A Hotspot of TTX Contamination in the Adriatic Sea: Study on the Origin and Causative Factors.

Tetrodotoxins (TTXs), the pufferfish venom traditionally associated with Indo-Pacific area, has been reported during last decades in ever wider range of marine organisms and ever more geographical areas, including shellfish in Europe. Wild mussels (Mytilus galloprovincialis) grown in the Marche Region (N Adriatic Sea, Italy) were shown to be prone to TTX contamination during the warm season, with a suspected role of Vibrio alginolyticus characterized by non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS)-encoding genes. This work aimed to deepen the knowledge about the toxin's origin and the way through which it accumulates in mussels. A two-year study (spring-summer 2020-2021) confirmed the recurrent presence of TTX (11-68 µg kg-1) in the official monitored natural mussel beds of the Conero Riviera. During 2021, a supplementary nonroutine monitoring of a natural mussel bed in the same area was carried out weekly from June until August for TTXs and/or the presence of V. alginolyticus. Biotic (mussels, mesozooplankton, worms and phytoplankton); abiotic (water and sediment) matrices and phytoplankton assemblage characterizations were studied. Mussels showed relevant TTX contamination levels (9-296 µg kg-1) with extremely rapid TTX accumulation/depletion rates. The toxin presence in phytoplankton and its distribution in the different mussel tissues supports its possible exogenous origin. The V. alginolyticus count trend overlaps that of TTX contamination in mussels, and similar trends were reported also for some phytoplankton species. The role of V. alginolyticus carrying NRPS or PKS genes as a possible TTX source and of phytoplankton as a "potential vector" should therefore be further investigated.

A High Throughput Screening HPLC-FLD Method for Paralytic Shellfish Toxins (PSTs) Enabling Effective Official Control.

Paralytic Shellfish Toxins (PSTs) are marine biotoxins, primarily produced by dinoflagellates of the genera Gymnodinium spp., Alexandrium spp. They can accumulate in shellfish and, through the food chain, be assimilated by humans, giving rise to Paralytic Shellfish Poisoning. The maximum permitted level for PSTs in bivalves is 800 µg STX·2HCl eqv/kg (Reg. EC N° 853/2004). Until recently, the reference analytical method was the Mouse Bioassay, but Reg. EU N° 1709/2021 entered into force on 13 October 2021 and identified in the Standard EN14526:2017 or in any other internationally recognized validated method not entailing the use of live animals as official methods. Then the official control laboratories had urgently to fulfill the new requests, face out the Mouse Bioassay and implement instrumental analytical methods. The "EURLMB SOP for the analysis of PSTs by pre-column HPLC-FLD according to OMA AOAC 2005.06" also introduced a simplified semiquantitative approach to discriminate samples above and below the regulatory limit. The aim of the present paper is to present a new presence/absence test with a cut-off at 600 µg STX·2HCl eqv/kg enabling the fast discrimination of samples with very low PSTs levels from those to be submitted to the full quantitative confirmatory EN14526:2017 method. The method was implemented, avoiding the use of a large number of certified reference standards and long quantification procedures, resulting in an efficient, economical screening instrument available for official control laboratories. The protocol was fully validated, obtaining good performances in terms of repeatability (<11%) and recovery (53−106%) and accredited according to ISO/IEC 17025. The method was applied to mollusks collected from March 2021 to February 2022 along the Marche region in the frame of marine toxins official control.

Tetrodotoxins (TTXs) and Vibrio alginolyticus in Mussels from Central Adriatic Sea (Italy): Are They Closely Related?

