Effects of oriented substrates on cell morphology, the cell cycle, and the cytoskeleton in Ros 17/2.8 cells

Absence of gravity or microgravity influences the cellular functions of bone forming osteoblasts. The underlying mechanism, however, of cellular sensing and responding to the gravity vector is poorly understood. This work quantified the impact of vector-directional gravity on the biological responses of Ros 17/2.8 cells grown on upward-, downward- or edge-on-oriented substrates. Cell morphology and nuclear translocation, cell proliferation and the cell cycle, and cytoskeletal reorganization were found to vary significantly in the three orientations. All of the responses were duration-dependent. These results provide a new insight into understanding how osteoblasts respond to static vector-directional gravity.

A model study on carbon cycle and phytoplankton dynamical processes in the Bohai Sea

The carbon cycle of lower trophic level in the Bohai Sea is studied with a three-dimension-al biological and physical coupled model. The influences of the processes (including horizontal advection,river nutrient load, active transport etc. ) on the phytoplankton biomass and its evolution are estimated.The Bohai Sea is a weak sink of the CO2 in the atmosphere. During the cycle, 13.7% of the gross pro-duction of the phytoplankton enter the higher trophic level and 76.8 % of it are consumed by the respira-tion itself. The nutrient reproduction comes mainly from the internal biogeochemical loop and the rem-ineralization is an important mechanism of the nutrient transfer from organic form to inorganic. Horizon-tal advection decreases the total biomass and the eutrophication in some sea areas. Change in the nutrientload of a river can only adjust the local system near its estuary. Controlling the input of the nutrient,which limits the alga growth, can be very useful in lessening the phytoplankton biomass.

Preparation, Surface Properties and Biological Activity of the Composite of Poly (lactide-co-glycolide) and Bioglass Nanoparticles Surface Grafted with Poly(L-lactide)

SiO2-CaO-P2O5 gel bioglass (BG) nanoparticles with the diameter of 40 nm were synthesized by sol-gel approach. The surface of BG nanoparticles was grafted through the ring-open polymerization of the L-lactide to yield poly (L-lactide) (PLLA) grafted gel particle (PLLA-g-BG). The PLLA-g-BG was further blended with poly(lactide-co-glycolide) (PLGA) to prepare the nanocomposites of PLLA-g-BG/PLGA with the various blend ratios of two phases. PLLA-g-BG accounted 10%, 20% and 40% in the composite, respectively. TGA, ESEM and EDX were used to analyze the graft ratio of PLLA-g-BG, the dispersion of nano-particles and the surface elements of the composites respectively. The rabbit osteoblasts were seeded and cultured on the thin films of composites in vitro. The cell adhesion, spreading and growth of osteoblasts were analyzed with FITC staining, NIH Image J software and MTT assay. The change of cell cycle was monitored by flow cytometry (FCM). The results demonstrated that the Surface modification of BG with PLLA could significantly improve the dispersing of the particles in the matrix of PLGA. The nanocomposite with 20% PLLA-g-BG exhibited superior surface properties, including roughness and plenty of silicon, calcium and phosper, to enhance the adhesion, spreading and proliferation of osteoblasts.

Involvement of p90 ribosomal S6 kinase in termination of cell cycle arrest during development of Artemia-encysted embryos

Artemia has evolved a unique developmental pattern of encysted embryos to cope with various environmental threats. Cell divisions totally cease during the preemergence developmental stage from gastrula to prenauplius. The molecular mechanism of this, however, remains unknown. Our study focuses on the involvement of p90 ribosomal S6 kinase (RSK), a family of serine/threonine kinase-mediating signal transduction downstream of mitogen-activated protein kinase cascades, in the termination of cell cycle arrest during the post-embryonic development of Artemia-encysted gastrula. With immunochemistry, morphology, and cell cycle analysis, the identified Artemia RSK was established to be specifically activated during the post-embryonic and early larval developmental stages when arrested cells of encysted embryos resumed mitoses. In vivo knockdown of RSK activity by RNA interference, kinase inhibition, and antibody neutralization consistently induced defective larvae with distinct gaps between the exoskeleton and internal tissues. In these abnormal individuals, mitoses were detected to be largely inhibited in the affected regions. These results display the requirement of RSK activity during Artemia development and suggest its role in termination of cell cycle (G(2)/M phase) arrest and promotion of mitogenesis. Our findings may, thus, provide insights into the regulation of cell division during Artemia post-embryonic development and reveal further aspects of RSK functions.

