Helichrysum microphyllum subsp. tyrrhenicum: a possible correlation between small volatile compounds and phloroglucinol content.

The chemical composition of the flowered aerial parts of four samples of Helichrysum microphyllum subsp. tyrrhenicum collected in South-West Sardinia was investigated with a combined focus on volatile constituents and phloroglucinols to find a possible correlation with the presence of arzanol endowed with the major anti-inflammatory activity. The volatile constituents were analysed by GC-MS as EO-HD and with HS-SPME identifying a total of 95 compounds of which 70 and 77 by EO-HD and HS-SPME respectively. The profile of the non-volatile phloroglucinols was investigated by HPLC-MS/MS. Arzanol concentrations ranged from 2.79 to 21.87 mg/g, helipyrone showed the same trend but in lower concentration. Surprisingly, leaves and stems contain higher concentration of phloroglucinols than the flowers. The concentration of arzanol was positively correlated to the one of γ-curcumene and ethylpyrone in the EO, while a negative correlation was observed with the monoterpene limonene and linalool as well as with the sesquiterpene 5-eudesmen-11-ol.

Thermodynamic Balance vs. Computational Fluid Dynamics Approach for the Outlet Temperature Estimation of a Benchtop Spray Dryer.

The use of design space (DS) is a key milestone in the quality by design (QbD) of pharmaceutical processes. It should be considered from early laboratory development to industrial production, in order to support scientists with making decisions at each step of the product's development life. Presently, there are no available data or methodologies for developing models for the implementation of design space (DS) on laboratory-scale spray dryers. Therefore, in this work, a comparison between two different modeling approaches, thermodynamics and computational fluid dynamics (CFD), to a laboratory spray dryer model have been evaluated. The models computed the outlet temperature (Tout) of the process with a new modeling strategy that includes machine learning to improve the model prediction. The model metrics calculated indicate how the thermodynamic model fits Tout data better than CFD; indeed, the error of the CFD model increases towards higher values of Tout and feed rate (FR), with a final mean absolute error of 10.43 K, compared to the 1.74 K error of the thermodynamic model. Successively, a DS of the studied spray dryer equipment has been implemented, showing how Tout is strongly affected by FR variation, which accounts for about 40 times more than the gas flow rate (Gin) in the DS. The thermodynamic model, combined with the machine learning approach here proposed, could be used as a valid tool in the QbD development of spray-dried pharmaceutical products, starting from their early laboratory stages, replacing traditional trial-and-error methodologies, preventing process errors, and helping scientists with the following scale-up.

Lipoaspirate Shows In Vitro Potential for Wound Healing.

Mesenchymal stem cells (MSCs) are a promising therapy in wound healing, although extensive time and manipulation are necessary for their use. In our previous study on cartilage regeneration, we demonstrated that lipoaspirate acts as a natural scaffold for MSCs and gives rise to their spontaneous outgrowth, together with a paracrine effect on resident cells that overcome the limitations connected to MSC use. In this study, we aimed to investigate in vitro whether the microfragmented adipose tissue (lipoaspirate), obtained with Lipogems® technology, could promote and accelerate wound healing. We showed the ability of resident cells to outgrow from the clusters of lipoaspirate encapsulated in a 3D collagen substrate as capability of repopulating a culture of human skin. Moreover, we demonstrated that the in vitro lipoaspirate paracrine effect on fibroblasts and keratinocytes proliferation, migration, and contraction rate is mediated by the release of trophic/reparative proteins. Finally, an analysis of the paracrine antibacterial effect of lipoaspirate proved its ability to secrete antibacterial factors and its ability to modulate their secretion in culture media based on a bacterial stimulus. The results suggest that lipoaspirate may be a promising approach in wound healing showing in vitro regenerative and antibacterial activities that could improve current therapeutic strategies.

Vaccination Strategies against Seasonal Influenza in Long Term Care Setting: Lessons from a Mathematical Modelling Study.

