Somatic Copy Number Alterations in Colorectal Cancer Lead to a Differentially Expressed ceRNA Network (ceRNet).

Colorectal cancer (CRC) represents the second deadliest malignancy worldwide. Around 75% of CRC patients exhibit high levels of chromosome instability that result in the accumulation of somatic copy number alterations. These alterations are associated with the amplification of oncogenes and deletion of tumor-ppressor genes and contribute to the tumoral phenotype in different malignancies. Even though this relationship is well known, much remains to be investigated regarding the effect of said alterations in long non-coding RNAs (lncRNAs) and, in turn, the impact these alterations have on the tumor phenotype. The present study aimed to evaluate the role of differentially expressed lncRNAs coded in regions with copy number alterations in colorectal cancer patient samples. We downloaded RNA-seq files of the Colorectal Adenocarcinoma Project from the The Cancer Genome Atlas (TCGA) repository (285 sequenced tumor tissues and 41 non-tumor tissues), evaluated differential expression, and mapped them over genome sequencing data with regions presenting copy number alterations. We obtained 78 differentially expressed (LFC > 1|< -1, padj < 0.05) lncRNAs, 410 miRNAs, and 5028 mRNAs and constructed a competing endogenous RNA (ceRNA) network, predicting significant lncRNA-miRNA-mRNA interactions. Said network consisted of 30 lncRNAs, 19 miRNAs, and 77 mRNAs. To understand the role that our ceRNA network played, we performed KEGG and GO analysis and found several oncogenic and anti-oncogenic processes enriched by the molecular players in our network. Finally, to evaluate the clinical relevance of the lncRNA expression, we performed survival analysis and found that C5orf64, HOTAIR, and RRN3P3 correlated with overall patient survival. Our results showed that lncRNAs coded in regions affected by SCNAs form a complex gene regulatory network in CCR.

Organotypic 3D Cell-Architecture Impacts the Expression Pattern of miRNAs-mRNAs Network in Breast Cancer SKBR3 Cells.

BACKGROUND: Currently, most of the research on breast cancer has been carried out in conventional two-dimensional (2D) cell cultures due to its practical benefits, however, the three-dimensional (3D) cell culture is becoming the model of choice in cancer research because it allows cell-cell and cell-extracellular matrix (ECM) interactions, mimicking the native microenvironment of tumors in vivo. METHODS: In this work, we evaluated the effect of 3D cell organization on the expression pattern of miRNAs (by Small-RNAseq) and mRNAs (by microarrays) in the breast cancer SKBR3 cell line and analyzed the biological processes and signaling pathways regulated by the differentially expressed protein-coding genes (DE-mRNAs) and miRNAs (DE-microRNAs) found in the organoids. RESULTS: We obtained well-defined cell-aggregated organoids with a grape cluster-like morphology with a size up to 9.2 × 105 µm3. The transcriptomic assays showed that cell growth in organoids significantly affected (all p < 0.01) the gene expression patterns of both miRNAs, and mRNAs, finding 20 upregulated and 19 downregulated DE-microRNAs, as well as 49 upregulated and 123 downregulated DE-mRNAs. In silico analysis showed that a subset of 11 upregulated DE-microRNAs target 70 downregulated DE-mRNAs. These genes are involved in 150 gene ontology (GO) biological processes such as regulation of cell morphogenesis, regulation of cell shape, regulation of canonical Wnt signaling pathway, morphogenesis of epithelium, regulation of cytoskeleton organization, as well as in the MAPK and AGE-RAGE signaling KEGG-pathways. Interestingly, hsa-mir-122-5p (Fold Change (FC) = 15.4), hsa-mir-369-3p (FC = 11.4), and hsa-mir-10b-5p (FC = 20.1) regulated up to 81% of the 70 downregulated DE-mRNAs. CONCLUSION: The organotypic 3D cell-organization architecture of breast cancer SKBR3 cells impacts the expression pattern of the miRNAs-mRNAs network mainly through overexpression of hsa-mir-122-5p, hsa-mir-369-3p, and hsa-mir-10b-5p. All these findings suggest that the interaction between cell-cell and cell-ECM as well as the change in the culture architecture impacts gene expression, and, therefore, support the pertinence of migrating breast cancer research from conventional cultures to 3D models.

