findings were confirmed in xenograft tumor growth experiments including an approach using growth factor-supplemented Matrigel. In vivo, the delivery of EGFR and ErbB3 antibodies significantly reduced tumor growth in cetuximab-resistant FaDu and CAL27 xenografts. In summary, this work demonstrates that autocrine NRG ligand secretion is a mechanism for therapeutic resistance to cetuximab and radiotherapy. This cross-resistance to both therapeutic modalities identifies NRG as an actionable therapeutic target for improving treatment regimens in HNSCC.

Oligosaccharyltransferase Inhibition Reduces Receptor Tyrosine Kinase Activation and Enhances Glioma Radiosensitivity.

PURPOSE: Parallel signaling reduces the effects of receptor tyrosine kinase (RTK)-targeted therapies in glioma. We hypothesized that inhibition of protein N-linked glycosylation, an endoplasmic reticulum co- and posttranslational modification crucial for RTK maturation and activation, could provide a new therapeutic approach for glioma radiosensitization.Experimental Design: We investigated the effects of a small-molecule inhibitor of the oligosaccharyltransferase (NGI-1) on EGFR family receptors, MET, PDGFR, and FGFR1. The influence of glycosylation state on tumor cell radiosensitivity, chemotherapy-induced cell toxicity, DNA damage, and cell-cycle arrest were determined and correlated with glioma cell receptor expression profiles. The effects of NGI-1 on xenograft tumor growth were tested using a nanoparticle formulation validated by in vivo molecular imaging. A mechanistic role for RTK signaling was evaluated through the expression of a glycosylation-independent CD8-EGFR chimera. RESULTS: NGI-1 reduced glycosylation, protein levels, and activation of most RTKs. NGI-1 also enhanced the radiosensitivity and cytotoxic effects of chemotherapy in those glioma cells with elevated ErbB family activation, but not in cells without high levels of RTK activation. NGI-1 radiosensitization was associated with increases in both DNA damage and G1 cell-cycle arrest. Combined treatment of glioma xenografts with fractionated radiotherapy and NGI-1 significantly reduced tumor growth compared with controls. Expression of the CD8-EGFR eliminated the effects of NGI-1 on G1 arrest, DNA damage, and cellular radiosensitivity, identifying RTK inhibition as the principal mechanism for the NGI-1 effect. CONCLUSIONS: This study suggests that oligosaccharyltransferase inhibition with NGI-1 is a novel approach to radiosensitize malignant gliomas with enhanced RTK signaling.See related commentary by Wahl and Lawrence, p. 455.

Oligosaccharyltransferase Inhibition Overcomes Therapeutic Resistance to EGFR Tyrosine Kinase Inhibitors.

Asparagine (N)-linked glycosylation is a posttranslational modification essential for the function of complex transmembrane proteins. However, targeting glycosylation for cancer therapy has not been feasible due to generalized effects on all glycoproteins. Here, we perform sensitivity screening of 94 lung cancer cell lines using NGI-1, a small-molecule inhibitor of the oligosaccharyltransferase (OST) that partially disrupts N-linked glycosylation, and demonstrate a selective loss of tumor cell viability. This screen revealed NGI-1 sensitivity in just 11 of 94 (12%) cell lines, with a significant correlation between OST and EGFR inhibitors. In EGFR-mutant non-small cell lung cancer with EGFR tyrosine kinase inhibitor (TKI) resistance (PC9-GR, HCC827-GR, and H1975-OR), OST inhibition maintained its ability to induce cell-cycle arrest and a proliferative block. Addition of NGI-1 to EGFR TKI treatment was synthetic lethal in cells resistant to gefitinib, erlotinib, or osimertinib. OST inhibition invariably disrupted EGFR N-linked glycosylation and reduced activation of receptors either with or without the T790M TKI resistance mutation. OST inhibition also dissociated EGFR signaling from other coexpressed receptors like MET via altered receptor compartmentalization. Translation of this approach to preclinical models was accomplished through synthesis and delivery of NGI-1 nanoparticles, confirmation of in vivo activity through molecular imaging, and demonstration of significant tumor growth delay in TKI-resistant HCC827 and H1975 xenografts. This therapeutic strategy breaks from kinase-targeted approaches and validates N-linked glycosylation as an effective target in tumors driven by glycoprotein signaling.Significance:EGFR-mutant NSCLC is incurable despite the marked sensitivity of these tumors to EGFR TKIs. These findings identify N-linked glycosylation, a posttranslational modification common to EGFR and other oncogenic signaling proteins, as an effective therapeutic target that enhances tumor responses for EGFR-mutant NSCLC. Cancer Res; 78(17); 5094-106. ©2018 AACR.

Simvastatin Enhances the Effects of Radiotherapy and Cetuximab on a Cell Line (FaDu) Derived from a Squamous Cell Carcinoma of Head and Neck.

