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Human papillomavirus (HPV) infection is a major cause of cervical cancer. Studies showed HPV carcinogenesis may be induced by oxidative stress affecting the host immune system. The objective of this study is to evaluate levels of four circulating oxidative stress biomarkers associated with the HPV infection, persistence, and cervical lesion status in women. The three serum biomarkers measuring oxidative damage to biomolecules (8-oxodG, 8-oxo-7,8-dihydro-2'-deoxyguanosine [8-oxodG] for DNA, 4-hydroxy-2-nonenal [4-HNE] for lipid, and protein carbonyl [PC] for protein) and one antioxidant (glutathione, GSH) collected from 38 women were evaluated. The PC levels were significantly higher for women with oncogenic HPV infection (p = 0.047) and persistence (p = 0.053) based on the unadjusted linear model. In particular, women with ≥3 oncogenic HPV types had a higher PC level than those without HPV infection (p = 0.041). Women with low-grade squamous intraepithelial lesions showed an elevated PC (p = 0.058). These trends remained similar after adjusting for age. The GSH levels were lower for women infected with ≥3 oncogenic HPV types based on age-adjusted results (p = 0.061). This study supported that serum PC was associated with HPV infection, persistence, and cervical lesions, so it can potentially be used to monitor HPV carcinogenesis. Further large-scale studies will be needed to confirm these findings.
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Infecções por Papillomavirus , Infecções Sexualmente Transmissíveis , Feminino , Humanos , Infecções por Papillomavirus/complicações , 8-Hidroxi-2'-Desoxiguanosina , Biomarcadores , Carcinogênese , Glutationa , Estresse Oxidativo , GenitáliaRESUMO
People living with HIV (PLWH) represent a vulnerable population to adverse musculoskeletal outcomes due to HIV infection, antiretroviral therapy (ART), and at-risk alcohol use. Developing measures to prevent skeletal degeneration in this group requires a grasp of the relationship between alcohol use and low bone mass in both the PLWH population and its constituents as defined by sex, age, and race. We examined the association of alcohol use with serum biochemical markers of bone health in a diverse cohort of PLWH enrolled in the New Orleans Alcohol Use in HIV (NOAH) study. To explore the effects of alcohol on bone in the context of HIV and ART and the role of estrogen, we conducted a parallel, translational study using simian immunodeficiency virus (SIV)+/ART+ female rhesus macaques divided into four groups: vehicle (Veh)/Sham; chronic binge alcohol (CBA)/Sham; Veh/ovariectomy (OVX); and CBA/OVX. Clinical data showed that both osteocalcin (Ocn) and procollagen type I N-propeptide (PINP) levels were inversely associated with multiple measures of alcohol consumption. Age (>50 years) significantly increased susceptibility to alcohol-associated suppression of bone formation in both female and male PLWH, with postmenopausal status appearing as an additional risk factor in females. Serum sclerostin (Scl) levels correlated positively with measures of alcohol use and negatively with Ocn. Micro-CT analysis of the macaque tibias revealed that although both CBA and OVX independently decreased trabecular number and bone mineral density, only OVX decreased trabecular bone volume fraction and impacted cortical geometry. The clinical data implicate circulating Scl in the pathogenesis of alcohol-induced osteopenia and suggest that bone morphology can be significantly altered in the absence of net change in osteoblast function as measured by serum markers. Inclusion of sophisticated tools to evaluate skeletal strength in clinical populations will be essential to understand the impact of alcohol-induced changes in bone microarchitecture. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
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OBJECTIVES: Rheumatoid arthritis (RA) is a chronic autoimmune disease, that involves both pro- and anti-inflammatory mechanisms. The purpose of the present study is to investigate T-cell immunoglobulin and mucin domain 3 (Tim-3) in RA. METHODS: Plasma levels of soluble (s) Tim-3 in early RA (n=98), were followed, to evaluate association with treatment and disease activity, acquired from a prospective collected biobank (clinicaltrials.gov (NCT00660647)). We also investigate the influence of Tim-3 on spontaneous cytokine production in synovial fluid mononuclear cells (SFMC) from RA patients after addition of neutralizing anti-Tim-3's antibodies, either alone or in combination with neutralizing anti-Programmed Cell death protein 1 (PD-1) antibodies. RESULTS: Long-time stimulated CD4 T-cells expressed high levels of Tim-3, but tended to decrease their PD-1 expression. Tim-3 expression was exclusively seen co-expressed with PD-1 by CD3, CD4, CD45RO positive cells in the inflamed RA joint. Addition of neutralizing Tim-3 antibodies increased the secretion of IFNγ and MCP-1, in SFMC cultures from RA. Whereas neutralizing anti-PD-1 antibodies showed a broader impact on cytokine production. Finally, we observed that soluble Tim-3 is increased in plasma and is associated with disease activity in early RA. CONCLUSION: Taken together, our findings indicate disease-suppressive functions of Tim-3 in RA.
