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A comparison of chronic multi-channel cortical implantation techniques: manual versus mechanical insertion.

High-density multi-channel intra-cortical electrode arrays allow researchers to record simultaneously from populations of neurons for the purpose of understanding neural coding and plasticity. These devices have tens to hundreds of electrodes spaced within a few square millimeters. During insertion, the high-density probes can compress the cortex several millimeters prior to breaking through the pia. Compression of cortical tissue has been demonstrated to result in traumatic brain injury (TBI) which may be a major contributor to low electrode yield and decreased recording longevity. Two insertion techniques for chronically implanting multi-wire electrode arrays in layer IV of primary auditory cortex were compared. A mechanical insertion device, capable of rapidly inserting the electrode array without visible compression of the brain, was constructed. The neural responses to broadband clicks and pure tones recorded from the arrays inserted with the mechanical device were compared to the results from a manual insertion method using a micromanipulator. Both techniques result in a similar number of active channels directly following surgery with a mean signal-to-noise ratio of approximately 4.5. Over 60% of the animals implanted with the mechanical insertion device had driven activity at week 6 whereas none of the animals with manually inserted arrays exhibited functional responses after 3 weeks. This report provides initial evidence that mechanical insertion devices, which prevent cortical compression, increase electrode recording longevity.

Acoustic Stimulation↗

Improved superfusion technique for rapid cooling or heating of cultured cells under patch-clamp conditions.

We have developed an improved technique for fast cooling and heating of solutions superfusing isolated cells under patch-clamp or calcium imaging conditions. The system meets the requirements for studying temperature dependency of all kinds of ion channels, in particular temperature-gated ion channels. It allows the application of temperature changes within a range of 5-60 degrees C at maximum rates of -40 degrees C/s to 60 degrees C/s. Barrels filled with different solutions are connected to a manifold consisting of seven silica capillaries (320 microm inner diameter, i.d.). A common outlet consists of a glass capillary through which the solutions are applied onto the cell surface. The upper part of this capillary is embedded in a temperature exchanger driven by a miniature Peltier device which preconditions the temperature of the passing solution. The lower part of the capillary carries an insulated copper wire, densely coiled over a length of 7 mm, and connected to a dc current source for resistive heating. The Peltier device and the heating element are electrically connected to the headstage probe which is fixed on to a micromanipulator for positioning of the manifold. The temperature of the flowing solution is measured by a miniature thermocouple inserted into the common outlet capillary near to its orifice which is placed at a distance of less than 100 microm from the surface of the examined cell. The temperature is either manually controlled by voltage commands or adjusted via the digital-to-analog converter of a conventional data acquisition interface. Examples are given of using the device in patch-clamp studies on heterologously expressed TRPV1, TRPM8, and on cultured rat sensory neurons.

Action Potentials↗

Quantifying the contribution of actin networks to the elastic strength of fibroblasts.

The structural models created to understand the cytoskeletal mechanics of cells in suspension are described here. Suspended cells can be deformed by well-defined surface stresses in an Optical Stretcher [Guck, J., Ananthakrishnan, R., Mahmood, H., Moon, T.J., Cunningham, C.C., Käs, J., 2001. The optical stretcher: a novel laser tool to micromanipulate cells. Biophys. J. 81(2), 767-784], a two-beam optical trap designed for the contact-free deformation of cells. Suspended cells have a well-defined cytoskeleton, displaying a radially symmetric actin cortical network underlying the cell membrane with no actin stress fibers, and microtubules and intermediate filaments in the interior. Based on experimental data using suspended fibroblasts, we create two structural models: a thick shell actin cortex model that describes cell deformation for a localized stress distribution on these cells and a three-layered model that considers the entire cytoskeleton when a broad stress distribution is applied. Applying the models to data, we obtain a (actin) cortical shear moduli G of approximately 220 Pa for normal fibroblasts and approximately 185 Pa for malignantly transformed fibroblasts. Additionally, modeling the cortex as a transiently crosslinked isotropic actin network, we show that actin and its crosslinkers must be co-localized into a tight shell to achieve these cortical strengths. The similar moduli values and cortical actin and crosslinker densities but different deformabilities of the normal and cancerous cells suggest that a cell's structural strength is not solely determined by cytoskeletal composition but equally importantly by (actin) cytoskeletal architecture via differing cortical thicknesses. We also find that although the interior structural elements (microtubules, nucleus) contribute to the deformed cell's exact shape via their loose coupling to the cortex, it is the outer actin cortical shell (and its thickness) that mainly determines the cell's structural response.

