Size-dependent accumulation of particles in lysosomes modulates dendritic cell function through impaired antigen degradation.

INTRODUCTION Nanosized particles may enable therapeutic modulation of immune responses by targeting dendritic cell (DC) networks in accessible organs such as the lung. To date, however, the effects of nanoparticles on DC function and downstream immune responses remain poorly understood. METHODS Bone marrow-derived DCs (BMDCs) were exposed in vitro to 20 or 1,000 nm polystyrene (PS) particles. Particle uptake kinetics, cell surface marker expression, soluble protein antigen uptake and degradation, as well as in vitro CD4(+) T-cell proliferation and cytokine production were analyzed by flow cytometry. In addition, co-localization of particles within the lysosomal compartment, lysosomal permeability, and endoplasmic reticulum stress were analyzed. RESULTS The frequency of PS particle-positive CD11c(+)/CD11b(+) BMDCs reached an early plateau after 20 minutes and was significantly higher for 20 nm than for 1,000 nm PS particles at all time-points analyzed. PS particles did not alter cell viability or modify expression of the surface markers CD11b, CD11c, MHC class II, CD40, and CD86. Although particle exposure did not modulate antigen uptake, 20 nm PS particles decreased the capacity of BMDCs to degrade soluble antigen, without affecting their ability to induce antigen-specific CD4(+) T-cell proliferation. Co-localization studies between PS particles and lysosomes using laser scanning confocal microscopy detected a significantly higher frequency of co-localized 20 nm particles as compared with their 1,000 nm counterparts. Neither size of PS particle caused lysosomal leakage, expression of endoplasmic reticulum stress gene markers, or changes in cytokines profiles. CONCLUSION These data indicate that although supposedly inert PS nanoparticles did not induce DC activation or alteration in CD4(+) T-cell stimulating capacity, 20 nm (but not 1,000 nm) PS particles may reduce antigen degradation through interference in the lysosomal compartment. These findings emphasize the importance of performing in-depth analysis of DC function when developing novel approaches for immune modulation with nanoparticles.

Accumulation of FOXP3+T-cells in the tumor microenvironment is associated with an epithelial-mesenchymal-transition-type tumor budding phenotype and is an independent prognostic factor in surgically resected pancreatic ductal adenocarcinoma.

Here we explore the role of the interplay between host immune response and epithelial-mesenchymal-transition (EMT)-Type tumor-budding on the outcome of pancreatic adenocarcinoma (PDAC).CD4+, CD8+, and FOXP3+T-cells as well as iNOS+ (M1) and CD163+- macrophages (M2) were assessed on multipunch tissue-microarrays containing 120 well-characterized PDACs, precursor lesions (PanINs) and corresponding normal tissue. Counts were normalized for the percentage of tumor/spot and associated with the clinico-pathological features, including peritumoral (PTB) and intratumoral (ITB) EMT-Type tumor-budding and outcome.Increased FOXP3+T-cell-counts and CD163-macrophages and decreased CD8+T-cell-counts were observed in PDACs compared with normal tissues and PanINs (p < 0.0001). Increased peritumoral FOXP3+T-cell-counts correlated significantly with venous invasion, distant metastasis, R1-status, high-grade ITB, PTB and independently with reduced survival. Increased intratumoral FOXP3+T-cells correlated with lymphatic invasion, N1-stage, PTB and marginally with adverse outcome. High peritumoral CD163-counts correlated with venous invasion, PTB and ITB. High intratumoral CD163-counts correlated with higher T-stage and PTB.PDAC-microenvironment displays a tumor-favoring immune-cell composition especially in the immediate environment of the tumor-buds that promotes further growth and indicates a close interaction of the immune response with the EMT-process. Increased peritumoral FOXP3+T-cell density is identified as an independent adverse prognostic factor in PDAC. Patients with phenotypically aggressive PDACs may profit from targeted immunotherapy against FOXP3.

Elevated temperature differentially affects virulence, VirB protein accumulation, and T-pilus formation in different Agrobacterium tumefaciens and Agrobacterium vitis strains.

