Auranofin and reactive oxygen species inhibit protein synthesis and regulate the level of the PLK1 protein in Ewing sarcoma cells.

Novel therapeutic approaches are needed for the treatment of Ewing sarcoma tumors. We previously identified that Ewing sarcoma cell lines are sensitive to drugs that inhibit protein translation. However, translational and therapeutic approaches to inhibit protein synthesis in tumors are limited. In this work, we identified that reactive oxygen species, which are generated by a wide range of chemotherapy and other drugs, inhibit protein synthesis and reduce the level of critical proteins that support tumorigenesis in Ewing sarcoma cells. In particular, we identified that both hydrogen peroxide and auranofin, an inhibitor of thioredoxin reductase and regulator of oxidative stress and reactive oxygen species, activate the repressor of protein translation 4E-BP1 and reduce the levels of the oncogenic proteins RRM2 and PLK1 in Ewing and other sarcoma cell lines. These results provide novel insight into the mechanism of how ROS-inducing drugs target cancer cells via inhibition of protein translation and identify a mechanistic link between ROS and the DNA replication (RRM2) and cell cycle regulatory (PLK1) pathways.

Standardized amino acid digestibility and nitrogen-corrected true metabolizable energy of frozen raw, freeze-dried raw, fresh, and extruded dog foods using precision-fed cecectomized and conventional rooster assays.

Processing conditions, particularly temperature and duration of heating, impact pet food digestibility. Various commercial pet food formats are now available, but few have been tested thoroughly. The objective of this study was to determine the amino acid (AA) digestibilities and nitrogen-corrected true metabolizable energy (TMEn) values of frozen raw, freeze-dried raw, fresh (mildly cooked), and extruded dog foods using the precision-fed cecectomized and conventional rooster assays. The diets tested were Chicken and Barley Recipe [Hill's Science Diet, extruded diet (EXT)], Chicken and White Rice Recipe [Just Food for Dogs, fresh diet (FRSH)], Chicken Formula [Primal Pet Foods, frozen raw diet (FRZN)], Chicken and Sorghum Hybrid Freeze-dried Formula [Primal Pet Foods, hybrid freeze-dried raw diet (HFD)], and Chicken Dinner Patties [Stella & Chewy's, freeze-dried raw diet (FD)]. Two precision-fed rooster assays utilizing Single Comb White Leghorn roosters were conducted. Cecectomized roosters (n = 4/treatment) and conventional roosters (n = 4/treatment) were used to determine standardized AA digestibilities and TMEn, respectively. All roosters were crop intubated with 12 g of test diet and 12 g of corn, with excreta collected for 48 h. In general, FD had the highest, while EXT had the lowest AA digestibilities; however, all diets performed relatively well and few differences in AA digestibility were detected among the diets. Lysine digestibility was higher (P < 0.05) in FD and FRZN than EXT, with other diets being intermediate. Threonine digestibility was higher (P < 0.05) in FD than EXT, with other diets being intermediate. Digestibilities of the other indispensable AA were not different among diets. The reactive lysine:total lysine ratios were 0.94, 0.96, 0.93, 0.93, and 0.95 for EXT, FRSH, FRZN, HFD, and FD, respectively. TMEn was higher (P < 0.05) in FRZN than FD, FRSH, and EXT, higher (P < 0.05) in HFD than FRSH and EXT, and higher (P < 0.05) in FD than EXT. In conclusion, our results support the notion that AA digestibilities are affected by diet processing, with FD, HFD, FRZN, and FRSH diets having higher AA digestibility coefficients and greater TMEn values, than the EXT diet; however, other factors such as ingredient inclusion and macronutrient composition may also have affected these results. More research in dogs is necessary to test the effects of format on diet palatability, digestibility, stool quality, and other physiologically relevant outcomes.

