Hydrogels based on galactomannan and κ-carrageenan containing immobilized biomolecules for in vivo thermal-burn wound treatment.

Hydrogels based on natural polysaccharides have demonstrated efficacy in epithelial recovery from cutaneous burn wounds. Here, we prepared a double-network hydrogel consisting of galactomannan (from Cassia grandis seeds) and κ-carrageenan (commercially sourced), cross-linked with CaCl2, as a matrix for immobilizing lactoferrin and/or Cramoll, aiming at its applicability as dressings for second-degree burn wounds. The formulations obtained [H - hydrogel, HL - hydrogel + lactoferrin, HC - hydrogel + Cramoll and HLC - hydrogel + lactoferrin + Cramoll] were analyzed rheologically as well as in terms of their stability (pH, color, microbial contamination) for 90 days. The burn was created with an aluminum bar (97 ± 3 °C) in the dorsal region of Wistar rats and subsequently treated with hydrogels (H, HL, HC, HLC) and control saline solution (S). The burn was monitored for 3, 7 and 14 days to evaluate the efficacy of the hydrogels in promoting wound healing. The hydrogels did not reveal significant pH or microbiological changes; there was an increase in brightness and a reduction in opacity for H. The rheological analysis confirmed the gel-like viscoelastic signature of the systems without substantial modification of the basic rheological characteristics, however HLC proved to be more rigid, due to rheological synergy when combining protein biomolecules. Macroscopic analyses confirmed centripetal healing with wound contraction: S < H < HC < HL < HLC. Histopathological analyses showed that hydrogel-treated groups reduced inflammation, tissue necrosis and fibrosis, while promoting re-epithelialization with focal acanthosis, especially in HLC due to a positive synergistic effect, indicating its potential as a promising therapy in the repair of burns.

Sustainable trehalose lipid production by Rhodotorula sp.: a promising bio-based alternative.

Global environmental concerns drive research toward the development of new eco-friendly compounds to replace pollutant chemicals. This study focuses on optimizing the production of trehalose lipids (TLs), which are glycolipid biosurfactants (BS) with various applications like antimicrobial or surface tension reduction. New microorganism sources, growth conditions, medium composition, purification conditions, and physicochemical properties of TLs are studied. Addressing a microscale approach, TLs production was successfully achieved using Rhodotorula sp. and Rhodococcus erythropolis to compare, with different media compositions including glucose-based and salt media supplemented with glycerol, glucose, n-hexadecane, n-dodecane. Liquid-liquid extraction using ethyl acetate and methanol was employed for compound extraction, followed by characterization using analytical methods such as Thin layer chromatography (TLC), High performance liquid chromatography (HPLC), and UHPLC. The produced TLs exhibited a minimum surface tension of 47 mN/m and a critical micellar concentration of 4.4 mg/mL. This study also identified Rhodotorula sp. as a new sustainable producer of TLs with improved productivity.

Induced Pluripotent Stem Cell-Derived Cardiomyocytes: From Regulatory Status to Clinical Translation.

Cardiovascular diseases, considered the deadliest worldwide by the World Health Organization (WHO), lack effective therapies for regenerating cardiomyocytes. With their self-renewal and pluripotency capabilities, stem cell therapies are increasingly used in precision medicine. Induced pluripotent stem cells (iPSCs) are a promising alternative to embryonic stem cells. Good Manufacturing Practice (GMP) principles are not yet adapted for large-scale production of iPSCs. Additionally, the quality risk for iPSC products may not always be possible to eliminate, potentially jeopardizing the health of patients. This review aims to identify critical quality attributes (CQAs) for iPSC-derived cardiomyocytes (iPSC-CMs) for the development of cardiovascular therapy to ensure compliance with regulations and safety for patients. To attain these goals, the literature review was conducted with articles related to iPSCs and iPSC-CM therapies, legislation, and regulatory guidelines of the European Medicines Agency (EMA), Food and Drug Administration (FDA), and Pharmaceuticals and Medical Devices Agency (PMDA). In conclusion, additional regulations and guidelines are needed to monitor differentiation, maturation, and tumorigenicity. GMP-compliant cell banks and fast-track approval systems may increase accessibility for patients.

