In Vivo Assessment of Decellularized Porcine Myocardial Slice as an Acellular Cardiac Patch.

Acellular cardiac patches made of various biomaterials have shown to improve heart function after myocardial infarction (MI). Extracellular matrix scaffold derived from a decellularized tissue has unique advantages to serve as an acellular cardiac patch due to its biomimetic nature. In this study, we examined the therapeutic outcomes of using a decellularized porcine myocardium slice (dPMS) as an acellular patch in a rat acute MI model. dPMSs with two different thicknesses (300 and 600 µm) were patched to the infarcted area of the rat myocardium, and their effects on cardiac function and host interactions were assessed. We found that the implanted dPMS firmly attached to host myocardium after implantation and prevented thinning of the left ventricular (LV) wall after an MI. A large number of host cells were identified to infiltrate into the implanted dPMS, and a significant number of vessel structures was observed in the dPMS and infarcted area. We detected a significantly higher density of M2 macrophages in the groups treated with dPMSs as compared to the MI group. Contraction of the LV wall and cardiac functional parameters (left ventricular ejection fraction and fractional shortening) was significantly improved in the treatment groups (300 and 600 µm dPMS) 4 weeks after surgery. Our results proved the therapeutic benefits of using dPMS as an acellular cardiac patch for the treatment of acute myocardial infarction.

Does Sericin, as a Novel Pleurodesis Agent, Have Higher Effectiveness Compared to Talcum Powder, Doxycycline, and Silver Nitrate Pleurodesis?

INTRODUCTION: The usefulness of sericin as pleurodesis agent has previously been described. Present study aims to compare sericin pleurodesis regarding success, effectiveness, tolerability, and side-effects. METHODS: Adult, 12-week-old Wistar-albino rats (n=60), divided to five groups as sericin, talcum-powder, doxycycline, silver-nitrate and control. Agents were administrated through left thoracotomy, rats sacrificed twelve-days after. RESULTS: Highest ratio of collagen fibers was observed in sericin group, and the intensity was higher than talcum-powder group (p<0.05). Compared to silver nitrate, sericin group displayed better mesothelial reaction, and multi-layer mesothelium was also better (p<0.05). Foreign body reaction and emphysema were less frequent in sericin group (p<0.05). The presence of biological tissue in parenchyma was less prominent in sericin group (p<0.05). Foreign body reaction on thoracic wall was less common in sericin group (p<0.05). Presence of biological tissue glue in thoracic wall was less prominent in sericin group (p<0.05). Glomerular degeneration was lower in sericin group compared to the silver nitrate group (p<0.05), and tubular degeneration was less common in sericin group than talcum group (p<0.05). Pericarditis was less common in sericin group compared to the other groups (p<0.05). CONCLUSION: As an intrinsic, natural glue protein, sericin protects the lung parenchyma and tissues, and its glue-like characteristics enable pleurodesis. The success of sericin in pleurodesis was demonstrated in the present study based on investigations of the pleurae. Being cost-effective and better tolerated agent associated with a low potential of side effects, sericin is more effective, less expensive and provides more lung parenchyma protection.

Simplified HPLC methodology for quantifying biological pterins by selective oxidation.

Tetrahydrobiopterin (BH4) has become a potential therapeutic tool to treat cardiovascular diseases, since it is an essential cofactor of nitric oxide synthase. In order to quantify the amount of BH4 and its related biopterins, a procedure that involves differential oxidation is currently used, which measures biopterin (the product of the oxidation of BH4 and BH2) at two different pH conditions to calculate the quantity of BH2 and BH4, using high performance liquid chromatography (HPLC). In this work, a method was established in order to quantify BH4 and BH2 by adapting previously described procedures. Several chromatographic conditions were evaluated to define the most convenient methodology. Four types of mobile phases and two different analytical columns were used for HPLC. Additionally, calibration curves were made in acid and basic pH compatible with the differential oxidation method. Each method was suitable for quantification purposes, but the choice was based on an economic factor. The selected condition was a mobile phase of 95% water/5% methanol using a C18 column at 35°C at a flow rate of 0.9mL/min. Then, it was calculated the recovery rate, which was about 80% using the chosen method. The aim of this work was to establish a simplified method of differential oxidation, compatible with matrixes such as cardiac tissue in order to facilitate the assessment of the BH4/BH2 ratio in biological samples.

