Colony,hanging drop,and methylcellulose three dimensional hypoxic growth optimization of renal cell carcinoma cell lines

Renal cell carcinoma (RCC) is the most lethal of the common urologic malignancies, comprising 3% of all human neoplasms, and the incidence of kidney cancer is rising annually. We need new approaches to target tumor cells that are resistant to current therapies and that give rise to recurrence and treatment failure. In this study, we focused on low oxygen tension and three-dimensional (3D) cell culture incorporation to develop a new RCC growth model. We used the hanging drop and colony formation methods, which are common in 3D culture, as well as a unique methylcellulose (MC) method. For the experiments, we used human primary RCC cell lines, metastatic RCC cell lines, human kidney cancer stem cells, and human healthy epithelial cells. In the hanging drop assay, we verified the potential of various cell lines to create solid aggregates in hypoxic and normoxic conditions. With the semi-soft agar method, we also determined the ability of various cell lines to create colonies under different oxygen conditions. Different cell behavior observed in the MC method versus the hanging drop and colony formation assays suggests that these three assays may be useful to test various cell properties. However, MC seems to be a particularly valuable alternative for 3D cell culture, as its higher efficiency of aggregate formation and serum independency are of interest in different areas of cancer biology.     

Identification of Differentially Expressed Genes in Breast Muscle and Skin Fat of Postnatal Pekin Duck

Lean-type Pekin duck is a commercial breed that has been obtained through long-term selection. Investigation of the differentially expressed genes in breast muscle and skin fat at different developmental stages will contribute to a comprehensive understanding of the potential mechanisms underlying the lean-type Pekin duck phenotype. In the present study, RNA-seq was performed on breast muscle and skin fat at 2-, 4- and 6-weeks of age. More than 89% of the annotated duck genes were covered by our RNA-seq dataset. Thousands of differentially expressed genes, including many important genes involved in the regulation of muscle development and fat deposition, were detected through comparison of the expression levels in the muscle and skin fat of the same time point, or the same tissue at different time points. KEGG pathway analysis showed that the differentially expressed genes clustered significantly in many muscle development and fat deposition related pathways such as MAPK signaling pathway, PPAR signaling pathway, Calcium signaling pathway, Fat digestion and absorption, and TGF-beta signaling pathway. The results presented here could provide a basis for further investigation of the mechanisms involved in muscle development and fat deposition in Pekin duck.     

Detection of double-stranded RNA molecules and virus-like particles in different Mucor species

The presence of double-stranded RNA elements was examined in 123 strains representing 18 Mucor species. These genetic elements were found to be present in 6 strains: 1 M. aligarensis, 1 M. hiemalis, 2 M. corticolus, 1 M. mucedo and 1 M. ramannianus. Electrophoretic separation of the nucleic acids revealed 4 different RNA patterns, with 1 to 5 discrete dsRNA bands. The molecular weights corresponding to these bands were 1.42–4.15 × 10⁶ D. Using electronmicroscopy, for the first time the presence of virus like particles in Mucor species has been revealed.     

Mycobacterium bovis identification by a molecular method from post-mortem inspected cattle obtained in abattoirs of Mato Grosso do Sul, Brazil

The presence of Mycobacterium bovis in bovine carcasses with lesions suggestive of tuberculosis was evaluated. Seventy-two carcass samples were selected during slaughter inspection procedures in abattoirs in the state of Mato Grosso do Sul, Brazil. Seventeen (23.6%) of samples showed colonies suggestive of mycobacteria that were confirmed to be acid-fast bacilli by Ziehl-Neelsen staining. Polymerase chain reaction (PCR) using primers specific for M. bovis identified M. bovis in 13 (76.5%) isolates. The PCR-restriction enzyme pattern analysis using gene encoding for the 65-kDa protein and two restriction enzymes identified the remaining four isolates that were represented by two M. tuberculosis complex and two nontuberculous mycobacteria. The results are indicative of infection of slaughter cattle by M. bovis and other mycobacteria in the state of Mato Grosso do Sul.     

Neuromedin U elicits cytokine release in murine Th2-type T cell clone D10.G4.1

Neuromedin U (NmU), originally isolated from porcine spinal cord and later from other species, is a novel peptide that potently contracts smooth muscle. NmU interacts with two G protein-coupled receptors designated as NmU-1R and NmU-2R. This study demonstrates a potential proinflammatory role for NmU. In a mouse Th2 cell line (D10.G4.1), a single class of high affinity saturable binding sites for (125)I-labeled NmU (K(D) 364 pM and B(max) 1114 fmol/mg protein) was identified, and mRNA encoding NmU-1R, but not NmU-2R, was present. Competition binding analysis revealed equipotent, high affinity binding of NmU isopeptides to membranes prepared from D10.G4.1 cells. Exposure of these cells to NmU isopeptides resulted in an increase in intracellular Ca(2+) concentration (EC(50) 4.8 nM for human NmU). In addition, NmU also significantly increased the synthesis and release of cytokines including IL-4, IL-5, IL-6, IL-10, and IL-13. Studies using pharmacological inhibitors indicated that maximal NmU-evoked cytokine release required functional phospholipase C, calcineurin, MEK, and PI3K pathways. These data suggest a role for NmU in inflammation by stimulating cytokine production by T cells.     

Isolation and characterization of a new fructophilic <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> FPL strain from honeydew

In the present study, a Lactobacillus plantarum FPL strain exhibiting fructophilic behavior has been isolated for the first time from honeydew. It is a probably syntrophic bacterium inhabiting the gastrointestinal tract of Coccus hesperidum L. and taking part in sugar metabolism. The promising growth characteristics and biochemical properties of Lb. plantarum FPL indicate that this may be a facultatively fructophilic species, whose properties are not associated with the loss of the alcohol/acetaldehyde dehydrogenase gene. The article attempts to classify the peculiar behavior of this strain by means of tests that are characteristic for FLAB as well as through a classic identification approach. In this study, we used a reference strain Lb. plantarum NRRL B-4496, which showed no fructophilic properties. With the FLAB group, the new strain shares the habit, such as a fructose-rich environment, the preference of this sugar for growth, and similar growth curves. However, it exceeds FLAB in terms of osmotolerance to high sugar content. The fructophilic Lb. plantarum FPL strain can proliferate and grow on a medium wherein the sugar concentration is 45 and 50% (w/v). Our findings indicate that honeydew can be a promising source of new fructophilic lactic acid bacteria.     

Studies on microplasmodia ofPhysarum polycephalum

Summary The new technique of fluorescent analog cytochemistry was used to investigate the cell surface morphology (RITC-WGA staining), the organization of the microfilament system (Rh-phalloidin staining) and the spatial distribution of mitochondria (Rh-123 staining) in the various growth stages of axenically cultured living and fixed microplasmodia ofPhysarum polycephalum. The differentiation degree of the cell surface is generally size- and age-dependent: the invagination system develops by degrees from small spherical stages (50–100 m) without invaginations to large vein-like or dumbbell-shaped specimens (300–1,000 (m long) with extensive invagination systems. The microfilaments are always organized in a cortical system along the entire cell surface and sometimes in a fibrillar system as well, extending throughout the cytoplasmic matrix. Results on living microplasmodia demonstrate that the cortical microfilament system is mainly involved in motive force generation for changes of cell surface morphology and protoplasmic streaming activity, whereas the fibrillar system rather serves a stabilizing and adhering function. Moreover, the functional differences of the two microfilament systems are indicated by the position of a large population of stationary mitochondria in close vicinity to the cell surface, thus pointing to a reasonable arrangement of the energy-supplying and energy-transforming system.