Tetrodotoxins (TTXs), potent neurotoxins, have become an increasing concern in Europe in recent decades, especially because of their presence in mollusks. The European Food Safety Authority published a Scientific Opinion setting a recommended threshold for TTX in mollusks of 44 µg equivalent kg-1 and calling all member states to contribute to an effort to gather data in order to produce a more exhaustive risk assessment. The objective of this work was to assess TTX levels in wild and farmed mussels (Mytilus galloprovincialis) harvested in 2018-2019 along the coastal area of the Marche region in the Central Adriatic Sea (Italy). The presence of Vibrio spp. carrying the non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes, which are suspected to be involved in TTX biosynthesis, was also investigated. Out of 158 mussel samples analyzed by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (HILIC-MS/MS), 11 (7%) contained the toxins at detectable levels (8-26 µg kg-1) and 3 (2%) contained levels above the EFSA safety threshold (61-76 µg kg-1). Contaminated mussels were all harvested from natural beds in spring or summer. Of the 2019 samples, 70% of them contained V. alginolyticus strains with the NRPS and/or PKS genes. None of the strains containing NRPS and/or PKS genes showed detectable levels of TTXs. TTXs in mussels are not yet a threat in the Marche region nor in Europe, but further investigations are surely needed.

New Nanometric Solid Dispersions of Glibenclamide in Neusilin(®) UFL2.

To improve the poor water solubility and dissolution rate of the oral hypoglycemic drug glibenclamide, it was molecularly dispersed in Neusilin(®) UFL2, an amorphous synthetic form of magnesium aluminometasilicate, at different proportions; the physicochemical and biopharmaceutical properties, as well as the stability of the four different batches recovered were characterised, and it was determined that complete dispersion of glibenclamide in the amorphous polymer was obtained at the drug to Neusilin ratio of 1 to 2.5. Completely amorphous dispersion was proven by Thermal Analysis and X-Ray Powder Diffractometry. Very small particles were obtained, ranging from approximately 200 to 400 nm. The amorphous batches were physically and chemically stable for the entire duration of experiments. The physicochemical properties of the four batches were compared to those of the starting materials and physical mixtures of Neusilin(®) UFL2 and glibenclamide, the latter showing the typical behaviour of simple mixes, i.e., the additivity of properties of single components. The dissolution studies of the four solid dispersions revealed a very high dissolution rate of the completely amorphous batches (Batches 3 and 4), behaviour that was ascribed to their high Intrinsic dissolution rate due to the amorphous characteristics of the solid dispersions, to their very small particle size, and to the presence of polysorbate 80 that improved solid wettability. The technique under investigation thus proved effective for recovering stable amorphous dispersions of very small particle sizes.

Brominated flame retardants (PBDEs and HBCDs) and perfluoroalkyl substances (PFASs) in wild boars (Sus scrofa) from Central Italy.

Twenty-six samples of wild boar liver and muscle from the Central Apennine Mountain (Italy) were analysed for 19 perfluoro-alkyl substances (PFASs), 10 polybrominated diphenylethers (PBDEs) and 3 hexabromocyclododecanes (HBCDs). All samples were analysed by gas chromatography-tandem mass spectrometry for PBDEs and liquid chromatography-tandem mass spectrometry for PFASs and HBCDs, using an in-house developed analytical procedure. The brominated flame retardants (BFR) levels in livers were negligible: Σ10PBDEs reached a maximum value of 0.079 µg/kg, whereas HBCDs were not quantified in almost all of the samples analysed. BFR concentrations in muscles were higher, but not significantly therefore, for Σ10PBDEs lower bound, a mean value of 0.045 µg/kg (0.005-0.155 µg/kg range) was measured, while α-HBCD was quantified with a maximum of 0.084 µg/kg in 9 of the samples. Only two muscles contained all 3 HBCD isomers at concentrations of approximately 0.200 µg/kg. Σ19PFAS in the 26 wild boar livers was in the range 31.9-228 µg/kg, with a mean value of 87.7 µg/kg, reaching levels significantly higher than in muscles, which exhibited a mean concentration of 3.08 µg/kg (0.59-9.12 µg/kg range). Perfluorooctanesulfonic acid (PFOS) was the most prevalent compound in all liver samples, accounting for more than half of the total PFASs contamination, confirming that the liver is the primary target organ for PFOS exposure Perfluorotridecanoic acid (PFTrDA), which accounts for 25-30-% of the total contamination, was the most abundant compound in the muscle, followed by PFOS. The estimated daily intake (EDIs) of BFRs remained below the estimated chronic human daily dietary intake (Dr,h) defined from European Food Safety Authority (EFSA). Furthermore, the exposure to PFASs in muscle was 7.7 times lower than the EFSA's tolerable daily intake (TDI). In contrast, exposure due to liver consumption was significant: the EDI exceeded the EFSA's 2020 TDI by approximately 7 times.

