Changes In Cell Length Research Articles

Improved Membrane Permeability via Hypervesiculation for In Situ Recovery of Lycopene in Escherichia coli.

Lycopene biosynthesis is frequently hampered by downstream processing hugely due to its inability to be secreted out from the producing chassis. Engineering cell factories can resolve this issue by secreting this hydrophobic compound. A highly permeable E.coli strain was developed for a better release rate of lycopene. Specifically, the heterologous mevalonate pathway and crtEBI genes from Corynebacterium glutamicum were overexpressed in Escherichia coli BL21 (DE3) for lycopene synthesis. To ensure in situ lycopene production, murein lipoprotein, lipoprotein NlpI, inner membrane permease protein, and membrane-anchored protein in TolA-TolQ-TolR were deleted for improved membrane permeability. The final strain, LYC-8, produced 438.44 ± 8.11 and 136.94 ± 1.94 mg/L of extracellular and intracellular lycopene in fed-batch fermentation. Both proteomics and lipidomics analyses of secreted outer membrane vesicles were perfect indicators of hypervesiculation. Changes in the ratio of saturated fatty acids, unsaturated fatty acids, and cyclopropane fatty acids coupled with the branching and acyl chain lengths altered the membrane fatty acid composition. This ensured membrane fluidity and permeability for in situ lycopene release. The combinatorial deletion of these genes altered the cellular morphology. The structural and morphological changes in cell shape, size, and length were associated with changes in the mechanical strength of the cell envelope. The enhanced lycopene production and secretion mediated by improved membrane permeability established a cell lysis-free system for an efficient releasing rate and downstream processing, demonstrating the importance of vesicle-associated membrane permeability in efficient lycopene production.

Vibratory responses to outer hair cell-generated distortion products along the width of the mouse organ of Corti

Mammalian hearing sensitivity depends on the amplification of cochlear vibrations by outer hair cells (OHCs). OHCs are thought to generate amplifying forces through electromotility, i.e., voltage-driven changes in cell length, though how exactly these forces influence the motion of the surrounding organ of Corti structures remains incompletely understood. Here, in the mouse cochlear apex, optical coherence tomography was used to characterize electromotility-induced vibrations along the axis of the OHCs and along the widths of both the basilar membrane (BM) and reticular lamina (RL). Two-tone stimuli were used to elicit OHC-driven vibrations at a variety of intermodulation distortion-product (DP) frequencies. For a wide range of DP frequencies, out-of-phase motions were observed between the bottoms and tops of the OHCs, as well as between the tops of the OHCs and the lateral supporting cells. However, along the BM, phase variations were only observed at frequencies above the characteristic frequency (CF), with in-phase motions being observed at lower frequencies. The data indicate that OHCs can rapidly deform the top of the organ of Corti, while the BM response is more complex, likely being dominated by traveling-wave energy at and below the CF. [Work supported by NIH/NIDCD R21 DC019209 and the Hearing Health Foundation.]

Two kinematic gains underlying the organ of Corti mechanotransduction

The organ of Corti (OoC) is the sensory epithelium in the mammalian hearing organ where vibrations are encoded into neural signals. The OoC vibrates due to external and internal agitations. Externally, differential fluid pressures across the OoC cause vibrations. Internally, actuator cells (outer hair cells) are known to boost the vibrations. These two agitations result in distinct vibration patterns. Eventually, these vibrations deflect the stereocilia bundle to activate mechanotransduction channels in them. How OoC vibrations result in the hair cell mechanotransduction is critical to hearing research, but a fundamental information (kinematic gain) has not been quantified clearly. Using the optical coherence tomography, we measured 2-D vibrations of the OoC in cochlear turns acutely excised from young Mongolian gerbils. The excised tissues were stimulated either mechanically or electrically, which are comparable to external or internal agitations, respectively. The stereocilia deflection was estimated from relative motion between overlying and underlying structures. Two kinematic gains were defined. Passive kinematic gain represents the amplitude ratio between the stereocilia deflection and the basilar membrane transverse vibration. Active kinematic gain represents the amplitude ratio between the stereocilia deflection and the outer hair cell length change. (Work supported by NIH NIDCD R01 DC014685.)

Photo Phenosizer, a rapid machine learning-based method to measure cell dimensions in fission yeast.