That gene transfer to plant cells is a temperature-sensitive process has been known for more than 50 years. Previous work indicated that this sensitivity results from the inability to assemble a functional T pilus required for T-DNA and protein transfer to recipient cells. The studies reported here extend these observations and more clearly define the molecular basis of this assembly and transfer defect. T-pilus assembly and virulence protein accumulation were monitored in Agrobacterium tumefaciens strain C58 at different temperatures ranging from 20 degrees C to growth-inhibitory 37 degrees C. Incubation at 28 degrees C but not at 26 degrees C strongly inhibited extracellular assembly of the major T-pilus component VirB2 as well as of pilus-associated protein VirB5, and the highest amounts of T pili were detected at 20 degrees C. Analysis of temperature effects on the cell-bound virulence machinery revealed three classes of virulence proteins. Whereas class I proteins (VirB2, VirB7, VirB9, and VirB10) were readily detected at 28 degrees C, class II proteins (VirB1, VirB4, VirB5, VirB6, VirB8, VirB11, VirD2, and VirE2) were only detected after cell growth below 26 degrees C. Significant levels of class III proteins (VirB3 and VirD4) were only detected at 20 degrees C and not at higher temperatures. Shift of virulence-induced agrobacteria from 20 to 28 or 37 degrees C had no immediate effect on cell-bound T pili or on stability of most virulence proteins. However, the temperature shift caused a rapid decrease in the amount of cell-bound VirB3 and VirD4, and VirB4 and VirB11 levels decreased next. To assess whether destabilization of virulence proteins constitutes a general phenomenon, levels of virulence proteins and of extracellular T pili were monitored in different A. tumefaciens and Agrobacterium vitis strains grown at 20 and 28 degrees C. Levels of many virulence proteins were strongly reduced at 28 degrees C compared to 20 degrees C, and T-pilus assembly did not occur in all strains except "temperature-resistant" Ach5 and Chry5. Virulence protein levels correlated well with bacterial virulence at elevated temperature, suggesting that degradation of a limited set of virulence proteins accounts for the temperature sensitivity of gene transfer to plants.

Drosophila valois encodes a divergent WD protein that is required for Vasa localization and Oskar protein accumulation

valois (vls) was identified as a posterior group gene in the initial screens for Drosophila maternal-effect lethal mutations. Despite its early genetic identification, it has not been characterized at the molecular level until now. We show that vls encodes a divergent WD domain protein and that the three available EMS-induced point mutations cause premature stop codons in the vls ORF. We have generated a null allele that has a stronger phenotype than the EMS mutants. The vlsnull mutant shows that vls+ is required for high levels of Oskar protein to accumulate during oogenesis, for normal posterior localization of Oskar in later stages of oogenesis and for posterior localization of the Vasa protein during the entire process of pole plasm assembly. There is no evidence for vls being dependent on an upstream factor of the posterior pathway, suggesting that Valois protein (Vls) instead acts as a co-factor in the process. Based on the structure of Vls, the function of similar proteins in different systems and our phenotypic analysis, it seems likely that vls may promote posterior patterning by facilitating interactions between different molecules.

Practice in lucid dreams enhances performance during wakefulness

Motor practice in lucid dreams is a form of mental rehearsal where the dreamer can con-sciously rehearse motor skills in the dream state while being physically asleep (Erlacher, Stumbrys & Schredl, 2011). A previous pilot study showed that practice in lucid dreams can improve subsequent performance (Erlacher & Schredl, 2010). This study aimed to replicated those findings with a different (serial reaction) task (finger-tapping; e.g. Walker et al., 2002) and compare the effectiveness of lucid dream practice not only to physical but also to mental practice in wakefulness.

Epigallocatechin gallate (EGCG) affects glucose metabolism and enhances fitness and life span in Drosophila melanogaster

In this study, we tested whether a standardized epigallocatechin-3-gallate (EGCG) rich green tea extract (comprising > 90% EGCG) affects fitness and lifespan as well as parameters of glucose metabolism and energy homeostasis in the fruit fly, Drosophila melanogaster. Following the application of the green tea extract a significant increase in the mean lifespan (+ 3.3 days) and the 50% survival (+ 4.3 days) as well as improved fitness was detected. These effects went along an increased expression of Spargel, the homolog of mammalian PGC1α, which has been reported to affect lifespan in flies. Intriguingly, in flies, treatment with the green tea extract decreased glucose concentrations, which were accompanied by an inhibition of α-amylase and α-glucosidase activity. Computational docking analysis proved the potential of EGCG to dock into the substrate binding pocket of α-amylase and to a greater extent into α-glucosidase. Furthermore, we demonstrate that EGCG downregulates insulin-like peptide 5 and phosphoenolpyruvate carboxykinase, major regulators of glucose metabolism, as well as the Drosophila homolog of leptin, unpaired 2. We propose that a decrease in glucose metabolism in connection with an upregulated expression of Spargel contribute to the better fitness and the extended lifespan in EGCG-treated flies.