Cyclic Imines (CIs) in Mussels from North-Central Adriatic Sea: First Evidence of Gymnodimine A in Italy.

Cyclic imines (CIs) are emerging marine lipophilic toxins (MLTs) occurring in microalgae and shellfish worldwide. The present research aimed to study CIs in mussels farmed in the Adriatic Sea (Italy) during the period 2014-2015. Twenty-eight different compounds belonging to spirolides (SPXs), gymnodimines (GYMs), pinnatoxins (PnTXs) and pteriatoxins (PtTXs) were analyzed by the official method for MLTs in 139 mussel samples collected along the Marche coast. Compounds including 13-desmethyl spirolide C (13-desMe SPX C) and 13,19-didesmethyl spirolide C (13,19-didesMe SPX C) were detected in 86% of the samples. The highest levels were generally reported in the first half of the year reaching 29.2 µg kg-1 in January/March with a decreasing trend until June. GYM A, for the first time reported in Italian mussels, was found in 84% of the samples, reaching the highest concentration in summer (12.1 µg kg-1). GYM A and SPXs, submitted to tissue distribution studies, showed the tendency to accumulate mostly in mussel digestive glands. Even if SPX levels in mussels were largely below the European Food Safety Authority (EFSA) reference of 400 µg SPXs kg-1, most of the samples contained CIs for the large part of the year. Since chronic toxicity data are still missing, monitoring is surely recommended.

Thermosensitive hybrid hydrogels for the controlled release of bioactive vancomycin in the treatment of orthopaedic implant infections.

The purpose of this work was the development of antibacterial delivery systems for vancomycin, with potential application in the prevention or treatment of orthopedic implant infections. Previous studies have shown tandem thermal gelling and Michael addition cross-linking of hydrogels based on methacrylate, acrylate or vinylsulfone triblock copolymers of PEG-p(HPMAm-lac1-2) and thiolated hyaluronic acid. In this work we exploited these α-ß unsaturated derivatives of PEG-p(HPMAm-lac1-2) triblock copolymers and used them in combination with thiolated hyaluronic acid as controlled delivery systems for vancomycin. It was found that the antibiotic was sustainably released from the hydrogel networks for at least 5 days with release kinetics depending on diffusion and dissociation of the positively charged vancomycin from the negatively charged hyaluronic acid. The release of vancomycin could be tailored mainly by HA-SH solid content and degree of thiolation. The developed hydrogels were demonstrate efficacious in preserving the structural and functional integrity of the encapsulated drug by physical immobilization within the gel network and ionic interaction with hyaluronic acid, thereby preventing vancomycin deamidation processes. Furthermore, the antimicrobial activity of vancomycin loaded hydrogels was assessed, demonstrating retention of inhibitory activity towards Staphylococcus aureus during formulation and release, with slightly increased activity of vancomycin encapsulated in hydrogels of higher HA-SH content as compared to controls.

In vivo biocompatibility of p(HPMAm-lac)-PEG hydrogels hybridized with hyaluronan.

