The activin receptor is stimulated in the skeleton, vasculature, heart, and kidney during chronic kidney disease.

We examined activin receptor type IIA (ActRIIA) activation in chronic kidney disease (CKD) by signal analysis and inhibition in mice with Alport syndrome using the ActRIIA ligand trap RAP-011 initiated in 75-day-old Alport mice. At 200 days of age, there was severe CKD and associated Mineral and Bone Disorder (CKD-MBD), consisting of osteodystrophy, vascular calcification, cardiac hypertrophy, hyperphosphatemia, hyperparathyroidism, elevated FGF23, and reduced klotho. The CKD-induced bone resorption and osteoblast dysfunction was reversed, and bone formation was increased by RAP-011. ActRIIA inhibition prevented the formation of calcium apatite deposits in the aortic adventitia and tunica media and significantly decreased the mean aortic calcium concentration from 0.59 in untreated to 0.36 mg/g in treated Alport mice. Aortic ActRIIA stimulation in untreated mice increased p-Smad2 levels and the transcription of sm22α and αSMA. ActRIIA inhibition reversed aortic expression of the osteoblast transition markers Runx2 and osterix. Heart weight was significantly increased by 26% in untreated mice but remained normal during RAP-011 treatment. In 150-day-old mice, GFR was significantly reduced by 55%, but only by 30% in the RAP-011-treated group. In 200-day-old mice, the mean BUN was 100 mg/dl in untreated mice compared to 60 mg/dl in the treated group. In the kidneys of 200-day-old mice, ActRIIA and p-Smad2 were induced and MCP-1, fibronectin, and interstitial fibrosis were stimulated; all were attenuated by RAP-011 treatment. Hence, the activation of ActRIIA signaling during early CKD contributes to the CKD-MBD components of osteodystrophy and cardiovascular disease and to renal fibrosis. Thus, the inhibition of ActRIIA signaling is efficacious in improving and delaying CKD-MBD in this model of Alport syndrome.

Ligand trap of the activin receptor type IIA inhibits osteoclast stimulation of bone remodeling in diabetic mice with chronic kidney disease.

Dysregulation of skeletal remodeling is a component of renal osteodystrophy. Previously, we showed that activin receptor signaling is differentially affected in various tissues in chronic kidney disease (CKD). We tested whether a ligand trap for the activin receptor type 2A (RAP-011) is an effective treatment of the osteodystrophy of the CKD-mineral bone disorder. With a 70% reduction in the glomerular filtration rate, CKD was induced at 14 weeks of age in the ldlr-/- high fat-fed mouse model of atherosclerotic vascular calcification and diabetes. Twenty mice with CKD, hyperphosphatemia, hyperparathyroidism, and elevated activin A were treated with RAP-011, wherease 19 mice were given vehicle twice weekly from week 22 until the mice were killed at 28 weeks of age. The animals were then evaluated by skeletal histomorphometry, micro-computed tomography, mechanical strength testing, and ex vivo bone cell culture. Results in the CKD groups were compared with those of the 16 sham-operated ldlr-/- high fat-fed mice. Sham-operated mice had low-turnover osteodystrophy and skeletal frailty. CKD stimulated bone remodeling with significant increases in osteoclast and osteoblast numbers and bone resorption. Compared with mice with CKD and sham-operated mice, RAP-011 treatment eliminated the CKD-induced increase in these histomorphometric parameters and increased trabecular bone fraction. RAP-011 significantly increased cortical bone area and thickness. Activin A-enhanced osteoclastogenesis was mediated through p-Smad2 association with c-fos and activation of nuclear factor of activated T cells c1 (NFATc1). Thus, an ActRIIA ligand trap reversed CKD-stimulated bone remodeling, likely through inhibition of activin-A induced osteoclastogenesis.

Ligand trap for the activin type IIA receptor protects against vascular disease and renal fibrosis in mice with chronic kidney disease.

