In recent years the introduction of target therapies with BRAF and MEK inhibitors (MAPKi) and of immunotherapy with anti-CTLA-4 and anti-PD-1 monoclonal antibodies have dramatically improved survival of metastatic melanoma patients. Despite these changes drug resistance remains a major hurdle. Several mechanisms are at the basis of drug resistance. Particular attention has been devoted over the last years to unravel mechanisms at the basis of adaptive/non genetic resistance occurring in BRAF mutated melanomas upon treatment with to MAPKi. In this paper we focus on the involvement of activation of ErbB3 receptor following early exposure of melanoma cells to BRAF or MEK inhibitors, and the following induction of PI3K/AKT pathway. Although different mechanisms have been invoked in the past at the basis of this activation we show here with a combination of approaches that autocrine production of neuregulin by melanoma cells is a major factor responsible for ErbB3 phosphorylation and downstream AKT activation. Interestingly the kinetic of neuregulin production and of the ensuing ErbB3 phosphorylation is different in different melanoma cell lines which underscores the high degree of tumor heterogeneity. Moreover, heterogeneity is further highlighted by the evidence that in different cell lines neuregulin upregulation can occur at the transcriptional or at the post-transcritpional level. Finally we complement our study by showing with a liquid biopsy assay that circulating tumor cells (CTCs) from melanoma patients undergo upregulation of ErbB3 phosphorylation in vivo shortly after initiation of therapy.

The aim of this split-mouth study is to compare the results of 24 months' clinical performance of primary molar Class I restorations with a nano-ceramic composite, Ceram•X mono (Dentsply) with a flowable bulk-fill material regular viscosity, SDR (Dentsply).

Daidzein (DZ) is a polyphenolic compound belonging to Biopharmaceutical Classification System class IV, which shows that it may have limited therapeutic effects due to its low solubility and poor bioavailability. This study aimed to obtain high-purity DZ and prepare and characterize different types of solid dispersions (SDs) in order to enhance aqueous solubility and bioavailability. Excipients were investigated in order to manufacture different types of solid dispersions (SDs). Second-generation solid dispersions (SG), third-generation solid dispersions (TG), and second- and third-generation pH-modulated solid dispersions (SD and TG pHM-SD) were produced via spray drying. The SDs were characterized and tested for in vitro DZ release and oral bioavailability. SDs have shown increased aqueous solubility and in vitro release rate. Solid-state characterization showed that DZ was in an amorphous state in most of the formulations. The enhanced aqueous solubility of TG-pHM SD was reflected by an increase in oral bioavailability, which significantly increased the maximum plasma concentration approximately 20-fold and decreased the time to reach the maximum plasma concentration. The production of pHM SDs that contain DZ via spray drying is a simple and effective approach for oral drug delivery, which has the potential to greatly reduce the dose and enhance therapeutics effects.

Pain is recognized as one of the main symptoms in knee osteoarthritis and is the main reason why patients seek medical attention. Scoparia dulcis has been popularly used to relieve discomfort caused by various painful conditions. The objective of the study is to evaluate the analgesic and anti-inflammatory effect of the crude extract of S. dulcis, in an experimental model of osteoarthritis. The experiment was performed with Wistar rats divided into 4 groups with 5 animals each: healthy, saline, crude extract, and meloxicam groups. Knee osteoarthritis was induced by intra-articular injection of sodium mono-iodoacetate. First, clinical parameters of pain were assessed at days 0, 5, 10, 15, and 20 after induction. Second, the potential cyclooxygenase inhibition was evaluated, and the cytokines of the synovial fluid were quantified. An in silico test and Molecular Docking tests were performed. A histopathological evaluation was made on articular cartilage with safranin O staining. The results showed that a 15-day treatment with crude extract reduced edema, spontaneous pain, peripheral nociceptive activity, and proinflammatory cytokines in the synovial fluid. The highest inhibition of cyclooxygenase 2 in the crude extract occurred at 50 µg/mL. The crude extract of S. dulcis presents therapeutic potential for the treatment of osteoarthritis due to its anti-inflammatory and anti-nociceptive action.

