Publications by authors named "Paula Paccielli Freire"

24 Publications

  • Page 1 of 1

Severe COVID-19 Shares a Common Neutrophil Activation Signature with Other Acute Inflammatory States.

Cells 2022 03 1;11(5). Epub 2022 Mar 1.

Department of Imunology, Institute of Biomedical Sciences, University of São Paulo, São Paulo 05508-000, Brazil.

Severe COVID-19 patients present a clinical and laboratory overlap with other hyperinflammatory conditions such as hemophagocytic lymphohistiocytosis (HLH). However, the underlying mechanisms of these conditions remain to be explored. Here, we investigated the transcriptome of 1596 individuals, including patients with COVID-19 in comparison to healthy controls, other acute inflammatory states (HLH, multisystem inflammatory syndrome in children [MIS-C], Kawasaki disease [KD]), and different respiratory infections (seasonal coronavirus, influenza, bacterial pneumonia). We observed that COVID-19 and HLH share immunological pathways (cytokine/chemokine signaling and neutrophil-mediated immune responses), including gene signatures that stratify COVID-19 patients admitted to the intensive care unit (ICU) and COVID-19_nonICU patients. Of note, among the common differentially expressed genes (DEG), there is a cluster of neutrophil-associated genes that reflects a generalized hyperinflammatory state since it is also dysregulated in patients with KD and bacterial pneumonia. These genes are dysregulated at the protein level across several COVID-19 studies and form an interconnected network with differentially expressed plasma proteins that point to neutrophil hyperactivation in COVID-19 patients admitted to the intensive care unit. scRNAseq analysis indicated that these genes are specifically upregulated across different leukocyte populations, including lymphocyte subsets and immature neutrophils. Artificial intelligence modeling confirmed the strong association of these genes with COVID-19 severity. Thus, our work indicates putative therapeutic pathways for intervention.
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http://dx.doi.org/10.3390/cells11050847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8909161PMC
March 2022

Autoantibodies targeting GPCRs and RAS-related molecules associate with COVID-19 severity.

Nat Commun 2022 03 9;13(1):1220. Epub 2022 Mar 9.

School of Medicine and Health Sciences, George Washington University, Washington, DC, USA.

COVID-19 shares the feature of autoantibody production with systemic autoimmune diseases. In order to understand the role of these immune globulins in the pathogenesis of the disease, it is important to explore the autoantibody spectra. Here we show, by a cross-sectional study of 246 individuals, that autoantibodies targeting G protein-coupled receptors (GPCR) and RAS-related molecules associate with the clinical severity of COVID-19. Patients with moderate and severe disease are characterized by higher autoantibody levels than healthy controls and those with mild COVID-19 disease. Among the anti-GPCR autoantibodies, machine learning classification identifies the chemokine receptor CXCR3 and the RAS-related molecule AGTR1 as targets for antibodies with the strongest association to disease severity. Besides antibody levels, autoantibody network signatures are also changing in patients with intermediate or high disease severity. Although our current and previous studies identify anti-GPCR antibodies as natural components of human biology, their production is deregulated in COVID-19 and their level and pattern alterations might predict COVID-19 disease severity.
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http://dx.doi.org/10.1038/s41467-022-28905-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8907309PMC
March 2022

Transcriptomic Response to Acidosis Reveals Its Contribution to Bone Metastasis in Breast Cancer Cells.

Cells 2022 02 4;11(3). Epub 2022 Feb 4.

Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of São Paulo, São Paulo 05508-000, Brazil.

Bone is the most common site of metastasis in breast cancer. Metastasis is promoted by acidosis, which is associated with osteoporosis. To investigate how acidosis could promote bone metastasis, we compared differentially expressed genes (DEGs) in MDA-MB-231 cancer cells in acidosis, bone metastasis, and bone metastatic tumors. The DEGs were identified using Biojupies and GEO2R. The expression profiles were assessed with Morpheus. The overlapping DEGs between acidosis and bone metastasis were compared to the bulk of the DEGs in terms of the most important genes and enriched terms using CytoHubba and STRING. The expression of the genes in this overlap filtered by secreted proteins was assessed in the osteoporosis secretome. The analysis revealed that acidosis-associated transcriptomic changes were more similar to bone metastasis than bone metastatic tumors. Extracellular matrix (ECM) organization would be the main biological process shared between acidosis and bone metastasis. The secretome genes upregulated in acidosis, bone metastasis, and osteoporosis-associated mesenchymal stem cells are enriched for ECM organization and angiogenesis. Therefore, acidosis may be more important in the metastatic niche than in the primary tumor. Acidosis may contribute to bone metastasis by promoting ECM organization. Untreated osteoporosis could favor bone metastasis through the increased secretion of ECM organization proteins.
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http://dx.doi.org/10.3390/cells11030544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8834614PMC
February 2022

Aging whole blood transcriptome reveals candidate genes for SARS-CoV-2-related vascular and immune alterations.

J Mol Med (Berl) 2022 02 6;100(2):285-301. Epub 2021 Nov 6.

Department of Structural and Functional Biology, Institute of Biosciences, UNESP-São Paulo State University, 510, Rubião Júnior, s/n, P.O. Box, Botucatu, São Paulo, 18618-689, Brazil.

