Cell Major Axis Research Articles

Abstract Tu051: ROR2 Drives Right Ventricular Failure Via Proteostatic Imbalances

Background: No therapies exist to treat right ventricular failure (RVF), in part because RVF molecular drivers have been incompletely studied. We recently identified that the developmentally restricted noncanonical WNT receptor ROR2 is re-expressed in a severity-dependent manner in human RVF. Here we test in mice if, and how, the re-expression of Ror2 causes RVF. Methods/Results: We find in neonatal rat ventricular myocytes (NRVMs) that Ror2 overexpression (Ror2 OE ) both activates protein translation and reduces Hspa1a/b mRNA and Hsp70 protein, resulting in increased protein turnover, fragmented sarcomeres, intercalated disc disruption, reduced cell major axis, impairment of contractile and relaxation kinetics, and ectopy. Ror2 knockdown (Ror2 KD ) results in opposing phenotypes. Hsp70 overexpression or proteasome inhibition rescues many of the Ror2 OE phenotypes, demonstrating their causal involvement. NRVM proteomic analysis indicates that Ror2 OE activates an ERK/p90RSK/Eef2 pathway that is known to regulate translation extension. In vivo, cardiac Ror2 OE in mice causes RV dilation and dysfunction, intercalated disc disruption, reduced Hspa1b (encoding for Hsp70), proteasome activation, and reduced cardiomyocyte ubiquitin staining. The mouse pulmonary artery banding (PAB) model of RVF reveals similar Ror2 induction and proteasome activation. Ror2 KD by AAV9 in PAB reduces RV dilation and improves RV systolic and diastolic function. Finally, in cardiac samples from human RVF, we find evidence for a similar ROR2-responsive proteostatic imbalance with increased proteasome activity and ubiquitin as well as markers of increased ERK and p90RSK. Conclusions: ROR2 is induced in human and mouse RVF, and mechanistic studies in NRVMs and mice show that the induction of ROR2 causes RVF by disrupting the proteostatic balance of translation, folding, and turnover. Knockdown or suppression of ROR2 may serve as a novel RVF therapeutic target.

Triploidy verification of Schizothorax richardsonii using erythrocyte cell and nucleus measurement

The individuals of diploid and triploid Schizothorax richardsonii (Gray, 1832) were identified using erythrocyte indices. The Erythrocyte cell's major axis of the triploid was 17% higher than the diploid. The increase in the nucleus major axis size of the erythrocyte of the triploid was also greater by 8% concerning the diploid fishes. The erythrocyte measurement ranged between 13.29±1.06 to 14.45±0.97 for diploid 15.91±1.32 to 16.49±1.23 for triploid erythrocyte nucleus 6.16±0.67 to 6.40±0.63 and 6.70±0.71 to 6.94±0.56 for diploid and triploid respectively. The data were analyzed by One Way of Analysis of Variance (ANOVA) for both diploid (control) and triploid (treated) erythrocyte and nucleus measurement which showed significant differences with p<0.05. These parameters, thus, can successfully be used in discriminating diploid and triploid S. richadrsonii.

Fabrication of cell plastics composed only of unicellular green alga Chlamydomonas reinhardtii as a raw material

Cell plastics in this study were fabricated with only unicellular green alga Chlamydomonas reinhardtii as raw materials. The sizes of cell-major axis as structures were 8.4 ± 1.2 µm, and the aspect ratios of those were 1.2 ± 0.1, showing homogeneous particle size. After optimizing extraction condition of intracellular contents, cell plastics were fabricated with the cells as ingredient components and the intracellular contents as matrix components. Those cell plastics were observed with scanning electron microscopy, displaying the smooth surfaces of the cell plastics at a low magnification level. However, the surface, especially exposed surface, were rough at high magnification level. Tensile strength test revealed that increasing the ratio of intracellular contents in the cell plastics until 21% led enhancing mechanical properties of Young’s modulus and tensile strength; however, 25% of intracellular contents displayed decreases of those properties. As the optimal point, the cell plastic (21%), which contained 21% (w/w) of intracellular contents in cell plastics, showed 764 ± 100 MPa and 8.6 ± 5.2 MPa of Young’s modulus and tensile strength. The cell plastics showed few plastic region and soon fractured, indicating the possibility that cells and intracellular contents could be electrostatically connected. Additionally, cells were shown as a negative charge and displayed the possibility to contribute electrically cell-gathering with intracellular ionic components. Therefore, cells and intracellular contents containing ionic metabolites could be electrostatically connected for giving the mechanical strength to cell plastics. In this study, we successfully demonstrated fabricating cell plastics with only cells for the first time and also showed the high possibility of conjugating each cell with the intracellular contents.Key points• Cell plastics are fabricated with unicellular green algal cell directly.• Unicellular cells required to be conjugated for the fabrication with matrix.• Cells were conjugated with intracellular contents for cell-plastic fabrication.