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Tightly regulated and cell-specific NADPH-oxidases (Nox) represent one of the major sources of reactive oxygen species (ROS) signaling molecules that are involved in tissue development and stem cell self-renewal. We have characterized the role of Nox4 in osteo-progenitors during postnatal bone development. Nox4 expression in bone and ROS generation were increased during early osteoblast differentiation and bone development. Stromal osteoblastic cell self-renewal, proliferation and ROS production were significantly lower in samples from whole-body Nox4 knockout mice (Nox4-/-) and conditional knockout (CKO) mice with depletion of Nox4 in the limb bud mesenchyme compared with those from control mice (Nox4fl/fl), but they were reversed after 9 passages. In both sexes, bone volume, trabecular number and bone mineral density were significantly lower in 3-week old CKO and Nox4-/- mice compared with Nox4fl/fl controls. This was reflected in serum levels of bone formation markers alkaline phosphatase (ALP) and procollagen 1 intact N-terminal propeptide (P1NP). However, under-developed bone formation in 3-week old CKO and Nox4-/- mice quickly caught up to levels of control mice by 6-week of age, remained no different at 13-week of age, and was reversed in 32-week old male mice. Osteoclastogenesis showed no differences among groups, however, CTX1 reflecting osteoclast activity was significantly higher in 3-week old male CKO and Nox4-/- mice compared with control mice, and significantly lower in 32-week old Nox4-/- mice compared with control mice. These data suggest that Nox4 expression and ROS signaling in bone and osteoblastic cells coordinately play an important role in osteoblast differentiation, proliferation and maturation.
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Desenvolvimento Ósseo , NADPH Oxidase 4 , Osteogênese , Animais , Desenvolvimento Ósseo/fisiologia , Feminino , Masculino , Camundongos , Camundongos Knockout , NADPH Oxidase 4/biossíntese , NADPH Oxidase 4/genética , NADPH Oxidase 4/metabolismo , Osteogênese/fisiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Objective: The programmed death-1 (PD-1) pathway is essential for maintaining self-tolerance and plays an important role in autoimmunity, including rheumatoid arthritis (RA). Here, we investigated how membrane-bound and soluble (s)PD-1 influence bone homeostasis during chronic inflammation, exemplified in RA. Methods: Bone mineral density and bone microstructure were examined in PD-1 and PD-L1 knockout (KO) mice and compared with wild-type (WT) mice. Receptor activator of nuclear factor kappa-B ligand (RANKL) was measured in serum, and the expression examined on activated bone marrow cells. Osteoclast formation was examined in cells from murine spleen and bone marrow and from human synovial fluid cells. sPD-1 was measured in chronic and early (e)RA patients and correlated to markers of disease activity and radiographic scores. Results: PD-1 and PD-L1 KO mice showed signs of osteoporosis. This was supported by a significantly reduced trabecular bone volume fraction and deteriorated microstructure, as well as increased osteoclast formation and an increased RANKL/OPG ratio. The recombinant form of sPD-1 decreased osteoclast formation in vitro, but was closely associated with disease activity markers in eRA patients. Sustained elevated sPD-1 levels indicated ongoing inflammation and were associated with increased radiographic progression. Conclusion: The PD-1 pathway is closely associated with bone homeostasis, and lacking members of this pathway causes a deteriorated bone structure. The immunological balance in the microenvironment determines how the PD-1 pathway regulates osteoclast formation. In eRA patients, sPD-1 may serve as a biomarker, reflecting residual but clinically silent disease activity and radiographic progression.
