Stimulating Neoblast-Like Cell Proliferation in Juvenile Fasciola hepatica Supports Growth and Progression towards the Adult Phenotype In Vitro

Fascioliasis (or fasciolosis) is a socioeconomically important parasitic disease caused by liver flukes of the genus Fasciola. Flukicide resistance has exposed the need for new drugs and/or a vaccine for liver fluke control. A rapidly improving 'molecular toolbox' for liver fluke encompasses quality genomic/transcriptomic datasets and an RNA interference platform that facilitates functional genomics approaches to drug/vaccine target validation. The exploitation of these resources is undermined by the absence of effective culture/maintenance systems that would support in vitro studies on juvenile fluke development/biology. Here we report markedly improved in vitro maintenance methods for Fasciola hepatica that achieved 65% survival of juvenile fluke after 6 months in standard cell culture medium supplemented with 50% chicken serum. We discovered that this long-term maintenance was dependent upon fluke growth, which was supported by increased proliferation of cells resembling the "neoblast" stem cells described in other flatworms. Growth led to dramatic morphological changes in juveniles, including the development of the digestive tract, reproductive organs and the tegument, towards more adult-like forms. The inhibition of DNA synthesis prevented neoblast-like cell proliferation and inhibited growth/development. Supporting our assertion that we have triggered the development of juveniles towards adult-like fluke, mass spectrometric analyses showed that growing fluke have an excretory/secretory protein profile that is distinct from that of newly-excysted juveniles and more closely resembles that of ex vivo immature and adult fluke. Further, in vitro maintained fluke displayed a transition in their movement from the probing behaviour associated with migrating stage worms to a slower wave-like motility seen in adults. Our ability to stimulate neoblast-like cell proliferation and growth in F. hepatica underpins the first simple platform for their long-term in vitro study, complementing the recent expansion in liver fluke resources and facilitating in vitro target validation studies of the developmental biology of liver fluke.

Epitaxially Grown Strained Pentacene Thin Film on Graphene Membrane

Organic-graphene system has emerged as a new platform for various applications such as flexible organic photovoltaics and organic light emitting diodes. Due to its important implication in charge transport, the study and reliable control of molecular packing structures at the graphene-molecule interface are of great importance for successful incorporation of graphene in related organic devices. Here, an ideal membrane of suspended graphene as a molecular assembly template is utilized to investigate thin-film epitaxial behaviors. Using transmission electron microscopy, two distinct molecular packing structures of pentacene on graphene are found. One observed packing structure is similar to the well-known bulk-phase, which adapts a face-on molecular orientation on graphene substrate. On the other hand, a rare polymorph of pentacene crystal, which shows significant strain along the c-axis, is identified. In particular, the strained film exhibits a specific molecular orientation and a strong azimuthal correlation with underlying graphene. Through ab initio electronic structure calculations, including van der Waals interactions, the unusual polymorph is attributed to the strong graphene-pentacene interaction. The observed strained organic film growth on graphene demonstrates the possibility to tune molecular packing via graphene-molecule interactions.

Autonomous growth of BALB/MK heratinocytes transfected with a retroviral vector carrying the human epidermal growth factor gene

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Determining day length and temperature regulation of flowering: a molecular and modelling approach

Thesis (Ph.D.)--University of Washington, 2016-08

Functions of the SLC36 transporter Pathetic in growth control of Drosophila sensory neurons

Thesis (Ph.D.)--University of Washington, 2016-08

Relative contribution of natural productivity and compound feed to tissue growth in blue shrimp (Litopenaeus stylirostris) reared in biofloc: Assessment by C and N stable isotope ratios and effect on key digestive enzymes