Actins↗

Accuracy and reproducibility of a subpixel extended phase correlation method to determine micron level displacements in the heart.

Future treatment of heart disease may involve local perturbations of mechanical function, such as intramyocardial injections of angiogenic growth factors or progenitor cells. This necessitates an accurate measurement technique to determine regional heart function. We have previously developed a method to determine regional heart function using a phase correlation algorithm. However, in determining regional function over a single heartbeat it is necessary to sum displacements between many images. We have therefore incorporated a subpixel algorithm that models the result of phase correlation as a sinc function in order to increase the accuracy of our technique. This method, which we have named high density mapping (HDM), determines the subpixel displacement of 64 x 64 pixel regions from images of the heart. To determine the accuracy and precision of the technique, a high contrast image of a heart was digitally shifted 1, 2 or 3 pixels. The original and shifted images were then downsampled four times resulting in 0.25, 0.50 or 0.75 pixel shifts between the original and shifted images. The average accuracy of HDM in the digitally shifted images was 0.06 pixels, with a precision of 0.08 pixels. Effectiveness of HDM in characterization of deformation was also assessed in digitally stretched images. Error in quantification of strain was found to be less than 3.5% of the calculated strain. In an additional set of experiments, in which accuracy was determined using physical motion instead of digital shifting and downsampling, a speckle pattern was displaced by known distances using a micromanipulator, such that the displacement between the captured images was 0.5 pixels. These data demonstrated an accuracy of 0.09 pixels and a precision of 0.02 pixels. Finally, as HDM is used to determine the regional stroke work index (RSW) in beating hearts, the repeatability of using this method to compute RSW was assessed. RSW, the integral of intraventricular pressure with respect to unitless regional area, where end diastolic area was normalized to unity, was assessed in consecutive beats from four different hearts. The average standard deviation of RSW was 0.098 mmHg. Uncertainty analysis determined the maximum error of RSW to be +/-0.41 mmHg, approximately two-thirds of the measured biologic variability. These data demonstrate the ability of HDM to accurately and reproducibly measure displacement and regional function in the beating heart.

Algorithms↗

Unveiling tumor heterogeneity by single cell RNA-sequencing: From basic considerations to clinical applications.

Tumor heterogeneity-encompassing diverse cellular phenotypes, genomic alterations, and microenvironmental contexts-is a principal barrier to effective cancer therapy. Single-cell RNA sequencing (scRNA-seq) has transformed our ability to resolve this complexity by capturing transcriptomes at single-cell resolution. Here, we review the technical foundations required for high-quality scRNA-seq studies. We then trace the evolution of scRNA-seq platforms from manual micromanipulation to high-throughput systems, and describe the computational pipelines that enable reliable data interpretation. The application of scRNA-seq is exemplarily shown in the context of lung cancer, where single-cell profiling has revealed (i) the clonal and sub-clonal architecture of tumors, (ii) extensive remodeling of the immune microenvironment, iii) key mechanisms underlying resistance to targeted agents and immune-checkpoint blockade, and (iv) the dynamics of neo-antigen-specific T-cell responses. Integrating machine-learning techniques-such as deep-learning classifiers and graph-based models-with single-cell transcriptomic data has markedly sped up biomarker discovery, produced more accurate risk-stratification scores, and enabled the generation of patient-specific therapeutic predictions. We surveyed the major trial registry ClinicalTrials.gov and identified ∼380 ongoing or completed studies that explicitly incorporate scRNA-seq as a correlative or pharmacodynamic endpoint. Overall, the analysis shows that scRNA-seq becomes an increasingly important component of modern trials, providing high-resolution cellular and molecular readouts that complement conventional imaging and bulk-omics endpoints. While key challenges remain, ranging from costs, scalability and need for rigorous validation before routine clinical deployment, ongoing technological advances continue to expand the potential of scRNA-seq as a cornerstone of precision medicine.

