Ejection Force Research Articles

Single-step Coprocessing of Cohesive Powder via Mechanical Dry Coating for Direct Tablet Compression

This study aims at testing the feasibility of a single-step coating process to produce a powder formulation of active and inactive ingredients for direct compression. A cohesive ibuprofen powder was coprocessed with a coating material, a binder (polyvinylpyrrolidone K25), and a superdisintegrant (crospovidone). Magnesium stearate (MgSt), l-leucine, and silica were selected as coating materials (1% w/w). A coprocessed powder without any coating material was employed as a control. Coating with MgSt, l-leucine, or silica produced significantly improved powder flow in comparison to the control batch. Robust tablets were produced from the processed powders for each coating material. The tablets compacted using the coated powders with MgSt or l-leucine also exhibited significantly lower tablet ejection forces than the control batch, demonstrating their lubrication effect. Furthermore, the disintegration time and dissolution rates of these tablets made of the formulations coprocessed with lubricants were enhanced, even for those coated with the hydrophobic material such as MgSt that has been previously reported to inhibit dissolution. However, the tablets made with silica-coated powders would not disintegrate. This study indicated the feasibility of a single-step dry coating process to produce powders with both flow-aid and lubrication effects, which are suitable for direct compression.

Evaluation of Small-Scale Powder Flow Characterization Tests in the Prediction of Large-Scale Process Failures

In pharmaceutical development, powder flow characteristics are notoriously difficult to predict. Although tests exist that aim to determine the flowability of powders, many have not been definitively correlated with a powder’s performance in downstream processes. Part of the challenge is that powder flow must occur over a broad range of consolidating stresses, including low-stress flow regimes such as the filling of a die cavity in a tablet press and high-stress flow regimes such as the deformation associated with tablet compression. The objective of this work was to characterize several placebo formulations utilizing a variety of standardized and non-standardized experiments and contrasting those results with downstream tablet compression performance. Angle of repose, bulk and tap density, Hausner ratio, Carr’s compressibility index, a visual powder flow assessment, a mass flow rate test, Flodex, and powder shear testing were evaluated on several different blends of placebo powders to determine a predictive test for downstream process performance. The placebo powders consisted of a standard base unit formula, mixed with various levels of four different “pseudo-active pharmaceutical ingredients (APIs)” (i.e., excipients that mimic poorly flowing active pharmaceutical ingredients). All formulations were evaluated by the individual flow tests and then compressed on a rotary tablet press. Formulations were deemed failures if the relative standard deviation of tablet weight was >2 %, material stuck to punches (low lubrication), or exhibited ejection forces high enough to trigger automatic shutdown of tablet press. The 2 % RSD limit is based on internally developed best practices for transferring tablet compression processes from development into manufacturing. The results of the tablet compression process were then compared to the results of the flowability tests to detect any correlations. The Hausner ratio and powder shear test results discriminated marginally between successful and failed batches and were highly correlated with each other.

Effects of die core treatments and surface finishes on the sticking and galling tendency of Al–Si alloy casting during ejection

In the process of high pressure die casting of aluminium alloys tool surfaces are severely damaged by cast alloy sticking and soldering which promote galling during casting ejection. As a consequence, casting quality is lowered and production efficiency is reduced. Although die casting tools are greatly improved by application of diffusion layers and surface coatings, effects of surface topography on performance of such treated tools are scarcely recognized. This investigation gives an insight in the effects of surface topography on the sticking tendency and galling of Al–Si alloy toward the H11 steel, plasma nitrided steel, and duplex treated steel with CrN and TiAlN PVD top coatings. The sticking tendency was evaluated by ejection test during which the force required to eject the sample from the casting was measured. It was found that the ejection force does not depend on chemical composition of the investigated materials. However, the ejection force of coated samples strongly depends on their surface topography. The coated samples which were polished after the deposition exhibited the highest ejection force. Wear conditions inherent in this specific tribo-system are recognised and interpreted for surfaces with different topography.

Excessive interatrial adiposity is associated with left atrial remodeling, augmented contractile performance in asymptomatic population.

