A compressomyograph train of four monitoring device.

The monitoring of the neuromuscular blockade is critical for patient's safety during and after surgery. The monitoring of neuromuscular blockade often requires the use of Train of Four (TOF) technique. During a TOF test two electrodes are attached to the ulnar nerve, and a series of four electric pulses are applied. The electrical stimulation causes the thumb to twitch, and the amount of twitch varies depending on the amount of neuromuscular blockade in patient's system. Current medical devices used to assist anesthesiologists to perform TOF monitoring often require free hand movement and do not provide accurate or reliable results. The goal of this work is to design, prototype and test a new medical device that provides reliable TOF results when thumb movement is restricted. A medical device that uses a pressurized catheter balloon to detect the response thumb twitch of the TOF test is created. An analytical model, numerical study, and mechanical finger testing were employed to create an optimum design. The design is tested through a pilot human subjects study. No significant correlation is reported with subjects' properties, including hand size.

Genetic analysis of PALB2 gene WD40 domain in canine mammary tumour patients.

BACKGROUND: DNA repair mechanisms are essential for tumorigenesis and disruption of HR mechanism is an important predisposing factor of human breast cancers (BC). PALB2 is an important part of the HR. There are similarities between canine mammary tumours (CMT) and BCs. As its human counterpart, PALB2 mutations could be a predisposing factor of CMT. OBJECTIVES: In this study, we aimed to investigate the impacts of PALB2 variants on tumorigenesis and canine mammary tumor (CMT) malignancy. METHODS: We performed Sanger sequencing to detect germline mutations in the WD40 domain of the canine PALB2 gene in CMT patients. We conducted in silico analysis to investigate the variants, and compared the germline PALB2 mutations in humans that cause breast cancer (BC) with the variants detected in dogs with CMT. RESULTS: We identified an intronic (c.3096+8C>G) variant, two exonic (p.A1050V and p.R1354R) variants, and a 3' UTR variant (c.4071T>C). Of these, p.R1354R and c.4071T>C novel variants were identified for the first time in this study. We found that the p.A1050V mutation had a significant effect. However, we could not determine sufficient similarity due to the differences in nucleotide/amino acid sequences between two species. Nonetheless, possible variants of human sequences in the exact location as their dog counterparts are associated with several cancer types, implying that the variants could be crucial for tumorigenesis in dogs. Our results did not show any effect of the variants on tumor malignancy. CONCLUSIONS: The current project is the first study investigating the relationship between the PALB2 gene WD40 domain and CMTs. Our findings will contribute to a better understanding of the pathogenic mechanism of the PALB2 gene in CMTs. In humans, variant positions in canines have been linked to cancer-related phenotypes such as familial BC, endometrial tumor, and hereditary cancer predisposition syndrome. The results of bioinformatics analyses should be investigated through functional tests or case-control studies.

ß-Dispersion of blood during sedimentation.

Aggregation of human red blood cells (RBC) is central to various pathological conditions from bacterial infections to cancer. When left at low shear conditions or at hemostasis, RBCs form aggregates, which resemble stacks of coins, known as 'rouleaux'. We experimentally examined the interfacial dielectric dispersion of aggregating RBCs. Hetastarch, an RBC aggregation agent, is used to mimic conditions leading to aggregation. Hetastrach concentration is incrementally increased in blood from healthy donors to measure the sensitivity of the technique. Time lapse electrical impedance measurements were conducted as red blood cells form rouleaux and sediment in a PDMS chamber. Theoretical modeling was used for obtaining complex permittivity of an effective single red blood cell aggregate at various concentrations of hetastarch. Time response of red blood cells' impedance was also studied to parametrize the time evolution of impedance data. Single aggregate permittivity at the onset of aggregation, evolution of interfacial dispersion parameters, and sedimentation kinetics allowed us to distinguish differential aggregation in blood.

Efficacy of inactivated Parapoxvirus ovis paraimmune activator as a prophylaxis against mastitis and therapy for subclinical mastitis in dairy cattle.

