Publications by authors named "Hernandes F Carvalho"

91 Publications

A modular, reversible sealing, and reusable microfluidic device for drug screening.

Anal Chim Acta 2021 Nov 17;1185:339068. Epub 2021 Sep 17.

University of Campinas, School of Chemical Engineering, Department of Materials and Bioprocesses Engineering, Campinas, Brazil. Electronic address:

Preclinical tests for evaluating potential drug candidates using conventional protocols can be exhaustive and high-cost processes. Microfluidic technologies that can speed up this process and allow fast screening of drugs are promising alternatives. This work presents the design, concept, and operational conditions of a simple, modular, and reversible sealing microdevice useful for drug screening. This microdevice allows for the operation of 4 parallel simultaneous conditions and can also generate a diffusive concentration gradient in sextuplicates. We used laminated polydimethylsiloxane (PDMS) and glass as building materials as proof of concept. The PDMS parts can be reused since they can be easily sterilized. We cultured MCF-7 (Michigan Cancer Foundation-7) breast cancer cells. Cells were exposed to a doxorubicin diffusive concentration gradient for 3 h. They were monitored by automated microscopy, and after data processing, it was possible to determine cell viability as a function of doxorubicin concentration. The reversible sealing enabled the recovery of the tested cells and image acquisition. Therefore, this microdevice is a promising tool for drug screening that allows assessing the cellular behavior in dynamic conditions and the recovery of cells for afterward processing and imaging.
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http://dx.doi.org/10.1016/j.aca.2021.339068DOI Listing
November 2021

Exposure to Bacteriophages T4 and M13 Increases Integrin Gene Expression and Impairs Migration of Human PC-3 Prostate Cancer Cells.

Antibiotics (Basel) 2021 Oct 3;10(10). Epub 2021 Oct 3.

Laboratory of Extracellular Matrix Biology, Department of Structural and Functional Biology, Institute of Biosciences of Botucatu, Sao Paulo State University (UNESP), Botucatu 18618-689, SP, Brazil.

The interaction between bacteriophages and integrins has been reported in different cancer cell lines, and efforts have been undertaken to understand these interactions in tumor cells along with their possible role in gene alterations, with the aim to develop new cancer therapies. Here, we report that the non-specific interaction of T4 and M13 bacteriophages with human PC-3 cells results in differential migration and varied expression of different integrins. PC-3 tumor cells (at 70% confluence) were exposed to 1 × 10 pfu/mL of either lytic T4 bacteriophage or filamentous M13 bacteriophage. After 24 h of exposure, cells were processed for a histochemical analysis, wound-healing migration assay, and gene expression profile using quantitative real-time PCR (qPCR). qPCR was performed to analyze the expression profiles of integrins , , , , and . Our findings revealed that PC-3 cells interacted with T4 and M13 bacteriophages, with significant upregulation of , , , genes after phage exposure. PC-3 cells also exhibited reduced migration activity when exposed to either T4 or M13 phages. These results suggest that wildtype bacteriophages interact non-specifically with PC-3 cells, thereby modulating the expression of integrin genes and affecting cell migration. Therefore, bacteriophages have future potential applications in anticancer therapies.
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http://dx.doi.org/10.3390/antibiotics10101202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8532711PMC
October 2021

Bacteriophages M13 and T4 Increase the Expression of Anchorage-Dependent Survival Pathway Genes and Down Regulate Androgen Receptor Expression in LNCaP Prostate Cell Line.

Viruses 2021 Sep 2;13(9). Epub 2021 Sep 2.

Laboratory of Extracellular Matrix Biology, Department of Structural and Functional Biology, Institute of Biosciences of Botucatu, Sao Paulo State University (UNESP), Botucatu 18618-689, SP, Brazil.

Wild-type or engineered bacteriophages have been reported as therapeutic agents in the treatment of several types of diseases, including cancer. They might be used either as naked phages or as carriers of antitumor molecules. Here, we evaluate the role of bacteriophages M13 and T4 in modulating the expression of genes related to cell adhesion, growth, and survival in the androgen-responsive LNCaP prostatic adenocarcinoma-derived epithelial cell line. LNCaP cells were exposed to either bacteriophage M13 or T4 at a concentration of 1 × 10 pfu/mL, 1 × 10 pfu/mL, and 1 × 10 pfu/mL for 24, 48, and 72 h. After exposure, cells were processed for general morphology, cell viability assay, and gene expression analyses. Neither M13 nor T4 exposure altered cellular morphology, but both decreased the MTT reduction capacity of LNCaP cells at different times of treatment. In addition, genes , , , , , , and were significantly up-regulated, whilst the genes , , , and were down-regulated. Our results show that bacteriophage M13 and T4 interact with LNCaP cells and effectively promote gene expression changes related to anchorage-dependent survival and androgen signaling. In conclusion, phage therapy may increase the response of PCa treatment with pathway inhibitors.
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http://dx.doi.org/10.3390/v13091754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8473360PMC
September 2021

Syndecan Family Gene and Protein Expression and Their Prognostic Values for Prostate Cancer.

Int J Mol Sci 2021 Aug 12;22(16). Epub 2021 Aug 12.

Department of Structural and Functional BIology, Institute of Bioscience of Botucatu (IBB), São Paulo State University, Botucatu 18618-689, SP, Brazil.