The risk of severe COVID-19 increases with age as older patients are at highest risk. Thus, there is an urgent need to identify how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) interacts with blood components during aging. We investigated the whole blood transcriptome from the Genotype-Tissue Expression (GTEx) database to explore differentially expressed genes (DEGs) translated into proteins interacting with viral proteins during aging. From 22 DEGs in aged blood, FASLG, CTSW, CTSE, VCAM1, and BAG3 were associated with immune response, inflammation, cell component and adhesion, and platelet activation/aggregation. Males and females older than 50 years old overexpress FASLG, possibly inducing a hyperinflammatory cascade. The expression of cathepsins (CTSW and CTSE) and the anti-apoptotic co-chaperone molecule BAG3 also increased throughout aging in both genders. By exploring single-cell RNA-sequencing data from peripheral blood of SARS-CoV-2-infected patients, we found FASLG and CTSW expressed in natural killer cells and CD8 + T lymphocytes, whereas BAG3 was expressed mainly in CD4 + T cells, naive T cells, and CD14 + monocytes. In addition, T cell exhaustion was associated with increased expression of CCL4L2 and DUSP4 over blood aging. LAG3, PDCD1, TIGIT, VCAM1, HLA-DRA, and TOX also increased in individuals aged 60-69 years old; conversely, the RGS2 gene decreased with aging. We further identified a distinct gene expression profile associated with type I interferon signaling following blood aging. These results revealed changes in blood molecules potentially related to SARS-CoV-2 infection throughout aging, emphasizing them as therapeutic candidates for aggressive clinical manifestation of COVID-19. KEY MESSAGES: • Prediction of host-viral interactions in the whole blood transcriptome during aging. • Expression levels of FASLG, CTSW, CTSE, VCAM1, and BAG3 increase in aged blood. • Blood interactome reveals targets involved with immune response, inflammation, and blood clots. • SARS-CoV-2-infected patients with high viral load showed FASLG overexpression. • Gene expression profile associated with T cell exhaustion and type I interferon signaling were affected with blood aging.
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http://dx.doi.org/10.1007/s00109-021-02161-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8571664PMC
February 2022

The network interplay of interferon and Toll-like receptor signaling pathways in the anti-Candida immune response.

Sci Rep 2021 10 13;11(1):20281. Epub 2021 Oct 13.

Department of Immunology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.

Fungal infections represent a major global health problem affecting over a billion people that kills more than 1.5 million annually. In this study, we employed an integrative approach to reveal the landscape of the human immune responses to Candida spp. through meta-analysis of microarray, bulk, and single-cell RNA sequencing (scRNA-seq) data for the blood transcriptome. We identified across these different studies a consistent interconnected network interplay of signaling molecules involved in both Toll-like receptor (TLR) and interferon (IFN) signaling cascades that is activated in response to different Candida species (C. albicans, C. auris, C. glabrata, C. parapsilosis, and C. tropicalis). Among these molecules are several types I IFN, indicating an overlap with antiviral immune responses. scRNA-seq data confirmed that genes commonly identified by the three transcriptomic methods show cell type-specific expression patterns in various innate and adaptive immune cells. These findings shed new light on the anti-Candida immune response, providing putative molecular pathways for therapeutic intervention.
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http://dx.doi.org/10.1038/s41598-021-99838-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8514550PMC
October 2021

The clinical spectrum and immunopathological mechanisms underlying ZIKV-induced neurological manifestations.

PLoS Negl Trop Dis 2021 08 5;15(8):e0009575. Epub 2021 Aug 5.

Department of Clinical Research, Instituto Fernandes Figueira, Fiocruz, Rio de Janeiro, Brazil.

Since the 2015 to 2016 outbreak in America, Zika virus (ZIKV) infected almost 900,000 patients. This international public health emergency was mainly associated with a significant increase in the number of newborns with congenital microcephaly and abnormal neurologic development, known as congenital Zika syndrome (CZS). Furthermore, Guillain-Barré syndrome (GBS), a neuroimmune disorder of adults, has also been associated with ZIKV infection. Currently, the number of ZIKV-infected patients has decreased, and most of the cases recently reported present as a mild and self-limiting febrile illness. However, based on its natural history of a typical example of reemerging pathogen and the lack of specific therapeutic options against ZIKV infection, new outbreaks can occur worldwide, demanding the attention of researchers and government authorities. Here, we discuss the clinical spectrum and immunopathological mechanisms underlying ZIKV-induced neurological manifestations. Several studies have confirmed the tropism of ZIKV for neural progenitor stem cells by demonstrating the presence of ZIKV in the central nervous system (CNS) during fetal development, eliciting a deleterious inflammatory response that compromises neurogenesis and brain formation. Of note, while the neuropathology of CZS can be due to a direct viral neuropathic effect, adults may develop neuroimmune manifestations such as GBS due to poorly understood mechanisms. Antiganglioside autoantibodies have been detected in multiple patients with ZIKV infection-associated GBS, suggesting a molecular mimicry. However, further additional immunopathological mechanisms remain to be uncovered, paving the way for new therapeutic strategies.
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http://dx.doi.org/10.1371/journal.pntd.0009575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8341629PMC
August 2021

Are cachexia-associated tumors transmitTERS of ER stress?

Biochem Soc Trans 2021 08;49(4):1841-1853

Department of Immunology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.