Assessment of cardiac fibrosis: a morphometric method comparison for collagen quantification.

Fibrotic remodeling of the heart is a frequent condition linked to various diseases and cardiac dysfunction. Collagen quantification is an important objective in cardiac fibrosis research; however, a variety of different histological methods are currently used that may differ in accuracy. Here, frequently applied collagen quantification techniques were compared. A porcine model of early stage heart failure with preserved ejection fraction was used as an example. Semiautomated threshold analyses were imprecise, mainly due to inclusion of noncollagen structures or failure to detect certain collagen deposits. In contrast, collagen assessment by automated image analysis and light microscopy (LM)-stereology was more sensitive. Depending on the quantification method, the amount of estimated collagen varied and influenced intergroup comparisons. PicroSirius Red, Masson's trichrome, and Azan staining protocols yielded similar results, whereas the measured collagen area increased with increasing section thickness. Whereas none of the LM-based methods showed significant differences between the groups, electron microscopy (EM)-stereology revealed a significant collagen increase between cardiomyocytes in the experimental group, but not at other localizations. In conclusion, in contrast to the staining protocol, section thickness and the quantification method being used directly influence the estimated collagen content and thus, possibly, intergroup comparisons. EM in combination with stereology is a precise and sensitive method for collagen quantification if certain prerequisites are considered. For subtle fibrotic alterations, consideration of collagen localization may be necessary. Among LM methods, LM-stereology and automated image analysis are appropriate to quantify fibrotic changes, the latter depending on careful control of algorithm and comparable section staining.NEW & NOTEWORTHY Direct comparison of frequently applied histological fibrosis assessment techniques revealed a distinct relation of measured collagen and utilized quantification method as well as section thickness. Besides electron microscopy-stereology, which was precise and sensitive, light microscopy-stereology and automated image analysis proved to be appropriate for collagen quantification. Moreover, consideration of collagen localization might be important in revealing minor fibrotic changes.

EPR spectroscopy solutions for assessment of decellularization of intrathoracic organs and tissues.

Using EPR spectroscopy it was established that the determination of the concentration of paramagnetic centers in lyophilized tissues allows indirect evaluation of the quality of decellularization of intrathoracic organs (diaphragm, heart, and lungs), since the content of paramagnetic particles in them can serve as a criterion of cell viability and points to the necessity to repeat decellularization. Experiments in rats showed that the EPR spectra of the native thoracic organs contained paramagnetic centers with g-factor values ranging from 2.007 to 2.011 at a concentration of 10(-8) to 6.62 × 10(-7) mol/g of lyophilized tissue, whereas in all decellularized tissues of the same organs paramagnetic particles were not detected.

Characterization of diffuse fibrosis in the failing human heart via diffusion tensor imaging and quantitative histological validation.