Cell metrics such as area, length, and width provide informative data about cell cycle dynamics. Factors that affect these dimensions include environmental conditions and genotypic differences. Fission yeast ( Schizosaccharomyces pombe ) is a rod-shaped ascomycete fungus in which cell cycle progression is linked to changes in cell length. Microscopy work to obtain these metrics places considerable burdens on time and effort. We now report on Photo Phenosizer (PP), a machine learning-based methodology that measures cell dimensions in fission yeast. It does this in an unbiased, automated manner and streamlines workflow from image acquisition to statistical analysis. Using this new approach, we constructed an efficient and flexible pipeline for experiments involving different growth media (YES and EMM) and treatments (Untreated and MMS) as well as different genotypes ( cut6-621 versus wildtype). This methodology allows for the analysis of larger sample sizes and faster image processing relative to manual segmentation. Our findings suggest that researchers using PP can quickly and efficiently determine cell size differences under various conditions that highlight genetic or environmental disruptions.

Abstract P413: Stretch-induced Caveolae-mediated Disruption Of Cyclic AMP Microdomains Leads To Arrhythmogenic Ca 2+ Mishandling In Mouse Atrial Myocytes

Background: Atrial fibrillation (AF) often occurs during hypertension and is associated with an increase in cardiomyocyte stretch. Mechanism of ectopic beats, that trigger AF, has been linked to Ca 2+ mishandling and leaky hyperphosphorylated ryanodine receptors (RyRs), while the underlying mechanisms remain elusive. Caveolae membrane structures are involved in cell mechanosensing processes and control the cAMP signaling pathway. We hypothesized that mechanical stretch disrupts caveolae, promoting cAMP production and sarcoplasmic reticulum Ca 2+ leak via augmentation of RyRs phosphorylation. Methods and Results: Cell size analysis and Ca 2+ dynamics measurements were performed by confocal imaging of isolated mouse atrial myocytes. Cell stretch was modeled by hypoosmotic swelling (from 310 mOsM to 220 mOsM to flatten caveolae structures) resulting in a ~30% increase in cell width (p<0.05) with no changes in cell length. Swelling resulted in a biphasic effect on Ca 2+ spark activity: a fast (<10 min of exposure) ~50% increase (p<0.001) followed by a slow decrease to the level observed in isotonic conditions (>30 min of exposure). Similarly, caveolae disruption via cholesterol depletion by 10 mM methyl-β-cyclodextrin (MβCD) led to 2-fold increase in Ca 2+ sparks frequency (p<0.001). Swelling- and MβCD-induced increases in atrial Ca 2+ spark activity were prevented via inhibition of cAMP production by adenylyl cyclases by 0.1mM SQ22536 or cAMP-dependent protein kinase A (PKA) by 1μM H-89. Then, we tested if this mechanism is present in atrial myocytes from pressure-overloaded (4-weeks transaortic constriction, TAC) mice. Atrial myocytes from TAC mice showed a 1.6 times higher Ca 2+ sparks frequency than wild-type myocytes (p<0.01), which was significantly reduced (p<0.01) to wild-type level after incubation with SQ22536. Conclusions: Our findings suggest that cell stretch increases spontaneous Ca 2+ spark activity through the disruption of caveolae and cAMP-mediated augmentation of PKA activity. This mechanism could be involved in the Ca 2+ mishandling and AF in pressure overloaded hearts.

Hyperglycemia and hyperlipidemia can induce morphophysiological changes in rat cardiac cell line

H9c2 cardiac cells were incubated under the control condition and at different hyperglycemic and hyperlipidemic media, and the following parameters were determined and quantified: a) cell death, b) type of cell death, and c) changes in cell length, width and height. Of all the proven media, the one that showed the greatest differences compared to the control was the medium glucose (G) 33 mM + 500 μM palmitic acid. This condition was called the hyperglycemic and hyperlipidemic condition (HHC). Incubation of H9c2 cells in HHC promoted 5.2 times greater total cell death when compared to the control. Of the total death ofthe HHC cells, 38.6% was late apoptotic and 8.3% early apoptotic. HHC also changes cell morphology.The reordering of the actin cytoskeleton and cell stiffness was also studied in control and HHC cells. The actin cytoskeleton was quantified and the number and distance of actin bundles were not the same in the control as under HHC. Young's modulus images show a map of cell stiffness. Cells incubated in HHC with the reordered actin cytoskeleton were stiffer than those incubated in control. The region of greatest stiffness was the peripheral zone of HHC cells (where the number of actin bundles was higher and the distance between them smaller).Our results suggest a correlation between the reordering of the actin cytoskeleton and cell stiffness. Thus, our study showed that HHC can promote morphophysiological changes in rat cardiac cells confirming that gluco-and lipotoxicity may play a central role in the development of diabetic cardiomyopathy.

