Three-dimensional multicellular cell culture for anti-melanoma drug screening: focus on tumor microenvironment

AbstractThe development of new treatments for malignant melanoma, which has the worst prognosis among skin neoplasms, remains a challenge. The tumor microenvironment aids tumor cells to grow and resist to chemotherapeutic treatment. One way to mimic and study the tumor microenvironment is by using three-dimensional (3D) co-culture models (spheroids). In this study, a melanoma heterospheroid model composed of cancer cells, fibroblasts, and macrophages was produced by liquid-overlay technique using the agarose gel. The size, growth, viability, morphology, cancer stem-like cells population and inflammatory profile of tumor heterospheroids and monospheroids were analyzed to evaluate the influence of stromal cells on these parameters. Furthermore, dacarbazine cytotoxicity was evaluated using spheroids and two-dimensional (2D) melanoma model. After finishing the experiments, it was observed the M2 macrophages induced an anti-inflammatory microenvironment in heterospheroids; fibroblasts cells support the formation of the extracellular matrix, and a higher percentage of melanoma CD271 was observed in this model. Additionally, melanoma spheroids responded differently to the dacarbazine than the 2D melanoma culture as a result of their cellular heterogeneity and 3D structure. The 3D model was shown to be a fast and reliable tool for drug screening, which can mimic the in vivo tumor microenvironment regarding interactions and complexity.Graphic abstract      

Influence of Paleolithic range contraction,admixture and long‐distance dispersal on genetic gradients of modern humans in Asia

Cavalli‐Sforza and coauthors originally explored the genetic variation of modern humans throughout the world and observed an overall east‐west genetic gradient in Asia. However, the specific environmental and population genetics processes causing this gradient were not formally investigated and promoted discussion in recent studies. Here we studied the influence of diverse environmental and population genetics processes on Asian genetic gradients and identified which could have produced the observed gradient. To do so, we performed extensive spatially‐explicit computer simulations of genetic data under the following scenarios: (a) variable levels of admixture between Paleolithic and Neolithic populations, (b) migration through long‐distance dispersal (LDD), (c) Paleolithic range contraction induced by the last glacial maximum (LGM), and (d) Neolithic range expansions from one or two geographic origins (the Fertile Crescent and the Yangzi and Yellow River Basins). Next, we estimated genetic gradients from the simulated data and we found that they were sensible to the analysed processes, especially to the range contraction induced by LGM and to the number of Neolithic expansions. Some scenarios were compatible with the observed east‐west genetic gradient, such as the Paleolithic expansion with a range contraction induced by the LGM or two Neolithic range expansions from both the east and the west. In general, LDD increased the variance of genetic gradients among simulations. We interpreted the obtained gradients as a consequence of both allele surfing caused by range expansions and isolation by distance along the vast east‐west geographic axis of this continent.     

Effect of two flow regimes on the upstream movements of the Iberian barbel (Luciobarbus bocagei) in an experimental pool‐type fishway

Literature related to pool‐type fishways has seen a recent upsurge of interest in the placement of instream structures for improving fish passage. However, there is still no information on how different flow regimes created by boulder placement have an influence on upstream fish movements. The main goal of this study was to assess the performance of two different flow regimes, characterized by the relative depth of flow (d/h), where d is the water depth and h is the height of artificial bottom substrata, in assisting fish passage in an experimental full‐scale pool‐type fishway. Two series of experiments consisting of 20 replicates each and representing distinct flow regimes created by boulder placement in the flume bottom – d/h > 4 (regime 1) and 1.3 < d/h < 4 (regime 2) – were carried out to analyse the proportion and timing of successful upstream movements of a potamodromous cyprinid, the Iberian barbel (Luciobarbus bocagei). Although no significant differences (P > 0.05) in passage success were observed between regimes (55% and 60%, respectively), fish transit time was significantly lower (P < 0.05) in regime 2 (mean ± SD: 2.6 ± 1.6 min.) than in regime 1 (7.1 ± 5.8 min.). The results of these experiments show that lower relative depths can be more beneficial to fish passage because they reduce the transit time for successful negotiation, thus providing a useful indication on how to improve fish passage through pool‐type fishways.     

Association of the C242T polymorphism in the NADPH oxidase p22 phox gene with carotid atherosclerosis in Slovenian patients with type 2 diabetes

Oxidative stress plays an important role in the pathogenesis of diabetes and its complications. Genetic variations of enzymes producing reactive oxygen species could change their activity, thus contributing to the susceptibility to oxidative stress. The aim of this study was to examine the role of the NADPH oxidase C242T polymorphism in the development of carotid atherosclerosis in patients with type 2 diabetes. 286 diabetic patients and 150 healthy controls were enrolled in the study. Carotid atherosclerosis was quantified ultrasonographically as carotid intima-media thickness, plaque score (0–6) and plaque type (1–5). Diabetic patients were divided into low and high risk groups based on ultrasound phenotypes of carotid atherosclerosis. Genotypes were determined by real-time PCR. Levels of 8-hydroxy-2-deoxyguanosine (8-OHdG) were measured by enzyme-linked immunosorbent assay (ELISA). Diabetic patients demonstrated a statistically significant difference compared to healthy controls in the following parameters: age, BMI, waist circumference, smoking prevalence, glucose, triglyceride and 8-OHdG serum levels. Control subjects had significantly higher levels of HDL, LDL and total cholesterol than diabetics (p?<?0.001). The NADPH C242T polymorphism was not related with clinical characteristics, lipid parameters and 8-OHdG serum levels. We found no significant difference in the NADPH genotype distribution between diabetics and controls (p?=?0.19) nor between low and high risk subgroups of diabetics (mean CIMT: p?=?0.67; plaque score: p?=?0.49, plaque type: p?=?0.56). In the present study the NADPH C242T polymorphism was not associated with the degree of oxidative stress and carotid atherosclerosis. Further studies will show if it can be used as a genetic marker for carotid atherosclerosis in diabetic patients.