Mastitis is the most important disease in dairy cattle industry because of its high economic losses both in herd management, milk and milk products. The aim of this study was to determine the efficacy of inactivated Parapoxvirus ovis (IPPVO) Para immune activator as a prophylaxis against mastitis and as the therapy for subclinical mastitis in dairy cattle. The prophylactic effects of IPPVO were investigated in California mastitis test (CMT). Healthy Holstein cows were divided into A1 (n = 30) and A2 (n = 30) subgroups. In addition, 90 subclinical mastitis Holstein cows were divided into subgroups of B1, B2, and B3 to investigate the efficacy of IPPVO treatment. A significant difference in CMT levels was observed (p ˂ 0.01) 30 day after treatment in A groups. The difference in somatic cell count (SCC) levels between the A groups 15, 30 day after treatment was significant (p ˂ 0.01). The results of the CMT among the B groups showed no statistically significant difference (p > 0.05). The results of the SCC tests showed no statistically significant difference (p > 0.05) among the B groups on days 0, 9 and 15 after treatment. Coagulase-negative Staphylococcus (n = 53) and Escherichia coli (n = 30) were the most prevalent bacteria isolated in this study. In conclusion, IPPVO, although had no additional effect when used in combination with antibiotics could possibly be used instead of antibiotics and to protect cattle from subclinical mastitis, however, it is not known how long this prophylaxis effect could last.

Multiple consecutive recapture of rigid nanoparticles using a solid-state nanopore sensor.

Solid-state nanopore sensors have been used to measure the size of a nanoparticle by applying a resistive pulse sensing technique. Previously, the size distribution of the population pool could be investigated utilizing data from a single translocation, however, the accuracy of the distribution is limited due to the lack of repeated data. In this study, we characterized polystyrene nanobeads utilizing single particle recapture techniques, which provide a better statistical estimate of the size distribution than that of single sampling techniques. The pulses and translocation times of two different sized nanobeads (80 nm and 125 nm in diameter) were acquired repeatedly as nanobeads were recaptured multiple times using an automated system controlled by custom-built scripts. The drift-diffusion equation was solved to find good estimates for the configuration parameters of the recapture system. The results of the experiment indicated enhancement of measurement precision and accuracy as nanobeads were recaptured multiple times. Reciprocity of the recapture and capacitive effects in solid state nanopores are discussed. Our findings suggest that solid-state nanopores and an automated recapture system can also be applied to soft nanoparticles, such as liposomes, exosomes, or viruses, to analyze their mechanical properties in single-particle resolution.

Dielectrophoresis assisted loading and unloading of microwells for impedance spectroscopy.

Dielectric spectroscopy (DS) is a noninvasive, label-free, fast, and promising technique for measuring dielectric properties of biological cells in real time. We demonstrate a microchip that consists of electro-activated microwell arrays for positive dielectrophoresis assisted cell capture, DS measurements, and negative dielectrophoresis driven cell unloading; thus, providing a high-throughput cell analysis platform. To the best of our knowledge, this is the first microfluidic chip that combines electro-activated microwells and DS to analyze biological cells. Device performance is tested using Saccharomyces cerevisiae (yeast) cells. DEP response of yeast cells is determined by measuring their Clausius-Mossotti factor using biophysical models in parallel plate microelectrode geometry. This information is used to determine the excitation frequency to load and unload wells. Effect of yeast cells on the measured impedance spectrum was examined both experimentally and numerically. Good match between the numerical and experimental results establishes the potential use of the microchip device for extracting subcellular properties of biological cells in a rapid and nonexpensive manner.

Enhancement of dielectrophoresis using fractal gold nanostructured electrodes.