Annual cycle of carbon, nitrogen and phosphorus in the Bohai Sea: A model study

The annual cycle of nutrient-phytoplankton dynamics in Bohai Sea (BS) is simulated using a coupled physical-biological model in this study. By comparison, the modeled seasonal variations of nutrients and primary productivity agree with observations rather well. Although the annual cycles of chlorophyll a and primary production are both characterized by a double-peak configuration, a structural difference is still apparent: the phytoplankton biomass reaches the highest value in spring while summer is characterized by the most productivity in the BS, which can be ascribed to the combined impact of seawater temperature and zooplankton-grazing pressure on the growth of algae. Based on the validated simulations, the annual budgets of carbon, nitrogen and phosphorus are estimated, and are about 0.82 mt C surplus, 39 kt N deficit and 12kt P surplus, respectively, implying that the BS ecosystem is somewhat nitrogen limited. The contribution of two external nutrient sources, namely river discharges and resuspended sediments, to the growth of algae is also examined numerically, and it is found that the influence of river-borne nutrients mainly concentrates in estuaries, whereas the reduction of sediment-borne nutrients may significantly inhibit the onset of algae bloom in the whole BS. (c) 2007 Elsevier Ltd. All rights reserved.

Micro and nanostructured impedance sensors for biological and biomedical applications

This thesis work covered the fabrication and characterisation of impedance sensors for biological applications aiming in particular to the cytotoxicity monitoring of cultured cells exposed to different kind of chemical compounds and drugs and to the identification of different types of biological tissue (fat, muscles, nerves) using a sensor fabricated on the tip of a commercially available needle during peripheral nerve block procedures. Gold impedance electrodes have been successfully fabricated for impedance measurement on cells cultured on the electrode surface which was modified with the fabrication of gold nanopillars. These nanostructures have a height of 60nm or 100nm and they have highly ordered layout as they are fabricated through the e-beam technique. The fabrication of the threedimensional structures on the interdigitated electrodes was supposed to improve the sensitivity of the ECIS (electric cell-substrate impedance sensing) measurement while monitoring the cytotoxicity effects of two different drugs (Antrodia Camphorata extract and Nicotine) on three different cell lines (HeLa, A549 and BALBc 3T3) cultured on the impedance devices and change the morphology of the cells growing on the nanostructured electrodes. The fabrication of the nanostructures was achieved combining techniques like UV lithography, metal lift-off, evaporation and e-beam lithography techniques. The electrodes were packaged using a pressure sensitive, medical grade adhesive double-sided tape. The electrodes were then characterised with the aid of AFM and SEM imaging which confirmed the success of the fabrication processes showing the nanopillars fabricated with the right layout and dimensions figures. The introduction of nanopillars on the impedance electrodes, however, did not improve much the sensitivity of the assay with the exception of tests carried out with Nicotine. HeLa and A549 cells appeared to grow in a different way on the two surfaces, while no differences where noticed on the BALBc 3T3 cells. Impedance measurements obtained with the dead cells on the negative control electrodes or the test electrodes with the drugs can be compared to those done on the electrodes containing just media in the tested volume (as no cells are attached and cover the electrode surface). The impedance figures recorded using these electrodes were between 1.5kΩ and 2.5 kΩ, while the figures recorded on confluent cell layers range between 4kΩ and 5.5kΩ with peaks of almost 7 kΩ if there was more than one layer of cells growing on each other. There was then a very clear separation between the values of living cell compared to the dead ones which was almost 2.5 - 3kΩ. In this way it was very easy to determine whether the drugs affected the cells normal life cycle on not. However, little or no differences were noticed in the impedance analysis carried out on the two different kinds of electrodes using cultured cells. An increase of sensitivity was noticed only in a couple of experiments carried out on A549 cells growing on the nanostructured electrodes and exposed to different concentration of a solution containing Nicotine. More experiments to achieve a higher number of statistical evidences will be needed to prove these findings with an absolute confidence. The smart needle project aimed to reduce the limitations of the Electrical Nerve Stimulation (ENS) and the Ultra Sound Guided peripheral nerve block techniques giving the clinicians an additional tool for performing correctly the peripheral nerve block. Bioimpedance, as measured at the needle tip, provides additional information on needle tip location, thereby facilitating detection of intraneural needle placement. Using the needle as a precision instrument and guidance tool may provide additional information as to needle tip location and enhance safety in regional anaesthesia. In the time analysis, with the frequency fixed at 10kHz and the samples kept at 12°C, the approximate range for muscle bioimpedance was 203 – 616 Ω, the approximate bioimpedance range for fat was 5.02 - 17.8 kΩ and the approximate range for connective tissue was 790 Ω – 1.55 kΩ. While when the samples were heated at 37°C and measured again at 10kHz, the approximate bioimpedance range for muscle was 100-175Ω. The approximate bioimpedance range of fat was 627 Ω - 3.2 kΩ and the range for connective tissue was 221-540Ω. In the experiments done on the fresh slaughtered lamb carcass, replicating a scenario close to the real application, the impedance values recorded for fat were around 17 kΩ, for muscle and lean tissue around 1.3 kΩ while the nervous structures had an impedance value of 2.9 kΩ. With the data collected during this research, it was possible to conclude that measurements of bioimpedance at the needle tip location can give valuable information to the clinicians performing a peripheral nerve block procedure as the separation (in terms of impedance figures) was very marked between the different type of tissues. It is then feasible to use an impedance electrode fabricated on the needle tip to differentiate several tissues from the nerve tissue. Currently, several different methods are being studied to fabricate an impedance electrode on the surface of a commercially available needle used for the peripheral nerve block procedure.