FC027: Uric Acid Stimulates Cytoskeleton Pathways in Vascular Smooth Muscle Cells Through F-ACTIN Polymerization and Atrogin, Αsma and SM22 up Regulation

Abstract BACKGROUND AND AIMS Hyperuricaemia (HU) has been identified as a risk factor for hypertension and renal disease. The most widely documented pathogenetic mechanisms for the uric acid (UA) mediated vascular and renal damage are vascular inflammation and remodeling. Vascular smooth muscle cells (VSMCs) possess a distinctive property of plasticity that allows the phenotypic transition and contributes to vascular remodeling. The aim of this study was to investigate UA effects on VSMC cell line, focusing on phenotypic transition and searching for possible signals involved in this process. METHOD MOVAS, a mouse VSMC cell line, was exposed for short (0–6 h) or long time (24–48 h) to 0 (No Treated cells: NT), 6, 9 and 12 mg/dL of UA, respectively. We evaluated cell viability by MTT test, migration property in a 48-well microchemotaxis chamber (using an 8 μm pore size, polycarbonate polyvinylpyrrolidone-free filters) and by phalloidin staining. Changes in cytoskeleton proteins [Smoothelin (SMT), alpha-Smooth Muscle Actin (αSMA), Smooth Muscle 22 Alpha (SM 22α)] were detected by real time polymerase chain reaction (rt-PCR) and Western blot. In addition, we evaluated angiotensin receptor 1 (AT1) and atrogin 1 expression by rt-PCR and Map kinase activation (Erk 1,2) by western blot. Lastly, we tested the UA effects on cellular changes through a prior treatment with angiotensin receptor blockers, Valsartan (V) and Losartan (L) 10 µmol. RESULTS A small increment in cell proliferation was observed at 24 h (+11%–15%; P < 0.05). UA promoted an increased migratory rate in UA treated VSMCs at 24 and 48 h respect to untreated cells (P < 0.001). These results were confirmed by F-actin intracellular distribution: the AlexaFluor 594-conjugate-phalloidin staining revealed a compact polymerization of F-actin in stress fibers along the major cell axis in untreated cells, while a re-arrangement in thinner and poorly oriented fibers localized at cortical level were found in UA treated cells (Fig. 1). When we evaluated cytoskeleton components, we found out that 24 h UA exposition rose up, SMT (2.5–3.4-fold; P < 0.05), αSMA (1.3–1.5-fold; P < 0.05) and SM 22α levels (1.3–2.5-fold P < 0.05). Conversely, we found a 48 h UA treatment caused them to drop (SMT = –20%–40%, P < 0.05; αSMA = –20%–40%, P < 0.05; SM 22α = –30%–43%, P < 0.05). Atrogin-1 was 2-fold up regulated at 48 h in UA treated VMSCs compared with NT (P = 0.04), suggesting a possible role for UA in cytoskeleton remodeling (Fig. 2). Furthermore, we observed a significant increase in VSMC area (+30%; P < 0.001) regardless of UA concentration and time exposition. Supposing a key role of Angiotensin involvement in UA induced VSMCs changes, AT1 expression was assessed. We found AT1 was up regulated in UA treated VSMCs compared with NT (P = 0.04). As expected, V and L had inhibitory effects on AT1 upregulation. Moreover, the two angiotensin receptors blockers inhibited all the phenotypic changes induced by UA. Lastly, UA induced a time dependent Erk 1, 2 phosphorylation (1.5–2.5-fold versus T0; P < 0.05–0.01), which was reversed by both L and V. CONCLUSION The results of this study show for the first time as UA-induced cytoskeletal changes involve polymerization of F-actin, Atrogin, αSMA and up-regulation of SM22.These results reveal the pathways by which UA induces an increase in VSMC area and in migratory rate, suggesting UA as a key player in vascular remodeling.