The causes of cardiovascular mortality associated with chronic kidney disease (CKD) are partly attributed to the CKD-mineral bone disorder (CKD-MBD). The causes of the early CKD-MBD are not well known. Our discovery of Wnt (portmanteau of wingless and int) inhibitors, especially Dickkopf 1, produced during renal repair as participating in the pathogenesis of the vascular and skeletal components of the CKD-MBD implied that additional pathogenic factors are critical. In the search for such factors, we studied the effects of activin receptor type IIA (ActRIIA) signaling by using a ligand trap for the receptor, RAP-011 (a soluble extracellular domain of ActRIIA fused to a murine IgG-Fc fragment). In a mouse model of CKD that stimulated atherosclerotic calcification, RAP-011 significantly increased aortic ActRIIA signaling assessed by the levels of phosphorylated Smad2/3. Furthermore, RAP-011 treatment significantly reversed CKD-induced vascular smooth muscle dedifferentiation as assessed by smooth muscle 22α levels, osteoblastic transition, and neointimal plaque calcification. In the diseased kidneys, RAP-011 significantly stimulated αklotho levels and it inhibited ActRIIA signaling and decreased renal fibrosis and proteinuria. RAP-011 treatment significantly decreased both renal and circulating Dickkopf 1 levels, showing that Wnt activation was downstream of ActRIIA. Thus, ActRIIA signaling in CKD contributes to the CKD-MBD and renal fibrosis. ActRIIA signaling may be a potential therapeutic target in CKD.

CKD-induced wingless/integration1 inhibitors and phosphorus cause the CKD-mineral and bone disorder.

In chronic kidney disease, vascular calcification, renal osteodystrophy, and phosphate contribute substantially to cardiovascular risk and are components of CKD-mineral and bone disorder (CKD-MBD). The cause of this syndrome is unknown. Additionally, no therapy addresses cardiovascular risk in CKD. In its inception, CKD-MBD is characterized by osteodystrophy, vascular calcification, and stimulation of osteocyte secretion. We tested the hypothesis that increased production of circulating factors by diseased kidneys causes the CKD-MBD in diabetic mice subjected to renal injury to induce stage 2 CKD (CKD-2 mice). Compared with non-CKD diabetic controls, CKD-2 mice showed increased renal production of Wnt inhibitor family members and higher levels of circulating Dickkopf-1 (Dkk1), sclerostin, and secreted klotho. Neutralization of Dkk1 in CKD-2 mice by administration of a monoclonal antibody after renal injury stimulated bone formation rates, corrected the osteodystrophy, and prevented CKD-stimulated vascular calcification. Mechanistically, neutralization of Dkk1 suppressed aortic expression of the osteoblastic transcription factor Runx2, increased expression of vascular smooth muscle protein 22-α, and restored aortic expression of klotho. Neutralization of Dkk1 did not affect the elevated plasma levels of osteocytic fibroblast growth factor 23 but decreased the elevated levels of sclerostin. Phosphate binder therapy restored plasma fibroblast growth factor 23 levels but had no effect on vascular calcification or osteodystrophy. The combination of the Dkk1 antibody and phosphate binder therapy completely treated the CKD-MBD. These results show that circulating Wnt inhibitors are involved in the pathogenesis of CKD-MBD and that the combination of Dkk1 neutralization and phosphate binding may have therapeutic potential for this disorder.

BAP1 deficiency causes loss of melanocytic cell identity in uveal melanoma.

BACKGROUND: Uveal melanoma is a highly aggressive cancer with a strong propensity for metastasis, yet little is known about the biological mechanisms underlying this metastatic potential. We recently showed that most metastasizing uveal melanomas, which exhibit a class 2 gene expression profile, contain inactivating mutations in the tumor suppressor BAP1. The aim of this study was to investigate the role of BAP1 in uveal melanoma progression. METHODS: Uveal melanoma cells were studied following RNAi-mediated depletion of BAP1 using proliferation, BrdU incorporation, flow cytometry, migration, invasion, differentiation and clonogenic assays, as well as in vivo tumorigenicity experiments in NOD-SCID-Gamma mice. RESULTS: Depletion of BAP1 in uveal melanoma cells resulted in a loss of differentiation and gain of stem-like properties, including expression of stem cell markers, increased capacity for self-replication, and enhanced ability to grow in stem cell conditions. BAP1 depletion did not result in increased proliferation, migration, invasion or tumorigenicity. CONCLUSIONS: BAP1 appears to function in the uveal melanocyte lineage primarily as a regulator of differentiation, with cells deficient for BAP1 exhibiting stem-like qualities. It will be important to elucidate how this effect of BAP1 loss promotes metastasis and how to reverse this effect therapeutically.

