Phospholipase C-Zeta Reveals the Underlying Pathological Influence after Density Gradient Centrifugation, Microfluidic Sorting, and Cryopreservation of Human Sperm

Objective:Sperm preparation techniques and cryopreservation are widely used in assisted reproductive techniques(ART).How to improve the quality of sperm management is a matter of great concern.Phospholipase C-zeta(PLCζ)is considered a sperm-specific agent that activates oocyte activation and thus playing a crucial role in male fertility.However,the potential mechanisms by which semen processing and cryopreservation on PLCζcontribute to keyhole have not been addressed.Methods:In this study,semen samples were taken from have not been addressed 10 normozoospermic men.Each semen sample was assigned to the following groups:density gradient centrifugation(DGC)as control,microfluidic sorting,and cryopreservation.Sperm parameters of molity,viability,membrane integrity,and intracellular ROS were evaluated during sperm preparation and cryopreservation.The expression of PLCζin human sperm was determined by immunofluorescence and western blotting.Results:The results showed that molity,viability,and membrane integrity decreased in cryopreservation group.Intracellular ROS were also significantly increased compared to the the control group.There was no significant difference between DGC and microfluidic sorting group.Our investigation revealed that total levels of PLCζwere comparable between DGC and microfluidic sorting,but there were significantly reduced levels of PLCζafter cryopreservation as quantified by both immunofluorescenceand immunoblotting.PLCζimmunofluorescence in sperm revealed different PLCζlocalization patterns around the acrosomal(Ac),equatorial(Eq),post-acrosomal(PA)areas of sperm heads,and their combination.The predominant patterns of PLCζlocalization in DGC were similar to that of microfluidic sorting,with strong,with staining.In contrast,PLCζstaining in freeze-thawed sperm was considerably weaker fluorescence intensity.Conclusion:This study clarified the mechanism of sperm preparation and cryopreservation underlying effect on sperm characteristic,accompanied with PLCζexpresion.We demonstrated that microfluidic sorting provides a highly efficient preparation method for clinical selection of PLCζ-expressing sperm comparable to DGC gene expression.It is suggested that the cryopreservation of sperm has a significant detrimental effect on PLCζ.

The Nanoscale Density Gradient as a Structural Stabilizer for Glass Formation

The rapid cooling of a metallic liquid(ML)results in short-range order(SRO)among the atomic arrangements and a disordered structure in the resulting metallic glass(MG).These phenomena cause various possible features in the microscopic structure of the MG,presenting a puzzle about the nature of the MGs’microscopic structure beyond SRO.In this study,the nanoscale density gradient(NDG)originating from a sequential arrangement of clusters with different atomic packing densities(APDs),representing the medium-range structural heterogeneity in Zr_(60)Cu_(30)Al_(10)MG,was characterized using electron tomography(ET)combined with image simulations based on structure modeling.The coarse polyhedrons with distinct facets identified in the three-dimensional images coincide with icosahedron-like clusters and represent the spatial positions of clusters with high APDs.Rearrangements of the different clusters according to descending APD order in the glass-forming process are responsible for the NDG that stabilizes both the supercooled ML and the amorphous states and acts as a hidden rule in the transition from ML to MG.

DIAGNOSTIC VALUE OF DENSITY GRADIENT CENTRIFU-GATION FOR EXFOLIATIVE TUMOR CELLS IN MALIG-NANT PLEURAL EFFUSIONS

Objective To find out a specific method for diagnosis of malignant pleural effusions (MPEs)with higher sensitivity and practicality. Methods The diagnosis of MPEs were made using density gradient cen-trifugation(DGC) , smear cytologic examination( SCE) and pleural needle biopsy (PNB). Comparisons between these results and those of benign pleural effusions were also made. Results The positive rates of DGC,SCE and PNB for diagnosing MPEs were 94. 3% ,62. 9% and 44. 6% , respectively, and the positive rate of SCE combined with PNB for diagnosing MPEs was 73. 2%. The positive rate of the exfoliative tumor cells ( ETCs ) by DGC was much higher than that of SCE or/and PNB with no false-positive. Conclusion The ETCs isolated by DGC from the MPEs is quite specific for the diagnosis of malignant tumors with higher sensitivity and practicality in clinico-patho-logical practice.