PurposePericardial adipose tissue had been shown to exert local effects on adjacent cardiac structures. Data regarding the mechanistic link between such measures and left atrial (LA) structural/functional remodeling, a clinical hallmark of early stage heart failure (HF) and atrial fibrillation (AF) incidence, in asymptomatic population remain largely unexplored.MethodsThis retrospective analysis includes 356 subjects free from significant valvular disorders, atrial fibrillation, or clinical HF. Regional adipose tissue including pericardial and periaortic fat volumes, interatrial septal (IAS), and left atrioventricular groove (AVG) fat thickness were all measured by multidetector computed tomography (MDCT) (Aquarius 3D Workstation, TeraRecon, San Mateo, CA, USA). We measured LA volumes, booster performance, reservoir capacity as well as conduit function, and analyzed their association with adiposity measures.ResultsAll four adiposity measures were positively associated with greater LA volumes (all P < 0.05), while IAS and AVG fat were also related to larger LA kinetic energy and worse reservoir capacity (both P < 0.01). In multivariate models, IAS fat thickness remained independently associated with larger LA volumes, increased LA kinetic energy and ejection force (β-coef: 0.17 & 0.15, both P < 0.05), and impaired LA reservoir and conduit function (β-coef: −0.20 & −0.12, both P < 0.05) after adjusting for clinical variables.ConclusionAccumulated visceral adiposity, especially interatrial fat depots, was associated with certain LA structural/functional remodeling characterized by impaired LA reservoir and conduit function though augmented kinetic energy and ejection performance. Our data suggested that interatrial fat burden may be associated with certain detrimental LA functions with compensatory LA adaptation in an asymptomatic population.

NuSAP governs chromosome oscillation by facilitating the Kid-generated polar ejection force.

In vertebrate cells, chromosomes oscillate to align precisely during metaphase. NuSAP, a microtubule-associated protein, plays a critical role in stabilizing spindle microtubules. In this study, we utilize 3D time-lapse live-cell imaging to monitor the role of NuSAP in chromosome oscillation and identify NuSAP as a novel regulator of the chromokinesin, Kid. Depletion of NuSAP significantly suppresses the amplitude and velocity of chromosome oscillation. We analyse the effects of NuSAP and Kid depletion in monopolar and bipolar cells with or without kinetochore microtubule depletion. Twelve postulated conditions are deciphered to reveal the contribution of NuSAP to the polar force generated at kinetochore microtubules and to the regulation of the polar ejection force generated by Kid, thus revealing a pivotal role of NuSAP in chromosome oscillation.

Left atrial ejection force correlates with left atrial strain and volume-based functional properties as assessed by three-dimensional speckle tracking echocardiography (from the MAGYAR-Healthy Study)

Introduction and ObjectiveThree-dimensional (3D) speckle tracking echocardiography (3DSTE) is a novel method for assessment of left atrial (LA) volumes and function without geometrical assumptions. 3DSTE allows detailed assessment of LA features including volume measurements, strain assessments and calculation of LA ejection force (LAEF). LA strain and volume-based functional parameters originate from the same 3D dataset, but assessment of LAEF requires more data including measurement of mitral annular dimensions and Doppler-derived inflow velocities. The present study was designed to find correlations between LAEF and 3DSTE-derived LA volume-based functional properties and strain parameters in healthy subjects. MethodsThe study population comprised 34 randomly selected healthy subjects (age 36.1±11.2 years, 15 men) in sinus rhythm, all of whom had undergone standard two-dimensional transthoracic Doppler echocardiographic study extended with 3DSTE. ResultsMitral annulus diameter-based LAEF correlated with global LA peak circumferential (r=0.39, p=0.02), longitudinal (r=0.32, p=0.05) and area (r=0.43, p=0.01) strain, total atrial stroke volume (r=0.30, p=0.05) and total atrial emptying fraction (r=0.31, p=0.05) characterizing (systolic) LA reservoir function and global LA 3D strain at atrial contraction (r=−0.44, p=0.01) and active atrial emptying fraction (r=0.36, p=0.04) characterizing (diastolic) LA contraction function (booster pump phase). ConclusionsComplex LA functional assessment can be provided by 3DSTE, including calculation of LAEF and volume-based and strain functional properties, with significant correlations between these parameters.