Tetraploid induction in Clarias gariepinus using cold shock protocol

This study was aimed at optimizing the induction of tetraploid in African catfish Clarias gariepinus eggs using cold shock protocol. Three temperatures (0, 5, and 10 °C) were applied at three durations (2, 5, 10 min) to suppress the first mitotic cell division at about 1.4 τ0 after fertilization. Thereafter, the treatment and control groups were incubated at room temperature till the eggs were hatched and the performance characteristics of the progenies evaluated. The result obtained showed that the 0 and 10 °C respectively resulted in poor hatchability and low tetraploid percentage of the induced eggs. However, 5 °C cold shock for 2 or 5 min gave the highest hatchability (70%) and tetraploid percentage (35%); beyond this time, performance significantly reduces. The cytogenetically confirmed tetraploid progenies (4n = 112) in this study had higher erythrocyte axis length compared to their diploid counterparts (2n = 56). The exclusive tetraploid range of 12.5–17.4 μm and 10.2–14.1 μm was identified for the cell major and minor axis respectively, hence can be used to discriminate the progenies of the two ploidy groups. However, the progenies were similar in terms of morphological characterization and growth performance. It was concluded that 5 °C cold shock for 2–5 min at about 1.4 τ0 after fertilization is sufficient to induce reasonable percentages of tetraploids in C. gariepinus.

Cell orientation under stretch: Stability of a linear viscoelastic model

The sensitivity of cells to alterations in the microenvironment and in particular to external mechanical stimuli is significant in many biological and physiological circumstances. In this regard, experimental assays demonstrated that, when a monolayer of cells cultured on an elastic substrate is subject to an external cyclic stretch with a sufficiently high frequency, a reorganization of actin stress fibres and focal adhesions happens in order to reach a stable equilibrium orientation, characterized by a precise angle between the cell major axis and the largest strain direction. To examine the frequency effect on the orientation dynamics, we propose a linear viscoelastic model that describes the coupled evolution of the cellular stress and the orientation angle. We find that cell orientation oscillates tending to an angle that is predicted by the minimization of a very general orthotropic elastic energy, as confirmed by a bifurcation analysis. Moreover, simulations show that the speed of convergence towards the predicted equilibrium orientation presents a changeover related to the viscous–elastic transition for viscoelastic materials. In particular, when the imposed oscillation period is lower than the characteristic turnover rate of the cytoskeleton and of adhesion molecules such as integrins, reorientation is significantly faster.

Separation and Enrichment of Yeast Saccharomyces cerevisiae by Shape Using Viscoelastic Microfluidics.

Yeast Saccharomyces cerevisiae (S. Cerevisiae) is one of the most attractive microbial species used for industrial production of value-added products and is an important model organism to understand the biology of the eukaryotic cells and humans. S. Cerevisiae has different shapes, such as spherical singlets, budded doublets, and clusters, corresponding to phases of the cell cycle, genetic, and environmental factors. The ability to obtain high-purity populations of uniform-shaped S. Cerevisiae cells is of significant importance for a wide range of applications in basic biological research and industrial processes. In this work, we demonstrate shape-based separation and enrichment of S. Cerevisiae using a coflow of viscoelastic and Newtonian fluids in a straight rectangular microchannel. Due to the combined effects of lift inertial and elastic forces, this label-free and continuous separation arises from shape-dependent migration of cells from the Newtonian to the non-Newtonian viscoelastic fluid. The lateral position of S. Cerevisiae cells with varying morphologies is found to be dependent on cell major axis. We also investigate the effects of sheath and sample flow rate, poly(ethylene oxide) (PEO) concentration and channel length on the performance of the viscoelastic microfluidic device for S. Cerevisiae enrichment and separation by shape. Moreover, the separation efficiency, cell extraction yield, and cell viability after sorting operations are studied.