SOX9 knockout decreases stemness properties in colorectal cancer cells.

Background: Colorectal cancer (CRC) is a leading cause of death worldwide. SRY-box transcription factor 9 (SOX9) participates in organogenesis and cell differentiation in normal tissues but has been involved in carcinogenesis development. Cancer stem cells (CSCs) are a small population of cells present in solid tumors that contribute to increased tumor heterogeneity, metastasis, chemoresistance, and relapse. CSCs have properties such as self-renewal and differentiation, which can be modulated by many factors. Currently, the role of SOX9 in the maintenance of the stem phenotype has not been well elucidated, thus, in this work we evaluated the effect of the absence of SOX9 in the stem phenotype of CRC cells. Methods: We knockout (KO) SOX9 in the undifferentiated CRC cell line HCT116 and evaluated their stemness properties using sphere formation assay, differentiation assay, and immunophenotyping. Results: SOX9-KO affected the epithelial morphology of HCT116 cells and stemness characteristics such as its pluripotency signature with the increase of SOX2 as a compensatory mechanism to induce SOX9 expression, the increase of KLF4 as a differentiation feature, as well as the inhibition of the stem cell markers CD44 and CD73. In addition, SOX9-KO cells gain the epithelial-mesenchymal transition (EMT) phenotype with a significant upregulation of CDH2. Furthermore, our results showed a remarkable effect on first- and second-sphere formation, being SOX9-KO cells less capable of forming high-size-resistant spheres. Nevertheless, CSCs surface markers were not affected during the differentiation assay. Conclusions: Collectively, our findings supply evidence that SOX9 promotes the maintenance of stemness properties in CRC-CSCs.

Replication of Dengue Virus 4 in Plodia interpunctella (Lepidoptera: Pyralidae) Larvae Under Laboratory Conditions.

Background: Developing methods for the isolation and replication of dengue virus (DENV), based on nonhematophagous insect models to assess virus-host interaction, would contribute, for instance, to the creation of drugs or vaccines and eventually to the control of the disease. In this regard, nonhematophagous mosquitoes have been used as biological hosts for the isolation of DENV because they are specific and sensitive to a low viral load and viral particles with low infectivity. However, implementation of these models is mainly affected by the complexity of the establishment of the entomological colonies. Materials and Methods: In this study, the susceptibility of DENV-4 infection in Plodia interpunctella larvae was evaluated. Ten larvae, previously inoculated with supernatant from DENV-4-infected C6/36 cells, were processed to determine viral replication by the optical density and 2-ΔΔCt methods at different time intervals (1 and 7 days postinoculation). Results: A prospective increase in viral replication was observed, which did not influence the survival and development of P. interpunctella. Conclusion: These results demonstrate the infectivity of DENV-4 in P. interpunctella, thus becoming an option as a biological model for the study of this etiological agent.

Evaluation of biocompatibility and angiogenic potential of extracellular matrix hydrogel biofunctionalized with the LL-37 peptide.

BACKGROUND: Biomaterials must allow revascularization for a successful tissue regeneration process. Biomaterials formulated from the extracellular matrix (ECM) have gained popularity in tissue engineering because of their superior biocompatibility, and due to their rheological properties, ECM-hydrogels can be easily applied in damaged areas, allowing cell colonization and integration into the host tissue. Porcine urinary bladder ECM (pUBM) retains functional signaling and structural proteins, being an excellent option in regenerative medicine. Even some small molecules, such as the antimicrobial cathelicidin-derived LL-37 peptide have proven angiogenic properties. OBJECTIVE: The objective of this study was to evaluate the biocompatibility and angiogenic potential of an ECM-hydrogel derived from the porcine urinary bladder (pUBMh) biofunctionalized with the LL-37 peptide (pUBMh/LL37). METHODS: Macrophages, fibroblasts, and adipose tissue-derived mesenchymal stem cells (AD-MSC) were exposed pUBMh/LL37, and the effect on cell proliferation was evaluated by MTT assay, cytotoxicity by quantification of lactate dehydrogenase release and the Live/Dead Cell Imaging assays. Moreover, macrophage production of IL-6, IL-10, IL-12p70, MCP-1, INF-γ, and TNF-α cytokines was quantified using a bead-based cytometric array. pUBMh/LL37 was implanted directly by dorsal subcutaneous injection in Wistar rats for 24 h to evaluate biocompatibility, and pUBMh/LL37-loaded angioreactors were implanted for 21 days for evaluation of angiogenesis. RESULTS: We found that pUBMh/LL37 did not affect cell proliferation and is cytocompatible to all tested cell lines but induces the production of TNF-α and MCP-1 in macrophages. In vivo, this ECM-hydrogel induces fibroblast-like cell recruitment within the material, without tissue damage or inflammation at 48 h. Interestingly, tissue remodeling with vasculature inside angioreactors was seen at 21 days. CONCLUSIONS: Our results showed that pUBMh/LL37 is cytologically compatible, and induces angiogenesis in vivo, showing potential for tissue regeneration therapies.