Prostate cancer (PCa) is the leading cause of cancer-associated mortality in men, and new biomarkers are still needed. The expression pattern and protein tissue localization of proteoglycans of the syndecan family (SDC 1-4) and syntenin-1 (SDCBP) were determined in normal and prostatic tumor tissue from two genetically engineered mouse models and human prostate tumors. Studies were validated using SDC 1-4 and SDCBP mRNA levels and patient survival data from The Cancer Genome Atlas and CamCAP databases. RNAseq showed increased expression of in mouse Pca and upregulation of expression and downregulation of and when compared to the normal prostatic tissue in mouse tumors. These changes were confirmed by immunohistochemistry. In human PCa, SDC 1-4 and SDCBP immunostaining showed variable localization. Furthermore, Kaplan-Meier analysis showed that patients expressing SDC3 had shorter prostate-specific survival than those without SDC3 expression (log-rank test, = 0.0047). Analysis of the MSKCC-derived expression showed that and overexpression is predictive of decreased biochemical recurrence-free survival ( = 0.0099 and = 0.045, respectively), and overexpression is predictive of increased biochemical recurrence-free survival ( = 0.035). SDC4 overexpression was associated with a better prognosis, while SDC1 and SDC3 were associated with more aggressive tumors and a worse prognosis.
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http://dx.doi.org/10.3390/ijms22168669DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8395474PMC
August 2021

Stromal cell interplay in prostate development, physiology, and pathological conditions.

Prostate 2021 Sep 12;81(13):926-937. Epub 2021 Jul 12.

Department of Structural and Functional Biology, Institute of Biology, State University of Campinas (UNICAMP), Campinas, Brazil.

Advances in prostatic stroma studies over the past few decades have demonstrated that the stroma not only supports and nourishes the gland's secretory epithelium but also participates in key aspects of morphogenesis, in the prostate's hormonal metabolism, and in the functionality of the secretory epithelium. Furthermore, the stroma is implicated in the onset and progression of prostate cancer through the formation of the so-called reactive stroma, which corresponds to a tumorigenesis-permissive microenvironment. Prostatic stromal cells are interconnected and exchange paracrine signals among themselves in a gland that is highly sensitive to endocrine hormones. There is a growing body of evidence that telocytes, recently detected interstitial cells that are also present in the prostate, are involved in stromal organization, so that their processes form a network of interconnections with both the epithelium and the other stromal cells. The present review provides an update on the different types of prostate stromal cells, their interrelationships and implications for prostate development, physiology and pathological conditions.
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http://dx.doi.org/10.1002/pros.24196DOI Listing
September 2021

Telocytes of the male urogenital system: Interrelationships, possible functions, and pathological implications.

Cell Biol Int 2021 Aug 3;45(8):1613-1623. Epub 2021 May 3.

Department of Structural and Functional Biology, Institute of Biology, State University of Campinas (UNICAMP), Campinas, São Paulo, Brazil.

The male urogenital system is composed of the reproductive system and the urinary tract; they have an interconnected embryonic development and share one of their anatomical components, the urethra. This system has a highly complex physiology deeply interconnected with the circulatory and nervous systems, as well as being capable of adapting to environmental variations; it also undergoes changes with aging and, in the case of the reproductive system, with seasonality. The stroma is an essential component in this physiological plasticity and its complexity has increased with the description in the last decade of a new cell type, the telocyte. Several studies have demonstrated the presence of telocytes in the organs of the male urogenital system and other systems; however, their exact function is not yet known. The present review addresses current knowledge about telocytes in the urogenital system in terms of their locations, interrelationships, possible functions and pathological implications. It has been found that telocytes in the urogenital system possibly have a leading role in stromal tissue organization/maintenance, in addition to participation in stem cell niches and an association with the immune system, as well as specific functions in the urogenital system, lipid synthesis in the testes, erythropoiesis in the kidneys and the micturition reflex in the bladder. There is also evidence that telocytes are involved in pathologies in the kidneys, urethra, bladder, prostate, and testes.
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http://dx.doi.org/10.1002/cbin.11612DOI Listing
August 2021

Fibrin and Transforming Growth Factor Alpha Affect Prostatic Smooth Muscle Cell's Phenotype and Motility.

Microsc Microanal 2021 Mar 11:1-8. Epub 2021 Mar 11.

Department of Structural and Functional Biology, Institute of Biology, State University of Campinas (UNICAMP), Campinas, SP, Brazil.

Smooth muscle cells (SMCs) are dynamic and transition from a contractile to a synthetic phenotype under different circumstances. Plasma factors (fibrin and transforming growth factors, TGFs) are possible components affecting SMCs differentiation and behavior. Thus, the objective of this work was to investigate how the fibrin matrix and TGFs affect SMCs differentiation and motility behavior. SMCs invaded the fibrin gel and adopted a stellate phenotype while reducing the expression of differentiation markers (Acta2, Myh11, and Smtn). At the ultrastructural level, SMCs did not assemble a basal lamina and showed numerous blebs along the entire cell surface. This transition was not associated with changes in focal adhesion kinase (FAK) content and phosphorylation status but reflected a marked change in FAK distribution in the cytoplasm. After 48 h in culture, SMCs caused an active degradation of the fibrin gel. Additionally, we tested the SMCs response to TGFs in a cell layer wound repair assay. TGFα, but not TGFβ1 or TGFβ3, had significantly increased motility. In conclusion, prostatic SMCs present a phenotypical transition when cultured on fibrin, adopting a micro-blebbing based motility behavior and increasing migration in response to TGFα.
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http://dx.doi.org/10.1017/S1431927621000209DOI Listing
March 2021

Dietary Fat Quality in Normolipidic Diets Affects Hepatocyte's Nuclear Phenotypes.

Nutr Metab Insights 2020 18;13:1178638820982003. Epub 2020 Dec 18.

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

Dietary fat quality affects overall systemic parameters and produce hepatic accumulation of fat and inflammation (steatohepatitis). In this communication we have assessed how mouse liver nuclear phenotypes are influenced by diets containing 7% lipid prepared with lard, linseed oil or soybean oil for 32 weeks. Liver specimens were imprinted on glass slides, fixed and stained with DAPI. 3D confocal images were obtained and employed for the calculation of nuclear thickness, nuclear volume and DAPI-DNA intensity. Hepatocytes' nuclei could be classified as diploid A, diploid B, tetraploid and higher ploidy levels. Linseed oil in the diet resulted in increased frequency of diploid A (more compact) and less polyploidy, while lard caused increased volume and more polyploidy. Soybean oil produced intermediate nuclear sizes. The results suggest a high demand on liver physiology promoted by lard, which has a predominance of saturated fatty acids, while linseed oil promoted the opposite effect.
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http://dx.doi.org/10.1177/1178638820982003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7750749PMC
December 2020

Recent advances in co-delivery nanosystems for synergistic action in cancer treatment.