Cancer cachexia is associated with deficient response to chemotherapy. On the other hand, the tumors of cachectic patients remarkably express more chemokines and have higher immune infiltration. For immunogenicity, a strong induction of the unfolded protein response (UPR) is necessary. UPR followed by cell surface exposure of calreticulin on the dying tumor cell is essential for its engulfment by macrophages and dendritic cells. However, some tumor cells upon endoplasmic reticulum (ER) stress can release factors that induce ER stress to other cells, in the so-called transmissible ER stress (TERS). The cells that received TERS produce more interleukin 6 (IL-6) and chemokines and acquire resistance to subsequent ER stress, nutrient deprivation, and genotoxic stress. Since ER stress enhances the release of extracellular vesicles (EVs), we suggest they can mediate TERS. It was found that ER stressed cachexia-inducing tumor cells transmit factors that trigger ER stress in other cells. Therefore, considering the role of EVs in cancer cachexia, the release of exosomes can possibly play a role in the process of blunting the immunogenicity of the cachexia-associated tumors. We propose that TERS can cause an inflammatory and immunosuppressive phenotype in cachexia-inducing tumors.
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http://dx.doi.org/10.1042/BST20210496DOI Listing
August 2021

Long-term persistence of supernumerary B chromosomes in multiple species of Astyanax fish.

BMC Biol 2021 03 19;19(1):52. Epub 2021 Mar 19.

Departamento de Genética, Universidad de Granada, 18071, Granada, Spain.

Background: Eukaryote genomes frequently harbor supernumerary B chromosomes in addition to the "standard" A chromosome set. B chromosomes are thought to arise as byproducts of genome rearrangements and have mostly been considered intraspecific oddities. However, their evolutionary transcendence beyond species level has remained untested.

Results: Here we reveal that the large metacentric B chromosomes reported in several fish species of the genus Astyanax arose in a common ancestor at least 4 million years ago. We generated transcriptomes of A. scabripinnis and A. paranae 0B and 1B individuals and used these assemblies as a reference for mapping all gDNA and RNA libraries to quantify coverage differences between B-lacking and B-carrying genomes. We show that the B chromosomes of A. scabripinnis and A. paranae share 19 protein-coding genes, of which 14 and 11 were also present in the B chromosomes of A. bockmanni and A. fasciatus, respectively. Our search for B-specific single-nucleotide polymorphisms (SNPs) identified the presence of B-derived transcripts in B-carrying ovaries, 80% of which belonged to nobox, a gene involved in oogenesis regulation. Importantly, the B chromosome nobox paralog is expressed > 30× more than the A chromosome paralog. This indicates that the normal regulation of this gene is altered in B-carrying females, which could potentially facilitate B inheritance at higher rates than Mendelian law prediction.

Conclusions: Taken together, our results demonstrate the long-term survival of B chromosomes despite their lack of regular pairing and segregation during meiosis and that they can endure episodes of population divergence leading to species formation.
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http://dx.doi.org/10.1186/s12915-021-00991-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7976721PMC
March 2021

Tumor Microenvironment Autophagic Processes and Cachexia: The Missing Link?

Front Oncol 2020 2;10:617109. Epub 2021 Feb 2.

Cancer Metabolism Research Group, Department of Surgery, LIM26-HC, Faculdade de Medicina, and Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.

Cachexia is a syndrome that affects the entire organism and presents a variable plethora of symptoms in patients, always associated with continuous and involuntary degradation of skeletal muscle mass and function loss. In cancer, this syndrome occurs in 50% of all patients, while prevalence increases to 80% as the disease worsens, reducing quality of life, treatment tolerance, therapeutic response, and survival. Both chronic systemic inflammation and immunosuppression, paradoxically, correspond to important features in cachexia patients. Systemic inflammation in cachexia is fueled by the interaction between tumor and peripheral tissues with significant involvement of infiltrating immune cells, both in the peripheral tissues and in the tumor itself. Autophagy, as a process of regulating cellular metabolism and homeostasis, can interfere with the metabolic profile in the tumor microenvironment. Under a scenario of balanced autophagy in the tumor microenvironment, the infiltrating immune cells control cytokine production and secretion. On the other hand, when autophagy is unbalanced or dysfunctional within the tumor microenvironment, there is an impairment in the regulation of immune cell's inflammatory phenotype. The inflammatory phenotype upregulates metabolic consumption and cytokine production, not only in the tumor microenvironment but also in other tissues and organs of the host. We propose that cachexia-related chronic inflammation can be, at least, partly associated with the failure of autophagic processes in tumor cells. Autophagy endangers tumor cell viability by producing immunogenic tumor antigens, thus eliciting the immune response necessary to counteract tumor progression, while preventing the establishment of inflammation, a hallmark of cachexia. Comprehensive understanding of this complex functional dichotomy may enhance cancer treatment response and prevent/mitigate cancer cachexia. This review summarizes the recent available literature regarding the role of autophagy within the tumor microenvironment and the consequences eliciting the development of cancer cachexia.
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http://dx.doi.org/10.3389/fonc.2020.617109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7884816PMC
February 2021

The authors reply: Comment on "The expression landscape of cachexia-inducing factors in human cancers" by Freire et al.

J Cachexia Sarcopenia Muscle 2020 12 30;11(6):1854-1857. Epub 2020 Sep 30.

Department of Structural and Functional Biology, Institute of Biosciences, São Paulo State University, UNESP, Botucatu, Brazil.

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http://dx.doi.org/10.1002/jcsm.12635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749551PMC
December 2020

MicroRNA-mRNA Co-sequencing Identifies Transcriptional and Post-transcriptional Regulatory Networks Underlying Muscle Wasting in Cancer Cachexia.

Front Genet 2020 29;11:541. Epub 2020 May 29.

Department of Structural and Functional Biology, Institute of Biosciences, São Paulo State University, Botucatu, Brazil.

