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Collagen-based membranes are class III-medical devices widely used in dental surgical procedures to favour bone regeneration. Here, we aimed to provide biophysical and biochemical data on this type of devices to support their optimal use and design/manufacturing. To the purpose, four commercial, non-crosslinked collagen-based-membranes, obtained from various sources (equine tendon, pericardium or cortical bone tissues, and porcine skin), were characterized in vitro. The main chemical, biophysical and biochemical properties, that have significant clinical implications, were evaluated. Membranes showed similar chemical features. They greatly differed in morphology as well as in porosity and density and showed a diverse ranking in relation to these latter two parameters. Samples highly hydrated in physiological medium (swelling-ratio values in the 2.5-6.0 range) and, for some membranes, an anisotropic expansion during hydration was, for the first time, highlighted. Rheological analyses revealed great differences in deformability (150-1500kPa G') also alerting about the marked variation in membrane mechanical behaviour upon hydration. Samples proved diverse sensitivity to collagenase, with the cortical-derived membrane showing the highest stability. Biological studies, using human-bone-derived cells, supported sample ability to allow cell proliferation and to prompt bone regeneration, while no relevant differences among membranes were recorded. Prediction of relative performance based on the findings was discussed. Overall, results represent a first wide panel of chemical/biophysical/biochemical data on collagen-based-membranes that 1) enhances our knowledge of these products, 2) aids their optimal use by providing clinicians with scientific basis for selecting products based on the specific clinical situation and 3) represents a valuable reference for optimizing their manufacturing., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Barrino et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: B. animalis subsp. lactis HN019 is a commercially available well-characterized probiotic with documented effects on human health, such as the ability to enhance the immune function and to balance the intestinal microbiome. Therefore, optimizing the manufacturing process to improve sustainability, increasing biomass yields and viability, and avoiding animal -derived nutrients in the medium to meet vegan consumer's needs, is currently of interest. Besides the established use of live probiotic cells, alternative supplements indicated as postbiotics, like non-viable cells and/or probiotics derived bioactive molecules might be considered as potential next generation biotherapeutics. In fact, advantages of postbiotics include fewer technological limitations, such as easier production processes and scale-up, and even higher specificity. Methods: In this work, medium design together with different fermentation strategies such as batch, fed-batch and in situ product removal on lab-scale bioreactors were combined. Medium pretreatment by ultrafiltration and protease digestion was performed to reduce polysaccharidic contaminants and facilitate the purification of secreted exopolysaccharides (EPS). The latter were isolated from the fermentation broth and characterized through NMR, GC-MS and SEC-TDA analyses. The expression of TLR-4, NF-kb and IL-6 in LPS challenged differentiated CaCo-2 cells treated with EPS, live and heat-killed B. lactis cells/broth, was evaluated in vitro by western blotting and ELISA. Zonulin was also assessed by immunofluorescence assays. Results and Discussion: The titer of viable B. lactis HN019 was increased up to 2.9 ± 0.1 x 10 10 on an animal-free semidefined medium by applying an ISPR fermentation strategy. Medium pre-treatment and a simple downstream procedure enriched the representativity of the EPS recovered (87%), the composition of which revealed the presence of mannuronic acid among other sugars typically present in polysaccharides produced by bifidobacteria. The isolated EPS, live cells and whole heat inactivated broth were compared for the first up to date for their immunomodulatory and anti-inflammatory properties and for their ability to promote intestinal barrier integrity. Interestingly, EPS and live cells samples demonstrated immune-stimulating properties by downregulating the expression of TLR-4 and NF-kb, and the ability to promote restoring the integrity of the intestinal barrier by up-regulating the expression of zonulin, one of the tight junctions forming proteins. Postbiotics in the form of heat killed broth only reduced NF-kb expression, whereas they did not seem effective in the other tested conditions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 D’ambrosio, Dabous, Sadiq, Casillo, Schiraldi, Cassese, Bedini, Corsaro and Cimini.)

Flavonoids exhibit various bioactivities including anti-oxidant, anti-tumor, anti-inflammatory, and anti-viral properties. Methylated flavonoids are particularly significant due to their enhanced oral bioavailability, improved intestinal absorption, and greater stability. The heterologous production of plant flavonoids in bacterial factories involves the need for enough biosynthetic precursors to allow for high production levels. These biosynthetic precursors are malonyl-CoA and l-tyrosine. In this work, to enhance flavonoid biosynthesis in Streptomyces albidoflavus , we conducted a transcriptomics study for the identification of candidate genes involved in l-tyrosine catabolism. The hypothesis was that the bacterial metabolic machinery would detect an excess of this amino acid if supplemented with the conventional culture medium and would activate the genes involved in its catabolism towards energy production. Then, by inactivating those overexpressed genes (under an excess of l-tyrosine), it would be possible to increase the intracellular pools of this precursor amino acid and eventually the final flavonoid titers in this bacterial factory. The RNAseq data analysis in the S. albidoflavus wild-type strain highlighted the hppD gene encoding 4-hydroxyphenylpyruvate dioxygenase as a promising target for knock-out, exhibiting a 23.2-fold change (FC) in expression upon l-tyrosine supplementation in comparison to control cultivation conditions. The subsequent knock-out of the hppD gene in S. albidoflavus resulted in a 1.66-fold increase in the naringenin titer, indicating enhanced flavonoid biosynthesis. Leveraging the improved strain of S. albidoflavus , we successfully synthesized the methylated flavanones hesperetin, homoeriodictyol, and homohesperetin, achieving titers of 2.52 mg/L, 1.34 mg/L, and 0.43 mg/L, respectively. In addition, the dimethoxy flavanone homohesperetin was produced as a byproduct of the endogenous metabolism of S. albidoflavus. To our knowledge, this is the first time that hppD deletion was utilized as a strategy to augment the biosynthesis of flavonoids. Furthermore, this is the first report where hesperetin and homoeriodictyol have been synthesized from l-tyrosine as a precursor. Therefore, transcriptomics is, in this case, a successful approach for the identification of catabolism reactions affecting key precursors during flavonoid biosynthesis, allowing the generation of enhanced production strains.

Cells and extracts derived from adipose tissue are gaining increasing attention not only in plastic surgery and for aesthetic purposes but also in regenerative medicine. The ability of hyaluronan (HA) to support human adipose stromal cell (hASC) viability and differentiation has been investigated. However, the compatibility of adipose tissue with HA-based formulation in terms of biophysical and rheological properties has not been fully addressed, although it is a key feature for tissue integration and in vivo performance. In this study, the biophysical and biochemical properties of highly concentrated (45 mg/mL) high/low-molecular-weight HA hybrid cooperative complex were assessed with a further focus on the potential application in adipose tissue augmentation/regeneration. Specifically, HA hybrid complex rheological behavior was observed in combination with different adipose tissue ratios, and hyaluronidase-catalyzed degradation was compared to that of a high-molecular-weight HA (HHA). Moreover, the HA hybrid complex's ability to induce in vitro hASCs differentiation towards adipose phenotype was evaluated in comparison to HHA, performing Oil Red O staining and analyzing gene/protein expression of PPAR-γ, adiponectin, and leptin. Both treatments supported hASCs differentiation, with the HA hybrid complex showing better results. These outcomes may open new frontiers in regenerative medicine, supporting the injection of highly concentrated hybrid formulations in fat compartments, eventually enhancing residing staminal cell differentiation and improving cell/growth factor persistence towards tissue regeneration districts.