Stochastic E2F activation and reconciliation of phenomenological cell-cycle models.

The transition of the mammalian cell from quiescence to proliferation is a highly variable process. Over the last four decades, two lines of apparently contradictory, phenomenological models have been proposed to account for such temporal variability. These include various forms of the transition probability (TP) model and the growth control (GC) model, which lack mechanistic details. The GC model was further proposed as an alternative explanation for the concept of the restriction point, which we recently demonstrated as being controlled by a bistable Rb-E2F switch. Here, through a combination of modeling and experiments, we show that these different lines of models in essence reflect different aspects of stochastic dynamics in cell cycle entry. In particular, we show that the variable activation of E2F can be described by stochastic activation of the bistable Rb-E2F switch, which in turn may account for the temporal variability in cell cycle entry. Moreover, we show that temporal dynamics of E2F activation can be recast into the frameworks of both the TP model and the GC model via parameter mapping. This mapping suggests that the two lines of phenomenological models can be reconciled through the stochastic dynamics of the Rb-E2F switch. It also suggests a potential utility of the TP or GC models in defining concise, quantitative phenotypes of cell physiology. This may have implications in classifying cell types or states.

Biological and ecological mechanisms supporting marine self-governance: The Seri Callo de Hacha Fishery in Mexico

My goal was to describe how biological and ecological factors give shape to fishing practices that can contribute to the successful self-governance of a small-scale fishing system in the Gulf of California, Mexico. The analysis was based on a comparison of the main ecological and biological indicators that fishers claim to use to govern their day-to-day decision making about fishing and data collected in situ. I found that certain indicators allow fishers to learn about differences and characteristics of the resource system and its units. Fishers use such information to guide their day-to-day fishing decisions. More importantly, these decisions appear unable to shape the reproductive viability of the fishery because no indicators were correlated to the reproductive cycle of the target species. As a result, the fishing practices constitute a number of mechanisms that might provide short-term buffering capacity against perturbations or stress factors that otherwise would threaten the overall sustainability and self-governance of the system. The particular biological circumstances that shape the harvesting practices might also act as a precursor of self-governance because they provide fishers with enough incentives to meet the costs of organizing the necessary rule structure that underlies a successful self-governance system.

Human-chimpanzee differences in a FZD8 enhancer alter cell-cycle dynamics in the developing neocortex.

The human neocortex differs from that of other great apes in several notable regards, including altered cell cycle, prolonged corticogenesis, and increased size [1-5]. Although these evolutionary changes most likely contributed to the origin of distinctively human cognitive faculties, their genetic basis remains almost entirely unknown. Highly conserved non-coding regions showing rapid sequence changes along the human lineage are candidate loci for the development and evolution of uniquely human traits. Several studies have identified human-accelerated enhancers [6-14], but none have linked an expression difference to a specific organismal trait. Here we report the discovery of a human-accelerated regulatory enhancer (HARE5) of FZD8, a receptor of the Wnt pathway implicated in brain development and size [15, 16]. Using transgenic mice, we demonstrate dramatic differences in human and chimpanzee HARE5 activity, with human HARE5 driving early and robust expression at the onset of corticogenesis. Similar to HARE5 activity, FZD8 is expressed in neural progenitors of the developing neocortex [17-19]. Chromosome conformation capture assays reveal that HARE5 physically and specifically contacts the core Fzd8 promoter in the mouse embryonic neocortex. To assess the phenotypic consequences of HARE5 activity, we generated transgenic mice in which Fzd8 expression is under control of orthologous enhancers (Pt-HARE5::Fzd8 and Hs-HARE5::Fzd8). In comparison to Pt-HARE5::Fzd8, Hs-HARE5::Fzd8 mice showed marked acceleration of neural progenitor cell cycle and increased brain size. Changes in HARE5 function unique to humans thus alter the cell-cycle dynamics of a critical population of stem cells during corticogenesis and may underlie some distinctive anatomical features of the human brain.