Astronauts are reported to have experienced some impairment in visual acuity during their mission on the International Space Station (ISS) and after they returned to Earth. There is emerging evidence that changes in vision may involve alterations in ocular structure and function. To investigate possible mechanisms, changes in protein expression profiles and oxidative stress-associated apoptosis were examined in mouse ocular tissue after spaceflight. Nine-week-old male C57BL/6 mice (n = 12) were launched from the Kennedy Space Center on a SpaceX rocket to the ISS for a 35-day mission. The animals were housed in the mouse Habitat Cage Unit (HCU) in the Japan Aerospace Exploration Agency (JAXA) “Kibo” facility on the ISS. The flight mice lived either under an ambient microgravity condition (µg) or in a centrifugal habitat unit that produced 1 g artificial gravity (µg + 1 g). Habitat control (HC) and vivarium control mice lived on Earth in HCUs or normal vivarium cages, respectively. Quantitative assessment of ocular tissue demonstrated that the µg group induced significant apoptosis in the retina vascular endothelial cells compared to all other groups (p < 0.05) that was 64% greater than that in the HC group. Proteomic analysis showed that many key pathways responsible for cell death, cell repair, inflammation, and metabolic stress were significantly altered in µg mice compared to HC animals. Additionally, there were more significant changes in regulated protein expression in the µg group relative to that in the µg + 1 g group. These data provide evidence that spaceflight induces retinal apoptosis of vascular endothelial cells and changes in retinal protein expression related to cellular structure, immune response and metabolic function, and that artificial gravity (AG) provides some protection against these changes. These retinal cellular responses may affect blood–retinal barrier (BRB) integrity, visual acuity, and impact the potential risk of developing late retinal degeneration.
p66Shc is an oxidoreductase that responds to cell stress by translocating to mitochondria, where p66Shc produces pro-apoptotic reactive oxygen species (ROS). This study identifies ROS-active p66Shc as a monomer that produces superoxide anion independent of metal ions, inhibits cytochrome c peroxidase, and is regulated by environmental condition-induced structural changes. p66Shc anti-apoptotic functions, including: cytochrome c reduction, increased electron transport chain activity, and caspase cascade inhibition were also discovered. This study also demonstrates that p66Shc is a stress-dependent rheostat of apoptosis, regulated by p66Shc-mortalin complexes. These complexes decrease pro-apoptotic ROS production, without blocking p66Shc-mediated cytochrome c reduction. However, stress disrupts p66Shc-mortalin interactions, promoting apoptosis. Tipping p66Shc apoptotic balance toward anti-apoptotic functions by genetic knockdown or p66Shc-selective ROS inhibition decreased pro-apoptotic effects and improved outcomes in zebrafish myocardial infarction models, representing a potential new myocardial infarction treatment with promising results.
Background: Neoadjuvant chemotherapy (NACT) is an increasingly used approach for treatment of breast cancer. The pathological complete response (pCR) is considered a good predictor of disease-specific survival. This study investigated whether circulating exosomal microRNAs could predict pCR in breast cancer patients treated with NACT. Method: Plasma samples of 20 breast cancer patients treated with NACT were collected prior to and after the first cycle. RNA sequencing was used to determine microRNA profiling. The Cancer Genome Atlas (TCGA) was used to explore the expression patterns and survivability of the candidate miRNAs, and their potential targets based on the expression levels and copy number variation (CNV) data. Results: Three miRNAs before that NACT (miR-30b, miR-328 and miR-423) predicted pCR in all of the analyzed samples. Upregulation of miR-127 correlated with pCR in triple-negative breast cancer (TNBC). After the first NACT dose, pCR was predicted by exo-miR-141, while miR-34a, exo-miR182, and exo-miR-183 predicted non-pCR. A significant correlation between the candidate miRNAs and the overall survival, subtype, and metastasis in breast cancer, suggesting their potential role as predictive biomarkers of pCR. Conclusions: If the miRNAs identified in this study are validated in a large cohort of patients, they might serve as predictive non-invasive liquid biopsy biomarkers for monitoring pCR to NACT in breast cancer.
Cancer immunotherapy provides durable clinical benefit in only a small fraction of patients, and identifying these patients is difficult due to a lack of reliable biomarkers for prediction and evaluation of treatment response. Here, we demonstrate the first application of label-free Raman spectroscopy for elucidating biomolecular changes induced by anti–CTLA4 and anti–PD-L1 immune checkpoint inhibitors (ICI) in the tumor microenvironment (TME) of colorectal tumor xenografts. Multivariate curve resolution–alternating least squares (MCR-ALS) decomposition of Raman spectral datasets revealed early changes in lipid, nucleic acid, and collagen content following therapy. Support vector machine classifiers and random forests analysis provided excellent prediction accuracies for response to both ICIs and delineated spectral markers specific to each therapy, consistent with their differential mechanisms of action. Corroborated by proteomics analysis, our observation of biomolecular changes in the TME should catalyze detailed investigations for translating such markers and label-free Raman spectroscopy for clinical monitoring of immunotherapy response in cancer patients. Significance: This study provides first-in-class evidence that optical spectroscopy allows sensitive detection of early changes in the biomolecular composition of tumors that predict response to immunotherapy with immune checkpoint inhibitors.
