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Supplementary Figure 3: Association of chemokine expression with a T cell-inflamed phenotype was validated in a second independent PDAC patient cohort

Supplementary Table 1: Top 99 genes associating with CD8+ TIL counts in primary resected cases

Purpose:The molecular drivers of antitumor immunity in pancreatic ductal adenocarcinoma (PDAC) are poorly understood, posing a major obstacle for the identification of patients potentially amenable for immune-checkpoint blockade or other novel strategies. Here, we explore the association of chemokine expression with effector T-cell infiltration in PDAC.Experimental Design:Discovery cohorts comprised 113 primary resected PDAC and 107 PDAC liver metastases. Validation cohorts comprised 182 PDAC from The Cancer Genome Atlas and 92 PDACs from the Australian International Cancer Genome Consortium. We explored associations between immune cell counts by immunohistochemistry, chemokine expression, and transcriptional hallmarks of antitumor immunity by RNA sequencing (RNA-seq), and mutational burden by whole-genome sequencing.Results:Among all known human chemokines, a coregulated set of four (CCL4, CCL5, CXCL9, and CXCL10) was strongly associated with CD8+ T-cell infiltration (P < 0.001). Expression of this “4-chemokine signature” positively correlated with transcriptional metrics of T-cell activation (ZAP70, ITK, and IL2RB), cytolytic activity (GZMA and PRF1), and immunosuppression (PDL1, PD1, CTLA4, TIM3, TIGIT, LAG3, FASLG, and IDO1). Furthermore, the 4-chemokine signature marked tumors with increased T-cell activation scores (MHC I presentation, T-cell/APC costimulation) and elevated expression of innate immune sensing pathways involved in T-cell priming (STING and NLRP3 inflammasome pathways, BATF3-driven dendritic cells). Importantly, expression of this 4-chemokine signature was consistently indicative of a T-cell–inflamed phenotype across primary PDAC and PDAC liver metastases.Conclusions:A conserved 4-chemokine signature marks resectable and metastatic PDAC tumors with an active antitumor phenotype. This could have implications for the appropriate selection of PDAC patients in immunotherapy trials.

The development of progressive osteopenia and osteoporosis (OP) is due to the imbalance between bone resorption and bone formation, determining a lower bone resistance, major risks of fractures, with consequent pain and functional limitations. Flavonoids, a class of polyphenols, have been extensively studied for their therapeutic activities against bone resorption, but less attention has been given to a whole series of molecules belonging to the polyphenolic compounds. However, these classes have begun to be studied for the treatment of OP. In this systematic review, comprehensive information is provided on non-flavonoid polyphenolic compounds, and we highlight pathways implicated in the action of these molecules that act often epigenetically, and their possible use for OP treatment and prevention.

Human bones are biological examples of functionally graded lattice capable to withstand large in vivo loading and allowing optimal stress distribution. Disruption of bone integrity may require biocompatible implants capable to restore the original bone structure and properties. This study aimed at comparing mechanical properties and biological behavior in vitro of uniform (POR-FIX) and graded (POR-VAR) Cobalt-chrome alloy lattice structures manufactured via Selective Laser Melting. In compression, the POR-VAR equivalent maximum stress was about 2.5 times lower than that of the POR-FIX. According to the DIC analysis, the graded lattice structures showed a stratified deformation associated to unit cells variation. At each timepoint, osteoblast cells were observed to colonize the surface and the first layer of both scaffolds. Cell activity was always significantly higher in the POR-VAR (p< 0.0005). In terms of gene expression, the OPG/RANKL ratio increased significantly over time (p< 0.0005) whereas IL1β and COX2 significantly decreased (7 day vs 1 day; p< 0.0005) in both scaffolds. Both uniform- and graded-porosity scaffolds provided a suitable environment for osteoblasts colonization and proliferation, but graded structures seem to represent a better solution to improve stress distribution between implant and bone of orthopedic implants.

A correct differentiation between hepatocellular carcinoma (HCC) and intracellular cholangiocarcinoma (ICC) is essential for clinical management and prognostic prediction. However, non-invasive differential diagnosis between HCC and ICC remains highly challenging. Dynamic contrast-enhanced ultrasound (D-CEUS) with standardized software is a valuable tool in the diagnostic approach to focal liver lesions and could improve accuracy in the evaluation of tumor perfusion. Moreover, the measurement of tissue stiffness could add more information concerning tumoral environment. To explore the diagnostic performance of multiparametric ultrasound (MP-US) in differentiating ICC from HCC. Our secondary aim was to develop an US score for distinguishing ICC and HCC. Between January 2021 and September 2022 consecutive patients with histologically confirmed HCC and ICC were enrolled in this prospective monocentric study. A complete US evaluation including B mode, D-CEUS and shear wave elastography (SWE) was performed in all patients and the corresponding features were compared between the tumor entities. For better inter-individual comparability, the blood volume-related D-CEUS parameters were analyzed as a ratio between lesions and surrounding liver parenchyma. Univariate and multivariate regression analysis was performed to select the most useful independent variables for the differential diagnosis between HCC and ICC and to establish an US score for non-invasive diagnosis. Finally, the diagnostic performance of the score was evaluated by receiver operating characteristic (ROC) curve analysis. A total of 82 patients (mean age ± SD, 68 ± 11 years, 55 men) were enrolled, including 44 ICC and 38 HCC. No statistically significant differences in basal US features were found between HCC and ICC. Concerning D-CEUS, blood volume parameters (peak intensity, PE; area under the curve, AUC; and wash-in rate, WiR) showed significantly higher values in the HCC group, but PE was the only independent feature associated with HCC diagnosis at multivariate analysis (p = 0.02). The other two independent predictors of histological diagnosis were liver cirrhosis (p < 0.01) and SWE (p = 0.01). A score based on those variables was highly accurate for the differential diagnosis of primary liver tumors, with an area under the ROC curve of 0.836 and the optimal cut-off values of 0.81 and 0.20 to rule in or rule out ICC respectively. MP-US seems to be a useful tool for non-invasive discrimination between ICC and HCC and could prevent the need for liver biopsy at least in a subgroup of patients.