Coumarins are widely present in a variety of plants and have a variety of pharmacological activities. In this study, we isolated a coumarin compound from Microsorium fortunei (Moore) Ching; the compound was identified as esculetin by hydrogen and carbon spectroscopy. Its anti-hepatitis B virus (HBV) activity was investigated in vitro and in vivo. In the human hepatocellular liver carcinoma 2.2.15 cell line (HepG2.2.15) transfected with HBV, esculetin effecting inhibited the expression of the HBV antigens and HBV DNA in vitro. Esculetin inhibited the expression of Hepatitis B virus X (HBx) protein in a dose-dependent manner. In the ducklings infected with duck hepatitis B virus (DHBV), the levels of DHBV DNA, duck hepatitis B surface antigen (DHBsAg), duck hepatitis B e-antigen (DHBeAg), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) decreased significantly after esculetin treatment. Summing up the above, the results suggest that esculetin efficiently inhibits HBV replication both in vitro and in vivo, which provides an opportunity for further development of esculetin as antiviral drug.

The olive fruit fly, Bactrocera oleae, is considered the main olive pest worldwide, and has been the target of biological control programmes through the release of the braconid parasitoid Psyttalia concolor. Laboratory tests were performed to evaluate the influence of distance from the host on parasitisation, placing larvae of the substitute host Ceratitis capitata at seven distances (0, 0.5, 1, 1.5, 2, 2.5, 3 mm) and four different time periods (7, 15, 30, 60 min). Moreover, field collected olives of Ogliarola Barese cultivar infested by B. oleae were exposed to P. concolor females to confirm its ability to parasitise B. oleae in small olives. Psyttalia concolor oviposition was inhibited at 2.5 and 3 mm due to the ovipositor length of the parasitoid females (2.7 mm). Hosts were easily parasitised at distances between 0 and 1.5 mm. The thin fruit pulp (up to 3.5 mm) of field collected olives allowed the parasitisation to occur also in mature fruits. At the best combination distance/time (0 mm, 30 min), tests performed with different larvae/parasitoid female ratio showed an increasing emergence of P. concolor (from 20% to 57%) with larvae/parasitoid ratio increasing from 0.11 to 0.74. The results of the present study might optimise the mass rearing of P. concolor, through a proper setting of its parameters, such as the host/parasitoid ratio, exposure distances, and interaction time.

In this work, composites of high density polyethylene (HDPE) with chitosan were prepared by melt compounding in a laboratory internal mixer. Maleic anhydride grafted HDPE (PE-g-MA) in a concentration up to 25 phr was used as a compatibilizer to enhance the dispersing effect of chitosan in the HDPE matrix. The degree of crystallinity was investigated by X-ray diffraction (XRD) and the thermal properties were analyzed by differential scanning calorimetry (DSC) and thermogravimetry (TG). The morphology was investigated by optical microscopy (OM) and scanning electron microscopy (SEM). The integrity of composites was evaluated by mechanical properties and antibacterial properties were assessed against Escherichia coli (DH5a). Neither crystallinity nor HDPE's melting parameters changed upon addition of chitosan and PE-g-MA. Chitosan aggregates were observed, which were dispersed upon addition of PE-g-MA, which also provided improved mechanical performance. Chitosan significantly improved the bacteriostatic effect of HDPE compounds preventing bacteria to colonize thus reducing the number of viable colony-forming units (CFU). This study revealed that HDPE/chitosan composites could be obtained by melt compounding, at lower cost and additionally having antibacterial properties, which might provide a new formulation option for developing antimicrobial film for food packaging.