Design, synthesis, in silico, and in vitro evaluation of benzylbenzimidazolone derivatives as potential drugs on α-glucosidase and glucokinase as pharmacological targets.

Benzimidazolones have shown biological activities, including antihyperglycemic and hypoglycemic, by inhibiting or activating of α-glu and GK. The aim of this study is the rational design of compounds using in silico assays to delimitate the selection of structures to synthesize and the in vitro evaluation of benzimidazolone derivatives in blood glucose control. A docking of 23 benzimidazolone derivatives was performed; selecting the compounds with better in silico profiles to synthesize by microwave-irradiation/conventional heat and evaluate in enzymatic in vitro evaluation. Compounds 2k, 2m, 2r, and 2s presented the best in silico profiles, showing good affinity energy (-10.9 to -8.6 kcal mol-1) and binding with catalytic-amino acids. They were synthesized at 70 °C and 24 h using DMF as the solvent and potassium carbonate (yield: 22-38%). The results with α-glu showed moderate inhibition of 2k (14 ± 1.23-29 ± 0.45), 2m (12 ± 2.21-36 ± 0.30), 2r (7 ± 2.21-13 ± 1.34), and 2s (11 ± 0.74-35 ± 2.95) at evaluated concentrations (0.1 to 100 µg mL-1). The GK activation assay showed an enzymatic activity increase; compound 2k increased 1.31 and 2.83 more than normal activity, 2m (2.13-fold), 2s (2.86 and 3.74-fold) at 100 and 200 µg mL-1 respectively. The present study showed that the 2s derivative presents moderate potential as an α-glu inhibitor and a good activator potential of GK, suggesting that this compound is a good candidate for blood glucose control through antihyperglycemic and hypoglycemic mechanisms.

An extracellular matrix hydrogel from porcine urinary bladder for tissue engineering: In vitro and in vivo analyses.

BACKGROUND: The necessity to manufacture scaffolds with superior capabilities of biocompatibility and biodegradability has led to the production of extracellular matrix (ECM) scaffolds. Among their advantages, they allow better cell colonization, which enables its successful integration into the hosted tissue, surrounding the area to be repaired and their formulations facilitate placing it into irregular shapes. The ECM from porcine urinary bladder (pUBM) comprises proteins, proteoglycans and glycosaminoglycans which provide support and enable signals to the cells. These properties make it an excellent option to produce hydrogels that can be used in regenerative medicine. OBJECTIVE: The goal of this study was to assess the biocompatibility of an ECM hydrogel derived from the porcine urinary bladder (pUBMh) in vitro using fibroblasts, macrophages, and adipose-derived mesenchymal stem cells (AD-MCSs), as well as biocompatibility in vivo using Wistar rats. METHODS: Effects upon cells proliferation/viability was measured using MTT assay, cytotoxic effects were analyzed by quantifying lactate dehydrogenase release and the Live/Dead Cell Imaging assay. Macrophage activation was assessed by quantification of IL-6, IL-10, IL-12p70, MCP-1, and TNF-α using a microsphere-based cytometric bead array. For in vivo analysis, Wistar rats were inoculated into the dorsal sub-dermis with pUBMh. The specimens were sacrificed at 24 h after inoculation for histological study. RESULTS: The pUBMh obtained showed good consistency and absence of cell debris. The biocompatibility tests in vitro revealed that the pUBMh promoted cell proliferation and it is not cytotoxic on the three tested cell lines and induces the production of pro-inflammatory cytokines on macrophages, mainly TNF-α and MCP-1. In vivo, pUBMh exhibited fibroblast-like cell recruitment, without tissue damage or inflammation. CONCLUSION: The results show that pUBMh allows cell proliferation without cytotoxic effects and can be considered an excellent biomaterial for tissue engineering.