This research study sought to evaluate aimed at evaluating the possible advantages of using Fuzzy Logic as opposed to Boolean Logic to assess environmental contamination and source appraisal for polycyclic aromatic hydrocarbons (PAH). Results obtained through traditional assessment tools for two different tropical coastal areas through using traditional clustering and principal components analysis were compared with those derived from the Fuzzy Logic, using the by Fuzzy C-means algorithm. The feedings achieved through Fuzzy Logic showed a greater qualitative detail than those derived from traditional tools. The abrupt and unnatural changes obtained from the usual classification methods were avoided by having membership values varying continuously in space, providing a more accurate picture of environmental contamination in complex and multiple sources environments. Furthermore, by not depending on statistic suppositions distribution of data like other methods, becomes more suitable for environmental data. Although Fuzzy Logic does not produce quantitative interpretations, its application generates adequate the data needed to avoid environmental management bias in the inference of contamination sources.

This research aimed at implementing and validating a method for analysis of pesticide residues in crops. QuEChERS extraction method with PSA purification was used following analyzes by gas chromatography with tandem mass spectrometry in Selected Reaction Monitoring mode. A short run method was successfully developed for the determination of 41 pesticides, confirmed by two precursor-products for each analyte. The calibration curve for each analyte was linear at concentration range from 1 to 500 μg kg

Osteoarthritis (OA) is a degenerative bone disease that involves the microenvironment and macroenvironment of joints. Progressive joint tissue degradation and loss of extracellular matrix elements, together with different grades of inflammation, are important hallmarks of OA disease. Therefore, the identification of specific biomarkers to distinguish the stages of disease becomes a primary necessity in clinical practice. To this aim, we investigated the role of miR203a-3p in OA progression starting from the evidence obtained by osteoblasts isolated from joint tissues of OA patients classified according to different Kellgren and Lawrence (KL) grading (KL ≤ 3 and KL > 3) and hMSCs treated with IL-1β. Through qRT-PCR analysis, it was found that osteoblasts (OBs) derived from the KL ≤ 3 group expressed high levels of miR203a-3p and low levels of ILs compared with those of OBs derived from the KL > 3 group. The stimulation with IL-1β improved the expression of miR203a-3p and the methylation of the IL-6 promoter gene, favoring an increase in relative protein expression. The gain and loss of function studies showed that the transfection with miR203a-3p inhibitor alone or in co-treatments with IL-1β was able to induce the expression of CX-43 and SP-1 and to modulate the expression of TAZ, in OBs derived from OA patients with KL ≤ 3 compared with KL > 3. These events, confirmed also by qRT-PCR analysis, Western blot, and ELISA assay performed on hMSCs stimulated with IL-1β, supported our hypothesis about the role of miR203a-3p in OA progression. The results suggested that during the early stage, miR203a-3p displayed a protective role reducing the inflammatory effects on CX-43, SP-1, and TAZ. During the OA progression the downregulation of miR203a-3p and consequently the upregulation of CX-43/SP-1 and TAZ expression improved the inflammatory response and the reorganization of the cytoskeleton. This role led to the subsequent stage of the disease, where the aberrant inflammatory and fibrotic responses determined the destruction of the joint.

The existence of a tight relationship between inflammation and epigenetics that in primary breast tumor cells can lead to tumor progression and the formation of bone metastases was investigated. It was highlighted how the induction of tumor progression and bone metastasis by Interleukin-1 beta, in a non-metastatic breast cancer cell line, MCF-7, was dependent on the de-methylating actions of ten-eleven translocation proteins (TETs). In fact, the inhibition of their activity by the Bobcat339 molecule, an inhibitor of TET enzymes, determined on the one hand, the modulation of the epithelial-mesenchymal transition process, and on the other hand, the reduction in the expression of markers of bone metastasis, indicating that the epigenetic action of TETs is a prerequisite for IL-1β-dependent tumor progression and bone metastasis formation.

