Laccase-Treated Polystyrene Surfaces with Caffeic Acid, Dopamine, and L-3,4-Dihydroxyphenylalanine Substrates Facilitate the Proliferation of Melanocytes and Embryonal Carcinoma Cells NTERA-2.

This study presents the effects of treating polystyrene (PS) cell culture plastic with oxidoreductase enzyme laccase and the catechol substrates caffeic acid (CA), L-DOPA, and dopamine on the culturing of normal human epidermal melanocytes (NHEMs) and human embryonal carcinoma cells (NTERA-2). The laccase-substrate treatment improved PS hydrophilicity and roughness, increasing NHEM and NTERA-2 adherence, proliferation, and NHEM melanogenesis to a level comparable with conventional plasma treatment. Cell adherence dynamics and proliferation were evaluated. The NHEM endpoint function was quantified by measuring melanin content. PS surfaces treated with laccase and its substrates demonstrated the forming of polymer-like structures. The surface texture roughness gradient and the peak curvature were higher on PS treated with a combination of laccase and substrates than laccase alone. The number of adherent NHEM and NTERA-2 was significantly higher than on the untreated surface. The proliferation of NHEM and NTERA-2 correspondingly increased on treated surfaces. NHEM melanin content was enhanced 6-10-fold on treated surfaces. In summary, laccase- and laccase-substrate-modified PS possess improved PS surface chemistry/hydrophilicity and altered roughness compared to untreated and plasma-treated surfaces, facilitating cellular adherence, subsequent proliferation, and exertion of the melanotic phenotype. The presented technology is easy to apply and creates a promising custom-made, substrate-based, cell-type-specific platform for both 2D and 3D cell culture.

Aging processes in dental thermoplastics - Thermoanalytical investigations and effects on Vickers as well as Martens hardness.

OBJECTIVE: The influence of various aging protocols, representing and accelerating influences present in the dental context, on possible changes in the microstructure and mechanical properties of thermoplastics was investigated. In order to minimize the complexity of the systems, first pure polymers and then later the equivalent dental polymeric materials were analyzed. MATERIALS AND METHODS: Pure polymers (Poly(methyl methacrylate) - PMMA, Polyoxymethylene homopolymer - POM-H, Polyether ether ketone - PEEK, Nylon 12 - PA12, Polypropylene - PP) were analyzed before as well as after applying different aging protocols relevant to the oral environment (ethanol, thermocycling, alkaline and acidic setting) by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The thermoanalytical parameters used were glass transition temperature (Tg), melting peak and crystallization peak temperature (Tpm, Tpc) and decomposition behavior. In a second step selected commercially available dental products (Telio CAD - PMMAD, Zirlux Acetal - POMD, Juvora Natural Dental Disc - PEEKD) aged by the protocol that previously showed strong effects were examined and additionally tested for changes in their Vickers and Martens hardness by Mann-Whitney-U test. RESULTS: The combinations of pure polymers and viable aging protocols analyzed within this study were identified via TGA or DSC as PA12 & thermocycling, POM-H & denture cleanser/lactic acid/ethanol, PP & lactic acid. The dental polymeric materials PMMAD and POMD due to aging in lactic acid showed slight but significantly (p < 0.01) reduced Vickers and partly Martens hardness. PEEK showed the greatest material resistance within this study.

Reproductive hormones mediate changes in the gut microbiome during pregnancy and lactation in Phayre's leaf monkeys.

Studies in multiple host species have shown that gut microbial diversity and composition change during pregnancy and lactation. However, the specific mechanisms underlying these shifts are not well understood. Here, we use longitudinal data from wild Phayre's leaf monkeys to test the hypothesis that fluctuations in reproductive hormone concentrations contribute to gut microbial shifts during pregnancy. We described the microbial taxonomic composition of 91 fecal samples from 15 females (n = 16 cycling, n = 36 pregnant, n = 39 lactating) using 16S rRNA gene amplicon sequencing and assessed whether the resulting data were better explained by overall reproductive stage or by fecal estrogen (fE) and progesterone (fP) concentrations. Our results indicate that while overall reproductive stage affected gut microbiome composition, the observed patterns were driven by reproductive hormones. Females had lower gut microbial diversity during pregnancy and fP concentrations were negatively correlated with diversity. Additionally, fP concentrations predicted both unweighted and weighted UniFrac distances, while reproductive state only predicted unweighted UniFrac distances. Seasonality (rainfall and periods of phytoprogestin consumption) additionally influenced gut microbial diversity and composition. Our results indicate that reproductive hormones, specifically progestagens, contribute to the shifts in the gut microbiome during pregnancy and lactation.