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Artrite Reumatoide , Osteoclastos , Animais , Artrite Reumatoide/metabolismo , Antígeno B7-H1 , Biomarcadores , Humanos , Inflamação , Camundongos , Osteoclastos/metabolismo , Receptor de Morte Celular Programada 1/genéticaRESUMO
Excessive ethanol consumption is a risk factor for osteopenia. Since a previous study showed that transgenic female mice with overexpression of catalase are partially protected from ethanol-mediated trabecular bone loss, we investigated the role of endogenous catalase in skeletal ethanol toxicity comparing catalase knockout to wild-type mice. We hypothesized that catalase depletion would exacerbate ethanol effects. The mice were tested in a newly designed binge ethanol model, in which 12-week-old mice were exposed to 4 consecutive days of gavage with ethanol at 3, 3, 4, and 4.5 g ethanol/kg body weight. Binge ethanol decreased the concentration of serum osteocalcin, a marker of bone formation. The catalase genotype did not affect the osteocalcin levels. RNA sequencing of femoral shaft RNA from males was conducted. Ethanol exposure led to significant downregulation of genes expressed in cells of the osteoblastic lineage with a role in osteoblastic function and collagen synthesis, including the genes encoding major structural bone proteins. Binge ethanol further induced a smaller set of genes with a role in osteoclastic differentiation. Catalase depletion affected genes with expression in erythroblasts and erythrocytes. There was no clear interaction between binge ethanol and the catalase genotype. In an independent experiment, we confirmed that the binge ethanol effects on gene expression were reproducible and occurred throughout the skeleton in males. In conclusion, the binge ethanol exposure, independently of endogenous catalase, reduces expression of genes involved in osteoblastic function and induces expression of genes involved in osteoclast differentiation throughout the skeleton in males.
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Etanol , Osteoclastos , Animais , Catalase/genética , Catalase/metabolismo , Catalase/farmacologia , Etanol/metabolismo , Etanol/toxicidade , Feminino , Masculino , Camundongos , Camundongos Transgênicos , OsteoblastosRESUMO
Activating mutations in some isoforms of RAS or RAF are drivers of a substantial proportion of cancers. The main Raf effector, MEK1/2, can be targeted with several highly specific inhibitors. The clinical activity of these inhibitors seems to be mixed, showing efficacy against mutant BRAF-driven tumors but not KRAS-driven tumors, such as pancreatic adenocarcinomas. To improve our understanding of this context-dependent efficacy, we generated pancreatic cancer cells resistant to MEK1/2 inhibition, which were also resistant to KRAS and ERK1/2 inhibitors. Compared with parental cells, inhibitor-resistant cells showed several phenotypic changes including increased metastatic ability in vivo. The transcription factor SLUG, which is known to induce epithelial-to-mesenchymal transition, was identified as the key factor responsible for both resistance to MEK1/2 inhibition and increased metastasis. Slug, but not similar transcription factors, predicted poor prognosis of pancreatic cancer patients and induced the transition to a cellular phenotype in which cell-cycle progression becomes independent of the KRAS-RAF-MEK1/2-ERK1/2 pathway. SLUG was targeted using two independent strategies: (i) inhibition of the MEK5-ERK5 pathway, which is responsible for upregulation of SLUG upon MEK1/2 inhibition, and (ii) direct PROTAC-mediated degradation. Both strategies were efficacious in preclinical pancreatic cancer models, paving the path for the development of more effective therapies against pancreatic cancer. SIGNIFICANCE: This study demonstrates that SLUG confers resistance to MEK1/2 inhibitors in pancreatic cancer by uncoupling tumor progression from KRAS-RAF-MEK1/2-ERK1/2 signaling, providing new therapeutic opportunities. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/14/3849/F1.large.jpg.
