Publications by authors named "Leonardo Nazário de Moraes"

10 Publications

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Degree of piRNA sharing and Piwi gene expression in the skeletal muscle of Piaractus mesopotamicus (pacu), Colossoma macropomum (tambaqui), and the hybrid tambacu.

Comp Biochem Physiol A Mol Integr Physiol 2021 Nov 23;264:111120. Epub 2021 Nov 23.

Department of Structural and Functional Biology, Institute of Biosciences, Sao Paulo State University - UNESP, Botucatu, Sao Paulo, Brazil. Electronic address:

PiRNAs are a class of small noncoding RNAs that, in their mature form, bind to Piwi proteins to repress transposable element activity. Besides their role in gametogenesis and genome integrity, recent evidence indicates their action in non-germinative tissues. We performed a global analysis of piRNA and Piwi gene expression in the skeletal muscle of juveniles pacu (Piaractus mesopotamicus), tambaqui (Colossoma macropomum), and the hybrid tambacu to evaluate the degree of piRNA sharing among these three genotypes. Total RNA was sequenced and analyzed using specific parameters of piRNAs by bioinformatics tools. piRNA and Piwi gene expression was analyzed by RT-qPCR. We detected 24 piRNA clusters common to the three genotypes, with eight shared between pacu and tambacu, three between pacu and tambaqui, and five between tambaqui and tambacu; seven, five, and four clusters were unique to pacu, tambacu, and tambaqui, respectively. Genomic localization and fold change values showed two clusters and 100 piRNAs shared among the three genotypes. The gene expression of four piRNAs was evaluated to validate our bioinformatics results. piRNAs from cluster 17 were higher in tambacu than pacu and piRNAs from cluster 18 were more highly expressed in tambacu than tambaqui and pacu. In addition, the expression of Piwis 1 and 2 was higher in tambacu and tambaqui than pacu. Our results open an important window to investigate whether these small noncoding RNAs benefit the hybrid in terms of faster growth and offer a new perspective on the function of piRNAs and Piwis in fish skeletal muscle.
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http://dx.doi.org/10.1016/j.cbpa.2021.111120DOI Listing
November 2021

Virucidal Activity of the Antiseptic Mouthwash and Dental Gel Containing Anionic Phthalocyanine Derivative: In vitro Study.

Clin Cosmet Investig Dent 2021 28;13:269-274. Epub 2021 Jun 28.

Bauru School of Dentistry, University of São Paulo, Department of Surgery, Stomatology, Pathology and Radiology, Bauru, Brazil.

Aim: This research suggested an in vitro virucidal action of a dental gel and a mouthwash with phthalocyanine derivative.

Purpose: The aim of this study was to report an in vitro study evaluating the virucidal capacity of mouthwash and dental gel containing anionic phthalocyanine derivate (APD).

Methods: The research followed the recommendations of the National Health Surveillance Agency (ANVISA) and adapted methodology, described in the standards EN14776: 2015; ASTM E1053-11 and the Robert Koch Institute - RKI, in addition to good laboratory practices (GLP). The determination of the percentage of inactivation of the SARS-CoV-2 virus particles was carried out by imposing the viral solution in contact with the respective tested products, with intervals of 30 seconds, 1 and 5 minutes, with subsequent submission of the aliquots, recovered in cell culture microplates following virus titration using the TCID50 (50% Median Tissue Culture Infectious Dose).

Results: The Mouthwash APD presented 90% of viral inactivation percentage, while the dental gel APD demonstrated 99.99% of viral inactivation.

Conclusion: In vitro analyses showed that mouthwash and dental gel APD can reduce the viability of SARS-CoV-2 virus particles.
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http://dx.doi.org/10.2147/CCIDE.S315419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8254183PMC
June 2021

Comparison of microRNA Expression Profile in Chronic Myeloid Leukemia Patients Newly Diagnosed and Treated by Allogeneic Hematopoietic Stem Cell Transplantation.

Front Oncol 2020 4;10:1544. Epub 2020 Sep 4.

Department of Internal Medicine, São Paulo State University (UNESP-FMB), Botucatu, Brazil.

Chronic myeloid leukemia (CML) results from a translocation between chromosomes 9 and 22, which generates the Philadelphia chromosome. This forms BCR/ABL1, an active tyrosine kinase protein that promotes cell growth and replication. Despite great progress in CML treatment in the form of tyrosine kinase inhibitors, allogeneic-hematopoietic stem cell transplantation (allo-HSCT) is currently used as an important treatment alternative for patients resistant to these inhibitors. Studies have shown that unregulated expression of microRNAs, which act as oncogenes or tumor suppressors, is associated with human cancers. This contributes to tumor formation and development by stimulating proliferation, angiogenesis, and invasion. Research has demonstrated the potential of microRNAs as biomarkers for cancer diagnosis, prognosis, and therapeutic targets. In the present study, we compared the circulating microRNA expression profiles of 14 newly diagnosed patients with chronic phase-CML and 14 Philadelphia chromosome-negative patients after allo-HSCT. For each patient, we tested 758 microRNAs by reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis. The global expression profile of microRNAs revealed 16 upregulated and 30 downregulated microRNAs. Target genes were analyzed, and key pathways were extracted and compared. Bioinformatics tools were used to analyze data. Among the downregulated miRNA target genes, some genes related to cell proliferation pathways were identified. These results reveal the comprehensive microRNA profile of CML patients and the main pathways related to the target genes of these miRNAs in cytogenetic remission after allo-HSCT. These results provide new resources for exploring stem cell transplantation-based CML treatment strategies.
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http://dx.doi.org/10.3389/fonc.2020.01544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7500210PMC
September 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