Non-invasive imaging techniques are highly desirable as an alternative to conventional biopsy for the characterization of the remodeling of tissues associated with disease progression, including end-stage heart failure. Cardiac diffusion tensor imaging (DTI) has become an established method for the characterization of myocardial microstructure. However, the relationships between diffuse myocardial fibrosis, which is a key biomarker for staging and treatment planning of the failing heart, and measured DTI parameters have yet to be investigated systematically. In this study, DTI was performed on left ventricular specimens collected from patients with chronic end-stage heart failure as a result of idiopathic dilated cardiomyopathy (n = 14) and from normal donors (n = 5). Scalar DTI parameters, including fractional anisotropy (FA) and mean (MD), primary (D1 ), secondary (D2 ) and tertiary (D3 ) diffusivities, were correlated with collagen content measured by digital microscopy. Compared with hearts from normal subjects, the FA in failing hearts decreased by 22%, whereas the MD, D2 and D3 increased by 12%, 14% and 24%, respectively (P < 0.01). No significant change was detected for D1 between the two groups. Furthermore, significant correlation was observed between the DTI scalar indices and quantitative histological measurements of collagen (i.e. fibrosis). Pearson's correlation coefficients (r) between collagen content and FA, MD, D2 and D3 were -0.51, 0.59, 0.56 and 0.62 (P < 0.05), respectively. The correlation between D1 and collagen content was not significant (r = 0.46, P = 0.05). Computational modeling analysis indicated that the behaviors of the DTI parameters as a function of the degree of fibrosis were well explained by compartmental exchange between myocardial and collagenous tissues. Combined, these findings suggest that scalar DTI parameters can be used as metrics for the non-invasive assessment of diffuse fibrosis in failing hearts.

A case of arrhythmogenic right ventricular cardiomyopathy (ARVC/D) in which tenascin C immunostaining made the assessment of myocardial remodeling possible.

Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is characterized by progressive fatty and fibrous replacement. A female in her 70s, suddenly found in cardiopulmonary arrest. The heart weighed 452 g and yellow discoloration was observed. Histological examination revealed the replacement of the right ventricular myocardium by adipose tissue and fibrosis. The cause of death was fatal arrhythmia caused by ARVC/D. Tenascin C staining was useful in evaluating myocardial remodeling.

Heavy metals levels in fish from aquaculture farms and risk assessment in Lhasa, Tibetan Autonomous Region of China.

Fish is consumed as a common food by humans due to its nutritional and therapeutic benefits. However, they can accumulate toxic chemicals (such as heavy metals, persistent organic pollutants) from water and food chain. Very few studies have been investigated on heavy metal contents in fish from Tibetan Autonomous Region of China. In order to study heavy metals levels in fish from aquaculture farms and evaluate the risk that human consume fish in this area, we collected four types of aquaculture fish species (6 big-head carps, 5 grass carps, 5 carps and 5 tilapias) from fisheries around Lhasa city in this study. 9 heavy metals (Cr, As, Cd, Pb, Cu, Ba, Co, Mn and V) in different tissues of fish were determined by an inductively coupled plasma mass spectrometer. Cr, Ba, Co, Mn and V could easily accumulate in the gill, and Cu was detected in the hearts of all the fishes. Toxic metal (As, Cd and Pb) contents were higher in the liver than those in other tissues, heavy metal levels were the lowest in the muscle among all tissues. Most of heavy metal concentrations in the tilapia tissues were higher than those in other fish tissues, especially arsenic. Arsenic content in the tilapia samples was ~2-4 times higher than the maximum levels (MLs) of contaminants in the national standard, and other metals were all lower than the MLs. Compared the estimated daily intake of heavy metals through fish consumption with tolerable daily intakes recommended by FAO, the metals daily intake of As, Cd and Pb from fish consumption might not pose serious health risk to the local inhabitants. It is therefore necessary to determine the dose level for human, which is considered to be taken daily over a lifetime without adverse effects.

In-vivo assessment of normal T1 values of the right-ventricular myocardium by cardiac MRI.

To test feasibility of myocardial T1 mapping of the right ventricle (RV) at systole when myocardium is more compact and to determine the most appropriate imaging plane. 20 healthy volunteers (11 men; 33 ± 8 years) were imaged on a 1.5T scanner (MAGNETOM Avanto, Siemens AG, Erlangen, Germany). A modified look-locker inversion-recovery sequence was acquired at mid-ventricular short axis (SAX), as horizontal long-axis view and as transversal view at systole (mean trigger time 363 ± 37 ms). Myocardial T1 time of the left-ventricular and RV myocardium was measured within a region of interest (ROI) on generated T1-maps. The most appropriate imaging plane for the RV was determined by the ability to draw a ROI including the largest amount of myocardium without including adjacent tissue or blood. At systole, when myocardium is thicker, measurements of the RV myocardium were feasible in 18/20 subjects. Average size of the ROI was 0.42 ± 0.28 cm(2). In 10/18 subjects, short axis was the most appropriate imaging plane to obtain measurements (p = 0.034). Average T1 time of the RV myocardium was 1,016 ± 61 ms, and average T1 of the left-ventricular (LV) was 956 ± 25 ms (p < 0.001). T1 mapping of the RV myocardium is feasible during systole in the majority of healthy subjects but with a small ROI only. SAX plane was the optimal imaging plane in the majority of subjects. Native myocardial T1 time of the RV is significantly longer compared to the LV, which might be explained by the naturally higher collagen content of the RV.

