Human iPSC-Derived 3D Hepatic Organoids in a Miniaturized Dynamic Culture System

The process of identifying and approving a new drug is a time-consuming and expensive procedure. One of the biggest issues to overcome is the risk of hepatotoxicity, which is one of the main reasons for drug withdrawal from the market. While animal models are the gold standard in preclinical drug testing, the translation of results into therapeutic intervention is often ambiguous due to interspecies differences in hepatic metabolism. The discovery of human induced pluripotent stem cells (hiPSCs) and their derivatives has opened new possibilities for drug testing. We used mesenchymal stem cells and hepatocytes both derived from hiPSCs, together with endothelial cells, to miniaturize the process of generating hepatic organoids. These organoids were then cultivated in vitro using both static and dynamic cultures. Additionally, we tested spheroids solely composed by induced hepatocytes. By miniaturizing the system, we demonstrated the possibility of maintaining the organoids, but not the spheroids, in culture for up to 1 week. This timeframe may be sufficient to carry out a hypothetical pharmacological test or screening. In conclusion, we propose that the hiPSCderived liver organoid model could complement or, in the near future, replace the pharmacological and toxicological tests conducted on animals

Extremely Low-Frequency Electromagnetic Fields Affect Myogenic Processes in C2C12 Myoblasts: Role of Gap-Junction-Mediated Intercellular Communication

Extremely low-frequency electromagnetic fields (ELF-EMFs) can interact with biological systems. Although they are successfully used as therapeutic agents in physiatrics and rehabilitative practice, they might represent environmental pollutants and pose a risk to human health. Due to the lack of evidence of their mechanism of action, the effects of ELF-EMFs on differentiation processes in skeletal muscle were investigated. C2C12 myoblasts were exposed to ELF-EMFs generated by a solenoid. The effects of ELF-EMFs on cell viability and on growth and differentiation rates were studied using colorimetric and vital dye assays, cytomorphology, and molecular analysis of MyoD and myogenin expression, respectively. The establishment of functional gap junctions was investigated analyzing connexin 43 expression levels and measuring cell permeability, using microinjection/dye-transfer assays. The ELF-EMFs did not affect C2C12 myoblast viability or proliferation rate. Conversely, at ELF-EMF intensity in the mT range, the myogenic process was accelerated, through increased expression of MyoD, myogenin, and connexin 43. The increase in gap-junction function suggests promoting cell fusion and myotube differentiation. These data provide the first evidence of the mechanism through which ELF-EMFs may provide therapeutic benefits and can resolve, at least in part, some conditions of muscle dysfunction

Cell cycle-dependent phosphorylation of pRb-like protein in root meristem cells of Vicia faba

The retinoblastoma tumor suppressor protein (pRb) regulates cell cycle progression by controlling the G1-to-S phase transition. As evidenced in mammals, pRb has three functionally distinct binding domains and interacts with a number of proteins including the E2F family of transcription factors, proteins with a conserved LxCxE motif (D-type cyclin), and c-Abl tyrosine kinase. CDK-mediated phosphorylation of pRb inhibits its ability to bind target proteins, thus enabling further progression of the cell cycle. As yet, the roles of pRb and pRb-binding factors have not been well characterized in plants. By using antibody which specifically recognizes phosphorylated serines (S807/811) in the c-Abl tyrosine kinase binding C-domain of human pRb, we provide evidence for the cell cycle-dependent changes in pRb-like proteins in root meristems cells of Vicia faba. An increased phosphorylation of this protein has been found correlated with the G1-to-S phase transition

Seroprevalence and Risk Factors Associated to Mycobacterium bovis in Wild Artiodactyl Species from Southern Spain, 2006–2010

The control of bovine tuberculosis (bTB) is at a critical point in the last stage of eradication in livestock. Wildlife species recently have emerged infected with TB in Europe, particularly ungulates in the Iberian Peninsula. Epidemiological information regarding TB in wild ungulates including affected species, prevalence, associated risk factors and appropriate diagnostic methods need to be obtained in these countries

PsRBR1 encodes a pea retinoblastoma-related protein that is phosphorylated in axillary buds during dormancy-to-growth transition

In intact plants, cells in axillary buds are arrested at the G1 phase of the cell cycle during dormancy. In mammalian cells, the cell cycle is suppressed at the G1 phase by the activities of retinoblastoma tumor suppressor gene (RB) family proteins, depending on their phosphorylation state. Here, we report the isolation of a pea cDNA clone encoding an RB-related protein (PsRBR1, Accession No. AB012024) with a high degree of amino acid conservation in comparison with RB family proteins. PsRBR1 protein was detected as two polypeptides using an anti-PsRBR1 antibody in dormant axillary buds, whereas it was detected as three polypeptides, which were the same two polypeptides and another larger polypeptide 2 h after terminal decapitation. Both in vitro-synthesized PsPRB1 protein and lambda protein phosphatase-treated PsRBR1 protein corresponded to the smallest polypeptide detected by anti-PsRBR1 antibody, suggesting that the three polypeptides correspond to non-phosphorylated form of PsRBR1 protein, and lower- and higher-molecular mass forms of phosphorylated PsRBR1 protein. Furthermore, in vivo labeling with [32P]-inorganic phosphate indicated that PsRBR1 protein was more phosphorylated before mRNA accumulation of cell cycle regulatory genes such as PCNA. Together these findings suggest that dormancy-to-growth transition in pea axillary buds is regulated by molecular mechanisms of cell cycle control similar to those in mammals, and that the PsRBR1 protein has an important role in suppressing the cell cycle during dormancy in axillary buds

Histological and immunohistochemical features suggesting aetiological differences in lymph node and (muco)cutaneous feline tuberculosis lesions

Objectives To identify and describe histological and immunohistochemical criteria that may differentiate between skin and lymph node lesions associated with Mycobacterium (M.) bovis and M. microti in a diagnostic pathology setting.Materials and Methods<jats:p/>Archived skin and lymph node biopsies of tuberculous lesions were stained with haematoxylin and eosin, Ziehl‐Neelsen and Masson's Trichrome. Immunohistochemistry was performed to detect the expression of calprotectin, CD3 and Pax5. Samples were scored for histological parameters (i.e. granulomas with central necrosis versus small granulomas without central necrosis, percentage necrosis and/or multinucleated giant cells), number of acid‐fast bacilli (bacterial index) and lesion percentage of fibrosis and positive immunohistochemical staining.Results Twenty‐two samples were examined (M. bovis n=11, M. microti n=11). When controlling for age, gender and tissue, feline M. bovis‐associated lesions more often featured large multi‐layered granulomas with central necrosis. Conversely, this presentation was infrequent in feline M. microti‐associated lesions, where small granulomas without central necrosis predominated. The presence of an outer fibrous capsule was variable in both groups, as was the bacterial index. There were no differences in intralesional expression of immunohistochemical markers.Clinical Significance Differences in the histological appearance of skin and lymph node lesions may help to infer feline infection with either M. bovis or M. microti at an earlier stage when investigating these cases, informing clinicians of the potential zoonotic risk. Importantly, cases of tuberculosis can present with numerous acid‐fast bacilli. This implies that a high bacterial index does not infer infection with non‐zoonotic non‐tuberculous mycobacteria