Silencing OsHI-LOX makes rice more susceptible to chewing herbivores, but enhances resistance to a phloem feeder

The jasmonic acid (JA) pathway plays a central role in plant defense responses against insects. Some phloem-feeding insects also induce the salicylic acid (SA) pathway, thereby suppressing the plant’s JA response. These phenomena have been well studied in dicotyledonous plants, but little is known about them in monocotyledons. We cloned a chloroplast-localized type 2 13-lipoxygenase gene of rice, OsHI-LOX, whose transcripts were up-regulated in response to feeding by the rice striped stem borer (SSB) Chilo suppressalis and the rice brown planthopper (BPH) Niaparvata lugens, as well as by mechanical wounding and treatment with JA. Antisense expression of OsHI-LOX (as-lox) reduced SSB- or BPH-induced JA and trypsin protease inhibitor (TrypPI) levels, improved the larval performance of SBB as well as that of the rice leaf folder (LF) Cnaphalocrocis medinalis, and increased the damage caused by SSB and LF larvae. In contrast, BPH, a phloem-feeding herbivore, showed a preference for settling and ovipositing on WT plants, on which they consumed more and survived better than on as-lox plants. The enhanced resistance of as-lox plants to BPH infestation correlated with higher levels of BPH-induced H2O2 and SA, as well as with increased hypersensitive response-like cell death. These results imply that OsHI-LOX is involved in herbivore-induced JA biosynthesis, and plays contrasting roles in controlling rice resistance to chewing and phloem-feeding herbivores. The observation that suppression of JA activity results in increased resistance to an insect indicates that revision of the generalized plant defense models in monocotyledons is required, and may help develop novel strategies to protect rice against insect pests.

Activation of RARα induces autophagy in SKBR3 breast cancer cells and depletion of key autophagy genes enhances ATRA toxicity

All-trans retinoic acid (ATRA), a pan-retinoic acid receptor (RAR) agonist, is, along with other retinoids, a promising therapeutic agent for the treatment of a variety of solid tumors. On the one hand, preclinical studies have shown promising anticancer effects of ATRA in breast cancer; on the other hand, resistances occurred. Autophagy is a cellular recycling process that allows the degradation of bulk cellular contents. Tumor cells may take advantage of autophagy to cope with stress caused by anticancer drugs. We therefore wondered if autophagy is activated by ATRA in mammary tumor cells and if modulation of autophagy might be a potential novel treatment strategy. Indeed, ATRA induces autophagic flux in ATRA-sensitive but not in ATRA-resistant human breast cancer cells. Moreover, using different RAR agonists as well as RARα-knockdown breast cancer cells, we demonstrate that autophagy is dependent on RARα activation. Interestingly, inhibition of autophagy in breast cancer cells by either genetic or pharmacological approaches resulted in significantly increased apoptosis under ATRA treatment and attenuated epithelial differentiation. In summary, our findings demonstrate that ATRA-induced autophagy is mediated by RARα in breast cancer cells. Furthermore, inhibition of autophagy results in enhanced apoptosis. This points to a potential novel treatment strategy for a selected group of breast cancer patients where ATRA and autophagy inhibitors are applied simultaneously.

Accumulation rates and predominant atmospheric sources of natural and anthropogenic Hg and Pb on the Faroe Islands

A monolith representing 5420 14C yr of peat accumulation was collected from a blanket bog at Myrarnar, Faroe Islands. The maximum Hg concentration (498 ng/g at a depth of 4.5 cm) coincides with the maximum concentration of anthropogenic Pb (111 μg/g). Age dating of recent peat accumulation using 210Pb (CRS model) shows that the maxima in Hg and Pb concentrations occur at AD 1954 ± 2. These results, combined with the isotopic composition of Pb in that sample (206Pb/207Pb = 1.1720 ± 0.0017), suggest that coal burning was the dominant source of both elements. From the onset of peat accumulation (ca. 4286 BC) until AD 1385, the ratios Hg/Br and Hg/Se were constant (2.2 ± 0.5 × 10-4 and 8.5 ± 1.8 × 10-3, respectively). Since then, Hg/Br and Hg/Se values have increased, also reaching their maxima in AD 1954. The age date of the maximum concentrations of anthropogenic Hg and Pb in the Faroe Islands is consistent with a previous study of peat cores from Greenland and Denmark (dated using the atmospheric bomb pulse curve of 14C), which showed maximum concentrations in AD 1953. The average rate of atmospheric Hg accumulation from 1520 BC to AD 1385 was 1.27 ± 0.38 μg/m2/yr. The Br and Se concentrations and the background Hg/Br and Hg/Se ratios were used to calculate the average rate of natural Hg accumulation for the same period, 1.32 ± 0.36 μg/m2/yr and 1.34 ± 0.29 μg/m2/yr, respectively. These fluxes are similar to the preanthropogenic rates obtained using peat cores from Switzerland, southern Greenland, southern Ontario, Canada, and the northeastern United States. Episodic volcanic emissions and the continual supply of marine aerosols to the Faroe Islands, therefore, have not contributed significantly to the Hg inventory or the Hg accumulation rates, relative to these other areas. The maximum rate of Hg accumulation was 34 μg/m2/yr. The greatest fluxes of anthropogenic Hg accumulation calculated using Br and Se, respectively, were 26 and 31 μg/m2/yr. The rate of atmospheric Hg accumulation in 1998 (16 μg/m2/yr) is comparable to the values recently obtained by atmospheric transport modeling for Denmark, the Faroe Islands, and Greenland.

