Creating tissue on chip constructs in microtitre plates for drug discovery.

We report upon a novel coplanar dielectrophoresis (DEP) based cell patterning system for generating transferrable hepatic cell constructs, resembling a liver-lobule, in culture. The use of paper reinforced gel substrates provided sufficient strength to enable these constructs to be transfered into 96-well plates for long term functional studies, including in the future, drug development studies. Experimental results showed that hepatic cells formed DEP field-induced structures corresponding to an array of lobule-mimetic patterns. Hepatic viability was observed over a period of 3 days by the use of a fluorescent cell staining technique, whilst the liver specific functionality of albumin secretion showed a significant enhancement due to the layer patterning of cell lines (HepG2/C3A), compared to 2D patterned cells and un-patterned control. This "build and transfer" concept could, in future, also be adapted for the layer-by-layer construction of organs-on-chip in microtitre formats.

Visualization of Surface Acoustic Waves in Thin Liquid Films.

We demonstrate that the propagation path of a surface acoustic wave (SAW), excited with an interdigitated transducer (IDT), can be visualized using a thin liquid film dispensed onto a lithium niobate (LiNbO3) substrate. The practical advantages of this visualization method are its rapid and simple implementation, with many potential applications including in characterising acoustic pumping within microfluidic channels. It also enables low-cost characterisation of IDT designs thereby allowing the determination of anisotropy and orientation of the piezoelectric substrate without the requirement for sophisticated and expensive equipment. Here, we show that the optical visibility of the sound path critically depends on the physical properties of the liquid film and identify heptane and methanol as most contrast rich solvents for visualization of SAW. We also provide a detailed theoretical description of this effect.

Assessment of biocompatibility of 3D printed photopolymers using zebrafish embryo toxicity assays.

3D printing has emerged as a rapid and cost-efficient manufacturing technique to enable the fabrication of bespoke, complex prototypes. If the technology is to have a significant impact in biomedical applications, such as drug discovery and molecular diagnostics, the devices produced must be biologically compatible to enable their use with established reference assays and protocols. In this work we demonstrate that we can adapt the Fish Embryo Test (FET) as a new method to quantify the toxicity of 3D printed microfluidic devices. We assessed the biocompatibility of four commercially available 3D printing polymers (VisiJetCrystal EX200, Watershed 11122XC, Fototec SLA 7150 Clear and ABSplus P-430), through the observation of key developmental markers in the developing zebrafish embryos. Results show all of the photopolymers to be highly toxic to the embryos, resulting in fatality, although we do demonstrate that post-printing treatment of Fototec 7150 makes it suitable for zebrafish culture within the FET.

Microfluidic resonant cavities enable acoustophoresis on a disposable superstrate.

We demonstrate surface acoustic wave (SAW) induced microparticle manipulation in a microstructured disposable glass-polymer composite superstrate, positioned on a piezoelectric substrate with a single, slanted SAW transducer. An excited SAW was coupled from the piezoelectric substrate into the superstrate, which acted as a transversal resonator structure. We show that the energy transmitted into the superstrate allowed acoustophoretic particle manipulation, while the wide frequency response of the SAW transducer enabled tuneable pressure distributions confined by the microchannel layout. The configuration provides a significant tolerance in positioning - making assembly easy.

From chip-in-a-lab to lab-on-a-chip: towards a single handheld electronic system for multiple application-specific lab-on-a-chip (ASLOC).

We present a portable, battery-operated and application-specific lab-on-a-chip (ASLOC) system that can be easily configured for a wide range of lab-on-a-chip applications. It is based on multiplexed electrical current detection that serves as the sensing system. We demonstrate different configurations to perform most detection schemes currently in use in LOC systems, including some of the most advanced such as nanowire-based biosensing, surface plasmon resonance sensing, electrochemical detection and real-time PCR. The complete system is controlled by a single chip and the collected information is stored in situ, with the option of transferring the data to an external display by using a USB interface. In addition to providing a framework for truly portable real-life developments of LOC systems, we envisage that this system will have a significant impact on education, especially since it can easily demonstrate the benefits of integrated microanalytical systems.