BACKGROUND: seasonal influenza in nursing homes is a major public health concern, since in EU 43,000 long term care (LTC) facilities host an estimated 2.9 million elderly residents. Despite specific vaccination campaigns, many outbreaks in such institutions are occasionally reported. We explored the dynamics of seasonal influenza starting from real data collected from a nursing home located in Italy and a mathematical model. Our aim was to identify the best vaccination strategy to minimize cases (and subsequent complications) among the guests. MATERIALS AND METHODS: after producing the contact matrices with surveys of both the health care workers (HCW) and the guests, we developed a mathematical model of the disease. The model consists of a classical SEIR part describing the spreading of the influenza in the general population and a stochastic agent based model that formalizes the dynamics of the disease inside the institution. After a model fit of a baseline scenario, we explored the impact of varying the HCW and guests parameters (vaccine uptake and vaccine efficacy) on the guest attack rates (AR) of the nursing home. RESULTS: the aggregate AR of influenza like illness in the nursing home was 36.4% (ward1 = 56%, ward2 = 33.3%, ward3 = 31.7%, ward4 = 34.5%). The model fit to data returned a probability of infection of the causal contact of 0.3 and of the shift change contact of 0.2. We noticed no decreasing or increasing AR trend when varying the HCW vaccine uptake and efficacy parameters, whereas the increase in both guests vaccine efficacy and uptake parameter was accompanied by a slight decrease in AR of all the wards of the LTC facility. CONCLUSION: from our findings we can conclude that a nursing home is still an environment at high risk of influenza transmission but the shift change room and the handover situation carry no higher relative risk. Therefore, additional preventive measures in this circumstance may be unnecessary. In a closed environment such as a LTC facility, the vaccination of guests, rather than HCWs, may still represent the cornerstone of an effective preventive strategy. Finally, we think that the extensive inclusion of real life data into mathematical models is promising and may represent a starting point for further applications of this methodology.

Diverse Effects of Natural and Synthetic Surfactants on the Inhibition of Staphylococcus aureus Biofilm.

A major challenge in the biomedical field is the creation of materials and coating strategies that effectively limit the onset of biofilm-associated infections on medical devices. Biosurfactants are well known and appreciated for their antimicrobial/anti-adhesive/anti-biofilm properties, low toxicity, and biocompatibility. In this study, the rhamnolipid produced by Pseudomonas aeruginosa 89 (R89BS) was characterized by HPLC-MS/MS and its ability to modify cell surface hydrophobicity and membrane permeability as well as its antimicrobial, anti-adhesive, and anti-biofilm activity against Staphylococcus aureus were compared to two commonly used surfactants of synthetic origin: Tween® 80 and TritonTM X-100. The R89BS crude extract showed a grade of purity of 91.4% and was composed by 70.6% of mono-rhamnolipids and 20.8% of di-rhamnolipids. The biological activities of R89BS towards S. aureus were higher than those of the two synthetic surfactants. In particular, the anti-adhesive and anti-biofilm properties of R89BS and of its purified mono- and di-congeners were similar. R89BS inhibition of S. aureus adhesion and biofilm formation was ~97% and 85%, respectively, and resulted in an increased inhibition of about 33% after 6 h and of about 39% after 72 h when compared to their chemical counterparts. These results suggest a possible applicability of R89BS as a protective coating agent to limit implant colonization.

Inhibitory Effects of Lipopeptides and Glycolipids on C. albicans-Staphylococcus spp. Dual-Species Biofilms.

Microbial biofilms strongly resist host immune responses and antimicrobial treatments and are frequently responsible for chronic infections in peri-implant tissues. Biosurfactants (BSs) have recently gained prominence as a new generation of anti-adhesive and antimicrobial agents with great biocompatibility and were recently suggested for coating implantable materials in order to improve their anti-biofilm properties. In this study, the anti-biofilm activity of lipopeptide AC7BS, rhamnolipid R89BS, and sophorolipid SL18 was evaluated against clinically relevant fungal/bacterial dual-species biofilms (Candida albicans, Staphylococcus aureus, Staphylococcus epidermidis) through quantitative and qualitative in vitro tests. C. albicans-S. aureus and C. albicans-S. epidermidis cultures were able to produce a dense biofilm on the surface of the polystyrene plates and on medical-grade silicone discs. All tested BSs demonstrated an effective inhibitory activity against dual-species biofilms formation in terms of total biomass, cell metabolic activity, microstructural architecture, and cell viability, up to 72 h on both these surfaces. In co-incubation conditions, in which BSs were tested in soluble form, rhamnolipid R89BS (0.05 mg/ml) was the most effective among the tested BSs against the formation of both dual-species biofilms, reducing on average 94 and 95% of biofilm biomass and metabolic activity at 72 h of incubation, respectively. Similarly, rhamnolipid R89BS silicone surface coating proved to be the most effective in inhibiting the formation of both dual-species biofilms, with average reductions of 93 and 90%, respectively. Scanning electron microscopy observations showed areas of treated surfaces that were free of microbial cells or in which thinner and less structured biofilms were present, compared to controls. The obtained results endorse the idea that coating of implant surfaces with BSs may be a promising strategy for the prevention of C. albicans-Staphylococcus spp. colonization on medical devices, and can potentially contribute to the reduction of the high economic efforts undertaken by healthcare systems for the treatment of these complex fungal-bacterial infections.

