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The organization of microtubules (MTs) within cells is critical for its internal organization during interphase and mitosis. During mitotic spindle assembly, MTs are made and organized around chromosomes in a process regulated by RanGTP. The role of RanGTP has been explored inXenopusegg extracts, which are not limited by a cell membrane. Here, we investigated whether cell-sized confinements affect the assembly of RanGTP-induced MT networks inXenopusegg extracts. We used microfluidics to encapsulate extract within monodisperse extract-in-oil droplets. Importantly, we find that the architecture of Ran-induced MT networks depends on the droplet diameter and the Ran concentration, and differs from structures formed in bulk extract. Our results highlight that both MT nucleation and physical confinement play critical roles in determining the spatial organization of the MT cytoskeleton.STATEMENT OF SIGNIFICANCEDuring cell division, chromosomes are segregated by the mitotic spindle, whose framework consists of up to hundreds of thousands of microtubules (MTs). Spindle MTs are generated via several pathways, one of which is regulated by RanGTP. Yet, how Ran-induced MTs self-organize within cell-sized confinement remains unclear. This work reports unexpected architectures of Ran-induced MT networks confined in cell-sized droplets, which depend on the droplet diameter and the RanGTP concentration. Thus, MT nucleation and confinement together give rise to specific MT network architectures, which are otherwise not observed in unconfined assays. The findings provide a simple strategy to engineer the architectures of MT networks and could have direct implications in nucleation-controlled soft material processing.

When granular materials, colloidal suspensions, and even animals and crowds exit through a narrow outlet, clogs can form spontaneously when multiple particles or entities attempt to exit simultaneously, thereby obstructing the outlet and ultimately halting the flow. Counterintuitively, the presence of an obstacle upstream of the outlet has been found to suppress clog formation. For soft particles such as emulsion drops, clogging has not been observed in the fast flow limit due to their deformability and vanishing interparticle friction. Instead, they pinch off each other and undergo break up when multiple drops attempt to exit simultaneously. Similar to how an obstacle reduces clogging in a rigid particle system, we hypothesize and demonstrate that an obstacle could suppress break up in the two-dimensional hopper flow of a microfluidic crystal consisting of dense emulsion drops by preventing the simultaneous exit of multiple drops. A regime map plotting the fraction of drops that undergo break up in a channel with different obstacle sizes and locations delineates the geometrical constraints necessary for effective break up suppression. When optimally placed, the obstacle induced an unexpected ordering of the drops, causing them to alternate and exit the outlet one at a time. Droplet break up is suppressed drastically by almost three orders of magnitude compared to when the obstacle is absent. This result can provide a simple, passive strategy to prevent droplet break up and can find use in improving the robustness and integrity of droplet microfluidic biochemical assays as well as in extrusion-based three-dimensional printing of emulsion or foam-based materials.

The timescale of droplet rearrangement in a 2D concentrated emulsion---a microfluidic crystal---displays 3 regimes with 2 transitions with increasing capillary number. The loss of order in the crystal flow corresponds to the second transition involving a solid to liquid-like transition of the emulsion.

This paper describes the dimensionless groups that determine the break-up probability of droplets in a concentrated emulsion during its flow in a tapered microchannel consisting of a narrow constriction. Such channel geometry is commonly used in droplet microfluidics to investigate the content of droplets from a concentrated emulsion. In contrast to solid wells in multi-well plates, drops are metastable, and are prone to break-up which compromises the accuracy and the throughput of the assay. Unlike single drops, the break-up process in a concentrated emulsion is stochastic. Analysis of the behavior of a large number of drops (N > 5000) shows that the probability of break-up increases with applied flow rate, the size of the drops relative to the size of the constriction, and the viscosity ratio of the emulsion. This paper shows that the break-up probability collapses into a single curve when plotted as a function of the product of capillary number, viscosity ratio, and confinement factor defined as the un-deformed radius of the drop relative to the hydraulic radius of the constriction. Fundamentally, the results represent a critical step towards the understanding of the physics governing instability in concentrated emulsions. Practically, the results provide a direct guide for the rational design of microchannels and the choice of operation parameters to increase the throughput of the droplet interrogation step while preserving droplet integrity and assay accuracy.