Agri-food residues offer significant potential as a raw material for the production of L-lactic acid through microbial fermentation. Weizmannia coagulans, previously known as Bacillus coagulans, is a spore-forming, lactic acid-producing, gram-positive, with known probiotic and prebiotic properties. This study aimed to evaluate the feasibility of utilizing untreated citrus waste as a sustainable feedstock for the production of L-lactic acid in a one-step process, by using the strain W. coagulans MA-13. By employing a thermophilic enzymatic cocktail (Cellic CTec2) in conjunction with the hydrolytic capabilities of MA-13, biomass degradation was enhanced by up to 62%. Moreover, batch and fed-batch fermentation experiments demonstrated the complete fermentation of glucose into L-lactic acid, achieving a concentration of up to 44.8 g/L. These results point to MA-13 as a microbial cell factory for one-step production of L-lactic acid, by combining cost-effective saccharification with MA-13 fermentative performance, on agri-food wastes. Moreover, the potential of this approach for sustainable valorization of agricultural waste streams is successfully proven. KEY POINTS: • Valorization of citrus waste, an abundant residue in Mediterranean countries. • Sustainable production of the L-( +)-lactic acid in one-step process. • Enzymatic pretreatment is a valuable alternative to the use of chemical., (© 2024. The Author(s).)

Lactobacillus paracasei IMC502® is a commercially successful probiotic strain. However, there are no reports that investigate growth medium composition in relation to improved biomass production for this strain. The major outcome of the present study is the design and optimization of a growth medium based on vegan components to be used in the cultivation of Lactobacillus paracasei IMC502®, by using Design of Experiments. Besides comparing different carbon sources, the use of plant-based peptones as nitrogen sources was considered. In particular, the use of guar peptone as the main nitrogen source, in the optimization of fermentation media for the production of probiotics, could replace other plant peptones (e.g. potato, rice, wheat, and soy) which are part of the human diet, thereby avoiding an increase in product and process prices. A model with R2 and adjusted R2 values higher than 95% was obtained. Model accuracy was equal to 94.11%. The vegan-optimized culture medium described in this study increased biomass production by about 65% compared to growth on De Man-Rogosa-Sharpe (MRS) medium. Moreover, this approach showed that most of the salts and trace elements generally present in MRS are not affecting biomass production, thus a simplified medium preparation can be proposed with higher probiotic biomass yield and titer. The possibility to obtain viable lactic acid bacteria at high density from vegetable derived nutrients will be of great interest to specific consumer communities, opening the way to follow this approach with other probiotics of impact for human health., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

The application of adult mesenchymal stem cells (MSCs) in the field of tissue regeneration is of increasing interest to the scientific community. In particular, scaffolds and/or hydrogel based on glycosaminoglycans (GAGs) play a pivotal role due to their ability to support the in vitro growth and differentiation of MSCs toward a specific phenotype. Here, we describe different possible approaches to develop GAGs-based biomaterials, hydrogel, and polymeric viscous solutions in order to assess/develop a suitable biomimetic environment. To sustain MSCs viability and promote their differentiation for potential therapeutic applications., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Obesity is a pathophysiological condition, dependent on body fat accumulation, that progressively induces systemic oxidative stress/inflammation leading to a set of associated clinical manifestations, including male infertility. CircRNAs, covalently closed RNA molecules, are key regulators of sperm quality. Recently, we have characterized a complete profile of high-fat diet (HFD) spermatic circRNA cargo, predicting paternal circRNA dependent networks (ceRNETs), potentially involved in sperm oxidative stress and motility anomalies. In the current work, using HFD C57BL6/J male mice, orally treated with a mix of bioactive molecules (vitamin C; vitamin B12; vitamin E; selenium-L-methionine; glutathione-GSH) for 4 weeks, a reversion of HFD phenotype was observed. In addition, the functional action of the proposed formulations on circRNA biogenesis was evaluated by assessing the endogenous spermatic FUS-dependent backsplicing machinery and related circRNA cargo. After that, spermatic viability and motility were also analyzed. Paternal ceRNETs, potentially involved in oxidative stress regulation and sperm motility defects, were identified and used to suggest that the beneficial action of the food supplements here conveniently formulated on sperm motility was likely due to the recovery of circRNA profile. Such a hypothesis was, then, verified by an in vitro assay., Competing Interests: The authors declare that AMG is an employee of IBSA. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mele, Chioccarelli, Finamore, D’Agostino, d’Agostino, Cimini, Mattia, Porreca, Giori, Fasano, Cobellis, Schiraldi, Chianese and Manfrevola.)

Currently, chondroitin sulfate (CS) and hyaluronic acid (HA) pharma-grade forms are used for osteoarthritis (OA) management, CS as an oral formulations component, and HA as intra-articular injective medical devices. Recently, unsulfated chondroitin, obtained through biofermentative (BC) manufacturing, has been proposed for thermally stabilized injective preparation with HA. This study aimed to highlight the specific properties of two commercial injective medical devices, one based on HA/BC complexes and the other containing HA, extractive CS, and cyclodextrins, in order to provide valuable information for joint disease treatments. Their biophysical and biomechanical features were assayed; in addition, biological tests were performed on human pathological chondrocytes. Rheological measurements displayed similar behavior, with a slightly higher G' for HA/BC, which also proved superior stability to the hyaluronidase attack. Both samples reduced the expression of specific OA-related biomarkers such as NF-kB, interleukin 6 (IL-6), and metalloprotease-13 (MMP-13). Moreover, HA/BC better ensured chondrocyte phenotype maintenance by up-regulating collagen type 2A1 (COLII) and aggrecan (AGN). Notwithstanding, the similarity of biomolecule components, the manufacturing process, raw materials characteristics, and specific concentration resulted in affecting the biomechanical and, more interestingly, the biochemical properties, suggesting potential better performances of HA/BC in joint disease treatment.

Chondroitin sulfate (CS) is a well-known glycosaminoglycan present in a large variety of animal tissues, with an outstanding structural heterogeneity mainly related to molecular weight and sulfation pattern. Recently, few microorganisms, eventually engineered, proved able to synthesize the CS biopolymer backbone, composed of d-glucuronic acid and N-acetyl-d-galactosamine linked through alternating β-(1-3)- and β-(1-4)-glycosidic bonds, and secrete the biopolymers generally unsulfated and possibly decorated with other carbohydrates/molecules. Enzyme catalyzed/assisted methods and chemical tailored protocols allowed to obtain a variety of macromolecules not only resembling the natural extractive ones, but even enlarging the access to unnatural structural features. These macromolecules have been investigated for their bioactivity in vitro and in vivo establishing their potentialities in an array of novel applications in the biomedical field. This review aims to present an overview of the advancements in: i) the metabolic engineering strategies and the biotechnological processes towards chondroitin manufacturing; ii) the chemical approaches applied to obtain specific structural features and targeted decoration of the chondroitin backbone; iii) the biochemical and biological properties of the diverse biotechnological-sourced chondroitin polysaccharides reported so far, unraveling novel fields of applications., Competing Interests: Declaration of Competing Interest The authors declare there is no conflict of interest regarding the present review article. The authors also declare they are inventors but not assignee of patents inherent to the topic of the review article, these have been filed few years ago and reported as references in the paper., (Copyright © 2023. Published by Elsevier Inc.)

This paper sets up a new route for producing non-covalently crosslinked bio-composites by blending poly-γ-glutamic acid (γ-PGA) of microbial origin and chitosan (CH) through poly-electrolyte complexation under specific experimental conditions. CH and two different molecular weight γ-PGA fractions have been blended at different mass ratios (1/9, 2/8 and 3/7) under acidic pH. The developed materials seemed to behave like moldable hydrogels with a soft rubbery consistency. However, after dehydration, they became exceedingly hard, glass-like materials completely insoluble in water and organic solvents. The native biopolymers and their blends underwent comprehensive structural, physicochemical, and thermal analyses. The study confirmed strong physical interactions between polysaccharide and polyamide chains, facilitated by electrostatic attraction and hydrogen bonding. The materials exhibited both crystalline and amorphous structures and demonstrated good thermal stability and degradability. Described as thermoplastic and saloplastic, these bio-composites offer vast opportunities in the realm of polyelectrolyte complexes (PECs). This unique combination of properties allowed the bio-composites to function as glass-like materials, making them highly versatile for potential applications in various fields. They hold potential for use in regenerative medicine, biomedical devices, food packaging, and 3D printing. Their environmentally friendly properties make them attractive candidates for sustainable material development in various industries.

