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Skeletal disorders, caused by trauma, disease, or carcinoma, may result in tissue loss and, finally, in endoprosthesis. Tissue engineering offers an alternative - tissue scaffolds. Its constructs may be seeded with autologous cells or, alternatively, attract cells from the surrounding tissues. Such a scaffold must meet several requirements, such as biocompatibility, biodegradability and suitable morphology for cell attachment and proliferation. Nonetheless, scaffold should stimulate cells migrated from the surrounding tissues to infiltrate the scaffold, proliferate and differentiate to the required cell type. In the current study, we developed a fibrous scaffold with 3D structure using emulsion centrifugal spinning. The scaffold from poly-ɛ-caprolactone contained a cocktail of growth factors, i.e. TGF-β, IGF and bFGF. The released growth factors enhanced cell proliferation and chondrogenic differentiation. The scaffold is a promising material for skeletal tissue engineering.

Suitable scaffolds for tissue engineering should promote several features that enable regeneration of the damaged tissue in vivo. In general, nanoto microfibrous meshes resemble extracellular matrix and support cell adhesion; three dimensional scaffolds, together with interconnected pores, promote cell migration into the volume of the scaffolds. Furthermore, the scaffold should be biodegradable with no harmful byproducts and easy to produce. Centrifugal spinning is an alternative method, to widely used electrospinning method, to produce 3D scaffolds suitable for use in tissue engineering. In this study, we tested different molecular weights and solvent systems of poly-ε-caprolactone (PCL) that were produced by the centrifugal spinning method. The produced scaffolds were characterized and seeded with Saos2 osteosarcoma cell line to verify their biocompatibility. We concluded from the results that group 4 scaffold, produced from a mixture of two molecular weights of PCL dissolved in acetic acid/formic acid, supported cell adhesion, proliferation and metabolic activity the most out of all the tested scaffolds. The other PCL scaffolds were prepared either from one type of molecular weight of PCL or chloroform was solely used to produce the scaffolds.

Osteochondral defects develop as a result of trauma, microtrauma, avascular necrosis or cancer. These are usually pre-arthrotic conditions, accompanied by chronic pain and limited joint mobility leading to decreased quality of life of the affected patients. The bone itself has self-repair potential facilitated by mesenchymal stem cells and other cells present in the bone tissue. On the other hand, mature cartilage has very low regenerative capacity due to limited mitotic potential of chondrocytes and lack of vascularization. Therefore, there is an effort to develop an alternative treatment strategy supporting and accelerating natural healing processes. We have designed nanofibrous scaffolds made of poly‑ε‑caprolactone/hyaluronic acid and enriched with specific growth factors – “osteogenic” part with BMP‑2 and “chondrogenic” part with bFGF and TGF‑β. These two parts are meant to be combined in one biphasic non‑cellular scaffold which would be possible to implant in the site of injury and serve as a mechanical support for the cells. We examined proliferation and viability of cells, depth of their penetration into scaffold, cell distribution, alkaline phosphatase activity and extracellular matrix proteins expression. We showed both “osteogenic” and “chondrogenic” scaffold was suitable for cell growth. Moreover, in comparison to the control samples, these two scaffolds exhibited positive effect on chondrogenic and osteogenic differentiation, respectively.

ABSTRACTIntroduction: Immunotherapy with checkpoint inhibitors is beginning to be recognized as a valid weapon for the treatment of metastatic prostate cancer (PCa) when chemotherapy fails. Ipilimumab (ipi) is a fully humanized monoclonal antibody that blocks the activity of CTLA4. It also has a molecular weight of 148 kDa and is water-soluble at physiological pH. Ipi was first approved by the FDA for the treatment of malignant melanoma and is currently being studied in metastatic castration-resistant prostate cancer, with promising early results.Areas covered: The aim of this review is to collate the most significant preclinical and clinical studies available that look at ipi to propose new strategies for the future.Expert opinion: Additional studies are required to reduce toxicity and increase the activity of ipi in PCa. A possible strategy is to combine ipi with standard anti-cancer therapeutics such as vaccines, PDL1 inhibitors, antiandrogen drugs, and chemotherapy agents. Several initial results have suggested that combination strategies are useful to increase the activity in mCRPC, even if the toxicity of the treatment can increase. The activity of combined treatments is still not predictable, but considering the ongoing studies, we believe that they have good potential that will lead to the discovery of an optimal therapeutic strategy.