Scaffolds Based on Silk Fibroin with Decellularized Rat Liver Microparticles: Investigation of the Structure, Biological Properties and Regenerative Potential for Skin Wound Healing.

The development of advanced biomaterials and constructs for accelerated recovery of damaged tissues is a key direction in regenerative medicine. Biocompatible scaffolds based on natural biopolymers are widely used for these tasks. Organ decellularization enables obtaining a cell-free extracellular matrix (ECM) with preserved composition and biological activity. The objectives of the present work were combining these two approaches for the development of a composite scaffold based on silk fibroin and ECM microparticles and assessing its structure, biological properties, and regenerative potential. ECM microparticles were obtained by grinding the decellularized matrix of Wistar rat liver in liquid nitrogen. Scaffolds in the form of films were prepared by the casting method. The sinuous and rough topography of the scaffold surface was assessed by the scanning probe nanotomography (SPNT) technique. The inclusion of ECM microparticles in the composition did not affect the elasticity and tensile strength of the scaffolds. The obtained scaffold was non-toxic to cells, maintained high levels of adhesion and proliferation of mouse 3T3 fibroblast and Hep-G2 cells, and showed high regenerative potential, which was studied in the experimental model of full-thickness rat skin wound healing. The wound healing was accelerated by 1.74 times in comparison with the control.

A Comparative Analysis of the Structure and Biological Properties of Films and Microfibrous Scaffolds Based on Silk Fibroin.

A comparative analysis of the structure and biological properties of silk fibroin constructions was performed. Three groups of constructions were obtained: films obtained by casting an aqueous solution of silk fibroin and electrospun microfibrous scaffolds based on silk fibroin, with the addition of 30% gelatin per total protein weight. The internal structures of the films and single fibers of the microfibrous scaffolds consisted of densely packed globule structures; the surface area to volume ratios and volume porosities of the microfibrous scaffolds were calculated. All constructions were non-toxic for cells and provide high levels of adhesion and proliferation. The high regenerative potential of the constructions was demonstrated in a rat full-thickness skin wound healing model. The constructions accelerated healing by an average of 15 days and can be considered to be promising constructions for various tasks of tissue engineering and regenerative medicine.

Silk Fibroin/Spidroin Electrospun Scaffolds for Full-Thickness Skin Wound Healing in Rats.

The main goal of our research was to fabricate electrospun scaffolds from three different silk proteins-silk fibroin from Bombyx mori silkworm cocoons and two recombinant spidroins, rS2/12 and rS2/12-RGDS-and to perform a comparative analysis of the structure, biological properties, and regenerative potential of the scaffolds in a full-thickness rat skin wound model. The surface and internal structures were investigated using scanning electron microscopy and scanning probe nanotomography. The structures of the scaffolds were similar. The average fiber diameter of the scaffolds was 315 ± 26 nm, the volume porosity was 94.5 ± 1.4%, the surface-to-volume ratio of the scaffolds was 25.4 ± 4.2 µm-1 and the fiber surface roughness was 3.8 ± 0.6 nm. The scaffolds were characterized by a non-cytotoxicity effect and a high level of cytocompatibility with cells. The scaffolds also had high regenerative potential-the healing of the skin wound was accelerated by 19 days compared with the control. A histological analysis did not reveal any fragments of the experimental constructions or areas of inflammation. Thus, novel data on the structure and biological properties of the silk fibroin/spidroin electrospun scaffolds were obtained.

Relation between micro- and nanostructure features and biological properties of the decellularized rat liver.