J Mater Chem B 2021 02;9(5):1208-1237

Department of Materials and Bioprocesses Engineering, School of Chemical Engineering, University of Campinas, Campinas, Brazil.

Nanocarrier delivery systems have been widely studied to carry unique or dual chemical drugs. The major challenge of chemotherapies is to overcome the multidrug-resistance (MDR) of cells to antineoplastic medicines. In this context, nano-scale technology has allowed researchers to develop biocompatible nano-delivery systems to overcome the limitation of chemical agents. The development of nano-vehicles may also be directed to co-deliver different agents such as drugs and genetic materials. The delivery of nucleic acids targeting specific cells is based on gene therapy principles to replace the defective gene, correct genome errors or knock-down a particular gene. Co-delivery systems are attractive strategies due to the possibility of achieving synergistic therapeutic effects, which are more effective in overcoming the MDR of cancer cells. These combined therapies can provide better outcomes than separate delivery approaches carrying either siRNA, miRNA, pDNA, or drugs. This article reviews the main design features that need to be associated with nano-vehicles to co-deliver drugs, genes, and gene-drug combinations with efficacy. The advantages and disadvantages of co-administration approaches are also overviewed and compared with individual nanocarrier systems. Herein, future trends and perspectives in designing novel nano-scale platforms to co-deliver therapeutic agents are also discussed.
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http://dx.doi.org/10.1039/d0tb02168gDOI Listing
February 2021

Polarization, migration, and homotypical interactions among prostatic smooth muscle cells in a laminin 111-rich extracellular matrix.

Cell Biol Int 2021 Apr 7;45(4):882-889. Epub 2021 Jan 7.

Department of Structural and Functional Biology, Institute of Biology, State University of Campinas (UNICAMP), Campinas, São Paulo, Brazil.

Prostate cancer is a life-threatening condition worldwide. As the tumor progresses, smooth muscle cells (SMCs) become atrophic/dedifferentiated, within a series of stromal changes named stromal reaction. Here, we tested whether a laminin 111-rich extracellular matrix (Lr-ECM) could affect SMCs phenotype and differentiation status. Using time-lapse microscopy, image analyses, quantitative real-time reverse transcription polymerase chain reaction, immunohistochemistry and immunoblotting, and transmission electron microscopy, we showed that SMCs acquires a migratory behavior with a decreased expression of differentiation markers and relocation of focal adhesion kinase. SMCs set homotypic cell junctions and were active in autophagy/phagocytosis. Analysis of the migratory behavior showed that SMCs polarized and migrated toward each other, recognizing long-distance signals such as matrix tensioning. However, half of the cell population were immotile, irrespective of the nearest neighbor distance, suggesting they do not engage in productive interactions, possibly as a result of back-to-back positioning. In conclusion, the Lr-ECM, mimics the effects of the proliferating and infiltrating tumor epithelium, causing SMCs phenotypical change similar to that observed in the stromal reaction, in addition to a hitherto undescribed, stereotyped pattern of cell motility resulting from cell polarization.
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http://dx.doi.org/10.1002/cbin.11535DOI Listing
April 2021

Heparan sulfate fine-tunes stromal-epithelial communication in the prostate gland.

Dev Dyn 2021 05 24;250(5):618-628. Epub 2020 Dec 24.

Department of Structural and Functional Biology, Institute of Biology, State University of Campinas, Campinas, Brazil.

Several studies reported the concerted and mutual communication between the prostate epithelium and stroma, which determines the final organ architecture and function, but gets awry in cancer. Deciphering the mechanisms involved in this communication is crucial to find new therapeutic strategies. HS sequesters a number of secreted growth factors and cytokines, controlling their bioavailability to the target cells, suggesting that HS is an important regulator of the extracellular matrix (ECM) and a key player in the cell-cell and cell-microenvironment communication during prostate morphogenesis and physiology. We propose that by controlling HS biosynthesis and sulfation pattern, as well as the cleavage of the HS chain and/or the shedding of proteoglycans, epithelial and stromal cells are able to precisely tune the availability of signaling molecules and modulate ligand-receptor interaction and intracellular signal transduction.
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http://dx.doi.org/10.1002/dvdy.281DOI Listing
May 2021

Telocytes contribute to aging-related modifications in the prostate.

Sci Rep 2020 12 7;10(1):21392. Epub 2020 Dec 7.

Department of Structural and Functional Biology, Institute of Biology, State University of Campinas (UNICAMP), Bertrand Russel Av., Campinas, São Paulo, Brazil.

Telocytes are interstitial cells present in the stroma of several organs, including the prostate. There is evidence that these cells are present during prostate alveologenesis, in which these cells play a relevant role, but there is no information about the presence of and possible changes in telocytes during prostate aging. Throughout aging, the prostate undergoes several spontaneous changes in the stroma that are pro-pathogenic. Our study used histochemistry, 3D reconstructions, ultrastructure and immunofluorescence to compare the adult prostate with the senile prostate of the Mongolian gerbil, in order to investigate possible changes in telocytes with senescence and a possible role for these cells in the age-associated alterations. It was found that the layers of perialveolar smooth muscle become thinner as the prostatic alveoli become more dilated during aging, and that telocytes form a network that involves smooth muscle cells, which could possibly indicate a role for telocytes in maintaining the integrity of perialveolar smooth muscles. On the other hand, with senescence, VEGF+ telocytes are seen in stroma possibly contributing to angiogenesis, together with TNFR1+ telocytes, which are associated with a pro-inflammatory microenvironment in the prostate. Together, these data indicate that telocytes are important both in understanding the aging-related changes that are seen in the prostate and also in the search for new therapeutic targets for pathologies whose frequency increases with age.
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http://dx.doi.org/10.1038/s41598-020-78532-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7721742PMC
December 2020

Enoxacin induces oxidative metabolism and mitigates obesity by regulating adipose tissue miRNA expression.

