Today, hydrogel dressings that can protect injury sites and effectively promote healing have become highly desirable in wound management. Therefore, multifunctional silver-poli(N-isopropylacrylamide/itaconic acid) (Ag-P(NiPAAm/IA)) hydrogel nanocomposites were developed for potential application as topical treatment dressings. The radiolytic method, used for the crosslinking of the polymer matrix as well as for the in situ incorporation of silver nanoparticles (AgNPs) into the polymer matrix, enables the preparation of hydrogel nanocomposites without introducing harmful and toxic agents. Moreover, materials produced using γ-irradiation are simultaneously sterilized, thus fulfilling one of the basic requirements regarding their potential biomedical applications. The NiPAAm/IA ratio and the presence of AgNPs influenced the microstructural parameters of the investigated systems. Increasing the IA content leads to the formation of a more porous polymer matrix with larger pores, while the incorporated AgNPs act as additional junction points, decreasing the porosity and pore size of the resulting nanocomposite hydrogels. Swelling studies showed that most investigated systems uptake the fluids from their surroundings by non-Fick diffusion. Further, the Ag+ ion release, antibacterial activity, and cytotoxicity of Ag-P(NiPAAm/IA) hydrogel nanocomposites were examined to evaluate their biomedical potential. All hydrogel nanocomposites showed an initial burst release of Ag+ ions (useful in preventing bacteria adherence and biofilm formation), followed by a slower release of the same (ensuring sterility for longer use). An antibacterial activity test against Escherichia coli and Staphylococcus aureus showed that Ag-P(NiPAAm/IA) hydrogel nanocomposites, with silver concentrations around 10 ± 1 ppm, successfully prevent bacterial growth. Finally, it was shown that the investigated hydrogel nanocomposites do not exhibit a cytotoxic effect on human keratinocyte HaCaT cells. Therefore, these multifunctional hydrogel nanocomposites may promote wound repair and show promising potential for application as functional wound dressing.

We examined the role of endoplasmic reticulum (ER) stress and the ensuing unfolded protein response (UPR) in the development of the central nervous system (CNS)-directed immune response in the rat model of experimental autoimmune encephalomyelitis (EAE). The induction of EAE with syngeneic spinal cord homogenate in complete Freund's adjuvant (CFA) caused a time-dependent increase in the expression of ER stress/UPR markers glucose-regulated protein 78 (GRP78), X-box-binding protein 1 (XBP1), C/EBP homologous protein (CHOP), and phosphorylated eukaryotic initiation factor 2α (eIF2α) in the draining lymph nodes of both EAE-susceptible Dark Agouti (DA) and EAE-resistant Albino Oxford (AO) rats. However, the increase in ER stress markers was more pronounced in AO rats. CFA alone also induced ER stress, but the effect was weaker and less sustained compared to full immunization. The ultrastructural analysis of DA lymph node tissue by electron microscopy revealed ER dilatation in lymphocytes, macrophages, and plasma cells, while immunoblot analysis of CD3-sorted lymph node cells demonstrated the increase in ER stress/UPR markers in both CD3+ (T cell) and CD3− (non-T) cell compartments. A positive correlation was observed between the levels of ER stress/UPR markers in the CNS-infiltrated mononuclear cells and the clinical activity of the disease. Finally, the reduction of EAE clinical signs by ER stress inhibitor ursodeoxycholic acid was associated with the decrease in the expression of mRNA encoding pro-inflammatory cytokines TNF and IL-1β, and encephalitogenic T cell cytokines IFN-γ and IL-17. Collectively, our data indicate that ER stress response in immune cells might be an important pathogenetic factor and a valid therapeutic target in the inflammatory damage of the CNS.