Radiotherapy (XRT) delivered with the antibody cetuximab is a standard treatment option for squamous cell carcinomas of head and neck (SCCNH). Cetuximab acts by blocking epidermal growth factor receptor (EGFR) signaling to inhibit cancer progression. However, a significant percentage of patients will not respond to XRT and cetuximab. Statins reduce the synthesis of cholesterol and isoprenoid derivates that may be required for efficient EGFR signaling. We assessed whether the statin simvastatin could improve this combined therapy. In vitro, simvastatin enhanced the effects of XRT alone and in combination with cetuximab in wound healing, cell proliferation, and clonogenic assays in FaDu cells. These results were reflected in xenoimplanted tumors growing into subcutaneous tissue of athymic mice where concomitant treatment with simvastatin decreased tumor growth. Consistently, lower levels of phosphorylated extracellular signal-regulated kinases 1 and 2, phosphatidylinositol 3-kinase/AKT-protein kinase B, and signal transducer and activator of transcription 3 oncoproteins and higher levels of caspase-3 and apoptosis in cell cultures and xenografts were observed. The EGFR-overexpressing A431 cell line was used to reproduce these antitumor effects of simvastatin. Our findings suggest that simvastatin may improve the efficiency of concomitant XRT and cetuximab. Further investigation in the treatment of SCCNH is warranted.

Development and characterization of an isogenic cell line with a radioresistant phenotype.

INTRODUCTION: Radiation resistance is a major cause of death in cancer patients. Cancer cells react during radiotherapy by re-programming specific cell functions that may confer resistance to radiation. The understanding of this complex process is hindered due to the lack of appropriate study models. We describe an experimental development of a radioresistant isogenic cancer cell line, and its molecular characterization. MATERIALS AND METHODS: A431-cultured cells were irradiated for 7 month until 85 Gy. Then, a selected single cell was left to grow as stable A431-R cell line. Clonogenic assay was used to determine cell survival, the α and ß parameters of the LQ model, and the mean inactivation dose. The DNA repair ability of cells was evaluated by pulsed-field electrophoresis method. Differential effect of fractionated radiation was ultimately tested in xenografts. Furthermore, we used a wound healing assay, Western blot for EGFR, AKT and ERK1/2 and ELISA test for vascular endothelial growth factor (VEGF) secretion. Finally we explored CD44 marker and cell cycle distribution. RESULTS: The established A431-R cell line showed radiation resistance in clonogenic assays, repair of radiation-induced DNA fragmentation and xenografted tumours. The radiation resistance was associated with in vitro higher cell growth and migration, increased levels of former oncoproteins, and secretion of VEGF. CONCLUSIONS: In this model, the emergence of radiation resistance was associated with the acquisition of biological traits that support more aggressive behaviour of cancer cells. We have generated a model that will be useful for mechanistic studies and development of rational treatments against radiation resistance in cancer.

Development and refinement of a technique using a medical radiation therapy facility to irradiate immunodeficient mice bearing xenografted human tumours.

The need for using immunodeficient mice for xenoimplantation of tumours is increasing in translational research in radiation oncology. However, adverse effects of radiation and infectious diseases may ruin the experimental work, in particular when appropriate facilities are not available. In this report, we describe a procedure to deliver fractionated radiotherapy to xenoimplanted tumours in immunodeficient mice using a medical linear accelerator, a method that was devised as an alternative to the lack of facilities devoted to radiation research. The mice were irradiated under anaesthesia and aseptic conditions. Thirty Gray in 10 days using a 6 MV photon beam were delivered only to the right thigh of the mice where tumours were implanted. The mice were evaluated twice a week up to planned euthanasia. The follow-up of mice was completed without premature interruption due to toxicities or infectious diseases, an observation which demonstrates the feasibility of the method.

The use of caspase inhibitors in pulsed-field gel electrophoresis may improve the estimation of radiation-induced DNA repair and apoptosis.

BACKGROUND: Radiation-induced DNA double-strand break (DSB) repair can be tested by using pulsed-field gel electrophoresis (PFGE) in agarose-encapsulated cells. However, previous studies have reported that this assay is impaired by the spontaneous DNA breakage in this medium. We investigated the mechanisms of this fragmentation with the principal aim of eliminating it in order to improve the estimation of radiation-induced DNA repair. METHODS: Samples from cancer cell cultures or xenografted tumours were encapsulated in agarose plugs. The cell plugs were then irradiated, incubated to allow them to repair, and evaluated by PFGE, caspase-3, and histone H2AX activation (γH2AX). In addition, apoptosis inhibition was evaluated through chemical caspase inhibitors. RESULTS: We confirmed that spontaneous DNA fragmentation was associated with the process of encapsulation, regardless of whether cells were irradiated or not. This DNA fragmentation was also correlated to apoptosis activation in a fraction of the cells encapsulated in agarose, while non-apoptotic cell fraction could rejoin DNA fragments as was measured by γH2AX decrease and PFGE data. We were able to eliminate interference of apoptosis by applying specific caspase inhibitors, and improve the estimation of DNA repair, and apoptosis itself. CONCLUSIONS: The estimation of radiation-induced DNA repair by PFGE may be improved by the use of apoptosis inhibitors. The ability to simultaneously determine DNA repair and apoptosis, which are involved in cell fate, provides new insights for using the PFGE methodology as functional assay.

