Membrane-bound heat shock protein mHsp70 is required for migration and invasion of brain tumors.

Molecular chaperones, especially 70 kDa heat shock protein, in addition to their intracellular localization in cancer cells, can be exposed on the surface of the plasma membrane. We report that the membrane-associated chaperone mHsp70 of malignant brain tumors is required for high migratory and invasive activity of cancer cells. Live-cell inverted confocal microscopy of tumor samples from adult (n=23) and pediatric (n=9) neurooncological patients showed pronounced protein expression on the membrane, especially in the perifocal zone. Mass-spectrometry analysis of lipid rafts isolated from tumor cells confirmed the presence of the protein in chaperone cluster (including representatives of other families such as Hsp70, Hsc70, Hsp105, Hsp90), which in turn, during interactome analysis, was associated with proteins involved in cell migration (e.g., Rac1, RhoC, myosin-9). The use of small-molecule inhibitors of HSP70 (PES, JG-98) led to a significant decrease in the invasive potential of cells isolated from a tumor sample of patients, which indicates the role of the chaperone in invasion. Moreover, the use of HSP70 inhibitors in animal models of orthotopic brain tumors significantly delayed tumor progression, which was accompanied by an increase in overall survival. Data demonstrate that chaperone inhibitors, particularly JG-98, disrupt the function of mHsp70, thereby providing an opportunity to better understand the diverse functions of this protein and offer aid in the development of novel cancer therapies.

Mesenchymal stem cells-derived extracellular vesicles for therapeutics of renal tuberculosis.

Extrapulmonary tuberculosis with a renal involvement can be a manifestation of a disseminated infection that requires therapeutic intervention, particularly with a decrease in efficacy of conventional regimens. In the present study, we investigated the therapeutic potency of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) in the complex anti-tuberculosis treatment (ATT). A rabbit model of renal tuberculosis (rTB) was constructed by injecting of the standard strain Mycobacterium tuberculosis H37Rv into the cortical layer of the kidney parenchyma. Isolated rabbit MSC-EVs were intravenously administered once as an addition to standard ATT (isoniazid, pyrazinamide, and ethambutol). The therapeutic efficacy was assessed by analyzing changes of blood biochemical biomarkers and levels of anti- and pro-inflammatory cytokines as well as by renal computed tomography with subsequent histological and morphometric examination. The therapeutic effect of therapy with MSC-EVs was shown by ELISA method that confirmed a statistically significant increase of the anti-inflammatory and decrease of pro-inflammatory cytokines as compared to conventional treatment. In addition, there is a positive trend in increase of ALP level, animal weigh, and normalization of ADA activity that can indicate an improvement of kidney state. A significant reduction of the area of specific and interstitial inflammation indicated positive affect of MSC-EVs that suggests a shorter duration of ATT. The number of MSC-EVs proteins (as identified by mass-spectometry analysis) with anti-microbial, anti-inflammatory and immunoregulatory functions reduced the level of the inflammatory response and the severity of kidney damage (further proved by morphometric analysis). In conclusion, MSC-EVs can be a promising tool for the complex treatment of various infectious diseases, in particularly rTB.

Identification and Study of the Action Mechanism of Small Compound That Inhibits Replication of Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is known to cause annual epidemics of respiratory infections; however, the lack of specific treatment options for this disease poses a challenge. In light of this, there has been a concerted effort to identify small molecules that can effectively combat RSV. This article focuses on the mechanism of action of compound K142, which was identified as a primary screening leader in the earlier stages of the project. The research conducted demonstrates that K142 significantly reduces the intensity of virus penetration into the cells, as well as the formation of syncytia from infected cells. These findings show that the compound's interaction with the surface proteins of RSV is a key factor in its antiviral activity. Furthermore, pharmacological modeling supports that K142 effectively interacts with the F-protein. However, in vivo studies have shown only weak antiviral activity against RSV infection, with a slight decrease in viral load observed in lung tissues. As a result, there is a need to enhance the bioavailability or antiviral properties of this compound. Based on these findings, we hypothesize that further modifications of the compound under study could potentially increase its antiviral activity.

Passing of Nanocarriers across the Histohematic Barriers: Current Approaches for Tumor Theranostics.

Over the past several decades, nanocarriers have demonstrated diagnostic and therapeutic (i.e., theranostic) potencies in translational oncology, and some agents have been further translated into clinical trials. However, the practical application of nanoparticle-based medicine in living organisms is limited by physiological barriers (blood-tissue barriers), which significantly hampers the transport of nanoparticles from the blood into the tumor tissue. This review focuses on several approaches that facilitate the translocation of nanoparticles across blood-tissue barriers (BTBs) to efficiently accumulate in the tumor. To overcome the challenge of BTBs, several methods have been proposed, including the functionalization of particle surfaces with cell-penetrating peptides (e.g., TAT, SynB1, penetratin, R8, RGD, angiopep-2), which increases the passing of particles across tissue barriers. Another promising strategy could be based either on the application of various chemical agents (e.g., efflux pump inhibitors, disruptors of tight junctions, etc.) or physical methods (e.g., magnetic field, electroporation, photoacoustic cavitation, etc.), which have been shown to further increase the permeability of barriers.