Glioblastomas are aggressive and usually incurable high-grade gliomas without adequate treatment. In this study, we aimed to investigate the potential of desloratadine to induce apoptosis/autophagy as genetically regulated processes that can seal cancer cell fates. All experiments were performed on U251 human glioblastoma cell line and primary human glioblastoma cell culture. Cytotoxic effect of desloratadine was investigated using MTT and CV assays, while oxidative stress, apoptosis, and autophagy were detected by flow cytometry and immunoblot. Desloratadine treatment decreased cell viability of U251 human glioblastoma cell line and primary human glioblastoma cell culture (IC50 value 50 µM) by an increase of intracellular reactive oxygen species and caspase activity. Also, desloratadine decreased the expression of main autophagy repressor mTOR and its upstream activator Akt and increased the expression of AMPK. Desloratadine exerted dual cytotoxic effect inducing both apoptosis- and mTOR/AMPK-dependent cytotoxic autophagy in glioblastoma cells and primary glioblastoma cell culture.

Mott cells are plasma cells that have multiple spherical Russell bodies packed in their cytoplasm. Russell bodies are dilated endoplasmic reticulum cisternae filled with aggregates of immunoglobulins that are neither secreted nor degraded. Mott cells were observed in our study by light and electron microscope in the lymph nodes of rats with experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Mott cells were detected on hematoxylin and eosin (HE)-stained lymph node sections as vacuolated cells with eccentrically positioned nuclei and large number of faint blue spherical inclusions in the cytoplasm. Electron microscopic investigation revealed the presence of Russell bodies of the "medusa" form inside Mott cells in lymph node ultra-thin sections of EAE animals. Mott cells expressed the plasma cell marker CD138 and either kappa or lambda immunoglobulin light chains, indicating their origin from polyclonally activated B cells. Finally, Mott cells were associated with active EAE, as they were not found in the lymph nodes of EAE-resistant Albino Oxford rats. The presence of Russell bodies implies an excessive production of immunoglobulins in EAE, thus further emphasizing the role of B cells, and among them Mott cells, in the pathogenesis of this animal model of multiple sclerosis.

BACKGROUND:The aim of this study was to examine the influence of intraluminal thrombus (ILT) volume on the level of proteolytic activity and the content of abdominal aortic aneurysm (AAA) wall. METHODS:The research was designed as a cross-sectional study at the Clinic for Vascular and Endovascular Surgery, Clinical Center of Serbia in the period from April 2017 to February 2018. During this period, a total of 155 patients with asymptomatic AAA underwent open surgical treatment and 50 were included in the study based on inclusion and exclusion criteria. Before surgery, patients included in the study were examined by MRI. During the operation, samples of ILT and AAA wall were taken for biochemical analysis. RESULTS:A statistically significant correlation was found between the volume of the ILT and largest AAA diameter (ρ = 0.56; P < 0.001). The correlation of the ILT volume on the anterior wall and the concentration of MMP-9, MMP-2 and NE/ELA in the wall did not find statistical significance. Also, no statistically significant association was found between the volume of ILT and the concentration of ECM proteins (collagen type 3, elastin, proteoglycan) in the corresponding part of the wall. The association of ILT volume with MDA was also of no statistical significance. There was a positive statistical significance found in correlation of volume of ILT and catalase activity in the wall of AAA (ρ = 0.28, P = 0.049). CONCLUSIONS:The volume of ILT in the aneurysmal sac seemed not to affect the level of proteolytic activity and the content of the aneurysm wall. However, a positive correlation was found between the ILT and the catalase activity. The effect of ILT on the aneurysm wall and its role in the progression of aneurysmal disease should be examined in future studies.

Dear Editor, We read with great interest the commentary by Lorzadeh and Ghavami1 on our article that reported AMP-activated protein kinase (AMPK)/mechanistic target of rapamycin (mTOR)-independent upregulation of autophagy in the tumor tissue of clear cell renal cell carcinoma (ccRCC) patients.2 We agree with their observation that the somewhat surprising absence of a link between AMPK/mTOR signaling and autophagy induction in ccRCC …