Organ decellularization is one of the promising technologies of regenerative medicine, which allows obtaining cell-free extracellular matrix (ECM), which provide preservation of the composition, architecture, vascular network and biological activity of the ECM. The method of decellularization opens up wide prospects for its practical application not only in the field of creating full-scale bioengineered structures, but also in the manufacture of vessels, microcarriers, hydrogels, and coatings. The main goal of our work was the investigation of structure and biological properties of lyophilized decellularized Wistar rat liver fragments (LDLFs), as well as we assessed the regenerative potential of the obtained ECM. We obtained decellularized liver of a Wistar rat, the vascular network and the main components of the ECM of tissue were preserved. H&E staining of histological sections confirmed the removal of cells. DNA content of ECM is equal to 0.7% of native tissue DNA content. Utilizing scanning probe nanotomogrphy method, we showed sinuous, rough topography and highly nanoporous structure of ECM, which provide high level of mouse 3T3 fibroblast and Hep-G2cells biocompatibility. Obtained LDLF had a high regenerative potential, which we studied in an experimental model of a full-thickness rat skin wound healing: we observed the acceleration of wound healing by 2.2 times in comparison with the control.

Controlling Charge Transfer from Quantum Dots to Polyelectrolyte Layers Extends Prospective Applications of Magneto-Optical Microcapsules.

The layer-by-layer (LbL) deposition approach allows combined incorporation of fluorescent, magnetic, and plasmonic nanoparticles into the shell of polyelectrolyte microcapsules to obtain stimulus-responsive systems whose imaging and drug release functions can be triggered by external stimuli. The combined use of fluorescent quantum dots (QDs) and magnetic nanoparticles (MNPs) yields magnetic-field-driven imaging tools that can be tracked and imaged even deep in tissue when the appropriate type of QDs and wavelength of their excitation are used. QDs are excellent photonic labels for microcapsule encoding due to their close-to-unity photoluminescence (PL) quantum yields, narrow PL emission bands, and tremendous one- and two-photon extinction coefficients. However, the presence of MNPs and electrically charged polyelectrolyte molecules used for the LbL fabrication of magneto-optical microcapsules provokes alterations of the QD optical properties because of the photoinduced charge and energy transfer resulting in QD photodarkening or photobrightening. These lead to variation of the microcapsule PL signal under illumination, which hampers their tracking and quantitative analysis in cells and tissues. Here, we have studied the effects of the structure and spatial arrangement of the nanoparticles within the microcapsule polyelectrolyte shell, the total shell thickness, and the shell surface charge on their PL properties under continuous illumination. The roles of the charge transfer and its main driving forces in the stability of the microcapsules PL signal have been established, and the design of the microcapsules dually encoded with QDs and MNPs providing the strongest and most stable PL has been determined. Controlling the energy transfer from the QDs and MNPs and the charge transfer from QDs to polyelectrolyte layers in the engineering of magneto-optical microcapsules with a bright and stable PL signal extends their applications to long-lasting quantitative fluorescence imaging.

Bioimaging Tools Based on Polyelectrolyte Microcapsules Encoded with Fluorescent Semiconductor Nanoparticles: Design and Characterization of the Fluorescent Properties.

Fluorescent imaging is a widely used technique for detecting and monitoring the distribution, interaction, and transformation processes at molecular, cellular, and tissue level in modern diagnostic and other biomedical applications. Unique photophysical properties of fluorescent semiconductor nanocrystals "quantum dots" (QDs) make them advanced fluorophores for fluorescent labeling of biomolecules or optical encoding of microparticles to be used as bioimaging and theranostic agents in targeted delivery, visualization, diagnostics, and imaging. This paper reports on the results of development of an improved approach to the optical encoding of polyelectrolyte microcapsules with stable, covered with the multifunctional polyethyleneglycol derivatives water-soluble QDs, as well as characterization of the optical properties, morphological and structural properties of the encoded microcapsules. The embedding of QDs into the polymer microcapsule membrane through layer-by-layer deposition on a preliminarily formed polymeric polyelectrolyte shell makes it possible to obtain bright fluorescent particles with an adapted charge and size distribution that are distinctly discernible by flow cytometry as individual homogeneous populations. The fluorescent microcapsules developed can be used in further designing bioimaging and theranostic agents sensitive to various external stimuli along with photoexcitation.