Yield Performance of Shade Tolerant Soybean Cultivars under Shaded Environment at Various Planting Densities

Expanding soybean [Glycine max (L.) Merr.] production under sub-optimum environment faces the problem of production under a shaded environment. The study aimed to evaluate the yield performance of shade-tolerant soybean cultivars under shaded environment conditions at various planting densities and to determine its related traits. A nested design field experiment was conducted at Banjar Sub-district, Pandeglang District, Banten Province Indonesia (lat. 6.37° S, long. 106.11° S) during the dry season from April to July 2019. Two field conditions, under coconut and banana plantation, which were identified as light-shaded (10-20 % shaded) and deep-shaded (40-50 % shaded) were used as main plots. Three soybean cultivars, Dena-1, as identified as shade-tolerant soybean cultivar, Detap-1 and Devon-1 were arranged at each environment with a single row (40 x 20 cm; normal density; 12.5 plants m−2) or double row (30 x 20 x 50 cm; high density; 14.5 plants m−2). Research results showed that seed yield decreased from 248 g m−2 to 205 g m−2 by the difference of shading. The yield decrease was due to the decrease of total biomass (TDW), branch number, node number, pod number, and seed number, and stomatal density (Nstoma) without any change in harvest index (HI) and guard cell length (Lguard). In plant arrangement, a better seed yield and TDW were resulted from normal density as compared to high density. Under the deep-shaded condition, Dena-1 under normal density (230 g m−2) was superior in seed yield but declined significantly when it was grown under high density (161 g m−2). Based on the above results, combining shade-tolerant soybean cultivar (Dena-1) and planting density (single row; 12.5 plants m−2) is recommended to maintain soybean seed yield under a shaded environment.

On the adhesion–velocity relation and length adaptation of motile cells on stepped fibronectin lanes

The biphasic adhesion-velocity relation is a universal observation in mesenchymal cell motility. It has been explained by adhesion-promoted forces pushing the front and resisting motion at the rear. Yet, there is little quantitative understanding of how these forces control cell velocity. We study motion of MDA-MB-231 cells on microlanes with fields of alternating Fibronectin densities to address this topic and derive a mathematical model from the leading-edge force balance and the force-dependent polymerization rate. It reproduces quantitatively our measured adhesion-velocity relation and results with keratocytes, PtK1 cells, and CHO cells. Our results confirm that the force pushing the leading-edge membrane drives lamellipodial retrograde flow. Forces resisting motion originate along the whole cell length. All motion-related forces are controlled by adhesion and velocity, which allows motion, even with higher Fibronectin density at the rear than at the front. We find the pathway from Fibronectin density to adhesion structures to involve strong positive feedbacks. Suppressing myosin activity reduces the positive feedback. At transitions between different Fibronectin densities, steady motion is perturbed and leads to changes of cell length and front and rear velocity. Cells exhibit an intrinsic length set by adhesion strength, which, together with the length dynamics, suggests a spring-like front-rear interaction force. We provide a quantitative mechanistic picture of the adhesion-velocity relation and cell response to adhesion changes integrating force-dependent polymerization, retrograde flow, positive feedback from integrin to adhesion structures, and spring-like front-rear interaction.

Characterization of Heterogeneity and Dynamics of Lysis of Single Bacillus subtilis Cells upon Prophage Induction During Spore Germination, Outgrowth, and Vegetative Growth Using Raman Tweezers and Live-Cell Phase-Contrast Microscopy.