Dataset for the morphological and erythrocytes parameters of Clarias gariepinus, Pangasianodon hypophthalmus, and their reciprocal hybrids.

Discrimination of different fishes can be done through different means which includes morphological appearance. When two fishes are successfully hybridized, they produce progenies that have shared morphology between their pure parent, hence, making morphometric characterization an important aspect of hybrid discrimination. However, erythrocyte characterization is also a simpler method for characterization. The dataset presented in this article represents the traditional morphological data, truss network data and erythrocyte data of pure and novel hybrids from reciprocal crosses of African catfish Clarias gariepinus and Asian catfish Pangasianodon hypophthalmus. Breeding of the broodstocks was done to produce pure and hybrid progenies which were maintained for a period of four to six months. Based on the cross combinations and morphotypes, traditional measurement of twenty-five morphological characters and five meristic counts were recorded. Thereafter pictures of the different fish groups were used to determine values of thirty-six distances between ten landmark points. The morphological abnormality of the hybrids at market size is also presented in this data article for the very first time. Blood was then collected from the caudal peduncle of ten fish per group and smeared on a slide for observation under a compound microscope (at 100 × magnification). Data gotten included erythrocytes parameters such as cell major axis, cell minor axis, nucleus major axis, nucleus minor axis cell area, nucleus area, cell volume, and nucleus volume. Data recording was through the Microsoft excel spreadsheet; which was also used to process the data to get the exclusive ranges of values for paired progenies. The data as presented is associated with the research article “Morphological characterization of the progenies of pure and reciprocal crosses of Pangasianodon hypophthalmus (Sauvage, 1878) and Clarias gariepinus (Burchell, 1822)” [1]. The dataset presented in this article can be used for easy identification of the novel hybrid progenies of the African Catfish and Asian Catfish.

Effect of electric induced triploidization on sex ratio, growth and gonad histology of red hybrid tilapia

Some effects of electric induced triploidization on Red Hybrid Tilapia were investigated in this study. With the aid of 12 V battery, electric current was used to induce shock in fertilized eggs of red hybrid tilapia for a duration of 10 min (at about 4 min after fertilization). Triploidy was confirmed to be 93 ± 1.6% using the exclusive triploid range of cell major axis previously reported for the same species and cytogenetic analysis (3n = 66 and 2n = 44). After four months of culture, it was observed that triploid induced progenies were male biased (%male = 77 ± 1.2%) compared to their diploid counterparts (%male = 46 ± 0.8%). The triploid female progenies observed in this study also showed significantly low values of gonadosomatic index which were thought to have been a result of the underdevelopment of the ovaries as revealed by gonad histology. Also, the final weight of the male triploid progenies was significantly higher compared to those of the diploids. It was concluded that the induction of triploidy in Red Hybrid Tilapia through electric shock among other things may have skewed sex ratio in favour of the males.

Efficiency and Robustness of Processes Driven by Nucleoid Exclusion in Escherichia coli.

The internal spatial organization of prokaryotic organisms, including Escherichia coli, is essential for the proper functioning of processes such as cell division. One source of this organization in E. coli is the nucleoid, which causes the exclusion of macromolecules - e.g. protein aggregates and the chemotaxis network - from midcell. Similarly, following DNA replication, the nucleoid(s) assist in placing the Z-ring at midcell. These processes need to be efficient in optimal conditions and robust to suboptimal conditions. After reviewing recent findings on these topics, we make use of past data to study the efficiency of the spatial constraining of Z-rings, chemotaxis networks, and protein aggregates, as a function of the nucleoid(s) morphology. Also, we compare the robustness of these processes to nonoptimal temperatures. We show that Z-rings, Tsr clusters, and protein aggregates have temperature-dependent spatial distributions along the major cell axis that are consistent with the nucleoid(s) morphology and the volume-exclusion phenomenon. Surprisingly, the consequences of the changes in nucleoid size with temperature are most visible in the kurtosis of these spatial distributions, in that it has a statistically significant linear correlation with the mean nucleoid length and, in the case of Z-rings, with the distance between nucleoids prior to cell division. Interestingly, we also find a negative, statistically significant linear correlation between the efficiency of these processes at the optimal condition and their robustness to suboptimal conditions, suggesting a trade-off between these traits.