Effects of pH, light and temperature on (1→3,1→4)-β-glucanase stability in wheat leaves

Changes in (1→3,1→4)-β-D-glucan endohydrolase (EC 3.2.1.73) protein levels were investigated in segments from second leaves of wheat (Triticum aestivum L.). The abundance of the enzyme protein markedly increased when leaf segments were incubated in the dark whereas the enzyme rapidly disappeared when dark-incubated segments were illuminated or fed with sucrose. Addition of cycloheximide (CHI) to the incubation medium led to the disappearance of previously synthesized (1→3,1→4)-β-glucanase and suppressed the dark-induced accumulation indicating that the enzyme was rather unstable. The degradation of (1→3,1→4)-β-glucanase was analyzed without the interference of de-novo synthesis in intercellular washing fluid (IWF). The loss of the enzyme protein during incubation of IWF (containing naturally present peptide hydrolases) indicated that the stability increased from pH 4 to pH 7 and that an increase in the temperature from 25 to 35 °C considerably decreased the stability. Chelating divalent cations in the IWF with o-phenanthroline also resulted in a lowered stability of the enzyme. A strong temperature effect in the range from 25 to 35 °C was also observed in wheat leaf segments. Diurnal changes in (1→3,1→4)-β-glucanase activity were followed in intact second leaves from young wheat plants. At the end of the dark period, the activity was high but constantly decreased during the light phase and remained low if the light period was extended. Activity returned to the initial level during a 10-h dark phase. During a diurnal cycle, changes in (1→3,1→4)-β-glucanase activity were associated with reciprocal changes in soluble carbohydrates. The results suggest that the synthesis and the proteolytic degradation of an apoplastic enzyme may rapidly respond to changing environmental conditions.

Oral Lutein Supplementation Enhances Macular Pigment Density and Contrast Sensitivity but Not in Combination With Polyunsaturated Fatty Acids.

Purpose It has been shown that lutein and zeaxanthin accumulate in the macula where they enhance contrast sensitivity and may reduce the risk of progression to advanced age-related macular degeneration (AMD). Furthermore, omega-3 long-chain polyunsaturated fatty acids (PUFA) might further reduce this risk. However, controversy exists regarding whether PUFA may reduce the bioavailability of lutein. Methods This was a prospective 12-month, randomized, open label study evaluating the effect of supplementation with lutein, other antioxidants, and minerals on contrast sensitivity (CS) and macular pigment optical density (MPOD) in patients with age-related maculopathy. A total of 79 patients were randomized to either lutein (10 mg) and antioxidant supplement or lutein and antioxidant supplement in combination with PUFA. Patients received supplementation for a period of 6 months and were followed for a total of 12 months. Results Serum lutein and zeaxanthin increased significantly by the first follow-up visit at 1 month, and remained elevated throughout the intervention period of 6 months in the lutein-only group but not in the lutein+PUFA group. Macular pigment optical density and CS increased significantly in the lutein-only group (P < 0.005) but not in the lutein+PUFA group (P = 0.059) compared to baseline. Best-corrected visual acuity remained unchanged during the entire study period in both groups. Conclusions Addition of PUFA may reduce the bioavailability of lutein and therefore lessen the beneficial effect on macular pigment and CS. This needs to be considered when prescribing lutein supplements to patients with low lutein levels. (ClinicalTrials.gov number, NCT00563979.).