Investigation were carried out targeting distribution and source apportioning of hydrocarbons in surface sediments from shallow to deep waters in the Campos Basin, one of the most important oil-producing provinces in Brazil. The observed levels of aliphatic (≤ 124 μg g−1) and polycyclic aromatic hydrocarbons (PAHs ≤ 599 ng g−1) are lower than those considered relevant for environmental risk to the benthic habitats. Higher median concentrations of aliphatic hydrocarbons (8.49 μg g−1) and PAHs (84.8 ng g−1) in the middle slope sediments (700–1000-m water depths) showed the influence of hydrodynamics upon hydrocarbon accumulation in the sediments. Diagnostic ratios and conventional statistical analysis applied to hydrocarbon data produced insufficient information on the contribution of different sources. These traditional approaches do not consider the potential changes affecting source assignment and therefore cannot deal with the uncertainties. The fuzzy logic applied as an alternative method for data treatment successfully incorporated the uncertainties in the differentiation between petrogenic and pyrolytic sources, including those due to degradation. Moreover, by using fuzzy logic, it was possible to identify that water circulation patterns, mass transport, deposition, and degradation processes are more relevant factors in determining hydrocarbon composition than source proximity.

Two sediment cores collected in the shelf off Rio de Janeiro (RJ13-01B: 10.8 cal kyr BP, and RJ13-02B: 4.7 cal kyr BP) were investigated in high-resolution to evaluate changes in sedimentary processes and paleoclimatic and paleoceanographic variability during the Holocene in the Southeastern Brazil. Information from inorganic proxies of continental input (Al/Ca and Fe/Ca), redox-sensitive elements (Fe/Al, V/Al and Mn/Al), paleoproductivity (Sr/Al, Cd/Al and Ba/Al), as well as data for grain size, geophysical properties (p-wave velocity, gamma-ray density, acoustic impedance, magnetic susceptibility and porosity), Si, total organic carbon and calcium carbonate were considered. The data revealed three main periods of sediment accumulation: (i) from 10.8 cal kyr BP to 7.6 cal kyr BP the sediment was coarse, Si content was high (27%), the magnetic susceptibility was low and the presence of shell fragments all suggest a period of low continental input and the deposition site was shallow (i.e, lower sea level); (ii) from 7.5 cal kyr BP to 4.6 cal kyr BP the elevated ratios of Al/Ca (0.69 ± 0.08), Fe/Ca (0.27 ± 0.04), Sr/Al (31.57 ± 4.47) ×10−4, Cd/Al (0.09 ± 0.03) ×10−5 and Ba/Al (0.70 ± 0.16) ×10−4 are consistent with a period of maximum sea transgression and elevated influence of the nutrient-rich South Atlantic Central Water (SACW); (iii) from 4.5 cal kyr BP to the present, events of higher fluxes of TOC, Ni, Cu and Zn were observed from ca 3.2 cal kyr BP to 3.4 cal kyr BP, in both cores, related to humid climate. Overall, the multi-proxy approach shed light on the effects of climate and oceanographic variability on sediment input and accumulation in a less-studied portion of the Brazilian shelf, which are consistent with other shelf areas and with changes in regional climate systems like the Intertropical Convergence Zone (ITCZ), South American Monsoon Systems (SAMS) and South Atlantic Convergence Zone (SACZ).

Lower bone resistance to load is due to the imbalance of bone homeostasis, where excessive bone resorption, compared with bone formation, determines a progressive osteopenia, leading to a high risk of fractures and consequent pain and functional limitations. Terpenoids, with their activities against bone resorption, have recently received increased attention from researchers. They are potentially more suitable for long-term use compared with traditional therapeutics. In this review of the literature of the past 5 years, we provide comprehensive information on terpenoids, with their anti-osteoporotic effects, highlighting molecular mechanisms that are often in epigenetic key and a possible pharmacological use in osteoporosis prevention and treatment.

Osteoarthritis (OA) is a degenerative bone disease that involved micro and macro-environment of joints. To date, there are no radical curative treatments for OA and novel therapies are mandatory. Recent evidence suggests the role of miRNAs in OA progression. In our previous studies, we demonstrated the role of miR-31-5p and miR-33a families in different bone regeneration signaling. Here, we investigated the role of miR-31-5p and miR-33a-5p in OA progression. A different expression of miR-31-5p and miR-33a-5p into osteoblasts and chondrocytes isolated from joint tissues of OA patients classified in based on different Kellgren and Lawrence (KL) grading was highlighted, and through a bioinformatic approach the common miRNAs target Specificity proteins (Sp1) were identified. Sp1 regulates the expression of gap junction protein Connexin43 (Cx43), which in OA drives the modification of i) osteoblasts and chondrocytes genes expression, ii) joint inflammation cytokines releases and iii) cell functions. Concerning this, thanks to gain and loss of function studies, the possible role of Sp1 as a modulator of CX43 expression through miR-31-5p and miR-33a-5p action was also evaluated. Finally, we hypothesize that both miRNAs cooperate to modulate the expression of SP1 in osteoblasts and chondrocytes and interfering, consequently, with CX43 expression, and they might be further investigated as new possible biomarkers for OA.

Summary Here we present baseline data of δ¹³C of n-alkanes (δ¹³Cn-alkanes) from leaf waxes of mangrove trees – Avicennia schaueriana, Rhizophora mangle and Laguncularia racemosa – distributed along a latitudinal gradient in Brazil. The objective was to evaluate the influence of trees physiology, local hydrology and climatic factors upon the molecular and isotopic signature of the leaf wax n-alkanes. Higher weighted average δ¹³Cn-alkanes (δ¹³CWA), average chain lenght (ACL25-35) and carbon preference index (CPI24-36) for R. mangle and A. schaueriana contrasting with lower values for L. racemosa seem to be linked to physiological characteristics of each species regarding intrinsic water use efficiency under the specific tidal flooding regime of mangrove forests. About the plant’s response to local environment influence, inverse correlations between all δ¹³Cn-alkanes values (δ¹³C₂₇, δ¹³C₂₉, δ¹³C₃₁, δ¹³C₃₃ and δ¹³CWA) and mean annual precipitation were found for the A. schaueriana samples. For the R. mangle species, a relationship between ACL25-35 and mean annual potential evapotranspiration and between CPI24-36 and mean annual temperature were observed. The different molecular and isotopic footprints of the three mangrove species presented here have potential to be used in future interpretations of n-alkanes as biomarkers in biogeochemical and paleoclimate studies near mangrove-dominated coastal regions.

