Viral adventitious agent detection using laser force cytology: Intrinsic cell property changes with infection and comparison to in vitro testing

Adventitious agent testing in biomanufacturing requires assays of broad detection capability to screen for as many infectious agents as possible. The current gold standard for general infectious adventitious virus screening is the in vitro assay in which test articles are cultured onto a panel of different cell lines and observed for cytopathic effect (CPE). However, this assay is inherently subjective due to the nature of visual observation of cell morphology and labor and time intensive, requiring highly trained personnel to identify CPE. Laser force cytology (LFC) is an alternative, automated analytical method that uses a combination of optical and fluidic forces along with imaging to objectively and quantitatively assess CPE in cell culture. Importantly, because LFC uses no labels or antibodies, the assay is appropriate for general adventitious agent testing. Using LFC, changes in cellular features associated with virally infected cells were identified using principal component analysis. Using these features of infected cells, the sensitivity and earliness of detection with LFC was directly compared with the in vitro assay for a diverse panel of viruses incubated with chinese hamster ovary (CHO), Vero, and Medical Research Council cell strain 5 (MRC-5) cells. LFC detected viral infection with a sensitivity equal to the in vitro assay on average, but in certain virus and cell combinations including mouse minute virus (MMV) and reovirus 3 in CHO cells, detection was 4 days earlier and for MMV, the limit of detection was 10-fold lower. Overall, these results demonstrate the ability of LFC to serve as a biopharmaceutical adventitious agent testing methodology with sensitivity equivalent to the in vitro assay, but in an objective and automated manner.     

Nanoscale nucleosome dynamics assessed with time-lapse AFM

A fundamental challenge associated with chromosomal gene regulation is accessibility of DNA within nucleosomes. Recent studies performed by various techniques, including single-molecule approaches, led to the realization that nucleosomes are dynamic structures rather than static systems, as was once believed. Direct data are required in order to understand the dynamics of nucleosomes more clearly and to answer fundamental questions, including: What is the range of nucleosome dynamics? Does a non-ATP-dependent unwrapping process of nucleosomes exist? What are the factors facilitating the large-scale opening and unwrapping of nucleosomes? This review summarizes the results of nucleosome dynamics obtained with time-lapse AFM, including a high-speed version (HS-AFM) capable of visualizing molecular dynamics on the millisecond time scale. With HS-AFM, the dynamics of nucleosomes at a sub-second time scale was observed, allowing one to visualize various pathways of nucleosome dynamics, such as sliding and unwrapping, including complete dissociation. Overall, these findings reveal new insights into the dynamics of nucleosomes and the novel mechanisms controlling spontaneous chromatin dynamics.     

原子力显微技术在观测微生物表面形态中的应用

原子力显微技术作为一门新发展起来的显微成像技术,不仅具有在近生理条件下对样本实时、高分辨率三维成像等特点,而且能通过力矩测量探知样本物理性状。即给人们认识微生物的表面结构提供又一平台,也为揭示微生物表面结构与功能之间的关系提供一种新方法。介绍了对微生物表面形态观测中常用测量模式和某些样品固定方法:多孔膜技术、凹陷技术,概括近年来原子力显微技术在微生物学中的应用情况。     

AFM characterization of tilt and intrinsic flexibility of Rhodobacter sphaeroides light harvesting complex 2 (LH2)

Atomic force microscopy (AFM) has developed into a powerful tool to investigate membrane protein surfaces in a close-to-native environment. Here we report on the surface topography of Rhodobacter sphaeroides light harvesting complex 2 (LH2) reconstituted into two-dimensional crystals. These photosynthetic trans-membrane proteins formed cylindrical oligomeric complexes, which inserted tilted into the lipid membrane. This peculiar packing of an integral membrane protein allowed us to determine oligomerization and tilt of the LH2 complexes, but also protrusion height and intrinsic flexibility of their individual subunits. Furthermore the surface contouring reliability and limits of the atomic force microscopy could be studied. The two-dimensional crystals examined had sizes of up to 5 microm and, as revealed by a 10 A cryo electron microscopy projection map, p22(1)2(1) crystal symmetry. The unit cell had dimensions of a = b = 150 A and gamma = 90 degrees, and housed four nonameric complexes, two pointing up and two pointing down. AFM topographs of these 2D crystals had a lateral resolution of 10 A. Further, the high vertical resolution of approximately 1 A, allowed the protrusion height of the cylindrical LH2 complexes over the membrane to be determined. This was maximally 13.1 A on one side and 3.8 A on the other. Interestingly, the protrusion height varied across the LH2 complexes, showing the complexes to be inserted with a 6.2 degree tilt with respect to the membrane plane. A detailed analysis of the individual subunits showed the intrinsic flexibility of the membrane protruding peptide stretches to be equal and independent of their protrusion height. Furthermore, our analysis of membrane proteins within this peculiar packing confirmed the high vertical resolution of the atomic force microscopy on biological samples, and led us to conclude that the image acquisition function was equally accurate for contouring protrusions with heights up to approximately 15 A.     