A prophage comprises a bacteriophage genome that has integrated into a host bacterium's DNA, which generally permits the cell to grow and divide normally. However, the prophage can be induced by various stresses, or induction can occur spontaneously. After prophage induction, viral replication and production of endolysins begin until the cell lyses and phage particles are released. However, the heterogeneity of prophage induction and lysis of individual cells in a population and the dynamics of a cell undergoing lysis by prophage induction have not been fully characterized. Here, we used Raman tweezers and live-cell phase-contrast microscopy to characterize the Raman spectral and cell length changes that occur during the lysis of individual Bacillus subtilis cells from spores that carry PBSX prophage during spores' germination, outgrowth, and then vegetative growth. Major findings of this work are as follows: (i) After addition of xylose to trigger prophage induction, the intensities of Raman spectral bands associated with nucleic acids of single cells in induced cultures gradually fell to zero, in contrast to the much smaller changes in protein band intensities and no changes in nucleic acid bands in uninduced cultures; (ii) the nucleic acid band intensities from an individual induced cell exhibited a rapid decrease, following a long lag period; (iii) after the addition of nutrient-rich medium with xylose, single spores underwent a long period (228 ± 41.4 min) for germination, outgrowth, and vegetative growth, followed by a short period of cell burst in 1.5 ± 0.8 min at a cell length of 8.2 ± 5.5 μm; (iv) the latent time (Tlatent) between the addition of xylose and the start of cell burst was heterogeneous in cell populations; however, the period (ΔTburst) from the latent time to the completion of cell lysis was quite small; (v) in a poor medium with l-alanine alone, addition of xylose caused prophage induction following spore germination but with longer Tlatent and ΔTburst times and without cell elongation; (vi) spontaneous prophage induction and lysis of individual cells from spores in a minimal nutrient medium were observed without xylose addition, and cell length prior to cell lysis was ∼4.1 μm, but spontaneous prophage induction was not observed in a rich medium; (vii) in a rich medium, addition of xylose at a time well after spore germination and outgrowth significantly shortened the average Tlatent time. The results of this study provide new insights into the heterogeneity and dynamics of lysis of individual B. subtilis cells derived from spores upon prophage induction.

Measurement of Skeletal Muscle Fiber Contractility with High-Speed Traction Microscopy

We describe a technique for simultaneous quantification of the contractile forces and cytosolic calcium dynamics of muscle fibers embedded in three-dimensional biopolymer gels under auxotonic loading conditions. We derive a scaling law for linear elastic matrices such as basement membrane extract hydrogels (Matrigel) that allows us to measure contractile force from the shape of the relaxed and contracted muscle cell and the Young’s modulus of the matrix without further knowledge of the matrix deformations surrounding the cell and without performing computationally intensive inverse force reconstruction algorithms. We apply our method to isolated mouse flexor digitorum brevis (FDB) fibers that are embedded in 10 mg/mL Matrigel. Upon electrical stimulation, individual FDB fibers show twitch forces of 0.37 ± 0.15 μN and tetanic forces (100-Hz stimulation frequency) of 2.38 ± 0.71 μN, corresponding to a tension of 0.44 ± 0.25 kPa and 2.53 ± 1.17 kPa, respectively. Contractile forces of FDB fibers increase in response to caffeine and the troponin-calcium stabilizer tirasemtiv, similar to responses measured in whole muscle. From simultaneous high-speed measurements of cell length changes and cytosolic calcium concentration using confocal line scanning at a frequency of 2048 Hz, we show that twitch and tetanic force responses to electric pulses follow the low-pass filtered calcium signal. In summary, we present a technically simple high-speed method for measuring contractile forces and cytosolic calcium dynamics of single muscle fibers. We expect that our method will help to reduce preparation time, costs, and the number of sacrificed animals needed for experiments such as drug testing.

Effect of starch/CNT on biodesulfurization using molecular dynamic simulation.

Nowadays, fossil fuel is the most important source of energy. However, the sulfur oxide release through oxidation of the available sulfur and the resultant air pollution has turned into an issue. In biodesulfurization (BDS) process, the sulfur from the recalcitrant organic compounds dissolved in crude oil fractions will be removed biologically. Carbon nanotubes (CNTs) exhibit good catalytic performance in dibenzothiophene (DBT) oxidation. Molecular dynamic simulation is the best and the only way to reach this end. Through this study, molecular dynamic simulations are applied to compute the effects of starch/CNTs on BDS process of DBT during 5 ns. The changes of cell length, energy, dynamic temperature, relative concentration of DBT, and radial distribution function (RDF) in the absence and presence of starch/CNTs were investigated. Regarding to the energy diagram, the fluctuation because of temperature fluctuations reaches the stable state. The high level of the first peak in RDF diagram showed the high and long desulfurization by microorganism. All the results showed stable and reasonable equilibrium state of the system. According to the graphs, the simultaneous in the presence of starch/CNT and Rhodococcus erythropolis increase the removal efficiency of nitrate reached 85% and in the absence of nanoparticle was 35.44%.