Left atrial ejection force correlates with left atrial strain and volume-based functional properties as assessed by three-dimensional speckle tracking echocardiography (from the MAGYAR-Healthy Study)

Introduction and ObjectiveThree-dimensional (3D) speckle tracking echocardiography (3DSTE) is a novel method for assessment of left atrial (LA) volumes and function without geometrical assumptions. 3DSTE allows detailed assessment of LA features including volume measurements, strain assessments and calculation of LA ejection force (LAEF). LA strain and volume-based functional parameters originate from the same 3D dataset, but assessment of LAEF requires more data including measurement of mitral annular dimensions and Doppler-derived inflow velocities. The present study was designed to find correlations between LAEF and 3DSTE-derived LA volume-based functional properties and strain parameters in healthy subjects. MethodsThe study population comprised 34 randomly selected healthy subjects (age 36.1±11.2 years, 15 men) in sinus rhythm, all of whom had undergone standard two-dimensional transthoracic Doppler echocardiographic study extended with 3DSTE. ResultsMitral annulus diameter-based LAEF correlated with global LA peak circumferential (r=0.39, p=0.02), longitudinal (r=0.32, p=0.05) and area (r=0.43, p=0.01) strain, total atrial stroke volume (r=0.30, p=0.05) and total atrial emptying fraction (r=0.31, p=0.05) characterizing (systolic) LA reservoir function and global LA 3D strain at atrial contraction (r=−0.44, p=0.01) and active atrial emptying fraction (r=0.36, p=0.04) characterizing (diastolic) LA contraction function (booster pump phase). ConclusionsComplex LA functional assessment can be provided by 3DSTE, including calculation of LAEF and volume-based and strain functional properties, with significant correlations between these parameters.

Chromokinesins NOD and KID use Alternative Nucleotide Mechanisms and One-Dimensional Diffusion to Target Microtubule Plus Ends

Chromokinesins have been found to associate with chromosomes during cell division and play a variety of functions including chromosome positioning, chromosome condensation, spindle bipolarity, and cytokinesis. These motors are thought to generate the elusive polar ejection force that drives chromatin away from the spindle poles. Kinesin-10's are chromokinesins that include human KID (also known as KIF22) and Drosophila melanogaster NOD, which share an N-terminal kinesin-like catalytic domain, a central stalk domain, and a C-terminal DNA-binding domain. Due to their common in vivo physiologies, we propose that kinesin-10's work together in teams of motors to translocate/target to spindle microtubule plus-ends. Using steady-state kinetics, we have characterized the motor domain constructs of NOD and KID by measuring the basal, unpolymerized tubulin-stimulated, and MT-stimulated steady-state ATPase and GTPase kinetics under both low (∼21 mM) and physiologically relevant (∼111 mM) ionic strength conditions. These results suggest both purine nucleotides may be utilized by kinesin-10's in vivo and set the foundation for comparing the ATPase and GTPase mechanisms of these two unconventional kinesins. Using GFP-tagged kinesin-10's and total internal reflection fluorescence (TIRF) microscopy, we observed the microtubule-bound NOD either remaining stationary or engaging in a one-dimensional random walk across the microtubule lattice. These two modes of microtubule binding are interchangeable and movement along the microtubule is rapid (similar to kinesin-13 and other microtubule end-binding proteins) and without directionality preference. Under these conditions, we observe significantly higher incidence of microtubule end-binding events, which is increased after removal of the negatively-charge C-terminal tails of tubulin using subtilisin. Our continued studies reveal mechanistic and functional details of kinesin-10 force generation and therefore provide new insights into the molecular motion that drives the polar ejection force during cell division.

Assessment of left ventricular ejection force and sympathetic skin response in normotensive and hypertensive subjects: A double-blind observational comparative case–control study