Evaluation of the t-system of rat cardiomyocytes during early stages of streptozotocin-induced diabetes

Disorganization of the T-system of cardiomyocytes is considered an early and critical step in the development of Diabetic Cardiomyopathy (DCM). To test this suggestion, male Wistar rats were injected with streptozotocin (STZ, 30 or 45 mg/kg) and studied one month later. STZ-rats that developed and maintained hyperglycemia (random blood glucose > 11 mM) were designated as hyperglycemic (STZ-HG) rats, while the remaining STZ-rats – as normoglycemic (STZ-NG) animals. The structural integrity of the T-system was investigated using an analysis of confocal images of the left ventricle (LV) sub-epicardium of isolated hearts, stained with the Di-8-ANEPPS. In control, T-system was organized into regular networks of t-tubules aligned with Z-discs of cardiomyocyte’s sarcomeres. Accordingly, the frequency distributions of intervals between neighboring t-tubules (INT, measured along the major cell axis) peaked at a 2 µm value with not more than 21% of INT (per cell) exceeding the 3 µm cut-off. Only 4±3% of the control cardiomyocytes (274 cells, 4 rats) could be considered as deficient, according to this parameter (>21% occurrence of long INT). Compared to control, in the hearts of STZ-NG and STZ-HG rats, the fractions of such deficient cardiomyocytes were statistically significantly higher: 48±13% (STZ-NG, 8 rats, 573 cells) and 76±8% (STZ-HG, 4 rats, 247 cells). Thus, structural changes in the T-system of the rat heart LV cardiomyocytes develop early during chronic hyperglycemia (overt diabetes) as well as during near-normoglycemic stages of diabetes (prediabetes). The relevance of these changes to the development of DCM in subjects with prediabetes remains to be studied.

Accuracy of electromagnetic models to estimate cardiomyocyte membrane polarization.

External electric fields (E) induce a spatially heterogeneous variation in the membrane potential (ΔVm) of cardiomyocytes that, if sufficiently large, results in an action potential and contraction. Insights into the phenomenon of ΔVm induction by E have been classically gained with electromagnetic models due to the lack of adequate experimental approaches. However, it is not clear yet how reliable these models are. To assess the accuracy of commonly used models, a reference 3D numerical model for cardiomyocytes (NMReal) was developed, consisting of the cell membrane shell reconstructed from rendered confocal microscopy images of freshly isolated ventricular myocytes. NMReal was used to estimate the E-induced maximum ΔVm values (ΔVmax), which were compared with estimates from seven other electromagnetic models. Accurate ΔVmax estimates (average error < 2%) were obtained with a less complex 3D model (NM3D) based on the extruded 2D image of the cell longitudinal section. Acceptable ΔVmax estimates (average error < 5%) were obtained with the prolate spheroid analytical model (PSAM) when the angle of E incidence and the cell major axis was < 30°. In this case, PSAM, a much simpler model requiring only the measurement of the longitudinal and transversal cell dimensions, can be a suitable alternative for ΔVmax calculation. Graphical abstract (A) Confocal images of the cell were used to reconstruct the realistic geometry of cardiomyocytes (NMReal). (B) NMReal was used to estimate the maximum variation in the transmembrane potential (ΔVmax) induced by an external electric field (E) applied at different angles with respect to the cell major axis. Plus (anode) and minus (cathode) signs indicate electrode position (E direction is from minus to plus). (C) Relative error (vs. NMReal) of ΔVmax estimation with simplified electromagnetic models, presented in descending order of accuracy (left-to-right, top-to-bottom). NM2D: 2D numerical model based on the longitudinal cell image; NM3D: numerical model based on the z extrusion of NM2D; EAM, PSAM, and CAM: ellipsoidal, prolate spheroidal, and cylindrical analytical models, respectively; PNM and CNM: parallelepipedal and cylindrical numerical models, respectively.