Comparative study on density gradients and swim-up preparation techniques utilizing neat and cryopreserved spermatozoa

Aim: To 1) compare post-wash and post-thaw parameters of sperm processed with PureSperm density gradient technique and swim-up method; and 2) test the efficacy of two commonly available density gradient media PureSperm and ISolate. Methods: This prospective study used semen specimens from 22 patients. Specimens from nine patients were processed by both PureSperm density gradient and swim-up method. These specimens were then cryopreserved. Thirteen specimens were processed by both PureSperm (40 % and 80 %) and Isolate (50 % and 90 %) double density gradient techniques. The two fractions processed by both PureSperm and swim-up were analyzed for post-wash sperm characteristics. Post-thaw analysis was done after 24 hours. Sperm fractions obtained after processing with PureSperm and ISolate were compared for post-wash sperm characteristics and ROS levels. Results: Specimens prepared with PureSperm had significantly higher median total motile sperm counts (TMSC) (32.2 x 10~6 vs. 17.6 x 10~6), recovery rates (69.2 % vs. 50.0 %), and longevity at 4 hours (83.0 % vs. 55.0 %) compared to specimen prepared by swim-up. Post-thaw specimens also had a higher recovery and longevity at 4 hours with PureSperm as compared to the swim-up. Semen specimens processed by PureSperm had significantly higher total sperm count, TMSC, and percentage recovery rates (30.0 % vs. 19.7 %) than ISolate. Conclusion: Semen quality is better preserved in fresh and cryopreserved semen prepared with PureSperm density gradient compared to swim-up. A significant enrichment of sperm is observed with PureSperm compared to ISolate. Higher recovery rates of mature motile sperm obtained after PureSperm sperm preparation may be beneficial for successful ART. i

Effects of substrate-ion density gradients on light-ion acceleration from ultraintense laser pulse irradiated thin-foils

A general solution of the electrostatic potential that determines the maximum light-ion energy is derived for the test-particle acceleration model by taking into account the influence of the substrate-ion density gradient. It is shown that the substrate-ion density structure is also dependent on laser pulse duration. In the picosecond or sub-picosecond regime, the decreasing density gradient of the substrate-ions leads to an evident reduction in the acceleration efficiency of the light-ions. However, this kind of influence is negligible in the ultrashort regime.

Tunable Separation of Single-Walled Carbon Nanotubes by Dual-Surfactant Density Gradient Ultracentrifugation

We present a systematic study of the effects of surfactants in the separation of single-walled carbon nanotubes （SWNTs） by density gradient ultracentrifugation （DGU）. Through analysis of the buoyant densities, layer positions, and optical absorbance spectra of SWNT separations using the bile salt sodium deoxycholate （DOC） and the anionic salt sodium dodecyl sulfate （SDS）, we clarify the roles and interactions of these two surfactants in yielding different DGU outcomes. The separation mechanism described here can also help in designing new DGU experiments by qualitatively predicting outcomes of different starting recipes, improving the efficacy of DGU and simplifying post-DGU fractionation.

Changes in human sperm motility and DNA fragmentation index after incubation at different temperatures following density gradient centrifugation and swim-up procedures

Objective:The purpose of this study was to evaluate the sperm motility and DNA integrity at different temperatures to analyze whether the sperms are suitable on the second day for insemination of in vitro matured oocytes by intra-cytoplasmic sperm injection(ICSI)following density gradient centrifugation(DGC)and swim-up(SU)procedures.Methods:Semen samples were collected from 30 outpatients who visited the Center for Reproductive Medicine for semen analyses.Following sperm selection by DGC and SU procedures,the liquified semen samples were divided into three groups and incubated at 4,25,and 37°C,respectively.Following incubation for 24,48,and 72 hours,the sperm motility and sperm DNA fragmentation index(DFI)were analyzed.Results:Following the combination of DGC and SU procedures,the sperm motility(91.8%±8.6%vs.50.8%±13.1%)and DFI(5.1%±7.9%vs.13.0%±11.6%)were significantly improved(P<0.01)compared to those without any treatment.The sperm motility of the 3 groups significantly declined(P<0.05)post-incubation compared to that of the groups prior incubation.However,sperm motility significantly increased(76.9%±10.4%)(P<0.05)at 25°C compared to that of the other 2 groups(53.5%±11.0%and 47.6%±10.2%).Sperm DFI significantly increased(P<0.05)at 37°C following incubation for 24 and 72 hours in comparison to that of the other 2 groups.However,the sperm DFI did not significantly increase when the sperm samples were incubated at 4(5.7%±5.9%)and 25°C(6.8%±5.6%)for 24 hours compared to that before incubation(5.1%±7.9%).Conclusions:These results indicate that the sperm quality,in terms of motility and DFI,can be efficiently improved by DGC in combination with SU.Following which,the sperm samples can be incubated at 25°C and be used on the second day for insemination of in vitro matured oocytes by ICSI.