The present study reports on the biocompatibility in vivo after intramuscular and subcutaneous administration in Balb/c mice of vinyl sulphone bearing p(HPMAm-lac1-2)-PEG-p(HPMAm-lac1-2)/thiolated hyaluronic acid hydrogels, designed as novel injectable biomaterials for potential application in the fields of tissue engineering and regenerative medicine. Ultrasonography, used as a method to study hydrogel gelation and residence time in vivo, showed that, upon injection, the biomaterial efficiently formed a hydrogel by simultaneous thermal gelation and Michael Addition cross-linking forming a viscoelastic spherical depot at the injection site. The residence time in vivo (20 days) was found to be shorter than that observed in vitro (32 days), indicating that the injected hydrogel was resorbed not only by chemical hydrolysis but also by cellular metabolism and/or enzymatic activity. Systemic biocompatibility was tested by analysing routine haematological parameters at different time-points (7, 14 and 21 days after administration) and histology of the main organs, including the haematopoietic system. No statistically significant difference between parameters of the saline-treated group and those of the hydrogel-treated group was found. Importantly, a time-dependent decrease of important pro-inflammatory cytokines (TREM1 (Triggering Receptor Expressed on Myeloid cells-1), tumour necrosis factor-α and interleukin-1ß) in cultured bone marrow cells extracted from hydrogel treated mice was observed, possibly correlated to the anti-inflammatory effect of hyaluronic acid released in time as hydrogel degraded. Copyright © 2016 John Wiley & Sons, Ltd.

Bioactive hydrogel scaffolds - advances in cartilage regeneration through controlled drug delivery.

The importance of growth factor delivery in cartilage tissue engineering is nowadays widely recognized. However, when growth factors are administered by a bolus injection, they undergo rapid clearance before they could stimulate the cells of interest at promoting cartilage repair. Their short half-lives make growth factors ineffective, unless administered at supraphysiological doses, with potentially harmful consequences on patient safety. Recently, new tissue engineering strategies relying on the combination of biodegradable scaffolds and specific biological cues, such as growth or adhesive factors or genetic material, have demonstrated that controlled release is the key factor for achieving effective cartilage repair at lower drug doses. Among all biomaterials, hydrogels have emerged as promising cartilage tissue engineering scaffolds for simultaneous cell growth and drug delivery. In fact, hydrogels can be easily loaded with cells and drugs, that are subsequently released in a controlled fashion. The success of hydrogels in controlled drug delivery for tissue engineering originates from their biocompatibility and capacity to integrate well with the host tissue. This review overviews the hydrogels technologies now available for the regeneration of cartilage that base their efficacy on the controlled release of bioactive substances able to modulate cellular behavior and to eventually lead to successful tissue repair.

Influence of pH and method of crystallization on the solid physical form of indomethacin.

The purpose of this study was to investigate the effect of pH and method of crystallization on the solid physical form of indomethacin (IDM). IDM, a non steroidal anti-inflammatory drug poorly soluble in water, underwent two different crystallization methods: crystallization by solvent evaporation under reduced pressure at 50.0°C (method A), and crystallization by cooling of solution from 50.0 to 5.0°C (method B). In both cases, several aqueous ethanolic solutions of IDM of different pHs were prepared. pHs were adjusted by adding acidic solutions (HCl 2M) or alkali (NaOH or NH4OH 2M) to an aqueous ethanolic solution of IDM. Thus, several batches were recovered after crystallization. The chemical stability of IDM was verified through (1)H NMR and mass spectroscopy (FIA-ESI-MS), that revealed that IDM degraded in strong alkali media (pH ≥ 12). Crystals obtained under different crystallization conditions at pHs of 1.0, 4.5, 7.0, 8.0, 10.0 and chemically stable were thus characterized for crystal habit by scanning electron microscopy, for thermal behaviour by differential scanning calorimetry, and thermogravimetry, and for solid state by X-ray powder diffractometry. Under the Method A, IDM always crystallized into pure metastable alpha form when solutions were acidified or alkalized respectively with HCl and NH4OH. On the contrary, in presence of NaOH, IDM crystallized under a mixture of alpha and sodium trihydrate form, because the presence of the sodium counter ion orientates the crystallization towards the formation of the trihydrate salt. Under the method B, at pH of 1.0, IDM crystallized under the alpha form; at pH 4.5, IDM crystallized under the form alpha in presence of some nuclei of gamma form; at pH 7.0, 8.0, and 10.0 for NH4OH, IDM crystallized under the most stable polymorph gamma form, whereas in presence of NaOH, a mix of alpha, and salt forms was formed whatever the pH of the solution.