Sci Adv 2020 Dec 2;6(49). Epub 2020 Dec 2.

Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas, Campinas, Brazil.

MicroRNAs (miRNAs) have been implicated in oxidative metabolism and brown/beige adipocyte identity. Here, we tested whether widespread changes in miRNA expression promoted by treatment with the small-molecule enoxacin cause browning and prevent obesity. Enoxacin mitigated diet-induced obesity in mice, and this was associated with increased energy expenditure. Consistently, subcutaneous white and brown adipose tissues and skeletal muscle of enoxacin-treated mice had higher levels of markers associated with thermogenesis and oxidative metabolism. These effects were cell autonomous since they were recapitulated in vitro in murine and human cell models. In preadipocytes, enoxacin led to a reduction of expression and up-regulation of its target genes (e.g., , , and ), thus increasing FGF21 signaling and promoting beige adipogenesis. Our data demonstrate that enoxacin counteracts obesity by promoting thermogenic signaling and inducing oxidative metabolism in adipose tissue and skeletal muscle in a mechanism that involves, at least in part, miRNA-mediated regulation.
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http://dx.doi.org/10.1126/sciadv.abc6250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710362PMC
December 2020

Quantum biochemistry in cancer immunotherapy: New insights about CTLA-4/ipilimumab and design of ipilimumab-derived peptides with high potential in cancer treatment.

Mol Immunol 2020 11 1;127:203-211. Epub 2020 Oct 1.

Department of Physics, Federal University of Ceará, Fortaleza, Ceará, CEP 60.440-970, Brazil.

Cancer is a group of diseases involving disordered growth of abnormal cells with the potential to invade and spread to other parts of the body. Today, immunotherapy is the most efficient treatment, with fewer side effects. Notably, the employment of monoclonal antibodies to inhibit checkpoint proteins, such as CTLA-4, has caused much excitement among cancer immunotherapy researchers. Thus, in-depth analysis through quantum biochemistry and molecular dynamics simulations was performed to understand the complex formed by ipilimumab and its target CTLA-4. Our computational results provide a better understanding of the binding mechanisms and new insights about the CTLA-4: ipilimumab interaction, identifying essential amino acid residues to support the complex. Additionally, we report new interactions such as aromatic-aromatic, aromatic-sulfur, and cation-pi interactions to stabilize the CTLA-4:ipilimumab complex. Finally, quantum biochemistry analyses reveal the most important amino acid residues involved in the CTLA-4:ipilimumab interface, which were used to design synthetic peptides to inhibit CTLA-4. The computational results presented here provide a better understanding of the CTLA-4:ipilimumab binding mechanisms, and can support the development of alternative antibody-based drugs with high relevance in cancer immunotherapy.
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http://dx.doi.org/10.1016/j.molimm.2020.09.013DOI Listing
November 2020

Prostate immunology: A challenging puzzle.

J Reprod Immunol 2020 11 18;142:103190. Epub 2020 Aug 18.

Department of Structural and Functional Biology, State University of Campinas - UNICAMP, Campinas, SP, Brazil; National Institute of Science and Technology of Photonics Applied to Cell Biology - INFABiC, Campinas, SP, Brazil. Electronic address:

Mucosal immunity defines the relationship of surfaces in contact with the environment and integrates diverse tissues such as epidermis, gum, nose, gut, uterus and prostate with the immune system. Although considered part of a system, each mucosa presents specific immune features beyond the barrier and secretory functions. Information regarding the mucosal immunology of the male reproductive tract and the prostate gland in particular is scarce. In this review, we approach the prostate as an epithelial barrier and as part of the mucosal immune system. Finally, we also raise a series of questions that will improve the understanding of this gland, its role in reproduction and its sensitivity/resistance to disease.
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http://dx.doi.org/10.1016/j.jri.2020.103190DOI Listing
November 2020

Explant culture: A relevant tool for the study of telocytes.

Cell Biol Int 2020 Dec 31;44(12):2395-2408. Epub 2020 Aug 31.

Department of Structural and Functional Biology, Institute of Biology, State University of Campinas (UNICAMP), São Paulo, Brazil.

Telocytes are cells present in the stroma of various tissues including the prostate. The detection of telocytes is still very much dependent on obtaining ultrastructural data that show the presence of telopodes, which are cytoplasmic projections that alternate between dilated regions, the podoms, and thin segments, the podomers. These structures are the distinctive characteristics of the telocytes. Thus, in vitro assays are important for the study of telocytes, which are more easily identified in culture, which also enables the experimental manipulation of these cells. The isolation of telocytes per se does not allow the analysis of the behavior of these cells in relation to other cell types in a given organ. In this sense, in the prostate, explants could be a useful tool for the study of telocytes. The present study obtained prostatic explants and evaluated the influence of recombinant proteins, scattering factor (SCF) and stromal-derived factor 1 (SDF-1), which could impact on the migration of CD34-positive cells. Telocytes migrate out of explants and SDF-1 stimulates the proliferation and formation of telocyte networks in vitro. Telocytes are not smooth muscle cell progenitors in the prostate; on the contrary, they are CD90- and CD44-negative cells and, hence, have limited progenitor capacity. The present study demonstrated that explants are useful tools to elucidate the nature of telocytes and their functions.
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http://dx.doi.org/10.1002/cbin.11446DOI Listing
December 2020

Polysaccharide Multilayer Films in Sensors for Detecting Prostate Tumor Cells Based on Hyaluronan-CD44 Interactions.

Cells 2020 06 26;9(6). Epub 2020 Jun 26.