Theoretical simulation of the effect of deformation on local gravity in a density gradient zone

We modeled the effect of the deformation of a Density Gradient Zone （DGZ） on a local gravity field using a cubical model and introduced a new method to simulate a complex DGZ （CDGZ）. Then, we analyzed the features of the model for the influence of the deformation of the DGZ on the local gravity field. We concluded that land-based gravity is not sensitive to the thickness of the DGZ and that the magnitude of the contribution of the DGZ is one order less than that of the volume strain with the same displacement.

Research on the Correlation of the Fluctuating Density Gradient of the Compressible Flows

This work is to study a role of the fluctuating density gradient in the compressible flows tbr the computational fluid dynamics （CFD）. A new anisotropy tensor with the fluctuating density gradient is introduced, and is used for an invariant modeling technique to model the turbulent density gradient correlation equation derived from the continuity equation. The modeling equation is decomposed into three groups proportional to the mean velocity, proportional to the mean strain rate, and proportional to the mean density. The characteristics of the correlation in a wake are extracted from the results by the two dimensional direct simulation, and shows the strong correlation with the vortices in the wake near the body. Thus, it can be concluded that the correlation of the density gradient is a significant parameter to describe the quick generation of the turbulent property in the compressible flows.

Separation of Gold Nanorods Using Density Gradient Ultracentrifugation

High quality gold nanorods （NRs） with a monodisperse size and aspect ratio are essential for many applications. Here, we describe how nearly monodisperse gold NRs can be separated from polydisperse samples using density gradient ultracentrifugation. Size and dimension analysis by transmission electron microscopy （TEM） and absorption spectroscopy revealed that the Au NRs were separated mainly as a function of their aspect ratio The surface-enhanced Raman scattering （SERS） activity of Au NRs with lower aspect ratio is notably stronger than that of NRs with higher aspect ratio under 633 nm laser excitation, due to the size-dependent absorption of the longitudinal plasmon band. The separation approach provides a method to improve the quality of NRs produced by large scale synthetic methods.

Single and double layer centrifugation improve the quality of cryopreserved bovine sperm from poor quality ejaculates

Background： Density gradient centrifugation was reported as a technique of semen preparation in assisted reproductive techniques in humans and animals. This technique was found to be efficient in improving semen quality after harmful techniques such as cryopreservation. Recently a modified technique, single layer centrifugation,was proposed as a technique providing a large amount of high quality spermatozoa, and this treatment was performed before conservation. Single layer centrifugation has been studied prevalently in stallions and in boars,but limited data were available for bulls. Occasionally bulls are known to experience a transient reduction in semen quality, thus techniques that allow improvement in semen quality could be applied in this context. The aim of this study was the evaluation of single layer and double layer centrifugation by the use of iodixanol, compared with conventional centrifugation and non-centrifuged semen, on the sperm characteristics during the cryopreservation process in bulls with normal and poor semen quality.Results： Single layer centrifugation and double layer centrifugation both significantly increased the percentage of normal spermatozoa and decreased the percentage of non-sperm cells in poor quality samples, while both were ineffective in those of normal quality. Sperm characteristics in poor quality samples increased after single layer centrifugation and double layer centrifugation, reaching values similar to those recorded in normal samples, and this trend is maintained after equilibration and after cryopreservation. On the other hand, SLC and DLC resulted in a consistent reduction in the spermatozoa recovered, and this resulted in a reduction of the absolute amount of spermatozoa cryopreserved in the normal samples, without a clear improvement in sperm characteristics in this type of sample.Conclusions： These data suggested that both SLC and DLC could be performed in practice, but their application should be limited to the cases in which the quality of the spermatozoa recovered is more important than the total amount of spermatozoa.