The FOXO1 transcription factor plays a central role in the proliferation and survival of B cells at several stages of differentiation. B cell malignancies, with exception of classical Hodgkin lymphoma, maintain expression of FOXO1 at levels characteristic for their non-malignant counterparts. Extensive expression profiling had revealed that Burkitt lymphoma (BL) show many characteristics of the dark zone (DZ) germinal center (GC) B cell program. Here we show that FOXO1 knockdown inhibits proliferation of human BL cell lines. The anti-proliferative effect of the FOXO1 knockdown is associated with the repression of the DZ B cell program including expression of MYB, CCND3, RAG2, BACH2, and CXCR4. In addition, the induction of signaling pathways of the light zone (LZ) program like NF-κB and PI3K-AKT was observed. Using a rescue experiment we identified downregulation of the proto-oncogene MYB as a critical factor contributing to the antiproliferative effect of FOXO1 knockdown. In an attempt to estimate the feasibility of pharmacological FOXO1 repression, we found that the small molecular weight FOXO1 inhibitor AS1842856 induces cell death and growth arrest in BL cell lines at low concentrations. Interestingly, we found that overactivation of FOXO1 also induces growth inhibition in BL cell lines, indicating the importance of a tight regulation of FOXO1 activity in BL.

An increased rate of cellular proliferation is a hallmark of cancer and may be accompanied by an increase in ribosome biogenesis and dysregulation in rRNA synthesis. In this regard, CX-5461 has been developed as a novel RNA polymerase I inhibitor and is currently in Phase I/II clinical trials for solid and hematological malignancies. In the present study, interactions between CX-5461 and single-dose X-ray exposure were assessed using isobologram analysis using MTS assay and drug-induced cell death was assessed using flow cytometric, confocal microscopy and Western blot analysis. Combination treatments involving CX-5461 and single-dose X-ray exposure highlighted increased effectiveness compared to individual treatment alone in the CaSki cervical cancer line, with marked synergistic interaction occurring within the low-drug (50 nM) and low-dose radiation range (2-6 Gy). Cell lines challenged with CX-5461 demonstrated the presence of DNA damage, induction of apoptosis, autophagy and senescence alongside high percentages of G2/M cell cycle arrest. In addition, we report preferential sensitivity of ovarian cancer cells with BRCA2 mutation to this novel agent. Taken together, CX-5461 displayed a broad spectrum of activity in a panel of solid cancer cell lines with IC50 values ranging from 35 nM to >1 µM. The work described herein identifies the synergistic effects of CX-5461 in combination with X-rays in solid cancers and may also aid in the design of clinical trials involving this novel agent.

Second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) triggers Ca2+ release via two-pore channels (TPCs) localized in endolysosomal vesicles. The aim of the present work is to evaluate the role of TPCs in the action of norepinephrine (NE), angiotensin II (AngII), vasopressin (AVP), and 5-hydroxytriptamine (5-HT) on free cytoplasmic calcium concentration ([Ca2+]i) in smooth muscle cells (SMCs) isolated from rat aorta and on aorta contraction. To address this issue, the NAADP structural analogue and inhibitor of TPCs, NED 19, was applied. We have demonstrated a high degree of colocalization of the fluorescent signals of cis-NED 19 and endolysosmal probe LysoTracker in SMCs. Both cis- or trans-NED 19 inhibited the rise of [Ca2+]i in SMCs induced by 100 μM NE by 50-60%. IC50 for cis- and trans-NED 19 were 2.7 and 8.9 μM, respectively. The inhibition by NED 19 stereoisomers of the effects of AngII, AVP, and 5-HT was much weaker. Both forms of NED 19 caused relaxation of aortic rings preconstricted by NE, with relative potency of cis-NED 19 several times higher than that of trans-NED 19. Inhibition by cis-NED 19 of NE-induced contraction was maintained after intensive washing and slowly reversed within an hour of incubation. Cis- and trans-NED 19 did not cause decrease in the force of aorta contraction in response to Ang II and AVP, and only slightly relaxed aorta preconstricted by 5-HT and by KCl. Suppression of TPC1 in SMCs with siRNA caused a 40% decrease in [Ca2+]i in response to NE, whereas siRNA against TPC2 did not change NE calcium signaling. These data suggest that TPC1 is involved in the NE-stimulated [Ca2+]i rise in SMCs. Inhibition of TPC1 activity by NED 19 could be the reason for partial inhibition of aortic rings contraction in response to NE.