Cancer cachexia is a metabolic syndrome with alterations in gene regulatory networks that consequently lead to skeletal muscle wasting. Integrating microRNAs-mRNAs omics profiles offers an opportunity to understand transcriptional and post-transcriptional regulatory networks underlying muscle wasting. Here, we used RNA sequencing to simultaneously integrate and explore microRNAs and mRNAs expression profiles in the tibialis anterior (TA) muscles of the Lewis Lung Carcinoma (LLC) model of cancer cachexia. We found 1,008 mRNAs and 18 microRNAs differentially expressed in cachectic mice compared with controls. Although our transcriptomic analysis demonstrated a high heterogeneity in mRNA profiles of cachectic mice, we identified a reduced number of differentially expressed genes that were uniformly regulated within cachectic muscles. This set of uniformly regulated genes is associated with the extracellular matrix (ECM), proteolysis, and inflammatory response. We also used transcriptomic data to perform enrichment analysis of transcriptional factor binding sites in promoter sequences, which revealed activation of the atrophy-related transcription factors NF-κB, Stat3, AP-1, and FoxO. Furthermore, the integration of mRNA and microRNA expression profiles identified post-transcriptional regulation by microRNAs of genes involved in ECM organization, cell migration, transcription factors binding, ion transport, and the FoxO signaling pathway. Our integrative analysis of microRNA-mRNA co-profiles comprehensively characterized regulatory relationships of molecular pathways and revealed microRNAs targeting ECM-associated genes in cancer cachexia.
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http://dx.doi.org/10.3389/fgene.2020.00541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272700PMC
May 2020

Increased angiotensin II from adipose tissue modulates myocardial collagen I and III in obese rats.

Life Sci 2020 Jul 12;252:117650. Epub 2020 Apr 12.

Department of Internal Medicine, Botucatu Medical School, São Paulo State University (UNESP), Botucatu, São Paulo 18618687, Brazil. Electronic address:

It has been described that the cardiac dysfunction in the obesity model is because of collagen imbalance and that angiotensin II (Ang II) contributes to myocardial fibrosis. However, it remains undefined if changes in collagen I and III metabolism in obesity is due to the renin-angiotensin system (RAS) dysregulation from myocardium or excessive adipose tissue.

Aim: This study aimed to verify whether the changes in myocardial collagen metabolism result from RAS deregulation of cardiac or adipose tissue in an obesity model.

Main Methods: Wistar rats were fed with control (CD) and high-fat (HFD) diets for 30 weeks. After the dietary intervention, animals were assigned to be treated with losartan at the 30 mg/kg/day dosage or kept untreated for an additional five weeks.

Key Findings: HFD induced obesity, comorbidities, and cardiac collagen overexpression. The HFD group presented an increase in Ang II levels in both adipose tissue and plasma, as well as AT1 receptor expression in cardiac tissue. Of note, the myocardial Ang II was not changed in the HFD group. Losartan administration reduced some obesity-induced comorbidities regardless of weight loss. The AT1 receptor blockade also decreased the release of Ang II from adipose tissue and myocardial AT1 receptor and collagen.

Significance: It was seen that excessive adipose tissue is responsible for the exacerbated circulating Ang II, which induced cardiac fibrosis development.
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http://dx.doi.org/10.1016/j.lfs.2020.117650DOI Listing
July 2020

Decreased Collagen Type I is Associated with Increased Metalloproteinase-2 Activity and Protein Expression of Leptin in the Myocardium of Obese Rats.

Arq Bras Cardiol 2020 07 7;115(1):61-70. Epub 2020 Aug 7.

Faculdade de Medicina de Botucatu, Universidade Estadual Paulista, Botucatu, SP, Brasil.

Background Obesity is a risk factor for medical complications, including the cardiovascular system. There is limited information on collagen in the heart in obesity. Our previous study showed decreased protein levels of myocardial collagen type I in obese rats fed a high-fat diet for 34 weeks. However, the mechanisms responsible for low levels are not fully elucidated. Objective The purpose of this study was to test the hypothesis that the reduction in collagen type I is associated with increased metalloproteinase-2 (MMP-2) activity, which is linked to elevated leptin in the myocardium of obese rats. Methods Thirty-day-old male Wistar rats were randomized into two groups, control (standard diet) and obese (high-fat diet), and fed for 34 weeks. The general animal characteristics and metabolic and endocrine profiles were evaluated. Myocardial protein expressions of collagen I, leptin, tissue inhibitors of metalloproteinases (TIMP), and MMP-2 activity were assessed. Pearson correlation was employed to determine the associations between variables. The level of significance was 5%. Results The obese animals had increased adiposity index compared to control. Comorbidities such as glucose intolerance, hyperinsulinemia, insulin resistance, hyperleptinemia, and hypertension were observed in obese rats. Obesity reduced collagen I, TIMP-1, and TIMP-2, and it increased leptin and MMP-2 in the myocardium. There was a negative correlation between collagen I and MMP-2 and a positive correlation between leptin and MMP-2. Conclusion The hypothesis was confirmed; the reduction in collagen type I is associated with increased MMP-2 activity and leptin expression in the myocardium of obese rats. (Arq Bras Cardiol. 2020; 115(1):61-70).
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http://dx.doi.org/10.36660/abc.20180143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8384327PMC
July 2020

An Integrated Meta-Analysis of Secretome and Proteome Identify Potential Biomarkers of Pancreatic Ductal Adenocarcinoma.

Cancers (Basel) 2020 03 18;12(3). Epub 2020 Mar 18.