Analysis of skeletal muscle has potential value in the assessment of cocaine-related deaths.

This study assesses the interpretive value of cocaine, benzoylecgonine (BZE) and cocaethylene (COET) in skeletal muscle (rectus femoris) in cocaine-using decedents. The distribution of these analytes in cardiac muscle (CM), vitreous humour (VH), femoral blood (FB) and cardiac blood (CB) is also reported. In rectus femoris muscle, the spatial distribution of the analytes was examined across the whole rectus femoris muscle collected from seven fatalities in which cocaine was detected. In six of these cases, death was attributed to trauma and in one case the cause of death was undetermined but suspected to be drug related. In two additional cases analytes were detected in the blood and/or VH but not in the muscle. The muscle was sectioned into 12-15 approximately equal segments, each of which was analysed after homogenisation. Tissue and bio-fluid samples were extracted by solid phase extraction with confirmation and quantification by GC-ion trap-MS/MS. No significant variation was observed in the concentration of any analyte throughout the muscle in the 7 cases analysed. The results reported here are in contrast to a previous study in which great variation in the concentration of some basic drugs (mainly tricyclic antidepressants and benzodiazepines) was observed throughout the thigh muscle bulk (Williams and Pounder, 1997). Analyte concentrations in skeletal muscle (SM) correlated well with those in FB (p<0.01). In general, the concentration of cocaine and COET followed the order VH > CM > SM > FB ≥ CB. Cocaine concentrations measured in VH were significantly higher than in blood and muscle. Inter-matrix variations in the concentrations of BZE and COET were less marked. The concentration of BZE exceeded that of cocaine in all matrices and in all cases except one where the time between death and drug intake was suspected to be short. In this case, the cocaine to BZE ratio measured in SM (2.66), CM (2.91) and VH (2.19) was higher than that measured in FB (0.97). Given that the concentrations of cocaine and its metabolites were uniformly distributed throughout the muscle and considering the good correlation observed between muscle and blood, muscle could be of interpretive value in cocaine related deaths. Further, since cocaine is known to have greater post-mortem stability in muscle than blood, concentrations measured in muscle may reflect more closely those at the time of death and might be of particular value in cases with an extended period between death and tissue sampling.

Rapid quantitative analysis of carnitine and acylcarnitines by ultra-high performance-hydrophilic interaction liquid chromatography-tandem mass spectrometry.

l-Carnitine and its acyl esters (acylcarnitines) play an important role in the metabolism of fatty acids. However, most of the present methods for the quantitative analysis of acylcarnitines have restrictions both in sample preparation and in chromatographic separation. Herein we present a validated method for determination of carnitine and eleven acylcarnitines in human serum and rat tissue biopsies by using ultra-high performance-hydrophilic interaction liquid chromatography-tandem mass spectrometry (UHP-HILIC-MS/MS). The procedure uses minimal sample preparation including only addition of organic solvent, labeled internal standard, incubation and centrifugation. The separation is performed without derivatization or addition of ion-pairing reagent within 7min on a hydrophilic interaction liquid chromatographic column with mass spectrometric detection. The method is linear in response over the concentration range from 20 to 600ng/ml for carnitine and acetylcarnitine and 5-200ng/ml for the other acylcarnitines, with correlation coefficients higher than 0.994. Recoveries were higher than 88% for most of the compounds. Limits of detection were 5ng/ml for carnitine and acetylcarnitine and approximately 0.5ng/ml for other acylcarnitines. The method was applied to the analysis of serum and tissue samples.