BackgroundPathophysiology of essential hypertension remains obscure. Correlation among ventricular ejection force, sympathetic activity, and hypertension is less clearly narrated in hypertensive subjects. Aims and objectivesTo assess correlation among ventricular ejection force, sympathetic activity, and hypertension in hypertensive subjects, and to be compared with normotensive subjects. MethodsThis is a case–control study to assess left ventricular ejection force (LVEF) and sympathetic skin response, in normotensive (group 1; control), and hypertensive subjects (group 2; cases). 100 cases were selected. Subjects having stages 1 and 2 hypertension were categorized in groups 2A and 2B, respectively. LVEF was calculated by using echocardiography observing aortic acceleration time (AT) and peak systolic velocity. Comparison among groups was done by using one-way ANOVA. ResultsBoth groups were comparable. In group 2, 60 cases had stage 1 hypertension and 40 had stage 2 hypertension. Significantly short AT and significantly high LVEF were found in hypertension (groups 2A and 2B) (p<0.0001). Sympathetic activity was high in group 2A (p<0.0001). Stroke volume (SV) was high in group 2B (p<0.0001). ConclusionStage 1 hypertension is a stage of increased sympathetic activity, leading to increased LVEF and hypertension (resetting of baroreceptors); stage 2 hypertension is a stage of normal sympathetic activity, increased LVEF, increased SV, and hypertension (possibly a stage of shift of renal equilibrium curve/renal output curve and blood pressure to a newer level).

FE optimization of the ejection force of a spring-maintained connector

The connector studied herein is a self-ejecting microconnector that features a circular compression spring. This type of circular connector ensures reliable connections for systems requiring reduced weight and miniaturization. Being implemented in various military and aerospace applications, the connector is liable to undergo extreme vibratory loadings and shocks; but even in more common applications (medical sensors, power control systems, communications, etc.) the manufacturer has to guarantee that the two ends will remain properly assembled to ensure electrical continuity. The main purpose of this work is to simulate the whole insertion-extraction process using the finite element method (FEM) and to optimize the ejection force required to unplug the connector. The shape of the groove machined on the male part of the connector is investigated, allowing determination of the geometric parameters of interest. Results will provide manufacturers with useful data, which may also help their customers choose their interconnection solution.

Would Left Atrial Strain Provide a Role as a New Prognostigator for Atrial Fibrillation?

REFER TO THE PAGE 20-27 Atrial fibrillation (AF) is the most common arrhythmia in clinical practice, which increases in prevalence with advancing age. Importantly, AF is associated with increased stroke as well as heart failure and mortality. Accordingly, medical costs related to this arrhythmia have been increasing in modern society Cardiovascular imaging plays a central role in assessing the etiology, pathophysiology and risk stratification in the patients with AF and provides therapeutic guide in certain circumstances.1),2) Undoubtedly, echocardiography is the most widely used tool in evaluating these patients. Evaluation of left atrial (LA) size and function is now feasible in clinical practice. Many studies have utilized volumetric or functional assessment of LA for correlation with or prediction of cardiovascular events such as AF or heart failure.3) However, there is currently no accepted gold standard parameter for evaluation of LA function in contrast to left ventricular function. Conventional parameters such as assessment of phasic atrial activities using LA volumes, transmitral Doppler peak late diastolic velocity and its time velocity integral, LA ejection force and LA appendage function can be used in assessing LA function. More recently, development of new echocardiographic techniques has enhanced the ability to assess myocardial properties in detail.4) Tissue Doppler imaging and strain imaging have been incorporated in evaluating LA global and segmental function, which might provide a better insight into LA function with higher sensitivity. Deformation measurement by speckle tracking can offer advantages over the Doppler-derived measurement given its angle independency and avoidance of tethering by the left ventricle. In this issue of the Journal, Kim et al.5) nicely demonstrated that global LA longitudinal strain by speckle tracking echocardiography can predict AF occurrence in 228 patients with acute ischemic stroke independently of LA size. They suggested global LA longitudinal strain < 14.5% for post-stroke AF development with sensitivity of 60% and specificity of 95%. While the exact pathogenesis of AF still remains unclear, both hemodynamic and non-hemodynamic factors would contribute to AF development. Previous studies have shown that LA strain is a predictor of adverse events over CHA2DS2-VASc score and it can be an important determinant for restoration and maintenance of sinus rhythm by catheter ablation or cardioversion in AF patients.6),7),8) Impaired LA strain can indicate reduced LA compliance or reservoir function and may indirectly reflect high fibrosis content.9) Early detection of LA dysfunction might provide the opportunities to modify the risk and improve clinical outcome. However, the suggested value of LA strain by Kim et al.5) had somewhat low sensitivity to use in clinical practice. Also, global LA strain in this study was lower than other previous data as the authors mentioned in the discussion and it could not extrapolate to general population for AF development.10),11),12) Although there have been considerable advances in understanding LA function using non-invasive imaging, pitfalls as well as advantages of each method should be considered in clinical applications. Speckle tracking echocardiography also has a challenge to be applied to the LA which is located in a farfield and has a very thin wall with the appendage and pulmonary veins. Further studies would be warranted to increase our understanding of the pathophysiologic changes regarding LA function and detect its subclinical condition.