Background: Chemotherapy treatment for breast cancer can induce cognitive impairments often involving oxidative stress. The brain, as a whole, is susceptible to oxidative stress due to its high-energy requirements, limited anaerobic respiration capacities, and limited antioxidant defenses. The goal of the current study was to determine if the manganese porphyrin superoxide dismutase mimetic MnTnBuOE-2-PyP (MnBuOE) could ameliorate the effects of doxorubicin, cyclophosphamide, and paclitaxel (AC-T) on mature dendrite morphology and cognitive function. Methods: Four-month-old female C57BL/6 mice received intraperitoneal injections of chemotherapy followed by subcutaneous injections of MnBuOE. Four weeks following chemotherapy treatment, mice were tested for hippocampus-dependent cognitive performance in the Morris water maze. After testing, brains were collected for Golgi staining and molecular analyses. Results: MnBuOE treatment preserved spatial memory during the Morris water-maze. MnBuOE/AC-T showed spatial memory retention during all probe trials. AC-T treatment significantly impaired spatial memory retention in the first and third probe trial (no platform). AC-T treatment decreased dendritic length in the Cornu Ammonis 1 (CA1) and dentate gyrus (DG) areas of the hippocampus while AC-T/MnBuOE maintained dendritic length. Comparative proteomic analysis revealed affected protein networks associated with cell morphology and behavior functions in both the AC-T and AC-T/MnBuOE treatment groups.
The damaging effects of ionizing radiation (IR) on bone mass are well-documented in mice and humans and are most likely due to increased osteoclast number and function. However, the mechanisms leading to inappropriate increases in osteoclastic bone resorption are only partially understood. Here, we show that exposure to multiple fractions of low-doses (10 fractions of 0.4 Gy total body irradiation [TBI]/week, i.e., fractionated exposure) and/or a single exposure to the same total dose of 4 Gy TBI causes a decrease in trabecular, but not cortical, bone mass in young adult male mice. This damaging effect was associated with highly activated bone resorption. Both osteoclast differentiation and maturation increased in cultures of bone marrow-derived macrophages from mice exposed to either fractionated or singular TBI. IR also increased the expression and enzymatic activity of mitochondrial deacetylase Sirtuin-3 (Sirt3)—an essential protein for osteoclast mitochondrial activity and bone resorption in the development of osteoporosis. Osteoclast progenitors lacking Sirt3 exposed to IR exhibited impaired resorptive activity. Taken together, targeting impairment of osteoclast mitochondrial activity could be a novel therapeutic strategy for IR-induced bone loss, and Sirt3 is likely a major mediator of this effect.
Therapeutic approaches to treat melanoma include small molecule drugs that target activating protein mutations in pro-growth signaling pathways like the MAPK pathway. While beneficial to the approximately 50% of patients with activatingBRAFV600mutation, mono- and combination therapy with MAPK inhibitors is ultimately associated with acquired resistance. To better characterize the mechanisms of MAPK inhibitor resistance in melanoma, we utilize patient-derived xenografts and apply proteogenomic approaches leveraging genomic, transcriptomic, and proteomic technologies that permit the identification of resistance-specific alterations and therapeutic vulnerabilities. A specific challenge for proteogenomic applications comes at the level of data curation to enable multi-omics data integration. Here, we present a proteogenomic approach that uses custom curated databases to identify unique resistance-specific alternations in melanoma PDX models of acquired MAPK inhibitor resistance. We demonstrate this approach with aNRASQ61Lmelanoma PDX model from which resistant tumors were developed following treatment with a MEK inhibitor. Our multi-omics strategy addresses current challenges in bioinformatics by leveraging development of custom curated proteogenomics databases derived from individual resistant melanoma that evolves following MEK inhibitor treatment and is scalable to comprehensively characterize acquired MAPK inhibitor resistance across patient-specific models and genomic subtypes of melanoma. The computational workflow for curation of a proteogenomics database is described here.
Leishmania parasites cause cutaneous leishmaniasis (CL), a disease characterized by disfiguring, ulcerative skin lesions. Both parasite and host gene expression following infection with various Leishmania species has been investigated in vitro, but global transcriptional analysis following L. major infection in vivo is lacking. Thus, we conducted a comprehensive transcriptomic profiling study combining bulk RNA sequencing (RNA-Seq) and single-cell RNA sequencing (scRNA-Seq) to identify global changes in gene expression in vivo following L. major infection. Bulk RNA-Seq analysis revealed that host immune response pathways like the antigen processing and presentation pathway were significantly enriched amongst differentially expressed genes (DEGs) upon infection, while ribosomal pathways were significantly downregulated in infected mice compared to naive controls. scRNA-Seq analyses revealed cellular heterogeneity including distinct resident and recruited cell types in the skin following murine L. major infection. Within the individual immune cell types, several DEGs indicative of many interferon induced GTPases and antigen presentation molecules were significantly enhanced in the infected ears including macrophages, resident macrophages, and inflammatory monocytes. Ingenuity Pathway Analysis of scRNA-Seq data indicated the antigen presentation pathway was increased with infection, while EIF2 signaling is the top downregulated pathway followed by eIF4/p70S6k and mTOR signaling in multiple cell types including macrophages, blood and lymphatic endothelial cells. Altogether, this transcriptomic profile highlights known recruitment of myeloid cells to lesions and recognizes a potential role for EIF2 signaling in murine L. major infection in vivo.
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