Medical-Grade Silicone Coated with Rhamnolipid R89 Is Effective against Staphylococcus spp. Biofilms.

Staphylococcus aureus and Staphylococcus epidermidis are considered two of the most important pathogens, and their biofilms frequently cause device-associated infections. Microbial biosurfactants recently emerged as a new generation of anti-adhesive and anti-biofilm agents for coating implantable devices to preserve biocompatibility. In this study, R89 biosurfactant (R89BS) was evaluated as an anti-biofilm coating on medical-grade silicone. R89BS is composed of homologues of the mono- (75%) and di-rhamnolipid (25%) families, as evidenced by mass spectrometry analysis. The antimicrobial activity against Staphylococcus spp. planktonic and sessile cells was evaluated by microdilution and metabolic activity assays. R89BS inhibited S. aureus and S. epidermidis growth with minimal inhibitory concentrations (MIC99) of 0.06 and 0.12 mg/mL, respectively and dispersed their pre-formed biofilms up to 93%. Silicone elastomeric discs (SEDs) coated by R89BS simple adsorption significantly counteracted Staphylococcus spp. biofilm formation, in terms of both built-up biomass (up to 60% inhibition at 72 h) and cell metabolic activity (up to 68% inhibition at 72 h). SEM analysis revealed significant inhibition of the amount of biofilm-covered surface. No cytotoxic effect on eukaryotic cells was detected at concentrations up to 0.2 mg/mL. R89BS-coated SEDs satisfy biocompatibility requirements for leaching products. Results indicate that rhamnolipid coatings are effective anti-biofilm treatments and represent a promising strategy for the prevention of infection associated with implantable devices.

Salmeterol, a ß2 Adrenergic Agonist, Promotes Adult Hippocampal Neurogenesis in a Region-Specific Manner.

Neurogenesis persists in the subgranular zone of the hippocampal formation in the adult mammalian brain. In this area, neural progenitor cells (NPCs) receive both permissive and instructive signals, including neurotransmitters, that allow them to generate adult-born neurons which can be functionally integrated in the preexisting circuit. Deregulation of adult hippocampal neurogenesis (ahNG) occurs in several neuropsychiatric and neurodegenerative diseases, including major depression, and represents a potential therapeutic target. Of interest, several studies suggested that, both in rodents and in humans, ahNG is increased by chronic administration of classical monoaminergic antidepressant drugs, suggesting that modulation of this process may participate to their therapeutic effects. Since the established observation that noradrenergic innervations from locus coeruleus make contact with NPC in the dentate gyrus, we investigated the role of beta adrenergic receptor (ß-AR) on ahNG both in vitro and in vivo. Here we report that, in vitro, activation of ß2-AR by norepinephrine and ß2-AR agonists promotes the formation of NPC-derived mature neurons, without affecting NPC survival or differentiation toward glial lineages. Additionally, we show that a selective ß2-AR agonist able to cross the blood-brain barrier, salmeterol, positively modulates hippocampal neuroplasticity when chronically administered in adult naïve mice. Indeed, salmeterol significantly increased number, maturation, and dendritic complexity of DCX+ neuroblasts. The increased number of DCX+ cells was not accompanied by a parallel increase in the percentage of BrdU+/DCX+ cells suggesting a potential prosurvival effect of the drug on neuroblasts. More importantly, compared to vehicle, salmeterol promoted ahNG, as demonstrated by an increase in the actual number of BrdU+/NeuN+ cells and in the percentage of BrdU+/NeuN+ cells over the total number of newly generated cells. Interestingly, salmeterol proneurogenic effects were restricted to the ventral hippocampus, an area related to emotional behavior and mood regulation. Since salmeterol is commonly used for asthma therapy in the clinical setting, its novel pharmacological property deserves to be further exploited with a particular focus on drug potential to counteract stress-induced deregulation of ahNG and depressive-like behavior.