Humans↗

Development of embryos reconstructed by interspecies nuclear transfer of adult fibroblasts between buffalo (Bubalus bubalis) and cattle (Bos indicus).

The objective of this study was to explore the feasibility of employing adult fibroblasts as donor cells in interspecies nuclear transfer (NT) between buffaloes and cattle. Buffalo and bovine oocytes matured in vitro for 22 h were enucleated by micromanipulation using the Spindle View system. An ear fibroblast, pretreated with 0.1 microg/mL aphidicolin for 24 h, followed by culture for 2-9 days in Dulbecco's Modified Eagle's Media+0.5% fetal bovine serum, was introduced into the cytoplast by microinjection. Reconstructed oocytes were activated by exposure to 5 microM ionomycin for 5 min and 2 mM 6-dimethylaminopurine for 3 h. When buffalo adult fibroblasts were used as donor cells, there were no differences (P < 0.75) in the cleavage rate (66.2% versus 64.0%) between bovine and buffalo recipient oocytes, but more embryos derived from bovine cytoplasts developed to blastocysts than from buffalo cytoplasts (13.3% versus 3.0%, P < 0.05). When bovine adult fibroblasts were used as donor nuclei, both cleavage rate (45.3%) and blastocyst yield (4.5%) of NT embryos derived from buffalo cytoplasts were lower than those of NT embryos derived from bovine cytoplasts (65.5 and 11.9%, P < 0.05). The proportion of parthenogenetic buffalo (29.1%) or bovine (35.6%) oocytes developing to blastocysts was higher than those of NT embryos (P < 0.01). Interspecies NT embryos were derived from the donor cells and 55.0-61.9% of them possessed a normal diploid karyotype. In conclusion, embryos reconstructed by interspecies NT of adult fibroblasts between buffaloes and cattle developed to blastocysts, but bovine cytoplasts may direct embryonic development more effectively than buffalo cytoplasts, regardless of donor cell species.

Animals↗

Comparative aspects of somatic cell nuclear transfer with conventional and zona-free method in cattle, horse, pig and sheep.

Nuclear transfer (NT) is a complex procedure that requires considerable technical skills. Over the years attempts have been made to simplify the micromanipulations involved and to make the procedure more user-friendly. A significant step forwards has been the development of the zona-free NT methods. We have used zona-free NT with mechanical aspiration of the metaphase plate as a mean of enucleation, in a comparative approach with the conventional nuclear transfer zona-enclosed method in cattle, horse, sheep and pig. The absence of the zona considerably facilitates the enucleation step and significantly increases cell fusion success. On the other hand, the culture of zona-free NT embryos requires the embryos to be cultured individually or anyway separated from each other to avoid aggregation and also requires to prolong the in vitro culture up to the blastocyst stage before transfer. Blastocyst rate is equal or higher with zona-free method as compared to zona-enclosed method while survival after cryopreservation and development to term is comparable. In conclusion, our findings, together with published data, demonstrate that the zona-free system described in this paper can significantly increase the output of NT blastocysts over the conventional zona-enclosed system.

Animals↗

FK778: new cellular and molecular mechanisms of action.

