Dupilumab-associated ocular adverse events are predicted by low tear break-up time and correlate with high IL-33 tear concentrations in patients with atopic dermatitis.

Dupilumab, blocking IL-4 and IL-13 signals, improves atopic dermatitis and Quality of Life but might be also associated with the occurrence of ocular adverse events (OAEs). The main objective of our prospective study was to characterize the cytokine and chemokine profile in the tear fluid of dupilumab-treated patients with moderate-to- severe atopic dermatitis and to identify biomarkers predicting the occurrence of ocular adverse events. Patients with moderate-to-severe AD underwent dermatological and ophthalmological evaluation at the baseline (T0) and week 16 or at the time of an eventual ocular adverse events (T1). A multiplex immunoassay measuring multiple cytokines and chemokines in the tear fluid extracted during ocular examination at both T0 and T1 was performed. Thirty-nine patients with moderate-to-severe AD and treated with dupilumab were included in the study. Baseline tear fluid levels revealed a significantly higher concentration of type 2 cytokines and chemokines in AD patients than healthy controls. The occurrence of ocular adverse events during dupilumab therapy was associated with a significant increase of IL-33 tear fluid levels and a significantly lower tear break-up time, this latter also identified as predictive factor. Our findings suggest that the ophthalmological examination should be considered a valid support to identify patients at risk of developing OAEs and to provide their appropriate management.

Adipocytes sustain pancreatic cancer progression through a non-canonical WNT paracrine network inducing ROR2 nuclear shuttling.

BACKGROUND: Solid epidemiological evidences connect obesity with incidence, stage and survival in pancreatic cancer. However, the underlying mechanistic basis linking adipocytes to pancreatic cancer progression remain largely elusive. We hypothesized that factors secreted by adipocytes could be responsible for epithelial-to-mesenchymal transition (EMT) induction and, in turn, a more aggressive phenotype in models of pancreatic preneoplastic lesions. METHODS: We studied the role of factors secreted by two adipogenic model systems from primary human bone marrow stromal cells (hBMSCs) in an in vitro experimental cell transformation model system of human pancreatic ductal epithelial (HPDE) cell stably expressing activated KRAS (HPDE/KRAS),Results:We measured a significant induction of EMT and aggressiveness in HPDE and HPDE/KRAS cell lines when cultured with medium conditioned by fully differentiated adipocytes (ADIPOCM) if compared with the same cells cultured with medium conditioned by hBMSC (hBMSCCM) from two different healthy donors. Several genes coding for soluble modulators of the non-canonical WNT signaling pathway, including FRZB, SFRP2, RSPO1, WNT5A and 5B were significantly overexpressed in fully differentiated adipocytes than in their respective in hBMSC. ADIPOCM induced the overexpression and the nuclear translocation of the Frizzled family member receptor tyrosine kinase-like orphan receptor (Ror) 2 in HPDE and HPDE/KRAS cells. Vantictumab, an anti-Frizzled monoclonal antibody, reduced ROR2 nuclear translocation and in turn the EMT and aggressiveness in HPDE and HPDE/KRAS cells. CONCLUSIONS: We demonstrated that adipocytes could induce EMT and aggressiveness in models of pancreatic preneoplastic lesions by orchestrating a complex paracrine signaling of soluble modulators of the non-canonical WNT signaling pathway that determine, in turn, the activation and nuclear translocation of ROR2. This signaling pathway could represent a novel target for pancreatic cancer chemoprevention. Most importantly, these factors could serve as novel biomarkers to select a risk population among obese subjects for screening and, thus, early diagnosis of pancreatic cancer.

TAK1-regulated expression of BIRC3 predicts resistance to preoperative chemoradiotherapy in oesophageal adenocarcinoma patients.