Dielectrophoretic motions of Saccharomyces cerevisiae (yeast) cells and colloidal gold are investigated using electrochemically modified electrodes exhibiting fractal topology. Electrodeposition of gold on electrodes generated repeated patterns with a fern-leaf type self-similarity. A particle tracking algorithm is used to extract dielectrophoretic particle velocities using fractal and planar electrodes in two different medium conductivities. The results show increased dielectrophoretic force when using fractal electrodes. Strong negative dielectrophoresis of yeast cells in high-conductivity media (1.5 S/m) is observed using fractal electrodes, while no significant motion is present using planar electrodes. Electrical impedance at the electrode/electrolyte interface is measured using impedance spectroscopy technique. Stronger electrode polarization (EP) effects are reported for planar electrodes. Decreased EP in fractal electrodes is considered as a reason for enhanced dielectrophoretic response.

Rough Gold Electrodes for Decreasing Impedance at the Electrolyte/Electrode Interface.

Electrode polarization at the electrolyte/electrode interface is often undesirable for bio-sensing applications, where charge accumulated over an electrode at constant potential causes large potential drop at the interface and low measurement sensitivity. In this study, novel rough electrodes were developed for decreasing electrical impedance at the interface. The electrodes were fabricated using electrochemical deposition of gold and sintering of gold nanoparticles. The performances of the gold electrodes were compared with platinum black electrodes. A constant phase element model was used to describe the interfacial impedance. Hundred folds of decrease in interfacial impedance were observed for fractal gold electrodes and platinum black. Biotoxicity, contact angle, and surface morphology of the electrodes were investigated. Relatively low toxicity and hydrophilic nature of the fractal and granulated gold electrodes make them suitable for bioimpedance and cell electromanipulation studies compared to platinum black electrodes which are both hydrophobic and toxic.

Electrothermal flow on electrodes arrays at physiological conductivities.

AC electrothermal (ET) flow is inevitable for microfluidic systems dissipating electric energy in a conducting medium. Therefore, many practical applications of biomicrofluidics are prone to ET flow. Here, a series of observations are reported on ET flow in a microfluidic chamber that houses three electrode pairs. The observations indicate that the variations in liquid conductivity and channel height critically impact the structure and magnitude of the flow field. Observations indicate that after a critical conductivity a global ET flow is present in the chamber, while at lower conductivities a vortex is present at every electrode edge. In addition, no ET flow is observed when the chamber height is kept below a critical value at physiological conductivity (∼1.5 S/m). The experimental observations are compared with the numerical simulations of ET flow. The validity of the assumptions made in the current AC ET flow theory is also discussed in the light of the experimental data. The observations can be critical while designing microfluidic systems that involve power dissipation in conductive fluids.

Flexible Bioimpedance Sensor for Label-Free Detection of Cell Viability and Biomass.

We introduce a flexible microfluidic bioimpedance sensor that is capable of detecting biomass and cell viability variations in a cell suspension. The sensor is developed on indium tin oxide (ITO) coated polyethylene terephthalate (PET) substrate and is devoid of gold, silicon, PDMS, or glass. In conjugation with a custom built PCB read-out module, the impedance characteristics of a cell suspension can be measured within one minute of sample introduction using liquid volumes less than 5 µL. The portable sensor system occupies very little bench space and has the potential to be developed as a disposable electrical bioimpedance probe for rapid detection of dielectric variations in a biological suspension. The sensor is designed to generate a differential impedance spectra exclusive to a cell suspension with a dual-electrode-pair system. The potential of the sensor to discriminate between live and heat treated Saccharomyces cerevisiae is demonstrated in this study. The disposable sensor along with the distance variation technique is touted to be an inexpensive alternative to some of the existing online disposable biomass detection probes and electrochemical sensors.

Differential dielectric responses of chondrocyte and Jurkat cells in electromanipulation buffers.