During the year 2017, a research work was carried out at the Department of laboratory diagnostics - Toxicology and Biochemistry U.O.S. of ASP 6 in Palermo. The purpose of this investigation was to perform a second level analysis in order to look for abuse drugs. The sample used consisted of 73 serum tubes, which had previously tested positive for screen- ing tests. The result of the analysis of these samples revealed a false positivity in most cases. From the information gathered through the sampling reports, it appears that the age group with several drivers, who are subject to checks by the traffic police, is the one from 17 to 30 years, with 35 samples analysed (48%). It also emerged that 92% are male and only 8% female. The percentages of substances detected are THC + Cocaine 8%, Cocaine 15% and THC 77%. Thanks to the processing of collected data, a statistic about the consumption of illicit drugs in the city of Palermo has been produced. Through the findings, it was possible to make a comparison with the incidence of abuse of these substances in Italy. Moreover, it was found that Cannabis is the illicit substance mainly used in the Italian territory. Due to the growing increase in drug use on our territory, it is necessary that the methods used for confirmatory analysis be accurate and pre- cise, in order to provide reliable results on the presence of substances of abuse in biological matrices. In fact more and more sophisticated methods and analytical tools are being developed.

Bone disease severely affects the quality of life of over 70% of multiple myeloma (MM) patients, which daily experience pain, pathological fractures, mobility issues and an increased mortality. Recent data have highlighted the crucial role of the endoplasmic reticulum-associated unfolded protein response (UPR) in malignant transformation and tumor progression, therefore, targeting of UPR-related molecules may open novel therapeutic avenues. Endoplasmic reticulum (ER) stress and UPR pathways are constitutively activated in MM cells, which are characterized by an increased protein turnover as a consequence of high production of immunoglobulins and high rates of protein synthesis. A great deal of scientific data also evidenced that a mild activation of UPR pathway can regulate cellular differentiation. Our previous studies revealed that MM cell-derived small extracellular vesicle (MM-EV) modulated osteoclasts (OCs) function and induced OCs differentiation. Here, we investigated the role of the UPR pathway, and in particular of the IRE1&alpha, /XBP1 axis, in osteoclastogenesis induced by MM-EVs. By proteomic analysis, we identified UPR signaling molecules as novel MM-EV cargo, prompting us to evaluate the effects of the MM-EVs on osteoclastogenesis through UPR pathway. MM-EVs administration in a murine macrophage cell line rapidly induced activation of IRE1&alpha, by phosphorylation in S724, accordingly, Xbp1 mRNA splicing was increased and the transcription of NFATc1, a master transcription factor for OCs differentiation, was activated. Some of these results were also validated using both human primary OC cultures and MM-EVs from MM patients. Notably, a chemical inhibitor of IRE1&alpha, (GSK2850163) counteracted MM-EV-triggered OC differentiation, hampering the terminal stages of OCs differentiation and reducing bone resorption.

Osteolytic bone disease is the major complication associated with the progression of multiple myeloma (MM). Recently, extracellular vesicles (EVs) have emerged as mediators of MM-associated bone disease by inhibiting the osteogenic differentiation of human mesenchymal stem cells (hMSCs). Here, we investigated a correlation between the EV-mediated osteogenic inhibition and MM vesicle content, focusing on miRNAs. By the use of a MicroRNA Card, we identified a pool of miRNAs, highly expressed in EVs, from MM cell line (MM1.S EVs), expression of which was confirmed in EVs from bone marrow (BM) plasma of patients affected by smoldering myeloma (SMM) and MM. Notably,we found that miR-129-5p, which targets different osteoblast (OBs) differentiation markers, is enriched in MM-EVs compared to SMM-EVs, thus suggesting a selective packaging correlated with pathological grade. We found that miR-129-5p can be transported to hMSCs by MM-EVs and, by the use of miRNA mimics, we investigated its role in recipient cells. Our data demonstrated that the increase of miR-129-5p levels in hMSCs under osteoblastic differentiation stimuli inhibited the expression of the transcription factor Sp1, previously described as a positive modulator of osteoblastic differentiation, and of its target the Alkaline phosphatase (ALPL), thus identifying miR-129-5p among the players of vesicle-mediated bone disease.

Imbalance of bone homeostasis, with excessive bone resorption compared with bone formation, leads to the development of progressive osteopenia leading to lower bone resistance to load, with consequent pain and functional limitations. Phytochemicals with therapeutic and preventive effects against bone resorption have recently received increasing attention since they are potentially more suitable for long-term use than traditional therapeutic chemical compounds. In this systematic review of the literature of the past 5 years, comprehensive information is provided on flavonoids with potential antiresorption and pro-osteogenic effects. It aims to highlight the molecular mechanisms of these molecules, often epigenetic, and their possible pharmacological use, which is of great importance for the prevention and treatment of osteoporosis (OP).