PURPOSE: The new malononitrilamide FK778 is currently being evaluated as an immunosuppressant for organ transplantation. Its main mechanism is inhibition of a pivotal enzyme of pyrimidine biosynthesis. This report revealed new mechanisms of action on different cell types involved in acute and chronic allograft rejection. METHODS: Purified Brown-Norway rat aortic endothelial cell (EC) cultures were pretreated with several concentrations of FK778. Endothelial adhesion molecule expression (ICAM-1/VCAM-1) stimulated with TNF-alpha was quantified by immunofluorescence. Purified Lewis rat lymphocytes (LC) incubated with FK778 were stimulated via TCR/CD28 signals, and CD25 expression was quantified using FACS analysis. Uridine addition was used in all assays to reverse the pyrimidine synthesis blockade. Lymphocyte-EC interaction was assessed by micromanipulator-assisted single-cell adhesion assays. Finally, smooth muscle cell (SMC) proliferation and migration was analyzed. Uridine addition was used in all assays to reverse the pyrimidine synthesis blockade. RESULTS: TNF-alpha stimulation and TCR/CD28 co-stimulation significantly increased EC ICAM-1/VCAM-1-expression and LC CD25 surface expression, respectively. These effects were dose-dependently inhibited by FK778 and were not reversed by the addition of uridine. FK778 dose-dependently attenuated LC adhesion to allogeneic EC. The dose-dependent inhibition of SMC proliferation by FK778 was abolished by uridine addition, whereas the inhibitory effect on SMC migration was not affected by uridine supplementation. CONCLUSIONS: FK778 directly reduced endothelial adhesion molecule up-regulation, inhibited lymphocyte activation, and attenuated lymphocyte-endothelium interactions, critical early steps in graft rejection. These effects were separate from the blockade of pyrimidine synthesis. The antiproliferative potency of FK778 on SMC may be an important mechanism to inhibit the fibroproliferative lesions of chronic organ rejection.

Alkynes↗

Development of Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea) in bryozoan hosts (as examined by light microscopy) and quantitation of infective dose to rainbow trout (Oncorhynchus mykiss).

The myxozoan parasite Tetracapsuloides bryosalmonae is the causative agent of proliferative kidney disease (PKD), a highly damaging disease of cultured salmonid fish. Within this study, phylactolaemate bryozoans were collected from a river known to be endemic for PKD and subsequently cultured in the laboratory. Sequential developmental stages of T. bryosalmonae were studied by light microscopy within the living bryozoan colonies, allowing the identification of stages attached to host peritoneum, consistent with previous molecular evidence of cryptic stages. Infection resulted in the production of large numbers of spores, which were released from the bryozoans. Experimental exposure of rainbow trout (Oncorhynchus mykiss) to medium in which infected bryozoans were cultured resulted in clinical PKD. Rainbow trout were exposed to known numbers of T. bryosalmonae spores collected by micromanipulation, which had been released from mature spore sacs within colonies of the bryozoan Fredericella sultana. Exposure to one spore was sufficient to lead to development of PKD. These findings indicate that small numbers of bryozoans are capable of releasing sufficient spores to infect large numbers of fish, having implications for future control methods for PKD in salmonid farming.

Animals↗

A review of floc strength and breakage.

The main focus of the paper is to review current understanding of floc structure and strength. This has been done by reviewing current theoretical understanding of floc growth and breakage and an analysis of different techniques used for measuring floc strength. An overview has also been made of the general trends seen in floc strength analysis. The rate of floc formation is a balance between breakage and aggregation with flocs eventually reaching a steady-state size for a given shear rate. The steady-state floc size for a particular shear rate can, therefore, be a good indicator of floc strength. This has resulted in the development of a range of techniques to measure floc size at different applied shear levels using a combination of one or more of the following tools: light scattering and transmission; microscopy; photography; video and image analysis software. Floc strength may be simply quantified using the initial floc size for a given shear rate and the floc strength factor. More complex techniques have used theoretical modelling to determine whether flocs break by large-scale fragmentation or smaller-scale surface erosion effects, although this interpretation is open to debate. Impeller-based mixing, ultrasound and vibrating columns have all been used to provide a uniform, accurate and controllable dissipation of energy onto a floc suspension to determine floc strength. Other more recent techniques have used sensitive micromanipulators to measure the force required to break or compress individual flocs, although these techniques have been limited to the measurement of only a few hundred flocs. General trends emerge showing that smaller flocs tend to have greater strength than larger flocs, whilst the use of polymer seems to give increased strength to only some types of floc. Finally, a comparison of the strength of different types of floc (activated sludge flocs, organic matter flocs, sweep flocs and charge neutralised flocs) has been made highlighting differences in relative floc strength.