Impact of lipid rafts on transient receptor potential channel activities.

Members of the transient receptor potential (TRP) superfamily are cation channels that are expressed in nearly every mammalian cell type and respond as cellular sensors to various environmental stimuli. Light, pressure, osmolarity, temperature, and other stimuli can induce TRP calcium conductivity and correspondingly trigger many signaling processes in cells. Disruption of TRP channel activity, as a rule, harms cellular function. Despite numerous studies, the mechanisms of TRP channel regulation are not yet sufficiently clear, in part, because TRP channels are regulated by a broad set of ligands having diverse physical and chemical features. It is now known that some TRP members are located in membrane microdomains termed lipid rafts. Moreover, interaction between specific raft-associated lipids with channels may be a key regulation mechanism. This review examines recent findings related to the roles of lipid rafts in regulation of TRP channel activity. The mechanistic events of channel interactions with the main lipid raft constituent, cholesterol, are being clarified. Better understanding of mechanisms behind such interactions would help establish the key elements of TRP channel regulation and hence allow control of cellular responses to environmental stimuli.

Lipid raft integrity is required for human leukemia Jurkat T-cell migratory activity.

Lipid rafts are membrane microdomains featuring high cholesterol, sphingolipid, and protein content. These microdomains recruit various receptors, ion channels, and signaling molecules for coordination of various cellular functions, including synaptic transmission, immune response, cytoskeletal organization, adhesion, and migration. Many of these processes also depend on Ca2+ intake. We have previously shown in Jurkat cells that activity of transient receptor potential vanilloid, type 6 (TRPV6) calcium channel, and TRPV6-mediated Ca2+ influx, depend on lipid raft integrity. In this study, using the transwell cell migration assay and time-lapse video microscopy with Jurkat cells, we found that lipid raft destruction was associated with: inhibited cell adhesion and migration; and decreased mean speed, maximum speed, and trajectory length. Using String Server, we constructed a Protein Interaction Network (PIN). The network indicated that TRPV6 proteins interact with the highest probability (0.9) with Src family kinase members (SFKs) involved in processes related to cell migration. Analysis of detergent-resistant membrane fractions and immunoelectron microscopy data confirmed an association in lipid rafts between TRPV6 and Lck kinase, an SFKs member. Destruction of lipid rafts led to uncoupling of TRPV6 clusters with Lck and their departure from the plasma membrane into the cytosol of the cells. Src family kinases are generally associated with their roles in tumor invasion and progression, epithelial-mesenchymal transitions, angiogenesis, and metastatic development. We suggest that a functional interaction between TRPV6 calcium channels and SFKs members in lipid rafts is one of necessary elements of migration and oncogenic signaling in leukemia cells.

Replicative senescence in MSCWJ-1 human umbilical cord mesenchymal stem cells is marked by characteristic changes in motility, cytoskeletal organization, and RhoA localization.

Here, we document changes in cell motility and organization of the contractile apparatus of human umbilical cord Wharton's jelly mesenchymal stem cells (MSCWJ-1) in the process of replicative senescence. Colocalization dynamics of F-actin and actin-binding proteins (myosin-9, α-actinin-4, RhoA) were examined in the MSCWJ-1 cell line. The results show that nuclear-cytoplasmic redistribution of RhoA occurs during replicative senescence, with maximal RhoA/nucleus colocalization evident at passage 15. At that time point, decreases in colocalization, namely myosin-9/F-actin and α-actinin-4/F-actin, were seen and myosin-9 was found in cytosolic extracts in the assembly-incompetent form. Using an automated intravital confocal cytometry system and quantitative analysis of MSCWJ-1 movements, we found that changes in cytoskeletal organization correlate with cell motility characteristics over a time period from passages 9 to 38. The factors examined (cytoskeleton structure, cell motility) indicate that the process by which cells transition to replicative senescence is best represented as three stages. The first stage lasts from cell culture isolation to passage 15 and is characterized by: accumulation of actin-binding proteins in assembly-incompetent forms; nuclear RhoA accumulation; and an increase in movement tortuosity. The second stage extends from passages 15 to 28 and is characterized by: an increase in the structural integrity of the actin cytoskeleton; exit of RhoA and alpha-actinin-4 from the nucleus; and a decrease in path tortuosity. The third stage extends from passage 28 to 38 and is marked by: a plateau in actin cytoskeleton structural integrity; significant decreases in nuclear RhoA levels; and decreases in cell speed.