Department of Structural and Functional Biology, Institute of Biosciences, São Paulo State University (UNESP), Botucatu 18618-689, São Paulo, Brazil.

Pancreatic ductal adenocarcinoma (PDAC) is extremely aggressive, has an unfavorable prognosis, and there are no biomarkers for early detection of the disease or identification of individuals at high risk for morbidity or mortality. The cellular and molecular complexity of PDAC leads to inconsistences in clinical validations of many proteins that have been evaluated as prognostic biomarkers of the disease. The tumor secretome, a potential source of biomarkers in PDAC, plays a crucial role in cell proliferation and metastasis, as well as in resistance to treatments, which together contribute to a worse clinical outcome. The massive amount of proteomic data from pancreatic cancer that has been generated from previous studies can be integrated and explored to uncover secreted proteins relevant to the diagnosis and prognosis of the disease. The present study aimed to perform an integrated meta-analysis of PDAC proteome and secretome public data to identify potential biomarkers of the disease. Our meta-analysis combined mass spectrometry data obtained from two systematic reviews of the pancreatic cancer literature, which independently selected 20 studies of the secretome and 35 of the proteome. Next, we predicted the secreted proteins using seven in silico tools or databases, which identified 39 secreted proteins shared between the secretome and proteome data. Notably, the expression of 31 genes of these secretome-related proteins was upregulated in PDAC samples from The Cancer Genome Atlas (TCGA) when compared to control samples from TCGA and The Genotype-Tissue Expression (GTEx). The prognostic value of these 39 secreted proteins in predicting survival outcome was confirmed using gene expression data from four PDAC datasets (validation set). The gene expression of these secreted proteins was able to distinguish high- and low-survival patients in nine additional tumor types from TCGA, demonstrating that deregulation of these secreted proteins may also contribute to the prognosis in multiple cancers types. Finally, we compared the prognostic value of the identified secreted proteins in PDAC biomarkers studies from the literature. This analysis revealed that our gene signature performed equally well or better than the signatures from these previous studies. In conclusion, our integrated meta-analysis of PDAC proteome and secretome identified 39 secreted proteins as potential biomarkers, and the tumor gene expression profile of these proteins in patients with PDAC is associated with worse overall survival.
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http://dx.doi.org/10.3390/cancers12030716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140071PMC
March 2020

The expression landscape of cachexia-inducing factors in human cancers.

J Cachexia Sarcopenia Muscle 2020 08 3;11(4):947-961. Epub 2020 Mar 3.

Department of Structural and Functional Biology, Institute of Biosciences, São Paulo State University, UNESP, Botucatu, Brazil.

Background: Cachexia is a multifactorial syndrome highly associated with specific tumour types, but the causes of variation in cachexia prevalence and severity are unknown. While circulating plasma mediators (soluble cachectic factors) derived from tumours have been implicated with the pathogenesis of the syndrome, these associations were generally based on plasma concentration rather than tissue-specific gene expression levels. Here, we hypothesized that tumour gene expression profiling of cachexia-inducing factors (CIFs) in human cancers with different prevalence of cachexia could reveal potential cancer-specific cachexia mediators and biomarkers of clinical outcome.

Methods: First, we combined uniformly processed RNA sequencing data from The Cancer Genome Atlas and Genotype-Tissue Expression databases to characterize the expression profile of secretome genes in 12 cancer types (4651 samples) compared with their matched normal tissues (2737 samples). We systematically investigated the transcriptomic data to assess the tumour expression profile of 25 known CIFs and their predictive values for patient survival. We used the Xena Functional Genomics tool to analyse the gene expression of CIFs according to neoplastic cellularity in pancreatic adenocarcinoma, which is known to present the highest prevalence of cachexia.

Results: A comprehensive characterization of the expression profiling of secreted genes in different human cancers revealed pathways and mediators with a potential role in cachexia within the tumour microenvironment. Cytokine-related and chemokine-related pathways were enriched in tumour types frequently associated with the syndrome. CIFs presented a tumour-specific expression profile, in which the number of upregulated genes was correlated with the cachexia prevalence (r : 0.80; P value: 0.002) and weight loss (r : 0.81; P value: 0.002). The distinct gene expression profile, according to tumour type, was significantly associated with prognosis (P value ≤ 1.96 E-06). In pancreatic adenocarcinoma, the upregulated CIF genes were associated with tumours presenting low neoplastic cellularity and high leucocyte fraction and not with tumour grade.

Conclusions: Our results present a biological dimension of tumour-secreted elements that are potentially useful to explain why specific cancer types are more likely to develop cachexia. The tumour-specific profile of CIFs may help the future development of better targeted therapies to treat cancer types highly associated with the syndrome.
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http://dx.doi.org/10.1002/jcsm.12565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432594PMC
August 2020

Prolonged fasting followed by refeeding modifies proteome profile and parvalbumin expression in the fast-twitch muscle of pacu (Piaractus mesopotamicus).

PLoS One 2019 19;14(12):e0225864. Epub 2019 Dec 19.

Department of Morphology, Institute of Bioscience of Botucatu, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.