Bioreactors↗

Regulation of the paternal inheritance of centrosomes in starfish zygotes.

In most animals, fertilized eggs inherit one centrosome from a meiosis-II spindle of oocytes and another centrosome from the sperm. However, since first proposed by Boveri [Sitzungsber. Ges. Morph. Phys. Münch. 3 (1887) 151-164] at the turn of the last century, it has been believed that only the paternal (sperm) centrosome provides the division poles for mitosis in animal zygotes. This uniparental (paternal) inheritance of centrosomes is logically based on the premise that the maternal (egg) centrosome is lost before the onset of the first mitosis. For the processes of the selective loss of the maternal centrosome, three models have been proposed: One stresses the intrinsic factors within the centrosome itself; the other two emphasize external factors such as cytoplasmic conditions or the sperm centrosome. In the present study, we have examined the validity of one of the models in which the sperm centrosome overwhelms the maternal centrosomes. Because centrosomes cast off into both the first and the second polar bodies (PB) are known to retain the capacity for reproduction and cell-division pole formation, we observed the behavior of those PB centrosomes with reproductive capacity and the sperm centrosome in the same zygotic cytoplasm. We prepared two kinds of fertilized eggs that contain reproductive maternal centrosomes, (1) by micromanipulative transplantation of the PB centrosomes into fertilized eggs, and (2) by suppression of the PB extrusions of fertilized eggs with cytochalasin B. In both types of eggs, the PB centrosomes could double and form cell-division poles, indicating that they are not suppressed by the sperm centrosome, which in turn indicates that selective loss of the maternal centrosome is due to intrinsic factors within the centrosomes themselves.

Animals↗

Determination of first cleavage plane: the relationships between the orientation of the mitotic apparatus for first cleavage and the position of meiotic division-related structures in starfish eggs.

In order to understand when the orientation of the first cleavage plane is fixed along the animal-vegetal axis in starfish eggs, the behavior of the sperm aster was examined by indirect immunofluorescence staining. After duplication, the sperm aster organizes the mitotic apparatus for first cleavage perpendicular to the cleavage plane. The sperm aster located in the egg periphery just after fertilization and moved to the site close to the animal pole rather than the egg center by meiosis II. At early metaphase II, duplication of the sperm aster was detected but the axis of the resultant sperm diaster randomly pointed. Subsequently, its axis had already turned perpendicular to the animal-vegetal axis before pronucleus fusion. These results indicate that the orientation processes of the sperm diaster consist of positioning before its duplication and successive determining its azimuth. Furthermore, the azimuth and position of the mitotic apparatus for first cleavage did not change by shifting or eliminating the meiotic division-related structures such as the germinal vesicle, meiotic spindle, and female pronucleus by micromanipulation. These results show that none of them determines the first cleavage plane. Therefore, we discuss the pointing mechanism of the first cleavage plane without the influence of these meiotic division-related structures.

Animals↗

RhD genotype determination by single sperm cell analysis.

