A cost-effective tool to standardize the scratch assay for cell migration.

BACKGROUND: The scratch assay is commonly used in cell biology to evaluate cell migration; however, it is not a standardized method; it produces highly variable gap dimensions. We design a printable device, comprising a single wounding tool and a guide, and compared the gap produced by our device and the traditional method. The deviceis printable in a standard 3D printer. Cells were seeded on a 24-well plate. After reaching full confluency, a gap was created using the traditional method (scratch assay with a pipette tip), a pipette tip and the guide of the device, or the single wounding tool and the guide. The gaps were observed for up to 48 h under a light microscope and analyzed. RESULTS: The results show that the traditional method produces irregular and not straight gaps, and had the worst cell migration rates compared to the other groups. The wounding tool produced scrape signs at the well surface. CONCLUSION: The guide and pipette tip delivered the best results for the scratch assay. SIGNIFICANCE: The use of the guide and the pipette tip for the scratch assay allows allows to perform reproducible cell migration experiments.

The Long Non-Coding RNA MALAT1 Modulates NR4A1 Expression through a Downstream Regulatory Element in Specific Cancer Cell Types.

Long non-coding RNAs (lncRNAs) have been shown to modulate gene expression and are involved in the initiation and progression of various cancer types. Despite the wealth of studies describing transcriptome changes upon lncRNA knockdown, there is limited information describing lncRNA-mediated effects on regulatory elements (REs) modulating gene expression. In this study, we investigated how the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) lncRNA regulates primary target genes using time-resolved MALAT1 knockdown followed by parallel RNA-seq and ATAC-seq assays. The results revealed that MALAT1 primarily regulates specific protein-coding genes and a substantial decrease in the accessibility downstream of the NR4A1 gene that was associated with a decreased NR4A1 expression. Moreover, the presence of an NR4A1-downstream RE was demonstrated by CRISPR-i assays to define a functional MALAT1/NR4A1 axis. By analyzing TCGA data, we identified a positive correlation between NR4A1 expression and NR4A1-downstream RE accessibility in breast cancer but not in pancreatic cancer. Accordingly, this regulatory mechanism was experimentally validated in breast cancer cells (MCF7) but not in pancreatic duct epithelial carcinoma (PANC1) cells. Therefore, our results demonstrated that MALAT1 is involved in a molecular mechanism that fine-tunes NR4A1 expression by modulating the accessibility of a downstream RE in a cell type-specific manner.

Investigating cooperative effects in small cobalt and cobalt-nickel alloy clusters with attached ethanol.

Small cationic cobalt, and cobalt-nickel alloy clusters with ethanol attached are generated in a pulsed molecular beam experiment using a laser ablation source. While the metal center is successively varied with respect to size and composition, a full-size study of these transition metal clusters is possible. The clusters are investigated via IR photodissociation spectroscopy in the region of OH- and CH-stretching vibrations. The results are compared with theoretical data obtained from DFT calculations. Both frequency shifts and structural changes according to cluster size and composition are identified and discussed in detail, also with respect to cooperative effects. Trimeric metal clusters with an uneven number of nickel atoms show evidence for C-O cleavage of the ethanol molecule. This result is elucidated by further calculations concerning the reactivity, charge and energetic distributions.

Effects of Low-Level Laser Therapy and Purified Natural Latex (Hevea brasiliensis) Protein on Injured Sciatic Nerve in Rodents: Morpho-Functional Analysis.

The present study analyzed the effects of low-level laser therapy (LLLT) and the purified natural latex protein (Hevea brasiliensis, F1 protein) on the morpho-function of sciatic nerve crush injuries in rats. One-hundred and eight male Wistar rats were randomly allocated to six groups (n = 18): 1. Control; 2. Exposed (nerve exposed); 3. Injury (injured nerve without treatment); 4. LLLT (injured nerve irradiated with LLLT (15 J/cm2, 780 nm)); 5. F1 (injured nerve treated with F1 protein (0.1%)); and 6. LLLT + F1 (injured nerve treated with LLLT and F1). On the 1st, 7th, 14th, and 56th days after injury, a functional sensory analysis of mechanical allodynia and mechanical hyperalgesia and a motor analysis of grip strength and gait were performed. After 3, 15, and 57 days, the animals were euthanized for morphometric/ultrastructural analyses. The treatments applied revealed improvements in morphometric/ultrastructural parameters compared to the injured group. Sensory analyses suggested that the improvements observed were associated with time progression and not influenced by the treatments. Motor analyses revealed significant improvements in grip strength from the 7th day in the LLLT group and in gait from the 56th day in all treated groups. We concluded that even though the morphological analyses showed improvements with the treatments, they did not influence sensory recovery, and LLLT improved motor recovery.

Biological properties of stem cells from the apical papilla exposed to lipopolysaccharides: An in vitro study.