Integrating microfluidic generation, handling and analysis of biomimetic giant unilamellar vesicles.

The key roles played by phospholipids in many cellular processes, has led to the development of model systems, to explore both lipid-lipid and lipid-peptide interactions. Biomimetic giant unilamellar vesicles represent close facsimiles of in vivo cellular membranes, although currently their widespread use in research is hindered by difficulties involving their integration into high-throughput techniques, for exploring membrane biology intensively in situ. This paper presents an integrated microfluidic device for the production, manipulation and high-throughput analysis of giant unilamellar vesicles. Its utility is demonstrated by exploring the lipid interaction dynamics of the pore-forming antimicrobial peptide melittin, assessed through the release of fluorescent dyes from within biomimetic vesicles, with membrane compositions similar to mammalian plasma membranes.

Aerosol droplet optical trap loading using surface acoustic wave nebulization.

We demonstrate the use of surface acoustic wave nebulization (SAWN) to load optical traps. We show that the droplets sizes produced can be tuned by altering the RF frequency applied to the devices, which leads to more control over the sizes of trapped particles. Typically the size distribution of the liquid aerosols delivered using SAWN is smaller than via a standard commercial nebulization device. The ability to trap a range of liquids or small solid particles, not readily accessible using other ultrasonic devices, is also demonstrated both in optical tweezers and dual beam fiber traps.

Autoantibodies directed against ribosomal proteins in systemic lupus erythematosus and rheumatoid arthritis: a comparative study.

To assess the specificity of autoantibodies (aAbs) directed against the ribosomal P-proteins (RPPaAbs) in patients with systemic lupus erythematosus (SLE) and to investigate aAbs directed to other ribosomal proteins, 100 SLE, 100 rheumatoid arthritis (RA), 25 thyroiditis and 20 blood-donors were analyzed in a comparative study using an immunoblotting technique. Forty-eight percent of SLB sera contained aAbs directed against the ribosomal proteins of the 60 S subunit compared to 9% for RA, 5% for blood donors and 0% for thyroiditis. RPPaAbs were only found in SLE (25%) and aAbs directed to a 31 kDa and/or a 28 kDa protein of the 60 S subunit were found with a statistically higher frequency for SLE compared to RA (p < 0.0001). aAbs directed to proteins of the 40 S subunit were present in 63% of the SLE sera compared to 42% for RA, 4% for thyroiditis and 5% for blood donors. The number of positive sera was not statistically different between SLE and RA but a much more intense reactivity was observed for SLE sera. These data shows that the aAbs against the ribosomal proteins, especially the P-proteins along with the 28 and 31 kDa proteins of the 60 S subunit proteins, can be considered as useful biological markers for t he diagnosis of SLE inclinical practice.

Pivotal role of the P1 N-terminal domain in the assembly of the mammalian ribosomal stalk and in the proteosynthetic activity.

In the 60 S ribosomal subunit, the lateral stalk made of the P-proteins plays a major role in translation. It contains P0, an insoluble protein anchoring P1 and P2 to the ribosome. Here, rat recombinant P0 was overproduced in inclusion bodies and solubilized in complex with the other P-proteins. This method of solubilization appeared suitable to show protein complexes and revealed that P1, but not P2, interacted with P0. Furthermore, the use of truncated mutants of P1 and P2 indicated that residues 1-63 in P1 connected P0 to residues 1-65 in P2. Additional experiments resulted in the conclusion that P1 and P2 bound one another, either connected with P0 or free, as found in the cytoplasm. Accordingly, a model of association for the P-proteins in the stalk is proposed. Recombinant P0 in complex with phosphorylated P2 and either P1 or its (1-63) domain efficiently restored the proteosynthetic activity of 60 S subunits deprived of native P-proteins. Therefore, refolded P0 was functional and residues 1-63 only in P1 were essential. Furthermore, our results emphasize that the refolding principle used here is worth considering for solubilizing other insoluble proteins.

Evidence for a second nucleotide binding site in rat elongation factor eEF-2 specific for adenylic nucleotides.