The rapid development of new materials and their application in an extremely wide variety of research and technological fields has lead to the request of increasingly sophisticated characterization methods. In particular residual stress measurements by neutron diffraction, small angle scattering of X-rays and neutrons, as well as 3D imaging techniques with spatial resolution at the micron or even sub-micron scale, like micro-and nano-computerized tomography, have gained a great relevance in recent years.Residual stresses are autobalancing stresses existing in a free body not submitted to any external surface force. Several manufacturing processes, as well as thermal and mechanical treatments, leave residual stresses within the components. Bragg diffraction of X-rays and neutrons can be used to determine residual elastic strains (and then residual stresses by knowing the material elastic constants) in a non-destructive way. Small Angle Scattering of neutrons or X-rays, complementary to Transmission Electron Microscopy, allows the determination of structural features such as volume fraction, specific surface and size distribution of inhomogeneities embedded in a matrix, in a huge variety of materials of industrial interest. X-ray microtomography is similar to conventional Computed Tomography employed in Medicine, allowing 3D imaging of the investigated samples, but with a much higher spatial resolution, down to the sub-micron scale. Some examples of applications of the experimental techniques mentioned above are described and discussed.

Homology modeling of the complete structure of the large cytoplasmic loop between the fourth and fifth transmembrane segments (H4–H5 loop) of the α subunit of Na+/K+-ATPase is reported. The deduced amino acid sequence shows high sequence identity and homology to the Ca2+-ATPase (32.8% identity and 53.3% similarity in our alignment), whose tertiary structure has been solved recently at 2.6-Å resolution by X-ray crystallography. This high homology allowed the construction of a model structure using the MODELLER program. Refinement was achieved through interactive visual and algorithmic analysis and minimization with the TRIPOS force field included in the SYBYL/MAXIMIN2 module. The docking of ATP as a substrate into the active site of the model was explored with the AUTODOCK program followed by molecular mechanics optimization of the most interesting complexes. Thus, the docking of ATP into the resulting model of the H4–H5 loop gave evidence for the existence of one ATP binding site only. We were able to specify Cys549, Phe548, Glu505, Lys501, Gln482, Lys480, Ser477, Phe475 and Glu446 as parts of the ATP binding site with Lys501 located in the depth of the positively charged binding pocket.

Lifetime analysis of tryptophan fluorescence of the mitochondrial processing peptidase (MPP) from Saccharomyces cerevisiae clearly proved that substrate binding evoked a conformational change of the α-subunit while presence of substrate influenced neither the lifetime components nor the average lifetime of the tryptophan excited state of the β-MPP subunit. Interestingly, lifetime analysis of tryptophan fluorescence decay of the α-MPP subunit revealed about 11% of steady-state fractional intensity due to the long-lived lifetime component, indicating that at least one tryptophan residue is partly buried at the hydrophobic microenvironment. Computer modeling, however, predicted none of three tryptophans, which the α-subunit contains, as deeply buried in the protein matrix. We conclude this as a consequence of a possible dimeric (oligomeric) structure.