OBJECTIVE: The aim of this study was to analyze the effect of lipopolysaccharides (LPS) on the biological properties of stem cells from the apical papilla (SCAPs), such as viability, adhesion to dentin, odontoblast-like differentiation, mineralization, and release of immunomodulatory cytokines. DESIGN: SCAPs were isolated from immature teeth of three donors (10 to 15 years old) and cultured in mineralizing media with or without 1 µg/mL lipopolysaccharide (LPS). Cells were seeded and cultured under standardized conditions; viability was assessed by MTT assay on days 1, 3, 5, and 7; adhesion to dentin was analyzed using an environmental scanning electron microscope after 2 days; the expression of odontogenic and mineralization genes (DSPP, DMP-1, OCN, Col1A1) was evaluated through qPCR after 14 days, mineralization was evaluated with alizarin red staining after 21 days; and the release of immunomodulatory cytokines (IL-6 and IL-10) was measured by ELISA after 1 and 7 days. The Kruskal-Wallis test was performed to detect the effect of LPS on SCAPs, followed by the Dunn-Sidak test. RESULTS: LPS presence in the culture media affected SCAPs viability on day 5 and increased IL-6 secretion by day 7, however, SCAPs retained the adhesion to dentin and mineralization capacities, as well as the differentiation capacity into a mineralizing phenotype. CONCLUSION: In conclusion, within the limitations of this in vitro study, and under the inflammatory microenvironment simulated in this study, stem cells from the apical papilla were found with retained adhesion capacity to dentin, differentiation into a mineralizing phenotype, mineralization, and release of IL-10.

Comprehensive Development of a Cellulose Acetate and Soy Protein-Based Scaffold for Nerve Regeneration.

BACKGROUND: The elaboration of biocompatible nerve guide conduits (NGCs) has been studied in recent years as a treatment for total nerve rupture lesions (axonotmesis). Different natural polymers have been used in these studies, including cellulose associated with soy protein. The purpose of this report was to describe manufacturing NGCs suitable for nerve regeneration using the method of dip coating and evaporation of solvent with cellulose acetate (CA) functionalized with soy protein acid hydrolysate (SPAH). METHODS: The manufacturing method and bacterial control precautions for the CA/SPAH NGCs were described. The structure of the NGCs was analyzed under a scanning electron microscope (SEM); porosity was analyzed with a degassing method using a porosimeter. Schwann cell (SCL 4.1/F7) biocompatibility of cell-seeded nerve guide conduits was evaluated with the MTT assay. RESULTS: The method employed allowed an easy elaboration and customization of NGCs, free of bacteria, with pores in the internal surface, and the uniform wall thickness allowed manipulation, which showed flexibility; additionally, the sample was suturable. The NGCs showed initial biocompatibility with Schwann cells, revealing cells adhered to the NGC structure after 5 days. CONCLUSIONS: The fabricated CA/SPAH NGCs showed adequate features to be used for peripheral nerve regeneration studies. Future reports are necessary to discuss the ideal concentration of CA and SPAH and the mechanical and physicochemical properties of this biomaterial.

Biological Properties of Dental Pulp Stem Cells Isolated from Inflamed and Healthy Pulp and Cultured in an Inflammatory Microenvironment.

INTRODUCTION: The aim of this study was to assess whether the biological characteristics of dental pulp stem cells (DPSCs), such as viability, adhesion to dentin, mineralization, and release of immunomodulatory cytokines, are affected by the inflammatory status of the donor tissue and/or the sustained inflammatory environment. METHODS: DPSCs were isolated from pulps from 3 caries-free teeth (healthy or hDPSCs), and from 3 teeth with irreversible pulpitis or deep caries (unhealthy DPSCs or uDPSCs). The cells were cultured in odontogenic and osteogenic media with or without lipopolysaccharides. Viability was analyzed by MTT assay at days 1, 3, 5, and 7; adhesion to dentin was evaluated through an environmental scanning electron microscope after 48 hours and through MTT assay; mineralization was analyzed with alizarin red staining after 21 days; and the release of proinflammatory (interleukin 6) and immunosuppressive cytokines (interleukin 10) was measured with the enzyme-linked immunosorbent assay after 24 hours and 7 days. RESULTS: The inflammatory status of the pulp significantly reduced the viability and mineralization capacity of the DPSCs, although it did not affect the adhesion capacity to dentin or the secretion of the proinflammatory interleukin. The inflammatory microenvironment (lipopolysaccharide) only had a significant impact on the secretion of interleukin 6, which was augmented after 7 days. CONCLUSIONS: The inflammatory status of the dental pulp should be taken into account when the use of DPSCs is intended either for research and/or for application in reparative or regenerative therapies.

Exploring Schwann Cell Behavior on Electrospun Polyhydroxybutyrate Scaffolds with Varied Pore Sizes and Fiber Thicknesses: Implications for Neural Tissue Engineering.

The placement of a polymeric electrospun scaffold is among the most promising strategies to improve nerve regeneration after critical neurotmesis. It is of great interest to investigate the effect of these structures on Schwann cells (SCs), as these cells lead nerve regeneration and functional recovery. The aim of this study was to assess SC viability and morphology when cultured on polyhydroxybutyrate (PHB) electrospun scaffolds with varied microfiber thicknesses and pore sizes. Six electrospun scaffolds were obtained using different PHB solutions and electrospinning parameters. All the scaffolds were morphologically characterized in terms of fiber thickness, pore size, and overall appearance by analyzing their SEM images. SCs seeded onto the scaffolds were analyzed in terms of viability and morphology throughout the culture period through MTT assay and SEM imaging. The SCs were cultured on three scaffolds with homogeneous smooth fibers (fiber thicknesses: 2.4 µm, 3.1 µm, and 4.3 µm; pore sizes: 16.7 µm, 22.4 µm, and 27.8 µm). SC infiltration and adhesion resulted in the formation of a three-dimensional network composed of intertwined fibers and cells. The SCs attached to the scaffolds maintained their characteristic shape and size throughout the culture period. Bigger pores and thicker fibers resulted in higher SC viability.