OBJECTIVE:The aim of the study was to determine whether magnetic resonance imaging (MRI) can be used in assessment of biologic activity of intraluminal thrombus (ILT) and proteolytic processes of the abdominal aortic aneurysm wall. METHODS:Using MRI, 50 patients with asymptomatic infrarenal abdominal aortic aneurysm were analyzed at the maximum aneurysm diameter on T1-weighted images in the arterial phase after administration of contrast material. Relative ILT signal intensity (SI) was determined as the ratio between ILT SI and psoas muscle SI. During surgery, the full thickness of the ILT and the adjacent part of the aneurysm wall were harvested at the maximal diameter for biochemical analysis. The concentrations of matrix metalloproteinase 9 and neutrophil elastase (NE/ELA) were analyzed in harvested thrombi, and the concentrations of collagen type III, elastin, and proteoglycans were analyzed in harvested aneurysm walls. RESULTS:A significant positive correlation was found between the NE/ELA concentration of the ILT and the relative SI (ρ = 0.309; P = .029). Furthermore, a negative correlation was observed between the elastin content of the aneurysm wall and the relative SI (ρ = -0.300; P = .034). No correlations were found between relative SI and concentration of matrix metalloproteinase 9, NE/ELA, collagen type III, or proteoglycan 4 in the aneurysm wall. CONCLUSIONS:These findings indicate a potential novel use of MRI in prediction of thrombus proteolytic enzyme concentrations and the extracellular matrix content of the aneurysm wall, thus providing additional information for the risk of potential aneurysm rupture.

Anterior mesoderm (AM) and definitive endoderm (DE) progenitors represent the earliest embryonic cell types that are specified during germ layer formation at the primitive streak (PS) of the mouse embryo. Genetic experiments indicate that both lineages segregate from Eomes-expressing progenitors in response to different Nodal signaling levels. However, the precise spatiotemporal pattern of the emergence of these cell types and molecular details of lineage segregation remain unexplored. We combined genetic fate labeling and imaging approaches with single-cell RNA sequencing (scRNA-seq) to follow the transcriptional identities and define lineage trajectories of Eomes-dependent cell types. Accordingly, all cells moving through the PS during the first day of gastrulation express Eomes AM and DE specification occurs before cells leave the PS from Eomes-positive progenitors in a distinct spatiotemporal pattern. ScRNA-seq analysis further suggested the immediate and complete separation of AM and DE lineages from Eomes-expressing cells as last common bipotential progenitor.

Background: Main objective of this study was to evaluate the influence of statins and/or acetylsalicylic acid on biochemical characteristics of abdominal aortic aneurysm (AAA) wall and intraluminal thrombus (ILT). Patients and methods: Fifty patients with asymptomatic infrarenal AAA were analyzed using magnetic resonance imaging on T1w sequence. Relative ILT signal intensity (SI) was determined as a ratio between ILT and psoas muscle SI. Samples containing the full ILT thickness and aneurysm wall were harvested from the anterior surface at the level of the maximal diameter. The concentration of enzymes such as matrix metalloproteinase (MMP) 9, MMP2 and neutrophil elastase (NE/ELA) were analyzed in ILT and AAA wall; while collagen type III, elastin and proteoglycan 4 were analyzed in harvested AAA wall. Oxidative stress in the AAA wall was assessed by catalase and malondialdehyde activity in tissue samples. Results: Relative ILT signal intensity (1.09 ± 0.41 vs 0.89 ± 0.21, p = 0.013) were higher in non-statin than in statin group. Patients who were taking aspirin had lower relative ILT area (0.89 ± 0.19 vs 1.13. ± 0.44, p = 0.016), and lower relative ILT signal intensity (0.85 [0.73-1.07] vs 1.01 [0.84-1.19], p = 0.021) compared to non-aspirin group. There were higher concentrations of elastin in AAA wall among patients taking both of aspirin and statins (1.21 [0.77-3.02] vs 0.78 (0.49-1.05) ng/ml, p = 0.044) than in patients who did not take both of these drugs. Conclusions: Relative ILT SI was lower in patients taking statin and aspirin. Combination of antiplatelet therapy and statins was associated with higher elastin concentrations in AAA wall.