Bioinformatic and statistical analysis of the optic nerve head in a primate model of ocular hypertension.

BACKGROUND: The nonhuman primate model of glaucomatous optic neuropathy most faithfully reproduces the human disease. We used high-density oligonucleotide arrays to investigate whole genome transcriptional changes occurring at the optic nerve head during primate experimental glaucoma. RESULTS: Laser scarification of the trabecular meshwork of cynomolgus macaques produced elevated intraocular pressure that was monitored over time and led to varying degrees of damage in different samples. The macaques were examined clinically before enucleation and the myelinated optic nerves were processed post-mortem to determine the degree of neuronal loss. Global gene expression was examined in dissected optic nerve heads with Affymetrix GeneChip microarrays. We validated a subset of differentially expressed genes using qRT-PCR, immunohistochemistry, and immuno-enriched astrocytes from healthy and glaucomatous human donors. These genes have previously defined roles in axonal outgrowth, immune response, cell motility, neuroprotection, and extracellular matrix remodeling. CONCLUSION: Our findings show that glaucoma is associated with increased expression of genes that mediate axonal outgrowth, immune response, cell motility, neuroprotection, and ECM remodeling. These studies also reveal that, as glaucoma progresses, retinal ganglion cell axons may make a regenerative attempt to restore lost nerve cell contact.

Emerging insights into the molecular pathogenesis of uveal melanoma.

Uveal melanoma is the most common primary cancer of the eye, and often results not only in vision loss, but also in metastatic death in up to half of patients. For many years, the details of the molecular pathogenesis of uveal melanoma remained elusive. In the past decade, however, many of these details have emerged to reveal a fascinating and complex story of how the primary tumor evolves and progresses. Early events that disrupt cell cycle and apoptotic control lead to malignant transformation and proliferation of uveal melanocytes. Later, the growing tumor encounters a critical bifurcation point, where it progresses along one of two genetic pathways with very distinct genetic signatures (monosomy 3 vs 6p gain) and metastatic propensity. Late genetic events are characterized by increasing aneuploidy, most of which is nonspecific. However, specific chromosomal alterations, such as loss of chromosome 8p, can hasten the onset of metastasis in susceptible tumors. Taken together, this pathogenetic scheme can be used to construct a molecularly based and prognostically relevant classification of uveal melanomas that can be used clinically for personalized patient management.

High resolution 3D microscopy study of cardiomyocytes on polymer scaffold nanofibers reveals formation of unusual sheathed structure.

Building functional and robust scaffolds for engineered biological tissue requires a nanoscale mechanistic understanding of how cells use the scaffold for their growth and development. A vast majority of the scaffolds used for cardiac tissue engineering are based on polymer materials, the matrices of nanofibers. Attempts to load the polymer fibers of the scaffold with additional sophisticated features, such as electrical conductivity and controlled release of the growth factors or other biologically active molecules, as well as trying to match the mechanical features of the scaffold to those of the extracellular matrix, cannot be efficient without a detailed knowledge of how the cells are attached and strategically positioned with respect to the scaffold nanofibers at micro and nanolevel. Studying single cell - single fiber interactions with the aid of confocal laser scanning microscopy (CLSM), scanning probe nanotomography (SPNT), and transmission electron microscopy (TEM), we found that cardiac cells actively interact with substrate nanofibers, but in different ways. While cardiomyocytes often create a remarkable "sheath" structure, enveloping fiber and, thus, substantially increasing contact zone, fibroblasts interact with nanofibers in the locations of focal adhesion clusters mainly without wrapping the fiber. STATEMENTS OF SIGNIFICANCE: We found that cardiomyocytes grown on electrospun polymer nanofibers often create a striking "sheath" structure, enveloping fiber with the formation of a very narrow (∼22 nm) membrane gap leading from the fiber to the extracellular space. This wrapping makes the entire fiber surface available for cell attachment. This finding gives a new prospective view on how scaffold nanofibers may interact with growing cells. It may play a significant role in effective design of novel nanofiber scaffolds for tissue engineering concerning mechanical and electrical properties of scaffolds as well as controlled drug release from "smart" biomaterials.