Morphological and immunophenotypic characterization of perivascular interstitial cells in human glioma: Telocytes, pericytes, and mixed immunophenotypes.

Telocytes (Tcs) and pericytes (Pcs) are two types of perivascular interstitial cell known to be widespread in various organs and tissues, including the brain. We postulated that Tcs and Pcs may be involved in glioblastoma (GBM) neovascularization. OBJECTIVE: Morphological study of Tc and Pc roles in GBM. MATERIALS AND METHODS: Samples from 15 GBM, 10 diffuse astrocytoma, as well as 5 control samples were studied. We used immunohistochemistry (IHC) with antibodies (Abs) to GFAP, Ki-67, CD117, NeuroD1, NG2, CD34, and SMA. Confocal laser scanning microscopy (CLSM) of 4 glioma tissue cultures and 4 GBM sections was performed with GFAP, CD117, CD34/connexin43, NeuroD1/connexin43, CD34/NG2 and CD13/CD117 Abs. Electron microscopy (EM) of GBM was performed in 4 cases. RESULTS: The presence of Tcs and Pcs was shown in GBM (IHC, EM, CLSM) and glioma cultures (CLSM). The Tc immunophenotype was CD117+/CD34+/connexin43+/NeuroD1+. The Pc immunophenotype was SMA+/NG2+/CD13+. The number of Tcs in GBM specimens was 10 times higher than in astrocytoma. We also identified CD13/CD117 and CD34/NG2 co-expressing cells in GBM blood vessels. CONCLUSION: Four immunophenotypes were found in GBM vessels, corresponding to endotheliocytes, Pcs, Tcs, and a mixed Pc/Tc immunophenotype. These and forthcoming improvements in our understanding of the origin and function of Tcs, including their relationship with Pcs, are necessary steps in oncology. Study of these cell types (Tcs, Pcs) and their roles in brain tumor oncogenesis will likely enable improved targeted therapies and support development of new forms of anti-neoplastic drugs.

Injury affects coelomic fluid proteome of the common starfish, Asterias rubens.

Echinoderms, possessing outstanding regenerative capabilities, provide a unique model system for the study of response to injury. However, little is known about the proteomic composition of coelomic fluid, an important biofluid circulating throughout the animal's body and reflecting the overall biological status of the organism. In this study, we used LC-MALDI tandem mass spectrometry to characterize the proteome of the cell-free coelomic fluid of the starfish Asterias rubens and to follow the changes occurring in response to puncture wound and blood loss. In total, 91 proteins were identified, of which 61 were extracellular soluble and 16 were bound to the plasma membrane. The most represented functional terms were 'pattern recognition receptor activity' and 'peptidase inhibitor activity'. A series of candidate proteins involved in early response to injury was revealed. Ependymin, ß-microseminoprotein, serum amyloid A and avidin-like proteins, which are known to be involved in intestinal regeneration in the sea cucumber, were also identified as injury-responsive proteins. Our results expand the list of proteins potentially involved in defense and regeneration in echinoderms and demonstrate dramatic effects of injury on the coelomic fluid proteome.

Sub-cellular Electrical Heterogeneity Revealed by Loose Patch Recording Reflects Differential Localization of Sarcolemmal Ion Channels in Intact Rat Hearts.

The cardiac action potential (AP) is commonly recoded as an integral signal from isolated myocytes or ensembles of myocytes (with intracellular microelectrodes and extracellular macroelectrodes, respectively). These signals, however, do not provide a direct measure of activity of ion channels and transporters located in two major compartments of a cardiac myocyte: surface sarcolemma and the T-tubule system, which differentially contribute to impulse propagation and excitation-contraction (EC) coupling. In the present study we investigated electrical properties of myocytes within perfused intact rat heart employing loose patch recording with narrow-tip (2 µm diameter) extracellular electrodes. Using this approach, we demonstrated two distinct types of electric signals with distinct waveforms (single peak and multi-peak AP; AP1 and AP2, respectively) during intrinsic pacemaker activity. These two types of waveforms depend on the position of the electrode tip on the myocyte surface. Such heterogeneity of electrical signals was lost when electrodes of larger pipette diameter were used (5 or 10 µm), which indicates that the electric signal was assessed from a region of <5 µm. Importantly, both pharmacological and mathematical simulation based on transverse (T)-tubular distribution suggested that while the AP1 and the initial peak of AP2 are predominantly attributable to the fast, inward Na+ current in myocyte's surface sarcolemma, the late components of AP2 are likely representative of currents associated with L-type Ca2+ channel and Na+/Ca2+ exchanger (NCX) currents which are predominantly located in T-tubules. Thus, loose patch recording with narrow-tip pipette provides a valuable tool for studying cardiac electric activity on the subcellular level in the intact heart.