Osteosarcoma (OS) is the most common primary bone tumor mainly occurring in young adults and derived from primitive bone-forming mesenchyme. OS develops in an intricate tumor microenvironment (TME) where cellular function regulated by microRNAs (miRNAs) may affect communication between OS cells and the surrounding TME. Therefore, miRNAs are considered potential therapeutic targets in cancer and one of the goals of research is to accurately define a specific signature of a miRNAs, which could reflect the phenotype of a particular tumor, such as OS. Through NGS approach, we previously found a specific molecular profile of miRNAs in OS and discovered 8 novel miRNAs. Among these, we deepen our knowledge on the fifth candidate renamed now miR-CT3. MiR-CT3 expression was low in OS cells when compared with human primary osteoblasts and healthy bone. Through TargetScan, VEGF-A was predicted as a potential biological target of miR-CT3 and luciferase assay confirmed it. We showed that enforced expression of miR-CT3 in two OS cell lines, SAOS-2 and MG-63, reduced expression of VEGF-A mRNA and protein, inhibiting tumor angiogenesis. Enforced expression of miR-CT3 also reduced OS cell migration and invasion as confirmed by soft agar colony formation assay. Interestingly, we found that miR-CT3 behaves inducing the activation of p38 MAP kinase pathway and modulating the epithelial-mesenchymal transition (EMT) proteins, in particular reducing Vimentin expression. Overall, our study highlights the novel role of miR-CT3 in regulating tumor angiogenesis and progression in OS cells, linking also to the modulation of EMT proteins.

The applicability of two organic geochemical proxies ( U 37 K ' based on alkenones and T E X 86 H based on glycerol dialkyl glycerol tetraethers) for ocean temperatures was investigated on a regional scale in the SE Brazilian continental margin (Campos Basin) using 53 core-top sediments. In 71% of the samples for U 37 K ' and 85% for T E X 86 H , the estimated annual mean sea surface temperature (SST) is comparable to the climatological annual mean data, with deviations within the calibration error. The surface T E X 86 H estimates reflects the surface mixed layer, while the depth-integrated (0–200 m) T E X 86 H estimates showed better agreement with temperatures at 100–200 m depth. In addition, the SST- U 37 K ' results were more consistent (73% of the samples) using a winter calibration rather than an annual mean calibration. This seasonal effect might be ascribed to an increase in the abundance of haptophytes in the region during the winter. Exceptions to these general trends were observed for both proxies as a strong cold bias in reconstructed temperature in a few samples in the southern portion of the studied region, which may be related to (i) a predominant subsurface habitat of Thaumarchaeota combined with enhanced primary production and flux of particles during upwelling and (ii) alkenones being laterally transported from adjacent colder waters. Effects of river input of terrestrially sourced GDGTs on T E X 86 H are not evident. Overall, our findings show that U 37 K ' and T E X 86 H are suitable temperature proxies for paleoclimate studies at a regional scale in the SE Brazilian continental margin, but on a local scale the effects of upwelling must be taken in consideration.

Background Bone tissue is one of the main sites for breast metastasis; patients diagnosed with advanced breast cancer mostly develop bone metastasis characterized by severe osteolytic lesions, which heavily influence their life quality. Low Intensity Pulsed Ultrasound (LIPUS) is a form of mechanical energy able to modulate various molecular pathways both in cancer and in health cells. The purpose of the present study was to evaluate for the first time, the ability of LIPUS to modulate osteolytic capability of breast cancer cells. Methods Two different approaches were employed: a) Indirect method -conditioned medium obtained by MDA-MB-231 cell line treated or untreated with LIPUS was used to induce osteoclast differentiation of murine macrophage Raw264.7 cell line; and b) Direct method -MDA-MB-231 were co-cultured with Raw264.7 cells and treated or untreated with LIPUS. Results LIPUS treatment impaired MDA-MB-231 cell dependentosteoclast differentiation and produced a reduction of osteoclast markers such as Cathepsin K, Matrix Metalloproteinase 9 and Tartrate Resistant Acid Phosphatase, suggesting its role as an effective and safe adjuvant in bone metastasis management. Conclusion LIPUS treatment could be a good and safety therapeutic adjuvant in osteolyitic bone metastasis not only for the induction properties of bone regeneration, but also for the reduction of osteolysis.

Lipid biomarkers [fatty acids (FAs), sterols and alcohols] and carbon stable isotopes (δ13C values) in surface sediments from 9 cross shelf transects (25–3000 m water depth) from the Campos Basin, SE Brazilian continental margin were analyzed. The aim was to investigate the link between the prevailing regional specific oceanographic conditions (upwelling events, intrusion of cold and nutrient-rich water, low river input) and the nature and distribution of organic matter (OM) in the basin. A general predominance of OM from autochthonous processes, but with a relevant spatial gradient in the quality and quantity of the sedimentary OM, was observed. On the shelf (