Cetuximab may inhibit tumor growth and angiogenesis induced by ionizing radiation: a preclinical rationale for maintenance treatment after radiotherapy.

BACKGROUND: The benefits of radiotherapy and cetuximab have encouraged evaluation of cetuximab after radiotherapy. The aims of this study were to preclinically evaluate the efficacy of cetuximab maintenance after radiotherapy and eventually determine its mechanisms of action. METHODS: The A431 human carcinoma cell line was treated in culture with fractionated radiotherapy and cetuximab. The surviving cells were injected s.c. into nude mice to mimic microscopic residual disease. The animals were randomized to receive either cetuximab or saline solution. Tumor growth, cell proliferation (Ki-67), microvessel density (MVD), epidermal growth factor receptor (EGFR) and transforming growth factor (TGF-α) mRNA transcription, and vascular endothelial growth factor (VEGF) secretion were measured. RESULTS: Tumors from irradiated cells had a faster growth rate, higher Ki-67 index, and greater angiogenesis than tumors from untreated cells. This aggressive phenotype was associated with in vitro radiation-induced extracellular signal-related kinase (ERK)-1/2 and Akt activation, greater EGFR and TGF-α transcription, and augmented VEGF secretion, all of which were inhibited by cetuximab. In cetuximab-treated mice with tumors arising from irradiated cells, time to volume was longer by a factor of 3.52, whereas the Ki-67 index and MVD were 1.57 and 1.49 times lower, respectively, a larger enhancement than seen in tumors from untreated cells. These findings suggest that cells surviving radiation may express factors that promote cell survival and induce an aggressive phenotype that may potentially be blocked by cetuximab maintenance therapy. CONCLUSIONS: These results support the clinical evaluation of adjuvant therapy with cetuximab after radiotherapy in EGFR-dependent carcinomas.

Natural allelic variants of bovine ATP-binding cassette transporter ABCG2: increased activity of the Ser581 variant and development of tools for the discovery of new ABCG2 inhibitors.

ATP-binding cassette transporter ABCG2 [breast cancer resistance protein (BCRP)] is a member of the ABC transporter superfamily that actively extrudes xenotoxins from cells and is a major determinant of the bioavailability of many compounds. ABCG2 expression is strongly induced during lactation in the mammary gland and is related to the active secretion of drugs into the milk. The presence of drug residues and environmental pollutants in milk is an outstanding problem for human milk consumption and milk industrial processes, involving important risks to public health and the dairy industry. In cows, a single nucleotide polymorphism (SNP) in this protein has been described previously (Tyr581) and is associated with higher fat and protein percentages and lower milk yield. However, whether this amino acid substitution affects ABCG2-mediated drug transport in cows, including milk secretion, required further exploration. We cloned the two variants of bovine ABCG2 and evaluated the effect of this SNP on mitoxantrone accumulation assays performed in ovine primary fibroblasts transiently expressing either of the variants. It is interesting to note that statistically significant differences in activity between both variants were observed, and the Ser581 variant was related with an increased efflux activity. In addition, we demonstrated that genistein is a very good inhibitor of bovine ABCG2 and identified new inhibitors of the transporter, such as the macrocyclic lactones, ivermectin, and selamectin. Moreover, the inhibitory effect of these compounds on human and murine ABCG2 homologs was confirmed using transduced Marbin-Dabin canine kidney II cells. These findings may have important implications regarding the presence of drug residues in milk and drug interactions affecting the pharmacological behavior of ABCG2 substrates.

The first hydrophobic region of the HPV16 E5 protein determines protein cellular location and facilitates anchorage-independent growth.

The human papillomavirus type 16 E5 protein (HPV16 E5) is 83 amino acids in length and contains three well-defined hydrophobic regions. The protein is expressed at very limited amounts in transfected cells and the absence of specific antibodies has strongly hampered functional analyses. To investigate the relationship between structure and function we have synthesized a codon-adapted version of the gene (hE5) and prepared a series of N-terminal and C-terminal deletions. Immunofluorescence analyses show colocaliation of the protein with calnexin, an ER marker, EEA-1, an early endosomes marker, and Lamp-2, a lysosomal marker. No major colocalization was found between hE5 and the Golgi marker 58 K. Whereas deletions at the C-terminal end of the protein do not greatly alter the localisation pattern, deletion of the first hydrophobic region results in loss of colocalisation with the ER, early endosomes and lysosomes. Further, we show that while the complete E5 protein confers to HaCaT cells the property to grow in an anchorage-independent manner, deletion of the first hydrophobic region results in loss of growth in soft agar. We conclude that the first hydrophobic region of the E5 protein largely determines the biological properties of the viral protein.