Thermophilic biocatalysts for one-step conversion of citrus waste into lactic acid.

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.

Vegan grade medium component screening and concentration optimization for the fermentation of the probiotic strain Lactobacillus paracasei IMC 502® using Design of Experiments.

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.

Highly Concentrated Stabilized Hybrid Complexes of Hyaluronic Acid: Rheological and Biological Assessment of Compatibility with Adipose Tissue and Derived Stromal Cells towards Regenerative Medicine.

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.

Biosynthesis of Hesperetin, Homoeriodictyol, and Homohesperetin in a Transcriptomics-Driven Engineered Strain of Streptomyces albidoflavus.

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.

Multi-step Strategies Toward Regioselectively Sulfated M-Rich Alginates.

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.

Sustainable Exploitation of Posidonia oceanica Sea Balls (Egagropili): A Review.

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.

Physicochemical Characterization of Chitosan/Poly-γ-Glutamic Acid Glass-like Materials.

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.

Novel Insights into circRNA Saga Coming from Spermatozoa and Epididymis of HFD Mice.

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.

Enhanced Streptomyces roseochromogenes melanin production by using the marine renewable source Posidonia oceanica egagropili.

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.

Molecular Fingerprint of Human Pathological Synoviocytes in Response to Extractive Sulfated and Biofermentative Unsulfated Chondroitins.

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 (CSb), pig (CSp) and fish (CSf) chondroitins, in comparison to BC. As expected, all samples had anti-inflammatory properties, however CSb, CSf and especially BC affected more cytokines and chemokines. Based on these results and molecular weight similarity, CSf and BC were selected to further explore the synoviocytes' response. In fact, Western blot analyses showed CSf 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, CSf 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.

Argan (Argania spinosa L.) Seed Oil Cake as a Potential Source of Protein-Based Film Matrix for Pharmaco-Cosmetic Applications.

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.

Potential of Biofermentative Unsulfated Chondroitin and Hyaluronic Acid in Dermal Repair.

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.

Label-Free Quantitative Proteomics to Explore the Action Mechanism of the Pharmaceutical-Grade Triticum vulgare Extract in Speeding Up Keratinocyte Healing.

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.

Unsulfated biotechnological chondroitin by itself as well as in combination with high molecular weight hyaluronan improves the inflammation profile in osteoarthritis in vitro model.

Several studies suggest that inflammation has a pivotal role during the progression of osteoarthritis (OA) and cytokines have been identified as the main process mediators. This study aimed to explore the ability to modulate the main OA pro-inflammatory biomarkers of novel gels (H-HA/BC) based on high molecular weight hyaluronan (H-HA) and unsulfated biotechnological chondroitin (BC). For the first time, BC was tested also in combination with H-HA on human primary cells isolated from pathological knee joints. Specifically, the experiments were performed using an OA in vitro model based on human chondrocytes and synoviocytes. To evaluate the anti-inflammatory effects of H-HA/BC in comparison with H-HA and BC single gels, NF-kB, COMP-2, MyD88, MMP-13 and a wide range of cytokines, known to be specific biomarkers in OA (e.g., IL-6, IL-8, and TNF-α), were evaluated. In addition, cell morphology and proliferation occurring in the presence of either H-HA/BC or single components were assessed using time-lapse video microscopy. It was shown that synovial fluids and cells isolated from OA suffering patients, presented a cytokine pattern respondent to an ongoing inflammation status. H-HA and BC significantly reduced the levels of 23 biomarkers associated with cartilage damage. However, H-HA/BC decreased significantly 24 biological mediators and downregulated 19 of them more efficiently than the single components. In synoviocytes cultures, cytokine analyses proved that H-HA/BC gels re-established an extracellular environment more similar to a healthy condition reducing considerably the concentration of 11 analytes. Instead, H-HA and BC significantly modulated 7 (5 only with a longer treatment) and 8 biological cytokines, respectively. Our results suggest that H-HA/BC beyond the viscosupplementation effect typical for HA-based gels, can improve the inflammation status in joints and thus could be introduced as a valid protective and anti-inflammatory intraarticular device in the field of Class III medical devices for OA treatments.

Semisynthetic Isomers of Fucosylated Chondroitin Sulfate Polysaccharides with Fucosyl Branches at a Non-Natural Site.

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.

Streptomycetes as platform for biotechnological production processes of drugs.

Streptomyces is one of the most versatile genera for biotechnological applications, widely employed as platform in the production of drugs. Although streptomycetes have a complex life cycle and metabolism that would need multidisciplinary approaches, review papers have generally reported only studies on single aspects like the isolation of new strains and metabolites, morphology investigations, and genetic or metabolic studies. Besides, even if streptomycetes are extensively used in industry, very few review papers have focused their attention on the technical aspects of biotechnological processes of drug production and bioconversion and on the key parameters that have to be set up. This mini-review extensively illustrates the most innovative developments and progresses in biotechnological production and bioconversion processes of antibiotics, immunosuppressant, anticancer, steroidal drugs, and anthelmintic agents by streptomycetes, focusing on the process development aspects, describing the different approaches and technologies used in order to improve the production yields. The influence of nutrients and oxygen on streptomycetes metabolism, new fed-batch fermentation strategies, innovative precursor supplementation approaches, and specific bioreactor design as well as biotechnological strategies coupled with metabolic engineering and genetic tools for strain improvement is described. The use of whole, free, and immobilized cells on unusual supports was also reported for bioconversion processes of drugs. The most outstanding thirty investigations published in the last 8 years are here reported while future trends and perspectives of biotechnological research in the field have been illustrated. KEY POINTS: • Updated Streptomyces biotechnological processes for drug production are reported. • Innovative approaches for Streptomyces-based biotransformation of drugs are reviewed. • News about fermentation and genome systems to enhance secondary metabolite production.

Complete Lipooligosaccharide Structure from Pseudoalteromonas nigrifaciens Sq02-Rifr and Study of Its Immunomodulatory Activity.

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-Rifr, 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.

Q-switched Nd-YAG laser alone and in combination with innovative hyaluronic acid gels improve keratinocytes wound healing in vitro.

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/cm2, 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 ß3 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.

Hard-to-heal wound treated with Integra Flowable Wound Matrix: analysis and clinical observations.

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.

Lignin/Carbohydrate Complex Isolated from Posidonia oceanica Sea Balls (Egagropili): Characterization and Antioxidant Reinforcement of Protein-Based Films.

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.