The aim of the case series was to determine the efficacy of a new medical device developed for adipose tissue restoration in the face. The medical device used the patented NAHYCO ® Hybrid Technology to deliver 45 mg of high- (1400 ± 200 kDa) and 45 mg of low- (100 ± 20 kDa) molecular-weight hyaluronan, in 2 mL. Patients and methods: Twenty-two volunteers, aged 36-60 years. Two mL of Profhilo ® Structura was injected using a 25 G cannula for each hemiface, into superficial fat compartment along the line from the preauricular area to the mandibular angle. Two injections were performed, and Profhilo Structura's effect on restoring adipose tissue was evaluated immediately after treatment, and over a 6-month follow-up. The studied medical device revealed a pseudoplastic behavior and consistency that allowed easy extrusion from a syringe. It showed a lower viscosity compared to dermal fillers, based on crosslinked HA. Clinically, the soft tissue thickness increased immediately after injection, and the clinical improvement persisted across a 6-month follow-up. The self-reported satisfaction with the treatment showed an amelioration in the midface of all the subjects enrolled, with no adverse effects. Profhilo ® Structura demonstrated a peculiar fat compartment integration, with a regenerating effect on adipose tissue senescence. The skin thickening and compaction effects were similar to those obtained using chemically crosslinked dermal fillers, while a natural look was preserved, and the use of crosslinking agents was avoided., Competing Interests: D.C. and C.S. declare no conflicts of interest. C.C. and G.B. are currently employees of IBSA Farmaceutici Italia Srl.

c-MET/hepatocyte growth factor (HGF) system deregulation is a well-known feature of malignancy in several solid tumors, and for this reason this system and its pathway have been considered as potential targets for therapeutic purposes. In previous manuscripts we reported c-MET/HGF expression and the role in testicular germ cell tumors (TGCTs) derived cell lines. We demonstrated the key role of c-Src and phosphatidylinositol 3-kinase (PI3K)/AKT adaptors in the HGF-dependent malignant behavior of the embryonal carcinoma cell line NT2D1, finding that the inhibition of these onco-adaptor proteins abrogates HGF triggered responses such as proliferation, migration, and invasion. Expanding on these previous studies, herein we investigated the role of mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK) pathways in the HGF-dependent and HGF-independent NT2D1 cells biological responses. To inhibit MAPK/ERK pathways we chose a pharmacological approach, by using U0126 inhibitor, and we analyzed cell proliferation, collective migration, and chemotaxis. The administration of U0126 together with HGF reverts the HGF-dependent activation of cell proliferation but, surprisingly, does not exert the same effect on NT2D1 cell migration. In addition, we found that the use of U0126 alone significantly promotes the acquisition of NT2D1 «migrating phenotype», while collective migration of NT2D1 cells was stimulated. Notably, the inhibition of ERK activation in the absence of HGF stimulation resulted in the activation of the AKT-mediated pathway, and this let us speculate that the paradoxical effects obtained by using U0126, which are the increase of collective migration and the acquisition of partial epithelium-mesenchyme transition (pEMT), are the result of compensatory pathways activation. These data highlight how the specific response to pathway inhibitors, should be investigated in depth before setting up therapy.

Sulfated alginates (ASs), as well as several artificially sulfated polysaccharides, show interesting bioactivities. The key factors for structure-activity relationships studies are the degree of sulfation and the distribution of the sulfate groups along the polysaccharide backbone (sulfation pattern). The former parameter can often be controlled through stoichiometry, while the latter requires the development of suitable chemical or enzymatic, regioselective methods and is still missing for ASs. In this work, a study on the regioselective installation of several different protecting groups on a d-mannuronic acid enriched (M-rich) alginate is reported in order to develop a semi-synthetic access to regioselectively sulfated AS derivatives. A detailed structural characterization of the obtained ASs revealed that the regioselective sulfation could be achieved complementarily at the O-2 or O-3 positions of M units through multi-step sequences relying upon a silylating or benzoylating reagent for the regioselective protection of M-rich alginic acid, followed by sulfation and deprotection.

Hyaluronan-(HA) short half-life in vivo limits its benefits in tissue repair. Self-esterified-HA is of great interest because it progressively releases HA, promoting tissue-regeneration longer than the unmodified-polymer. Here, the 1-ethyl-3-(3-diethylaminopropyl)carbodiimide(EDC)-hydroxybenzotriazole(HOBt) carboxyl-activating-system was evaluated for self-esterifying HA in the solid state. The aim was to propose an alternative to the time-consuming, conventional reaction of quaternary-ammonium-salts of HA with hydrophobic activating-systems in organic media, and to the EDC-mediated reaction, limited by by-product formation. Additionally, we aimed to obtain derivatives releasing defined molecular-weight(MW)-HA that would be valuable for tissue renewal. A 250 kDa-HA(powder/sponge) was reacted with increasing EDC/HOBt amounts. HA-modification was investigated through Size-Exclusion-Chromatography-Triple-Detector-Array-analyses, FT-IR/ 1 H NMR and the products(XHAs) extensively characterized. Compared to conventional protocols, the set procedure is more efficient, avoids side-reactions, allows for an easier processing to diverse clinically-usable 3D-forms, leads to products gradually releasing HA under physiological conditions with the possibility to tune the MW of the biopolymer-released. Finally, the XHAs exhibit sound stability to Bovine-Testicular-Hyaluronidase, hydration/mechanical properties suitable for wound-dressings, with improvements over available matrices, and prompt in vitro wound-regeneration, comparably to linear-HA. To the best of our knowledge, the procedure is the first valid alternative to conventional protocols for HA self-esterification with advances in the process itself and in product performance., Competing Interests: Declaration of competing interest All the authors do not have any conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Posidonia oceanica (L.) Delile is the main seagrass plant in the Mediterranean basin that forms huge underwater meadows. Its leaves, when decomposed, are transported to the coasts, where they create huge banquettes that protect the beaches from sea erosion. Its roots and rhizome fragments, instead, aggregate into fibrous sea balls, called egagropili, that are shaped and accumulated by the waves along the shoreline. Their presence on the beach is generally disliked by tourists, and, thus, local communities commonly treat them as waste to remove and discard. Posidonia oceanica egagropili might represent a vegetable lignocellulose biomass to be valorized as a renewable substrate to produce added value molecules in biotechnological processes, as bio-absorbents in environmental decontamination, to prepare new bioplastics and biocomposites, or as insulating and reinforcement materials for construction and building. In this review, the structural characteristics, and the biological role of Posidonia oceanica egagropili are described, as well as their applications in different fields as reported in scientific papers published in recent years.

Obesity is a pathophysiological disorder associated with adiposity accumulation, oxidative stress, and chronic inflammation state that is progressively increasing in younger population worldwide, negatively affecting male reproductive skills. An emerging topic in the field of male reproduction is circRNAs, covalently closed RNA molecules produced by backsplicing, actively involved in a successful spermatogenesis and in establishing high-quality sperm parameters. However, a direct correlation between obesity and impaired circRNA cargo in spermatozoa (SPZ) remains unclear. In the current work, using C57BL6/J male mice fed with a high-fat diet (HFD, 60% fat) as experimental model of oxidative stress, we investigated the impact of HFD on sperm morphology and motility as well as on spermatic circRNAs. We performed a complete dataset of spermatic circRNA content by a microarray strategy, and differentially expressed (DE)-circRNAs were identified. Using a circRNA/miRNA/target network (ceRNET) analysis, we identified circRNAs potentially involved in oxidative stress and sperm motility pathways. Interestingly, we demonstrated an enhanced skill of HFD sperm in backsplicing activity together with an inefficient epididymal circRNA biogenesis. Fused protein in sarcoma (FUS) and its ability to recruit quaking (QKI) could be involved in orchestrating such mechanism.