Injectable Scaffolds Enriched with Silver to Inhibit Bacterial Invasion in Tissue Regeneration.

During wound healing, bacterial infections may prolong skin regeneration and tissue repair, causing delayed or incomplete healing. The therapeutic strategies currently used include general therapeutic modes, growth factors, skin substitutes, matrices and/or cell therapy. Among recent technologies, wound dressing materials comprising silver nitrate or silver sulfadiazine as the antimicrobial agent are widespread, despite their known cytotoxicity. The aim of this work was to develop and evaluate the efficacy of gelatinous injectable biomaterials composed of collagen and alginates, enriched with silver against bacterial pathogens commonly involved in wound infections. To reduce cytotoxicity, silver was used as lactate and saccharinated salts. Results show that silver-enriched beads were effective against both Gram-positive and Gram-negative strains in a concentration-dependent manner. Silver addition was more active against Staphylococcus epidermidis than against Pseudomonas aeruginosa. The antibacterial activity was localized only in the area of contact with the beads at concentrations lower than 0.3 mM, whereas at higher concentrations a larger inhibition halo was observed. No cytotoxic effect on eukaryotic cells was seen both testing the materials' extracts or the Ag-doped beads in contact tests. These results, although preliminary, suggest that these scaffolds are a promising approach for realizing injectable or spreadable functional biomaterials with antibacterial activity for applications in wound management.

Improvement of a mixture experiment model relating the component proportions to the size of nanonized itraconazole particles in extemporary suspensions.

A paper by Foglio Bonda et al. published previously in this journal (2016, Vol. 83, pp. 175-183) discussed the use of mixture experiment design and modeling methods to study how the proportions of three components in an extemporaneous oral suspension affected the mean diameter of drug particles (Zave). The three components were itraconazole (ITZ), Tween 20 (TW20), and Methocel® E5 (E5). This commentary addresses some errors and other issues in the previous paper, and also discusses an improved model relating proportions of ITZ, TW20, and E5 to Zave. The improved model contains six of the 10 terms in the full-cubic mixture model, which were selected using a different cross-validation procedure than used in the previous paper. Compared to the four-term model presented in the previous paper, the improved model fit the data better, had excellent cross-validation performance, and the predicted Zave of a validation point was within model uncertainty of the measured value.

Protumor Steering of Cancer Inflammation by p50 NF-κB Enhances Colorectal Cancer Progression.

Although tumor-associated macrophages (TAM) display a M2-skewed tumor-promoting phenotype in most cancers, in colorectal cancer, both TAM polarization and its impact remain controversial. We investigated the role of the M2-polarizing p50 NF-κB subunit in orchestrating TAM phenotype, tumor microenvironment composition, and colorectal cancer progression. We first demonstrated, by parallel studies in colitis-associated cancer (CAC) and in genetically driven ApcMin mouse models, that the p50-dependent inhibition of M1-polarized gut inflammation supported colorectal cancer development. In accordance with these studies, p50-/- mice displayed exacerbated CAC with fewer and smaller tumors, along with enhanced levels of M1/Th1 cytokines/chemokines, including IL12 and CXCL10, whose administration restrained CAC development in vivo The inflammatory profile supporting tumor resistance in colons from p50-/- tumor bearers correlated inversely with TAM load and positively with both recruitment of NK, NKT, CD8+ T cells and number of apoptotic tumor cells. In agreement, myeloid-specific ablation of p50 promoted tumor resistance in mice, whereas in colorectal cancer patients, a high number of p50+ TAMs at the invasive margin was associated with decreased IL12A and TBX21 expression and worse postsurgical outcome. Our findings point to p50 involvement in colorectal cancer development, through its engagement in the protumor activation of macrophages, and identify a candidate for prognostic and target therapeutic intervention. Cancer Immunol Res; 6(5); 578-93. ©2018 AACR.