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Sistema de Sinalização das MAP Quinases , Neoplasias Pancreáticas/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Animais , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Pancreáticas/genética , Quinases raf/metabolismoRESUMO
BACKGROUND: Around 15-20% of primary breast cancers are characterized by HER2 protein overexpression and/or HER2 gene amplification. Despite the successful development of anti-HER2 drugs, intrinsic and acquired resistance represents a major hurdle. This study was performed to analyze the RANK pathway contribution in HER2-positive breast cancer and anti-HER2 therapy resistance. METHODS: RANK and RANKL protein expression was assessed in samples from HER2-positive breast cancer patients resistant to anti-HER2 therapy and treatment-naive patients. RANK and RANKL gene expression was analyzed in paired samples from patients treated with neoadjuvant dual HER2-blockade (lapatinib and trastuzumab) from the SOLTI-1114 PAMELA trial. Additionally, HER2-positive breast cancer cell lines were used to modulate RANK expression and analyze in vitro the contribution of RANK signaling to anti-HER2 resistance and downstream signaling. RESULTS: RANK and RANKL proteins are more frequently detected in HER2-positive tumors that have acquired resistance to anti-HER2 therapies than in treatment-naive ones. RANK (but not RANKL) gene expression increased after dual anti-HER2 neoadjuvant therapy in the cohort from the SOLTI-1114 PAMELA trial. Results in HER2-positive breast cancer cell lines recapitulate the clinical observations, with increased RANK expression observed after short-term treatment with the HER2 inhibitor lapatinib or dual anti-HER2 therapy and in lapatinib-resistant cells. After RANKL stimulation, lapatinib-resistant cells show increased NF-κB activation compared to their sensitive counterparts, confirming the enhanced functionality of the RANK pathway in anti-HER2-resistant breast cancer. Overactivation of the RANK signaling pathway enhances ERK and NF-κB signaling and increases lapatinib resistance in different HER2-positive breast cancer cell lines, whereas RANK loss sensitizes lapatinib-resistant cells to the drug. Our results indicate that ErbB signaling is required for RANK/RANKL-driven activation of ERK in several HER2-positive cell lines. In contrast, lapatinib is not able to counteract the NF-κB activation elicited after RANKL treatment in RANK-overexpressing cells. Finally, we show that RANK binds to HER2 in breast cancer cells and that enhanced RANK pathway activation alters HER2 phosphorylation status. CONCLUSIONS: Our data support a physical and functional link between RANK and HER2 signaling in breast cancer and demonstrate that increased RANK signaling may contribute to the development of lapatinib resistance through NF-κB activation. Whether HER2-positive breast cancer patients with tumoral RANK expression might benefit from dual HER2 and RANK inhibition therapy remains to be elucidated.
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Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Receptor ErbB-2/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lapatinib/uso terapêutico , NF-kappa B/metabolismo , Terapia Neoadjuvante , Ligação Proteica , Receptor Ativador de Fator Nuclear kappa-B/genética , Receptor ErbB-2/antagonistas & inibidores , Transdução de Sinais , Trastuzumab/uso terapêuticoRESUMO
Estrogen deficiency and aging play critical roles in the pathophysiology of bone as a result of increased oxidative stress. It has been suggested that prevention of NADPH oxidase- (Nox-) dependent accumulation of ROS may be an approach to potentially minimize bone loss caused by these conditions. Using ovariectomized (OVX) and Nox4 gene-deletion mouse models, we investigated the role of Nox4 in OVX-induced bone loss and osteoblast senescence signaling. Six-month-old WT C57Bl6 mice were allocated to a sham control group, OVX, and OVX plus E2 treatment group for 8 weeks. Decreased bone mass including BMD and BMC were found in the OVX group compared with the sham control (p < 0.05); E2 treatment completely reversed OVX-induced bone loss. Interestingly, the prevention of OVX-induced bone loss by E2 was associated with the elimination of increased senescence signaling in bone osteoblastic cells from the OVX group. E2 blunted OVX-induced p53 and p21 overexpression, but not p16 and Nox4 in bone. In addition, 8- and 11-month-old Nox4 KO female mice were OVX for 8 weeks. Significant bone loss and increased bone osteoblastic cell senescence signaling occurred not only in Nox4 KO OVX mice compared with sham-operated animals, but also in 11-month-old Nox4 KO sham mice compared with 8-month-old Nox4 KO sham mice (p < 0.05). These data suggest that Nox4-mediated ROS in bone osteoblastic cells may be dispensable for sex steroid deficiency-induced bone loss and senescence. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.