The microtubule-binding and coiled-coil domains of Kid are required to turn off the polar ejection force at anaphase.

Mitotic chromosomes move dynamically along the spindle microtubules using the forces generated by motor proteins such as chromokinesin Kid (also known as KIF22). Kid generates a polar ejection force and contributes to alignment of the chromosome arms during prometaphase and metaphase, whereas during anaphase, Kid contributes to chromosome compaction. How Kid is regulated and how this regulation is important for chromosome dynamics remains unclear. Here, we address these questions by expressing mutant forms of Kid in Kid-deficient cells. We demonstrate that Cdk1-mediated phosphorylation of Thr463 is required to generate the polar ejection force on Kid-binding chromosomes, whereas dephosphorylation of Thr463 prevents generation of the ejection force on such chromosomes. In addition to activation of the second microtubule-binding domain through dephosphorylation of Thr463, the coiled-coil domain is essential in suspending generation of the polar ejection force, preventing separated chromosomes from becoming recongressed during anaphase. We propose that phosphorylation of Thr463 switches the mitotic chromosome movement from an anti-poleward direction to a poleward direction by converting the Kid functional mode from polar-ejection-force-ON to -OFF during the metaphase-anaphase transition, and that both the second microtubule-binding domain and the coiled-coil domain are involved in this switching process.

Assessment of Left Atrial Ejection Force in Patients with different Grades of Left Ventricular Diastolic Dysfunction

Assessment of Left Atrial Ejection Force in Patients with different Grades of Left Ventricular Diastolic Dysfunction Objective: To assess left atrial ejection force (LAEF) in patients with different grades of left ventricular (LV) diastolic dysfunction compared to traditional Doppler and tissue Doppler measurements. Methods: 120 patients enrolled into four equal groups (n=30), each group represented a different grade of LV diastolic dysfunction and 30 subjects with normal diastolic function. We assessed echocardiographic measurements of dimensions and diastolic function, LA volume index (LAVI), and LAEF and age-corrected %LAEF. Results: In comparison to control subjects, the study group had more diabetic and hypertensive patients, increased all LV dimensions, estimated LVEDP (p<0.0001), LAVI (p<0.0001), LAEF and %LAEF (p<0.0001). On comparing different grades of diastolic dysfunction, patients with worse grades of diastolic dysfunction were older, more hypertensive and diabetic. They had larger LV dimensions, estimated LVEDP (p<0.0001), and LAVI (p<0.0001). LAEF and %LAEF showed an increase from grade I (8.84 ± 3.09 Kdynes, 188.93 ± 40.12%) to Ia (10.66 ± 3.10 Kdynes, 239.7 ± 72.45%) to II (11.82 ± 45.9 Kdynes, 256.57 ± 45.92%) then demonstrated a significant reduction in grade III to 2.77 ± 1.71 Kdynes, 57.88 ± 32.49% (p<0.0001) dropping below normal values seen in the control subjects (4.98 ± 1.72 Kdynes, 93.30 ± 9.31%) (p<0.0001). Linear regression analysis showed factors affecting %LAEF were: age, E-wave velocity, average e’ velocity, E/A ratio and E/e’ ratio. Conclusions: LAEF is generally increased in patients with LV diastolic dysfunction compared to those with normal diastolic function. Patients in grade III diastolic dysfunction show marked impairment of LAEF dropping below those with normal diastolic function.