Breast Cancer Cells Reprogram the Oncogenic lncRNAs/mRNAs Coexpression Networks in Three-Dimensional Microenvironment.

Organotypic three-dimensional (3D) cell cultures more accurately mimic the characteristics of solid tumors in vivo in comparison with traditional two-dimensional (2D) monolayer cell models. Currently, studies on the regulation of long non-coding RNAs (lncRNAs) have not been explored in breast cancer cells cultured in 3D microenvironments. In the present research, we studied the expression and potential roles of lncRNAs in estrogen receptor-positive luminal B subtype BT-474 breast cancer cells grown over extracellular matrix proteins-enriched 3D cultures. Global expression profiling using DNA microarrays identifies 290 upregulated and 183 downregulated lncRNAs in 3D cultures relative to 2D condition. Using a co-expression analysis approach of lncRNAs and mRNAs pairs expressed in the same experimental conditions, we identify hundreds of regulatory axes modulating genes involved in cancer hallmarks, such as responses to estrogens, cell proliferation, hypoxia, apical junctions, and resistance to endocrine therapy. In addition, we identified 102 lncRNAs/mRNA correlations in 3D cultures, which were similar to those reported in TCGA datasets obtained from luminal B breast cancer patients. Interestingly, we also found a set of mRNAs transcripts co-expressed with LINC00847 and CTD-2566J3.1 lncRNAs, which were predictors of pathologic complete response and overall survival. Finally, both LINC00847 and CTD -2566J3.1 were co-expressed with essential genes for cancer genetic dependencies, such as FOXA1 y GINS2. Our experimental and predictive findings show that co-expressed lncRNAs/mRNAs pairs exhibit a high degree of similarity with those found in luminal B breast cancer patients, suggesting that they could be adequate pre-clinical tools to identify not only biomarkers related to endocrine therapy response and PCR, but to understand the biological behavior of cancer cells in 3D microenvironments.

Metabolomic Analysis of Phytochemical Compounds from Agricultural Residues of Eggplant (Solanum melongena L.).

The eggplant is a fruit rich in natural products and produced worldwide. However, its cultivation generates a large amount of scarcely used agricultural residues with poor chemical characterization. This study aimed to identify and quantify the metabolome and determine the composition of select phytochemicals and the overall antioxidant capacity of various anatomical parts of the plant. The plant's root, leaf, stem, and fruit were analyzed by quantitative mass spectrometry-based untargeted metabolomics and chemoinformatics, and phytochemicals were quantified by spectrophotometric analysis. Moreover, we determined the total antioxidant capacity of the distinct plant parts to infer a possible biological effect of the plant's metabolites. Various secondary metabolites were identified as terpenes, phenolic compounds, alkaloids, and saponins, distributed throughout the plant. The leaf and fruit presented the highest concentration of phenolic compounds, flavonoids, anthocyanins, and alkaloids, accompanied by the highest antioxidant capacity. Although the stem and root showed the lowest abundance of secondary metabolites, they provided around 20% of such compounds compared with the leaf and fruit. Overall, our study improved the understanding of the eggplant metabolome and concluded that the plant is rich in secondary metabolites, some with antioxidant properties, and shows potential nutraceutical and biopharmaceutical applications.

Cytokine-polymorphisms associated with Preeclampsia: A review.