Outer hair cell electromotility is low-pass filtered relative to the molecular conformational changes that produce nonlinear capacitance.

The outer hair cell (OHC) of the organ of Corti underlies a process that enhances hearing, termed cochlear amplification. The cell possesses a unique voltage-sensing protein, prestin, that changes conformation to cause cell length changes, a process termed electromotility (eM). The prestin voltage sensor generates a capacitance that is both voltage- and frequency-dependent, peaking at a characteristic membrane voltage (Vh), which can be greater than the linear capacitance of the OHC. Accordingly, the OHC membrane time constant depends upon resting potential and the frequency of AC stimulation. The confounding influence of this multifarious time constant on eM frequency response has never been addressed. After correcting for this influence on the whole-cell voltage clamp time constant, we find that both guinea pig and mouse OHC eM is low pass, substantially attenuating in magnitude within the frequency bandwidth of human speech. The frequency response is slowest at Vh, with a cut-off, approximated by single Lorentzian fits within that bandwidth, near 1.5 kHz for the guinea pig OHC and near 4.3 kHz for the mouse OHC, each increasing in a U-shaped manner as holding voltage deviates from Vh Nonlinear capacitance (NLC) measurements follow this pattern, with cut-offs about double that for eM. Macro-patch experiments on OHC lateral membranes, where voltage delivery has high fidelity, confirms low pass roll-off for NLC. The U-shaped voltage dependence of the eM roll-off frequency is consistent with prestin's voltage-dependent transition rates. Modeling indicates that the disparity in frequency cut-offs between eM and NLC may be attributed to viscoelastic coupling between prestin's molecular conformations and nanoscale movements of the cell, possibly via the cytoskeleton, indicating that eM is limited by the OHC's internal environment, as well as the external environment. Our data suggest that the influence of OHC eM on cochlear amplification at higher frequencies needs reassessment.

Light-emitting diode irradiation using 660 nm promotes human fibroblast HSP90 expression and changes cellular activity and morphology.

We evaluated changes in cell viability and morphology in response to low-level light irradiation and underlying variations in the levels of heat shock proteins (HSPs). Human fibroblasts were irradiated with a light-emitting diode (LED) array at 660 nm (50 mW for 15, 30, and 60 minutes). Cell viability and morphological changes were evaluated via epifluorescence analysis; we also assessed cell viability and length changes. The expression levels of adenosine triphosphate (ATP) and various HSPs (HSP27, 60, 70, and 90) were analyzed by immunohistochemical staining, Western blotting and microarray analysis. After LED irradiation, cellular viability and morphology changed. Of the several HSPs analyzed, the HSP90 level increased significantly, suggesting that this protein played roles in the morphological and cellular changes. Thus, low-level irradiation triggered cellular changes mediated by increased HSP90 expression; this may explain why skin irradiation enhances wound-healing.

OsMPH1 regulates plant height and improves grain yield in rice.

Plant height is a major trait affecting yield potential in rice. Using a large-scale hybrid transcription factor approach, we identified the novel MYB-like transcription factor OsMPH1 (MYB-like gene of Plant Height 1), which is involved in the regulation of plant height in rice. Overexpression of OsMPH1 leads to increases of plant height and grain yield in rice, while knockdown of OsMPH1 leads to the opposite phenotypes. Microscopy of longitudinal stem sections indicated that a change in internode cell length resulted in the change in plant height. RNA sequencing (RNA-seq) analysis of transgenic rice lines showed that multiple genes related to cell elongation and cell wall synthesis, which are associated with plant height and yield phenotypes, exhibited an altered expression profile. These results imply that OsMPH1 might be involved in specific recognition and signal transduction processes related to plant height and yield formation, providing further insights into the mechanisms underlying the regulation of plant height and providing a candidate gene for the efficient improvement of rice yield.

Distinct Domains of CheA Confer Unique Functions in Chemotaxis and Cell Length in Azospirillum brasilense Sp7.

