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.

The Role of ncRNAs in the Immune Dysregulation of Preeclampsia.

The main complications causing practically 75% of all maternal deaths are severe bleeding, infections, and high blood pressure during pregnancy (preeclampsia (PE) and eclampsia). The usefulness of ncRNAs as clinical biomarkers has been explored in an extensive range of human diseases including pregnancy-related diseases such as PE. Immunological dysregulation show that the Th1/17:Th2/Treg ratio is "central and causal" to PE. However, there is evidence of the involvement of placenta-expressed miRNAs and lncRNAs in the immunological regulation of crucial processes of placenta development and function during pregnancy. Abnormal expression of these molecules is related to immune physiopathological processes that occur in PE. Therefore, this work aims to describe the importance of miRNAs and lncRNAs in immune dysregulation in PE. Interestingly, multiple ncRNAS are involved in the immune dysregulation of PE participating in type 1 immune response regulation, immune microenvironment regulation in placenta promoting inflammatory factors, trophoblast cell invasion in women with Early-Onset PE (EOPE), placental development, and angiogenesis, promotion of population of M1 and M2, proliferation, invasion, and migration of placental trophoblast cells, and promotion of invasion and autophagy through vias such as PI3K/AKT/mTOR, VEGF/VEGFR1, and TLR9/STAT3.

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.

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.

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.

Nanoparticle Detection on SEM Images Using a Neural Network and Semi-Synthetic Training Data.

Processing images represents a necessary step in the process of analysing the information gathered about nanoparticles after characteristic material samples have been scanned with electron microscopy, which often requires the use of image processing techniques or general purpose image manipulation software to carry out tasks such as nanoparticle detection and measurement. In recent years, the use of networks has been successfully implemented to detect and classify electron microscopy images as well as the objects within them. In this work, we present four detection models using two versions of the YOLO neural network architectures trained to detect cubical and quasi-spherical particles in SEM images; the training datasets are a mixture of real images and synthetic ones generated by a semi-arbitrary method. The resulting models were capable of detecting nanoparticles in images different than the ones used for training and identifying them in some cases as the close proximity between nanoparticles proved a challenge for the neural networks in most situations.

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.

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.

More Evidence of the Link of Interleukin-6 and Interleukin-10 with Critical COVID-19: A Report in Mexican Patients.

BACKGROUND: According to the World Health Organization, Mexico presents one of the highest mortality rates due to coronavirus disease 2019 (COVID-19). The "cytokine storm" phenomenon has been proposed as a pathological hallmark of severe COVID-19. OBJECTIVE: To determine the association of serum cytokine levels with COVID-19 severity. METHODS: We studied the cytokines IL-2, IL-4, IL-6, IL-10, TNF-α, and the IFN-γ serum levels through flow cytometry in 56 COVID-19 patients (24 critical and 32 non-critical) from Northwest Mexico. RESULTS: We observed a significant increase in the IL-6 and the IL-10 levels in the sera of critical patients. These cytokines were also associated with mechanical ventilation necessity and death, IL-6 showing AUC values above 0.7 for both variables; and correlated with Na+, creatinine, and platelet levels. On the other hand, no association was found between IL-2, IL-4, TNF-α, and IFN-γ with tested variables. CONCLUSION: Our results corroborate previous observations regarding IL-6 and IL-10 involvement in the severity of COVID-19.

Estructura poblacional de la tortuga blanca, Dermatemys mawii Gray (Testudines, Dermatemydidae), en la Laguna El Perú, Petén, Guatemala / Population structure of the white turtle, Central American River Turtle Dermatemys mawii Gray (Testudines, Dermatemydidae) in Laguna El Peru, Peten, Guatemala

La tortuga blanca (Dermatemys mawii Gray) está considerada en peligro de extinción por UICN, y está en el apéndice II de CITES. Hasta la fecha pocos estudios se han realizado sobre la ecología de esta especie, especialmente sobre la estructura poblacional, siendo importante su estudio pues es la única especie sobreviviente de la familia Dermatemydidae. El presente trabajo analiza la estructura poblacional (razón de sexos, estructura de edades) y la abundancia relativa de dicha especie en la laguna El Perú, Petén, Guatemala. Se analizó la estructura mediante la determinación del sexo y clases de edades, y se calculó la abundancia relativa tomando en cuenta el número de individuos capturados por unidad de esfuerzo (metros de trasmallos por horas que estuvo instalado el trasmallo). El trabajo fue realizado de junio 2012 a julio 2013 utilizando el método de captura-marca-recaptura y trampeando por cuatro noches consecutivas cada mes, obteniendo un esfuerzo total de captura de 124,800 hrs/m/ trasmallo. Se capturaron, marcaron y liberaron 121 individuos, de los cuales 70 fueron machos y 51 fueron hembras. La razón de sexos se obtuvo mediante el cociente del total de capturas entre machos y hembras, obteniendo una razón de sexos de 1.37 machos por 1 hembra. Se encontró que cerca del 62% de los individuos eran sub-adultos seguido de un número alto de capturas de adultos (38%) y sin ningún reporte de juveniles. Con los resultados obtenidos se infiere que la laguna El Perú posee una población de tortugas blancas importante para la conservación, además de ser un sitio importante para la anidación, refugio y alimentación de esta especie. Por lo tanto es necesario llevar a cabo estudios a lo largo de varios ciclos anuales completos para poder plantear un diseño de manejo y conservación adecuado que tome en cuenta las fluctuaciones poblacionales

The white turtle (Dermatemys mawii Gray) is considered an endangered species by the IUCN and is listed in the Appendix II of CITES. To this date, few studies have been conducted on the ecology of this species, especially on the population structure, which is important because it is the only surviving species of the family Dermatemydidae. This work analyzes the population structure (sex ratio, age structure) and the relative abundance of this species in the lagoon El Perú, Petén, Guatemala. The structure was analyzed by determining the sex and age classes, and the relative abundance was calculated taking into account the number of individuals caught per unit effort (meters of trammel net per the amount of hours the net trammel was installed). The work was performed from June 2012 to July 2013 using the method of capture-markrecapture and tricking for four consecutive nights each month, obtaining a total of 124,800 hrs/m/trammel net. There were 121 individuals captured, markedand released, of whom 70 were males and 51 were females. The sex ratio was obtained using the quotient of the total captures between males and females, resulting in a sex ratio of 1.37 males to 1 female. We found that about 62% of individuals were sub-adult, followed by a large number of captured adults (38%) and no reports of juveniles. With the obtained results, we conclude that the lagoon Peru has a population of green turtles important for conservation, in addition to being an important nesting and feeding refuge site for this species. Therefore, it is necessary to conduct studies over several complete annual cycles to propose an appropriate management and conservation design that considers the population fluctuation