BACKGROUND: About 20% of resectable oesophageal carcinoma is resistant to preoperative chemoradiotherapy. Here we hypothesised that the expression of the antiapoptotic gene Baculoviral inhibitor of apoptosis repeat containing (BIRC)3 induced by the transforming growth factor ß activated kinase 1 (TAK1) might be responsible for the resistance to the proapoptotic effect of chemoradiotherapy in oesophageal carcinoma. METHODS: TAK1 kinase activity was inhibited in FLO-1 and KYAE-1 oesophageal adenocarcinoma cells using (5Z)-7-oxozeaenol. The BIRC3 mRNA expression was measured by qRT-PCR in 65 pretreatment frozen biopsies from patients receiving preoperatively docetaxel, cisplatin, 5-fluorouracil, and concurrent radiotherapy. Receiver operator characteristic (ROC) analyses were performed to determine the performance of BIRC3 expression levels in distinguishing patients with sensitive or resistant carcinoma. RESULTS: In vitro, (5Z)-7-oxozeaenol significantly reduced BIRC3 expression in FLO-1 and KYAE-1 cells. Exposure to chemotherapeutic agents or radiotherapy plus (5Z)-7-oxozeaenol resulted in a strong synergistic antiapoptotic effect. In patients, median expression of BIRC3 was significantly (P<0.0001) higher in adenocarcinoma than in the more sensitive squamous cell carcinoma subtype. The BIRC3 expression significantly discriminated patients with sensitive or resistant adenocarcinoma (AUC-ROC=0.7773 and 0.8074 by size-based pathological response or Mandard's tumour regression grade classifications, respectively). CONCLUSIONS: The BIRC3 expression might be a valid biomarker for predicting patients with oesophageal adenocarcinoma that could most likely benefit from preoperative chemoradiotherapy.

An Optimized Energy-Harvesting Transmission Scheme for Diffusion-Based Molecular Communications.

In this paper, we propose an optimized transmission scheme with energy-harvesting for a diffusion-based molecular communication system composed by nano-devices fed by piezoelectric nanogenerators. To this end, we firstly derive a system model that analytically describes the mean and the variance of the aggregated noise at the output of the receiver and the achievable Bit Error Rate. Then, we formulate an optimization problem that minimizes an objective function defined as a linear combination of the probability that the voltage across the ultra-nanocapacitor of the transmitter goes under a target value and the number of enqueued packets. We solve this problem by considering the actual energy budget, a target Bit Error Rate, and the need to achieve the simplicity of the transmitter as constraints. Finally, we use computer simulations to validate the formulated analytical models and demonstrate the unique ability of the proposed approach to guarantee BER = 5% and BER = 10% for communication distances up to 47 µm and 50 µm , respectively, while registering better results against baseline scenarios.

Vorinostat synergises with capecitabine through upregulation of thymidine phosphorylase.

BACKGROUND: Potentiation of anticancer activity of capecitabine is required to improve its therapeutic index. In colorectal cancer (CRC) cells, we evaluated whether the histone deacetylase-inhibitor vorinostat may induce synergistic antitumour effects in combination with capecitabine by modulating the expression of thymidine phosphorylase (TP), a key enzyme in the conversion of capecitabine to 5-florouracil (5-FU), and thymidylate synthase (TS), the target of 5-FU. METHODS: Expression of TP and TS was measured by real-time PCR, western blotting and immunohistochemistry. Knockdown of TP was performed by specific small interfering RNA. Antitumour activity of vorinostat was assessed in vitro in combination with the capecitabine active metabolite deoxy-5-fluorouridine (5'-DFUR) according to the Chou and Talay method and by evaluating apoptosis as well as in xenografts-bearing nude mice in combination with capecitabine. RESULTS: Vorinostat induced both in vitro and in vivo upregulation of TP as well as downregulation of TS in cancer cells, but not in ex vivo treated peripheral blood lymphocytes. Combined treatment with vorinostat and 5'-DFUR resulted in a synergistic antiproliferative effect and increased apoptotic cell death in vitro. This latter effect was impaired in cells where TP was knocked. In vivo, vorinostat plus capecitabine potently inhibited tumour growth, increased apoptosis and prolonged survival compared with control or single-agent treatments. CONCLUSIONS: Overall, this study suggests that the combination of vorinostat and capecitabine is an innovative antitumour strategy and warrants further clinical evaluation for the treatment of CRC.

Application of response surface methodology (RSM) for the optimization of supercritical CO2 extraction of oil from patè olive cake: Yield, content of bioactive molecules and biological effects in vivo.