Institute of Biology, University of Campinas, 13083-970 Campinas, Brazil.

The increasing need for point-of-care diagnosis has sparked the development of label-free sensing platforms, some of which are based on impedance measurements with biological cells. Here, interdigitated electrodes were functionalized with layer-by-layer (LbL) films of hyaluronan (HA) and chitosan (CHI) to detect prostatic tumor cells (PC3 line). The deposition of LbL films was confirmed with atomic force microscopy and polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS), which featured the vibrational modes of the HA top layer capable of interacting specifically with glycoprotein CD44 receptors overexpressed in tumor cells. Though the CHI/HA LbL films cannot be considered as a traditional biosensor due to their limited selectivity, it was possible to distinguish prostate tumor cells in the range from 50 to 600 cells/µL in in vitro experiments with impedance spectroscopy. This was achieved by treating the impedance data with information visualization methods, which confirmed the distinguishing ability of the films by observing the absence of false positives in a series of control experiments. The CD44-HA interactions may, therefore, be exploited in clinical analyses and point-of-care diagnostics for cancer, particularly if computational methods are used to process the data.
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http://dx.doi.org/10.3390/cells9061563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7349506PMC
June 2020

Evaluating internalization and recycling of folate receptors in breast cancer cells using quantum dots.

J Photochem Photobiol B 2020 Aug 31;209:111918. Epub 2020 May 31.

Departamento de Biofísica e Radiobiologia, Universidade Federal de Pernambuco, Recife, PE, Brazil. Electronic address:

Folic acid (FA) regulates metabolic activities essential to the human body. FA receptor (FR) overexpression has been reported for many cancers, but there are still few or conflicting data about FRs in breast cancer cells. Quantum dots (QDs) have arisen as tools to elucidate aspects on FRs, due to their unique physicochemical properties. Herein, QDs conjugated to FA were explored to study the internalization and recycling of FRs in breast cancer cells, using HeLa as an out-group control. QDs were covalently conjugated to FA under different conditions. The best conjugate was applied to study FRs in HeLa, MCF7, MDA-MB231, and T47D cells applying confocal microscopy and flow cytometry analyses. The conjugation efficiency and specificity were evaluated, respectively, using fluorescence correlation spectroscopy (FCS) and saturation assays. FCS confirmed the effectiveness of the conjugation. HeLa and T47D had/internalized a higher amount of FRs (95% and 90% of labeling, respectively) than MDA-MB231 cells (68%). MCF7 cells seem to have very low functional FRs (3%). Saturation assays proved the specificity of QD-FA conjugates and suggested that FR recycling rate is low in the majority of cells studied, except for T47D. QD-FA conjugates were successfully developed. Therapies targeting FRs may be more effective for HeLa, T47D, and MDA-MB231.
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http://dx.doi.org/10.1016/j.jphotobiol.2020.111918DOI Listing
August 2020

Transcriptional regulators and regulatory pathways involved in prostate gland adaptation to a hypoandrogen environment.

Genet Mol Biol 2020 14;42(4):e20180362. Epub 2020 Feb 14.

Departamento de Biologia Estrutural e Funcional, Instituto de Biologia, Universidade de Campinas (UNICAMP), Campinas, SP, Brazil.

Anti-androgen therapies, including orchiectomy, are effective at promoting prostate cancer remission, but are followed by progression to the more aggressive castration-resistant prostate cancer (CRPC). Castration promotes gland and tumor shrinkage. However, prostate adaptation to androgen deprivation involves striking parallel events, all requiring changes in gene expression. We hypothesized that transcription factors (TF) and other transcription-related genes are needed to orchestrate those changes. In this work, downstream analysis using bioinformatic tools and published microarray data allowed us to identify sixty transcriptional regulators (including 10 TF) and to integrate their function in physiologically relevant networks. Functional associations revealed a connection between Arnt, Bhlhe41 and Dbp circadian rhythm genes with the Ar circuitry and a small gene network centered in Pex14, which might indicate a previously unanticipated metabolic shift. We have also identified human homologs and mapped the corresponding genes to human chromosome regions commonly affected in prostate cancer, with particular attention to the PTEN/HHEX/MXI1 cluster at 10q23-25 (frequently deleted in PCa) and to MAPK1 at 22q11.21 (delete in intermediate risk but not in high risk PCa). Twenty genes were found mutated or with copy number alterations in at least five percent of three cancer cohorts and six of them (PHOX2A, NFYC, EST2, EIF2S1, SSRP1 and PARP1) associated with impacted patient survival. These changes are specific to the adaptation to the hypoandrogen environment and seem important for the progression to CRPC when mutated.
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http://dx.doi.org/10.1590/1678-4685-GMB-2018-0362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7198032PMC
February 2020

The urokinase plasminogen activator binding to its receptor: a quantum biochemistry description within an in/homogeneous dielectric function framework with application to uPA-uPAR peptide inhibitors.

Phys Chem Chem Phys 2020 Feb 29;22(6):3570-3583. Epub 2020 Jan 29.

Departamento de Biologia Estrutural e Funcional, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP, Brazil.