Sensitivity of a subject-specific musculoskeletal model to the uncertainties on the joint axes location

Subject-specific musculoskeletal models have become key tools in the clinical decision-making process. However, the sensitivity of the calculated solution to the unavoidable errors committed while deriving the model parameters from the available information is not fully understood. The aim of this study was to calculate the sensitivity of all the kinematics and kinetics variables to the inter-examiner uncertainty in the identification of the lower limb joint models. The study was based on the computer tomography of the entire lower-limb from a single donor and the motion capture from a body-matched volunteer. The hip, the knee and the ankle joint models were defined following the International Society of Biomechanics recommendations. Using a software interface, five expert anatomists identified on the donor's images the necessary bony locations five times with a three-day time interval. A detailed subject-specific musculoskeletal model was taken from an earlier study, and re-formulated to define the joint axes by inputting the necessary bony locations. Gait simulations were run using OpenSim within a Monte Carlo stochastic scheme, where the locations of the bony landmarks were varied randomly according to the estimated distributions. Trends for the joint angles, moments, and the muscle and joint forces did not substantially change after parameter perturbations. The highest variations were as follows: (a) 11° calculated for the hip rotation angle, (b) 1% BW × H calculated for the knee moment and (c) 0.33 BW calculated for the ankle plantarflexor muscles and the ankle joint forces. In conclusion, the identification of the joint axes from clinical images is a robust procedure for human movement modelling and simulation.     

Ontogenetic scaling of the morphology and biomechanics of the feeding apparatus in the Pacific hagfish Eptatretus stoutii

The form and function of the support skeleton, musculature and teeth were examined in an ontogenetic series of Pacific hagfish Eptatretus stoutii spanning about a six-fold range in total length (L(T)). Tooth area, feeding apparatus length, basal plate size, theoretical dental plate retractile force, penetration force and applied tooth stress were measured relative to body size. Morphological variables (e.g. tooth area and basal plate size) scaled with positive allometry and functional variables (e.g. retractile force and applied tooth stress) scaled isometrically with L(T). These results suggest that juveniles do not undergo ontogenetic dietary changes and consume functionally equivalent prey to adults, although adults can grasp proportionally larger portions of food. Low tooth stress in juveniles and adults imposes mechanical constraints to puncturing and tearing, which are circumvented by a preference for softer prey tissue or the inclusion of knotting behaviours for reducing tougher prey.     

Association between biomechanical alterations and migratory ability of semaphorin-3A-treated thymocytes

Background

Class 3 semaphorins are soluble proteins involved in cell adhesion and migration. Semaphorin-3A (Sema3A) was initially shown to be involved in neuronal guidance, and it has also been reported to be associated with immune disorders. Both Sema3A and its receptors are expressed by most immune cells, including monocytes, macrophages, and lymphocytes, and these proteins regulate cell function. Here, we studied the correlation between Sema3A-induced changes in biophysical parameters of thymocytes, and the subsequent repercussions on cell function.

Negatively Charged Lipids Are Essential for Functional and Structural Switch of Human 2-Cys Peroxiredoxin II

The function of ubiquitous 2-Cys peroxiredoxins (Prxs) can be converted alternatively from peroxidases to molecular chaperones. This conversion has been reported to occur by the formation of high-molecular-weight (HMW) complexes upon overoxidation of or ATP/ADP binding to 2-Cys Prxs, but its mechanism is not well understood. Here, we show that upon binding to phosphatidylserine or phosphatidylglycerol dimeric human 2-Cys PrxII (hPrxII) is assembled to trefoil-shaped small oligomers (possibly hexamers) with full chaperone and null peroxidase activities. Spherical HMW complexes are formed, only when phosphatidylserine or phosphatidylglycerol is bound to overoxidized or ATP/ADP-bound hPrxII. The spherical HMW complexes are lipid vesicles covered with trefoil-shaped oligomers arranged in a hexagonal lattice pattern. Thus, these lipids with a net negative charge, which can be supplied by increased membrane trafficking under oxidative stress, are essential for the structural and functional switch of hPrxII and possibly most 2-Cys Prxs.     

Effect of external torque on the ATP-driven rotation of F1-ATPase

F₁-ATPase is a rotary molecular motor powered by the torque generated by another rotary motor F₀ to synthesize ATP in vivo. Therefore elucidation of the behavior of F₁ under external torque is very important. Here, we applied controlled external torque by electrorotation and investigated the ATP-driven rotation for the first time. The rotation was accelerated by assisting torque and decelerated by hindering torque, but F₁ rarely showed rotations in the ATP synthesis direction. This is consistent with the prediction by models based on the assumption that the rotation is tightly coupled to ATP hydrolysis and synthesis. At low ATP concentrations (2 and 5 μM), 120° stepwise rotation was observed. Due to the temperature rise during experiment, quantitative interpretation of the data is difficult, but we found that the apparent rate constant of ATP binding clearly decreased by hindering torque and increased by assisting torque.