Background The incidence rates of colorectal cancer (CRC) in young individuals are increasing. There has been a significant improvement in overall survival in CRC because of advances in adjuvant chemotherapy and chemoradiotherapy over the past decades. However, these procedures may compromise the function of the reproductive system, and ovarian failure and premature menopause may occur. The objective of this analysis was to determine the incidence of long-term amenorrhea (≥ 12 months) in women with CRC aged 40 years and younger after adjuvant treatment. Patients and Methods The authors identified 162 premenopausal women with CRC aged 40 years or younger who were treated with adjuvant chemotherapy and chemoradiotherapy at Fudan University Shanghai Cancer Center from January 2008 to December 2012. One hundred twenty-three patients met all eligibility criteria and had sufficient follow-up for evaluation. The median age at diagnosis in patients with colon and rectal cancers was, respectively, 36 and 35 years (range, 17-40 and 24-40 years). Results All patients had regular menses before treatment; 3 patients with colon cancer (4.2%) experienced long-term amenorrhea, and 48 patients with rectal cancer (94.1%) experienced long-term amenorrhea. The incidence of amenorrhea was significantly lower in patients with colon cancer (4.2%; 3 of 72) than in patients with rectal cancer (94.1%; 48 of 51) ( P Conclusion In this retrospective series, the incidence of amenorrhea in patients with colon and rectal cancers was 4.2% and 94.1%, respectively. We believe our data support the fact that young female patients with CRC, especially those with rectal cancer who are scheduled to undergo pelvic irradiation, should be counseled regarding fertility preservation options, including ovarian transposition and cryopreservation of ovarian tissue, embryo, or oocyte.

Wound repair is a key feature distinguishing living from nonliving matter. Single cells are increasingly recognized to be capable of healing wounds. The lack of reproducible, high-throughput wounding methods has hindered single-cell wound repair studies. This work describes a microfluidic guillotine for bisecting single Stentor coeruleus cells in a continuous-flow manner. Stentor is used as a model due to its robust repair capacity and the ability to perform gene knockdown in a high-throughput manner. Local cutting dynamics reveals two regimes under which cells are bisected, one at low viscous stress where cells are cut with small membrane ruptures and high viability and one at high viscous stress where cells are cut with extended membrane ruptures and decreased viability. A cutting throughput up to 64 cells per minute—more than 200 times faster than current methods—is achieved. The method allows the generation of more than 100 cells in a synchronized stage of their repair process. This capacity, combined with high-throughput gene knockdown in Stentor, enables time-course mechanistic studies impossible with current wounding methods.

This paper describes the break-up behavior of a concentrated emulsion comprising drops stabilized by amphiphilic silica nanoparticles flowing in a tapered microchannel. Such geometry is often used in serial droplet interrogation and sorting processes in droplet microfluidics applications. When exposed to high viscous stresses, drops can undergo break-up and compromise their physical integrity. As these drops are used as micro-reactors, such compromise leads to a loss in the accuracy of droplet-based assays. Here, we show droplet break-up is suppressed by replacing the fluoro-surfactant similar to the one commonly used in current droplet microfluidics applications with amphiphilic nanoparticles as droplet stabilizer. We identify parameters that influence the break-up of these drops and demonstrate that break-up probability increases with increasing capillary number and confinement, decreasing nanoparticle size, and is insensitive to viscosity ratio within the range tested. Practically, our results reveal two key advantages of nanoparticles with direct applications to droplet microfluidics. First, replacing surfactants with nanoparticles suppresses break-up and increases the throughput of the serial interrogation process to 3 times higher than that in surfactant system under similar flow conditions. Second, the insensitivity of break-up to droplet viscosity makes it possible to process samples having different composition and viscosities without having to change the channel and droplet geometry in order to maintain the same degree of break-up and corresponding assay accuracy.

To investigate the effect of piperine on the disorder of glucose metabolism in the cell model with insulin resistance (IR) and explore the molecules mechanism on intervening the upstream target of AMPK signaling pathway. The insulin resistance models in HepG2 cells were established by fat emulsion stimulation. Then glucose consumption in culture supernatant was detected by GOD-POD method. Enzyme-linked immunosorbent assay(ELISA) was used to measure the levels of leptin(LEP) and adiponectin(APN) in culture supernatant; Real-time quantitative PCR was used to assess the mRNA expression of APN and LEP; and the protein expression levels of LepR, AdipoR1, AdipoR2 and the activation of AMPK signaling pathway were detected by Western blot analysis. The results showed that piperine, rosiglitazone and AMPK agonist AICAR could significantly elevate the glucose consumption in insulin resistance cell models, enhance the level of APN, promote APN mRNA transcripts and increase the protein expression of Adipo receptor. Meanwhile,AMPKα mRNA and р-AMPKα protein expressions were also increased in piperine treated cells, but both LEP mRNA expression and LepR protein expressions were decreased in piperine treated group. The results indicated that piperine could significantly ameliorate the glucose metabolism disorder in insulin resistance cell models through regulating upstream molecules (APN and LEP) of AMPK signaling pathway, and thus activate the AMPK signaling pathway.

