Springer Science and Business Media LLC

Determining in vivo force–velocity relationships of motor proteins is a critical step toward clarifying how they accomplish intracellular transport. We show that in vivo force–velocity curves corresponding to an estimated 1, 2, and 3 motors-per-vesicle can be constructed by tracking and sizing transported vesicles. The force range for these curves would normally be constrained by diffraction limited diameter measurements. However, we present a new method that uses the image intensity obtained with differential interference contrast microscopy as a proxy for vesicle diameters smaller than the diffraction limit. We calibrate this novel sizing method in vitro with polystyrene microsphere standards and apply it in vivo to vesicles. The resulting diameter vs. velocity data for large, small, and sub-diffraction limited vesicles is used to construct force–velocity curves that extend the force range of our previous curves. These extended 1-, 2-, and 3-motor in vivo curves qualitatively agree with a simple model of load sharing for motors that jointly transport a single vesicle.

The relationship between substrate properties and cell behavior is complex, including roles for both mechanics and biochemistry. Here we investigate the role of viscous dissipation on cell adhesion behaviors, using polymer films of tunable lateral mobility. We find that fibroblasts selectively use $$\alpha _v \beta _3$$ and $$\alpha _5 \beta _1$$ integrin receptors to control their spreading area and polarization on low and high mobility films, respectively. In addition, the dynamics of cell spreading and polarization are well described by a semi-empirical sigmoidal relationship. Analysis of cell dynamic behavior reveals that spreading dynamics are controlled by the availability of integrins, whereas the polarization dynamics are controlled by intracellular signaling. The result that cells preferentially use specific integrin receptors in response to substrate mechanical properties has broad implications for processes in dynamic environments such as wound healing and cancer metastasis.

Mechanical loads are essential toward maintaining bone mass and skeletal integrity. Such loads generate various stimuli at the cellular level, including cyclic hydraulic pressure (CHP) and fluid shear stress (FSS). To gain insight into the anabolic responses of osteoblasts to CHP and FSS, we subjected MC3T3-E1 preosteoblasts to either FSS (12 dynes/cm2) or CHP varying from 0 to 68 kPa at 0.5 Hz. As with FSS, CHP produced a significant increase in ATP release over static controls within 5 min of onset. Cell stiffness examined by atomic force microscopy increased after 15 min of either CHP or FSS stimulation, which was attenuated when extracellular ATP was hydrolyzed with apyrase. As previously shown FSS induced polymerization of actins into stress fibers. However, the microtubule network was completely disrupted under FSS. In contrast, CHP appeared to maintain strong microtubule and f-actin networks. The purinergic signaling was found to be involved in the remodeling of f-actin, but not microtubule. Both CHP and FSS applied for 1 h increased expression of COX-2. These data indicate that, while CHP and FSS produce similar anabolic responses, these stimuli have very different effects on the cytoskeleton remodeling and could contribute to loss of mechanosensitivity with extended loading.

The development of an endothelialized membrane oxygenator requires solution strategies combining the knowledge of oxygenators with endothelial cells’ biology. Since it is well known that exposing cells towards pure oxygen causes oxidative stress, this aspect has to be taken into account in the development of a biohybrid oxygenator system. N-Acetylcysteine (NAC) is known for its antioxidant properties in cells. We tested its applicability for the development of an endothelialized oxygenator model. Cultivating human umbilical vein derived endothelial cells (HUVEC) up to 6 days with increasing concentrations of NAC from 1 to 30 mM revealed NAC toxicity at concentrations from 20 mM. Cell density clearly decreased after radical oxygen species exposure in non-NAC pretreated cells compared to 20 mM NAC precultured HUVEC after 3 and 6 days. Also the survival rate after ROS treatment could be restored by incubation with NAC from 15 to 25 mM for all time points. NAC treated cells changed their morphology from typical endothelial cells’ cobblestone pattern to a fusiform, elongated configuration. Transformed cells were still positive for typical endothelial cell markers. Our present results show the potential of NAC for the protection of an endothelial cell layer in an endothelialized membrane oxygenator due to its antioxidative properties. Moreover, NAC induces a morphological change in HUVEC similar to dynamic cultivation procedures.