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We have previously demonstrated promotion of diethylnitrosamine (DEN) initiated liver tumorigenesis after feeding diets high in fat or ethanol (EtOH) to male mice. This was accompanied by hepatic induction of the proto-oncogene PIKE (Agap2). Switch of dietary protein from casein to soy protein isolate (SPI) significantly reduced tumor formation in these models. We have linked EtOH consumption in mice to microbial dysbiosis. Adoptive transfer studies demonstrate that microbiota from mice fed ethanol can induce hepatic steatosis in the absence of ethanol suggesting that microbiota or the microbial metabolome play key roles in development of fatty liver disease. Feeding SPI significantly changed gut bacteria in mice increasing alpha diversity (P < 0.05) and levels of Clostidiales spp. Feeding soy formula to piglets also resulted in significant changes in microbiota, the pattern of bile acid metabolites and in inhibition of the intestinal-hepatic FXR/FGF19-SHP pathway which has been linked to both steatosis and hepatocyte proliferation. Moreover, feeding SPI also resulted in induction of hepatic PPARα signaling and inhibition of PIKE mRNA expression coincident with inhibition of steatosis and cancer prevention. Feeding studies in the DEN model with differing dietary fats demonstrated tumor promotion specific to the saturated fat, cocoa butter relative to diets containing olive oil or corn oil associated with microbial dysbiosis including dramatic increases in Lachnospiraceae particularly from the genus Coprococcus. Immunohistochemical analysis demonstrated that tumors from EtOH-fed mice and patients with alcohol-associated HCC also expressed high levels of a novel cytochrome P450 enzyme CYP2W1. Additional adoptive transfer experiments and studies in knockout mice are required to determine the exact relationship between soy effects on the microbiota, expression of PIKE, CYP2W1, PPARα activation and prevention of tumorigenesis.
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Família 2 do Citocromo P450/metabolismo , Microbioma Gastrointestinal , Neoplasias Hepáticas/complicações , Neoplasias Hepáticas/prevenção & controle , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Hepatopatia Gordurosa não Alcoólica/complicações , Proteínas de Soja/farmacologia , Animais , Carcinogênese/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Epidemiological evidence links polychlorinated biphenyls (PCBs) to skeletal toxicity, however mechanisms whereby PCBs affect bone are poorly studied. In this study, coplanar PCB 126 (5 µmol/kg) or corn oil vehicle was administered to N = 5 and 6 male and female, wild type (WT) or AhR -/- rats via intraperitoneal injection. Animals were sacrificed after 4 weeks. Bone length was measured; bone morphology was assessed by microcomputed tomography and dynamic histomorphometry. Reduced bone length was the only genotype-specific effect and only observed in males (p < .05). WT rats exposed to PCB 126 had reduced serum calcium, and smaller bones with reduced tibial length, cortical area, and medullary area relative to vehicle controls (p < .05). Reduced bone formation rate observed in dynamic histomorphometry was consistent with inhibition of endosteal and periosteal bone growth. The effects of PCB 126 were abolished in AhR -/- rats. Gene expression in bone marrow and shaft were assessed by RNA sequencing. Approximately 75% of the PCB-regulated genes appeared AhR dependent with 89 genes significantly (p < .05) regulated by both PCB 126 and knockout of the AhR gene. Novel targets significantly induced by PCB 126 included Indian hedgehog (Ihh) and connective tissue growth factor (Ctgf/Ccn2), which regulate chondrocyte proliferation and differentiation in the bone growth plate and cell-matrix interactions. These data suggest the toxic effects of PCB 126 on bone are mediated by AhR, which has direct effects on the growth plate and indirect actions related to endocrine disruption. These studies clarify important mechanisms underlying skeletal toxicity of dioxin-like PCBs and highlight potential therapeutic targets.