Population differences in elastin maturation in optic nerve head tissue and astrocytes.

PURPOSE: Glaucomatous optic neuropathy is characterized by remodeling of the extracellular matrix with disorganization of elastic fibers in the optic nerve head (ONH). There are significant differences in prevalence of glaucomatous optic neuropathy between African Americans (AAs) and Caucasian Americans (CAs). The goal of this study was to evaluate differences in elastin synthesis and maturation in ONH tissue and cells of AA and CA donors with no eye disease, to provide a basis for underlying racial differences in susceptibility to elevated intraocular pressure. METHODS: The amount of mature elastin in ONHs from each group of donors was evaluated by desmosine radioimmunoassay. The distribution of elastic fibers in ONH tissue was investigated by immunofluorescent staining. Elastin and lysyl oxidase mRNA levels and alternative splicing of elastin in ONH astrocytes were investigated by quantitative PCR. Tropoelastin protein expression was assessed by immunoblot analysis. RESULTS: ONHs from AA donors had significantly reduced levels of desmosine compared with those of CAs. In contrast, elastin mRNA and tropoelastin synthesis were elevated in ONH astrocytes from AA individuals. The inclusion of exon 23 in elastin mRNA and lysyl oxidase-like 2 mRNA levels was significantly reduced in astrocytes from AA compared with CA donors. CONCLUSIONS: A reduced number of cross-linking domains in elastin and decreased lysyl oxidase-like 2 expression leads to decreased amount of mature elastin in ONHs from healthy AA individuals compared with CA donors. These results suggest ELN and LOXL2 as candidate susceptibility genes for population-specific genetic risk of primary open-angle glaucoma (POAG).

Expression of ephrinB1 and its receptor in glaucomatous optic neuropathy.

OBJECTIVE: To determine ephrinB1, ephrinB2 and EphB1 expression in the optic nerve head (ONH) and retina of monkeys with glaucoma and in human ONH astrocytes. METHODS: Using immunohistochemistry, the localisation of ephrinB1, ephrinB2 and EphB1 was determined in the ONH and retina bilaterally in monkeys with monocular laser-induced glaucoma. RT-PCR, western blot and immunocytochemistry were used to study ephrinB1, ephrinB2 and EphB1 expression in cultured human ONH astrocytes from donors with and without glaucoma. RESULTS: There was an increase in ephrinB1 and EphB1 expression in mild to moderate glaucoma. In the ONH, both ephrinB1 and EphB1 were localised to astrocytes and EphB1 was also localised to lamina cribrosa cells and perivascular cells. In the retina, ephrinB1 localised to Muller cells and astrocytes, and EphB1 was found in retinal ganglion cells. In ONH astrocytes in humans with glaucoma, ephrinB1 and EphB1 were up-regulated but barely present in donors without glaucoma. CONCLUSIONS: Ephrins are activated in early and moderate glaucoma in the ONH and retina. We postulate that the up-regulation of Eph/ephrin pathway may play a protective role by limiting axonal damage and inflammatory cell invasion. Loss of ephrin signalling in advanced glaucoma may explain macrophage activation.

The chronic kidney disease - Mineral bone disorder (CKD-MBD): Advances in pathophysiology.

The causes of excess cardiovascular mortality associated with chronic kidney disease (CKD) have been attributed in part to the CKD-mineral bone disorder syndrome (CKD-MBD), wherein, novel cardiovascular risk factors have been identified. New advances in the causes of the CKD-MBD are discussed in this review. They demonstrate that repair and disease processes in the kidneys release factors to the circulation that cause the systemic complications of CKD. The discovery of WNT inhibitors, especially Dickkopf 1 (Dkk1), produced during renal repair as participating in the pathogenesis of the vascular and skeletal components of the CKD-MBD implied that additional pathogenic factors are critical. This lead to the discovery that activin A is a second renal repair factor circulating in increased levels during CKD. Activin A derives from peritubular myofibroblasts of diseased kidneys, wherein it stimulates fibrosis, and decreases tubular klotho expression. Activin A binds to the type 2 activin A receptor, ActRIIA, which is variably affected by CKD in the vasculature. In diabetic/atherosclerotic aortas, specifically in vascular smooth muscle cells (VSMC), ActRIIA signaling is inhibited and contributes to CKD induced VSMC dedifferentiation, osteogenic transition and neointimal atherosclerotic calcification. In nondiabetic/nonatherosclerotic aortas, CKD increases VSMC ActRIIA signaling, and vascular fibroblast signaling causing the latter to undergo osteogenic transition and stimulate vascular calcification. In both vascular situations, a ligand trap for ActRIIA prevented vascular calcification. In the skeleton, activin A is responsible for CKD stimulation of osteoclastogenesis and bone remodeling increasing bone turnover. These studies demonstrate that circulating renal repair and injury factors are causal of the CKD-MBD and CKD associated cardiovascular disease.