New insight on the relationship between lethal electrical fields versus cardiomyocyte orientation

Abstract Introduction Cardiovascular diseases represent a major cause of death world-wide and one of their greatest complications is the development of cardiac arrhythmias, in which ventricular fibrillation (VF) stands out as the most severe one. The only therapy that reverses VF is defibrillation. However defibrillatory shock is capable of killing heart cells and it is known that the orientation of the cell major axis with respect to the electrical field (E) direction is a determining factor for cellular excitation and injury, which is leading to the development of new defibrillation protocols. The aim of this work is to fill the gap in information about cell lethality for intermediate cell orientation angles. Methods Ventricular myocytes were extracted from adult male Wistar rats and the cells were plated in a chamber for perfusion and stimulation with bipolar voltage pulses to determine the stimulation threshold (ET). Then, monopolar stimulus was applied and amplitude was increased until cell lethal injury. This protocol was performed on four experimental groups: cells oriented at 0°, 30°, 60° and 90°, with respect to E direction. Results 87 cells were analyzed and an increase in amplitude of E associated with 50% lethality (E50) was verified as the direction of E application and cell major axis orientation departed. Conclusion Taken the same probability of lethality, our data suggest a nonlinear increase of E amplitude from 0° to 90° similar to that of ET. These in-between data had not yet been shown and are important for service-based future defibrillation protocols.

Triploidy induction by electric shock in red hybrid tilapia

The induction of triploidy in red hybrid tilapia (Oreochromis mossambicus × Oreochromis niloticus) using electric-shock at different duration (3, 5, 10, 15 and 20 min) is reported in this study. Triploidy induction was attempted by exposing fertilized egg to electric field maintained at 12 V at approximately 4 min after fertilization. The efficiency of triploidization was confirmed by counting the number of chromosomes and affirmed by assessing erythrocyte biometrics of one month old fingerlings. The result obtained shows that shock duration of 10 min was most effective as it induced triploidy in 87% of the fish in this group with a hatching rate of 68%. The chromosome count of induced triploids was 3n = 66 while diploid fish had 2n = 44. Also, many erythrocyte biometrics of the triploid hybrid tilapia were significantly larger than those of the diploid groups (between 12 and 56% increments). Out of all the erythrocyte parameters measured or calculated, the cell major axis had the highest percentage of erythrocytes (91.4%) that were within the exclusive triploid range (12.61–15.98 μm). Hence, despite the dominant oval shape of both diploid and triploid erythrocytes, discrimination of the entire fish groups was accurately done by solely accessing the exclusive range of the cell major axis. It was concluded that application of an electric field of 12 V for 10 mins was better for triploidization of the red hybrid tilapia eggs.

Cardiomyocyte lethality by multidirectional stimuli.

Multidirectional defibrillation protocols have shown better efficiency than monodirectional; still, no testing was performed to assess cell lethality. We investigated lethality of multidirectional defibrillator-like shocks on isolated cardiomyocytes. Cells were isolated from adult male Wistar rats and plated into a perfusion chamber. Electrical field stimulation threshold (ET) was obtained, and cells were paced with suprathreshold bipolar electrical field (E) pulses. Either one monodirectional high-intensity electrical field (HEF) pulse aligned at 0° (group Mono0) or 60° (group Mono60) to cell major axis or a multidirectional sequence of three HEF pulses aligned at 0°, 60°, and 120° each was applied. If cell recovered from shock, pacing was resumed, and a higher amplitude HEF, proportional to ET, was applied. The sequence was repeated until cell death. Lethality curves were built by means of survival analysis from sub-lethal and lethal E. Non-linear fit was performed, and E values corresponding to 50% probability of lethality (E50) were compared. Multidirectional groups presented lethality curves similar to Mono0. Mono60 displayed the highest E50. The novel data endorse the idea of multidirectional stimuli being safer because their effects on lethality of individual cells were equal to a single monodirectional stimulus, while their defibrillatory threshold is lower. Graphical abstract Monodirectional and multidirectional lethality protocol comparison on isolated rat cardiomyocytes. The heart image is a derivative of "3D Heart in zBrush" ( https://vimeo.com/65568770 ) by Laloxl, used under CC BY 3.0 ( https://creativecommons.org/licenses/by/3.0/legalcode )/image extracted from original video.