When a many-body system is driven away from equilibrium, order can spontaneously emerge in places where disorder might be expected. Here we report an unexpected order in the flow of a concentrated emulsion in a tapered microfluidic channel. The velocity profiles of individual drops in the emulsion show periodic patterns in both space and time. Such periodic patterns appear surprising from both a fluid and a solid mechanics point of view. In particular, when the emulsion is considered as a soft crystal under extrusion, a disordered scenario might be expected based on the stochastic nature of dislocation dynamics in microscopic crystals. However, an orchestrated sequence of dislocation nucleation and migration is observed to give rise to a highly ordered deformation mode. This discovery suggests that nanocrystals can be made to deform more controllably than previously thought. It can also lead to novel flow control and mixing strategies in droplet microfluidics.

BACKGROUND Puerarin has protective effects on ischemia-reperfusion injury, but the underlying mechanisms are not fully revealed. This study explored the effect of puerarin on the expression of Bcl-2 associated athanogene 3 (BAG3) in an in vitro model of anoxia/reoxygenation injury (A/RI) in neonate rat primary cardiomyocytes and the functions of BAG3 in A/RI. MATERIAL AND METHODS BAG3 expression in cardiomyocytes with or without puerarin pre-treatment was quantified using qRT-PCR and Western blot analysis. The effects of BAG3 on A/RI were studied by measuring the activity of lactate dehydrogenase (LDH) and creatine phosphate kinase (CPK), the concentration of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). The effects of BAG3 on autophagy and apoptosis of the cardiomyocytes after A/RI were further studied. RESULTS Puerarin significantly promoted BAG3 expression in the rat primary cardiomyocytes after A/RI. Enforced BAG3 expression presented similar effects as puerarin pre-treatment in attenuating A/RI in terms of CPK, LDH, MDA, SOD, GSH-Px, ROS generation, and cell viability. BAG3 overexpression significantly stimulated autophagy in cardiomyocytes after A/RI, which presented protective effects on A/RI in terms of cell viability and apoptosis. Autophagy inhibition partly abrogated the protective effects of BAG3. CONCLUSIONS Puerarin can directly increase BAG3 transcription and translation in cardiomyocytes after A/RI. The elevated BAG3 expression presents protective effects on A/RI at least through enhancing autophagy and reducing apoptosis, which is a novel protective mechanism of puerarin in ARI.

NC machine plays an irreplaceable role in the modern manufacturing because of its high machining processing accuracy, quality stable, flexibility. Through using the Renishaw ML10 laser interferometer detect the positioning accuracy and repositioning accuracy of X axis and Z axis of the HTC20 series of NC machine tools. According to the detection result compensate NC system to meet the machining accuracy requirement. The result shows that the error compensation of NC system is a effective method to improve the position accuracy of NC machine.

Droplet microfluidics, in which each droplet serves as a micro-reactor, has found widespread use in high-throughput biochemical screening applications. These droplets are often concentrated at various steps to form a concentrated emulsion. As part of a serial interrogation and sorting process, such concentrated emulsions are typically injected into a tapered channel leading to a constriction that fits one drop at a time for the probing of droplet content in a serial manner. The flow physics inside the droplets under these flow conditions are not well understood but are critical for predicting and controlling the mixing of reagents inside the droplets as reactors. Here we investigate the flow field inside droplets of a concentrated emulsion flowing through a tapered microchannel using micro-particle image velocimetry. The confining geometry of the channel forces the number of rows of drops to reduce by one at specific and uniformly spaced streamwise locations, which are referred to as droplet rearrangement...

This paper describes an optofluidic droplet interrogation device capable of counting fluorescent drops at a throughput of 254,000 drops per second. To our knowledge, this rate is the highest interrogation rate published thus far. Our device consists of 16 parallel microfluidic channels bonded directly to a filter-coated two-dimensional Complementary Metal-Oxide-Semiconductor (CMOS) sensor array. Fluorescence signals emitted from the drops are collected by the sensor that forms the bottom of the channel. The proximity of the drops to the sensor facilitates efficient collection of fluorescence emission from the drops, and overcomes the trade-off between light collection efficiency and field of view in conventional microscopy. The interrogation rate of our device is currently limited by the acquisition speed of CMOS sensor, and is expected to increase further as high-speed sensors become increasingly available.

