RNA-Mediated Gene Silencing of Nicotinamide N-Methyltransferase Is Associated with Decreased Tumorigenicity in Human Oral Carcinoma Cells

Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer. Despite progress in the treatment of OSCC, overall survival has not improved substantially in the last three decades. Therefore, identification of reliable biomarkers becomes essential to develop effective anti-cancer therapy. In this study, we focused on the enzyme Nicotinamide N-methyltransferase (NNMT), which plays a fundamental role in the biotransformation of many xenobiotics. Although several tumors have been associated with abnormal NNMT expression, its role in cancer cell metabolism remains largely unknown. In this report, 7 human oral cancer cell lines were examined for NNMT expression by Real-Time PCR, Western blot and HPLC-based catalytic assay. Subsequently, we evaluated the in vitro effect of shRNA-mediated silencing of NNMT on cell proliferation. In vivo tumorigenicity of oral cancer cells with stable knockdown of NNMT was assayed by using xenograft models. High expression levels of NNMT were found in PE/CA PJ-15 cells, in keeping with the results of Western blot and catalytic activity assay. PE/CA PJ-15 cell line was stably transfected with shRNA plasmids against NNMT and analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and soft agar Assays. Transfected and control cells were injected into athymic mice in order to evaluate the effect of NNMT silencing on tumor growth. NNMT downregulation resulted in decreased cell proliferation and colony formation ability on soft agar. In athymic mice, NNMT silencing induced a marked reduction in tumour volume. Our results show that the downregulation of NNMT expression in human oral carcinoma cells significantly inhibits cell growth in vitro and tumorigenicity in vivo. All these experimental data seem to suggest that NNMT plays a critical role in the proliferation and tumorigenic capacity of oral cancer cells, and its inhibition could represent a potential molecular approach to the treatment of oral carcinoma.     

Identification of a Novel Zinc Metalloprotease through a Global Analysis of Clostridium difficile Extracellular Proteins

Clostridium difficile is a major cause of infectious diarrhea worldwide. Although the cell surface proteins are recognized to be important in clostridial pathogenesis, biological functions of only a few are known. Also, apart from the toxins, proteins exported by C. difficile into the extracellular milieu have been poorly studied. In order to identify novel extracellular factors of C. difficile, we analyzed bacterial culture supernatants prepared from clinical isolates, 630 and R20291, using liquid chromatography-tandem mass spectrometry. The majority of the proteins identified were non-canonical extracellular proteins. These could be largely classified into proteins associated to the cell wall (including CWPs and extracellular hydrolases), transporters and flagellar proteins. Seven unknown hypothetical proteins were also identified. One of these proteins, CD630_28300, shared sequence similarity with the anthrax lethal factor, a known zinc metallopeptidase. We demonstrated that CD630_28300 (named Zmp1) binds zinc and is able to cleave fibronectin and fibrinogen in vitro in a zinc-dependent manner. Using site-directed mutagenesis, we identified residues important in zinc binding and enzymatic activity. Furthermore, we demonstrated that Zmp1 destabilizes the fibronectin network produced by human fibroblasts. Thus, by analyzing the exoproteome of C. difficile, we identified a novel extracellular metalloprotease that may be important in key steps of clostridial pathogenesis.     

H/ACA Small RNA Dysfunctions in Disease Reveal Key Roles for Noncoding RNA Modifications in Hematopoietic Stem Cell Differentiation

1. Download : Download full-size image

     

Development of large engineered cartilage constructs from a small population of cells

Confronted with articular cartilage's limited capacity for self‐repair, joint resurfacing techniques offer an attractive treatment for damaged or diseased tissue. Although tissue engineered cartilage constructs can be created, a substantial number of cells are required to generate sufficient quantities of tissue for the repair of large defects. As routine cell expansion methods tend to elicit negative effects on chondrocyte function, we have developed an approach to generate phenotypically stable, large‐sized engineered constructs (≥3 cm²) directly from a small amount of donor tissue or cells (as little as 20,000 cells to generate a 3 cm² tissue construct). Using rabbit donor tissue, the bioreactor‐cultivated constructs were hyaline‐like in appearance and possessed a biochemical composition similar to native articular cartilage. Longer bioreactor cultivation times resulted in increased matrix deposition and improved mechanical properties determined over a 4 week period. Additionally, as the anatomy of the joint will need to be taken in account to effectively resurface large affected areas, we have also explored the possibility of generating constructs matched to the shape and surface geometry of a defect site through the use of rapid‐prototyped defect tissue culture molds. Similar hyaline‐like tissue constructs were developed that also possessed a high degree of shape correlation to the original defect mold. Future studies will be aimed at determining the effectiveness of this approach to the repair of cartilage defects in an animal model and the creation of large‐sized osteochondral constructs. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Oxidative Stress EPR Measurement in Human Liver by Radical-probe Technique. Correlation with Etiology,Histology and Cell Proliferation