Despite being recognized as a therapeutic target in the processes of cancer cell proliferation and metastasis for over 50 years, the interaction of the urokinase plasminogen activator uPA with its receptor uPAR still needs an improved understanding. High resolution crystallographic data (PDB ) of the uPA-uPAR binding geometry was used to perform quantum biochemistry computations within the density functional theory (DFT) framework. A divide to conquer methodology considering a mixed homogeneous/inhomogeneous dielectric model and explicitly taking water molecules into account was employed to obtain a large set of uPA-uPAR residue-residue interaction energies. In order of importance, not only were Phe25 > Tyr24 > Trp30 > Ile28 shown to be the most relevant uPA residues binding it to uPAR, but the residues Lys98 > His87 > Gln40 > Asn22 > Lys23 > Val20 also had significant interaction energies, which helps to explain published experimental mutational data. Furthermore, the results obtained with the uPA-uPAR in/homogeneous dielectric function show that a high dielectric constant value ε = 40 is adequate to take into account the electrostatic environment at the interface between the proteins, while using a smaller value of ε (<10) leads to an overestimation of the uPA-uPAR binding energy. Hot spots of the uPA-uPAR binding domain were identified and a quantum biochemistry description of the uPAR blockers uPA and cyclouPA[(S21C;H29C)] was performed, demonstrating that cyclization improves the stability of mimetic peptides without compromising their binding energies to uPAR.
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http://dx.doi.org/10.1039/c9cp06530jDOI Listing
February 2020

Dietary fatty acid quality affects systemic parameters and promotes prostatitis and pre-neoplastic lesions.

Sci Rep 2019 12 17;9(1):19233. Epub 2019 Dec 17.

Department of Structural and Functional Biology, State University of Campinas, Campinas, SP, Brazil.

Environmental and nutritional factors, including fatty acids (FA), are associated with prostatitis, benign prostate hyperplasia and prostate cancer. We hypothesized that different FA in normolipidic diets (7%) affect prostate physiology, increasing the susceptibility to prostate disorders. Thus, we fed male C57/BL6 mice with normolipidic diets based on linseed oil, soybean oil or lard (varying saturated and unsaturated FA contents and ω-3/ω-6 ratios) for 12 or 32 weeks after weaning and examined structural and functional parameters of the ventral prostate (VP) in the systemic metabolic context. Mongolian gerbils were included because they present a metabolic detour for low water consumption (i.e., oxidize FA to produce metabolic water). A linseed oil-based diet (LO, 67.4% PUFAs, ω-3/ω-6 = 3.70) resulted in a thermogenic profile, while a soybean oil-based diet (SO, 52.7% PUFAs, ω-3/ω-6 = 0.11) increased body growth and adiposity. Mice fed lard (PF, 13.1% PUFA, ω-3/ω-6 = 0.07) depicted a biphasic growth, resulting in decreased adiposity in adulthood. SO and PF resulted in hepatic steatosis and steatohepatitis, respectively. PF and SO increased prostate epithelial volume, and lard resulted in epithelial hyperplasia. Animals in the LO group had smaller prostates with predominant atrophic epithelia and inflammatory loci. Inflammatory cells were frequent in the VP of PF mice (predominantly stromal) and LO mice (predominantly luminal). RNAseq after 12 weeks revealed good predictors of a later-onset inflammation. The transcriptome unveiled ontologies related to ER stress after 32 weeks on PF diets. In conclusion, different FA qualities result in different metabolic phenotypes and differentially impact prostate size, epithelial volume, inflammation and gene expression.
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http://dx.doi.org/10.1038/s41598-019-55882-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6917739PMC
December 2019

Ghrelin effects on mitochondrial fitness modulates macrophage function.

Free Radic Biol Med 2019 12 14;145:61-66. Epub 2019 Sep 14.

Laboratory of Immunometabolism, Department of Genetics, Evolution, Microbiology and Immunology, University of Campinas, Brazil; Department of Immunology, Institute of Biomedical Sciences, University of Sao Paulo, Brazil. Electronic address:

Over the past years, systemic derived cues that regulate cellular metabolism have been implicated in the regulation of immune responses. Ghrelin is an orexigenic hormone produced by enteroendocrine cells in the gastric mucosa with known immunoregulatory roles. The mechanism behind the function of ghrelin in immune cells, such as macrophages, is still poorly understood. Here, we explored the hypothesis that ghrelin leads to alterations in macrophage metabolism thus modulating macrophage function. We demonstrated that ghrelin exerts an immunomodulatory effect over LPS-activated peritoneal macrophages, as evidenced by inhibition of TNF-α and IL-1β secretion and increased IL-12 production. Concomitantly, ghrelin increased mitochondrial membrane potential and increased respiratory rate. In agreement, ghrelin prevented LPS-induced ultrastructural damage in the mitochondria. Ghrelin also blunted LPS-induced glycolysis. In LPS-activated macrophages, glucose deprivation did not affect ghrelin-induced IL-12 secretion, whereas the inhibition of pyruvate transport and mitochondria-derived ATP abolished ghrelin-induced IL-12 secretion, indicating a dependence on mitochondrial function. Ghrelin pre-treatment of metabolic activated macrophages inhibited the secretion of TNF-α and enhanced IL-12 levels. Moreover, ghrelin effects on IL-12, and not on TNF-α, are dependent on mitochondria elongation, since ghrelin did not enhance IL-12 secretion in metabolic activated mitofusin-2 deficient macrophages. Thus, ghrelin affects macrophage mitochondrial metabolism and the subsequent macrophage function.
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http://dx.doi.org/10.1016/j.freeradbiomed.2019.09.012DOI Listing
December 2019

Differences between male and female prostates in terms of physiology, sensitivity to chemicals and pathogenesis-A review in a rodent model.

Cell Biol Int 2019 Aug 8. Epub 2019 Aug 8.

Department of Structural and Functional Biology, State University of Campinas-UNICAMP, Bertrand Russel Av., Campinas, São Paulo, Brazil.

The prostate is a gland that is not exclusively present in males, being also found in females of several mammalian species, including humans. There is evidence that the prostate in both sexes is affected by the same pathologies such as prostatitis, benign alterations and even cancer. In view of the difficulties of manipulating the prostate gland, the Mongolian gerbil (Meriones unguiculatus), a rodent species with high incidence of functional prostates in females, is widely used in studies of the female prostate. However, despite knowing much about the similarities between the female and male prostate, little emphasis has been placed on the differences between them. This review investigates the intersex differences in prostate development, physiology and pathogenesis. The female prostate develops earlier than in males and studies indicate that it is more sensitive to oestrogens than the male prostate, as well as being more sensitive to exposure to xenoestrogens, such as Bisphenol A and methylparaben, with a higher susceptibility to benign lesions in the adult and senile prostate than in males. In addition, the female prostate is impacted by pregnancy and the oestrous cycle, and is also dependent on progesterone. The peculiarities of the female prostate raise concerns about the risk of it undergoing neglected changes as a result of environmental chemicals, since safe dosages are established exclusively for the male prostate.
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http://dx.doi.org/10.1002/cbin.11214DOI Listing
August 2019

Laminin-derived peptide C16 regulates Tks expression and reactive oxygen species generation in human prostate cancer cells.