Background : The integrity of the intestinal barrier is fundamental to gut health and homeostasis; its damage can increase intestinal permeability, with translocation of bacteria and/or endotoxins from gut, and the onset of various intestinal diseases. Lactobacillus spp. is one of the most common probiotics normally found in fermented foods and dairy products and is known for its anti-inflammatory and immunomodulatory properties and for its ability to protect and enhance the intestinal barrier functions. The aim of this work was to evaluate the ability of different strains of Lactobacillus spp. to improve in vitro the integrity of the intestinal barrier, to exert anti-inflammatory and immunomodulatory activity and to prevent Salmonella Typhimurium and enteroinvasive Escherichia coli (EIEC) infections. Methods : We analyzed the cellular expression of tight junctions, antimicrobial peptide HBD-2, pro-inflammatory cytokines and the inhibition of pathogens adhesion and invasion in a model of co-cultured epithelial cells treated with Lactobacillus spp. Results : L. brevis, L. reuteri and L. rhamnosus proved to be more effective in protecting the intestinal epithelium. Conclusions: These in vitro studies can help select strains particularly active in their intended use to obtain consortia formulations that can have as much maximum yield as possible in terms of patient benefit.

Sea and environmental pollution due to microplastics are global problems that in recent years have attracted particular interest in the scientific community. The increase in the world population and the consequent consumerism of non-reusable materials are amplifying these problems. In this manuscript, we present novel bioplastics, which are completely biodegradable, for their potential use in food packaging, to replace fossil-fuel-derived plastic films and slow food degradation due to oxidative processes or microbial contamination. In this study, thin films based on polybutylene succinate (PBS) were prepared to reduce pollution, and different percentages by weight (1, 2 and 3 wt%) of extra virgin olive oil (EVO) and coconut oil (CO) were included to improve the chemico-physical properties of the polymer and possibly improve the functionality of the films in terms of prolonged food preservation. Attenuated total reflectance Fourier transform infrared (ATR/FTIR) spectroscopy was used to evaluate the interactions between the polymer and the oil. Furthermore, the mechanical properties and thermal behavior of the films were evaluated as a function of the oil content. A scanning electron microscopy (SEM) micrograph showed the surface morphology and the thickness of the materials. Finally, apple and kiwi were selected for a food-contact test, and the wrapped sliced fruit was monitored and evaluated for 12 days to macroscopically evaluate the oxidative process and/or eventually occurring contamination. The films were shown to reduce the browning of sliced fruit due to oxidation, and no molds were evidenced up to 10/12 days of observation with the addition of PBS, with 3 wt% of EVO achieving the best outcomes.

Pharma-grade extractive chondroitin sulfate (CS) is widely used for osteoarthritis (OA) treatment. Recently, unsulfated biofermentative chondroitin (BC) proved positive effects in OA in vitro model. This study, based on primary pathological human synoviocytes, aimed to analyze, by a multiplex assay, a panel of OA-related biomarkers in response to short-term treatments with bovine (CS b ), pig (CS p ) and fish (CS f ) chondroitins, in comparison to BC. As expected, all samples had anti-inflammatory properties, however CS b , CS f and especially BC affected more cytokines and chemokines. Based on these results and molecular weight similarity, CS f and BC were selected to further explore the synoviocytes' response. In fact, Western blot analyses showed CS f and BC were comparable, downregulating OA-related biomarkers such as the proteins mTOR, NF-kB, PTX-3 and COMP-2. Proteomic analyses, performed by applying a nano-LC-MS/MS TMT isobaric labelling-based approach, displayed the modulation of both common and distinct molecules to chondroitin treatments. Thus, CS f and BC modulated the biological mediators involved in the inflammation cascade, matrix degradation/remodeling, glycosaminoglycans' synthesis and cellular homeostasis. This study helps in shedding light on different molecular mechanisms related to OA disease that may be potentially affected not only by animal-source chondroitin sulfate but also by unsulfated biofermentative chondroitin.

Bacterial extracellular membrane vesicles (EMVs) play an active role in many physiological and pathogenic processes. Here, we report the identification and the detailed structural characterization of the capsular polysaccharide from both cells and EMVs from Shewanella vesiculosa by NMR and chemical analysis. The polysaccharide consists of a pentasaccharide repeating unit containing neutral monosaccharides together with amino sugars, of which one has never been isolated from a natural source. The adhesion ability of the polymer both on synthetic surfaces, such as polystyrene nanoparticles and on vesicles with a bilayer mimicking the bacterial membrane in the presence and absence of lipopolysaccharide was investigated. In both cases, a "CPS-corona" that could be the first stage of biofilm formation was observed. The polymer also activates Caspases on colon cancer cells, making S. vesiculosa EMVs as natural nanocarriers for drug delivery., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Osteoarthritis is a very disabling disease that can be treated with both non-pharmacological and pharmacological approaches. In the last years, pharmaceutical-grade chondroitin sulfate (CS) and glucosamine emerged as symptomatic slow-acting molecules, effective in pain reduction and improved function in patients affected by osteoarthritis. CS is a sulfated glycosaminoglycan that is currently produced mainly by extraction from animal tissues, and it is commercialized as a pharmaceutical-grade ingredient and/or food supplement. However, public concern on animal product derivatives has prompted the search for alternative non-extractive production routes. Thus, different approaches were established to obtain animal-free natural identical CS. On the other hand, the unsulfated chondroitin, which can be obtained via biotechnological processes, demonstrated promising anti-inflammatory properties in vitro , in chondrocytes isolated from osteoarthritic patients. Therefore, the aim of this study was to explore the potential of chondroitin, with respect to the better-known CS, in an in vivo mouse model of knee osteoarthritis. Results indicate that the treatment with biotechnological chondroitin (BC), similarly to CS, significantly reduced the severity of mechanical allodynia in an MIA-induced osteoarthritic mouse model. Decreased cartilage damage and a reduction of inflammation- and pain-related biochemical markers were also observed. Overall, our data support a beneficial activity of biotechnological unsulfated chondroitin in the osteoarthritis model tested, thus suggesting BC as a potential functional ingredient in pharmaceuticals and nutraceuticals with the advantage of avoiding animal tissue extraction., Competing Interests: DC and CS are inventors but not assignees of the patent WO/2010/136435, International Patent Application PCT/EP2010/057129. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cimini, Boccella, Alfano, Stellavato, Paino, Schiraldi, Guida, Perrone, Donniacuo, Tirino, Desiderio and Rinaldi.)

Since the possibility to biotechnologically produce melanin by Streptomycetes using plant biomass has been so far poorly investigated, Posidonia oceanica egagropili, a marine waste accumulating along the Mediterranean Sea coasts, was explored as a renewable source to enhance extracellular melanin production by Streptomyces roseochromogenes ATCC 13400. Therefore, different amounts of egagropili powder were added to a culture medium containing glucose, malt extract, and yeast extract, and their effect on the melanin biosynthesis was evaluated. A 2.5 g·L -1 supplementation in 120-h shake flask growths at 26 °C, at pH 6.0 and 250 rpm, was found to enhance the melanin production up to 3.94 ± 0.12 g·L -1 , a value 7.4-fold higher than the control. Moreover, 2-L batches allowed to reach a concentration of 9.20 ± 0.12 g·L -1 in 96 h with a productivity of 0.098 g·L -1 ·h -1 . Further studies also demonstrated that the melanin production enhancement was due to the synergistic effect of both the lignin carbohydrate complex and the holocellulose components of the egagropili. Finally, the pigment was purified from the broth supernatant by acidic precipitation and reversed-phase chromatography, characterized by UV absorbance and one- and two-dimensional NMR, and also tested for its chemical, antioxidant, and photo-protective properties. KEY POINTS: • S. roseochromogenes ATCC 13400 produces extracellular soluble melanin. • Egagropili added to the growth medium enhances melanin production and productivity. • Both the lignin carbohydrate complex and the holocellulose egagropili components influence the melanin biosynthesis., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Several Levilactobacillus brevis strains have the potential to be used as probiotics since they provide health benefits due to the interaction of live cells, and of their secreted products, with the host (tissues). Therefore, the development of simple fermentation processes that improve cell viability to reduce industrial production costs, and at the same time the characterization and biological evaluation of cell-free postbiotics that can further promote application, are of great interest. In the present study, small scale batch fermentations on semi defined media, deprived of animal derived raw materials, were used to optimize growth of L. brevis SP48, reaching 1.2 ± 0.4 × 10 10  CFU/ml of viable cells after 16 h of growth. Displacement, competition, and inhibition assays compared the effect, on Helicobacter pylori , of L. brevis cells to that of its partially purified potentially postbiotic fraction rich in exopolysaccharides and proteins. The expression of pro and anti-inflammatory biochemical markers indicated that both samples activated antimicrobial defenses and innate immunity in a gastric model. Moreover, these compounds also acted as modulators of the inflammatory response in a gut in vitro model. These data demonstrate that the high molecular weight compounds secreted by L. brevis SP48 can contrast H. pylori and reduce inflammation related to intestinal bowel disease, potentially overcoming issues related to the preservation of probiotic viability., Competing Interests: AMG is employed by the company R&D—IBSA Farmaceutici Italia. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cimini, D’ambrosio, Stellavato, Fusco, Corsaro, Dabous, Casillo, Donnarumma, Giori and Schiraldi.)