The aim of the present study was to establish the best experimental conditions that lead to the extracts richest in polyphenolic compounds obtained from pomace and canes of Vitis vinifera. In this regard, a D-Optimal design of experiments (DoE) method was applied to investigate the extraction process parameters from each of three materials: red pomace (RP), white pomace (WP) and canes (C). The input variables were the extraction temperature and the ethanol ratio and as response, the total polyphenols content (TPC) was determined. A design space was generated for each of the plant materials and the most concentrated polyphenol extracts were obtained using 50% ethanol at a temperature of 80 °C. Further, the phenolic profiles of the concentrated extracts were detected by LC/MS/MS and the results showed that WP extract was richer in polyphenolic compounds, both flavonoid and phenolic acids, followed by the RP and C extracts. The antioxidant assays revealed that WP and RP extracts exhibited a higher antioxidant activity which correlated to the high content of polyphenols. These findings revealed that RP, WP and C, currently considered agricultural wastes from winery, may be valorized as an important source of natural antioxidants.

Connexin-36 (Cx36) electrical synapses strengthen transmission in a calcium/calmodulin (CaM)/calmodulin-dependent kinase II (CaMKII)-dependent manner similar to a mechanism whereby the N-methyl-D-aspartate (NMDA) receptor subunit NR2B facilitates chemical transmission. Since NR2B-microtubule interactions recruit receptors to the cell membrane during plasticity, we hypothesized an analogous modality for Cx36. We determined that Cx36 binding to tubulin at the carboxy-terminal domain was distinct from Cx43 and NR2B by binding a motif overlapping with the CaM and CaMKII binding motifs. Dual patch-clamp recordings demonstrated that pharmacological interference of the cytoskeleton and deleting the binding motif at the Cx36 carboxyl-terminal (CT) reversibly abolished Cx36 plasticity. Mechanistic details of trafficking to the gap-junction plaque (GJP) were probed pharmacologically and through mutational analysis, all of which affected GJP size and formation between cell pairs. Lys279, Ile280, and Lys281 positions were particularly critical. This study demonstrates that tubulin-dependent transport of Cx36 potentiates synaptic strength by delivering channels to GJPs, reinforcing the role of protein transport at chemical and electrical synapses to fine-tune communication between neurons.

Bacterial endophytes, a subset of a plant's microbiota, can facilitate plant growth by a number of different mechanisms. The aims of this study were to assess the diversity and functionality of endophytic bacterial strains from internal root tissues of native legume species grown in two distinct sites in South of Portugal and to evaluate their ability to promote plant growth. Here, 122 endophytic bacterial isolates were obtained from 12 different native legume species. Most of these bacteria possess at least one of the plant growth-promoting features tested in vitro, with indole acetic acid production being the most common feature among the isolates followed by the production of siderophores and inorganic phosphate solubilization. The results of in planta experiments revealed that co-inoculation of chickpea plants with specific endophytic bacteria along with N2-fixing symbionts significantly improved the total biomass of chickpea plants, in particular when these plants were grown under saline conditions. Altogether, this study revealed that Mediterranean native legume species are a reservoir of plant growth-promoting bacteria, that are also tolerant to salinity and to toxic levels of Mn. Thus, these bacterial endophytes are well adapted to common constraints present in soils of this region which constitutes important factors to consider in the development of bacterial inoculants for stressful conditions in the Mediterranean region.