Alkalinity and Its Consequences for the Performance of Steel-Reinforced Geopolymer Materials.

This paper investigates the development of the alkalinity and its impact on carbon steel reinforcement embedded in alkali-activated fly ashes (AAFA) and alkali-activated fly ashes with ten percentage mass (wt%) of blast furnace slag (AAFAS)-based materials (geopolymer-GP). The pH analysis of eluates indicates a remarkable decrease of alkalinity in AAFA and AAFAS in the first hours of the geopolymerization process. Phenolphthalein solution and pore solution tests on concretes also show a sharp decrease of alkalinity with increased Ca content in the binder due to carbonation. Micro X-ray computer tomography (µXCT) and electrochemical techniques indicate that the changed pH in the GP systems was accompanied by a decrease in the corrosion rates of steel reinforcement when compared to ordinary Portland cement (OPC) systems. In contrast to calcite and vaterite, which were detected in OPC and AAFAS after a carbonation process, only sodium carbonate natron was determined at lower levels in AAFA by X-ray diffraction (XRD).

Age-Associated Changes in Estrogen Receptor Ratios Correlate with Increased Female Susceptibility to Coxsackievirus B3-Induced Myocarditis.

Sexual bias is a hallmark in various diseases. This review evaluates sexual dimorphism in clinical and experimental coxsackievirus B3 (CVB3) myocarditis, and how sex bias in the experimental disease changes with increased age. Coxsackieviruses are major causes of viral myocarditis, an inflammation of the heart muscle, which is more frequent and severe in men than women. Young male mice infected with CVB3 develop heart-specific autoimmunity and severe myocarditis. Females infected during estrus (high estradiol) develop T-regulatory cells and when infected during diestrus (low estradiol) develop autoimmunity similar to males. During estrus, protection depends on estrogen receptor alpha (ERα), which promotes type I interferon, activation of natural killer/natural killer T cells and suppressor cell responses. Estrogen receptor beta has opposing effects to ERα and supports pro-inflammatory immunity. However, the sexual dimorphism of the disease is significantly ameliorated in aged animals when old females become as susceptible as males. This correlates to a selective loss of the ERα that is required for immunosuppression. Therefore, sex-associated hormones control susceptibility in the virus-mediated disease, but their impact can alter with the age and physiological stage of the individual.

Reactive oxygen species induce virus-independent MAVS oligomerization in systemic lupus erythematosus.

The increased expression of genes induced by type I interferon (IFN) is characteristic of viral infections and systemic lupus erythematosus (SLE). We showed that mitochondrial antiviral signaling (MAVS) protein, which normally forms a complex with retinoic acid gene I (RIG-I)-like helicases during viral infection, was activated by oxidative stress independently of RIG-I helicases. We found that chemically generated oxidative stress stimulated the formation of MAVS oligomers, which led to mitochondrial hyperpolarization and decreased adenosine triphosphate production and spare respiratory capacity, responses that were not observed in similarly treated cells lacking MAVS. Peripheral blood lymphocytes of SLE patients also showed spontaneous MAVS oligomerization that correlated with the increased secretion of type I IFN and mitochondrial oxidative stress. Furthermore, inhibition of mitochondrial reactive oxygen species (ROS) by the mitochondria-targeted antioxidant MitoQ prevented MAVS oligomerization and type I IFN production. ROS-dependent MAVS oligomerization and type I IFN production were reduced in cells expressing the MAVS-C79F variant, which occurs in 30% of sub-Saharan Africans and is linked with reduced type I IFN secretion and milder disease in SLE patients. Patients expressing the MAVS-C79F variant also had reduced amounts of oligomerized MAVS in their plasma compared to healthy controls. Together, our findings suggest that oxidative stress-induced MAVS oligomerization in SLE patients may contribute to the type I IFN signature that is characteristic of this syndrome.

Disabling Mitochondrial Peroxide Metabolism via Combinatorial Targeting of Peroxiredoxin 3 as an Effective Therapeutic Approach for Malignant Mesothelioma.