In glioma C6 cells, extracellular ATP generates inositol 1,4,5-trisphosphate (InsP3), indicating the presence of purinergic receptors coupled to phosphoinositide turnover. To identify the effect of ATP (acting via InsP3) and thapsigargin (acting without InsP3production as a specific inhibitor of the endoplasmic reticulum Ca2+-ATPase) on intracellular Ca2+pools we used video imaging of Fura-2 loaded into single, intact glioma C6 cells. It has been shown that ATP and thapsigargin initiate Ca2+response consistent with the capacitative model of Ca2+influx. When the cells were stimulated by increasing concentrations of ATP (1, 10, 50 and 100 μM) the graded, quantal Ca2+response was observed. In the absence of extracellular Ca2+thapsigargin and ionomycin-releasable Ca2+pools are overlapping, demonstrating that Ca2+stores are located mainly in the endoplasmic reticulum. After maximal Ca2+mobilization by ATP, thapsigargin causes further increase in cytosolic Ca2+concentration, whereas emptying of thapsigargin-sensitive intracellular stores prevents any further Ca2+release by ATP. Thus, the thapsigargin-sensitive intracellular pool of Ca2+in glioma C6 cells seems to be larger than that sensitive to InsP3. Two hypothesis to explain this result are proposed. One postulates a presence of two different Ca2+pools, sensitive and insensitive to InsP3and both discharged by thapsigargin, and the other, the same intrecellular pool of Ca2+completely emptying by thapsigargin and only partially by InsP3. These results may contribute to understanding the mechanism of Ca2+signalling mediated by ATP, the most potent intracellular Ca2+mobilizing agonist in all types of glial cells. © 1997 Elsevier Science Ltd

Summary The effect of phospholipase A2 and of related agents on ouabain binding and Na,K-ATPase activity were studied in intact and detergent-treated membrane preparations of rat brain cortex and pig kidney medulla. It was found that phospholipase A2 (PLA2) may distinguish or dissociate ouabain binding complexes I (ATP+Mg+Na) and II (Pi+Mg), stimulating the former and inhibiting the latter. Procedures which break the permeability barriers of vesicular membrane preparations, such as repeated freezing-thawing, sonication or hypoosmotic shock failed to mimic the effect of PLA2, indicating that it was not acting primarily by opening the inside-out oriented vesicles. The detergent digitonin exhibited similar effects on ouabain binding in both ATP+Mg+Na and Pi+Mg media. Other detergents were ineffective.

7‐Chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD‐Cl) labeled Na+/K+‐ATPase covalently with two different inactivation constants (Ki=2.5 μM; Ki′=10 μM). It apparently modified the two different ATP‐binding sites of the enzyme since it decreased the activity of the E2ATP site, i.e. the K+‐activated para‐nitrophenylphosphatase activity, in an enzyme whose high‐affinity E1ATP site had been blocked by fluorescein 5′isothiocyanate (FITC). It also reduced the activity of the E1ATP site, i.e. the Na+‐activated protein phosphorylation, in an enzyme whose low‐affinity E2ATP site had been blocked by Co(NH3)4PO4. Fluorescence quenching experiments with KI, CsCl and MnCl2of the NBD‐Cl‐labeled Na+/K+‐ATPase revealed two differently accessible types of fluorophores depending on the ATP site: The E2ATP site apparently differs from the E1ATP site in that it is more open because the fluorophore labeling in the E2ATP site was sterically better accessible for quenchers.

Fluorescein-5′-isothiocyanate (FITC) was used to study the high-affinity ATP-binding site of Na+/K+-ATPase. The molar ratio of specifically bound FITC per α-subunit of Na+/K+-ATPase was found to be 0.5 as followed from pretreatment experiments with another specific E1ATP-inhibitor Cr(H2O)4AdoPP[CH2]P.This indicated an existence of one high affinity ATP-binding site (E1ATP-binding site) in the native (αβ)2-diprotomer of Na+/K+-ATPase. Fluorescence dual-excitation ratio of specifically bound FITC revealed that at external pH 7.5, the pH value inside the E1ATP-binding site is 6.95 ± 0.18. In addition, FITC fluorescence quenching by anti-fluorescein and by iodide choline indicated the limited access of water into the small pocket of the E1ATP-binding site.

