Establishment and characterization of an in vitro 3D ovarian cancer model for drug screening assays

The acquired drug chemoresistance represents the main challenge of the ovarian cancer treatment. In addition, the absence of an adequate in vitro model able to reproduce the native tumor environment can contribute to the poor success rate of pre-clinical studies of new compounds. Three-dimensional (3D) culture models have been recently used for drug screening purposes due to their ability to reproduce the main characteristics of in vivo solid tumors. Here we describe the establishment and characterization of 3D ovarian cancer spheroids using different adenocarcinoma tumor cell lines (SKOV-3 and OVCAR-3 cells) in two different 3D scaffold-free methods: forced-floating in ultra-low attachment (ULA) plates and hanging drop (HD). Spheroids were evaluated in both 3D cultures in order to establish the best condition to perform the drug response analysis with Paclitaxel, a common drug used to treat ovarian cancer. SKOV-3 and OVCAR-3 spheroids with the desired characteristics (roundness close to 1.0 and diameter in the 200–500 μm range) were obtained using both methods after addition of the methylcellulose (MC) in the culture medium (0.25% and 0.5%, w/v). We also observed the presence of microvilli on the surface of the spheroids, higher presence of apoptotic cells and higher drug resistance, when compared with 2D cultures. The 3D cultures obtained seem to provide more reliable results in terms of drug response than those provided by 2D monolayer culture. The forced floating method was considered more suitable and straightforward to generate ovarian cancer spheroids for drug screening/cytotoxicity assays.     

Stable reference genes for expression studies in breast muscle of normal and white striping-affected chickens

The normalization with proper reference genes is a crucial step to obtain accurate mRNA expression levels in quantitative PCR (qPCR) studies. Therefore, in this study, 10 reference candidate genes were evaluated to determine their stability in normal pectoralis major muscle of broilers and those counterparts affected with White Striping (WS) myopathy at 42 days age. Four different tools were used for ranking the most stable genes: GeNorm, NormFinder, BestKeeper and Comparative Ct (ΔCt), and a general ranking was performed using the RankAggreg tool to select the best reference genes among all tools. From the 10 genes evaluated in the breast muscle of broilers, 8 were amplified. Most of the algorithms/tools indicated the same two genes, RPL30 and RPL5, as the most stable in the broilers breast muscle. In addition, there was agreement among the tools for the least stable genes: MRPS27, GAPDH and RPLP1 in the broilers breast muscle. Therefore, it is interesting to note that even with different tools for evaluating gene expression, there was consensus on the most and least stable genes. These results indicate that the Ribosomal protein L30 (RPL30) and Ribosomal protein L5 (RPL5) can be recommended for accurate normalization in qPCR studies with chicken pectoralis major muscle affected with White Striping and other myopathies.

     

The crustaceans burrow Sinusichnus sinuosus from the Oligocene-Miocene carbonate deposits of eastern Amazonia

Abstract

Sinusichnus Gibert 1996 Gibert, J. M. d. 1996. “A New Decapod Burrow System from the NW Mediterranean Pliocene.” Revista Española de Paleontología 11: 251–254. [Google Scholar] is recorded for the first time in the Oligocene-Miocene Pirabas Formation of Northern Brazil. In these Oligocene-Miocene carbonate deposits, Sinusichnus sinuosus is characterized by horizontal, highly regular sinusoidal burrow systems with T- and H-shaped branching points. The main difference between the S. sinuosus described herein and other occurrences worldwide is the anomalous diameter of the burrows (4 to 10?cm). These trace fossils occur in organic matter-rich, wackestone/packstone and laminated mudstones interbedded with boundstones deposited in an inner carbonate platform paleoenvironment. The exceptional size of the studied S. sinuosus could have been associated to the producer’s size, which may be attributed to construction by large crustaceans, similar to fossils found within the Pirabas Formation. Also, the sinusoidal morphology and retrusive spreiten could be a result of the fodinichnial/domichnial behavior.