J Cell Physiol 2020 01 28;235(1):587-598. Epub 2019 Jun 28.

Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil.

Laminin peptides influence cancer biology. We investigated the role of a laminin-derived peptide C16 regulating invadopodia molecules in human prostate cancer cells (DU145). C16 augmented invadopodia activity of DU145 cells, and stimulated expression Tks4, Tks5, cortactin, and membrane-type matrix metalloproteinase 1. Reactive oxygen species generation is also related to invadopodia formation. This prompted us to address whether C16 would induce reactive oxygen species generation in DU145 cells. Quantitative fluorescence and flow cytometry showed that the peptide C16 increased reactive oxygen species in DU145 cells. Furthermore, significant colocalization between Tks5 and reactive oxygen species was observed in C16-treated cells. Results suggested that the peptide C16 increased Tks5 and reactive oxygen species in prostate cancer cells. The role of C16 increasing Tks and reactive oxygen species are novel findings on invadopodia activity.
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http://dx.doi.org/10.1002/jcp.28997DOI Listing
January 2020

An improved acridine orange staining of DNA/RNA.

Acta Histochem 2019 May 3;121(4):450-454. Epub 2019 Apr 3.

Department of Anatomy, Cell Biology, Physiology and Biophysics, Institute of Biology, State University of Campinas (UNICAMP), Campinas SP, Brazil. Electronic address:

New tools are desirable to examine the metabolic state of individual cells within tissues. We proposed a fluorescence-based procedure consisting of acridine orange staining and fast green counterstaining (AO-FG) to improve the selectivity of the former for nucleic acids (acridine orange stains both DNA and RNA with different fluorescence colors), with no interference from proteins. We compared this test with the biochemical quantification of the relative amounts of RNA and DNA in selected rat ventral prostate samples and PC3 cells. The epithelium of the prostate gland is highly active metabolically for the production of secretions. Differences in AO-RNA staining were revealed and correlated with the metabolic state of the epithelium. Specificity was confirmed by RNase A. To assess how AO-FG staining correlates with the metabolic state of the cell, we cultured PC3 cells in different concentrations of glucose and measured the ratios between the amounts of RNA and DNA. In parallel, similar cultures were subjected to AO-FG, and the staining pattern correlated closely (r=0.886) with the obtained biochemical results. The results confirmed that the combined use of AO and FG is useful for detecting DNA and RNA simultaneously, as well as for assessing quantitatively the transcriptional activity of individual cells and their changes in response to experimental manipulation.
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http://dx.doi.org/10.1016/j.acthis.2019.03.010DOI Listing
May 2019

Castration-induced prostate epithelial cell apoptosis results from targeted oxidative stress attack of M1 -macrophages.

J Cell Physiol 2019 08 28;234(10):19048-19058. Epub 2019 Mar 28.

Department of Structural and Functional Biology, State University of Campinas, Campinas, Sao Paulo, Brazil.

Prostate development and function are regulated by androgens. Epithelial cell apoptosis in response to androgen deprivation is caspase-9-dependent and peaks at Day 3 after castration. However, isolated epithelial cells survive in the absence of androgens. Znf142 showed an on-off expression pattern in intraepithelial CD68-positive macrophages, with the on-phase at Day 3 after castration. Rats treated with gadolinium chloride to deplete macrophages showed a significant drop in apoptosis, suggesting a causal relationship between macrophages and epithelial cell apoptosis. Intraepithelial M1-polarization was also limited to Day 3, and the inducible nitric oxide synthase (iNOS) knockout mice showed significantly less apoptosis than wild-type controls. The epithelial cells showed focal DNA double-strand breaks (DSB), 8-oxoguanine, and protein tyrosine-nitrosylation, fingerprints of exposure to peroxinitrite. Cultured epithelial cells induced M1-polarization and showed focal DSB and underwent apoptosis. The same phenomena were reproduced in LNCaP cells cocultured with Raw 264.7 macrophages. In conclusion, the M1 -macrophage (named after Znf142) attack causes activation of the intrinsic apoptosis pathway in epithelial cells after castration.
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http://dx.doi.org/10.1002/jcp.28544DOI Listing
August 2019

Transcription regulators are transiently expressed during the prostate gland adaptation to the hypoandrogenic environment.

Histol Histopathol 2019 Sep 26;34(9):1025-1036. Epub 2019 Mar 26.

INFABiC - National Institute of Science and Technology in Photonics Applied to Cell Biology, Campinas SP, Brazil.

The high incidence of prostatic diseases, including malignant tumors, makes the understanding of prostate biology very important. Androgen deprivation, blockade by orchiectomy, or chemical castration causes prostate and tumor shrinkage. The gene networks involved in a cell type-specific fashion are rather unknown. This work was undertaken to identify genes with annotated function in transcription regulation that might define transitions in gene expression. A total of 15 potential regulatory genes were identified. Validation by qRT-PCR showed that Zfp703 and Arid1a exhibit expression maxima at day 1; Ash2l, Nelf, Pbx3, Eya2 at day 4; Dmrt2 at day 5 and Lbh and Sox1 at day 7 after castration. Using immunohistochemistry, we further determined that PBX3 was found in both stromal and epithelial cells, whereas ARID1A and NELF were restricted to the epithelium, and DMRT2 and EYA2 were exclusively found in the stroma. Though the proteins ZFP703 and ASH2l were not found in any experimental condition, their mRNAs were located by in situ hybridization in both epithelium and stroma. In conclusion, androgen deprivation triggers the expression of temporally regulated gene sets in both epithelial and stromal cells. These gene subsets will help establish the regulatory gene expression programs orchestrating the castration-induced remodeling of the prostate gland, and represent putative targets to increase the efficacy of androgen-deprivation to induce epithelial (and cancer) cell death.
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http://dx.doi.org/10.14670/HH-18-105DOI Listing
September 2019

Heparanase-1 activity and the early postnatal prostate development.