Dysregulation of signaling pathways and energy metabolism in cancer cells enhances production of mitochondrial hydrogen peroxide that supports tumorigenesis through multiple mechanisms. To counteract the adverse effects of mitochondrial peroxide many solid tumor types up-regulate the mitochondrial thioredoxin reductase 2--thioredoxin 2 (TRX2)--peroxiredoxin 3 (PRX3) antioxidant network. Using malignant mesothelioma cells as a model, we show that thiostrepton (TS) irreversibly disables PRX3 via covalent crosslinking of peroxidatic and resolving cysteine residues in homodimers, and that targeting the oxidoreductase TRX2 with the triphenylmethane gentian violet (GV) potentiates adduction by increasing levels of disulfide-bonded PRX3 dimers. Due to the fact that activity of the PRX3 catalytic cycle dictates the rate of adduction by TS, immortalized and primary human mesothelial cells are significantly less sensitive to both compounds. Moreover, stable knockdown of PRX3 reduces mesothelioma cell proliferation and sensitivity to TS. Expression of catalase in shPRX3 mesothelioma cells restores defects in cell proliferation but not sensitivity to TS. In a SCID mouse xenograft model of human mesothelioma, administration of TS and GV together reduced tumor burden more effectively than either agent alone. Because increased production of mitochondrial hydrogen peroxide is a common phenotype of malignant cells, and TS and GV are well tolerated in mammals, we propose that targeting PRX3 is a feasible redox-dependent strategy for managing mesothelioma and other intractable human malignancies.

The c-FLIPL cleavage product p43FLIP promotes activation of extracellular signal-regulated kinase (ERK), nuclear factor κB (NF-κB), and caspase-8 and T cell survival.

Caspase-8 is now appreciated to govern both apoptosis following death receptor ligation and cell survival and growth via inhibition of the Ripoptosome. Cells must therefore carefully regulate the high level of caspase-8 activity during apoptosis versus the modest levels observed during cell growth. The caspase-8 paralogue c-FLIP is a good candidate for a molecular rheostat of caspase-8 activity. c-FLIP can inhibit death receptor-mediated apoptosis by competing with caspase-8 for recruitment to FADD. However, full-length c-FLIPL can also heterodimerize with caspase-8 independent of death receptor ligation and activate caspase-8 via an activation loop in the C terminus of c-FLIPL. This triggers cleavage of c-FLIPL at Asp-376 by caspase-8 to produce p43FLIP. The continued function of p43FLIP has, however, not been determined. We demonstrate that acute deletion of endogenous c-FLIP in murine effector T cells results in loss of caspase-8 activity and cell death. The lethality and caspase-8 activity can both be rescued by the transgenic expression of p43FLIP. Furthermore, p43FLIP associates with Raf1, TRAF2, and RIPK1, which augments ERK and NF-κB activation, IL-2 production, and T cell proliferation. Thus, not only is c-FLIP the initiator of caspase-8 activity during T cell activation, it is also an initial caspase-8 substrate, with cleaved p43FLIP serving to both stabilize caspase-8 activity and promote activation of pathways involved with T cell growth.

Phytochemicals and reproductive function in wild female Phayre's leaf monkeys (Trachypithecus phayrei crepusculus).

Female reproduction is known to be influenced by food availability and its impact on energetic status. However, emerging evidence suggests that the phytochemical content of food may also be an important factor. Here, we investigated this hypothesis, presenting 20 months of data on fecal progestin (fP) patterns in wild female Phayre's leaf monkeys (Trachypithecus phayrei crepusculus). We examined whether (a) the availability of Vitex (a plant known to contain phytochemicals) might be linked to seasonal fP levels, (b) fP levels were associated with female reproductive performance, and (c) reproductive performance might also be linked with energetic status (as measured by physical condition). We collected fecal samples (N=2077) from 10 adult females to analyze estrogen (fE) and progestin (fP) metabolites, behavioral data from 7 cycling females to determine receptivity, and monthly data on Vitex availability and female physical condition. Seasonally elevated fP levels were found in all females, with higher levels when Vitex leaves and fruits were abundant. During the period of high progestins, females had longer cycle lengths and follicular phases, while receptive periods did not change. Nevertheless, when ovulations occurred, females were more likely to conceive. On the other hand, conceptions were also more likely when physical condition was improving, suggesting that the effects of phytochemicals and energetic status on reproduction may be difficult to separate. Although our results support the predicted effects of Vitex on endocrine and reproductive function, future studies with detailed feeding data and chemical analyses of plants are needed to confirm this finding.

Multicenter assessment of coronary allograft vasculopathy by intravascular ultrasound-derived analysis of plaque composition.