A smart pinless ejection mechanism using dual-resonance excitation Langevin piezoelectric transducers

This study investigated a smart pinless ejection mechanism comprising two dual-resonance excitation Langevin piezoelectric transducers (DRELPTs) for keeping the injection parts intact and protecting their top and bottom surfaces from scarring during plastic injection molding. The dimensions of each DRELPT were determined using longitudinal vibration models, and an optimization method was used to set the frequency ratio of the first to the second longitudinal mode to 1:2. This concept enables the driving of DRELPT in its two longitudinal modes consistent with the ejection direction in resonant-type smooth impact drive mechanisms. During the ejection process, DRELPT provides an ejection force, which is applied on the sidewalls of the injection parts to protect their top and bottom surfaces from scarring. Considering individual differences in the resonance frequencies of DRELPTs, a resonance frequency tracking circuit based on a phase-locked loop was designed to keep DRELPT actuating in resonance. The ejection velocity of the injection part was estimated using the kinetic models derived from the dynamic behavior of the mold cavity and injection parameters. A characteristic number S was defined to evaluate the average velocity of the injection part during ejection. Proof-of-concept experimental results of the pinless ejection mechanism are presented. The ejection time, that is, the time from triggering the composite wave to the full departure of the injection part from the mold cavity, was 72 ms.

Inferring the Forces Controlling Metaphase Kinetochore Oscillations by Reverse Engineering System Dynamics.

Kinetochores are multi-protein complexes that mediate the physical coupling of sister chromatids to spindle microtubule bundles (called kinetochore (K)-fibres) from respective poles. These kinetochore-attached K-fibres generate pushing and pulling forces, which combine with polar ejection forces (PEF) and elastic inter-sister chromatin to govern chromosome movements. Classic experiments in meiotic cells using calibrated micro-needles measured an approximate stall force for a chromosome, but methods that allow the systematic determination of forces acting on a kinetochore in living cells are lacking. Here we report the development of mathematical models that can be fitted (reverse engineered) to high-resolution kinetochore tracking data, thereby estimating the model parameters and allowing us to indirectly compute the (relative) force components (K-fibre, spring force and PEF) acting on individual sister kinetochores in vivo. We applied our methodology to thousands of human kinetochore pair trajectories and report distinct signatures in temporal force profiles during directional switches. We found the K-fibre force to be the dominant force throughout oscillations, and the centromeric spring the smallest although it has the strongest directional switching signature. There is also structure throughout the metaphase plate, with a steeper PEF potential well towards the periphery and a concomitant reduction in plate thickness and oscillation amplitude. This data driven reverse engineering approach is sufficiently flexible to allow fitting of more complex mechanistic models; mathematical models of kinetochore dynamics can therefore be thoroughly tested on experimental data for the first time. Future work will now be able to map out how individual proteins contribute to kinetochore-based force generation and sensing.

Consolidation of titanium hydride powders during the production of titanium PM parts: The effect of die wall lubricants

The effects of die wall lubricants on the cold compaction of titanium hydride powder are studied. Three commonly-used die wall powder metallurgy lubricants – zinc stearate, Acrawax® C dispersion and Mirror Glaze® – are compared. The influence of each lubricant on the cold compaction behaviour of titanium hydride powder was assessed with respect to the green density and strength, green compact ejection force, and the occurrence of green compact cracking. In addition, green compacts were sintered under vacuum at various sintering temperatures in order to determine the effect of the lubricants on oxygen content, phase compositions, microstructures and hardness of the sintered components. The results indicate that there is an immediate improvement in the cold compaction process when lubrication is used. The results further indicate that the lubricity of the lubricants is similar and most of the monitored variables are insensitive to the die wall lubricant used, the microstructures of the sintered components included. However, the use of Acrawax® dispersion resulted in more instances of green compact cracking, while zinc stearate tended to increase the oxygen content of sintered specimens due to the tentative decomposition of zinc oxide, a residual of the high temperature decomposition of zinc stearate.

Atrial dominance in the human embryonic heart: a study of cardiac function at 6-10 weeks of gestation.

To investigate the difference in forces developed by atrial and ventricular myocardium during contraction in human embryos using a novel method. This was a cross-sectional study of 115 pregnancies examined at 6-10 weeks of gestation. Ventricular inflow and outflow velocities were obtained by Doppler echocardiography and the relative force development was evaluated by calculating the ratio between atrial and ventricular ejection force (EF). Data from 97 embryos with monophasic ventricular filling were analyzed. Both inflow and outflow cardiac velocities increased with advancing gestational age (P < 0.001). The mean peak inflow velocity (31.7 ± 10.2 cm/s) was significantly higher (P < 0.0001) than the outflow velocity (19.6 ± 6.1 cm/s). The EFinflow /EFoutflow ratio was more than 1.0 in all but four (96%) cases. Our study indicates the possibility of atrial dominance in the embryonic period of heart development, which may have clinical implications, as deviations from this normal pattern may indicate human embryonic cardiac dysfunction.