The two-phase technology for olive oil extraction generates large amounts of patè olive cake (POC), a by-product that is rich in bioactive health-promoting compounds. Here, response surface methodology (RSM) was used to maximize supercritical-CO2 oil extraction from POC, while minimizing operative temperature, pressure and time. Under the optimal parameters (40.2 °C, 43.8 MPa and time 30 min), the oil yield was 14.5 g·100 g-1 dw (~65% of the total oil content of the freeze-dried POC matrix), as predicted by RSM. Compared with freeze-dried POC, the oil contained more phytosterols (13-fold), tocopherols (6-fold) and squalene (8-fold) and was a good source of pentacyclic triterpenes. When the biological effects of POC oil intake (20-40 µL·die-1) were evaluated in the livers of BALB/c mice, no significant influence on redox homeostasis was observed. Notably, a decline in liver triglycerides alongside increased activities of NAD(P)H:Quinone Oxidoreductase 1, Carnitine Palmitoyl-CoA Transferase and mitochondrial respiratory complexes suggested a potential beneficial effect on liver fatty acid oxidation.

Synthesis of cell-wall glycoproteins and their characterization in oat coleoptiles.

D-[U-14C]Glucosamine was rapidly taken up by oat coleoptile segments and metabolized to radioactive UDP-N-acetylglucosamine, which acted as specific glycosyl donor for the synthesis of glycolipids and cytosolic, membrane-bound and cell-wall glycoproteins. Cell-wall glycoproteins were solubilized from the walls by either cell-wall-degrading enzymes or chemical extractants. The solubilized cell-wall glycoproteins in the presence of peptide N-glycosidase F released oligosaccharide chains higher than seven glycosidic residues. The combined action of peptide N-glycosidase F and N-acetyl-beta-D-glucosaminidase on cell-wall glycoproteins indicated the presence of N-acetylglucosamine residues beta-1,2-linked to mannose. Less than 9% of the radioactive oligosaccharide chains was released from the solubilized cell-wall glycoproteins when treated with 0.5 M NaOH at 20 degrees, whereas more than 45% of the radioactivity was released in the presence of 1 M NaOH at 50 degrees. The high hydrolytic sensitivity of cell-wall glycoproteins to peptide N-glycosidase F, N-acetyl-beta-D-glucosaminidase and NaOH at 50 degrees indicated that most N-acetylglucosamine residues were incorporated into N-linked cell-wall glycoproteins. Further evidence of this was obtained by the use of inhibitors of biosynthesis and processing of N-linked glycoproteins.

Acquired resistance to zoledronic acid and the parallel acquisition of an aggressive phenotype are mediated by p38-MAP kinase activation in prostate cancer cells.

The nitrogen-containing bisphosphonates (N-BP) zoledronic acid (ZOL) inhibits osteoclast-mediated bone resorption, and it is used to prevent skeletal complications from bone metastases. ZOL has also demonstrated anticancer activities in preclinical models and, recently, in cancer patients, highlighting the interest in determining eventual mechanisms of resistance against this agent. In our study, we selected and characterised a resistant subline of prostate cancer (PCa) cells to better understand the mechanisms, by which tumour cells can escape the antitumour effect of ZOL. DU145R80-resistant cells were selected in about 5 months using stepwise increasing concentrations of ZOL from DU145 parental cells. DU145R80 cells showed a resistance index value of 5.5 and cross-resistance to another N-BP, pamidronate, but not to the non-nitrogen containing BP clodronate. Notably, compared with DU145 parental cells, DU145R80 developed resistance to apoptosis and anoikis, as well as overexpressed the anti-apoptotic protein Bcl-2 and oncoprotein c-Myc. Moreover, DU145R80 cells underwent epithelial to mesenchymal transition (EMT) and showed increased expression of the metalloproteases MMP-2/9, as well as increased invading capability. Interestingly, compared with DU145, DU145R80 cells also increased the gene expression and protein secretion of VEGF and the cytokines Eotaxin-1 and IL-12. At the molecular level, DU145R80 cells showed strong activation of the p38-MAPK-dependent survival pathway compared with parental sensitive cells. Moreover, using the p38-inhibitor SB203580, we completely reversed the resistance to ZOL, as well as EMT marker expression and invasion. Furthermore, SB203580 treatment reduced the expression of VEGF, Eotaxin-1, IL-12, MMP-9, Bcl-2 and c-Myc. Thus, for the first time, we demonstrate that the p38-MAPK pathway can be activated under continuous extensive exposure to ZOL in PCa cells and that the p38-MAPK pathway has a critical role in the induction of resistance, as well as in the acquisition of a more aggressive and invasive phenotype.

A membrane-bound enzyme complex synthesising glucan and glucomannan in pine tissues.