Comparative study of differential matrix and extended polar decomposition formalisms for polarimetric characterization of complex tissue-like turbid media.

Development of methodologies for quantification/unique interpretation of the intrinsic polarimetry characteristics of biological tissues are important for various applications involving tissue characterization/diagnosis. A detailed comparative evaluation of the polar decomposition and the differential matrix decomposition of Mueller matrices for extraction/quantification of the intrinsic polarimetry characteristics (with special emphasis on linear retardance δ, optical rotation Ψ and depolarization Δ parameters was performed, because these are the most prominent tissue polarimetry effects) from complex tissue-like turbid media exhibiting simultaneous scattering and polarization effects. The results suggest that for media exhibiting simultaneous linear retardance and optical rotation polarization events, the use of retarder polar decomposition with its associated analysis which assumes sequential occurrence of these effects, results in systematic underestimation of δ and overestimation of Ψ parameters. Analytical relationships between the polarization parameters (δ, Ψ) extracted from both the retarder polar decomposition and the differential matrix decomposition for either simultaneous or sequential occurrence of the linear retardance and optical rotation effects were derived. The self-consistency of both decompositions is validated on experimental Mueller matrices recorded from tissue-simulating phantoms (whose polarization properties are controlled, known a-priori, and exhibited simultaneously) of increasing biological complexity. Additional theoretical validation tests were performed on Monte Carlo-generated Mueller matrices from analogous turbid media exhibiting simultaneous depolarization (Δ), linear retardance (δ) and optical rotation (Ψ) effects. After successful evaluation, the potential advantage of the differential matrix decomposition over the polar decomposition formalism was explored for monitoring of myocardial tissue regeneration following stem cell therapy.

Left and right ventricular function and volume assessment in young thalassemia major patients with no related myocardial iron overload.

Thalassemia major (TM) patients have altered ventricular volumes and ejection fraction compared to normals, although evidence for these findings stem from restricted patient groups and has never been reproduced. We sought to evaluate cardiac parameters by cardiovascular magnetic resonance (CMR) in a group of young TM patients not covered by previous studies that are more representative of the TM population in many countries. Seventy patients including 40 TM with normal myocardial iron concentrations, and 30 age- and gender-matched normal (NL) volunteers underwent a CMR study for assessment of left and right ventricle volumes and function using a 1.5-T scanner. Left and right ventricle ejection fraction, indexed systolic and diastolic volumes, and indexed mass were compared between the two groups. Mean age of TM patients was 18.2 ± 7.1 versus 17.5 ± 8.5 years in NL with no significant differences (P = 0.73). There was no difference in left ventricular (LV) ejection fraction between the groups (TM 64.9 ± 5.7 %, NL 64.9 ± 5.2 %; P = 0.97). LV normalized end-diastolic and end-systolic volumes were significantly higher in patients with TM compared to NL volunteers (76.8 ± 19.4 versus 66.6 ± 11.7 mL/m², P = 0.008, and 27.0 ± 8.8 versus 23.6 ± 5.0 mL/m², P = 0.045). LV indexed mass was also higher in TM patients compared to NL (51.2 ± 11.9 versus 42.0 ± 8.5 g/m², P < 0.001). No significant differences were observed in right ventricular parameters. In conclusion, younger patients with TM do not present different left or right ventricular function values compared to normal controls despite having increased left ventricular volumes and mass.

Magnetic resonance Spectroscopy with Linear Algebraic Modeling (SLAM) for higher speed and sensitivity.