BACKGROUND: Preeclampsia (PE) is a syndromic disorder that affects 2% to 8% of pregnancies and is diagnosed principally when hypertension appears in the second-d half of pregnancy. WHO estimates the incidence of PE to be seven times higher in developing countries than in developed countries. Severe preeclampsia/eclampsia is one of the most important causes of maternal mortality, associated with 50,000 to 100,000 annual deaths globally as well as serious fetal and neonatal morbidity and mortality, especially in developing countries. Even though evidence from family-based studies suggest PE has a heritable component, its etiology, and specific genetic contributions remain unclear. Many studies examining the genetic factors contributing to PE have been conducted, most of them are focused on single nucleotide polymorphisms (SNPs). Given that PE has a very important inflammatory component, is mandatory to examine cytokine-SNPs for elucidating all mechanisms involved in this pathology. In this review, we describe the most important cytokine-polymorphisms associated with the onset and development of PE. We aim to provide current and relevant evidence in this regard. METHODS: We searched English databases such as PubMed and the National Center for Biotechnology Information. The publication time of the papers was set from the establishment of the databases to February 2022. All studies about Th1/Th2/Th17 cytokines polymorphisms were included in our study. RESULTS: SNPs in IFN-γ, TNF-α, IL-4, IL-6, IL-10, IL-17A, and IL-22 are associated with the development, early-onset and severity of PE, being the Th1/Th2/Th17 responses affected by the presence of these SNPs. CONCLUSIONS: The changes in Th1/Th2/Th17 response modify processes such as placentation, control of inflammation, and vascular function. Nonetheless, association studies have shown different results depending on sample size, diagnostic, and population.

The Chemokine MIG is Associated with an Increased Risk of COVID-19 Mortality in Mexican Patients.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is an emergent viral disease in which the host inflammatory response modulates the clinical outcome. Severe outcomes are associated with an exacerbation of inflammation in which chemokines play an important role as the attractants of immune cells to the tissues. OBJECTIVE: To evaluate the relationship of the chemokines IL-8, RANTES, MIG, MCP-1, and IP-10 with COVID-19 severity and outcomes in Mexican patients. METHODS: We analyzed the serum levels of IL-8, RANTES, MIG, MCP-1 and IP-10 in 148 COVID-19 hospitalized patients classified as mild (n=20), severe (n=61), and critical (n=67), as well as in healthy individuals (n=10), by flow cytometry bead array assay. RESULTS: Chemokine levels were higher in patients than in the healthy individuals, but only MIG, MCP-1, and IP-10 increased according to the disease severity, showing the highest levels in the critical group. MIG, MCP-1, and IP-10 levels were also higher in COVID-19 patients with comorbidities such as renal disease, type 2 diabetes, and hypertension. Moreover, elevated MIG levels seem to be related to organic failure/shock, and an increased risk of death. CONCLUSIONS: Our results suggest that the increased levels of MCP-1, IP-10, and especially MIG might be useful in predicting severe COVID-19 outcomes and could be promising therapeutic targets.

Antioxidant potential, cytokines regulation, and inflammation-related genes expression of phenolic extracts from Mexican oregano.

The Mexican population traditionally uses oregano infusions to treat oxidative and inflammation-related disorders. Therefore, this study was focused on the examination of the antioxidant capacity and potential against inflammation from three Mexican oregano species (Lippia graveolens [LG], Lippia palmeri [LP], and Hedeoma patens [HP]). The extracts from LG showed a superior total phenolic content. LG, LP, and HP exhibited a higher capacity to inhibit the radical DPPH (up to 90.33 ± 0.25%) and significantly lowered the release of MCP-1 and IL-6. At the same time, LG and HP increased the secretion of IL-10. Extracts from LG, LP, and HP did not significantly diminish the expression of il-1ß or inos, although a slight decrease in inos expression was observed. Our findings support that phenolic extracts from L. graveolens, L. palmeri, and H. patens possess antioxidant and anti-inflammatory properties and might be potential therapeutic candidates against oxidative and inflammation-related diseases. PRACTICAL APPLICATIONS: Oregano species have traditionally been exploited as remedies against inflammatory-related diseases, namely headaches, asthma, bowel disorders, and rheumatism. This study explored the antioxidant potential of three Mexican oregano species (Lippia graveolens, Lippia palmeri, and Hedeoma patens) and their anti-inflammatory effects in a murine cell model. Phenolic extracts from oregano showed antioxidant capacity and exerted activity against inflammation by improving anti-inflammatory cytokines secretion or negatively regulating pro-inflammatory cytokines. The results of our study demonstrate that the phenolic extracts from these Mexican oregano species have the potential in treating inflammation-related diseases.