Particulate membrane preparations isolated from cambial cells and differentiating and differentiated xylem cells of pine (Pinus sylvestris L.) trees synthesised [(14)C]glucans using either guanosine 5'-diphosphate (GDP)-D-[U-(14)C]glucose or uridine 5'-diphosphate (UDP)-D-[U-(14)C]glucose as glycosyl donors. Although these glucans had ß-(1→3) and ß-(1→4) linkages in an approximate ratio 1:1, the distribution of the linkages in the glucan synthesised from GDP-D-glucose was different from that synthesised from UDP-D-glucose. The synthesis of the mixed ß-(1→3) and ß-(1→4) glucan from GDP-D-[U-(14)C]glucose was changed to that of ß-(1→4) glucomannan in the presence of increasing concentrations of GDP-D-mannose. The glucan formed from UDP-D-[U-(14)C]glucose was not affected by any concentration of GDP-D-mannose. The membrane preparations epimerized GDP-D-glucose to GDP-D-mannose; however, the low amount of GDP-D-mannose formed was not incorporated into the polymer becaus the affinity of the synthase for GDP-D-glucose was much greater than that for GDP-D-mannose. The glucan formed from GDP-D-glucose and the glucomannan formed from GDP-D-glucose together with GDP-D-mannose were characterized. The apparent K m and V max of the glucan synthase for GDP-D-glucose were 6.38 µM and 5.08 µM·min(-1), respectively. No lipid intermediates were detected during the synthesis of either glucan or glucomannan. The results indicated that an enzyme complex for the formation of the glucomannan was bound to the membrane.

Glucomannan synthesis in pea epicotyls: the mannose and glucose transferases.

Membrane fractions and digitonin-solubilized enzymes prepared from stem segments isolated from the third internode of etiolated pea seedlings (Pisum sativum L. cv. Alaska) catalyzed the synthesis of a beta-1,4-[14C]mannan from GDP-D-[U-14C]-mannose, a mixed beta-1,3- and beta-1,4-[14C]glucan from GDP-D-[U-14C]-glucose and a beta-1,4-[14C]-glucomannan from both GDP-D-[U-14C]mannose and GDP-D-[U-14C]glucose. The kinetics of the membrane-bound and soluble mannan and glucan synthases were determined. The effects of ions, chelators, inhibitors of lipid-linked saccharides, polyamines, polyols, nucleotides, nucleoside-diphosphate sugars, acetyl-CoA, group-specific chemical probes, phospholipases and detergents on the membrane-bound mannan and glucan synthases were investigated. The beta-glucan synthase had different properties from other preparations which bring about the synthesis of beta-1,3-glucans (callose) and mixed beta-1,3- and beta-1,4- glucans and which use UDP-D-glucose as substrate. It also differed from xyloglucan synthase because in the presence of several concentrations of UDP-D-xylose in addition to GDP-D-glucose no xyloglucan was formed. Using either the membrane-bound or the soluble mannan synthase, GDP-D-glucose acted competitively in the presence of GDP-D-mannose to inhibit the incorporation of mannose into the polymer. This was not due to an inhibition of the transferase activity but was a result of the incorporation of glucose residues from GDP-D-glucose into a glucomannan. The kinetics and the composition of the synthesized glucomannan depended on the ratio of the concentrations of GDP-D-glucose and GDP-D-mannose that were available. Our data indicated that a single enzyme has an active centre that can use both GDP-D-mannose and GDP-D-glucose to bring about the synthesis of the heteropolysaccharide.

Glucomannan-synthase activity in differentiating cells of Pinus sylvestris L.

Particulate membrane preparations have been isolated from cambial cells, and from differentiating and differentiated xylem cells of the main stem of pine trees. These preparations synthesise a ß1→4 glucomannan from guanosine 5'-diphosphate-mannose. The polysaccharide and the synthase have been characterized and the Km and Vmax for the synthase determined as 85 µM and 52.9 µM·min(-1), respectively. The enzymic activity was inhibited by the addition of guanosine 5'-diphosphate-D-glucose so that the presence of an epimerase on the particulate fraction in conjunction with the synthase probably allowed the heteropolymer to be formed with the optimal ratio of the concentrations of the nucleoside-diphosphate sugar donors. No evidence for a polyprenyl-phosphate derivative as an intermediate during the polymer synthesis was obtained. Part of the control mechanism for the deposition of the large amounts of the glucomannan during the secondary thickening of the tracheids of the vascular system is by an increase in the amount of synthase activity at the endomembrane system of the cells. This probably occurs by an increase in the amount of enzyme which is modulated by gene regulation during differentiation.