A novel molecular mechanism to explain mutations of the HCV protease associated with resistance against covalently bound inhibitors.

Virus Res 2019 12 13;274:197778. Epub 2019 Oct 13.

Sao Paulo State University (UNESP), School of Agriculture, Department of Bioprocess and Biotechnology, Avenue Universitária, 3780, Botucatu, SP, Brazil; Sao Paulo State University (UNESP), Medical School, Blood Center, Avenue Prof. Mário Rubens Guimarães Montenegro, s/n, Botucatu, SP, Brazil. Electronic address:

NS3 is an important therapeutic target for direct-acting antiviral (DAA) drugs. However, many patients treated with DAAs have unsustained virologic response (UVR) due to the high mutation rate of HCV. The aim of this work was to shed some light on the puzzling molecular mechanisms of the virus's of patients who showed high viral loads even under treatment with DAA. Bioinformatics tools, molecular modelling analyses were employed to identify mutations associated with HCV resistance to boceprevir and possible structural features related to this phenomenon. We identified two mutations of NS3 that may be associated with HCV resistance: D168N and L153I. The substitution D168N was previously reported in the literature as related with drug failure. Additionally, we identified that its molecular resistance mechanism can be explained by the destabilization of receptor-ligand hydrogen bonds. For the L153I mutation, the resistance mechanism is different from previous models reported in the literature. The L153I substitution decreases the S139 deprotonation susceptibility, and consequently, this mutation impairs the covalent binding between the residue S139 from NS3 and the electrophilic trap on boceprevir, which can induce drug failure. These results were supported by the time course analysis of the mutations of the NS3 protease, which showed that boceprevir was designed for enzymes with an L residue at position 153; however, the sequences with I153 are predominant nowadays. The results presented here could be used to infer about resistance in others DAA, mainly protease inhibitors.
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http://dx.doi.org/10.1016/j.virusres.2019.197778DOI Listing
December 2019

Development and comparative analysis of yeast protein extraction protocols for mass spectrometry.

Anal Biochem 2019 02 2;567:90-95. Epub 2018 Nov 2.

São Paulo State University (UNESP), Department of Bioprocess and Biotechnology, School of Agriculture, Avenue Universitária, n 3780, 18610-034, Altos do Paraíso, Botucatu, Brazil. Electronic address:

Mass spectrometry is the most used method for protein identification and quantification. Here we developed four protein extraction protocols precisely for mass spectrometry, and we compared with other ones already published. The best protocol developed by us consists on a simple extraction solution, a heat-shock step, and does not use protease inhibitor; moreover, it is the most efficient and uniform among replicates, besides to be safe, cheap and fast. That method also provided the highest number of proteins uniquely identified and allows finding a diversity of protein classes, which their absence is a problem to be avoided.
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http://dx.doi.org/10.1016/j.ab.2018.10.028DOI Listing
February 2019

Ultrastructural analysis and residual DNA evaluation of rabbit vein scaffold.

Acta Cir Bras 2017 Sep;32(9):706-711

Associate Professor, Department of Urology, Botucatu Medical School, UNESP, Botucatu-SP, Brazil. Conception and design of the study, manuscript writing, critical revision.

Purpose: To investigate the ultrastructural characteristics and analysis of residual DNA in scaffold models, produced with decellularized vena cava in an experimental model with rabbits.

Methods: Three groups were created for ultrastructural and residual DNA analysis: group 1 - control, consisting of samples of vena cava in natura; group 2 - SD, consisting of vein fragments submitted to 2% sodium deoxycholate decellularization by shaking (160rpm - Shaker News Brunswick Scientific®) for 1 hour at controlled temperature shaker at 37°C; group 3 - SDS, consisting of vein fragments submitted to 1% sodium dodecyl sulfate decellularization under the same previous condition, for 2 hours.

Results: The ultrastructural matrix of the blood vessel maintained its vintegrity after either decellularization models. The results of the two quantification methods demonstrated a significant decrease in the DNA content of the decellularized vena cava samples as compared to the control samples and, differed statistically from each other, p <0.05.

Conclusion: The 2% DS protocol for vein decellularization, in this experimental model, was considered the best protocol because it presented less amount of residual DNA without causing substantial destruction of the extracellular matrix.
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http://dx.doi.org/10.1590/s0102-865020170090000003DOI Listing
September 2017

Food restriction increase the expression of mTORC1 complex genes in the skeletal muscle of juvenile pacu (Piaractus mesopotamicus).

