Biomedical subjects

Cristian Ionescu-Zanetti

Publications and source records attributed to Cristian Ionescu-Zanetti.

8 recordsLinked to original sources

PubMed2005

Mammalian electrophysiology on a microfluidic platform.

The recent development of automated patch clamp technology has increased the throughput of electrophysiology but at the expense of visual access to the cells being studied. To improve visualization and the control of cell position, we have developed a simple alternative patch clamp technique based on microfluidic junctions between a main chamber and lateral recording capillaries, all fabricated by micromolding of polydimethylsiloxane (PDMS). PDMS substrates eliminate the need for vibration isolation and allow direct cell visualization and manipulation using standard microscopy. Microfluidic integration allows recording capillaries to be arrayed 20 microm apart, for a total chamber volume of <0.5 nl. The geometry of the recording capillaries permits high-quality, stable, whole-cell seals despite the hydrophobicity of the PDMS surface. Using this device, we are able to demonstrate reliable whole-cell recording of mammalian cells on an inexpensive microfluidic platform. Recordings of activation of the voltage-sensitive potassium channel Kv2.1 in mammalian cells compare well with traditional pipette recordings. The results make possible the integration of whole-cell electrophysiology with easily manufactured microfluidic lab-on-a-chip devices.

Animals↗

PubMed2005

Alkaline hemolysis fragility is dependent on cell shape: results from a morphology tracker.

BACKGROUND: The morphometric analysis of red blood cells (RBCs) is an important area of study and has been performed previously for fixed samples. We present a novel method for the analysis of morphologic changes of live erythrocytes as a function of time. We use this method to extract information on alkaline hemolysis fragility. Many other toxins lyse cells by membrane poration, which has been studied by averaging over cell populations. However, no quantitative data are available for changes in the morphology of individual cells during membrane poration-driven hemolysis or for the relation between cell shape and fragility. METHODS: Hydroxide, a porating agent, was generated in a microfluidic enclosure containing RBCs in suspension. Automatic cell recognition, tracking, and morphometric measurements were done by using a custom image analysis program. Cell area and circular shape factor (CSF) were measured over time for individual cells. Implementations were developed in MATLAB and on Kestrel, a parallel computer that affords higher speed that approaches real-time processing. RESULTS: The average CSF went through a first period of fast increase, corresponding to the conversion of discocytes to spherocytes under internal osmotic pressure, followed by another period of slow increase until the fast lysis event. For individual cells, the initial CSF was shown to be inversely correlated to cell lifetime (linear regression factor R=0.44), with discocytes surviving longer than spherocytes. The inflated cell surface area to volume ratio was also inversely correlated to lifetime (R=0.43) but not correlated to the CSF. Lifetime correlated best to the ratio of cell inflation volume (Vfinal-Vinitial) to surface area (R=0.65). CONCLUSIONS: RBCs inflate at a rate proportional to their surface area, in agreement with a constant flux model, and lyse after attaining a spherical morphology. Spherical RBCs display increased alkaline hemolysis fragility (shorter lifetimes), providing an explanation for the increased osmotic fragility of RBCs from patients who have spherocytosis.

Cell Shape↗

PubMed2005

Mechanism of thioflavin T binding to amyloid fibrils.

Thioflavin T is a benzothiazole dye that exhibits enhanced fluorescence upon binding to amyloid fibrils and is commonly used to diagnose amyloid fibrils, both ex vivo and in vitro. In aqueous solutions, thioflavin T was found to exist as micelles at concentrations commonly used to monitor fibrils by fluorescence assay ( approximately 10-20 microM). Specific conductivity changes were measured at varying concentration of thioflavin T and the critical micellar concentration was calculated to be 4.0+/-0.5 microM. Interestingly, changes in the fluorescence excitation and emission of thioflavin T were also dependent on the micelle formation. The thioflavin T micelles of 3 nm diameter were directly visualized using atomic force microscopy, and bound thioflavin T micelles were observed along the fibril length for representative fibrils. Increasing concentration of thioflavin T above the critical micellar concentration shows increased numbers of micelles bound along the length of the amyloid fibrils. Thioflavin T micelles were disrupted at low pH as observed by atomic force microscopy and fluorescence enhancement upon binding of thioflavin T to amyloid fibrils also reduced by several-fold upon decreasing the pH to below 3. This suggests that positive charge on the thioflavin T molecule has a role in its micelle formation that then bind the amyloid fibrils. Our data suggests that the micelles of thioflavin T bind amyloid fibrils leading to enhancement of fluorescence emission.

Amyloid↗

PubMed2004

On-chip cell lysis by local hydroxide generation.