Animal origin chondroitin sulfate is employed as anti-inflammatory drug and food supplement against anti-osteoarthritis, but also as antioxidant, antitumor, anticoagulant, and immune-regulatory agent or as biomaterial in tissue engineering scaffolds and in drug-delivery systems. As its biological properties depend on the structural characteristics, multi-analytical approaches are necessary to correlate specific features of its heterogenic composition to the different bioactivities. This is of paramount importance to assess the efficacy of pharmaceuticals and food supplements, beyond safety quality control. This review would address the issue of chondroitin sulfate characterization according to the Pharmacopeia testing monograph point of view giving an update of the analytical novelties reported in the last ten years that might be employed for the product testing and releasing on the market. Not-instrumental (e.g. colorimetric assays) and instrumental techniques, most of them coupling diverse chromatographic separation methods with spectroscopic and spectrometry detection techniques, mono and bi-dimensional NMR approaches, are compared as tools to evaluate identity, titer, purity grade, monosaccharide and disaccharide composition, averaged molecular weight and viscosity, charge and sulfate content, impurities and related substances including the presence of other glycosaminoglycans., (Copyright © 2022. Published by Elsevier Ltd.)

Various different agri-food biomasses might be turned into renewable sources for producing biodegradable and edible plastics, potentially attractive for food, agricultural and cosmeceutical sectors. In this regard, different seeds utilized for edible and non-edible oil extraction give rise to high amounts of organic by-products, known as seed oil cakes (SOCs), potentially able to become protein-rich resources useful for the manufacturing of biodegradable films. This study reports the potential of SOC derived from Argania spinosa (argan), a well-known plant containing valuable non-refined oil suitable for food or cosmetic use, to be a promising valuable source for production of a protein-based matrix of biomaterials to be used in the pharmaco-cosmetic sector. Thus, glycerol-plasticized films were prepared by casting and drying using different amounts of argan seed protein concentrate, in the presence of increasing glycerol concentrations, and characterized for their morphological, mechanical, barrier, and hydrophilicity properties. In addition, their antioxidant activity and effects on cell viability and wound healing were investigated. The hydrophobic nature of the argan protein-based films, and their satisfying physicochemical and biological properties, suggest a biorefinery approach for the recycling of argan SOC as valuable raw material for manufacturing new products to be used in the cosmeceutical and food industries.

BDDE (1,4-butanediol-diglycidylether)-crosslinked hyaluronan (HA) hydrogels are widely used for dermo-aesthetic purposes. The rheology and stability of the gels under physiological conditions greatly affect their clinical indications and outcomes. To date, no studies investigating how these features are related to the chemistry of the polymeric network have been reported. Here, four available HA-BDDE hydrogels were studied to determine how and to what extent their rheology and stability with respect to enzymatic hydrolysis relate to the type and degree of HA structural modification. 1 H-/ 13 C-NMR analyses were associated for the quantification of the "true" HA chemical derivatization level, discriminating between HA that was effectively crosslinked by BDDE, and branched HA with BDDE that was anchored on one side. The rheology was measured conventionally and during hydration in a physiological medium. Sensitivity to bovine testicular hyaluronidase was quantified. The correlation between NMR data and gel rheology/stability was evaluated. The study indicated that (1) the gels greatly differed in the amounts of branched, crosslinked, and overall modified HA, with most of the HA being branched; (2) unexpectedly, the conventionally measured rheological properties did not correlate with the chemical data; (3) the gels' ranking in terms of rheology was greatly affected by hydration; (4) the rheology of the hydrated gels was quantitatively correlated with the amount of crosslinked HA, whereas the correlations with the total HA modification level and with the degree of branched HA were less significant; (5) increasing HA derivatization/crosslinking over 9/3 mol% did not enhance the stability with respect to hyaluronidases. These results broaden our knowledge of these gels and provide valuable information for improving their design and characterization.

Polycaprolactone nanofibers are used as scaffolds in the field of tissue engineering for tissue regeneration or drug delivery. Polycaprolactone (PCL) is a biodegradable hydrophobic polyester used to obtain implantable nanostructures, which are clinically applicable due to their biological safety. Polydatin (PD), a glycosidic precursor of resveratrol, is known for its antioxidant, antitumor, antiosteoporotic, and bone regeneration activities. We aimed to use the osteogenic capacity of polydatin to create a biomimetic innovative and patented scaffold consisting of PCL-PD for bone tissue engineering. Both osteosarcoma cells (Saos-2) and mesenchymal stem cells (MSCs) were used to test the in vitro cytocompatibility of the PD-PCL scaffold. Reverse-phase (RP) HPLC was used to evaluate the timing release of PD from the PCL-PD nanofibers and the MTT assay, scanning electron microscopy, and alkaline phosphatase (ALP) activity were used to evaluate the proliferation, adhesion, and cellular differentiation in both osteosarcoma and human mesenchymal stem cells (MSCs) seeded on PD-PCL nanofibers. The proliferation of osteosarcoma cells (Saos-2) on the PD-PCL scaffold decreased when compared to cells grown on PLC nanofibers, whereas the proliferation of MSCs was comparable in both PCL and PD-PCL nanofibers. Noteworthy, after 14 days, the ALP activity was higher in both Saos-2 cells and MSCs cultivated on PD-PCL than on empty scaffolds. Moreover, the same cells showed a spindle-shaped morphology after 14 days when grown on PD-PCL as shown by SEM. In conclusion, we provide evidence that nanofibers appropriately coated with PD support the adhesion and promote the osteogenic differentiation of both human osteosarcoma cells and MSCs.

Gelatin is widely proposed as scaffold for cartilage tissue regeneration due to its high similarities to the extracellular matrix. However, poor mechanical properties and high sensitivity to enzymatic degradation encouraged the scientific community to develop strategies to obtain better performing hydrogels. Gelatin networks, specifically gelatin-methacryloyl (GM), have been coupled to hyaluronan or chondroitin sulfate (CS). In this study, we evaluated the biophysical properties of an innovative photocross-linked hydrogel based on GM with the addition of CS or a new unsulfated biotechnological chondroitin (BC). Biophysical, mechanical, and biochemical characterization have been assessed to compare GM hydrogels to the chondroitin containing networks. Moreover, mesenchymal stem cells (MSCs) were seeded on these biomaterials in order to evaluate the differentiation toward the chondrocyte phenotype in 21 days. Rheological characterization showed that both CS and BC increased the stiffness (G' was about 2-fold), providing a stronger rigid matrix, with respect to GM alone. The biological tests confirmed the onset of MSCs differentiation process starting from 14 days of in vitro culture. In particular, the combination GM + BC resulted to be more effective than GM + CS in the up-regulation of key genes such as collagen type 2A1 (COLII), SOX-9, and aggrecan). In addition, the scanning microscope analyses revealed the cellular adhesion on materials and production of extracellular vesicles. Immunofluorescence staining confirmed an increase of COLII in presence of both chondroitins. Finally, the outcomes suggest that BC entangled within cross-linked GM matrix may represent a promising new biomaterial with potential applications in cartilage regeneration., (© 2022 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)