Here, we analyzed the fast-twitch muscle of juvenile Piaractus mesopotamicus (pacu) submitted to prolonged fasting (30d) and refeeding (6h, 24h, 48h and 30d). We measured the relative rate of weight and length increase (RRIlength and RRIweight), performed shotgun proteomic analysis and did Western blotting for PVALB after 30d of fasting and 30d of refeeding. We assessed the gene expression of igf-1, mafbx and pvalb after 30d of fasting and after 6h, 24h, 48h and 30d of refeeding. We performed a bioinformatic analysis to predict miRNAs that possibly control parvalbumin expression. After fasting, RRIlength, RRIweight and igf-1 expression decreased, while the mafbx expression increased, which suggest that prolonged fasting caused muscle atrophy. After 6h and 24h of refeeding, mafbx was not changed and igf-1 was downregulated, while after 48h of refeeding mafbx was downregulated and igf-1 was not changed. After 30d of refeeding, RRIlength and RRIweight were increased and igf-1 and mafbx expression were not changed. Proteomic analysis identified 99 proteins after 30d of fasting and 71 proteins after 30d of refeeding, of which 23 and 17, respectively, were differentially expressed. Most of these differentially expressed proteins were related to cytoskeleton, muscle contraction, and metabolism. Among these, parvalbumin (PVALB) was selected for further validation. The analysis showed that pvalb mRNA was downregulated after 6h and 24h of refeeding, but was not changed after 30d of fasting or 48h and 30d of refeeding. The Western blotting confirmed that PVALB protein was downregulated after 30d of fasting and 30d of refeeding. The downregulation of the protein and the unchanged expression of the mRNA after 30d of fasting and 30d of refeeding suggest a post-transcriptional regulation of PVALB. Our miRNA analysis predicted 444 unique miRNAs that may target pvalb. In conclusion, muscle atrophy and partial compensatory growth caused by prolonged fasting followed by refeeding affected the muscle proteome and PVALB expression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0225864PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6922423PMC
March 2020

Tumor Transcriptome Reveals High Expression of IL-8 in Non-Small Cell Lung Cancer Patients with Low Pectoralis Muscle Area and Reduced Survival.

Cancers (Basel) 2019 08 26;11(9). Epub 2019 Aug 26.

Department of Morphology, Institute of Biosciences, São Paulo State University (UNESP), Botucatu 18618-689, São Paulo, Brazil.

Cachexia is a syndrome characterized by an ongoing loss of skeletal muscle mass associated with poor patient prognosis in non-small cell lung cancer (NSCLC). However, prognostic cachexia biomarkers in NSCLC are unknown. Here, we analyzed computed tomography (CT) images and tumor transcriptome data to identify potentially secreted cachexia biomarkers (PSCB) in NSCLC patients with low-muscularity. We integrated radiomics features (pectoralis muscle, sternum, and tenth thoracic (T10) vertebra) from CT of 89 NSCLC patients, which allowed us to identify an index for screening muscularity. Next, a tumor transcriptomic-based secretome analysis from these patients (discovery set) was evaluated to identify potential cachexia biomarkers in patients with low-muscularity. The prognostic value of these biomarkers for predicting recurrence and survival outcome was confirmed using expression data from eight lung cancer datasets (validation set). Finally, C2C12 myoblasts differentiated into myotubes were used to evaluate the ability of the selected biomarker, interleukin (IL)-8, in inducing muscle cell atrophy. We identified 75 over-expressed transcripts in patients with low-muscularity, which included and . Also, we identified , , , , , , and as PSCB in the tumor secretome. These PSCB were capable of distinguishing worse and better prognosis (recurrence and survival) in NSCLC patients. was confirmed as a predictor of worse prognosis in all validation sets. In vitro assays revealed that IL-8 promoted C2C12 myotube atrophy. Tumors from low-muscularity patients presented a set of upregulated genes encoding for secreted proteins, including pro-inflammatory cytokines that predict worse overall survival in NSCLC. Among these upregulated genes, expression in NSCLC tissues was associated with worse prognosis, and the recombinant IL-8 was capable of triggering atrophy in C2C12 myotubes.
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http://dx.doi.org/10.3390/cancers11091251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6769884PMC
August 2019

The Pathway to Cancer Cachexia: MicroRNA-Regulated Networks in Muscle Wasting Based on Integrative Meta-Analysis.

Int J Mol Sci 2019 Apr 22;20(8). Epub 2019 Apr 22.

Department of Morphology, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, São Paulo 18.618-619, Brazil.

Cancer cachexia is a multifactorial syndrome that leads to significant weight loss. Cachexia affects 50%-80% of cancer patients, depending on the tumor type, and is associated with 20%-40% of cancer patient deaths. Besides the efforts to identify molecular mechanisms of skeletal muscle atrophy-a key feature in cancer cachexia-no effective therapy for the syndrome is currently available. MicroRNAs are regulators of gene expression, with therapeutic potential in several muscle wasting disorders. We performed a meta-analysis of previously published gene expression data to reveal new potential microRNA-mRNA networks associated with muscle atrophy in cancer cachexia. We retrieved 52 differentially expressed genes in nine studies of muscle tissue from patients and rodent models of cancer cachexia. Next, we predicted microRNAs targeting these differentially expressed genes. We also include global microRNA expression data surveyed in atrophying skeletal muscles from previous studies as background information. We identified deregulated genes involved in the regulation of apoptosis, muscle hypertrophy, catabolism, and acute phase response. We further predicted new microRNA-mRNA interactions, such as miR-27a/, miR-27a/, miR-27b/, miR-27b/, miR-140/, miR-199a/, and miR-199a/, which may contribute to muscle wasting in cancer cachexia. Finally, we found drugs targeting , , and , which may be considered for the development of novel therapeutic strategies for cancer cachexia. Our study has broadened the knowledge of microRNA-regulated networks that are likely associated with muscle atrophy in cancer cachexia, pointing to their involvement as potential targets for novel therapeutic strategies.
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http://dx.doi.org/10.3390/ijms20081962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6515458PMC
April 2019

Ascorbic acid stimulates the in vitro myoblast proliferation and migration of pacu (Piaractus mesopotamicus).