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Fatores de Transcrição Hélice-Alça-Hélice Básicos/agonistas , Disruptores Endócrinos/toxicidade , Lâmina de Crescimento/efeitos dos fármacos , Bifenilos Policlorados/toxicidade , Receptores de Hidrocarboneto Arílico/agonistas , Tíbia/efeitos dos fármacos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Lâmina de Crescimento/metabolismo , Lâmina de Crescimento/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , RNA-Seq , Ratos Sprague-Dawley , Ratos Transgênicos , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais , Tíbia/metabolismo , Tíbia/patologia , TranscriptomaRESUMO
Bone loss in response to alcohol intake has previously been hypothesized to be mediated by excessive production of reactive oxygen species via NADPH oxidase (Nox) enzymes. Nox4 is one of several Nox enzymes expressed in bone. We investigated the role of Nox4 in the chondro-osteoblastic lineage of the long bones in mice during normal chow feeding and during chronic ethanol feeding for 90 days. We generated mice with a genotype (PrxCre +/- Nox4 fl/fl) allowing conditional knockout of Nox4 in the limb bud mesenchyme. Adult mice had 95% knockdown of Nox4 expression in the femoral shafts. For mice on regular chow, only whole-body Nox4 knockout mice had clearly increased cortical thickness and bone mineral density in the tibiae. When chronically fed a liquid diet with and without ethanol, conditional Nox4 knockout mice had slightly reduced dimensions of the cortical and trabecular regions of the tibiae (P < 0.1). The ethanol diet caused a significant reduction in cortical bone area and cortical thickness relative to a control diet without ethanol (P < 0.05). The ethanol diet further reduced gene expression of Frizzled related protein (Frzb), myosin heavy chain 3, and several genes encoding collagen and other major structural bone proteins (P < 0.05), whereas the Nox4 genotype had no effects on these genes. In conclusion, Nox4 expression from both mesenchymal and nonmesenchymal cell lineages appears to exert subtle effects on bone. However, chronic ethanol feeding reduces cortical bone mass and cortical gene expression of major structural bone proteins in a Nox4-independent manner. SIGNIFICANCE STATEMENT: Excessive alcohol intake contributes to osteopenia and osteoporosis, with oxidative stress caused by the activity of NADPH oxidases hypothesized to be a mediator. We tested the role of NADPH oxidase (Nox) 4 in osteoblast precursors in the long bones of mice with a conditional Nox4 knockout model. We found that Nox4 exerted effects independent of alcohol intake, and ethanol effects on bone were Nox4-independent.
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Osso e Ossos/efeitos dos fármacos , Etanol/administração & dosagem , Expressão Gênica/efeitos dos fármacos , NADPH Oxidase 4/genética , Animais , Densidade Óssea/efeitos dos fármacos , Densidade Óssea/genética , Feminino , Genótipo , Masculino , Camundongos , Camundongos Knockout , NADPH Oxidases/genética , Osteoblastos/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
Accurate gene expression analysis of bone requires the ability to isolate RNA of good quality. Isolation of intact RNA from frozen bone tissue is problematic since RNA rapidly becomes degraded after thawing. Since we are interested in assessing gene expression from both bone marrow and mineralized bone, we aimed to develop improved simple, robust and statistically validated methods providing high-quality RNA from both mouse femur shaft and femur marrow. RNA integrity was quantified by the RNA Integrity Number (RIN) measured on a TapeStation. While the RNA stabilization reagent RNAlater is not commonly used or recommended for mineralized bone, we found that preservation methods with RNAlater significantly improved the RNA quality with a mean RIN for the femur shaft of 8.0 and a mean RIN for femur marrow of 9.6. With RNAlater, high quality RNA with a mean RIN of 9.3 could also be isolated from lumbar vertebral bone. A further advantage of using RNAlater is that the tissue can be allowed to thaw to room temperature before TRI Reagent lysis without any loss of RNA integrity. A comparison of the TRI Reagent method with a hybrid method combining TRI Reagent lysis with RNeasy column purification showed no difference in RNA integrity. However, the hybrid method seemed to give femur shaft RNA with fewer impurities inhibiting qRT-PCR.
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Liver cancer results in a high degree of mortality, especially among men. As fatty liver disease is a risk factor for development of hepatocellular carcinoma, we investigated the role of dietary fat type in tumor promotion by high-fat diets in mice after initiation with the chemical carcinogen diethyl nitrosamine. Tumor incidence and multiplicity were significantly greater in males than those in females. In males, fat type had complex effects on tumorigenesis. Preneoplastic foci were most prevalent in mice fed a polyunsaturated fat diet enriched in docosahexaenoic acid, whereas carcinomas and large visible liver tumors were significantly greater in mice fed a saturated fat diet made with cocoa butter relative to mice fed mono- or polyunsaturated fats. Different mechanisms thus seemed involved in early and late tumor promotion. The hepatic transcriptome and gut microbiome were assessed for traits associated with tumorigenesis. Hepatic expression of more than 20% of all genes was affected by sex, whereas fat type affected fewer genes. In males, the saturated fat diet induced expression of the proto-oncogene Agap2 and affected the expression of several cytochrome P450 genes, and genes involved in lipid, bile acid and fatty acid metabolism. The gut microbiome had a higher level of genus Akkermansia and a lower level of Firmicutes in females than in males. Males fed saturated fat had an altered microbiome, including an enrichment of the genus Coprococcus. In conclusion, sex and the dietary fat type affect the gut microbiome, the hepatic transcriptome and ultimately hepatic tumor growth.