Emerging role of lncRNAs in drug resistance mechanisms in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) originates in the squamous cell lining the mucosal surfaces of the head and neck region, including the oral cavity, nasopharynx, tonsils, oropharynx, larynx, and hypopharynx. The heterogeneity, anatomical, and functional characteristics of the patient make the HNSCC a complex and difficult-to-treat disease, leading to a poor survival rate and a decreased quality of life due to the loss of important physiologic functions and aggressive surgical injury. Alteration of driver-oncogenic and tumor-suppressing lncRNAs has recently been recently in HNSCC to obtain possible biomarkers for diagnostic, prognostic, and therapeutic approaches. This review provides current knowledge about the implication of lncRNAs in drug resistance mechanisms in HNSCC. Chemotherapy resistance is a major therapeutic challenge in HNSCC in which lncRNAs are implicated. Lately, it has been shown that lncRNAs involved in autophagy induced by chemotherapy and epithelial-mesenchymal transition (EMT) can act as mechanisms of resistance to anticancer drugs. Conversely, lncRNAs involved in mesenchymal-epithelial transition (MET) are related to chemosensitivity and inhibition of invasiveness of drug-resistant cells. In this regard, long non-coding RNAs (lncRNAs) play a pivotal role in both processes and are important for cancer detection, progression, diagnosis, therapy response, and prognostic values. As the involvement of more lncRNAs is elucidated in chemoresistance mechanisms, an improvement in diagnostic and prognostic tools could promote an advance in targeted and specific therapies in precision oncology.

Cytotoxic Activity of Polyphenol Extracts from Three Oregano Species: Hedeoma patens, Lippia graveolens and Lippia palmeri, and Antiproliferative Potential of Lippia graveolens against Two Types of Breast Cancer Cell Lines (MDA-MB-231 and MCF-7).

Oregano infusions have traditionally been used to treat some diseases related to inflammation and cancer; also, some species have shown antiproliferative activity on cancer cell lines, for example, colon and liver, and this has been attributed to its phytochemical profile, mainly its phenolic compounds. This study aimed to evaluate the cytotoxicity and antiproliferative potential of the polyphenols-rich extracts (PRE) of the oregano species H. patens, L. graveolens, and L. palmeri on breast cancer cell lines. The PRE of the three oregano species were obtained from dried leaves. The extract was characterized by determining antioxidant activity, total phenols content, and identifying the profile of phenolic acids and flavonoids by chromatography UPLC-MS/MS. Furthermore, the cytotoxicity of the extracts was evaluated in vitro on a non-cancer cell line of fibroblast NIH3T3 and the antiproliferative potential on the breast cancer cell lines MDA-MB-231 and MCF-7. L. graveolens showed the highest antioxidant capacity and significantly inhibited the proliferation of MCF-7 and MDA-MB-231 cells at non-cytotoxic concentrations in normal cells, with a similar effect to that cisplatin in MDA-MB-231 cells. Therefore, the polyphenol-rich extract from L. graveolens showed the greatest potential to guide future research on the antiproliferative mechanism of action.

HypoxaMIRs: Key Regulators of Hallmarks of Colorectal Cancer.

Hypoxia in cancer is a thoroughly studied phenomenon, and the logical cause of the reduction in oxygen tension is tumor growth itself. While sustained hypoxia leads to death by necrosis in cells, there is an exquisitely regulated mechanism that rescues hypoxic cells from their fatal fate. The accumulation in the cytoplasm of the transcription factor HIF-1α, which, under normoxic conditions, is marked for degradation by a group of oxygen-sensing proteins known as prolyl hydroxylases (PHDs) in association with the von Hippel-Lindau anti-oncogene (VHL) is critical for the cell, as it regulates different mechanisms through the genes it induces. A group of microRNAs whose expression is regulated by HIF, collectively called hypoxaMIRs, have been recognized. In this review, we deal with the hypoxaMIRs that have been shown to be expressed in colorectal cancer. Subsequently, using data mining, we analyze a panel of hypoxaMIRs expressed in both normal and tumor tissues obtained from TCGA. Finally, we assess the impact of these hypoxaMIRs on cancer hallmarks through their target genes.

