Zenon Rajfur

Inhibition of oxytocin and vasopressin neuron activity in rat hypothalamic paraventricular nucleus by relaxin‐3–RXFP3 signalling

Relaxin‐3 is a stress‐responsive neuropeptide that acts at its cognate receptor, RXFP3, to alter behaviours including feeding. In this study, we have demonstrated a direct, RXFP3‐dependent, inhibitory action of relaxin‐3 on oxytocin and vasopressin paraventricular nucleus (PVN) neuron electrical activity, a putative cellular mechanism of orexigenic actions of relaxin‐3. We observed a Gαi/o‐protein‐dependent inhibitory influence of selective RXFP3 activation on PVN neuronal activity in vitro and demonstrated a direct action of RXFP3 activation on oxytocin and vasopressin PVN neurons, confirmed by their abundant expression of RXFP3 mRNA. Moreover, we demonstrated that RXFP3 activation induces a cadmium‐sensitive outward current, which indicates the involvement of a characteristic magnocellular neuron outward potassium current. Furthermore, we identified an abundance of relaxin‐3‐immunoreactive axons/fibres originating from the nucleus incertus in close proximity to the PVN, but associated with sparse relaxin‐3‐containing fibres/terminals within the PVN.

Glutaraldehyde fixation preserves the trend of elasticity alterations for endothelial cells exposed to TNF-α.

Among the users of atomic force microscopy based techniques, there is an ongoing discussion, whether cell elasticity measurements performed on fixed cells could be used for determination of the relative elasticity changes of the native (unfixed) cells subjected to physiologically active external agents. In this article, we present a case, for which the legitimacy of cell fixation for elasticity measurements is justified. We provide an evidence that the alterations of cell elasticity triggered by tumor necrosis factor alpha (TNF‐α) in EA.hy926 endothelial cells are preserved after glutaraldehyde (GA) fixation. The value of post‐fixation elasticity parameter is a product of the elasticity parameter obtained for living cells and a constant value, dependent on the GA concentration. The modification of the initial value of elasticity parameter caused by remodeling of the cortical actin cytoskeleton is reflected in the elasticity measurements performed on fixed cells. Thus, such fixation procedure may be particularly helpful for experiments, where the influence of an external agent on the cell cortex should be assessed and AFM measurements of living cells are problematic or better statistics is needed.

Studies of unicellular microorganisms Saccharomyces cerevisiae by means of positron annihilation lifetime spectroscopy

Abstract Results of positron annihilation lifetime spectroscopy (PALS) and microscopic studies on simple microorganisms, brewing yeasts, are presented. Lifetime of ortho-positronium (o-Ps) were found to change from 2.4 to 2.9 ns (longer-lived component) for lyophilized and aqueous yeasts, respectively. Also hygroscopicity of yeasts in time was examined, allowing to check how water – the main component of the cell – affects PALS parameters, thus lifetime of o-Ps were found to change from 1.2 to 1.4 ns (shorter-lived component) for the dried yeasts. The time sufficient to hydrate the cells was found below 10 hours. In the presence of liquid water, an indication of reorganization of yeast in the molecular scale was observed. Microscopic images of the lyophilized, dried, and wet yeasts with best possible resolution were obtained using inverted microscopy (IM) and environmental scanning electron microscopy (ESEM) methods. As a result, visible changes to the surface of the cell me mbrane were observed in ESEM images.

AFM-based detection of glycocalyx degradation and endothelial stiffening in the db/db mouse model of diabetes

Degradation of the glycocalyx and stiffening of endothelium are important pathophysiological components of endothelial dysfunction. However, to our knowledge, these events have not been investigated in tandem in experimental diabetes. Here, the mechanical properties of the glycocalyx and endothelium in ex vivo mouse aorta were determined simultaneously in indentation experiments with an atomic force microscope (AFM) for diabetic db/db and control db/+ mice at ages of 11–19 weeks. To analyze highly heterogeneous aorta samples, we developed a tailored classification procedure of indentation data based on a bi-layer brush model supplemented with Hertz model for quantification of nanomechanics of endothelial regions with and without the glycocalyx surface. In db/db mice, marked endothelial stiffening and reduced glycocalyx coverage were present already in 11-week-old mice and persisted in older animals. In contrast, reduction of the effective glycocalyx length was progressive and was most pronounced in 19-week-old db/db mice. The reduction of the glycocalyx length correlated with an increasing level of glycated haemoglobin and decreased endothelial NO production. In conclusion, AFM nanoindentation analysis revealed that stiffening of endothelial cells and diminished glycocalyx coverage occurred in early diabetes and were followed by the reduction of the glycocalyx length that correlated with diabetes progression.

Early events during human coronavirus OC43 entry to the cell

The Coronaviridae family clusters a number of large RNA viruses, which share several structural and functional features. However, members of this family recognize different cellular receptors and exploit different entry routes, what affects their species specificity and virulence. The aim of this study was to determine how human coronavirus OC43 enters the susceptible cell. Using confocal microscopy and molecular biology tools we visualized early events during infection. We found that the virus employs caveolin-1 dependent endocytosis for the entry and the scission of virus-containing vesicles from the cell surface is dynamin-dependent. Furthermore, the vesicle internalization process requires actin cytoskeleton rearrangements. With our research we strove to broaden the understanding of the infection process, which in future may be beneficial for the development of a potential therapeutics.