The rat elongation factor eEF-2 catalyzes the translocation step of protein synthesis. Besides its well-characterized GTP/GDP binding properties, we have previously shown that ATP and ADP bind to eEF-2 [Sontag, B., Reboud, A. M., Divita, G., Di Pietro, A., Guillot, D., and Reboud, J. P. (1993) Biochemistry 32, 1976-1980]. However, whether the adenylic and guanylic nucleotide binding sites were different or not remained unclear. To further characterize these sites, eEF-2 was incubated in the presence of N-methylanthraniloyl (Mant) fluorescent derivatives of GTP, GDP, ATP, and ADP. This led to an increase in the probe fluorescence and to a partial quenching of eEF-2 tryptophans in each case. The Mant-derivatives and the unmodified corresponding nucleotides were shown to bind to eEF-2 with a similar affinity. Competition experiments between Mant-labeled and unmodified nucleotides suggested the presence of two different sites binding either guanylic or adenylic nucleotides. A Förster's transfer between tryptophan residues and the Mant-probe is obtained with both the adenylic and the guanylic Mant-nucleotides, and comparison of the transfer efficiencies confirmed the presence of a second binding site specific for adenylic nucleotides. A sequence alignment of EF-Gs with eEF-2s from different species suggests the presence of potential Walker A and B motifs in an insert of the G-domain of eEF-2s from higher eukaryotes. Our results raise the possibility that a site specific for adenylic nucleotides and located in this insert has appeared in the course of evolution although its physiological function is still unknown.

Autoantibodies directed against the ribosomal P proteins are not only directed against a common epitope of the P0, P1 and P2 proteins.

The autoantibodies (aAbs) directed against the ribosomal P proteins (RPP aAbs) are known to react mainly against epitopes localized within the common C-terminal sequence of the three acidic ribosomal P proteins, P0, P1 and P2. In order to investigate the opportunity to select short recombinant peptides of this common C-terminal sequence to detect the RPP-aAbs, the location of the epitopes recognized by ribosomal proteins (RP) aAb(+)sera of systemic lupus erythematosus patients (SLE) was investigated. Immunoblotting and ELISA techniques using extracted or recombinant, entire or cleaved RPP showed that 55% of the RP aAbs were directed against the three ribosomal P0, P1, and P2 proteins. The epitopes recognized by the RPP aAbs are located not only within the C-terminal sequence common to the three proteins but also within the N-terminal sequence of the P2 or P1 protein. The other RP aAbs sera (45%) did not react with all three proteins but with some of them, and showed the following pattern: P0(+)P1(+); P1(+); P2(+); P0(+)and P1(+). They recognized epitopes located in the region of the C-terminal sequence of the protein but not common to the three proteins. In addition two out of the six monoclonal Abs produced by immunization of mice using the P1 protein did not react with the peptide N-65 or N-71 of the P2 protein or with the C-terminal sequence of the three proteins. In conclusion, this study showed that the RPP aAb in SLE patients are not only directed against epitopes within the C-terminal sequence shared by the three acidic ribosomal P proteins. In view of these data it seems necessary to be cautious in using only a C-terminal peptide of ribosomal P proteins in tests performed to detect RPP aAb in human sera.

Interaction of elongation factor eEF-2 with ribosomal P proteins.

The eukaryotic P1 and P2 ribosomal proteins which constitute, with P0, a pentamer forming the lateral stalk of the 60 S ribosomal subunit, exhibit several differences from their prokaryotic equivalents L7 and L12; in particular, P1 does not have the same primary structure as P2 and both of them are phosphorylated, the significance of the latter remaining unclear. Rat liver P1 and P2 were overproduced in Escherichia coli cells and their interaction with elongation factor eEF-2 was studied. Both recombinant proteins were found to be required for the ribosome-dependent GTPase activity of eEF-2, with P2 in the phosphorylated form. The surface plasmon resonance technique revealed that, in vitro, both proteins interact specifically with eEF-2, with a higher affinity for P1 (Kd = 3.8 x 10-8 m) than for P2 (Kd = 2.2 x 10-6 m). Phosphorylation resulted in a moderate increase (two- to four-fold) in these affinities. The interaction of both P1 and P2 (phosphorylated or not) with eEF-2 resulted in a conformational change in the factor, revealed by an increase in the accessibility of Glu554 to proteinase Glu-C. This increase was observed in both the presence and absence of GTP and GDP, which themselves produced marked opposite effects on the conformation of eEF-2. Our results suggest that the two proteins P1 and P2 both interact with eEF-2 inducing a conformational transition of the factor, but have acquired some specific properties during evolution.