Background aims During bone formation, angiogenesis and osteogenesis are regulated by hypoxia, which is able to induce blood vessel formation, as well as recruit and differentiate human mesenchymal stromal cells (hMSCs). The molecular mechanisms involved in HIF-1α response and hMSC differentiation during bone formation are still unclear. This study aimed to investigate the synergistic role of hypoxia and hypoxia-mimetic microRNA miR-675-5p in angiogenesis response and osteo-chondroblast commitment of hMSCs. Methods By using a suitable in vitro cell model of hMSCs (maintained in hypoxia or normoxia), the role of HIF-1α and miR-675-5p in angiogenesis and osteogenesis coupling was investigated, using fluorescence-activated cell sorting (FACS), gene expression and protein analysis. Results Hypoxia induced miR-675-5p expression and a hypoxia-angiogenic response, as demonstrated by increase in vascular endothelial growth factor messenger RNA and protein release. MiR-675-5p overexpression in normoxia promoted the down-regulation of MSC markers and the up-regulation of osteoblast and chondroblast markers, as demonstrated by FACS and protein analysis. Moreover, miR-675-5p depletion in a low-oxygen condition partially abolished the hypoxic response, including angiogenesis, and in particular restored the MSC phenotype, demonstrated by cytofluorimetric analysis. In addition, current preliminary data suggest that the expression of miR-675-5p during hypoxia plays an additive role in sustaining Wnt/β-catenin pathways and the related commitment of hMSCs during bone ossification. Discussion MiR-675-5p may trigger complex molecular mechanisms that promote hMSC osteoblastic differentiation through a dual strategy: increasing HIF-1α response and activating Wnt/β-catenin signaling.

Low-intensity pulsed ultrasound (LIPUS) as an adjuvant therapy in in vitro and in vivo bone engineering has proven to be extremely useful. The present study aimed at investigating the effect of 30 mW/cm(2) LIPUS stimulation on commercially available human mesenchymal stem cells (hMSCs) cultured in basal or osteogenic medium at different experimental time points (7d, 14d, 21d). The hypothesis was that LIPUS would improve the osteogenic differentiation of hMSC and guarantying the maintenance of osteogenic committed fraction, as demonstrated by cell vitality and proteomic analysis. LIPUS stimulation (a) regulated the balance between osteoblast commitment and differentiation by specific networks (activations of RhoA/ROCK signaling and upregulation of Ribosome constituent/Protein metabolic process, Glycolysis/Gluconeogenesis, RNA metabolic process/Splicing and Tubulins); (b) allowed the maintenance of a few percentage of osteoblast precursors (21d CD73 + /CD90 + : 6%; OCT-3/4 + /NANOG + /SOX2 + : 10%); (c) induced the activation of osteogenic specific pathways shown by gene expression (early: ALPL, COL1A1, late: RUNX2, BGLAP, MAPK1/6) and related protein release (COL1a1, OPN, OC), in particular in the presence of osteogenic soluble factors able to mimic bone microenvironment. To summarize, LIPUS might be able to improve the osteogenic commitment of hMSCs in vitro, and, at the same time, enhance their osteogenic differentiation. This article is protected by copyright. All rights reserved.

Branched glycerol dialkyl glycerol tetraethers (brGDGTs) are membrane lipids produced by bacteria usually ascribed to soil and peat deposits. The presence of brGDGTs in marine sediments can thus be used to track terrigenous organic matter inputs to the continental margin and to infer the local continental mean annual air temperature (MAT) and soil pH. The proxy rationale is based on the degree methylation and cyclization of the brGDGTs from terrestrial bacteria, but recently evidence was found of river and oceanic production of brGDGTs with similar configuration, indicating the necessity to better constrain the applicability of the soil brGDGTs in the marine realm. Here we considered crenarchaeol and brGDGTs obtained in 46 core-top sediments from cross-margin transects in the Campos Basin in the Southwest Atlantic, with the goal to evaluate the effectiveness of the brGDGT-associated proxies in a region in the southeastern tropical Brazilian continental margin influenced by upwelling events and low terrigenous inputs. The separation of the 5- and 6-methyl brGDGTs proved to be essential for a better evaluation of the sources of brGDGTs in the environment. Direct evidence of input of terrigenous organic matter by the medium-sized Paraiba do Sul River and other small rivers in the region were observed. More importantly, the high proportions of ring-containing brGDGTs – and the consequent increased values of the #ringstetra – in the sediments deposited between 75 and 400 m water depths (mid-shelf to upper slope) were clear evidence of marine in situ production of brGDGTs. In some stations deeper than 1900 m, an increase in the acyclic 6-methyl hexamethylated compounds can also be ascribed to in situ production. Our results revealed that the initial soil signal is lost during transport and after river discharge in the Campos Basin, which compromise the use of brGDGTs to reconstruct the soil pH and MAT of the nearby land area.