The aim of this study was to assess the relative contribution of natural productivity and compound food to the growth of the juvenile blue shrimp Litopenaeus stylirostris reared in a biofloc system. Two experiments were carried out based on the same protocol with three treatments: clear water with experimental diet (CW), biofloc with experimental diet (BF) and biofloc unfed (BU). Shrimp survival was significantly higher in biofloc rearing than in CW rearing. The contribution of the biofloc to shrimp diet was estimated through measurement of carbon and nitrogen stable isotope ratios in shrimp and food sources. Different isotopic compositions between feeds were obtained by feeding natural productivity with a mixture rich in fish meal and the shrimps with a pellet containing a high level of soy protein concentrate. Using a two source one-isotope mixing model, we found that the natural productivity of the biofloc system contributed to shrimp growth at a level of 39.8% and 36.9%, for C and N, respectively. The natural food consumed by the shrimps reared in the biofloc system resulted in higher gene expression (mRNA transcript abundance) and activities of two digestive enzymes in their digestive gland: α-amylase and trypsin. The growth of shrimp biomass reared in biofloc was, on average, 4.4 times that of those grown in clear water. Our results confirmed the best survival and promoted growth of shrimps using biofloc technology and highlighted the key role of the biofloc in the nutrition of rearing shrimps. Statement of relevance In this study, we have applied an original protocol to determine the respective contribution of natural productivity and artificial feeds on the alimentation of the juvenile blue shrimp L. stylirostris reared in biofloc system by using C and N natural stable isotope analysis. Moreover, we have compared, in shrimp digestive gland, the α-amylase and trypsin enzyme activities at biochemical and molecular levels for two different shrimp rearing systems, biofloc and clear water. In our knowledge, the use of molecular tool to study the influence of biofloc consumption on digest process of shrimp was never carried out. We think that our research is new and important to increase knowledge on biofloc topic.

Molecular and biological characterisation of orthobunyaviruses

Orthobunyaviruses are the largest genus within the Bunyaviridae family, with over 170 named viruses classified into 18 serogroups (Elliott and Blakqori, 2001; Plyusnin et al., 2012). Orthobunyaviruses are transmitted by arthropods and have a tripartite negative sense RNA genome, which encodes 4 structural proteins and 2 non-structural proteins. The non-structural protein NSs is the primary virulence factor of orthobunyaviruses and potent antagonist of the type I interferon (IFN) response. However, sequencing studies have identified pathogenic viruses that lack the NSs protein (Mohamed et al., 2009; Gauci et al., 2010). The work presented in this thesis describes the molecular and biological characterisation of divergent orthobunyaviruses. Data on plaque morphology, growth kinetics, protein profiles, sensitivity to IFN and activation of the type I IFN system are presented for viruses in the Anopheles A, Anopheles B, Capim, Gamboa, Guama, Minatitlan, Nyando, Tete and Turlock serogroups. These are complemented with complete genome sequencing and phylogenetic analysis. Low activation of IFN by Tete serogroup viruses, which naturally lack an NSs protein, was also further investigated by the development of a reverse genetics system for Batama virus (BMAV). Recombinant viruses with mutations in the virus nucleocapsid protein amino terminus showed higher activation of type I IFN in vitro and data suggests that low levels of IFN are due to lower activation rather than active antagonism. The anti-orthobunyavirus activity of IFN-stimulated genes IFI44, IFITMs and human and ovine BST2 were also studied, revealing that activity varies not only within the orthobunyavirus genus and virus serogroups but also within virus species. Furthermore, there was evidence of active antagonism of the type I IFN response and ISGs by non-NSs viruses. In summary, the results show that pathogenicity in man and antagonism of the type I IFN response in vitro cannot be predicted by the presence, or absence, of an NSs ORF. They also highlight problems in orthobunyavirus classification with discordance between classical antigen based data and phylogenetic analysis.

Seed layer technique for high quality epitaxial manganite films

We introduce an innovative approach to the simultaneous control of growth mode and magnetotransport properties of manganite thin films, based on an easy-to-implement film/substrate interface engineering. The deposition of a manganite seed layer and the optimization of the substrate temperature allows a persistent bi-dimensional epitaxy and robust ferromagnetic properties at the same time. Structural measurements confirm that in such interface-engineered films, the optimal properties are related to improved epitaxy. A new growth scenario is envisaged, compatible with a shift from heteroepitaxy towards pseudo-homoepitaxy. Relevant growth parameters such as formation energy, roughening temperature, strain profile and chemical states are derived.

Nitric oxide synthase and NAD(P)H oxidase modulate coronary endothelial cell growth