Electromanipulation of cells as a label-free cell manipulation and characterization tool has gained particular interest recently. However, the applicability of electromanipulation, particularly dielectrophoresis (DEP), to biological cells is limited to cells suspended in buffers containing lower amounts of salts relative to the physiological buffers. One might question the use of low conductivity buffers (LCBs) for DEP separation, as cells are stressed in buffers lacking physiological levels of salt. In LCB, cells leak ions and undergo volume regulation. Therefore, cells exhibit time-dependent DEP response in LCB. In this work, cellular changes in LCB are assessed by dielectric spectroscopy, cell viability assay, and gene expression of chondrocytes and Jurkats. Results indicate leakage of ions from cells, increases in cytoplasmic conductivity, membrane capacitance, and conductance. Separability factor, which defines optimum conditions for DEP cell separation, for the two cell types is calculated using the cellular dielectric data. Optimum DEP separation conditions change as cellular dielectric properties evolve in LCB. Genetic analyses indicate no changes in expression of ionic channel proteins for chondrocytes suspended in LCB. Retaining cellular viability might be important during dielectrophoretic separation, especially when cells are to be biologically tested at a downstream microfluidic component.

Dielectric characterization of costal cartilage chondrocytes.

BACKGROUND: Chondrocytes respond to biomechanical and bioelectrochemical stimuli by secreting appropriate extracellular matrix proteins that enable the tissue to withstand the large forces it experiences. Although biomechanical aspects of cartilage are well described, little is known of the bioelectrochemical responses. The focus of this study is to identify bioelectrical characteristics of human costal cartilage cells using dielectric spectroscopy. METHODS: Dielectric spectroscopy allows non-invasive probing of biological cells. An in house computer program is developed to extract dielectric properties of human costal cartilage cells from raw cell suspension impedance data measured by a microfluidic device. The dielectric properties of chondrocytes are compared with other cell types in order to comparatively assess the electrical nature of chondrocytes. RESULTS: The results suggest that electrical cell membrane characteristics of chondrocyte cells are close to cardiomyoblast cells, cells known to possess an array of active ion channels. The blocking effect of the non-specific ion channel blocker gadolinium is tested on chondrocytes with a significant reduction in both membrane capacitance and conductance. CONCLUSIONS: We have utilized a microfluidic chamber to mimic biomechanical events through changes in bioelectrochemistry and described the dielectric properties of chondrocytes to be closer to cells derived from electrically excitably tissues. GENERAL SIGNIFICANCE: The study describes dielectric characterization of human costal chondrocyte cells using physical tools, where results and methodology can be used to identify potential anomalies in bioelectrochemical responses that may lead to cartilage disorders.

A separability parameter for dielectrophoretic cell separation.

In this study, a separability parameter is introduced to determine the selection of optimum operating parameters for DEP separation of a cell pair. The separability parameter is defined as a function of cells' Clausius-Mossotti (CM) factors. T-cell leukemia Jurkat and mouse melanoma B16 cells are tested to validate the separability parameter. CM factors of cells are measured using a recently developed microfluidic impedance spectroscopy device. Separability maps are generated for varying values of field frequency and buffer conductivity. Cell separation is tested using a planar interdigitated electrode array at different buffer conductivities. Impedance measurements of the DEP device are performed at various buffer conductivities. Electrode polarization effects and energy allocation for dielectrophoretic manipulation of cells are computed from the impedance data utilizing an equivalent circuit model. Cell separation results are explained in the light of the impedance measurements.

Enhanced killing effect of nanosecond pulse electric fields on PANC1 and Jurkat cell lines in the presence of Tween 80.

We investigated the effects of nanosecond pulse electric fields (nsPEFs) on Jurkat and PANC1 cells, which are human carcinoma cell lines, in the presence of Tween 80 (T80) at a concentration of 0.18% and demonstarted an enhanced killing effect. We used two biological assays to determine cell viability after exposing cells to nsPEFs in the presence of T80 and observed a significant increase in the killing effect of nsPEFs. We did not see a toxic effect of T80 when cells were exposed to surfactant alone. However, we saw a synergistic effect when cells exposed to T80 were combined with the nsPEFs. Increasing the time of exposure for up to 8 h in T80 led to a significant decrease in cell viability when nsPEFs were applied to cells compared to control cells. We also observed cell type-specific swelling in the presence of T80. We suggest that T80 acts as an adjuvant in facilitating the effects of nsPEFs on the cell membrane; however, the limitations of the viability assays were addressed. We conclude that T80 may increase the fragility of the cell membrane, which makes it more susceptible to nsPEF-mediated killing.