Processing conditions, particularly temperature and duration of heating, impact pet food digestibility. This study tested the standardized amino acid (AA) digestibilities and nitrogen-corrected true metabolizable energy (TMEn) values of five commercial dog diets: extruded diet (EXT), fresh (mildly cooked) diet (FRSH), frozen raw diet (FRZN), hybrid freeze-dried raw diet (HFD), and freeze-dried raw diet (FD). The first study, to determine AA digestibility, used 20 roosters who had their ceca (the main site of microbial fermentation in chickens) surgically removed. The second study used 20 conventional roosters to determine the TMEn of the diets. In general, FD had the highest AA digestibilities, while EXT had the lowest AA digestibilities. True metabolizable energy concentration was higher in the FRZN diet than the FD, FRSH, and EXT diets, higher in the HFD diet than the FRSH and EXT diets, and higher in the FD diet than the EXT diet. Our results support the notion that differences in diet processing, as well as factors such as macronutrient composition, and ingredient source, characteristics, and inclusion may impact AA digestibility and TMEn of dog diets. More research should be conducted to determine exactly how, and to what extent, these different factors impact digestibility in dogs.

Activator Protein-1 (AP-1) Signaling Inhibits the Growth of Ewing Sarcoma Cells in Response to DNA Replication Stress.

Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in the synthesis of deoxyribonucleosides and is required for DNA replication. Multiple types of cancer, including Ewing sarcoma tumors, are sensitive to RNR inhibitors or a reduction in the levels of either the RRM1 or RRM2 subunits of RNR. However, the polypharmacology and off-target effects of RNR inhibitors have complicated the identification of the mechanisms that regulate sensitivity and resistance to this class of drugs. Consequently, we used a conditional knockout (CRISPR/Cas9) and rescue approach to target RRM1 in Ewing sarcoma cells and identified that loss of the RRM1 protein results in the upregulation of the expression of multiple members of the activator protein-1 (AP-1) transcription factor complex, including c-Jun and c-Fos, and downregulation of c-Myc. Notably, overexpression of c-Jun and c-Fos in Ewing sarcoma cells is sufficient to inhibit cell growth and downregulate the expression of the c-Myc oncogene. We also identified that the upregulation of AP-1 is mediated, in part, by SLFN11, which is a replication stress response protein that is expressed at high levels in Ewing sarcoma. In addition, small-molecule inhibitors of RNR, including gemcitabine, and histone deacetylase inhibitors, which reduce the level of the RRM1 protein, also activate AP-1 signaling and downregulate the level of c-Myc in Ewing sarcoma. Overall, these results provide novel insight into the critical pathways activated by loss of RNR activity and the mechanisms of action of inhibitors of RNR. Significance: RNR is the rate-limiting enzyme in the synthesis of deoxyribonucleotides. Although RNR is the target of multiple chemotherapy drugs, polypharmacology and off-target effects have complicated the identification of the precise mechanism of action of these drugs. In this work, using a knockout-rescue approach, we identified that inhibition of RNR upregulates AP-1 signaling and downregulates the level of c-Myc in Ewing sarcoma tumors.

Effects of a mildly cooked human-grade dog diet on gene expression, skin and coat health measures, and fecal microbiota of healthy adult dogs.