Corynebacterium striatum ( C. striatum ) is an emerging multidrug-resistant (MDR) pathogen associated with nosocomial infections. In this scenario, we screened the antimicrobial activity of the anthelmintic drugs doramectin, moxidectin, selamectin and niclosamide against 20 C. striatum MDR clinical isolates. Among these, niclosamide was the best performing drug against C. striatum . Niclosamide cytotoxicity was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay on immortalized human keratinocyte cells (HaCaT). After 20 h of treatment, the recorded 50% cytotoxic concentration (CC 50 ) was 2.56 μg/mL. The antibacterial efficacy was determined via disc diffusion, broth microdilution method and time-killing. Against C. striatum , niclosamide induced a growth inhibitory area of 22 mm and the minimum inhibitory concentration that inhibits 90% of bacteria (MIC 90 ) was 0.39 μg/mL, exhibiting bactericidal action. The biofilm biomass eradicating action was investigated through crystal violet (CV), MTT and confocal laser scanning microscopy (CLSM). Niclosamide affected the biofilm viability in a dose-dependent manner and degraded biomass by 55 and 49% at 0.39 μg/mL and 0.19 μg/mL. CLSM images confirmed the biofilm biomass degradation, showing a drastic reduction in cell viability. This study could promote the drug-repurposing of the anthelmintic FDA-approved niclosamide as a therapeutic agent to counteract the C. striatum MDR infections.

Probiotics are living microorganisms that give beneficial health effects while consumed, and each strain possesses diverse and unique properties and also different technological characteristics that affect its ability to be produced at large scale. Limosilactobacillus fermentum is a widely studied member of probiotics, however, few data are available on the development of fermentation and downstream processes for the production of viable biomasses for potential industrial applications. In the present study a novel L. fermentum strain was isolated from buffalo milk and used as test example for biotechnological process development. The strain was able to produce up to 10 9 CFU/mL on a (glucose based) semi-defined medium deprived of animal-derived raw materials up to the pilot scale (150 L), demonstrating improved results compared to commonly used, although industrially not suitable, media rich of casein and beef extract. The study of strain behavior in batch experiments indicated that the highest concentration of viable cells was reached after only 8 h of growth, greatly shortening the process. Moreover, initial concentrations of glucose in the medium above 30 g/L, if not supported by higher nitrogen concentrations, reduced the yield of biomass and increased production of heterolactic fermentation by-products. Biomass concentration via microfiltration on hollow fibers, and subsequent spray-drying allowed to recover about 5.7 × 10 10 CFU/g powder of viable cells, indicating strain resistance to harsh processing conditions. Overall, these data demonstrate the possibility to obtain and maintain adequate levels of viable L. fermentum cells by using a simple approach that is potentially suitable for industrial development. Moreover, since often exopolysaccharides produced by lactobacilli contribute to the strain's functionality, a partial characterization of the EPS produced by the newly identified L. fermentum strain was carried out. Finally, the effect of L. fermentum versus H. pylori in a gastric epithelial cell model was evaluated demonstrating its ability to stimulate the response of the immune system and displace the infective agent., Competing Interests: The authors declare non conflict of interest., (© 2022 The Authors. Published by Elsevier B.V.)

Since the potentialities of applications of low molecular weight poly-γ-glutamic acid (γ-PGA) chains have been so far only partially explored, the separation of diverse molecular families of them, as well as their characterization for potential bioactivity and ability to form films, were investigated. Two different approaches based on organic solvent precipitation or on ultra- and nano-filtration membrane-based purification of inexpensive commercial material were employed to obtain size-specific γ-PGA fractions, further characterized by size exclusion chromatography equipped with a triple detector array and by ultra-high-performance liquid chromatography to assess their average molecular weight and their concentration. The γ-PGA low molecular weight fractions, purified by ultra-filtration, have been shown both to counteract the desiccation and the oxidative stress of keratinocyte monolayers. In addition, they were exploited to prepare novel hydrocolloid films by both solvent casting and thermal compression, in the presence of different concentrations of glycerol used as plasticizer. These biomaterials were characterized for their hydrophilicity, thermal and mechanical properties. The hot compression led to the attainment of less resistant but more extensible films. However, in all cases, an increase in elongation at break as a function of the glycerol content was observed. Besides, the thermal analyses of hot compressed materials demonstrated that thermal stability was increased with higher γ-PGA distribution po-lymer fractions. The obtained biomaterials might be potentially useful for applications in cosmetics and as vehicle of active molecules in the pharmaceutical field.

Among the widespread malignancies colorectal cancer is the most lethal. Treatments of this malignant tumor include surgery for lesions and metastases, radiotherapy, immunotherapy, and chemotherapy. Nevertheless, novel therapies to reduce morbidity and mortality are demanding. Natural products, such as polysaccharides, can be a valuable alternative to sometimes very toxic chemotherapeutical agents, also because they are biocompatible and biodegradable biomaterials. Microbial polysaccharides have been demonstrated to fulfill this requirement. In this paper, the results about the structure and the activity of a capsular polysaccharide isolated from the psychrotroph Pseudoalteromonas nigrifaciens Sq02-Rif r , newly isolated from the fish intestine, have been described. The characterization has been obtained by spectroscopic and chemical methods, and it is supported by the bioinformatic analysis. The polymer activates Caspases 3 and 9 on colon cancer cells CaCo-2 and HCT-116, indicating a promising antitumor effect, and suggesting a potential capacity of CPS to induce apoptosis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Plant extracts have shown beneficial properties in terms of skin repair, promoting wound healing through a plethora of mechanisms. In particular, the poly-/oligosaccharidic aqueous extract of Triticum vulgare (TVE), as well as TVE-based products, shows interesting biological assets, hastening wound repair. Indeed, TVE acts in the treatment of tissue regeneration mainly on decubitus and venous leg ulcers. Moreover, on scratched monolayers, TVE prompts HaCat cell migration, correctly modulating the expression of metalloproteases toward a physiological matrix remodeling. Here, using the same HaCat-based in vitro scratch model, the TVE effect has been investigated thanks to an LFQ proteomic analysis of HaCat secretomes and immunoblotting. Indeed, the unbiased TVE effect on secreted proteins has not yet been fully understood, and it could be helpful to obtain a comprehensive picture of its bio-pharmacological profile. It has emerged that TVE treatment induces significant up-regulation of several proteins in the secretome (153 to be exact) whereas only a few were down-regulated (72 to be exact). Interestingly, many of the up-regulated proteins are implicated in promoting wound-healing-related processes, such as modulating cell-cell interaction and communication, cell proliferation and differentiation, and prompting cell adhesion and migration.

Chondroitin obtained through biotechnological processes (BC) shares similarities with both chondroitin sulfate (CS), due to the dimeric repetitive unit, and hyaluronic acid (HA), as it is unsulfated. In the framework of this experimental research, formulations containing BC with an average molecular size of about 35 KDa and high molecular weight HA (HHA) were characterized with respect to their rheological behavior, stability to enzymatic hydrolysis and they were evaluated in different skin damage models. The rheological characterization of the HHA/BC formulation revealed a G' of 92 ± 3 Pa and a G″ of 116 ± 5 Pa and supported an easy injectability even at a concentration of 40 mg/mL. HA/BC preserved the HHA fraction better than HHA alone. BTH was active on BC alone only at high concentration. Assays on scratched keratinocytes (HaCaT) monolayers showed that all the glycosaminoglycan formulations accelerated cell migration, with HA/BC fastening healing 2-fold compared to the control. In addition, in 2D HaCaT cultures, as well as in a 3D skin tissue model HHA/BC efficiently modulated mRNA and protein levels of different types of collagens and elastin remarking a functional tissue physiology. Finally, immortalized human fibroblasts were challenged with TNF-α to obtain an in vitro model of inflammation. Upon HHA/BC addition, secreted IL-6 level was lower and efficient ECM biosynthesis was re-established. Finally, co-cultures of HaCaT and melanocytes were established, showing the ability of HHA/BC to modulate melanin release, suggesting a possible effect of this specific formulation on the reduction of stretch marks. Overall, besides demonstrating the safety of BC, the present study highlights the potential beneficial effect of HHA/BC formulation in different damage dermal models.