Purification of rectangle DNA origami by rate-zonal centrifugation

DNA origami technique, a breakthrough in DNA nanotechnology, has been widely used to prepare complex DNA nanostructures with nanoscale addressability. However, the purity and yield are generally the bottleneck to application of DNA nanostructures, and current methods for purifying DNA origami nanostructures in large quantities are time-consuming and laborious. This study aims to develop a scalable, cost-effective and contamination-free method of purifying DNA origami nanostructures. We employ an effective and convenient purification approach to purify planar rectangle DNA origami structures through rate-zonal centrifugation. By subjecting DNA origami samples to high centrifugal force in a density gradient media of glycerol, well-folded nanostructures and by-products are separated successfully, which are confirmed by agarose gel electrophoresis and atomic force microscopy(AFM). This method will aid the production of pure rectangle DNA origami nanostructures in large quantity.

Density gradient ultracentrifugation for colloidal nanostructures separation and investigation

In this article,we review the advancement in nanoseparation and concomitant purification of nanoparticles(NPs) by using density gradient ultracentrifugation technique(DGUC) and demonstrated by taking several typical examples.Study emphasizes the conceptual advances in classification,mechanism of DGUC and synthesis-structure-property relationships of NPs to provide the significant clue for the further synthesis optimization.Separation,concentration,and purification of NPs by DGUC can be achieved at the same time by introducing the water/oil interfaces into the separation chamber.We can develop an efficient method ‘‘lab in a tube" by introducing a reaction zone or an assembly zone in the gradient to find the surface reaction and assembly mechanism of NPs since the reaction time can be precisely controlled and the chemical environment change can be extremely fast.Finally,to achieve the best separation parameters for the colloidal systems,we gave the mathematical descriptions and computational optimized models as a new direction for making practicable and predictable DGUC separation method.Thus,it can be helpful for an efficient separation as well as for the synthesis optimization,assembly and surface reactions as a potential cornerstone for the future development in the nanotechnology and this review can be served as a plethora of advanced notes on the DGUC separation method.

Study of the axial density/impedance gradient composite long rod hypervelocity penetration into a four-layer Q345 target

Based on the dynamic shock response of the material and structure,the hypervelocity impact processes and mechanisms of long composite rods with axial density/impedance gradients penetration into fourlayer targets were studied through experiments and numerical simulation methods.The propagation law of the shock waves,together with the structural responses of the projectiles and targets,the formation and evolution of the fragment groups formed during the processes and their distributions were described.The damage of each target plate was quantitatively analysed by comparing the results of the experiment and numerical simulation.The results showed that the axial density/impedance gradient projectiles could decrease the impact pressure to a certain extent,and the degree of damage to the target plate decreased layer by layer when the head density/impedance of the projectile was high.When the head density/impedance of the projectile was low,the degree of target damage first increased layer by layer until the projectile was completely eroded and then it decreased.The results can provide a reference for the design and application of long rods with axial composite structure for velocities ranging from 6 to 10 Ma or greater.

Strong evidence for latitudinal diversity gradient in mosses across the world

Species richness generally decreases with increasing latitude,a biodiversity gradient that has long been considered as one of the few laws in ecology.This latitudinal diversity gradient has been observed in many major groups of organisms.In plants,the latitudinal diversity gradient has been observed in vascular plants,angiosperms,ferns,and liverworts.However,a conspicuous latitudinal diversity gradient in mosses at a global or continental scale has not been observed until now.Here,we analyze a comprehensive data set including moss species in each band of 20° in latitude worldwide.Our results show that moss species richness decreases strongly with increasing latitude,regardless of whether the globe is considered as a whole or different longitudinal segments(e.g.,Old World versus New World) are considered separately.This result holds when variation in area size among latitudinal bands is taken into account.Pearson's correlation coefficient between latitude and species richness is-0.99 for both the Northern and Southern Hemispheres.Because bryophytes are an extant lineage of early land plants and because mosses not only include most of extant species of bryophytes but also are important constituents of most terrestrial ecosystems,understanding geographic patterns of mosses is particularly important The finding of our study fills a critical knowledge gap.