We examined the status and role of autophagy, a process of lysosomal recycling of cellular material, in clear cell renal cell carcinoma (ccRCC). Paired samples of tumor and adjacent non-malignant tissue were collected from 20 patients with ccRCC after radical nephrectomy. The mRNA levels of apoptosis ( BAD, BAX, BCL2, BCLXL, BIM) and autophagy ( ATG4, BECN1, GABARAP, p62, UVRAG) regulators were measured by RT-qPCR. The protein levels of autophagosome-associated LC3-II, autophagy receptor p62, apoptotic marker PARP, as well as phosphorylation of autophagy initiator Unc 51-like kinase 1 (ULK1), its activator AMP-activated protein kinase (AMPK) and 4EBP1, the substrate of ULK1 inhibitor mechanistic target of rapamycin (mTOR), were analyzed by immunoblotting. The mRNA levels of pro-apoptotic BAX, anti-apoptotic BCLXL and pro-autophagic ATG4, p62 and UVRAG were higher in ccRCC tumors. Autophagy induction was confirmed by an increase in phospho-ULK1 and degradation of the autophagic target p62, while apoptotic PARP cleavage was unaltered. AMPK phosphorylation was reduced and 4EBP1 phosphorylation was increased in ccRCC tissue. The expression of apoptosis regulators did not correlate with clinicopathological features of ccRCC. Conversely, high mRNA levels of ATG4, GABARAP and p62 were associated with lower tumor stage, as well as with smaller tumor size and better disease-specific 5-year survival ( ATG4 and p62). Accordingly, low p62 protein levels, corresponding to increased autophagic flux, were associated with lower tumor stage, reduced metastasis and improved 5-year survival. These data demonstrate that transcriptional induction of autophagy in ccRCC is accompanied by AMPK/mTOR-independent increase in ULK1 activation and autophagic flux, which might slow tumor progression and metastasis independently of apoptosis.

Menopause is characterized by deep metabolic disturbances, including decreased insulin sensitivity, adiposity, and changes in lipid profiles. Estrogen replacement therapy can partially reverse these changes, and while it is safe in most healthy postmenopausal women, there are still existing concerns regarding an increased risk for breast and endometrial cancer as well as a risk for cardiovascular and thromboembolic disease. Therefore, certain natural compounds with positive metabolic effects may be considered as a possible alternative or adjunctive treatment in patients not willing to take estrogens or patients with contraindications for estrogens. The aim of this study was to investigate the influence of Sideritis scardica (mountain tea) extract on metabolic disturbances induced by ovariectomy in rats. The study included 24 rats divided into three groups: ovariectomized rats treated with 200 mg/kg S. scardica extract for 24 weeks (n=8), ovariectomized non-treated (n=8), and Sham-operated (n=8) rats. Food intake, weight gain, body composition, fasting glucose levels, response to oral glucose challenge, liver glycogen content, catalase activity, thiol groups, and malondialdehyde concentrations as well as AMP-activated protein kinase activity in liver cells were studied. Ovariectomized rats treated with S. scardica extract had lower blood triglycerides, reduced fasting glucose levels, as well lower glucose peaks after oral glucose challenge, increased liver glycogen content, and significantly higher catalase activity and thiol group concentration than non-treated ovariectomized rats. The ability of S. scardica extract to attenuate metabolic disturbances associated with ovariectomy was associated with the activation of AMP-activated protein kinase in liver cells.

In search for antitumor metal-based drugs that would mitigate the severe side-effects of cisplatin, Ru(II) complexes are gaining increasing recent interest. In this work, we report on the synthesis, characterization (H-1- and C-13-NMR, FT-IR), and cytotoxicity studies of two new half-sandwich organometallic Ru(II) complexes of the general formula [Ru(eta(6)-arene)(XY)Cl](PF6) where arene = benzene or toluene and XY = bidentates: dipyrido[3,2-a:2 ',3 '-c]phenazine (dppz) or 2-(9-anthryl)-1H-imidazo[4,5-f][1,10]phenanthroline (aip), which are bound to Ru(II) via two phenanthroline-N atoms in a characteristic "piano-stool" configuration of Ru(II)-arene complexes-as confirmed by vibrational and NMR spectra. In addition, cytotoxic studies were performed for similar half-sandwich organometallic [Ru(eta(6)-p-cymene)(Me(2)dppz)Cl]PF6 complex (Me(2)dppz = 11,12-dimethyl-dipyrido[3,2-a:2 ',3 '-c]phenazine). This study is complemented with elaborate modeling with density functional theory (DFT) calculations, which provided insight into reactive sites of Ru(II) structures, further detailed by molecular docking on the B-DNA dodecamer, which identified binding sites and affinities: most pronounced for the [Ru(eta(6)-benzene)(aip)Cl](PF6) in both A-T and G-C regions of the DNA minor groove. Cytotoxic activity was probed versus tumor cell lines B16, C6, and U251 (B16 mouse melanoma, C6 rat glioma, U251 human glioblastoma) and non-tumor cell line HACAT (HACAT normal human keratinocytes). [GRAPHICS] .