APOBEC3-mediated restriction of RNA virus replication

APOBEC3 family members are cytidine deaminases with roles in intrinsic responses to infection by retroviruses and retrotransposons, and in the control of other DNA viruses, such as herpesviruses, parvoviruses and hepatitis B virus. Although effects of APOBEC3 members on viral DNA have been demonstrated, it is not known whether they edit RNA genomes through cytidine deamination. Here, we investigated APOBEC3-mediated restriction of Coronaviridae. In experiments in vitro, three human APOBEC3 proteins (A3C, A3F and A3H) inhibited HCoV-NL63 infection and limited production of progeny virus, but did not cause hypermutation of the coronaviral genome. APOBEC3-mediated restriction was partially dependent on enzyme activity, and was reduced by the use of enzymatically inactive APOBEC3. Moreover, APOBEC3 proteins bound to the coronaviral nucleoprotein, and this interaction also affected viral replication. Although the precise molecular mechanism of deaminase-dependent inhibition of coronavirus replication remains elusive, our results further our understanding of APOBEC-mediated restriction of RNA virus infections.

Precise mass determination of single cell with cantilever-based microbiosensor system

Having determined the mass of a single cell of brewer yeast Saccharomyces cerevisiae by means of a microcantilever-based biosensor Cantisens CSR-801 (Concentris, Basel, Switzerland), it was found that its dry mass is 47,65 ± 1,05 pg. Found to be crucial in this mass determination was the cell position along the length of the cantilever. Moreover, calculations including cells positions on the cantilever provide a threefold better degree of accuracy than those which assume uniform mass distribution. We have also examined the influence of storage time on the single cell mass. Our results show that after 6 months there is an increase in the average mass of a single yeast cell.

Entry of Human Coronavirus NL63 into the Cell

ABSTRACT The first steps of human coronavirus NL63 (HCoV-NL63) infection were previously described. The virus binds to target cells by use of heparan sulfate proteoglycans and interacts with the ACE2 protein. Subsequent events, including virus internalization and trafficking, remain to be elucidated. In this study, we mapped the process of HCoV-NL63 entry into the LLC-Mk2 cell line and ex vivo three-dimensional (3D) tracheobronchial tissue. Using a variety of techniques, we have shown that HCoV-NL63 virions require endocytosis for successful entry into the LLC-MK2 cells, and interaction between the virus and the ACE2 molecule triggers recruitment of clathrin. Subsequent vesicle scission by dynamin results in virus internalization, and the newly formed vesicle passes the actin cortex, which requires active cytoskeleton rearrangement. Finally, acidification of the endosomal microenvironment is required for successful fusion and release of the viral genome into the cytoplasm. For 3D tracheobronchial tissue cultures, we also observed that the virus enters the cell by clathrin-mediated endocytosis, but we obtained results suggesting that this pathway may be bypassed. IMPORTANCE Available data on coronavirus entry frequently originate from studies employing immortalized cell lines or undifferentiated cells. Here, using the most advanced 3D tissue culture system mimicking the epithelium of conductive airways, we systematically mapped HCoV-NL63 entry into susceptible cells. The data obtained allow for a better understanding of the infection process and may support development of novel treatment strategies.

Canine respiratory coronavirus employs caveolin-1-mediated pathway for internalization to HRT-18G cells

Canine respiratory coronavirus (CRCoV), identified in 2003, is a member of the Coronaviridae family. The virus is a betacoronavirus and a close relative of human coronavirus OC43 and bovine coronavirus. Here, we examined entry of CRCoV into human rectal tumor cells (HRT-18G cell line) by analyzing co-localization of single virus particles with cellular markers in the presence or absence of chemical inhibitors of pathways potentially involved in virus entry. We also targeted these pathways using siRNA. The results show that the virus hijacks caveolin-dependent endocytosis to enter cells via endocytic internalization.

Cdk5-mediated phosphorylation regulates phosphatidylinositol 4-phosphate 5-kinase type I γ 90 activity and cell invasion

Phosphatidylinositol 4‐phosphate 5‐kinase type I γ (PIPKIγ90) regulates cell migration, invasion, and metastasis. However, it is unknown how cellular signals regulate those processes. Here, we show that cyclin‐dependent kinase 5 (Cdk5), a protein kinase that regulates cell migration and invasion, phosphorylates PIPKIγ90 at S453, and that Cdk5‐mediated PIPKIγ90 phosphorylation is essential for cell invasion. Moreover, Cdk5‐mediated phosphorylation down‐regulates the activity of PIPKIγ90 and the secretion of fibronectin, an extracellular matrix protein that regulates cell migration and invasion. Furthermore, inhibition of PIPKIγ activity with the chemical inhibitor UNC3230 suppresses fibronectin secretion in a dose‐dependent manner, whereas depletion of Cdk5 enhances fibronectin secretion. With total internal reflection fluorescence microscopy, we found that secreted fibronectin appears as round dots, which colocalize with Tks5 and CD9 but not with Zyxin. These data suggest that Cdk5‐mediated PIPKIγ90 phosphorylation regulates cell invasion by controlling PIPKIγ90 activity and fibronectin secretion.—Li, L., Kotodziej, T., Jafari, N., Chen, J., Zhu, H., Rajfur, Z., Huang, C. Cdk5‐mediated phosphorylation regulates phosphatidylinositol 4‐phosphate 5‐kinase type I γ 90 activity and cell invasion. FASEB J. 33, 631–642 (2019). www.fasebj.org