OBJECTIVE: An Rh-negative woman with preexisting anti-D antibodies may affect some or all subsequent fetuses, depending on the genotype of her Rh-positive partner. Currently, a reliable technique for an absolute determination of RhD genotype is not available. This study was initiated to develop an accurate method for RhD genotyping in men. STUDY DESIGN: RhD genotype was determined by deoxyribonucleic acid amplification of a D-specific sequence in single sperm cell samples. Micromanipulation techniques were used for sampling of single sperm cells, which were further amplified by multiplex nested polymerase chain reaction at the RhD locus. A RhD sequence amplification product was expected in all of the successfully amplified samples from Rh-positive homozygotes, in some of the samples from heterozygotes, and in none of the samples form Rh-negative subjects. RESULTS: RhD genotype was accurately determined in 10 of 10 donors. A total of 132 single sperm cells were analyzed (8 to 17 samples per donor), of which 96 were successfully amplified as assessed by an internal control. As expected, the specific region of the RhD gene was amplified in all, some, and none of the signal-positive sperm samples from Rh-positive homozygotes, heterozygotes, and Rh-negative subjects, respectively, allowing accurate determination of the genotype. CONCLUSION: An accurate diagnosis of the RhD genotype can be attained from single sperm cell analysis by means of polymerase chain reaction and may have major clinical applications in the management of Rh isoimmunization.

Base Sequence↗

Ultramicrosurgical removal of subretinal hemorrhage in cats.

Subretinal hemorrhages are associated with progressive degeneration of the outer retina and a corresponding poor visual prognosis. Mixed results have been reported in previous attempts to remove such subretinal hemorrhages. We developed an ultramicrosurgical system that used the control of a stereotactic micromanipulator to direct a micropipette tip through a small retinotomy into the subretinal space in three cat eyes. Low-dose recombinant tissue plasminogen activator was then introduced into the subretinal space around the subretinal hemorrhage via a controlled microinfusion system. The recombinant tissue plasminogen activator solution facilitated clot lysis and subsequent removal through the micropipette. Light- and electron-microscopic analysis of histopathologic specimens disclosed good preservation of retinal architecture in the three cat eyes in which experimental subretinal hemorrhages were removed. This was in contrast to the retinal degeneration observed in similar but untreated experimental subretinal hemorrhages.

Animals↗

Analysis of T-cell subpopulations in T-cell non-Hodgkin's lymphoma of angioimmunoblastic lymphadenopathy with dysproteinemia type by single target gene amplification of T cell receptor- beta gene rearrangements.

Angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) is defined in the current lymphoma classifications as a T-cell non-Hodgkin's lymphoma. However, in approximately one third of the cases of this lymphoproliferative disease rearrangements of T-cell receptor (TCR) genes indicating clonal expansion of T cells are not detectable. It is currently believed that these cases may represent early stages of a lymphoma with a minor oligoclonal T-cell population. In the present study, 18 lymph nodes with the characteristic histology of AILD were investigated for clonal T-cell receptor gene rearrangements by analysis of DNA extracted from whole tissue sections. Dominant T-cell clones were detected in 12 of these cases. Single CD4(+) and CD8(+) T cells and proliferating Ki67(+) cells of seven cases were micromanipulated from frozen tissue sections. TCRbeta gene rearrangements were amplified from these cells by polymerase chain reaction and sequenced. In all informative cases, the clonal gene rearrangements were only detected among CD4(+), and not among CD8(+) T cells, indicating that the tumor clones in AILD usually derive from CD4(+) T cells. Minor clonal T-cell populations in those cases in which no clone was found by whole-tissue DNA analysis were not detectable even at single cell resolution. T-cell clones in 4 of 10 cases were found to express similar TCRbeta chains, indicating a potential role of (super) antigen triggering in at least some cases of AILD.

Amino Acid Sequence↗

Recurrent chromosomal imbalances detected in biopsy material from oral premalignant and malignant lesions by combined tissue microdissection, universal DNA amplification, and comparative genomic hybridization.