Mesenchymal stromal cells (hMSCs) display a pleiotropic function in bone regeneration. The signaling involved in osteoblast commitment is still not completely understood, and that determines the failure of current therapies being used. In our recent studies, we identified two miRNAs as regulators of hMSCs osteoblast differentiation driving hypoxia signaling and cytoskeletal reorganization. Other signalings involved in this process are epithelial to mesenchymal transition (EMT) and epidermal growth factor receptor (EGFR) signalings through the regulation of Yes-associated protein (YAP)/PDZ-binding motif (TAZ) expression. In the current study, we investigated the role of miR-33a family as a (i) modulator of YAP/TAZ expression and (ii) a regulator of EGFR signaling during osteoblast commitments. Starting from the observation on hMSCs and primary osteoblast cell lines (Nh-Ost) in which EMT genes and miR-33a displayed a specific expression, we performed a gain and loss of function study with miR-33a-5p and 3p on hMSCs cells and Nh-Ost. After 24 h of transfections, we evaluated the modulation of EMT and osteoblast genes expression by qRT-PCR, Western blot, and Osteoimage assays. Through bioinformatic analysis, we identified YAP as the putative target of miR-33a-3p. Its role was investigated by gain and loss of function studies with miR-33a-3p on hMSCs, qRT-PCR and Western blot analyses were also carried out. Finally, the possible role of EGFR signaling in YAP/TAZ modulation by miR-33a-3p expression was evaluated. Human MSCs were treated with EGF-2 and EGFR inhibitor for different time points, and qRT-PCR and Western blot analyses were performed. The above-mentioned methods revealed a balance between miR-33a-5p and miR-33a-3p expression during hMSCs osteoblast differentiation. The human MSCs phenotype was maintained by miR-33a-5p, while the maintenance of the osteoblast phenotype in the Nh-Ost cell model was permitted by miR-33a-3p expression, which regulated YAP/TAZ through the modulation of EGFR signaling. The inhibition of EGFR blocked the effects of miR-33a-3p on YAP/TAZ modulation, favoring the maintenance of hMSCs in a committed phenotype. A new possible personalized therapeutic approach to bone regeneration was discussed, which might be mediated by customizing delivery of miR-33a in simultaneously targeting EGFR and YAP signaling with combined use of drugs.

One of the goals of personalized medicine is to understand and treat diseases with greater precision through the molecular profile of the patient. This profiling is becoming a powerful tool for the discovery of novel biomarkers that can guide physicians in assessing, in advance, the disease stage, and monitoring disease progression. Circulating miRNAs and exosomal miRNAs, a group of small non-coding RNAs, are considered the gold standard diagnostic biomarkers for human diseases. We have previously demonstrated that osteosarcoma-derived exosomes are able to influence crucial mechanisms inside tumor niches, inducing osteoclast differentiation, and sustaining bone resorption activity. Here we discovered, through Next-Generation Sequencing (NGS), eight novel microRNAs in three different osteosarcoma cell lines, and assessed the selective packaging into the exosomes released. We then investigated, as proof-of-principle, the presence of the novel microRNAs in osteosarcoma patient samples, and found that 5 of the 8 novel microRNAs were more present in circulating exosomes of osteosarcoma patients compared with the controls. These results raise a question: Could the 8 novel microRNAs play a role for osteosarcoma pathogenesis? Although still premature, the results are encouraging, and further studies with a validation in a larger cohort are needed.

Purpose: The molecular drivers of antitumor immunity in pancreatic ductal adenocarcinoma (PDAC) are poorly understood, posing a major obstacle for the identification of patients potentially amenable for immune-checkpoint blockade or other novel strategies. Here, we explore the association of chemokine expression with effector T-cell infiltration in PDAC. Experimental Design: Discovery cohorts comprised 113 primary resected PDAC and 107 PDAC liver metastases. Validation cohorts comprised 182 PDAC from The Cancer Genome Atlas and 92 PDACs from the Australian International Cancer Genome Consortium. We explored associations between immune cell counts by immunohistochemistry, chemokine expression, and transcriptional hallmarks of antitumor immunity by RNA sequencing (RNA-seq), and mutational burden by whole-genome sequencing. Results: Among all known human chemokines, a coregulated set of four (CCL4, CCL5, CXCL9, and CXCL10) was strongly associated with CD8+ T-cell infiltration (P < 0.001). Expression of this “4-chemokine signature” positively correlated with transcriptional metrics of T-cell activation (ZAP70, ITK, and IL2RB), cytolytic activity (GZMA and PRF1), and immunosuppression (PDL1, PD1, CTLA4, TIM3, TIGIT, LAG3, FASLG, and IDO1). Furthermore, the 4-chemokine signature marked tumors with increased T-cell activation scores (MHC I presentation, T-cell/APC costimulation) and elevated expression of innate immune sensing pathways involved in T-cell priming (STING and NLRP3 inflammasome pathways, BATF3-driven dendritic cells). Importantly, expression of this 4-chemokine signature was consistently indicative of a T-cell–inflamed phenotype across primary PDAC and PDAC liver metastases. Conclusions: A conserved 4-chemokine signature marks resectable and metastatic PDAC tumors with an active antitumor phenotype. This could have implications for the appropriate selection of PDAC patients in immunotherapy trials.

This abstract book contains the abstracts presented at the First symposium on experimental biology: sea and environment (Primo simposio di biologia sperimentale applicata al mare e all’ambiente), Trapani, Italy, 24-25 May 2019.