Polar Ejection Forces Promote the Conversion from Lateral to End-on Kinetochore-Microtubule Attachments on Mono-oriented Chromosomes.

SummaryChromosome bi-orientation occurs after conversion of initial lateral attachments between kinetochores and spindle microtubules into stable end-on attachments near the cell equator. After bi-orientation, chromosomes experience tension from spindle forces, which plays a key role in the stabilization of correct kinetochore-microtubule attachments. However, how end-on kinetochore-microtubule attachments are first stabilized in the absence of tension remains a key unanswered question. To address this, we generated Drosophila S2 cells undergoing mitosis with unreplicated genomes (SMUGs). SMUGs retained single condensed chromatids that attached laterally to spindle microtubules. Over time, laterally attached kinetochores converted into end-on attachments and experienced intra-kinetochore stretch/structural deformation, and SMUGs eventually exited a delayed mitosis with mono-oriented chromosomes after satisfying the spindle-assembly checkpoint (SAC). Polar ejection forces (PEFs) generated by Chromokinesins promoted the conversion from lateral to end-on kinetochore-microtubule attachments that satisfied the SAC in SMUGs. Thus, PEFs convert lateral to stable end-on kinetochore-microtubule attachments, independently of chromosome bi-orientation.

Effect of jet injection on infectivity of measles, mumps, and rubella vaccine in a bench model

Disposable-syringe jet injectors (DSJIs) with single-use, auto disable, needle-free syringes offer the opportunity to avoid hazards associated with injection using a needle and syringe. Clinical studies have evaluated DSJIs for vaccine delivery, but most studies have focused on inactivated, subunit, or DNA vaccines. Questions have been raised about possible damage to live attenuated viral vaccines by forces generated during the jet injection process. This study examines the effect of jet injection on the integrity of measles, mumps, and rubella vaccine (MMR), measured by viral RNA content and infectivity. Three models of DSJIs were evaluated, each generating a different ejection force. Following jet injection, the RNA content for each of the vaccine components was measured using RT-qPCR immediately after injection and following passage in Vero cells. Jet injection was performed with and without pig skin as a simulation of human skin. There was little to no reduction of RNA content immediately following jet injection with any of the three DSJIs. Samples passaged in Vero cells showed no loss in infectivity of the measles vaccine following jet injection. Mumps vaccine consistently showed increased replication following jet injection. Rubella vaccine showed no loss after jet injection alone but some infectivity loss following injection through pig skin with two of the devices. Overall, these data demonstrated that forces exerted on a live attenuated MMR vaccine did not compromise vaccine infectivity. The bench model and protocol used in this study can be applied to evaluate the impact of jet injection on other live virus vaccines.

Aurora A Kinase Contributes to a Pole-Based Error Correction Pathway

Chromosome biorientation, where sister kinetochores attach to microtubules (MTs) from opposing spindle poles, is the configuration that best ensures equal partitioning of the genome during cell division. Erroneous kinetochore-MT attachments are commonplace but are often corrected prior to anaphase. Error correction, thought to be mediated primarily by the centromere-enriched Aurora B kinase (ABK), typically occurs near spindle poles; however, the relevance of this locale is unclear. Furthermore, polar ejection forces (PEFs), highest near poles, can stabilize improper attachments by pushing mal-oriented chromosome arms away from spindle poles. Hence, there is a conundrum: erroneous kinetochore-MT attachments are weakened where PEFs are most likely to strengthen them. Here, we report that Aurora A kinase (AAK) opposes the stabilizing effect of PEFs. AAK activity contributes to phosphorylation of kinetochore substrates near poles and its inhibition results in chromosome misalignment and an increased incidence of erroneous kinetochore-MT attachments. Furthermore, AAK directly phosphorylates a site in the N-terminal tail of Ndc80/Hec1 that has been implicated in reducing the affinity of the Ndc80 complex for MTs when phosphorylated. We propose that an AAK activity gradient contributes to correcting mal-oriented kinetochore-MT attachments in the vicinity of spindle poles.