Purported benefits of human-grade pet foods include reduced inflammation, enhanced coat quality, and improved gut health, but research is scarce. Therefore, we compared gene expression, skin and coat health measures, and the fecal microbiome of dogs consuming a mildly cooked human-grade or extruded kibble diet. Twenty beagles (BW = 10.25 ± 0.82 kg; age = 3.85 ± 1.84 yr) were used in a completely randomized design. Test diets included: 1) chicken and brown rice recipe [feed-grade; extruded; blue buffalo (BB)]; and 2) chicken and white rice [human-grade; mildly cooked; Just Food for Dogs (JFFD)]. The study consisted of a 4-week baseline when all dogs ate BB, and a 12-week treatment phase when dogs were randomized to either diet (n = 10/group). After the baseline and treatment phases, fresh fecal samples were scored and collected for pH, dry matter (DM), and microbiome analysis; blood samples were collected for gene expression analysis; hair samples were microscopically imaged; and skin was analyzed for delayed-type hypersensitivity (DTH), sebum concentration, hydration status, and transepidermal water loss (TEWL). Data were analyzed as a change from baseline (CFB) using the Mixed Models procedure of SAS (version 9.4). At baseline, fecal pH was higher (P < 0.05) and hair surface score, superoxide dismutase (SOD) expression, and tumor necrosis factor-α (TNF-α) expression was lower (P < 0.05) in dogs allotted to JFFD. The decrease in CFB fecal pH and DM was greater (P < 0.05) in dogs fed JFFD, but fecal scores were not different. The increase in CFB hair surface score was higher (P < 0.05) in dogs fed JFFD. The decrease in CFB TEWL (back region) was greater (P < 0.05) in dogs fed JFFD, but TEWL (inguinal and ear regions), hydration status, and sebum concentrations in all regions were not different. Hair cortex scores and DTH responses were not affected by diet. The increase in CFB gene expression of SOD, COX-2, and TNF-α was greater (P < 0.05) in dogs fed JFFD. PCoA plots based on Bray-Curtis distances of bacterial genera and species showed small shifts over time in dogs fed BB, but dramatic shifts in those fed JFFD. JFFD increased (adj. P < 0.05) relative abundances of 4 bacterial genera, 11 bacterial species, 68 KEGG pathways, and 167 MetaCyc pathways, and decreased (adj. P < 0.05) 16 genera, 25 species, 98 KEGG pathways, and 87 MetaCyc pathways. In conclusion, the JFFD diet dramatically shifted the fecal microbiome but had minor effects on skin and coat measures and gene expression.

This study tested the effects of a mildly cooked human-grade diet and a feed-grade extruded kibble diet on the fecal microbiome, skin and coat health measures, and expression of genes related to inflammation and oxidative stress in healthy adult dogs. During a 4-week baseline, 20 beagles consumed the kibble diet. After baseline, 10 dogs continued to consume that diet, while 10 dogs consumed the mildly cooked diet for 12 weeks. After baseline and treatment phases, fresh fecal, blood, and hair samples were collected and skin was analyzed. The mildly cooked diet led to lower fecal pH and dry matter percentage, but fecal scores were not affected. The mildly cooked diet dramatically altered the fecal microbiome, shifting the relative abundances of over 30 bacterial species and 165 bacterial metabolic pathways. Measures of skin sebum content and hydration status were not different between groups, but skin water loss was lower in dogs consuming the mildly cooked diet. Baseline and post-treatment gene expression and hair surface scores were noted, but hair cortex and delayed-type hypersensitivity testing were not altered by diet. Our results demonstrate that mildly cooked diets dramatically change the fecal microbiome, but may not impact skin and coat in healthy adult dogs over a short time period.

Analysis of retinal sublayer thicknesses and rates of change in ABCA4-associated Stargardt disease.

Stargardt disease, the most common inherited macular dystrophy, is characterized by vision loss due to central retinal atrophy. Although clinical trials for Stargardt are currently underway, the disease is typically slowly progressive, and objective, imaging-based biomarkers are critically needed. In this retrospective, observational study, we characterize the thicknesses of individual retinal sublayers by macular optical coherence tomography (OCT) in a large cohort of patients with molecularly-confirmed, ABCA4-associated Stargardt disease (STGD1) relative to normal controls. Automated segmentation of retinal sublayers was performed with manual correction as needed, and thicknesses in various macular regions were compared using mixed effects models. Relative to controls (42 eyes, 40 patients), STGD1 patients (107 eyes, 63 patients) had slight thickening of the nerve fiber layer and retinal pigment epithelium-Bruch's membrane, with thinning in other sublayers, especially the outer nuclear layer (ONL) (p < 0.0015). When comparing the rate of retinal sublayer thickness change over time (mean follow-up 3.9 years for STGD1, 2.5 years for controls), STGD1 retinas thinned faster than controls in the outer retina (ONL to photoreceptor outer segments). OCT-based retinal sublayer thickness measurements are feasible in STGD1 patients and may provide objective measures of disease progression or treatment response.