Three-Dimensional 3D Culture Models in Gynecological and Breast Cancer Research.

Traditional two-dimensional (2D) monolayer cell cultures have long been the gold standard for cancer biology research. However, their ability to accurately reflect the molecular mechanisms of tumors occurring in vivo is limited. Recent development of three-dimensional (3D) cell culture models facilitate the possibility to better recapitulate several of the biological and molecular characteristics of tumors in vivo, such as cancer cells heterogeneity, cell-extracellular matrix interactions, development of a hypoxic microenvironment, signaling pathway activities depending on contacts with extracellular matrix, differential growth kinetics, more accurate drugs response, and specific gene expression and epigenetic patterns. In this review, we discuss the utilization of different types of 3D culture models including spheroids, organotypic models and patient-derived organoids in gynecologic cancers research, as well as its potential applications in oncological research mainly for screening drugs with major physiological and clinical relevance. Moreover, microRNAs regulation of cancer hallmarks in 3D cell cultures from different types of cancers is discussed.

Three-Dimensional Organotypic Cultures Reshape the microRNAs Transcriptional Program in Breast Cancer Cells.

The 3D organotypic cultures, which depend on the growth of cells over the extracellular matrix (ECM) used as a scaffold, can better mimic several characteristics of solid cancers that influence tumor biology and the response to drug therapies. Most of our current knowledge on cancer is derived from studies in 2D cultures, which lack the ECM-mediated microenvironment. Moreover, the role of miRNAs that is critical for fine-tuning of gene expression is poorly understood in 3D cultures. The aim of this study was to compare the miRNA expression profiles of breast cancer cells grown in 2D and 3D conditions. On an on-top 3D cell culture model using a basement membrane matrix enriched with laminin, collagen IV, entactin, and heparin-sulfate proteoglycans, the basal B (Hs578T) and luminal (T47D) breast cancer cells formed 3D spheroid-like stellate and rounded mass structures, respectively. Morphological changes in 3D cultures were observed as cell stretching, cell-cell, and cell-ECM interactions associated with a loss of polarity and reorganization on bulk structures. Interestingly, we found prolongations of the cytoplasmic membrane of Hs578T cells similar to tunneled nanotubes contacting between neighboring cells, suggesting the existence of cellular intercommunication processes and the possibility of fusion between spheroids. Expression profiling data revealed that 354 miRNAs were differentially expressed in 3D relative to 2D cultures in Hs578T cells. Downregulated miRNAs may contribute to a positive regulation of genes involved in hypoxia, catabolic processes, and focal adhesion, whereas overexpressed miRNAs modulate genes involved in negative regulation of the cell cycle. Target genes of the top ten modulated miRNAs were selected to construct miRNA/mRNA coregulation networks. Around 502 interactions were identified for downregulated miRNAs, including miR-935/HIF1A and miR-5189-3p/AKT that could contribute to cell migration and the response to hypoxia. Furthermore, the expression levels of miR-935 and its target HIF1A correlated with the expression found in clinical tumors and predicted poor outcomes. On the other hand, 416 interactions were identified for overexpressed miRNAs, including miR-6780b-5p/ANKRD45 and miR-7641/CDK4 that may result in cell proliferation inhibition and cell cycle arrest in quiescent layers of 3D cultures. In conclusion, 3D cultures could represent a suitable model that better resembles the miRNA transcriptional programs operating in tumors, with implications not only in the understanding of basic cancer biology in 3D microenvironments, but also in the identification of novel biomarkers of disease and potential targets for personalized therapies in cancer.

Extracellular matrix hydrogel derived from bovine bone is biocompatible in vitro and in vivo.