Lipopeptides from Bacillus subtilis AC7 inhibit adhesion and biofilm formation of Candida albicans on silicone.

Candida albicans is the major fungus that colonises medical implants, causing device-associated infections with high mortality. Antagonistic bacterial products with interesting biological properties, such as biosurfactants, have recently been considered for biofilm prevention. This study investigated the activity of lipopeptide biosurfactant produced by Bacillus subtilis AC7 (AC7 BS) against adhesion and biofilm formation of C. albicans on medical-grade silicone elastomeric disks (SEDs). Chemical analysis, stability, surface activities of AC7 BS crude extract and physicochemical characterisation of the coated silicone disk surfaces were also carried out. AC7 BS showed a good reduction of water surface tension, low critical micelle concentration, good emulsification activity, thermal resistance and pH stability. Co-incubation with 2 mg ml(-1) AC7 BS significantly reduced adhesion and biofilm formation of three C. albicans strains on SEDs in a range of 67-69 % and of 56-57 %, respectively. On pre-coated SEDs, fungal adhesion and biofilm formation were reduced by 57-62 % and 46-47 %, respectively. Additionally, AC7 BS did not inhibit viability of C. albicans strains in both planktonic and sessile form. Chemical analysis of the crude extract revealed the presence of two families of lipopeptides, principally surfactin and a lower percentage of fengycin. The evaluation of surface wettability indicated that AC7 BS coating of SEDs surface was successful although uneven. AC7 BS significantly prohibits the initial deposition of C. albicans and slows biofilm growth, suggesting a potential role of biosurfactant coatings for preventing fungal infection associated with silicone medical devices.

Nanonized itraconazole powders for extemporary oral suspensions: Role of formulation components studied by a mixture design.

Itraconazole (ITZ) nanocrystal-containing powders were prepared through the combined use of high pressure homogenization (HPH) and spray drying (SD). These powders were intended as base materials for the preparation of extemporary oral suspensions of the drug. The role and the effect of stabilizers on the size of re-dispersed particles were studied using a mixture design and a Scheffé model relating the dried nanosuspension composition to the mean particle diameters. The homogenization process required a surface active agent (Tween 20) to obtain the efficient comminution of itraconazole micronized powder. SD was carried out on ITZ nanosuspensions after addition of a cellulose derivative (Methocel(®) E5) that allowed the prompt re-dispersion of nanoparticles under "in use" conditions. The powders obtained by drying of homogenized systems showed in vitro dissolution profile faster than that of the micronized drug, suggesting a potential ameliorated GI absorption of itraconazole released from the nanosuspensions.

Intracellular lactate-mediated induction of estrogen receptor beta (ERß) in biphasic malignant pleural mesothelioma cells.

Biphasic malignant pleural mesothelioma (MPM) is the second most common histotype of MPM. It is histologically characterized by the concomitant presence of epithelioid and sarcomatoid features, the latter associated with worse prognosis. In this report we describe that silencing of AKT1 in spindle-shaped biphasic MPM cells promotes the shift toward an epithelioid phenotype. Furthermore, AKT1 silencing resulted in decreased expression of the lactate/H+ symporter MCT4 and its chaperone CD147/Basigin, and in the induction of estrogen receptor ß (ERß) expression. We provide evidence that ERß expression is induced by increased intracellular lactate concentration. Spheroid culturing and tumor growth of ERß negative biphasic MPM in nude mice resulted in the induction of ERß expression and response to the selective agonist KB9520. In both models, the treatment with the ERß agonist results in reduced cell proliferation, decreased expression of MCT4 and CD147/Basigin and increased acetylation and inactivation of AKT1. Collectively, in response to metabolic changes, ERß expression is induced and exerts an anti-tumor effect through selective agonist activation. The possibility to reverse the more aggressive biphasic mesothelioma histotype by targeting ERß with a selective agonist could represent a new effective treatment strategy.