We examined the effects of betaine, an endogenous and dietary methyl donor essential for the methionine-homocysteine cycle, on oxidative stress, inflammation, apoptosis, and autophagy in methionine-choline deficient diet (MCD)-induced non-alcoholic fatty liver disease (NAFLD). Male C57BL/6 mice received standard chow (control), standard chow and betaine (1.5% w/v in drinking water), MCD, or MCD and betaine. After six weeks, serum and liver samples were collected for analysis. Betaine reduced MCD-induced increase in liver transaminases and inflammatory infiltration, as well as hepatosteatosis and serum levels of low-density lipoprotein, while it increased that of high-density lipoprotein. MCD-induced hepatic production of reactive oxygen and nitrogen species was significantly reduced by betaine, which also improved liver antioxidative defense by increasing glutathione content and superoxide-dismutase, catalase, glutathione peroxidase, and paraoxonase activity. Betaine reduced the liver expression of proinflammatory cytokines tumor necrosis factor and interleukin-6, as well as that of proapoptotic mediator Bax, while increasing the levels of anti-inflammatory cytokine interleukin-10 and antiapoptotic Bcl-2 in MCD-fed mice. In addition, betaine increased the expression of autophagy activators beclin 1, autophagy-related (Atg)4 and Atg5, as well as the presence of autophagic vesicles and degradation of autophagic target sequestosome 1/p62 in the liver of NAFLD mice. The observed effects of betaine coincided with the increase in the hepatic phosphorylation of mammalian target of rapamycin (mTOR) and its activator Akt. In conclusion, the beneficial effect of betaine in MCD-induced NAFLD is associated with the reduction of liver oxidative stress, inflammation, and apoptosis, and the increase in cytoprotective Akt/mTOR signaling and autophagy.

The protective activity of dry olive leaf extract (DOLE) in carbon tetrachloride (CCl4)-induced liver damage and possible mechanisms involved in this protection were investigated in rats. Acute CCl4 intoxication resulted in a massive hepatic necrosis, in increased serum transaminases, and in a perturbation of oxidative stress parameters in liver tissue [malondyaldehide, glutathione (GSH), catalase]. CCl4 did not affect the expression of caspase-3 and cytochrome c as markers of apoptosis; however, CCl4 increased the AMP-activated protein kinase (AMPK) activity and the expression of autophagy-related protein LC3II and decreased the expression of p62 protein. The pre-treatment with DOLE significantly improved serum markers of liver damage, liver catalase activity, and GSH concentration, suggesting that antioxidative mechanism is responsible for hepatoprotection. Oral administration of DOLE did not influence LC3II conversion and p62 degradation in liver, but AMPK activity was significantly decreased, suggesting the energy balance perturbation as an additional potential mechanism of DOLE hepatoprotective effect.

The first lineage specification of pluripotent mouse epiblast segregates neuroectoderm (NE) from mesoderm and definitive endoderm (ME) by mechanisms that are not well understood. Here we demonstrate that the induction of ME gene programs critically relies on the T-box transcription factors Eomesodermin (also known as Eomes ) and Brachyury , which concomitantly repress pluripotency and NE gene programs. Cells deficient in these T-box transcription factors retain pluripotency and differentiate to NE lineages despite the presence of ME-inducing signals transforming growth factor β (TGF-β)/Nodal and Wnt. Pluripotency and NE gene networks are additionally repressed by ME factors downstream of T-box factor induction, demonstrating a redundancy in program regulation to safeguard mutually exclusive lineage specification. Analyses of chromatin revealed that accessibility of ME enhancers depends on T-box factor binding, whereas NE enhancers are accessible and already activation primed at pluripotency. This asymmetry of the chromatin landscape thus explains the default differentiation of pluripotent cells to NE in the absence of ME induction that depends on activating and repressive functions of Eomes and Brachyury .