Speed and signal-to-noise ratio (SNR) are critical for localized magnetic resonance spectroscopy (MRS) of low-concentration metabolites. Matching voxels to anatomical compartments a priori yields better SNR than the spectra created by summing signals from constituent chemical-shift-imaging (CSI) voxels post-acquisition. Here, a new method of localized Spectroscopy using Linear Algebraic Modeling (SLAM) is presented, that can realize this additional SNR gain. Unlike prior methods, SLAM generates spectra from C signal-generating anatomic compartments utilizing a CSI sequence wherein essentially only the C central k-space phase-encoding gradient steps with highest SNR are retained. After MRI-based compartment segmentation, the spectra are reconstructed by solving a sub-set of linear simultaneous equations from the standard CSI algorithm. SLAM is demonstrated with one-dimensional CSI surface coil phosphorus MRS in phantoms, the human leg and the heart on a 3T clinical scanner. Its SNR performance, accuracy, sensitivity to registration errors and inhomogeneity, are evaluated. Compared to one-dimensional CSI, SLAM yielded quantitatively the same results 4-times faster in 24 cardiac patients and healthy subjects. SLAM is further extended with fractional phase-encoding gradients that optimize SNR and/or minimize both inter- and intra-compartmental contamination. In proactive cardiac phosphorus MRS of six healthy subjects, both SLAM and fractional-SLAM (fSLAM) produced results indistinguishable from CSI while preserving SNR gains of 36-45% in the same scan-time. Both SLAM and fSLAM are simple to implement and reduce the minimum scan-time for CSI, which otherwise limits the translation of higher SNR achievable at higher field strengths to faster scanning.

Assessment of the functional diversity of human myoglobin.

Myoglobin is presumably the most studied protein in biology. Its functional properties as a dioxygen storage and facilitator of dioxygen transport are widely acknowledged. Experimental evidence also implicates an essential role for myoglobin in the heart in regulating nitric oxide homeostasis. Under normoxia, oxygenated myoglobin can scavenge excessive nitric oxide, while under hypoxia, deoxygenated myoglobin can reduce nitrite, an oxidative product of nitric oxide, to bioactive nitric oxide. Myoglobin-driven nitrite reduction can protect the heart from ischemia and reperfusion injury. While horse and mouse myoglobin have been previously described to reduce nitrite under these conditions, a comparable activity has not been detected in human myoglobin. We here show that human myoglobin is a fully functional nitrite reductase. To study the role of human myoglobin for nitric oxide homeostasis we used repeated photometric wavelength scans and chemiluminescence based experiments. The results revealed that oxygenated human myoglobin reacts with nitrite-derived nitric oxide to form ferric myoglobin and that deoxygenated human myoglobin acts as a nitrite reductase in vitro and in situ. Rates of both nitric oxide scavenging and nitrite reduction were significantly higher in human compared to horse myoglobin. These data extend the existing knowledge about the functional properties of human myoglobin and are the basis for further translational studies towards the importance of myoglobin for nitric oxide metabolism in humans.

Coupling of adjacent tropomyosins enhances cross-bridge-mediated cooperative activation in a markov model of the cardiac thin filament.

We developed a Markov model of cardiac thin filament activation that accounts for interactions among nearest-neighbor regulatory units (RUs) in a spatially explicit manner. Interactions were assumed to arise from structural coupling of adjacent tropomyosins (Tms), such that Tm shifting within each RU was influenced by the Tm status of its neighbors. Simulations using the model demonstrate that this coupling is sufficient to produce observed cooperativity in both steady-state and dynamic force-Ca(2+) relationships. The model was further validated by comparison with reported responses under various conditions including inhibition of myosin binding and the addition of strong-binding, non-force-producing myosin fragments. The model also reproduced the effects of 2.5 mM added P(i) on Ca(2+)-activated force and the rate of force redevelopment measured in skinned rat myocardial preparations. Model analysis suggests that Tm-Tm coupling potentiates the activating effects of strongly-bound cross-bridges and contributes to force-Ca(2+) dynamics of intact cardiac muscle. The model further predicts that activation at low Ca(2+) concentrations is cooperatively inhibited by nearest neighbors, requiring Ca(2+) binding to >25% of RUs to produce appreciable levels of force. Without excluding other putative cooperative mechanisms, these findings suggest that structural coupling of adjacent Tm molecules contributes to several properties of cardiac myofilament activation.