The role of reactive oxygen species (ROS) in liver disease is controversial. This mostly reflects the difficulties to quantify ROS in vivo, particularly in humans. We aimed to measure the presence of ROS in diseased human liver and identify possible relations between ROS levels and etiology, histology and hepatocyte proliferation. Liver biopsy specimens from 102 individuals: 18 healthy controls and 84 patients (42 HCV chronic hepatitis (CHC), 19 HBV chronic hepatitis (CHB), 7 PBC, 4 PSC, 4 HCV relapsing hepatitis after liver transplantation, 3 autoimmune hepatitis, 3 hepatocellular carcinoma, 2 alcoholic hepatitis) underwent analysis by radical-probe electron paramagnetic resonance (EPR). ROS in patients (median=5 &#50 10 &#109 6 mmol/mg) were higher than in controls (median=3 &#50 10 &#109 11 mmol/mg) ( p <0.001). Progressively increasing levels of ROS were recorded passing from control values to CHB (median=4 &#50 10 &#109 7 mmol/mg), CHC (median=3 &#50 10 &#109 6 mmol/mg) and PBC (median=2 &#50 10 &#109 5 mmol/mg), the differences being significant ( p <0.001). ROS in CHC positively correlated with histological disease activity ( r =0.92; p <0.001). No correlation was found between ROS and hepatocyte proliferation rate, presence/degree of steatosis, serum ferritin levels and aminotransferases. ROS overproduction in liver appears to be a common thread linking different pathologic conditions and seems to be influenced by diseases' etiologies.     

Discovery and synthesis of novel benzofurazan derivatives as inhibitors of influenza A virus

The identification of a novel hit compound inhibitor of the protein–protein interaction between the influenza RNA-polymerase PA and PB1 subunits has been accomplished by means of high-throughput screening. A small family of structurally related molecules has been synthesized and biologically evaluated with most of the compounds showing micromolar potency of inhibition against viral replication.     

New genetic maps for globe artichoke and wild cardoon and their alignment with an SSR-based consensus map

An F1 mapping population was bred by crossing an accession of wild cardoon with a single Argentinian globe artichoke plant of the variety Estrella del Sur FCA with a view to generating new Cynara cardunculus linkage maps. Genotyping was conducting using a set of 553 SRAP, SSR, AFLP and SNP markers. The 1,465.5 cM map based on the segregation of alleles present in the wild cardoon parent comprised 214 loci distributed across 16 linkage groups (LGs), while the 910.1 cM globe artichoke-based map featured 141 loci falling into 12 LGs covering the total length. Three of the morphological traits (head spininess, leaf spininess and head color) for which the parents contrasted were inherited monogenically, and the genes conditioning them were mapped. A set of 48 co-dominant loci was used to align the LGs with those derived from a reference SSR-based consensus map of the species.     

Quality by Design: Scale-Up of Freeze-Drying Cycles in Pharmaceutical Industry

This paper shows the application of mathematical modeling to scale-up a cycle developed with lab-scale equipment on two different production units. The above method is based on a simplified model of the process parameterized with experimentally determined heat and mass transfer coefficients. In this study, the overall heat transfer coefficient between product and shelf was determined by using the gravimetric procedure, while the dried product resistance to vapor flow was determined through the pressure rise test technique. Once model parameters were determined, the freeze-drying cycle of a parenteral product was developed via dynamic design space for a lab-scale unit. Then, mathematical modeling was used to scale-up the above cycle in the production equipment. In this way, appropriate values were determined for processing conditions, which allow the replication, in the industrial unit, of the product dynamics observed in the small scale freeze-dryer. This study also showed how inter-vial variability, as well as model parameter uncertainty, can be taken into account during scale-up calculations.     

To trim or not to trim: Progression and control of DSB end resection

DNA double-strand breaks (DSBs) are the most cytotoxic form of DNA damage, since they can lead to genome instability and chromosome rearrangements, which are hallmarks of cancer cells. To face this kind of lesion, eukaryotic cells developed two alternative repair pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). Repair pathway choice is influenced by the cell cycle phase and depends upon the 5′-3′ nucleolytic processing of the break ends, since the generation of ssDNA tails strongly stimulates HR and inhibits NHEJ. A large amount of work has elucidated the key components of the DSBs repair machinery and how this crucial process is finely regulated. The emerging view suggests that besides endo/exonucleases and helicases activities required for end resection, molecular barrier factors are specifically loaded in the proximity of the break, where they physically or functionally limit DNA degradation, preventing excessive accumulation of ssDNA, which could be threatening for cell survival.     

Shape Similarity,Better than Semantic Membership,Accounts for the Structure of Visual Object Representations in a Population of Monkey Inferotemporal Neurons

The anterior inferotemporal cortex (IT) is the highest stage along the hierarchy of visual areas that, in primates, processes visual objects. Although several lines of evidence suggest that IT primarily represents visual shape information, some recent studies have argued that neuronal ensembles in IT code the semantic membership of visual objects (i.e., represent conceptual classes such as animate and inanimate objects). In this study, we investigated to what extent semantic, rather than purely visual information, is represented in IT by performing a multivariate analysis of IT responses to a set of visual objects. By relying on a variety of machine-learning approaches (including a cutting-edge clustering algorithm that has been recently developed in the domain of statistical physics), we found that, in most instances, IT representation of visual objects is accounted for by their similarity at the level of shape or, more surprisingly, low-level visual properties. Only in a few cases we observed IT representations of semantic classes that were not explainable by the visual similarity of their members. Overall, these findings reassert the primary function of IT as a conveyor of explicit visual shape information, and reveal that low-level visual properties are represented in IT to a greater extent than previously appreciated. In addition, our work demonstrates how combining a variety of state-of-the-art multivariate approaches, and carefully estimating the contribution of shape similarity to the representation of object categories, can substantially advance our understanding of neuronal coding of visual objects in cortex.