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Carcinogênese/patologia , Dieta Hiperlipídica/efeitos adversos , Proteínas de Ligação ao GTP/metabolismo , Microbioma Gastrointestinal/fisiologia , Neoplasias Hepáticas/etiologia , Proto-Oncogenes/fisiologia , Animais , Ácidos e Sais Biliares/metabolismo , Carcinogênese/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/microbiologia , Carcinoma Hepatocelular/patologia , Gorduras na Dieta/efeitos adversos , Ácidos Docosa-Hexaenoicos/farmacologia , Ácidos Graxos/metabolismo , Feminino , Metabolismo dos Lipídeos/fisiologia , Fígado/metabolismo , Fígado/microbiologia , Fígado/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/microbiologia , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Molecular junctions offer unique opportunities for controlling charge transport on the atomic scale and for studying energy conversion. For example, quantum interference effects in molecular junctions have been proposed as an avenue for highly efficient thermoelectric power conversion at room temperature. Toward this goal, we investigated the effect of quantum interference on the thermoelectric properties of molecular junctions. Specifically, we employed oligo(phenylene ethynylene) (OPE) derivatives with a para-connected central phenyl ring ( para-OPE3) and meta-connected central ring ( meta-OPE3), which both covalently bind to gold via sulfur anchoring atoms located at their ends. In agreement with predictions from ab initio modeling, our experiments on both single molecules and monolayers show that meta-OPE3 junctions, which are expected to exhibit destructive interference effects, yield a higher thermopower (with â¼20 µV/K) compared with para-OPE3 (with â¼10 µV/K). Our results show that quantum interference effects can indeed be employed to enhance the thermoelectric properties of molecular junctions.
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BACKGROUND: Glutathione S-transferase A4-4 (GSTA4) is a key enzyme for removal of toxic lipid peroxidation products such as 4-hydroxynonenal (4-HNE). In this study, we examined the potential role of GSTA4 on protein carbonylation and progression of alcoholic liver disease by examining the development of liver injury in male wild-type (WT) SV/J mice and SV/J mice lacking functional GSTA4 (GSTA4-/- mice). METHODS: Adult male WT and GSTA4-/- mice were fed chow (N = 10 to 12) or high-fat Lieber-DeCarli liquid diets containing up to 28% calories as ethanol (EtOH) (N = 18 to 20) for 116 days. At the end of the study, half of the EtOH-fed mice were acutely challenged with an EtOH binge (3 g/kg given intragastrically) 12 hours before sacrifice. Carbonylation of liver proteins was assessed by immunohistochemical staining for 4-HNE adduction and by comprehensive liquid chromatography-tandem mass spectrometry (LC-MS/MS) of purified carbonylated proteins. RESULTS: Chronic EtOH intake significantly increased hepatic 4-HNE adduction and protein carbonylation, including carbonylation of ribosomal proteins. EtOH intake also resulted in steatosis and increased serum alanine aminotransferase. Hepatic infiltration with B cells, T cells, and neutrophils and mRNA expression of pro-inflammatory cytokines tumor necrosis factor (TNF)α and interferon (IFN)γ was modest in WT mice. However, an EtOH binge increased hepatic necrosis, hepatic cell proliferation, and expression of TNFα mRNA (p < 0.05). EtOH treatment of GSTA4-/- mice increased B-cell infiltration and increased mRNA expression of TNFα and IFNγ and of matrix remodeling markers MMP9, MMP13, and Col1A1 (p < 0.05). GSTA4-/- mice exhibited panlobular rather than periportal distribution of 4-HNE-adducted proteins and increased overall 4-HNE staining after EtOH binge. Comprehensive LC-MS of carbonylated proteins identified 1,022 proteins of which 189 were unique to the GSTA4-/- group. CONCLUSIONS: These data suggest long-term adaptation to EtOH in WT mice does not occur in GSTA4-/- mice. Products of lipid peroxidation appear to play a role in inflammatory responses due to EtOH. And EtOH effects on B-cell infiltration and autoimmune responses may be secondary to formation of carbonyl adducts.