Sci Rep 2019 02 18;9(1):2229. Epub 2019 Feb 18.

São Paulo State University (UNESP), Institute of Biosciences, Department of Morphology, Botucatu, São Paulo, Brazil.

The postembryonic growth of skeletal muscle in teleost fish involves myoblast proliferation, migration and differentiation, encompassing the main events of embryonic myogenesis. Ascorbic acid plays important cellular and biochemical roles as an antioxidant and contributes to the proper collagen biosynthesis necessary for the structure of connective and bone tissues. However, whether ascorbic acid can directly influence the mechanisms of fish myogenesis and skeletal muscle growth remains unclear. The aim of our work was to evaluate the effects of ascorbic acid supplementation on the in vitro myoblast proliferation and migration of pacu (Piaractus mesopotamicus). To provide insight into the potential antioxidant role of ascorbic acid, we also treated myoblasts in vitro with menadione, which is a powerful oxidant. Our results show that ascorbic acid-supplemented myoblasts exhibit increased proliferation and migration and are protected against the oxidative stress caused by menadione. In addition, ascorbic acid increased the activity of the antioxidant enzyme superoxide dismutase and the expression of myog and mtor, which are molecular markers related to skeletal muscle myogenesis and protein synthesis, respectively. This work reveals a direct influence of ascorbic acid on the mechanisms of pacu myogenesis and highlights the potential use of ascorbic acid for stimulating fish skeletal muscle growth.
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http://dx.doi.org/10.1038/s41598-019-38536-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379551PMC
February 2019

Recovery of Cardiac Remodeling and Dysmetabolism by Pancreatic Islet Injury Improvement in Diabetic Rats after Yacon Leaf Extract Treatment.

Oxid Med Cell Longev 2018 10;2018:1821359. Epub 2018 Apr 10.

Medical School, São Paulo State University (UNESP), Botucatu, SP, Brazil.

Yacon () is a native Andean plant rich in phenolic compounds, and its effects on dysmetabolism and cardiomyopathy in diabetic rats was evaluated. The rats (10/group) were allocated as follows: C, controls; C + Y, controls treated with Yacon leaf extract (YLE); DM, diabetic controls; and DM + Y, diabetic rats treated with YLE. Type 1 diabetes (T1DM) was induced by the administration of streptozotocin (STZ; 40 mg/kg body weight, single dose, i.p.), and treated groups received 100 mg/kg body weight YLE daily via gavage for 30 d. The YLE group shows an improvement in dysmetabolism and cardiomyopathy in the diabetic condition (DM versus DM + Y) promoting a significant reduction of glycemia by 63.39%, an increase in insulin concentration by 49.30%, and a decrease in serum triacylglycerol and fatty acid contents by 0.39- and 0.43-fold, respectively, by ameliorating the pancreatic islet injury, as well as increasing the activity of the antioxidant enzymes (catalase, superoxide dismutase, and glutathione peroxidase) and decreasing the fibrosis and cellular disorganization in cardiac tissue. The apparent benefits of YLE seem to be mediated by ameliorating dysmetabolism and oxidative stress in pancreatic and cardiac tissues.
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http://dx.doi.org/10.1155/2018/1821359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6051012PMC
October 2018

Osteoglycin post-transcriptional regulation by miR-155 induces cellular architecture changes in H9c2 cardiomyoblasts.

Gene 2018 Nov 7;676:9-15. Epub 2018 Jul 7.

Department of Morphology, Institute of Biosciences of Botucatu, São Paulo State University, Botucatu, São Paulo, Brazil. Electronic address:

Several studies have demonstrated dysregulated cardiac microRNAs (miRNAs) following cardiac stress and development of cardiac hypertrophy and failure. miRNAs are also differentially expressed in the inflammation that occurs in heart failure and, among these inflammatory-related miRNAs, the miR-155 has been implicated in the regulation of cardiac hypertrophy. Despite these data showing the role of miRNA-155 in cardiomyocyte hypertrophy under a hypertrophic stimulus, it is also important to understand the endogenous regulation of this miRNA without a hypertrophic stimulus to fully appreciate its function in this cell type. The first aim of the present study was to determine whether, without a hypertrophic stimulus, miR-155 overexpression induces H9c2 cardiac cells hypertrophy in vitro. The second objective was to determine whether osteoglycin (Ogn), a key regulator of heart mass in rats, mice, and humans, is post-transcriptionally regulated by miR-155 with a potential role in inducing H9c2 cells hypertrophy. Here, we show that, without a hypertrophic stimulus, miR-155 significantly repressed Ogn protein levels, but induce neither alteration in morphological phenotype nor in the expression of the molecular markers that fully characterize pathological hypertrophy of H9c2 cells. However, most importantly, Ogn silencing in H9c2 cells mimicked the effects of miR-155 overexpression in inducing cellular architecture changes that were characterized by a transition of the cell shape from fusiform to rounded. This is a new role of the post-transcriptional regulation of Ogn by miR-155 in the maintenance of the cardiac cell morphology in physiological and pathological conditions.
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http://dx.doi.org/10.1016/j.gene.2018.07.020DOI Listing
November 2018

Fractal dimension analysis reveals skeletal muscle disorganization in mdx mice.

Biochem Biophys Res Commun 2018 09 6;503(1):109-115. Epub 2018 Jun 6.