Agonist activation of estrogen receptor beta (ERß) sensitizes malignant pleural mesothelioma cells to cisplatin cytotoxicity.

BACKGROUND: Estrogen receptor (ER) ß acts as a tumor suppressor in malignant mesotheliomas. METHODS: Here we explored the anti-proliferative and anti-tumorigenic efficacy of the selective ERß agonist, KB9520, in human mesothelioma cell lines in vitro and in a mesothelioma mouse model in vivo. RESULTS: KB9520 showed significant anti-proliferative effect in ERß positive human malignant pleural mesothelioma cells in vitro. Selective activation of ERß with KB9520 sensitized the cells to treatment with cisplatin, resulting in enhanced growth inhibition and increased apoptosis. Furthermore, in CD1 nude mice mesothelioma tumor growth was significantly inhibited when KB9520 was added on top of the standard of care chemo combination cisplatin/pemetrexed, as compared to the cisplatin/pemetrexed alone group. Importantly, KB9520 exerted a protective effect to cisplatin toxicity in the non-malignant mesothelium derived MET5A cells. CONCLUSIONS: Together, the data presented suggest that selective targeting of ERß may be an efficacious stand-alone treatment option and/or become an important add-on to existing malignant mesothelioma therapy.

Improving the application of SSR polymorphism analysis coupled with Lab-on-a-chip® capillary electrophoresis to assess food authenticity: Italian pigmented rice as case study.

Genetic distances evaluated via SSR-based profiling can be usefully assessed by using capillary electrophoresis. In order to set up a method to distinguish pure Italian rice varieties from imported Asian blends, seven Italian rice genotypes and seven uncharacterized rice samples coming from outside Italy were studied using a classical SSR polymorphism analysis coupled with Lab-on-a-chip® microcapillary electrophoresis. A special algorithm for the elaboration of the raw outputs provided by the software was generated, thus overcoming the problems connected to the instrument intrinsic limits of resolution. The results showed that even considering just the smallest verifiable genetic distance between the employed samples, locally cultivated Italian rice varieties clustered separately from other foreign cultivars. Moreover, it was possible to clearly identify an artificial blend formed by Venere rice mixed with a black variety from Thailand, thus confirming the usefulness of this new post-analysis approach.

Bayesian inference from count data using discrete uniform priors.

We consider a set of sample counts obtained by sampling arbitrary fractions of a finite volume containing an homogeneously dispersed population of identical objects. We report a Bayesian derivation of the posterior probability distribution of the population size using a binomial likelihood and non-conjugate, discrete uniform priors under sampling with or without replacement. Our derivation yields a computationally feasible formula that can prove useful in a variety of statistical problems involving absolute quantification under uncertainty. We implemented our algorithm in the R package dupiR and compared it with a previously proposed Bayesian method based on a Gamma prior. As a showcase, we demonstrate that our inference framework can be used to estimate bacterial survival curves from measurements characterized by extremely low or zero counts and rather high sampling fractions. All in all, we provide a versatile, general purpose algorithm to infer population sizes from count data, which can find application in a broad spectrum of biological and physical problems.

Characterization of Muscat wines aroma evolution using comprehensive gas chromatography followed by a post-analytic approach to 2D contour plots comparison.

This study presents the application of a headspace solid-phase microextraction (HS-SPME) method on the analysis of Muscat-based wines volatiles by comprehensive two-dimensional gas chromatography (GC×GC) and Time-Of-Flight mass spectrometry (TOF-MS). The aroma patterns were established for different samples of Asti Spumante and Moscato d'Asti wines, stored in bottles for 6 months at different temperatures. Wines stored at 5 °C for 6 months did not show significant changes in flavor; otherwise, the samples stored at 15 and 25 °C, showed a significant decrease in linalool, ß-damascenone, ethyl hexanoate, and ethyl octanoate levels. In these last samples, α-terpineol, hotrienol, nerol oxide, furanic linalool oxides A/B and rose oxide concentrations significantly increased. A mathematical approach was developed and applied to raw data exported after the chromatographic course, in order (i) to normalise different 2D chromatograms, permitting their direct comparison and (ii) to automatically identify and calculate from pixel-to-pixel re-designed 2D chromatograms any differences among key volatile compounds.