To study the protein expression of sex hormone receptors in relation to failure of the testicular descent (cryptorchidism), cryptorchid rats were obtained with administrating flutamide, an androgen antagonist, to pregnant rats. Immunolocalization of the androgen receptor (AR) and estrogen receptors α (ER α) and β (ERβ) was examined in the gubernaculum and cremaster muscle in cryptorchid rats. Testicular descent was normally completed by 4 weeks of age in rats. In control rat fetuses, AR and ERβ were expressed at high levels in fetal gubernaculum but were downregulated after birth. ERα was undetectable in fetal gubernaculum, but its protein expression was upregulated after birth. Similar results were found in the cremaster muscle. These results suggest that expression of AR, ERα and ERβ is developmentally regulated in the gubernaculum and cremaster muscle during the period of testicular descent. In cryptorchid rats, however, remarkable levels of expression of ERα and ERβ were detected in the gubernaculum at 2 weeks after birth. These strong expressions of ERα and ERβ decreased to control levels at 4 weeks. Immunoexpression of AR in cryptorchid rats after birth was the same level as that of control rats. These results indicate that flutamide significantly affects the expression of ERs, but AR expression less intensely. It is assumed that sex hormone receptors have an important role during the period of testicular descent.

The fine structural alterations of hepatocytes of Japanese monkeys under 4 days of fasting stress were analyzed morphometrically. One of the conspicuous alterations was the enlargement of mitochondria. The average diameter of mitochondria in fasting group increased to approximately 1.89-fold of that in control group, though their number did not change. The number of peroxisomes was 1.36-fold of that in control, though their area did not change. In addition, many of r-ER were swollen and were vesiculated. The appearance of bundle of actin-like stress fiber also increased in the fasting animals. The glycogen area as well as liver weight decreased in fasting group.

The distribution of desmin filaments in neonatal swine muscle was identified by immunoelectron microscopy. As piglets grow from 0 hr to 10 days after birth, the diameter of myofibril gradually increases and the intermyofibril space becomes narrower. The quantitative analysis suggested that the largest number of desmin filaments was identified in 0 hr after birth. Desmin filaments were identified around the immature myofibrils and also distributed in the wide intermyofibril space. These desmin filaments might be involved in the growth and myofibrillogenesis in developing myocytes. Desmin filaments formed an internal framework to connect the myofibrils and cell organelles. The entire array of myofibrils was then anchored to the sarcolemma. Desmin filaments in neonatal skeletal muscle might play a role as like as connective tissue filaments between parenchymal cells. By the quantitative analysis from fetus, neonate to adult, desmin filaments appeared to relocate from the intermyofibril spaces to the Z-lines. As myocytes mature, desmin filaments gradually acted as the integrators between the adjacent myofibrils.

The distribution of desmin filaments in the skeletal and cardiac muscles of experimental animals and cultured cells have been extensively studied. The purpose of this report is to identify the distribution of desmin filaments in vivo in human fetal skeletal muscle. Using 10nm gold-particle-labeling immunoelectron microscopy, desmin intermediate filaments were localized in skeletal muscles of 12, 20 and 29-week-old human fetuses. A number of desmin filaments were closely associated with the nascent nonstriated myofibrils. Nascent myofibrils were distributed in the subsarcolemmal space at all three developmental stages. Desmin, accompanying the nascent myofibrils, was most abundant after 12 weeks of gestation. An irregular network of desmin filaments was conspicuous in the subsarcolemmal space after 20 weeks of gestation. Desmin filaments penetrated the myofibrils and the Z-discs after 29 weeks of gestation. The developmental change in fine structural localization of desmin between 12, 20 and 29 weeks of gestation in human fetuses indicated that desmin filaments were involved in myofibril assembly in the subsarcolemmal space, and also in myofibrillogenesis in the inner space of the myofibrils.

Droplet microfluidics has enabled a wide variety of high-throughput biotechnical applications through the use of monodisperse micro-droplets as bioreactors. Previous fluid dynamics studies of droplet microfluidics have focused on single droplets or emulsions at low volume fractions. The study of concentrated emulsions at high volume fractions is important for further increasing the throughput of droplet microfluidics, but the fluid dynamics of such emulsions in confined microchannels is not well understood. This paper describes the use of microscopic particle image velocimetry to quantify the flow inside individual droplets within a concentrated emulsion having volume fraction φ ∼ 85% flowing as a monolayer in a straight microfluidic channel. The effects of confinement (namely, the number of rows of droplets across the width of the channel) and viscosity ratio on the internal flow patterns inside the drops at a fixed capillary number of 10−3 and a Reynolds number of 10−2 to 10−1 are studied. The results s...