Department of Morphology, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil. Electronic address:

Duchenne Muscular Dystrophy (DMD) is characterized by muscle extracellular matrix disorganization due to the increased collagen deposition leading to fibrosis that significantly exacerbates disease progression. Fractal dimension analysis is a method that quantifies tissue/cellular disorganization and characterizes complex structures. The first objective of the present study was use fractal analysis to evaluate extracellular matrix disorganization in mdx mice soleus muscle. Next, we mimic a hyper-proliferation of fibrogenic cells by co-culturing NIH3T3 fibroblasts and C2C12 myoblasts to test whether fibroblasts induce disorganization in myoblast arrangement. Here, we show mdx presented high skeletal muscle disorganization as revealed by fractal analysis. Similarly, this method revealed that myoblasts co-cultured with fibroblast also presented cellular arrangement disorganization. We also reanalyzed skeletal muscle microarrays transcriptomic data from mdx and DMD patients that revealed transcripts related to extracellular matrix organization. This analysis also identified Osteoglycin, which was validated as a potential regulator of ECM organization in mdx dystrophic muscles. Our results demonstrate that fractal dimension is useful tool for the analysis of skeletal muscle disorganization in DMD and also reveal a fibroblast-myoblast cross-talk that contributes to "in vitro" myoblast disarrangement.
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http://dx.doi.org/10.1016/j.bbrc.2018.05.189DOI Listing
September 2018

Osteoglycin inhibition by microRNA miR-155 impairs myogenesis.

PLoS One 2017 21;12(11):e0188464. Epub 2017 Nov 21.

Department of Morphology, Institute of Biosciences, São Paulo State University, Botucatu, São Paulo, Brazil.

Skeletal myogenesis is a regulated process in which mononucleated cells, the myoblasts, undergo proliferation and differentiation. Upon differentiation, the cells align with each other, and subsequently fuse to form terminally differentiated multinucleated myotubes. Previous reports have identified the protein osteoglycin (Ogn) as an important component of the skeletal muscle secretome, which is expressed differentially during muscle development. However, the posttranscriptional regulation of Ogn by microRNAs during myogenesis is unknown. Bioinformatic analysis showed that miR-155 potentially targeted the Ogn transcript at the 3´-untranslated region (3´ UTR). In this study, we tested the hypothesis that miR-155 inhibits the expression of the Ogn to regulate skeletal myogenesis. C2C12 myoblast cells were cultured and miR-155 overexpression or Ogn knockdown was induced by transfection with miR-155 mimic, siRNA-Ogn, and negative controls with lipofectamine for 15 hours. Near confluence (80-90%), myoblasts were induced to differentiate myotubes in a differentiation medium. Luciferase assay was used to confirm the interaction between miR-155 and Ogn 3'UTR. RT-qPCR and Western blot analyses were used to confirm that the differential expression of miR-155 correlates with the differential expression of myogenic molecular markers (Myh2, MyoD, and MyoG) and inhibits Ogn protein and gene expression in myoblasts and myotubes. Myoblast migration and proliferation were assessed using Wound Healing and MTT assays. Our results show that miR-155 interacts with the 3'UTR Ogn region and decrease the levels of Ogn in myotubes. The overexpression of miR-155 increased MyoG expression, decreased myoblasts wound closure rate, and decreased Myh2 expression in myotubes. Moreover, Ogn knockdown reduced the expression levels of MyoD, MyoG, and Myh2 in myotubes. These results reveal a novel pathway in which miR-155 inhibits Ogn expression to regulate proliferation and differentiation of C2C12 myoblast cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0188464PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5697837PMC
December 2017

Obesity does not lead to imbalance between myocardial phospholamban phosphorylation and dephosphorylation.

Arq Bras Cardiol 2014 Jul;103(1):41-50

Departamento de Clínica Médica, Faculdade de Medicina de Botucatu, Universidade Estadual Paulista, Botucatu, SP, Brazil.

Background: The activation of the beta-adrenergic system promotes G protein stimulation that, via cyclic adenosine monophosphate (cAMP), alters the structure of protein kinase A (PKA) and leads to phospholamban (PLB) phosphorylation. This protein participates in the system that controls intracellular calcium in muscle cells, and it is the primary regulator of sarcoplasmic reticulum calcium pump activity. In obesity, the beta-adrenergic system is activated by the influence of increased leptin, therefore, resulting in higher myocardial phospholamban phosphorylation via cAMP-PKA.

Objective: To investigate the involvement of proteins which regulate the degree of PLB phosphorylation due to beta-adrenergic activation in obesity. In the present study, we hypothesized that there is an imbalance between phospholamban phosphorylation and dephosphorylation, with prevalence of protein phosphorylation.

Methods: Male Wistar rats were randomly distributed into two groups: control (n = 14), fed with normocaloric diet; and obese (n = 13), fed with a cycle of four unsaturated high-fat diets. Obesity was determined by the adiposity index, and protein expressions of phosphatase 1 (PP-1), PKA, PLB, phosphorylated phospholamban at serine16 (PPLB-Ser16) were assessed by Western blot.

Results: Obesity caused glucose intolerance, hyperinsulinemia, hypertriglyceridemia, hyperleptinemia and did not alter the protein expression of PKA, PP-1, PLB, PPLB-Ser16.

Conclusion: Obesity does not promote an imbalance between myocardial PLB phosphorylation and dephosphorylation via beta-adrenergic system.
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http://dx.doi.org/10.5935/abc.20140083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4126760PMC
July 2014
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