BACKGROUND: Coronary allograft vasculopathy is a severe complication of heart transplantation. We used virtual histology intravascular ultrasound to characterize plaque burden and tissue composition over time in heart transplant recipients. METHODS: We recruited patients undergoing heart transplantation in four centers in Europe and the US between 2004 and 2006. We used intravascular ultrasound to obtain morphological plaque measurements and to perform virtual histology in the left anterior descending coronary artery. Data were characterized according to the duration between transplantation and intravascular ultrasound assessment: 24-60, >60-120 and >120-192 months. RESULTS: We assessed vessels from 152 patients (mean age 58 +/- 12 years) a mean of 70 +/- 53 months (range 1 week to 16 years) after transplantation. Plaque burden of >40% was observed in 26% of vessels analyzed, with increases from baseline being seen in all time categories. If assessed >24 months after transplantation, necrotic core and dense calcified volumes were significantly greater than at baseline (P = 0.0005 and P = 0.01, respectively). Time since heart transplantation and donor age and recipient age were independent predictive factors of increased necrotic core content. Necrotic core volume >2.01 mm(3), diabetes mellitus, donor age older than 40 years, follow-up from transplantation longer than 5 years and recipient age older than 58 years were associated with the need for revascularization. CONCLUSIONS: In coronary allograft vasculopathy, plaque burden and composition change over time and seem to affect clinical outcome. This relationship might facilitate identification of high-risk patients in whom the value of more aggressive medical therapy should be tested.

Caspase-8 and c-FLIPL associate in lipid rafts with NF-kappaB adaptors during T cell activation.

Humans and mice lacking functional caspase-8 in T cells manifest a profound immunodeficiency syndrome due to defective T cell antigen receptor (TCR)-induced NF-kappaB signaling and proliferation. It is unknown how caspase-8 is activated following T cell stimulation, and what is the caspase-8 substrate(s) that is necessary to initiate T cell cycling. We observe that following TCR ligation, a small portion of total cellular caspase-8 and c-FLIP(L) rapidly migrate to lipid rafts where they associate in an active caspase complex. Activation of caspase-8 in lipid rafts is followed by rapid cleavage of c-FLIP(L) at a known caspase-8 cleavage site. The active caspase.c-FLIP complex forms in the absence of Fas (CD95/APO1) and associates with the NF-kappaB signaling molecules RIP1, TRAF2, and TRAF6, as well as upstream NF-kappaB regulators PKC theta, CARMA1, Bcl-10, and MALT1, which connect to the TCR. The lack of caspase-8 results in the absence of MALT1 and Bcl-10 in the active caspase complex. Consistent with this observation, inhibition of caspase activity attenuates NF-kappaB activation. The current findings define a link among TCR, caspases, and the NF-kappaB pathway that occurs in a sequestered lipid raft environment in T cells.

Cardiac magnetic resonance perfusion imaging for the functional assessment of coronary artery disease: a comparison with coronary angiography and fractional flow reserve.

AIMS: Cardiac magnetic resonance perfusion imaging (CMRI) is a promising technique for non-invasive measurement of myocardial perfusion reserve. Fractional flow reserve (FFR) is an established invasive method for functional assessment of coronary artery disease (CAD). To prospectively assess the diagnostic value of CMRI for the detection of haemodynamically significant coronary lesions, compared with coronary angiography (CA) and FFR. METHODS AND RESULTS: Forty-three patients with suspected or known CAD underwent CA, CMRI, and FFR measurement. First pass magnetic resonance perfusion examination was performed during hyperaemia (140 microg/kg/min adenosine over 6 min) and at rest. One hundred and twenty-nine perfusion territories were assessed by semi-quantitative evaluation of signal intensity-time curves using the myocardial perfusion reserve index (MPRI) [upslope(stress(corrected))/upslope(rest(corrected))]. Perfusion territories were categorized as normal (coronary stenosis < or = 50%), intermediate (stenosis > 50% and FFR > 0.75), or severe (stenosis > 50% and FFR < or = 0.75 or total occlusion). MPRI values (+/-SD) were significantly different between the three categories [normal, 2.2 +/- 0.5 vs. intermediate, 1.8 +/- 0.5 (P = 0.005) and intermediate vs. severe, 1.2 +/- 0.3 (P < 0.001)]. An MPRI cut-off value of 1.5 (derived from receiver operating characteristics analysis) distinguished haemodynamically relevant (severe) from non-relevant (normal and intermediate) stenoses with a sensitivity of 88% (CI 74-100%) and a specificity of 90% (CI 84-96%). CONCLUSION: In contrast to earlier studies that compared CMRI with morphological examination (CA) alone, the present study compared CMRI with CA plus a standard invasive functional assessment (FFR) and demonstrated that CMRI is able to distinguish haemodynamically relevant from non-relevant coronary lesions with a high sensitivity and specificity and may therefore contribute to clinical decision-making.