Exploring Functionalized Magnetic Hydrogel Polyvinyl Alcohol and Chitosan Electrospun Nanofibers.

Nanofibrous materials present interesting characteristics, such as higher area/mass ratio and reactivity. These properties have been exploited in different applications, such as drug-controlled release and site-specific targeting of biomolecules for several disease treatments, including cancer. The main goal of this study was to develop magnetized nanofiber systems of lysozyme (Lys) for biological applications. The system envisaged electrospun polyvinyl alcohol (PVA) and PVA/chitosan (CS) nanofibers, loaded with Lys, crosslinked with boronic acids [phenylboronic acid (PBA), including 2-acetylphenylboronic acid (aPBA), 2-formylphenylboronic (fPBA), or bortezomib (BTZ)] and functionalized with magnetic nanobeads (IONPs), which was successfully built and tested using a microscale approach. Evaluation of the morphology of nanofibers, obtained by electrospinning, was carried out using SEM. The biological activities of the Lys-loaded PVA/CS (90:10 and 70:30) nanofibers were evaluated using the Micrococcus lysodeikticus method. To evaluate the success of the encapsulation process, the ratio of adsorbed Lys on the nanofibers, Lys activity, and in vitro Lys release were determined in buffer solution at pH values mimicking the environment of cancer cells. The viability of Caco-2 cancer cells was evaluated after being in contact with electrospun PVA + Lys and PVA/CS + Lys nanofibers, with or without boronic acid functionalation, and all were magnetized with IONPs.

Effects including photobiomodulation of galactomannan gel from Cassia grandis seeds in the healing process of second-degree burns.

The epithelium recovery of skin-burned wounds has been currently achieved by several therapies, for example, hydrogel-based dressings and photobiomodulation therapy (PBMT). Thus, this work aimed to evaluate the healing activity of Cassia grandis seeds' galactomannan gel, associated or not with PBMT, in second-degree burns. Sixty male Wistar rats were assigned to four groups: Control (CG), Gel (GG), Laser/PBMT (LG), and Laser+Gel (GLG). Burns were made with an aluminum bar (90 °C), and submitted to clinical observations diary and area measurements at specific days. Microscopic analysis was based on histological criteria. The results showed that GG, LG, and GLG had a higher contraction rate (p < 0.05) than CG on the 14th experimental day, not differing from each other (∼95 %). At 21 days, all groups showed complete contraction (p > 0.05). Considering the histological results, LG and GLG showed excellent pro-wound healing properties after 14 days; at 21 days, all groups showed wound recovery compared to previous days. In view of the macroscopic and microscopic observations, the isolated treatments (Gel or Laser) effectively accelerated healing; however, the association (Laser+Gel) promoted re-epithelialization and stromal remodeling with better evolution of epithelium recovery due to the positive synergistic effect, thus emerging as a promising therapeutic alternative in the repair of burns.

Physicochemical, Nutritional, and Medicinal Properties of Opuntia ficus-indica (L.) Mill. and Its Main Agro-Industrial Use: A Review.

The cactus, Opuntia ficus-indica (L.) Mill. (OFI) belongs to the Cactaceae family, which contains about 130 genera and nearly 1600 species. This review aims to evaluate this plant from several perspectives, namely, botanic, physicochemical, nutritional, and medicinal properties, as well as agro-industrial use. The botanical aspects and morphological characteristics of OFI enable genetic variability, ecological adaptation, and broad geographic distribution. Due to its physicochemical and nutritional composition, it has several medicinal properties appropriate (or suitable) for several industries, such as pharmaceutical, food, and cosmetics. Its fruit, the prickly pear (PP), has potential agro-industrial expansion through the application of different conservation and transformation methods, making it possible to obtain a variety of products. The PP is a source of several nutrients and is an effective system to produce varied foods, which have several advantages from a nutritional, sensory, economic, and shelf-life point of view.

Application of statistical modeling for the production of highly pure rhamnolipids using magnetic biocatalysts: Evaluating its efficiency as a bioremediation agent.