PLoS One 2017 15;12(5):e0177679. Epub 2017 May 15.

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

Skeletal muscle is capable of phenotypic adaptation to environmental factors, such as nutrient availability, by altering the balance between muscle catabolism and anabolism that in turn coordinates muscle growth. Small noncoding RNAs, known as microRNAs (miRNAs), repress the expression of target mRNAs, and many studies have demonstrated that miRNAs regulate the mRNAs of catabolic and anabolic genes. We evaluated muscle morphology, gene expression of components involved in catabolism, anabolism and energetic metabolism and miRNAs expression in both the fast and slow muscle of juvenile pacu (Piaractus mesopotamicus) during food restriction and refeeding. Our analysis revealed that short periods of food restriction followed by refeeding predominantly affected fast muscle, with changes in muscle fiber diameter and miRNAs expression. There was an increase in the mRNA levels of catabolic pathways components (FBXO25, ATG12, BCL2) and energetic metabolism-related genes (PGC1α and SDHA), together with a decrease in PPARβ/δ mRNA levels. Interestingly, an increase in mRNA levels of anabolic genes (PI3K and mTORC1 complex: mTOR, mLST8 and RAPTOR) was also observed during food restriction. After refeeding, muscle morphology showed similar patterns of the control group; the majority of genes were slightly up- or down-regulated in fast and slow muscle, respectively; the levels of all miRNAs increased in fast muscle and some of them decreased in slow muscle. Our findings demonstrated that a short period of food restriction in juvenile pacu had a considerable impact on fast muscle, increasing the expression of anabolic (PI3K and mTORC1 complex: mTOR, mLST8 and RAPTOR) and energetic metabolism genes. The miRNAs (miR-1, miR-206, miR-199 and miR-23a) were more expressed during refeeding and while their target genes (IGF-1, mTOR, PGC1α and MAFbx), presented a decreased expression. The alterations in mTORC1 complex observed during fasting may have influenced the rates of protein synthesis by using amino acids from protein degradation as an alternative mechanism to preserve muscle phenotype and metabolic demand maintenance.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0177679PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5432107PMC
September 2017

Defining suitable reference genes for RT-qPCR analysis on human sertoli cells after 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure.

Mol Biol Rep 2014 Nov 31;41(11):7063-6. Epub 2014 Jul 31.

Department of Morphology, Institute of Biosciences, UNESP- Univ Estadual Paulista, Botucatu, SP, 18618-970, Brazil.

Quantitative real-time RT-PCR (qPCR) has proven to be a valuable molecular technique to quantify gene expression. There are few studies in the literature that describe suitable reference genes to normalize gene expression data. Studies of transcriptionally disruptive toxins, like tetrachlorodibenzo-p-dioxin (TCDD), require careful consideration of reference genes. The present study was designed to validate potential reference genes in human Sertoli cells after exposure to TCDD. 32 candidate reference genes were analyzed to determine their applicability. geNorm and NormFinder softwares were used to obtain an estimation of the expression stability of the 32 genes and to identify the most suitable genes for qPCR data normalization.
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http://dx.doi.org/10.1007/s11033-014-3637-0DOI Listing
November 2014

Involvement of renal corpuscle microRNA expression on epithelial-to-mesenchymal transition in maternal low protein diet in adult programmed rats.

PLoS One 2013 19;8(8):e71310. Epub 2013 Aug 19.

Fetal Programming Laboratory, Morphology Department, São Paulo State University, Botucatu, São Paulo, Brazil.

Prior study shows that maternal protein-restricted (LP) 16-wk-old offspring have pronounced reduction of nephron number and arterial hypertension associated with unchanged glomerular filtration rate, besides enhanced glomerular area, which may be related to glomerular hyperfiltration/overflow and which accounts for the glomerular filtration barrier breakdown and early glomerulosclerosis. In the current study, LP rats showed heavy proteinuria associated with podocyte simplification and foot process effacement. TGF-β1 glomerular expression was significantly enhanced in LP. Isolated LP glomeruli show a reduced level of miR-200a, miR-141, miR-429 and ZEB2 mRNA and upregulated collagen 1α1/2 mRNA expression. By western blot analyzes of whole kidney tissue, we found significant reduction of both podocin and nephrin and enhanced expression of mesenchymal protein markers such as desmin, collagen type I and fibronectin. From our present knowledge, these are the first data showing renal miRNA modulation in the protein restriction model of fetal programming. The fetal-programmed adult offspring showed pronounced structural glomerular disorders with an accentuated and advanced stage of fibrosis, which led us to state that the glomerular miR-200 family would be downregulated by TGF-β1 action inducing ZEB 2 expression that may subsequently cause glomeruli epithelial-to-mesenchymal transition.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0071310PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3747155PMC
April 2014
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