The effector proteins secreted by a pathogen not only promote virulence and infection of the pathogen, but also trigger plant defense response. Therefore, these proteins could be used as important genetic resources for transgenic improvement of plant disease resistance. Magnaporthe oryzae systemic defense trigger 1 (MoSDT1) is an effector protein. In this study, we compared the agronomic traits and blast disease resistance between wild type (WT) and MoSDT1 overexpressing lines in rice. Under control conditions, MoSDT1 transgenic lines increased the number of tillers without affecting kernel morphology. In addition, MoSDT1 transgenic lines conferred improved blast resistance, with significant effects on the activation of callose deposition, reactive oxygen species (ROS) accumulation and cell death. On the one hand, overexpression of MoSDT1 could delay biotrophy-necrotrophy switch through regulating the expression of biotrophy-associated secreted protein 4 (BAS4) and Magnaporthe oryzaecell death inducing protein 1 (MoCDIP1), and activate plant defense response by regulating the expression of Bsr-d1, MYBS1, WRKY45, peroxidase (POD), heat shock protein 90 (HSP90), allenoxide synthase 2 (AOS2), phenylalanine ammonia lyase (PAL), pathogenesis-related protein 1a (PR1a) in rice. On the other hand, overexpression of MoSDT1 could increase the accumulation of some defense-related primary metabolites such as two aromatic amino acids (L-tyrosine and L-tryptohan), 1-aminocyclopropane carboxylic acid, which could be converted to ethylene, vanillic acid and L-saccharopine. Taken together, overexpression of MoSDT1 confers improved rice blast resistance in rice, through modulation of callose deposition, ROS accumulation, the expression of defense-related genes, and the accumulation of some primary metabolites.

Ice-binding proteins (IBPs) are found in many organisms, such as fish and hexapods, plants, and bacteria that need to cope with low temperatures. Ice nucleation and thermal hysteresis are two attributes of IBPs. While ice nucleation is promoted by large proteins, known as ice nucleating proteins, the smaller IBPs, referred to as antifreeze proteins (AFPs), inhibit the growth of ice crystals by up to several degrees below the melting point, resulting in a thermal hysteresis (TH) gap between melting and ice growth. Recently, we showed that the nucleation capacity of two types of IBPs corresponds to their size, in agreement with classical nucleation theory. Here, we expand this finding to additional IBPs that we isolated from snow fleas (the arthropod Collembola), collected in northern Israel. Chemical analyses using circular dichroism and Fourier-transform infrared spectroscopy data suggest that these IBPs have a similar structure to a previously reported snow flea antifreeze protein. Further experiments reveal that the ice-shell purified proteins have hyperactive antifreeze properties, as determined by nanoliter osmometry, and also exhibit low ice-nucleation activity in accordance with their size.

Growing evidence exists for the benefits of adequate infant and young children feeding (IYCF) practices at the weaning stage (≥ 6 months), including optimal growth, building the immune system, cognitive development, healthy food preferences, and reduced mortality and morbidity rates. However, these outcomes are not universally experienced. To ensure that a developing country such as Malawi, where recent studies have shown high rates of food insecurity and malnutrition benefits from adequate IYCF, five nutrient-dense complementary foods (Recipes 1 to 5) were developed. Standardized food processing techniques were used in the preparation and combination of Malawian indigenous food samples. The developed food recipes were assessed for nutrient density and cultural acceptability through sensory evaluations. Recipe 5 emerged as the winning weaning food (WWF), with an overall acceptability rate of 65% (mean score of 5.82 ± 0.87). Unlike theoretical analysis with the ESHA Food Processor, statistical analysis did not show that Recipe 5 met the Codex Alimentarius recommendations for macro- and micronutrients. However, it showed that the micronutrient recommendations for iron (p = 0.0001; 95%CI) and zinc (p = 1.00; 95%CI) were partially met, but not those for calcium and vitamins A and D. The prototype and outcome of this pilot study will be invaluable for interventions aimed at combating food insecurity and malnutrition in Malawi.