Probing nanoparticle interactions in cell culture media.

Nanoparticle research is often performed in vitro with little emphasis on the potential role of cell culture medium. In this study, gold nanoparticle interactions with cell culture medium and two cancer cell lines (human T-cell leukemia Jurkat and human pancreatic carcinoma PANC1) were investigated. Gold nanoparticles of 10, 25, 50, and 100 nm in diameter at fixed mass concentration were tested. Size distributions and zeta potentials of gold nanoparticles suspended in deionized (DI) water and Dulbecco's Modified Eagle's Media (DMEM) supplemented with fetal calf serum (FCS) were measured using dynamic light scattering (DLS) technique. In DI water, particle size distributions exhibited peaks around their nominal diameters. However, the gold nanoparticles suspended in DMEM supplemented with FCS formed complexes around 100 nm, regardless of their nominal sizes. The DLS and UV-vis spectroscopy results indicate gold nanoparticle agglomeration in DMEM that is not supplemented by FCS. The zeta potential results indicate that protein rich FCS increases the dispersion quality of gold nanoparticle suspensions through steric effects. Cellular uptake of 25 and 50 nm gold nanoparticles by Jurkat and PANC1 cell lines were investigated using inductively coupled plasma-mass spectroscopy. The intracellular gold level of PANC1 cells was higher than that of Jurkat cells, where 50 nm particles enter cells at faster rates than the 25 nm particles.

Microfluidic impedance spectroscopy as a tool for quantitative biology and biotechnology.

A microfluidic device that is able to perform dielectric spectroscopy is developed. The device consists of a measurement chamber that is 250 µm thick and 750 µm in radius. Around 1000 cells fit inside the chamber assuming average quantities for cell radius and volume fraction. This number is about 1000 folds lower than the capacity of conventional fixtures. A T-cell leukemia cell line Jurkat is tested using the microfluidic device. Measurements of deionized water and salt solutions are utilized to determine parasitic effects and geometric capacitance of the device. Physical models, including Maxwell-Wagner mixture and double shell models, are used to derive quantities for sub-cellular units. Clausius-Mossotti factor of Jurkat cells is extracted from the impedance spectrum. Effects of cellular heterogeneity are discussed and parameterized. Jurkat cells are also tested with a time domain reflectometry system for verification of the microfluidic device. Results indicate good agreement of values obtained with both techniques. The device can be used as a unique cell diagnostic tool to yield information on sub-cellular units.

Acoustophoresis in shallow microchannels.

Acoustophoretic (AP) motion of spherical polystyrene particles in a steady pressure driven flow is investigated in shallow microchannels, where the channel height is comparable to the particle diameter. Particle trajectories at different ultrasonic actuation amplitudes are extracted by a particle tracking algorithm. Depths of the particles are predicted using the streamwise particle speed that is due to the pressure driven flow. The particle depths are shown to be influenced by the actuation voltage. The particle migration along the channel height is explained using the second-order perturbation theory. The particle equation of motion is employed to extract the AP force. Wall effects are included in the analysis of both particle depth and force predictions. Differences as large as 20% in the AP force magnitude due to the wall corrections are reported. The AP force is also calculated using the theoretical force expression, and compared with the experimental results. The focal length, which is the necessary distance to effectively concentrate particles in a microchannel, is calculated using the analytical solution of the particle equation of motion. The calculated force and the focusing length agree well with the experimental results. The focal length is critical for the design of micro sample concentration devices.

Dielectrophoretic separation of mouse melanoma clones.