Non-invasive assessment of tissue iron overload.

In recent years, there has been increasing interest in non-invasive iron measurement, especially of the liver and heart, in patients with iron overload. Serum ferritin still remains an essential monitoring parameter in intervals between liver iron measurements; however, confounding factors such as inflammation, chelation treatment changes and the specific disease have to be taken into account. Liver iron measurements can now routinely be performed in clinical applications either by quantitative magnetic resonance imaging (MRI) using the transverse magnetic relaxation rate R(2) or R(2)* (1/T(2)*) or by biomagnetic liver susceptometry. For iron measurements in the heart, the single-breathhold multi-echo MRI-R(2)* method has become a standard modality and is now applied in clinical settings beyond research studies. In other tissues like the pancreas, pituitary, and brain, different MRI methods are employed, but their clinical benefit has yet to be proven.

Velocity convergence of free energy surfaces from single-molecule measurements using Jarzynski's equality.

We studied the velocity dependence of mechanical unfolding of single protein molecules with the atomic force microscope. We showed that with enough realizations, the free energy surfaces reconstructed from Jarzynski's equality converge with respect to pulling velocity, in good agreement with theory. Using the I27 domain of titin as an example, we estimated the required number of realizations for a given pulling velocity, and suggested the optimal range of velocities for single-molecule experiments. The results demonstrate that Jarzynski's equality is a powerful and practical tool for reconstructing free energy landscapes.

Synergistic design of electric field and membrane in facilitating continuous adsorption for cleanup and enrichment of proteins in direct ESI-MS analysis.

We designed and fabricated a novel microdevice to facilitate continuous adsorption phenomena for biological sample preparation. Using the device, we also developed an online, highly integrated, multifunctional strategy, with a promise of accepting a large volume of crude tissue extracts with the end point generation of a reliable MS identification within 20 min. Under an external electric field, charged membranes can adsorb multiple layers of proteins, which exceed the capacity limit of common resins or membranes. It enlarges sample loading and trapping efficiency, thus bypasses the tradeoff between sample capacity and downstream detection sensitivity. This integrated approach, formed by synergistic utilization among electric field, membrane, and fluidic handling at the microscale, reduces the overall complexity of crude samples in one step for direct MS analysis. The sample preparation goals, including enrichment, desalting, removal of noncharged contaminants, and initial fractionation, can be rapidly performed in a single device. The strategy facilitates reproducible MS quantification by circumventing traditional laborious and time-consuming sample preparation steps. In addition, MEPD extended the ion trap linear dynamic range from 2 to at least 4 orders of magnitude by eliminating ion suppression effect, enriching target analyte(s), and decreasing sample loss during integrated sample preparation.

[Assessment of the mechanical activity of cardiac isomyosins V1 and V3 by an in vitro motility assay with regulated thin filaments].

A series of experiments in an in vitro motility assay with reconstructed thin filaments has been performed to determine the dependence of the velocity of thin filament movement on the concentration of calcium in solution (in the pCa range from 5 to 8) for rabbit cardiac isomyosins V1 and V3. The "pCa-velocity" curves had the sigmoid form. It was found for each isoform that sliding velocities of regulated thin filaments (at the saturating calcium concentration (pCa 5)) and actin filaments did not differ from each other. The Hill coefficient was 1.04 and 0.75 for isomyosins V1 and V3, respectively. The calcium sensitivity of V3 was found to be higher than that of V1. In the framework of the same method, the relationship between the velocity of thin filament sliding and the concentration of the actin-binding protein a-actinin (analog of the "force-velocity" relationship) has been estimated for each isoform V1 and V3 at the saturating calcium concentration. The results obtained suggest that the calcium regulation of the contractile activity of isomyosins V1 and V3 occurs by different mechanisms.