A specific role for the phosphorylation of mammalian acidic ribosomal protein P2.

The acidic ribosomal proteins P1-P2 from rat liver were overproduced for the first time by expression of their cDNA in Escherichia coli. They were tested for their ability to reactivate inactive P1-P2-deficient core particles derived from 60 S ribosomal subunits treated with dimethylmaleic anhydride, in poly(U)-directed poly(Phe) synthesis. The recombinant P1-P2 were unable to reactivate these core particles although they could bind to them. When recombinant P1-P2 had been phosphorylated first with casein kinase II, they were as efficient in the reactivation process as P1-P2 extracted with ethanol/KCl from the 60 S subunits. Reconstitution experiments were carried out using all possible combinations of the two recombinant proteins phosphorylated or not. Reactivation of the core particles required the presence of both P1 and P2 with the latter in its phosphorylated form. These experiments reveal a distinct role for P1 and P2 in protein synthesis. Phosphorylated P2 produced a partial quenching of the intrinsic fluorescence of eukaryotic elongation factor 2, which was not observed with the unphosphorylated protein. This result demonstrates the existence of an interaction between phosphorylated P2 and eukaryotic elongation factor 2. P2 also quenched part of the intrinsic fluorescence of P1, due to the interaction between the two proteins.

Photoaffinity labeling of elongation factor-2 with 8-azido derivatives of GTP and ATP.

Elongation factor 2 (eEF-2) can interact not only with guanylic nucleotides but also with adenylic ones, as was shown by intrinsic fluorescence quenching studies [Sontag, B., Reboud, A.M., Divita, G., Di Pietro, A., Guillot, D. & Reboud, J.P. (1993) Biochemistry 32, 1976-1980]. Here we studied sites of these interactions by using photoactivable 8-azido-[gamma-32P]GTP and 8-azido-[gamma-32P]ATP. Photoincorporation of the radioactive GTP derivative into eEF-2 was prevented by the previous addition of GTP and GDP. The addition of adenylic nucleotides (ATP, ADP) and some adenylic derivatives [NAD+, NADH,poly(A)] decreased the photoincorporation by only 40% at most. However, photoincorporation of the radioactive ATP derivative was prevented by the previous addition not only of adenylic compounds [ATP, ADP, NAD+, NADH, poly(A)] but also of GTP and GDP. Photoincorporation of radioactive nucleotide derivatives was not decreased by the addition of other nucleotidic compounds [UTP, poly(U), ITP, NADP+, NADPH]. ATP and GTP acted as non-competitive inhibitors of the photoincorporation of 8-azido-[gamma-32P]GTP and 8-azido-[gamma-32P]ATP, respectively. eEF-2 photolabeled with these radioactive nucleotide derivatives was submitted to trypsin digestion under different conditions and the labeled peptidic fragments identified after HPLC purification and gel electrophoresis by N-terminal sequencing. An octapeptide, Y264FDPANGK271, was the only peptide photolabeled with 8-azido-[gamma-32P]GTP whereas a N-terminal fragment of about 7 kDa was the only one photolabeled with 8-azido-[gamma-32P]ATP. The different results support the hypothesis that guanylic and adenylic nucleotides do not interact with the same site of eEF-2.

Properties of elongation factor-2 fragments obtained by partial proteolysis.

Rat liver elongation factor eEF-2 was treated with endoproteinase Glu-C. Two major fragments were obtained, which were identified by N-terminal sequencing and purified. The larger one (F61) contained 554 residues including the N-terminal end, and after a second cleavage released a N-terminal peptide (F7) of 62 residues. The smaller one (F34) contained the other 303 residues including the C terminal end. F61 and F34, either isolated or after combination, were unable to catalyze protein synthesis. However, we show by fluorimetry that F61 could still interact with GTP and GDP. This fragment was was able to participate into a ternary complex with ribosome and GDP, but not with ribosome and a GTP analogue. It was unable to protect the ribosome against ricin-inactivation and to be phosphorylated by the eEF-2-specific Ca(2+)-calmodulin-dependent kinase, though it contained Trp221 and Thr56 involved in these reactions. On the other hand, F34 could be ADP-ribosylated in the presence of NAD+ and diphtheria toxin, but this fragment was apparently unable to bind to ribosomes. These results and those obtained with other proteinases are discussed in the light of the data published recently which show the existence of five different domains in the three-dimensional structure of EF-G.