The several interesting activities detected for fucosylated chondroitin sulfate (fCS) have fueled in the last years several efforts toward the obtainment of fCS oligosaccharides and low molecular weight (LMW) polysaccharides with a well-defined structure, in order to avoid the problems associated with the potential employment of native, sea cucumber sourced fCSs as a drug. Total synthesis and controlled depolymerization of the natural fCS polysaccharides are the main approaches to this aim; nonetheless, they present some limitations. These could be circumvented by semisynthesis, a strategy relying upon the regioselective fucosylation and sulfation of a microbial sourced polysaccharide sharing the same chondroitin backbone of fCS but devoid of any fucose (Fuc) and sulfate decoration on it. This approach is highly versatile, as it could open access also to fCS isomers carrying Fuc and sulfate groups at non-natural sites. Here we prepare for the first time some structurally homogeneous fCS isomers through a multistep procedure with a glycosylation reaction between a LMW polysaccharide acceptor and three different Fuc donors as key step. The obtained products were subjected to a detailed structural characterization by 2D-NMR. The conformational behavior was also investigated by NMR and molecular dynamics simulation methods and compared with data reported for natural fCS.

Lipopolysaccharides (LPS) are surface glycoconjugates embedded in the external leaflet of the outer membrane (OM) of the Gram-negative bacteria. They consist of three regions: lipid A, core oligosaccharide (OS), and O-specific polysaccharide or O-antigen. Lipid A is the glycolipid endotoxin domain that anchors the LPS molecule to the OM, and therefore, its chemical structure is crucial in the maintenance of membrane integrity in the Gram-negative bacteria. In this paper, we reported the characterization of the lipid A and OS structures from Pseudoalteromonas nigrifaciens Sq02-Rif r , which is a psychrotrophic Gram-negative bacterium isolated from the intestine of Seriola quinqueradiata . The immunomodulatory activity of both LPS and lipid A was also examined.

Chondroitin sulfates (CS) are a class of sulfated glycosaminoglycans involved in many biological processes. Several studies reported their protective effect against neurodegenerative conditions like Alzheimer's disease. CS are commonly derived from animal sources, but ethical concerns, the risk of contamination with animal proteins, and the difficulty in controlling the sulfation pattern have prompted research towards non-animal sources. Here we exploited two microbiological-chemical sourced CS (i.e., CS-A,C and CS-A,C,K,L) and Carbopol 974P NF/agarose semi-interpenetrating polymer networks (i.e., P.NaOH.0 and P.Ethanol.0) to set up a release system, and tested the neuroprotective role of released CS against H 2 O 2 -induced oxidative stress. After assessing that our CS (1-100 µM) require a 3 h pre-treatment for neuroprotection with SH-SY5Y cells, we evaluated whether the autoclave type (i.e., N- or B-type) affects hydrogel viscoelastic properties. We selected B-type autoclaves and repeated the study after loading CS (1 or 0.1 mg CS/0.5 mL gel). After loading 1 mg CS/0.5 mL gel, we evaluated CS release up to 7 days by 1,9-dimethylmethylene blue (DMMB) assay and verified the neuroprotective role of CS-A,C (1 µM) in the supernatants. We observed that CS-A,C exhibits a broader neuroprotective effect than CS-A,C,K,L. Moreover, sulfation pattern affects not only neuroprotection, but also drug release.

Background: Hyaluronans exist in different forms, accordingly with molecular weight and degree of crosslinking. Here, we tested the capability to induce osteogenic differentiation in hDPSCs (human dental pulp stem cells) of three hyaluronans forms: linear pharmaceutical-grade hyaluronans at high and (HHA) low molecular weight (LHA) and hybrid cooperative complexes (HCC), containing both sizes., Methods: hDPSCs were treated with HHA, LHA, HCC for 7, 14 and 21 days. The effects of hyaluronans on osteogenic differentiation were evaluated by qRT-PCR and WB of osteogenic markers and by Alizarin Red S staining. To identify the involved pathway, CD44 was analyzed by immunofluorescence, and YAP/TAZ expression was measured by qRT-PCR. Moreover, YAP/TAZ inhibitor-1 was used, and the loss of function of YAP/TAZ was evaluated by qRT-PCR, WB and immunofluorescence., Results: We showed that all hyaluronans improves osteogenesis. Among these, HCC is the main inducer of osteogenesis, along with overexpression of bone related markers and upregulating CD44. We also found that this biological process is subordinate to the activation of YAP/TAZ pathway., Conclusions: We found that HA's molecular weight can have a relevant impact on HA performance for bone regeneration, and we unveil a new molecular mechanism by which HA acts on stem cells.

Curdlan is a bacterial sourced polysaccharide, consisting of a linear backbone of β-1 → 3-linked glucose (Glc) units. The high interest in pharmaceutical applications of curdlan and derivatives thereof is fueling the study of multi-step sequences for regioselective modifications of its structure. Here we have developed semi-synthetic sequences based on a regioselective protection-sulfation-deprotection approach, allowing the access to some, new, low molecular weight curdlan polysaccharide derivatives with unprecedented sulfation patterns. Three different semi-synthetic schemes were investigated, all relying upon the installation of a cyclic benzylidene protecting group on Glc O-4,6-diols, followed by either direct sulfation and deprotection, or some additional steps - including a hydrolytic or oxidative cleavage of the benzylidene rings - prior to sulfation and deprotection. The six obtained polysaccharides were subjected to a detailed structural characterization by 2D-NMR analysis, revealing that some of them showed the majority of Glc units along the polymeric backbone decorated by unprecedented sulfation motifs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In this research work, viscosupplements based on linear, derivatized, crosslinked and complexed HA forms were extensively examined, providing data on the hydrodynamic parameters for the water-soluble-HA-fraction, rheology, sensitivity to enzymatic hydrolysis and capacity to modulate specific biomarkers' expression in human pathological chondrocytes and synoviocytes. Soluble HA ranged from 0 to 32 mg/mL and from 150 to 1330 kDa MW. The rheological behavior spanned from purely elastic to viscoelastic, suggesting the diversity of the categories that are suitable for restoring specific/different features of the healthy synovial fluid. The rheological parameters were reduced in a diverse manner upon dilution and hyaluronidases action, indicating different durations of the viscosupplementation effect. Bioactivity was found for all the samples, increasing the expression of different matrix markers (e.g., hyaluronan-synthase); however, the hybrid cooperative complexes performed better in most of the experiments. Hybrid cooperative complexes improved COLII mRNA expression (~12-fold increase vs. CTR), proved the most effective at preserving cell phenotype. In addition, in these models, the HA samples reduced inflammation. IL-6 was down-regulated vs. CTR by linear and chemically modified HA, and especially by hybrid complexes. The results represent the first comprehensive panel of data directly comparing the diverse HA forms for intra-articular injections and provide valuable information for tailoring products' clinical use as well as for designing new, highly performing HA-formulations that can address specific needs.