BACKGROUND: Nowadays, biomaterials used as a scaffold must be easy to deliver in the bone defect area. Extracellular matrix (ECM) hydrogels are highly hydrated polymers that can fill irregular shapes and act as bioactive materials. OBJECTIVE: This work aims to show the effects of ECM hydrogels derived from bovine bone (bECMh) on proliferation, cytotoxicity and expression of pro-inflammatory cytokines in three cells types involved in tissue regeneration, as well as biocompatibility in vivo. METHODS: In vitro, we used an extract of bECMh to test it on macrophages, fibroblasts, and adipose-derived mesenchymal stem cells (AD-MCSs). Cell proliferation was measured using the MTT assay, cytotoxicity was measured by quantifying lactate dehydrogenase release and the Live/Dead Cell Imaging assays. Concentrations of IL-6, IL-10, IL-12p70, MCP-1 and TNF-α were quantified in the supernatants using a microsphere-based cytometric bead array. For in vivo analysis, Wistar rats were inoculated into the dorsal sub-dermis with bECMh, taking as reference the midline of the back. The specimens were sacrificed at 24 h for histological study. RESULTS: In vitro, this hydrogel behaves as a dynamic biomaterial that increases fibroblast proliferation, induces the production of pro-inflammatory cytokines in macrophages, among which MCP-1 and TNF-α stand out. In vivo, bECMh allows the colonization of host fibroblast-like and polymorphonuclear cells, without tissue damage or inflammation. CONCLUSIONS: The results indicate that bECMh is a biocompatible material that could be used as a scaffold, alone or in conjunction with cells or functional biomolecules, enhancing proliferation and allowing the filling of bone defects to its further regeneration.

The Usefulness of Peripheral Blood Cell Counts to Distinguish COVID-19 from Dengue during Acute Infection.

COVID-19 and dengue disease are challenging to tell apart because they have similarities in clinical and laboratory features during the acute phase of infection, leading to misdiagnosis and delayed treatment. The present study evaluated peripheral blood cell count accuracy to distinguish COVID-19 non-critical patients from non-severe dengue cases between the second and eleventh day after symptom onset. A total of 288 patients infected with SARS-CoV-2 (n = 105) or dengue virus (n = 183) were included in this study. Neutrophil, platelet, and lymphocyte counts were used to calculate the neutrophil-lymphocyte ratio (NLR), the platelet-lymphocyte ratio (PLR), and the neutrophil-lymphocyte*platelet ratio (NLPR). The logistic regression and ROC curves analysis revealed that neutrophil and platelet counts, NLR, LPR, and NLPR were higher in COVID-19 than dengue. The multivariate predictive model showed that the neutrophils, platelets, and NLPR were independently associated with COVID-19 with a good fit predictive value (p = 0.1041). The neutrophil (AUC = 0.95, 95% CI = 0.84-0.91), platelet (AUC = 0.89, 95% CI = 0.85-0.93) counts, and NLR (AUC = 0.88, 95% CI = 0.84-0.91) were able to discriminate COVID-19 from dengue with high sensitivity and specificity values (above 80%). Finally, based on predicted probabilities on combining neutrophils and platelets with NLR or NLPR, the adjusted AUC was 0.97 (95% CI = 0.94-0.98) to differentiate COVID-19 from dengue during the acute phase of infection with outstanding accuracy. These findings might suggest that the neutrophil, platelet counts, and NLR or NLPR provide a quick and cost-effective way to distinguish between dengue and COVID-19 in the context of co-epidemics in low-income tropical regions.

First Evidence of Horizontal Transmission by Fecal Shedding of Dengue Virus 4 Among Aedes aegypti Larvae (Diptera: Culicidae) Under Laboratory Conditions.

The transmission pathways of dengue virus (DENV) among mosquitoes are a topic that has gained relevance in recent years because they could explain the maintenance of the virus in the wild independently of the human-mosquito horizontal transmission cycle. In this regard, Aedes aegypti larvae exposed to supernatants of C6/36 cells infected with DENV-4 were evaluated for virus excretion in feces and viability of infection in immature stages (larvae). The results demonstrate that larvae excrete DENV-4 in their feces with the potential to at least infect immature individuals of the same species. A horizontal transmission pathway of larvae-larvae DENV-4 under laboratory conditions is suggested.