Mode of action of bolesatine, a cytotoxic glycoprotein from Boletus satanas Lenz. Mechanistic approaches.

Bolesatine is a potent cytotoxic glycoprotein purified from Boletus satanas Lenz, which has previously been shown to be an inhibitor of protein synthesis in several in vitro systems and in vivo. For a better understanding of its mechanism of action on protein synthesis at the ribosomal level, rat liver ribosomes were pretreated with bolesatine (1 to 10 micrograms) added to in vitro polyuridylic acid (poly(U)) translation systems before and after washing. The fact that ribosomes were still active confirmed that bolesatine cannot be included in the group of protein synthesis inhibitors of plant origin, known as ribosome-inactivating proteins (RIPs). The effect of bolesatine on the EF-2 elongation factor and post-ribosomal fraction was then studied in vitro. The results indicated that bolesatine does not have a direct effect on elongation factors, but hydrolyses the nucleoside triphosphates, GTP (80% to 90%, respectively for 1 to 10 micrograms) and ATP (10% to 40%, respectively for 1 to 10 micrograms), with consequent inhibition of protein synthesis. Thus, bolesatine should be classified as a nucleoside triphosphate phosphatase, rather than as a direct inhibitor of protein synthesis. The study of the effect of bolesatine on the EF-2 factor revealed that the mechanism whereby bolesatine affects protein synthesis probably involves GTP hydrolysis rather than EF-2 inhibition.

Interaction of phosphorylated elongation factor EF-2 with nucleotides and ribosomes.

The intrinsic fluorescence emission spectrum of elongation factor EF-2 due to the 7 Trp residues was not modified after complete phosphorylation of the factor by the specific Ca2+/Calmodulin-dependent kinase III. The effect of nucleotide binding on this fluorescence revealed differences between phosphorylated and unmodified EF-2. Low concentrations of GTP had a smaller quenching effect on the fluorescence of phosphorylated EF-2 than on the fluorescence of unmodified EF-2, whereas GDP had exactly the same quenching effect on the fluorescence of both samples. These results suggest that phosphorylation of EF-2 decreased its affinity for GTP but not for GDP. Ability of phosphorylated EF-2 to form a ternary complex with ribosomes in the presence of a non-hydrolysable GTP analog and its ability to protect ribosomes against ricin-inactivation were both decreased to the same extent. The lower affinity of phosphorylated EF-2 for GTP could be responsible for a weaker and/or incorrect interaction of the factor with the ribosome, in particular with the ricin-site of the 28-S rRNA assumed to be involved in translocation initiation.

Trp221 is involved in the protective effect of elongation factor eEF-2 on the ricin/alpha-sarcin site of the ribosome.

Elongation factor eEF-2 treated by N-bromosuccinimide under conditions which oxidize 2 Trp residues (Trp343 and Trp221) is inactivated in ribosome-dependent GTP hydrolysis and polyphenylalanine synthesis, and inactivation correlates with the specific oxidation of Trp221 (Guillot, D., Penin, F., Di Pietro, A., Sontag, B., Lavergne, J. P., and Reboud, J. P. (1993) J. Biol. Chem. 268, 20911-20916). It is shown here that this oxidation prevents neither GTP binding to eEF-2 nor the formation of the ribosome-eEF-2-GPP(NH)P complex, but that oxidized eEF-2 is no longer able to protect ribosomes against ricin inactivation. These observations suggest that Trp221 or an amino-acid sequence containing this residue interacts with the 28 S rRNA loop including the GAGA sequence, which is the target of ricin. Such a hypothesis is discussed in relation with data on RNA recognition motifs described in different proteins.