Complex I (CI) is the first enzyme of the mitochondrial respiratory chain and couples the electron transfer with proton pumping. Mutations in genes encoding CI subunits can frequently cause inborn metabolic errors. We applied proteome and metabolome profiling of patient-derived cells harboring pathogenic mutations in two distinct CI genes to elucidate underlying pathomechanisms on the molecular level. Our results indicated that the electron transfer within CI was interrupted in both patients by different mechanisms. We showed that the biallelic mutations in NDUFS1 led to a decreased stability of the entire N-module of CI and disrupted the electron transfer between two iron-sulfur clusters. Strikingly interesting and in contrast to the proteome, metabolome profiling illustrated that the pattern of dysregulated metabolites was almost identical in both patients, such as the inhibitory feedback on the TCA cycle and altered glutathione levels, indicative for reactive oxygen species (ROS) stress. Our findings deciphered pathological mechanisms of CI deficiency to better understand inborn metabolic errors.

Nanoparticles (NPs) are increasingly being used for drug delivery, as well as antigen carriers and immunostimulants for the purpose of developing vaccines. In this work, we examined how intranasal (i.n.) priming followed by i.n. or subcutaneous (s.c.) boosting immunization affects the humoral immune response to chicken ovalbumin (Ova) and Ova conjugated to 20 nm NPs (NP-Ova). We show that i.n. priming with 20 mg of soluble Ova, a dose known to trigger oral tolerance when administered via gastric gavage, induced substantial systemic IgG1 and IgG2c, as well as mucosal antibodies. These responses were further boosted following a s.c. immunization with Ova and complete Freund's adjuvant (Ova+CFA). In contrast, 100 µg of Ova delivered via NPs induced an IgG1-dominated systemic response, and primed the intestinal mucosa for secretion of IgA. Following a secondary s.c. or i.n. immunization with Ova+CFA or NP-Ova, systemic IgG1 titers significantly increased, and serum IgG2c and intestinal antibodies were induced in mice primed nasally with NP-Ova. Only Ova- and NP-Ova-primed mice that were s.c.-boosted exhibited substantial systemic and mucosal titers for up to 6 months after priming, whereas the antibodies of i.n.-boosted mice declined over time. Our results indicate that although the amount of Ova delivered by NPs was 1000-fold less than Ova delivered in soluble form, the antigen-specific antibody responses, both systemic and mucosal, are essentially identical by 6 months following the initial priming immunization. Additionally, both i.n.- and s.c.-boosting strategies for NP-Ova-primed mice were capable of inducing a polarized Th1/Th2 immune response, as well as intestinal antibodies; however, it is only by using a heterogeneous prime-boost strategy that long-lasting antibody responses were initiated. These results provide valuable insight for future mucosal vaccine development, as well as furthering our understanding of mucosal antibody responses.

Extracellular adenosine 5'-triphosphate (ATP) plays important mechanistic roles in pulmonary disorders in general and chronic obstructive pulmonary disease (COPD) and cough in particular. The effects of ATP in the lungs are mediated to a large extent by P2X2/3 receptors (P2X2/3R) localized on vagal sensory nerve terminals (both C and Aδ fibers). The activation of these receptors by ATP triggers a pulmonary-pulmonary central reflex, which results in bronchoconstriction and cough, and is also proinflammatory due to the release of neuropeptides from these nerve terminals via the axon reflex. These actions of ATP in the lungs constitute a strong rationale for the development of a new class of drugs targeting P2X2/3R. DT-0111 is a novel, small, water-soluble molecule that acts as an antagonist at P2X2/3R sites.

DNA methylation can regulate the role of long noncoding RNAs (lncRNAs) in the development of lung adenocarcinoma (LUAD). The present study aimed to identify methylation-driven lncRNAs and mRNAs as biomarkers in the prognosis of LUAD using bioinformatics analysis.