We present a novel method for on-chip cell lysis based on local hydroxide electro-generation. Hydroxide ions porate the cell membrane, leading to cell lysis. After lysis occurs, hydrogen ions, also generated on chip, react with excess hydroxide, creating a neutral pH lysate and eliminating the need for a wash step. Three different cell types are shown to be effectively lysed by this method: red blood cells, HeLa (human tumor line) and Chinese Hamster Ovary (CHO) cell lines. The release of cytoplasmic molecules from HeLa and CHO cells is demonstrated by monitoring the escape of a membrane impermeant dye from the cytoplasm. In the vicinity of the cathode, the hydroxide concentration is predicted by finite element simulations and shown to fit the lysis rates at different distances from the generating cathode. For flow-through experiments, a second device integrating a mechanical filter with hydroxide generation is fabricated and tested. The purpose of the filter is to trap whole cells and only allow lysate to pass through. The flow rate dependence of hydroxide concentration at the lysis filter is modeled and lysis efficiency is experimentally determined to be proportional to the hydroxide concentration for flow rates from 15 to 30 microl min(-1).

Animals↗

PubMed2004

A single cell electroporation chip.

Increasing the cell membrane's permeability can be accomplished via single cell electroporation. Polar substances that cannot otherwise permeate the plasma membrane (such as dyes, drugs, DNA, proteins, peptides, and amino acids) can thus be introduced into the cell. We developed a polymeric chip that can selectively immobilize and locally electroporate single cells. This easy-to-use chip focuses the electric field, eliminating the need to manipulate electrodes or glass pipettes. Moreover, this device allows parallel single cell electroporation. We demonstrate the effectiveness of our device design by electroporating HeLa cells using low applied voltages (< 1 V). We found the average transmembrane potential required for electroporation of HeLa cells to be 0.51 +/- 0.13 V. Membrane permeation is assessed electrically by measuring characteristic 'jumps' in current that correspond to drops in cell resistance, and microscopically by recording either the escape of cytoplasmic dye Calcein AM or the entrance of Trypan blue stain.

Cell Membrane Permeability↗

PubMed2003

A general model for amyloid fibril assembly based on morphological studies using atomic force microscopy.

Based on atomic force microscopy analysis of the morphology of fibrillar species formed during fibrillation of alpha-synuclein, insulin, and the B1 domain of protein G, a previously described model for the assembly of amyloid fibrils of immunoglobulin light-chain variable domains is proposed as a general model for the assembly of protein fibrils. For all of the proteins studied, we observed two or three fibrillar species that vary in diameter. The smallest, protofilaments, have a uniform height, whereas the larger species, protofibrils and fibrils, have morphologies that are indicative of multiple protofilaments intertwining. In all cases, protofilaments intertwine to form protofibrils, and protofibrils intertwine to form fibrils. We propose that the hierarchical assembly model describes a general mechanism of assembly for all amyloid fibrils.

Amyloid↗

PubMed2003

A model for amyloid fibril formation in immunoglobulin light chains based on comparison of amyloidogenic and benign proteins and specific antibody binding.

In an attempt to understand the mechanism of amyloid fibril formation in light chain amyloidosis, the properties of amyloidogenic (SMA) and benign (LEN) immunoglobulin light chain variable domains (VL) were compared. The conformations of LEN and SMA were measured using secondary and tertiary structural probes over the pH range from 2 and 8. At all pH values, LEN was more stable than SMA. The CD spectra of LEN at pH 2 were comparable to those of SMA at pH 7.5, indicating that the low pH conformation of LEN closely resembles that of SMA at physiological pH. At low pH, a relatively unfolded intermediate conformation is populated for SMA and rapidly leads to amyloid fibrils. The lack of such an intermediate with LEN, is attributed to sequence differences and accounts for the lack of LEN fibrils in the absence of agitation. A kappa IV-specific monoclonal antibody that recognizes the N-terminal of SMA caused unraveling of the fibrils to the protofilaments and was observed to bind to one end of SMA protofilaments by atomic force microscopy. The antibody result indicates that each protofilament is asymmetric with different ends. A model for the formation of fibrils by SMA is proposed.

Amyloid↗

PubMed2002

Surface-catalyzed amyloid fibril formation.

Light chain (or AL) amyloidosis is characterized by the pathological deposition of insoluble fibrils of immunoglobulin light chain fragments in various tissues, walls of blood vessels, and basement membranes. In the present investigation, the in vitro assembly of a recombinant amyloidogenic light chain variable domain, SMA, on various surfaces was monitored using atomic force microscopy. SMA formed fibrils on native mica at pH 5.0, conditions under which predominantly amorphous aggregates form in solution. Fibril formation was accelerated significantly on surfaces compared with solution; for example, fibrils grew on surfaces at significantly faster rates and at much lower concentrations than in solution. No fibrils were observed on hydrophobic or positively charged surfaces or at pH >7.0. Two novel types of fibril growth were observed on the surface: bidirectional linear assembly of oligomeric units, and linear growth from preformed amorphous cores. In addition to catalyzing the rate of fibrillation, the mechanism of fibril formation on the surfaces was significantly different from in solution, but it may be more physiologically relevant because in vivo the deposits are associated with surfaces.

Amyloid beta-Peptides↗