Microscale fermentation systems are important high throughput tools in clone selection, and bioprocess set up and optimization, since they provide several parallel experiments in controlled conditions of pH, temperature, agitation, and gas flow rate. In this work we evaluated the performance of biotechnologically relevant strains with different respiratory requirements in the micro-Matrix microbioreactor. In particular Escherichia coli K4 requires well aerated fermentation conditions to improve its native production of chondroitin-like capsular polysaccharide, a biomedically attractive polymer. Results from batch and fed-batch experiments demonstrated high reproducibility with those obtained on 2 L reactors, although highlighting a pronounced volume loss for longer-term experiments. Basfia succiniciproducens and Actinobacillus succinogenes need CO 2 addition for the production of succinic acid, a building block with several industrial applications. Different CO 2 supply modes were tested for the two strains in 24 h batch experiments and results well compared with those obtained on lab-scale bioreactors. Overall, it was demonstrated that the micro-Matrix is a useful scale-down tool that is suitable for growing metabolically different strains in simple batch process, however, a series of issues should still be addressed in order to fully exploit its potential., (© 2021 The Authors. Biotechnology Progress published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)

A lignin fraction (LF) was extracted from the sea balls of Posidonia oceanica (egagropili) and extensively dialyzed and characterized by FT-IR and NMR analyses. LF resulted water soluble and exhibited a brownish-to-black color with the highest absorbance in the range of 250-400 nm, attributed to the chromophore functional groups present in the phenylpropane-based polymer. LF high-performance size exclusion chromatography analysis showed a highly represented (98.77%) species of 34.75 kDa molecular weight with a polydispersity index of 1.10 and an intrinsic viscosity of 0.15. Quantitative analysis of carbohydrates indicated that they represented 28.3% of the dry weight of the untreated egagropili fibers and 72.5% of that of LF. In particular, eight different monosaccharides were detected (fucose, arabinose, rhamnose, galactose, glucose, xylose, glucosamine and glucuronic acid), glucuronic acid (46.6%) and rhamnose (29.6%) being the most present monosaccharides in the LF. Almost all the phenol content of LF (113.85 ± 5.87 mg gallic acid eq/g of extract) was water soluble, whereas around 22% of it consisted of flavonoids and only 10% of the flavonoids consisted of anthocyanins. Therefore, LF isolated from egagropili lignocellulosic material could be defined as a water-soluble lignin/carbohydrate complex (LCC) formed by a phenol polymeric chain covalently bound to hemicellulose fragments. LCC exhibited a remarkable antioxidant activity that remained quite stable during 6 months and could be easily incorporated into a protein-based film and released from the latter overtime. These findings suggest egagropili LCC as a suitable candidate as an antioxidant additive for the reinforcement of packaging of foods with high susceptibility to be deteriorated in aerobic conditions.

Skin healing defects severely impair the quality of life of millions of people and burden healthcare systems globally. The therapeutic approach to these pathologies still represents a challenge. Novel scaffolds, used as dermal substitutes, possibly represent a promising strategy in complex wound management. Integra Flowable Wound Matrix (IFWM) is composed of a lyophilised, micronised form of collagen/chondroitin sulphate matrix, already used in regenerative medicine and endorsed in the therapy of diabetic foot lesions. In this paper, IFWM was applied to a tunnelling hard-to-heal skin lesion in order to restore tissue integrity. Although the different phases of skin wound healing are well established, the molecular mechanism underpinning IFWM-induced tissue repair are almost unknown. Here, we report, for the first time, the comparative analysis of molecular, histological and clinical observations of the healing process of a hard-to-heal tunnelling skin wound. The therapeutic success of this clinical case allowed us to recommend the use of IFWM as a tissue substitute in this rare type of hard-to-heal wound in which the high inflammatory status hampered the natural healing process.

During the last years, several attempts have been accomplished to improve the wound healing. Device application aimed at enhancing skin ability to reconstruct its damaged sites through a proper dermal regenerative process. In particular, Q-switched Nd-YAG laser (Medlite C6 laser, Conbio, USA) applied with a fluence of 8 J/cm 2 , a pulse width of 5 ns, and a spot size of 4 mm exerts a photo-mechanical action that improve skin repair. Besides, hyaluronan hybrid cooperative complexes (HCC) widely exploited in dermoesthetic applications proved specific actions on keratinocytes and fibroblasts monolayer repair. We evaluated this specific laser treatment in vitro on a wound healing model based on human keratinocytes (HaCaT) alone and in combination with HCC. In addition, we evaluated key biomarkers of dermal repair. Scratched HaCaT monolayers were treated with laser and successively with HA-based formulations (HHA and HCC). For each treatment and the control samples, at least 3 different wells were analyzed. Wound closure was quantified, measuring five view filed for each well at increasing incubation time, exploiting time lapse videomicroscopy and image analysis, permitting to compare the different healing rate of treatments respect to control. By real-time PCR and western blotting, we evaluated biomarkers of wound regeneration, such as integrins, aquaporin three (AQP3), and proinflammatory cytokines. The ANOVA test was used to assess statistical significance of the results obtained. Laser-treated cells achieved wound closure in about 37 h, faster than the control, while when coupled to HCC, the complete reparation was obtained in 24 h. Integrin αV was upregulated by treatments, with in particular about four-fold increase respect to the control when HCC + laser was used. In addition, integrin β3 was upregulated by all treatments especially with the combination of laser and HCC proved more efficient than others (~ 14-folds). A slighter but significant increase of AQP3 gene expression of 61% was found for laser treatment while the latter combined with HCC determined an upregulation of 72%. By coupling laser treatment and HCC, further healing improvement and consistent biomarker modulation was observed. Our results may support clinical implementation of new dermatology protocols conjugating laser treatments with topical or injective HA formulations as a valid tool in treatments to repair scars or other skin defects.

Diabetic foot ulcer (DFU) is a diabetes complication which greatly impacts the patient's quality of life, often leading to amputation of the affected limb unless there is a timely and adequate management of the patient. DFUs have a high economic impact for the national health system. Data have indeed shown that DFUs are a major cause of hospitalization for patients with diabetes. Based on that, DFUs represent a very important challenge for the national health system. Especially in developed countries diabetic patients are increasing at a very high rate and as expected, also the incidence of DFUs is increasing due to longevity of diabetic patients in the western population. Herein, the surgical approach focused on the targeted use of the acellular dermal matrix has been integrated with biochemical and morphological/histological analyses to obtain evidence-based information on the mechanisms underlying tissue regeneration. In this research report, the clinical results indicated decreased postoperative wound infection levels and a short healing time, with a sound regeneration of tissues. Here we demonstrate that the key biomarkers of wound healing process are activated at gene expression level and also synthesis of collagen I, collagen III and elastin is prompted and modulated within the 28-day period of observation. These analyses were run on five patients treated with Integra ® sheet and five treated with the injectable matrix Integra ® Flowable, for cavitary lesions. In fact, clinical evaluation of improved healing was, for the first time, supported by biochemical and histological analyses. For these reasons, the present work opens a new scenario in DFUs treatment and follow-up, laying the foundation for a tailored protocol towards complete healing in severe pathological conditions.

Gelatin hydrogels by microbial-transglutaminase crosslinking are being increasingly exploited for tissue engineering, and proved high potential in bone regeneration. This study aimed to evaluate, for the first time, the combination of enzymatically crosslinked gelatin with hyaluronan and the newly developed biotechnological chondroitin in enhancing osteogenic potential. Gelatin enzymatic crosslinking was carried out in the presence of hyaluronan or of a hyaluronan-chondroitin mixture, obtaining semi-interpenetrating gels. The latter proved lower swelling extent and improved stiffness compared to the gelatin matrix alone, whilst maintaining high stability. The heteropolysaccharides were retained for 30 days in the hydrogels, thus influencing cell response over this period. To evaluate the effect of hydrogel composition on bone regeneration, materials were seeded with human dental pulp stem cells and osteogenic differentiation was assessed. The expression of osteocalcin (OC) and osteopontin (OPN), both at gene and protein level, was evaluated at 7, 15 and 30 days of culture. Scanning electron microscopy (SEM) and two-photon microscope observations were performed to assess bone-like extracellular matrix (ECM) deposition and to observe the cell penetration depth. In the presence of the heteropolysaccharides, OC and OPN expression was upregulated and a higher degree of calcified matrix formation was observed. Combination with hyaluronan and chondroitin improved both the biophysical properties and the biological response of enzymatically crosslinked gelatin, fastening bone deposition., (© The Author(s) 2021. Published by Oxford University Press.)