Dev Dyn 2019 03 4;248(3):211-220. Epub 2019 Feb 4.

Departamento de Biologia Estrutural e Funcional, Universidade Estadual de Campinas, Instituto de Biologia, Campinas, São Paulo, Brazil.

Ventral prostate (VP) morphogenesis starts during embryonic development and continues for the first three postnatal weeks. Heparan sulfate (HS) affects paracrine signaling. Heparanase-1 (HPSE) is the only enzyme capable of cleaving HS. HPSE releases the HS bioactive fragment and mobilizes growth factors. Little is known, however, about HS turnover and HPSE function during VP morphogenesis. In this study, we measured HSPG expression and analyzed the expression and distribution of HPSE in the rat VP. HPSE was predominantly expressed by the VP epithelium. The VP was treated with heparin in ex vivo cultures to interfere with HS and resulted in delayed epithelial growth. Hpse knockdown using siRNA delayed epithelial growth in the first postnatal week ex vivo, which was similar to treating with the lower concentration of heparin. Hpse silencing was related to changes in HS chain length (as determined by size-exclusion chromatography, up-regulation of Mmp9, and down-regulation of Mmp2 expression). It also down-modulated ERK1/2 phosphorylation, suggesting a reduction in signaling, likely due to decreased HS cleavage and growth factor bioavailability. Our results showed that HPSE played a role in early epithelial growth during the first week of VP postnatal development. Developmental Dynamics 248:211-220, 2019. © 2019 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/dvdy.12DOI Listing
March 2019

Phylogenetic, molecular evolution and structural analyses of the WFDC1/prostate stromal protein 20 (ps20).

Gene 2019 Feb 10;686:125-140. Epub 2018 Nov 10.

Department of Structural and Functional Biology, State University of Campinas, 13083-863 Campinas, São Paulo, Brazil. Electronic address:

The WFDC1 gene is frequently down-regulated or lost in prostate cancer, and the encoded protein, ps20, has been implicated in epithelial cell behaviour and angiogenesis. However, ps20 remains largely uncharacterised with respect to its structure and interacting partners. This study characterised the evolution, functionality and structural characteristics of WFDC1/ps20 using phylogenetic reconstruction and other computational approaches. Bayesian phylogenetic analyses suggested that ps20 appeared in a common ancestor of deuterostomes-protostomes. The rate of evolutionary change within the coding regions of vertebrate WFDC1 genes and the synteny conservation in mammals differed from that of other vertebrate clades, indicating a possible functional diversity of ps20 homologues. A gene set enrichment analysis of the genes around WFDC1 (conserved synteny) showed functional relationships between the WFDC1, CDH13, CRISPLD2, IRF8 and TFPI2 genes. The molecular evolution of ps20 has been driven by purifying selection, particularly in the segments corresponding to exons 3 and 4, which encode the most conserved regions of the protein. A co-evolution analysis showed that residues within these regions co-vary with each other during the evolution of ps20. These results show that the regions corresponding to exons 3 and 4 are ps20-specific structure-function modules. Homology modelling of the exon 2-encoded polypeptide and subsequent dynamics calculus using a Gaussian network model showed that residues with high conformational flexibility are part of a loop region involved in protein-protein recognition, given the similarity with other serine protease inhibitors. Residues C96, R94, L105, and C66 are critical for the integrity and functionality of this ps20 region.
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http://dx.doi.org/10.1016/j.gene.2018.10.046DOI Listing
February 2019

The role of SDF1 in prostate epithelial morphogenesis.

J Cell Physiol 2019 05 26;234(5):6886-6897. Epub 2018 Oct 26.

Departamento de Biologia Estrutural e Funcional, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, Brasil.

Androgens induce rat prostate induction from the urogenital sinus epithelium at embryonic day 17.5. Subsequent morphogenesis, including epithelial cord growth, branching, and canalization, results from concerted paracrine interactions with the stroma. A significant number of paracrine factors bind heparan sulfate (HS). We hypothesized that interfering with overall sulfation could disrupt the signaling mediated by HS-binding factors and that the undersulfated environment would allow investigation of individual exogenous morphogens. First, we investigated whether acinar morphogenesis involved HS-proteoglycan expression and found that syndecans 1 and 3 were upregulated in RWPE1 cells in the transition from two- to three-dimensional (3D) Matrigel, capable of promoting spheroid formation. We then investigated whether sodium chlorate, a general sulfation inhibitor, interfered with spheroid formation by RWPE1 cells and acinar morphogenesis in ex vivo ventral prostate (VP) organ culture. As expected, treatment with sodium chlorate inhibited spheroid formation by RWPE1 cells in 3D culture. Chlorate also inhibited ex vivo VP epithelial branching and canalization, resulting in long branchless epithelial structures. We then investigated whether the HS-binding factors, FGF10, TGFβ1, and SDF1, could reverse the effect of sodium chlorate. Although no effect was seen in the FGF10- and TGFβ1-treated samples, SDF1 promoted epithelial canalization in the low sulfated environment, highlighting its specific role in lumen formation. Altogether, the results show that sodium chlorate perturbed prostate morphogenesis and allowed investigation of factors involved in branching and/or canalization, implicating SDF1 signaling in epithelial canalization.
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http://dx.doi.org/10.1002/jcp.27447DOI Listing
May 2019
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