Chemotaxis is the movement of cells in response to gradients of diverse chemical cues. Motile bacteria utilize a conserved chemotaxis signal transduction system to bias their motility and navigate through a gradient. A central regulator of chemotaxis is the histidine kinase CheA. This cytoplasmic protein interacts with membrane-bound receptors, which assemble into large polar arrays, to propagate the signal. In the alphaproteobacterium Azospirillum brasilense, Che1 controls transient increases in swimming speed during chemotaxis, but it also biases the cell length at division. However, the exact underlying molecular mechanisms for Che1-dependent control of multiple cellular behaviors are not known. Here, we identify specific domains of the CheA1 histidine kinase implicated in modulating each of these functions. We show that CheA1 is produced in two isoforms: a membrane-anchored isoform produced as a fusion with a conserved seven-transmembrane domain of unknown function (TMX) at the N terminus and a soluble isoform similar to prototypical CheA. Site-directed and deletion mutagenesis combined with behavioral assays confirm the role of CheA1 in chemotaxis and implicate the TMX domain in mediating changes in cell length. Fluorescence microscopy further reveals that the membrane-anchored isoform is distributed around the cell surface while the soluble isoform localizes at the cell poles. Together, the data provide a mechanism for the role of Che1 in controlling multiple unrelated cellular behaviors via acquisition of a new domain in CheA1 and production of distinct functional isoforms.IMPORTANCE Chemotaxis provides a significant competitive advantage to bacteria in the environment, and this function has been transferred laterally multiple times, with evidence of functional divergence in different genomic contexts. The molecular principles that underlie functional diversification of chemotaxis in various genomic contexts are unknown. Here, we provide a molecular mechanism by which a single CheA protein controls two unrelated functions: chemotaxis and cell length. Acquisition of this multifunctionality is seemingly a recent evolutionary event. The findings illustrate a mechanism by which chemotaxis function may be co-opted to regulate additional cellular functions.

Mechanosensitive Adhesion Explains Stepping Motility in Amoeboid Cells

Cells employing amoeboid motility exhibit repetitive cycles of rapid expansion and contraction and apply coordinated traction forces to their environment. Although aspects of this process are well studied, it is unclear how the cell controls the coordination of cell length changes with adhesion to the surface. Here, we develop a simple model to mechanistically explain the emergence of periodic changes in length and spatiotemporal dynamics of traction forces measured in chemotaxing unicellular amoeba, Dictyostelium discoideum. In contrast to the biochemical mechanisms that have been implicated in the coordination of some cellular processes, we show that many features of amoeboid locomotion emerge from a simple mechanochemical model. The mechanism for interaction with the environment in Dictyostelium is unknown and thus, we explore different cell-environment interaction models to reveal that mechanosensitive adhesions are necessary to reproduce the spatiotemporal adhesion patterns. In this modeling framework, we find that the other motility modes, such as smooth gliding, arise naturally with variations in the physical properties of the surface. Thus, our work highlights the prominent role of biomechanics in determining the emergent features of amoeboid locomotion.

Serum from asthmatic rat treated with acupuncture inhibits acetylcholine-induced contractile responses of airway smooth muscle cells

OBJECTIVETo investigate the influence of serum from asthmatic rat treated with acupuncture (acupuncture serum) on the contractile responses of airway smooth muscle cells (ASMCs). METHODSOVA-induced asthmatic rats were subject to acupuncture treatment at the points of Dazhui (GV 14), Fengmen (BL 12), and Feishu (BL 13). The resulting serum was collected, as well as serum from normal animals. Contraction of ASMCs was induced with 10 µM acetylcholine (Ach), and acupuncture serum or normal serum was supplemented 15 min later. The effects of the sera on the changes in cell length, width, and surface area were monitored in real time via a live cell imaging system. RESULTSThe contraction rates of ASMCs 15 min and 30 min after Ach stimulation reached 38.2% ± 3.8% and 49.7% ± 4.6%, both of which were significantly higher than unstimulated control cells (P < 0.01). Acupuncture serum at the 1: 100 dosage reduced the contraction rate of ASMCs from 40.1% ± 5.1% to 17.6% ± 6.5% (P < 0.01). Thus, the treatment significantly inhibited the Ach-induced contractile responses of ASMCs and yielded a relaxation efficiency of 58%, which was greater than the effects obtained with a 1: 250 dosage. Treatment with acupuncture serum at 1: 500 dosage or normal serum displayed no clear effect of suppressing Ach-induced contraction of ASMCs. CONCLUSIONOur data suggest that acupuncture serum has the effect of inhibiting Ach-induced contraction of ASMCs, thereby promoting relaxation of the cells.