The roles of low-intensity pulsed ultrasound (LIPUS) and microRNAs (miRNAs) on hMSCs commitments have already been investigated, however, the effects of the application of their co-treatments in an in vitro cell model are still unknown. Our previous studies demonstrated that (i) LIPUS modulated hMSCs cytoskeletal organization and (ii) miRNA-675-5p have a role in HIF-1&alpha, signaling modulation during hMSCs osteoblast commitment. We investigated for the first time the role of LIPUS as promoter tool for miRNA expression. Thanks to bioinformatic analysis, we identified miR-31-5p as a LIPUS-induced miRNA and investigated its role through in vitro studies of gain and loss of function. Results highlighted that LIPUS stimulation induced a hypoxia adaptive cell response, which determines a reorganization of cell membrane and cytoskeleton proteins. MiR-31-5p gain and loss of function studies, demonstrated as miR-31-5p overexpression, were able to induce hypoxic and cytoskeletal responses. Moreover, the co-treatments LIPUS and miR-31-5p inhibitor abolished the hypoxic responses including angiogenesis and the expression of Rho family proteins. MiR-31-5p was identified as a LIPUS-mechanosensitive miRNAs and may be considered a new therapeutic option to promote or abolish hypoxic response and cytoskeletal organization on hMSCs during the bone regeneration process.

Osteosarcoma (OS) is a primary bone sarcoma, manifesting as osteogenesis by malignant cells. Nowadays, patients’ quality of life has been improved, however continuing high rates of limb amputation, pulmonary metastasis and drug toxicity, remain unresolved issues. Thus, effective osteosarcoma therapies are still required. Recently, the potentialities of biophysical treatments in osteosarcoma have been evaluated and seem to offer a promising future, thanks in this field as they are less invasive. Several approaches have been investigated such as hyperthermia (HT), high intensity focused ultrasound (HIFU), low intensity pulsed ultrasound (LIPUS) and sono- and photodynamic therapies (SDT, PDT). This review aims to summarize in vitro and in vivo studies and clinical trials employing biophysical stimuli in osteosarcoma treatment. The findings underscore how the technological development of biophysical therapies might represent an adjuvant role and, in some cases, alternative role to the surgery, radio and chemotherapy treatment of OS. Among them, the most promising are HIFU and HT, which are already employed in OS patient treatment, while LIPUS/SDT and PDT seem to be particularly interesting for their low toxicity.

Tissue engineering offers new approaches to repair bone defects, which cannot be repaired physiologically, developing scaffolds that mimic bone tissue architecture. Furthermore, biomechanical stimulation induced by bioreactor, provides biomechanical cues that regulate a wide range of cellular events especially required for cellular differentiation and function. The improvement of human mesenchymal stem cells (hMSCs) colonization in poly-L-lactic-acid (PLLA)/nano- hydroxyapatite (nHA) composite scaffold was evaluated in terms of cell proliferation (dsDNA content), bone differen- tiation (gene expression and protein synthesis) and ultrastructural analysis by comparing static (s3D) and dynamic (d3D) 3D culture conditions at 7 and 21 days. The colonization rate of hMSCs and osteogenic differentiation were amplified by d3D when physical stimulation was provided by a perfusion bioreactor. Increase in dsDNA content (p < 0.0005), up- regulation of RUNX2, ALPL, SPP1 (p < 0.0005) and SOX9 (p < 0.005) gene expression, and more calcium nodule forma- tion (p < 0.0005) were observed in d3D cultures in comparison to s3D ones over time. Dynamic 3D culture, mimicking the mechanical signals of bone environment, improved significantly osteogenic differentiation of hMSCs on PLLA/nHA scaffold, without the addition of growth factors, confirming this composite scaffold suitable for bone regeneration

Bone microenvironment provides growth and survival signals essential for osteosarcoma (OS) initiation and progression. OS cells regulate communications inside tumor microenvironment through different ways and, among all, tumor-derived exosomes support cancer progression and metastasis. To define the contribution of OS-derived exosomes inside the microenvironment, we investigated the effects induced in bone remodeling mechanism and tumor angiogenesis. We demonstrated that exosomes promoted osteoclasts differentiation and bone resorption activity. Furthermore, exosomes potentiated tube formation of endothelial cells and increased angiogenic markers expression. We therefore investigated the micro RNA (miRNA) cargo from exosomes and their parental cells by performing small RNA sequencing through NGS Illumina platform. Hierarchical clustering highlighted a unique molecular profile of exosomal miRNA; bioinformatic analysis by DIANA-mirPath revealed that miRNAs identified take part in various biological processes and carcinogenesis. Among these miRNAs, some were already known for their involvement in the tumor microenvironment establishment, as miR-148a and miR-21-5p. Enforced expression of miR-148a and miR-21-5p in Raw264.7 and hTert immortalized umbilical vein endothelial cells recapitulated the effects induced by exosomes. Overall, our study highlighted the importance of OS exosomes in tumor microenvironment also by a specific packaging of miRNAs.

Multiple myeloma (MM) is a hematologic malignancy of differentiated plasma cells that accumulate in the bone marrow, where a complex microenvironment made by different cell types supports proliferation, survival, and drug resistance of tumor cells. MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression at posttranscriptional level. Emerging evidence indicates that miRNAs are aberrantly expressed or functionally deregulated in MM cells as the result of multiple genetic or epigenetic mechanisms and that also the tumor microenvironment regulates MM cell functions by miRNAs. Consistently, modulation of miRNA levels in MM cells has been demonstrated to impair their functional interaction with the bone marrow microenvironment and to produce significant antitumor activity even able to overcome the protective bone marrowmilieu. This review will describe the most recent findings on miRNA function in the context of MM bone marrow microenvironment, focusing on the therapeutic potential of miRNA-based approaches.