Effect of the Density of Molten Metal on the Raining Phenomenon in Horizontal Centrifugal Casting

In this work the influence of the density of the molten metal on the emergence of the raining phenomenon in the horizontal centrifugal casting process is numerically studied. Transient 2D numerical simulations were carried out using Computational Fluid Dynamics software. Three molten metals with different density, namely aluminum, iron and lead, and three angular frequencies, namely 50, 66 and 77 rad/s were considered. It is found that the density of the molten metal significantly affects the emergence, transient or permanent, of the rain phenomenon. However, the magnitude and duration of the rain phenomenon depend on the angular frequency of the rotating mold. Likewise, since gravitational forces affect the metal according to its density, the value of the critical rotation speed of the mold is also affected.

Solvent switching and purification of colloidal nano- particles through water/oil Interfaces within a density gradient

Traditional post-treatment of colloidal nanoparticles （NPs） usually involves repeated centrifugation-wash-sonication processes to separate NPs from the original synthetic environment; however, such separation processes have either high energy cost or low efficiency and tend to cause aggregation. Here we show a general and scalable colloid post-processing technique based on density gradient centrifugation through water/oil interfaces. Such a one-step technique can switch the solvent in a colloid at almost any concentration without aggregation, and meanwhile purify colloidal nanoparticles by separating them from by-products and environmental impurities. Droplet sedimentation was shown to be the mechanism of this one-step concentration/purification process, and mathematical modeling was established to quantify the accumulation and sedimentation velocities of different NPs.

Lithosphere density structure beneath the eastern margin of the Tibetan Plateau and its surrounding areas derived from GOCE gradients data

A three-dimensional density model of the crust and uppermost mantle is determined by the inversion of a set of GOCE gravity and gradients residual anomalies beneath the eastern margin of the Tibetan Plateau and its surrounding areas. In our work, we choose five independent gravity gradients （Txx, Tzz, Txy, Txz, Tyz） to perform density inversion. Objective function is given based on Tikhonov regularization theory. Seismic S-wave velocities play the role of initial constraint for the inversion based on a relationship between density and S-wave velocity. Damped Least Square method is used during the inversion. The final density results offer some insights into understanding the underlying geodynamic processes： （1） Low densities in the margin of the Tibet, along with low wave velocity and resistivity results, yield conversions from soft and weak Tibet to the hard and rigid cratons. （2）The lowest densities are found in the boundary of the plateau, instead of the whole Tibet indicates that the effects of extrusion stress environment in the margin affect the changes of the substance there. The substances and environments conditioning for the earthquake preparations and strong deformation in this transitional zone. （3） Evident low-D anomaly in the upper and middle crust in the Lasha terrane and Songpan-Ganzi terrane illustrated the eastward sub-ducted of southeastern Tibet, which could be accounts for the frequent volcano and earthouakes there.

Hybrid vector beams with non-uniform orbital angular momentum density induced by designed azimuthal polarization gradient

Based on angular amplitude modulation of orthogonal base vectors in common-path interference method, we propose an interesting type of hybrid vector beams with unprecedented azimuthal polarization gradient and demonstrate in experiment. Geometrically, the configured azimuthal polarization gradient is indicated by intriguing mapping tracks of angular polarization states on Poincaré sphere, more than just conventional circles for previously reported vector beams. Moreover, via tailoring relevant parameters, more special polarization mapping tracks can be handily achieved. More noteworthily, the designed azimuthal polarization gradients are found to be able to induce azimuthally non-uniform orbital angular momentum density, while generally uniform for circle-track cases, immersing in homogenous intensity background whatever base states are. These peculiar features may open alternative routes for new optical effects and applications.

Assignment of terahertz vibrational modes of L-glutamine using density functional theory within generalized-gradient approximation

The crystal structure of L-glutamine is stabilized by a three-dimensional network of intermolecular hydrogen bonds.We utilize plane-wave density functional theory lattice-dynamics calculations within the generalized-gradient approximation（GGA）, Perdew–Burke–Ernzerhof（PBE）, PBE for solids（PBEsol）, PBE with Wu–Cohen exchange（WC）, and dispersion-corrected PBE, to investigate the effect of these intermolecular contacts on the absorption spectra of glutamine in the terahertz frequency range. Among these calculations, the solid-state simulated results obtained using the WC method exhibit a good agreement with the measured absorption spectra, and the absorption features are assigned with the help of WC. This indicates that the vibrational modes of glutamine were related to the combination of intramolecular and intermolecular motions, the intramolecular modes were dominated by rocking or torsion involving functional groups; the intermolecular modes mainly result from the translational motions of individual molecules, and the rocking of the hydrogenbonded functional groups.