Interrogating the Escherichia coli cell cycle by cell dimension perturbations

Bacteria tightly regulate and coordinate the various events in their cell cycles to duplicate themselves accurately and to control their cell sizes. Growth of Escherichia coli, in particular, follows a relation known as Schaechter's growth law. This law says that the average cell volume scales exponentially with growth rate, with a scaling exponent equal to the time from initiation of a round of DNA replication to the cell division at which the corresponding sister chromosomes segregate. Here, we sought to test the robustness of the growth law to systematic perturbations in cell dimensions achieved by varying the expression levels of mreB and ftsZ We found that decreasing the mreB level resulted in increased cell width, with little change in cell length, whereas decreasing the ftsZ level resulted in increased cell length. Furthermore, the time from replication termination to cell division increased with the perturbed dimension in both cases. Moreover, the growth law remained valid over a range of growth conditions and dimension perturbations. The growth law can be quantitatively interpreted as a consequence of a tight coupling of cell division to replication initiation. Thus, its robustness to perturbations in cell dimensions strongly supports models in which the timing of replication initiation governs that of cell division, and cell volume is the key phenomenological variable governing the timing of replication initiation. These conclusions are discussed in the context of our recently proposed "adder-per-origin" model, in which cells add a constant volume per origin between initiations and divide a constant time after initiation.

Rapid Detection of Antibiotic Resistance in Gram-Negative Bacteria Through Assessment of Changes in Cellular Morphology.

Rapid antimicrobial susceptibility testing has the potential to improve patient outcomes and reduce healthcare-associated costs. In this study, a novel assay based on bacterial cell elongation after exposure to an antibiotic (ceftazidime) was evaluated for its ability to rapidly detect resistance in Gram-negative bacteria. The assay was used to detect resistance in a large collection of strains containing 320 clinical isolates of Acinetobacter baumannii, 171 clinical isolates of Klebsiella pneumoniae, and 212 clinical isolates of Pseudomonas aeruginosa, and the results were compared to those obtained using standard antimicrobial susceptibility testing methods. The assay identified ceftazidime-resistant strains with 100% sensitivity and 100% specificity for A. baumannii, 100% sensitivity and 97.2% specificity for K. pneumoniae, and with 82.3% sensitivity and 100% specificity for P. aeruginosa. Importantly, results were obtained in 1 hour 15 minutes from exponentially growing cultures. This study demonstrates that changes in cell length are highly correlated with phenotypic antibiotic susceptibility determined using standard susceptibility testing methods. This study therefore provides proof-of-concept that changes in cell morphology can be used as the basis for rapid detection of antibiotic resistance and provides the basis for the development of novel rapid diagnostics for the detection of antibiotic resistance.

Accumulation of PHA granules in Cupriavidus necator as seen by confocal fluorescence microscopy.

Many bacteria are capable of accumulating intracellular granules of polyhydroxyalkanoates (PHA). In this work, we developed confocal microscopy analysis of bacterial cells to study changes in the diameters of cells as well as PHA granules during growth and PHA accumulation in the bacterium Cupriavidus necator H16 (formerly Ralstonia eutropha). The cell envelope was stained by DiD(®) fluorescent probe and PHA granules by Nile Red. Signals from both probes were separated based on their spectral and fluorescence life-time properties. During growth and PHA accumulation, bacterial cells increased their length but the width of the cells remained constant. The volume fraction of PHA granules in cells increased during PHA accumulation, nevertheless, its value did not exceed 40 vol. % regardless of the PHA weight content. It seems that bacterial cultures lengthen the cells in order to control the PHA volume portion. However, since similar changes in cell length were also observed in a PHA non-accumulating mutant, it seems that there is no direct control mechanism, which regulates the prolongation of the cells with respect to PHA granules volume. It is more likely that PHA biosynthesis and the length of cells are influenced by the same external stimuli such as nutrient limitation.