Effects of cell state and staining on femtosecond laser nanosurgery.

Femtosecond laser nanosurgery enables precise manipulation of subcellular elements to study regeneration. However, currently it is not frequently employed-probably because of its unknown consequences on the whole cell level. To better understand the associated biological response of the cell, especially in the context of different cell states and cell staining, we manipulated C2C12 myoblasts and myotubes, which were either unstained (nicotinamide adenine dinucleotide signal) or stained with MitoTracker Red. Both signals overlap well and stain similar areas in untreated cells. We chose 3 different cutting lengths and performed surgery in the cytosol along the major cell axis. The cuts resealed within several minutes independent of the cutting length. We analyzed cell area, perimeter, major and minor axis on long term. We observed significant changes in the cell area and perimeter, dependent on the staining and more pronounced in differentiated myotubes. We conclude, that laser parameters must be chosen carefully, depending on the staining of the cell, its (differentiation) state, and the extent of the cut region, such that unwanted cell responses can be avoided. Laser manipulation of C2C12 myotubes with small ablation (0.8 μm) and large ablation (3.0 μm). While small damages recover, larger damages lead to elimination from the syncytium. Scale bar: 20 μm.

System for open-chest, multidirectional electrical defibrillation

Abstract Introduction: Cardiomyocytes are more sensitive to stimulatory electrical fields when the latter are applied longitudinally to the cell major axis. In the whole heart, cells have different spatial orientations, which may limit the effectiveness of conventional electrical defibrillation (i.e., shock delivery in a single direction). This article describes the constructive aspects of a portable system for rapidly-switching, multidirectional stimulus delivery, composed of an electrical defibrillator and multielectrode-bearing paddles for direct cardiac defibrillation. Methods: The defibrillator delivers monophasic, truncated monoexponential waveforms with energy up to 7.3 J. Upon selection of the defibrillation modality (unidirectional or multidirectional), shock delivery is triggered through 1 or 3 outputs. In the latter case, triggering is sequentially switched to the outputs, without interval or temporal overlap. Each paddle contains 3 electrodes that define shock pathways spaced by 60°. The system was tested in vivo for reversal of experimentally-induced ventricular fibrillation in healthy swine, using 30- and 20-ms long shocks (N= 4 in each group). Results: The defibrillator delivers identical stimulus waveforms through all outputs in both stimulation modalities. In all animals, successful defibrillation required lower shock energy when 20 ms-long stimuli were applied in 3 directions, compared to a single direction. However, performance was poorer with multidirectional defibrillation for 30 ms-long shocks. Conclusion: The delivery of identical shock waveforms allowed confirmation that multidirectional defibrillation can promote restoration of sinus rhythm with lower shock energy, which may reduce myocardial electrical damage during defibrillation. Nevertheless, increase in shock duration greatly impairs the effectiveness of this defibrillation modality.