Accurate determination of solute diffusivities from fluorescence recovery after photobleaching (FRAP) experiments is often hindered by limitations of existing analytical models. This study describes the development and validation of a finite-element-based direct diffusion simulation parameter estimation (DDSPE) method for determining solute diffusivities from FRAP data. The DDSPE method improves on other models by accounting for experimentally measured post-bleaching fluorescence profiles and time-varying boundary conditions, and includes a reaction term to account for the detrimental effects of low level photobleaching produced by image acquisition during recovery. Analyses of simulated FRAP data demonstrate the advantages of this method over common analytical approaches, including a low sensitivity to variations in the spot radius and to the effects of photobleaching during scanning. As an example application, the effects of gel density and dextran size on the diffusivities of fluorescently labeled dextrans (10–250 kDa) in agarose gels (2–6%) were measured via FRAP. As with the simulated data, the DDSPE method was insensitive to spot radius while analytical models were strongly dependent on this experimental parameter. The diffusivities determined by the DDSPE method decreased with increasing solute size and gel density and were in excellent agreement with reference values based on a recent empirical model.

AddAB enzyme is a helicase–nuclease complex that initiates recombinational repair of double-stranded DNA breaks. It catalyzes processive DNA unwinding and concomitant resection of the unwound strands, which are modulated by the recognition of a recombination hotspot called Chi in the 3′-terminated strand. Despite extensive structural, biochemical and single molecule studies, the detailed molecular mechanism of DNA unwinding by the complex and modulation by Chi sequence remains unclear. A model of DNA unwinding by the AddAB complex and modulation by Chi recognition was presented, based on which the dynamics of AddAB complex was studied analytically. The theoretical results explain well the available experimental data on effect of DNA sequence on velocity, effect of Chi recognition on velocity, static disorder peculiar to the AddAB complex, and dynamics of pausing of wild-type and mutant AddAB complexes occurring at Chi or Chi-like sequence. Predictions were provided. Comparisons of AddAB complex with other helicase–nuclease complexes such as RecBCD and AdnAB were made. The study has strong implications for the molecular mechanism of DNA unwinding by the AddAB complex. The intriguing issues are addressed of why Chi recognition is an inefficient process, how AddAB complex pauses upon recognizing Chi sequence, how the paused state transits to the translocating state, why the mutant AddAB with a stronger affinity to Chi sequence has a shorter pausing lifetime, why the pausing lifetime is sensitive to the solution temperature, and so on.

SARS-CoV-2 is a SARS-like novel coronavirus strain first identified in December 2019 in Wuhan, China. The virus has since spread globally, resulting in the current ongoing coronavirus disease 19 (COVID-19) pandemic. SARS-CoV-2 spike protein is a critical factor in the COVID-19 pathogenesis via interactions with the host cell angiotensin-converting enzyme 2 (ACE2) PD domain. Worldwide, numerous efforts are being made to combat COVID19. In the current study, we identified potential peptidomimetics against the SARS-CoV-2 spike protein. We utilized the information from ACE2-SARS-CoV-2 binary interactions, and based on crucial interacting interface residues, novel peptidomimetics were designed. Top scoring peptidomimetics were found to bind at the ACE2 binding site of the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. The current studies could pave the way for further investigations of these novel and potent compounds against the SARS-CoV-2.

Nanoporous gold (np-Au) is a promising multifunctional material for neural electrodes. We have previously shown that np-Au nanotopography reduces astrocyte surface coverage (linked to undesirable gliosis) while maintaining high neuronal coverage in a cortical primary neuron-glia co-culture model as long as 2 weeks in vitro. Here, we investigate the potential influence of secreted soluble factors from cells grown on np-Au leading to the cell type-specific surface coverage on conventional tissue culture plastic. We then test the hypothesis that secretion of factors is responsible for inhibiting astrocyte coverage on np-Au. In order to assess whether factors secreted from cells grown on np-Au surfaces reduced surface coverage by astrocytes, we seeded fresh primary rat neuron-glia co-cultures on conventional polystyrene culture dishes, but maintained the cells in conditioned media from co-cultures grown on np-Au surfaces. After 1 week in vitro, a preferential reduction in astrocyte surface coverage was not observed, suggesting that soluble factors are not playing a role. In contrast, 4 h after cell seeding there were a significant number of non-adhered, yet still viable, cells for the cultures on np-Au surfaces. We hypothesize that the non-adherent cells are mainly astrocytes, because: (i) there was no difference in neuronal cell coverage between np-Au and pl-Au for long culture durations and (ii) neurons are post-mitotic and not expected to increase in number upon attaching to the surface. Overall, the results suggest that the np-Au topography leads to preferential neuronal attachment shortly after cell seeding and limits astrocyte-specific np-Au surface coverage at longer culture durations.