Dielectrophoresis (DEP) is employed to differentiate clones of mouse melanoma B16F10 cells. Five clones were tested on microelectrodes. At a specific excitation frequency, clone 1 showed a different DEP response than the other four. Growth rate, melanin content, recovery from cryopreservation, and in vitro invasive studies were performed. Clone 1 is shown to have significantly different melanin content and recovery rate from cryopreservation. This paper reports the ability of DEP to differentiate between two malignant cells of the same origin. Different DEP responses of the two clones could be linked to their melanin content.

Dispersion state and toxicity of mwCNTs in cell culture medium with different T80 concentrations.

In this study, size distribution, zeta potential, shape, and toxicity of multi-walled carbon nanotubes (mwCNTs), and effect of non-ionic detergent Tween 80 (T80) concentrations (0%, 0.2%, and 1%) on the dispersion quality and cell viability are investigated. Nanotubes are suspended in biological solutions (DMEM, RPMI) with three different concentrations (10, 50, and 100 microg/ml) and toxicological investigations are carried on human T-cell leukemia (Jurkat) and human pancreatic carcinoma (PANC1) cell lines. According to light and transmission electron microscopy results, mwCNTs form well-defined and regular bundles in the presence of 1% T80 surfactant; whereas more irregular structures are present in absence of T80. Dispersion quality is represented in terms of the size distribution from dynamic light scattering (DLS) experiments and its second moment. Dispersion quality of the mwCNTs decreases with decreasing T80 concentration, while the constituents of RPMI and DMEM increase the dispersion quality. No significant differences between the dispersive effects of RPMI and DMEM suspensions are observed. Zeta potential of the mwCNTs is measured using electrophoretic light scattering. Variations in the nanotube and T80 concentrations do not change the zeta potential significantly. T80 concentrations above 0.2% are found to be toxic for Jurkat andPANC1 cells.

Lambing rates and litter size following carazolol administration prior to insemination in Kivircik ewes.

The effect of carazolol on the ease of penetrating the cervix during artificial insemination, lambing rate and litter size was studied using 1.5-4.0-year old Kivircik ewes in an incomplete 3 x 2 x 2 experimental design. All of the ewes in this study were synchronized for oestrus by insertion of a progesterone impregnated vaginal sponge for 12 days and administration of 400 IU PMSG at sponge withdrawal. Three methods of service were compared: natural service, artificial insemination (AI) with fresh semen, or AI with frozen semen. Two times of insemination (fixed time AI versus AI at observed oestrus) were compared on the fresh and frozen AI treatments. The absence (control) or use of carazolol (carazolol; 0.5mg/ewe i.m. 30 min before mating) was the third factor in the design and penetration of the cervix by the insemination pipette was assessed as shallow (<10mm), middle (10-20mm) or deep (>20mm). Natural service ewes were only mated at observed oestrus. Consequently, the factorial design was incomplete and there were a total of 10 treatments each represented by 30 ewes. Natural service resulted in a significantly (P<0.05) higher lambing rate and litter size (86%; 2.0+/-0.05 lambs/ewe) than AI using fresh (65%; 1.6+/-0.1 lambs/ewe) or frozen (40%; 1.4+/-0.14 lambs/ewe) semen. For AI animals the lambing rate and litter size were not significantly different when service was at a fixed time (50%; 1.5+/-0.12 lambs/ewe) or at observed oestrus (56%; 1.5+/-0.12 lambs/ewe). Carazolol did not permit complete cervical penetration in any ewe. Deep penetration of the cervix at AI was achieved in 33% of untreated (control) and 48% of carazolol treated ewes (P<0.05). However, the proportion of ewes in which penetration of the cervix and semen deposition was greater than shallow was similar for control (82%) and carazolol (85%), and lambing rate and litter size were similar for both treatments. Over the three service methods, the lambing rate was 56% for control and 63% for carazolol (NS) and litter size was similar for both treatments. It was concluded that the carazolol treatment used prior to natural mating or AI in this experiment did not improve lambing rate or litter size in Kivircik ewes.