Focal adhesion kinase regulation in stem cell alignment and spreading on nanofibers

While electrospun nanofibers have demonstrated the potential for novel tissue engineering scaffolds, very little is known about the molecular mechanism of how cells sense and adapt to nanofibers. Here, we revealed the role of focal adhesion kinase (FAK), one of the key molecular sensors in the focal adhesion complex, in regulating mesenchymal stem cell (MSC) shaping on nanofibers. We produced uniaxially aligned and randomly distributed nanofibers from poly(l-lactic acid) to have the same diameters (about 130 nm) and evaluated MSC behavior on these nanofibers comparing with that on flat PLLA control. C3H10T1/2 murine MSCs exhibited upregulations in FAK expression and phosphorylation (pY397) on nanofibrous cultures as assessed by immunoblotting, and this trend was even greater on aligned nanofibers. MSCs showed significantly elongated and well-spread morphologies on aligned and random nanofibers, respectively. In the presence of FAK silencing via small hairpin RNA (shRNA), cell elongation length in the aligned nanofiber direction (cell major axis length) was significantly decreased, while cells still showed preferred orientation along the aligned nanofibers. On random nanofibers, MSCs with FAK-shRNA showed impaired cell spreading resulting in smaller cell area and higher circularity. Our study provides new data on how MSCs shape their morphologies on aligned and random nanofibrous cultures potentially via FAK-mediated mechanism.

Robustness of the Process of Nucleoid Exclusion of Protein Aggregates in Escherichia coli.

Escherichia coli segregates protein aggregates to the poles by nucleoid exclusion. Combined with cell divisions, this generates heterogeneous aggregate distributions in subsequent cell generations. We studied the robustness of this process with differing medium richness and antibiotics stress, which affect nucleoid size, using multimodal, time-lapse microscopy of live cells expressing both a fluorescently tagged chaperone (IbpA), which identifies in vivo the location of aggregates, and HupA-mCherry, a fluorescent variant of a nucleoid-associated protein. We find that the relative sizes of the nucleoid's major and minor axes change widely, in a positively correlated fashion, with medium richness and antibiotic stress. The aggregate's distribution along the major cell axis also changes between conditions and in agreement with the nucleoid exclusion phenomenon. Consequently, the fraction of aggregates at the midcell region prior to cell division differs between conditions, which will affect the degree of asymmetries in the partitioning of aggregates between cells of future generations. Finally, from the location of the peak of anisotropy in the aggregate displacement distribution, the nucleoid relative size, and the spatiotemporal aggregate distribution, we find that the exclusion of detectable aggregates from midcell is most pronounced in cells with mid-sized nucleoids, which are most common under optimal conditions. We conclude that the aggregate management mechanisms of E. coli are significantly robust but are not immune to stresses due to the tangible effect that these have on nucleoid size. Escherichia coli segregates protein aggregates to the poles by nucleoid exclusion. From live single-cell microscopy studies of the robustness of this process to various stresses known to affect nucleoid size, we find that nucleoid size and aggregate preferential locations change concordantly between conditions. Also, the degree of influence of the nucleoid on aggregate positioning differs between conditions, causing aggregate numbers at midcell to differ in cell division events, which will affect the degree of asymmetries in the partitioning of aggregates between cells of future generations. Finally, we find that aggregate segregation to the cell poles is most pronounced in cells with mid-sized nucleoids. We conclude that the energy-free process of the midcell exclusion of aggregates partially loses effectiveness under stressful conditions.

In Vivo Kinetics of Segregation and Polar Retention of MS2-GFP-RNA Complexes in Escherichia coli

The cytoplasm of Escherichia coli is a crowded, heterogeneous environment. From single cell live imaging, we investigated the spatial kinetics and heterogeneities of synthetic RNA-protein complexes. First, although their known tendency to accumulate at the cell poles does not appear to introduce asymmetries between older and newer cell poles within a cell lifetime, these emerge with cell divisions. This suggests strong polar retention of the complexes, which we verified in their history of positions and mean escape time from the poles. Next, we show that the polar retention relies on anisotropies in the displacement distribution in the region between midcell and poles, whereas the speed is homogeneous along the major cell axis. Afterward, we establish that these regions are at the border of the nucleoid and shift outward with cell growth, due to the nucleoid’s replication. Overall, the spatiotemporal kinetics of the complexes, which is robust to suboptimal temperatures, suggests that nucleoid occlusion is a source of dynamic heterogeneities of macromolecules in E. coli that ultimately generate phenotypic differences between sister cells.