A porcine antibody was used to clarify the localization of desmin filaments in the swine longissimus muscle by post-embedding immuno-electron microscopy. The major site of desmin localization was the bundle of filaments connecting Z-discs of adjacent myofibrils. These desmin filaments were localized not only between but also within myofibrils. This indicates that there exist more complicated networks rather than simple bundles of desmin filaments running parallel to the Z-discs. Clarification of desmin distribution within the myofibrils surrounding Z-discs provides a basic concept for consideration of the combination and transformation of vimentin to desmin in developing human embryonic muscles.

Suspended substance (SS) concentration is an important index of water quality evaluation. It is significant to build a quantitative remote sensing retrieval algorithm to inverse the SS concentration. With focus on the problems that empirical method is lack of the support of the physical theory and not robust in quantitative remote sensing monitor of SS concentration in inland water, the feasibility of spectrum and inherent optical properties analysis with clear physical meaning is discussed. Based on the experiment taken on 17th November 2007, the characteristic spectrum, absorption coefficient and SS concentration are taken on 30 samples distributed along the reservoir where is far away from the dam above 3 kilometers. The absorption characteristic of the suspended substance and the contribution of the main constituents of the water (chlorophyll, suspended substance, DOC) to the total absorption are analyzed. Based on the achievements we have acquired, a semi-analytical model using Gordon model is built to inverse the SS concentration. With the SS concentration on samples, the precision of the inversion model is tested. The results show that the error of SS concentration retrieved can satisfy the later research requirement; and the minimum retrieval error is -6.22%, the maximum retrieval error is 62.83%, the mean retrieval error is 25.65%. Further, the inversion method is applied on the multi-temporal image CHRIS to check the precision of the model, and the mean retrieval error is 21.81%.

The developmental relationship of myosin binding proteins (myomesin, connectin and C-protein) to myosin was studied in chicken cervical somites by immunofluorescence microscopy. Muscle and non-muscle myosins initially appeared as slender rods at the same sites, and then, fused to form non-striated fibrils. As muscle myosin formed striated structures (A bands), non-muscle myosin disappeared from this structure. Myomesin (reactive with monoclonal antibodies MyB4 and MyBB78) and connectin (carboxy terminal region, reactive with monoclonal antibody T51) were seen as dots in the center of these myosin rods. These proteins then formed characteristic mature striations on non-striated fibrils of myosin. Earlier alignment of these myosin binding proteins rather than myosin indicates that the correct assembly of these proteins seems to be related to the formation of initial myosin rods as well as subsequent linear and periodic alignment of myosin molecules to form early A bands. Connectin spots reactive with 9D10 were scattered around myosin rods/myomesin dots/connectin T51 dots. These spots may represent radiating connectin filaments from these rods/dots to link myosin rods to the I-Z-I structures of myofibrils to be incorporated. Since the slow isoform of C-protein formed its characteristic bands (“doublets”) prior to H zone formation within A bands by myosin, this isoform may help to precisely align myosin filaments within the A band region. The presence of the slow, then the slow and the cardiac, and finally the co-existence of the slow and the fast isoforms of C-protein may interfere with the incorporation and co-polymerization of non-adult isoforms into myofibrils.

Background Desmin and vimentin are the major intermediate filaments in muscle. They have been extensively studied in animal experiments. This study is the first to identify the distribution and to analyse the correlation of desmin and vimentin in human fetal skeletal muscle. Vimentin might be replaced by or transformed into desmin during myogenesis in chick embryo, although the precise process remains to be elucidated. The aim of this report is to evaluate the ratio of desmin to vimentin in human fetal muscle. Methods By double-labeling immunoelectron microscopy, desmin and vimentin intermediate filaments were localized in developing skeletal muscles of 20–29-week-old human fetuses. The ratio of desmin and vimentin was analyzed statistically. Results Two sizes of colloidal gold particles, 5 nm (vimentin) and 10 nm (desmin), were distributed along the intermediate filaments. The commonest distance between gold particles were approximately 40–50 nm. Desmin and vimentin labeled with gold particles were arranged very close together in the same intermediate filament. The ratio of vimentin to desmin varied but the amount of vimentin decreased progressively from the undifferentiated myoblast to the differentiated myocytes. As the fetuses developed, desmin increased and vimentin decreased. Desmin and vimentin intermediate filaments were identified in the intermyofibrils of differentiated myocytes, in subsarcolemmal space, and in myoblast. Conclusions Desmin and vimentin were colocalized in the same intermediate filaments. More vimentin existed in the less differentiated myocytes, although a small number of desmin filaments were already found in undifferentiated myoblasts. These intermediate filaments may not only connect myofibril bundles, cell organelles, and cell membrane but also provide a basis for myofibrillogenesis that is similar to relation between connective fibers and parenchymal cells. © 1996 Wiley-Liss, Inc.