ATP hydrolysis by Na+/K+-ATPase proceeds via the interaction of simultaneously existing and cooperating high (E1ATP) and low (E2ATP) substrate binding sites. It is unclear whether both ATP sites reside on the same or on different catalytic α-subunits. To answer this question, we looked for a fluorescent label for the E2ATP site that would be suitable for distance measurements by Förster energy transfer after affinity labeling of the E1ATP site by fluorescein 5′-isothiocyanate (FITC). Erythrosin 5′-isothiocyanate (ErITC) inactivated, in an E1ATP site-blocked enzyme (by FITC), the residual activity of the E2ATP site, namely K+-activated p-nitrophenylphosphatase in a concentration-dependent way that was ATP-protectable. The molar ratios of FITC/α-subunit of 0.6 and of ErITC/α-subunit of 0.48 indicate 2 ATP sites per (αβ)2diprotomer. Measurements of Förster energy transfer between the FITC-labeled E1ATP and the ErITC-labeled or Co(NH3)4ATP-inactivated E2ATP sites gave a distance of 6.45 ± 0.64 nm. This distance excludes 2 ATP sites per α-subunit since the diameter of α is 4–5 nm. Förster energy transfer between cardiac glycoside binding sites labeled with anthroylouabain and fluoresceinylethylenediamino ouabain gave a distance of 4.9 ± 0.5 nm. Hence all data are consistent with the hypothesis that Na+/K+-ATPase in cellular membranes is an (αβ)2diprotomer and works as a functional dimer (Thoenges, D., and Schoner, W. (1997) J. Biol. Chem.272, 16315–16321).

7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) labeled Na +/K +-ATPase covalently with two different inactivation constants ( Ki=2.5 μM; Ki′=10 μM). It apparently modified the two different ATP-binding sites of the enzyme since it decreased the activity of the E 2ATP site, i.e. the K +-activated para-nitrophenylphosphatase activity, in an enzyme whose high-affinity E 1ATP site had been blocked by fluorescein 5′isothiocyanate (FITC). It also reduced the activity of the E 1ATP site, i.e. the Na +-activated protein phosphorylation, in an enzyme whose low-affinity E 2ATP site had been blocked by Co(NH 3) 4PO 4. Fluorescence quenching experiments with KI, CsCl and MnCl 2of the NBD-Cl-labeled Na +/K +-ATPase revealed two differently accessible types of fluorophores depending on the ATP site: The E 2ATP site apparently differs from the E 1ATP site in that it is more open because the fluorophore labeling in the E 2ATP site was sterically better accessible for quenchers.

A Koshland-Némethy-Filmer model of two cooperating ATP sites has previously been shown to explain the kinetics of inhibition of Na+/K+-ATPase (EC3.6.1.37) by dansylated ATP (Thoenges, D., and Schoner, W. (1997)J. Biol. Chem.272, 16315–16321). The present work demonstrates that this model adequately describes all types of interactions and kinetics of a number of ATP analogs that differ in their cooperativity of the high and low affinity ATP binding sites of the enzyme. 2′,3′-O(2,4,6-trinitrophenyl)ATP binds in a negative cooperative way to the E1ATP site (Kd= 0.7 μm) and to the E2ATP site (Kd= 210 μm), but 3′(2′)-O-methylanthraniloyl-ATP in a positive cooperative way with a lower affinity to the E1ATP binding site (Kd= 200 μm) than to the E2ATP binding site (Kd= 80 μm). 3′(2′)-O(5-Fluor-2,4-dinitrophenyl)-ATP, however, binds in a noncooperative way, with equal affinities to both ATP binding sites (Kd= 10 μm). In a research for the structural parameters determining ATP site specificity and cooperativity, we became aware that structural flexibility of ribose is necessary for catalysis. Moreover, puckering of the ring atoms in the ribose is essential for the interaction between ATP sites in Na+/K+-ATPase. A number of derivatives of 2′(3′)-O-adenosine with bulky fluorescent substitutes bind with high affinity to the E2ATP site and inhibit Na+/K+-ATPase activity. Evidently, an increased number of interactions of such a bulky adenosine with the enzyme protein tightens binding to the E2ATP site.

The high‐affinity E1ATP site of Na+/K+‐ATPase labeled with fluorescein 5′‐isothiocyanate and its E2ATP site labeled with erythrosin 5′‐isothiocyanate (ErITC), as was shown recently [Linnertz et al. (1998) J. Biol. Chem. 273, 28813–28821], reside on separate and adjacent catalytic α subunits. This paper provides evidence that specific labeling of the E2ATP binding site with ErITC resulted in a modification of the Cys549residue in the tryptic fragment with the sequence Val545‐Leu‐Gly‐Phe‐Cys549‐His550. Hence, Cys549is part of or close to the low‐affinity E2ATP binding site of Na+/K+‐ATPase.