Biopsies routinely performed for the histopathological diagnosis of oral epithelial lesions before treatment were screened for chromosomal imbalances by comparative genomic hybridization. Comparative genomic hybridization was performed on 12 oral premalignant lesions (OPLs; dysplasias and carcinomas in situ) and 14 oral squamous cell carcinomas (OSCCs). Eight biopsies displayed areas of different histopathological appearance, so that OPLs and OSCCs from the same patient were analyzed. To avoid contamination with nonneoplastic cells, defined cell populations were isolated by micromanipulation with a glass needle. Before comparative genomic hybridization analysis, universal DNA amplification was performed using the DOP-polymerase chain reaction protocol. In the 14 OSCCs examined, the average number of chromosomal imbalances was significantly higher than in the 12 OPLs (mean +/- SEM: 11.9 +/- 1.9 versus 3.2 +/- 1.2; P = 0.003). The DNA copy number changes identified in more than one OPL were gains on 8q (3 of 12) and 16p (2 of 12), as well as losses on 3p (5 of 12); 5q (4 of 12); 13q (3 of 12); and 4q, 8p, and 9p (2 of 12 each). In more than 30% of OSCCs, gains of chromosomal material were identified on 20q (8 of 14); 8q, 11q, 22q (7 of 14 each); 3q, 15q, and 17p (6 of 14 each); and 14q, 17q, and 20p (5 of 14 each), and losses were identified on 3p and 4q (9 of 14 each), 5q (7 of 14), 13q (6 of 14), and 2q and 9p (5 of 14 each). These results were validated by positive and negative control comparative genomic hybridization experiments and microsatellite analysis for the detection of allelic loss. The vast majority of genomic alterations found in OPLs were again identified in OSCCs from the same biopsy, supporting the hypothesis that multiple lesions in the same patient are clonally related. In summary, we show that comprehensive information on the genomic alterations in oral epithelial lesions can be obtained from small biopsies. Such data may identify prognostic indicators that could eventually assist in designing therapeutic strategies.

Adult↗

Improved reproducibility of magnetic stimulation-evoked motor potentials in the human masseter by a new method for locating stimulation sites on the scalp.

In magnetic-stimulation studies where motor-evoked potentials (MEP) are measured on different occasions, accurate relocation of the stimulation site on the scalp is essential. Here a novel method of locating neural stimulation sites was tested and the reproducibility of MEPs in the human masseter assessed. The heads of the participants were immobilized in a plastic mask that incorporated a grid overlying the left cerebral hemisphere. A figure-of-eight coil was oriented with a micromanipulator. Electrodes were placed over the right masseter using a standardized method. Two discrete sites on the grid were stimulated alternately at threshold + 10% whilst participants clenched their teeth. Recordings were repeated after the mask and electrodes had been removed and replaced. Within individual participants, the latency and amplitude of the MEP were reproducible between trials and when the mask and electrodes were replaced. The new method appears to be accurate and practical when recording longitudinal MEP data in the masseter.

Adult↗

Direct measurement of the area expansion and shear moduli of the human red blood cell membrane skeleton.

The area expansion and the shear moduli of the free spectrin skeleton, freshly extracted from the membrane of a human red blood cell (RBC), are measured by using optical tweezers micromanipulation. An RBC is trapped by three silica beads bound to its membrane. After extraction, the skeleton is deformed by applying calibrated forces to the beads. The area expansion modulus K(C) and shear modulus mu(C) of the two-dimensional spectrin network are inferred from the deformations measured as functions of the applied stress. In low hypotonic buffer (25 mOsm/kg), one finds K(C) = 4.8 +/- 2.7 microN/m, mu(C) = 2.4 +/- 0.7 microN/m, and K(C)/mu(C) = 1.9 +/- 1.0. In isotonic buffer, one measures higher values for K(C), mu(C), and K(C)/mu(C), partly because the skeleton collapses in a high-ionic-strength environment. Some data concerning the time evolution of the mechanical properties of the skeleton after extraction and the influence of ATP are also reported. In the Discussion, it is shown that the measured values are consistent with estimates deduced from experiments carried out on the intact membrane and agree with theoretical and numerical predictions concerning two-dimensional networks of entropic springs.

Adenosine Triphosphate↗