A novel design of a scanning probe microscope integrated with an ultramicrotome for serial block-face nanotomography.

We present a new concept of a combined scanning probe microscope (SPM)/ultramicrotome apparatus. It enables "slice-and-view" scanning probe nanotomography measurements and 3D reconstruction of the bulk sample nanostructure from series of SPM images after consecutive ultrathin sections. The sample is fixed on a flat XYZ scanning piezostage mounted on the ultramicrotome arm. The SPM measuring head with a cantilever tip and a laser-photodiode tip detection system approaches the sample for SPM measurements of the block-face surface immediately after the ultramicrotome sectioning is performed. The SPM head is moved along guides that are also fixed on the ultramicrotome arm. Thereby, relative dysfunctional displacements of the tip, the sample, and the ultramicrotome knife are minimized. The design of the SPM head enables open frontal optical access to the sample block-face adapted for high-resolution optical lenses for correlative SPM/optical microscopy applications. The new system can be used in a wide range of applications for the study of 3D nanostructures of biological objects, biomaterials, polymer nanocomposites, and nanohybrid materials in various SPM and optical microscopy measuring modes.

SIRT6 deficiency culminates in low-turnover osteopenia.

Deficiency of Sirtuin 6 (SIRT6), a chromatin-related deacetylase, in mice reveals severe premature aging phenotypes including osteopenia. However, the underlying molecular mechanisms of SIRT6 in bone metabolism are unknown. Here we show that SIRT6 deficiency in mice produces low-turnover osteopenia caused by impaired bone formation and bone resorption, which are mechanisms similar to those of age-related bone loss. Mechanistically, SIRT6 interacts with runt-related transcription factor 2 (Runx2) and osterix (Osx), which are the two key transcriptional regulators of osteoblastogenesis, and deacetylates histone H3 at Lysine 9 (H3K9) at their promoters. Hence, excessively elevated Runx2 and Osx in SIRT6(-/-) osteoblasts lead to impaired osteoblastogenesis. In addition, SIRT6 deficiency produces hyperacetylation of H3K9 in the promoter of dickkopf-related protein 1 (Dkk1), a potent negative regulator of osteoblastogenesis, and osteoprotegerin, an inhibitor of osteoclastogenesis. Therefore, the resulting up-regulation of Dkk1 and osteoprotegerin levels contribute to impaired bone remodeling, leading to osteopenia with a low bone turnover in SIRT6-deficient mice. These results establish a new link between SIRT6 and bone remodeling that positively regulates osteoblastogenesis and osteoclastogenesis.

Functional analysis of the engineered cardiac tissue grown on recombinant spidroin fiber meshes.

In the present study, we examined the ability of the recombinant spidroin to serve as a substrate for the cardiac tissue engineering. For this purpose, isolated neonatal rat cardiomyocytes were seeded on the electrospun spidroin fiber matrices and cultured to form the confluent cardiac monolayers. Besides the adhesion assay and immunostaining analysis, we tested the ability of the cultured cardiomyocytes to form a functional cardiac syncytium by studying excitation propagation in the cultured tissue with the aid of optical mapping. It was demonstrated that recombinant spidroin fiber meshes are directly suitable for the adherence and growth of the cardiomyocytes without additional coating with the attachment factors, such as fibronectin.