Reactive oxygen species (ROS) including nitric oxide (NO) and superoxide anion (O2-) are associated with cell migration, proliferation and many growth-related diseases. The objective of this study was to determine whether there was a reciprocal relationship between rat coronary microvascular endothelial cell (CMEC) growth and activity/expressions (mRNA and protein) of endothelial NO synthase (eNOS) and NAD(P)H oxidase enzymes. Proliferating namely, 50% confluent CMEC possessed approximately three-fold increased activity and expression of both enzymes compared to 100% confluent cells. Treatment of CMEC with an inhibitor of eNOS (L-NAME, 100M) increased cell proliferation as assessed via three independent methods i.e. cell counting, determination of total cellular protein levels and [3H]thymidine incorporation. Similarly, treatment of CMEC with pyrogallol (0.3-3 mM), a superoxide anion (O2-)- generator, also increased CMEC growth while spermine NONOate (SpNO), a NO donor, significantly reduced cell growth. Co-incubation of CMEC with a cell permeable superoxide dismutase mimetic (Mn-III-tetrakis-4-benzoic acid-porphyrin; MnTBAP) plus either pyrogallol or NO did not alter cell number and DNA synthesis thereby dismissing the involvement of peroxynitrite (OONO-) in CMEC proliferation. Specific inhibitors of NAD(P)H oxidase but not other ROS-generating enzymes including cyclooxygenase and xanthine oxidase, attenuated cell growth. Transfection of CMEC with antisense p22-phox cDNA, a membrane-bound component of NAD(P)H oxidase, resulted in substantial reduction in [3H]thymidine incorporation, total cellular protein levels and expression of p22-phox protein. These data demonstrate a cross-talk between CMEC growth and eNOS and NAD(P)H oxidase enzyme activity and expression, thus suggesting that the regulation of these enzymes may be critical in preventing the initiation and/or progression of coronary atherosclerosis.

Physiological and molecular analysis of the interaction between aluminium toxicity and drought stress in common bean (Phaseolus vulgaris)

Aluminium (Al) toxicity and drought are two major factors limiting common bean (Phaseolus vulgaris) production in the tropics. Short-term effects of Al toxicity and drought stress on root growth in acid, Al-toxic soil were studied, with special emphasis on Al-drought interaction in the root apex. Root elongation was inhibited by both Al and drought. Combined stresses resulted in a more severe inhibition of root elongation than either stress alone. This result was different from the alleviation of Al toxicity by osmotic stress (-0.60 MPa polyethylene glycol) in hydroponics. However, drought reduced the impact of Al on the root tip, as indicated by the reduction of Al-induced callose formation and MATE expression. Combined Al and drought stress enhanced up-regulation of ACCO expression and synthesis of zeatin riboside, reduced drought-enhanced abscisic acid (ABA) concentration, and expression of NCED involved in ABA biosynthesis and the transcription factors bZIP and MYB, thus affecting the regulation of ABA-dependent genes (SUS, PvLEA18, KS-DHN, and LTP) in root tips. The results provide circumstantial evidence that in soil, drought alleviates Al injury, but Al renders the root apex more drought-sensitive, particularly by impacting the gene regulatory network involved in ABA signal transduction and cross-talk with other phytohormones necessary for maintaining root growth under drought.

Scaffolds for controlled release of cartilage growth factors

International audience

Low-Molecular-Weight Fucoidan Induces Endothelial Cell Migration via the PI3K/AKT Pathway and Modulates the Transcription of Genes Involved in Angiogenesis

Low-molecular-weight fucoidan (LMWF) is a sulfated polysaccharide extracted from brown seaweed that presents antithrombotic and pro-angiogenic properties. However, its mechanism of action is not well-characterized. Here, we studied the effects of LMWF on cell signaling and whole genome expression in human umbilical vein endothelial cells and endothelial colony forming cells. We observed that LMWF and vascular endothelial growth factor had synergistic effects on cell signaling, and more interestingly that LMWF by itself, in the absence of other growth factors, was able to trigger the activation of the PI3K/AKT pathway, which plays a crucial role in angiogenesis and vasculogenesis. We also observed that the effects of LMWF on cell migration were PI3K/AKT-dependent and that LMWF modulated the expression of genes involved at different levels of the neovessel formation process, such as cell migration and cytoskeleton organization, cell mobilization and homing. This provides a better understanding of LMWF's mechanism of action and confirms that it could be an interesting therapeutic approach for vascular repair.

Coarse-graining and renormalisation group methods for the elucidation of the kinetics of complex nucleation and growth processes

We review our work on generalisations of the Becker-Doring model of cluster-formation as applied to nucleation theory, polymer growth kinetics, and the formation of upramolecular structures in colloidal chemistry. One valuable tool in analysing mathematical models of these systems has been the coarse-graining approximation which enables macroscopic models for observable quantities to be derived from microscopic ones. This permits assumptions about the detailed molecular mechanisms to be tested, and their influence on the large-scale kinetics of surfactant self-assembly to be elucidated. We also summarise our more recent results on Becker-Doring systems, notably demonstrating that cross-inhibition and autocatalysis can destabilise a uniform solution and lead to a competitive environment in which some species flourish at the expense of others, phenomena relevant in models of the origins of life.