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Etanol/toxicidade , Glutationa Transferase/deficiência , Glutationa Transferase/genética , Hepatopatias Alcoólicas/genética , Hepatopatias Alcoólicas/metabolismo , Carbonilação Proteica/fisiologia , Animais , Etanol/administração & dosagem , Glutationa Transferase/química , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Hepatopatias Alcoólicas/patologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Carbonilação Proteica/efeitos dos fármacos , Estrutura Secundária de ProteínaRESUMO
Over 70% of Americans take some form of dietary supplement every day, and the supplement industry is currently big business, with a gross of over $28 billion. However, unlike either foods or drugs, supplements do not need to be registered or approved by the US Food and Drug Administration (FDA) prior to production or sales. Under the Dietary Supplement Health and Education Act of 1994, the FDA is restricted to adverse report monitoring postmarketing. Despite widespread consumption, there is limited evidence of health benefits related to nutraceutical or supplement use in well-nourished adults. In contrast, a small number of these products have the potential to produce significant toxicity. In addition, patients often do not disclose supplement use to their physicians. Therefore, the risk of adverse drug-supplement interactions is significant. An overview of the major supplement and nutraceutical classes is presented here, together with known toxic effects and the potential for drug interactions.
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Suplementos Nutricionais/efeitos adversos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/etiologia , Animais , Interações Medicamentosas/fisiologia , Humanos , Estados Unidos , United States Food and Drug AdministrationRESUMO
Angiotensin-converting enzyme 2 (ACE2) has protective effects on a wide range of morbidities associated with elevated angiotensin-II signaling. Most tissues, including pancreatic islets, express ACE2 mainly from the proximal promoter region. We previously found that hepatocyte nuclear factors 1α and 1ß stimulate ACE2 expression from three highly conserved hepatocyte nuclear factor 1 binding motifs in the proximal promoter region. We hypothesized that other highly conserved motifs would also affect ACE2 expression. By systematic mutation of conserved elements, we identified five regions affecting ACE2 expression, of which two regions bound transcriptional activators. One of these is a functional FOXA binding motif. We further identified the main protein binding the FOXA motif in 832/13 insulinoma cells as well as in mouse pancreatic islets as FOXA2.
RESUMO
Molecular electronics offers unique scientific and technological possibilities, resulting from both the nanometre scale of the devices and their reproducible chemical complexity. Two fundamental yet different effects, with no classical analogue, have been demonstrated experimentally in single-molecule junctions: quantum interference due to competing electron transport pathways, and the Kondo effect due to entanglement from strong electronic interactions. Here we unify these phenomena, showing that transport through a spin-degenerate molecule can be either enhanced or blocked by Kondo correlations, depending on molecular structure, contacting geometry and applied gate voltages. An exact framework is developed, in terms of which the quantum interference properties of interacting molecular junctions can be systematically studied and understood. We prove that an exact Kondo-mediated conductance node results from destructive interference in exchange-cotunneling. Nonstandard temperature dependences and gate-tunable conductance peaks/nodes are demonstrated for prototypical molecular junctions, illustrating the intricate interplay of quantum effects beyond the single-orbital paradigm.
RESUMO
Angiotensin-converting enzyme 2 (ACE2) is a component of the renin-angiotensin system (RAS) which plays an important role in the regulation of blood pressure and volume homeostasis. Accumulating evidence shows alterations in ACE2 expression and activity in several hypertensive animal models, as well as in patients with hypertension. In order to assess the role of brain ACE2 in hypertension, a specific ACE2 assay is required. Based on a quenched fluorescent substrate, we describe an easy-to-use method for determining ACE2 activity in brain tissue and cerebrospinal fluid. The method can further be adapted for other tissues, plasma, cell extracts, and cell culture supernatants.