We investigated the therapeutic capacity of nano-sized graphene sheets, called graphene quantum dots (GQD), in experimental autoimmune encephalomyelitis (EAE), an animal model of immune-mediated central nervous system (CNS) damage. Intraperitoneally administered GQD (10 mg/kg/day) accumulated in the lymph node and CNS cells of Dark Agouti rats in which EAE was induced by immunization with spinal cord homogenate in complete Freund's adjuvant. GQD significantly reduced clinical signs of EAE when applied throughout the course of the disease (day 0-32), while the protection was less pronounced if the treatment was limited to the induction (day 0-7 post-immunization) or effector (from day 8 onwards) phase of the disease. GQD treatment diminished immune infiltration, demyelination, axonal damage, and apoptotic death in the CNS of EAE animals. GQD also reduced the numbers of interferon-gamma-expressing T helper (Th)1 cells, as well as the expression of Th1 transcription factor T-bet and proinflammatory cytokines tumor necrosis factor, interleukin-1, and granulocyte macrophage colony-stimulating factor in the lymph nodes and CNS immune infitrates. The protective effect of GQD in EAE was associated with the activation of p38 and p42/44 mitogen-activated protein kinases (MAPK) and Akt in the lymph nodes and/or CNS. Finally, GQD protected oligodendrocytes and neurons from T cell mediated damage in the in vitro conditions. Collectively, these data demonstrate the ability of GQD to gain access to both immune and CNS cells during neuroinflammation, and to alleviate immune-mediated CNS damage by modulating MAPK/Akt signaling and encephalitogenic Th1 immune response.

The protective effect of antioxidant peptides from grass carp scale gelatin on hydrogen peroxide (H2O2)-mediated oxidative injured HepG2 cells was investigated, and the protective mechanism as well as peptide structure were studied. Pretreated with grass carp scale gelatin hydrolysates (GSGH) for 24 h significantly increased the survival rates and decreased the apoptosis rates of H2O2-mediated oxidative injured HepG2 cells. The increase in SOD, CAT and GPX activities, reduce in ROS level and MDA content, and weaken in damage on cell membrane and DNA could be responsible for the protective effect of GSGH on H2O2-mediated oxidative injured HepG2 cells. Peptide sequences of GSGH were analyzed by LC-ESI-Q-Orbitrap-MS/MS, and results showed that most of them were low molecular weight peptide at 358–986 Da. Synergistic effect existed among antioxidant peptides and contributed to the strong antioxidant activities of GSGH.

Here, we describe how to reprogram mouse embryonic broblasts into renal tubular epithelial cells \ (iRECs) by lentiviral transduction of four transcription factors.First, MEFs are isolated from limbs of E12.5 reporter mice.Second, lentivirus is generated to overexpress transcription factors.Finally, reprogrammed iRECs are selected by ow cytometry.

Pluripotency can be induced in vitro from adult somatic mammalian cells by enforced expression of defined transcription factors regulating and initiating the pluripotency network. Despite the substantial advances over the last decade to improve the efficiency of direct reprogramming, exact mechanisms underlying the conversion into the pluripotent stem cell state are still vaguely understood. Several studies suggested that induced pluripotency follows reversed embryonic development. For somatic cells of mesodermal and endodermal origin that would require the transition through a Primitive streak-like state, which would necessarily require an Eomesodermin ( Eomes ) expressing intermediate. We analyzed reprogramming in human and mouse cells of mesodermal as well as ectodermal origin by thorough marker gene analyses in combination with genetic reporters, conditional loss of function and stable fate-labeling for the broad primitive streak marker Eomes . We unambiguously demonstrate that induced pluripotency is not dependent on a transient primitive streak-like stage and thus does not represent reversal of mesendodermal development in vivo .