In the present study, highly pure rhamnolipids (RLs) was produced using biocatalysts immobilized on amino-functionalized chitosan coated magnetic nanoparticles. Upon immobilizing naringinase and Candida antarctica lipase B (CaLB) under the optimized conditions, an enhanced operational stability with biocatalytic loads of 935 ± 2.4 U/g (naringinase) and 825 ± 4.1 U/g (CaLB) were achieved. Subsequently, the immobilized biocatalysts were utilized sequentially in a two-step RLs synthesis process. The key parameters involved in RLs production were optimized using artificial neural network (ANN) coupled genetic algorithm (GA) and were compared with composite central design (CCD). On validating the efficiency of both models, mean square errors of 1.58% (CCD) and 1.04% (ANN) were obtained. Optimization of parameters by ANN-GA resulted in 1.2-fold increase in experimental RLs yield (80.53%), which was 1.05-fold higher when compared to CCD model. Further, to establish the efficiency of RLs as a bioremediation agent, it was utilized as washing agent. It was observed that at a soil to RLs volume of 1:05, RLs concentration of 0.4 mg/mL, a 95.35 ± 1.33% removal of Total Petroleum Hydrocarbons (TPHs) was obtained at 35 â„ƒ and 160 rpm in 75 min. Thus, this strategy provides an efficient biocatalytic toolbox for RLs synthesis, which can be effectively used as a bioremediation agent.

Triacylglycerols accumulation and glycolipids secretion by the oleaginous yeast Rhodotorula babjevae Y-SL7: Structural identification and biotechnological applications.

The newly isolated oleaginous yeast, Rhodotorula babjevae Y-SL7, was shown to accumulate high intracellular content of microbial oil (mainly triacylglycerols) and to secret, under the same culture conditions, an atypical glycolipid. This unusual behavior was induced when the strain was subjected to nitrogen limitation and high amount of carbon. A series of analytical methods was adopted in order to structurally characterize the secreted glycolipid. The latter consists of a mixture of 9 molecules formed by a polyol head group, bound through the carboxyl end of an acetylated 3-hydroxy fatty acid with C18 or C16 chain length. In addition of their physicochemical properties such as emulsifying activity on hydrophobic substrates, Y-SL7 glycolipids have shown a therapeutically promising cytotoxic effect against different cancer cell lines. In fact, Y-SL7 strain can be used for the production of triacylglycerols as energetic molecules and for the secretion of a biosurfactant of therapeutic and environmental interest.

Exploring magnetic and imprinted cross-linked enzyme aggregates of rhamnopyranosidase in microbioreactors.

The goal of this work was the development of magnetic cross link enzyme aggregates (mCLEAs) of rhamnopyranosidase (Rhmnase), prepared by chemical cross-linking with functionalized magnetite nanoparticles for glycompounds biosynthesis in microbioreactors (specially design 24-well microplate and mini-packed bed). Rhamnopyranosidase (EC number 3.2.1.40) present high potential in glycocompounds production, with applications in food and pharmaceutical industries. The influence of precipitants, cross-linkers, temperature and time on (m)CLEAs@Rhmnase development were optimized. Biocatalyst activity was accessed in the hydrolysis of 4',5,7-trihydroxyflavanone-7-rhamnoglucoside and kinetic constants in the deglycosylation reaction were evaluated. Rhmnase operational stability was enhanced in mCLEAs, retaining almost 90% initial activity after 7 cycles of 24 h each. In a mini-packed bed bioreactor a maximum volumetric productivity of 140 µmol/L.h was attained. In this bioreactor the operational stability of mCLEAs@Rhmnase were evaluated at a flow rate of 5 mL/h during 5 days and a residual activity of 95% was observed.

Healing activity evaluation of the galactomannan film obtained from Cassia grandis seeds with immobilized Cratylia mollis seed lectin.

Galactomannan films from Cassia grandis seeds, associated or not with Cramoll 1,4, were used on topical wounds of rats for the evaluation of the healing process during 14days. All of the films were evaluated by cytotoxic assay, FTIR and lectin hemagglutinating activity (HA). Forty-five male rats were submitted to aseptic dermal wounds (Ø=0.8cm) and divided in groups (n=15): control, test 1, and test 2, treated respectively with saline, galactomannan film and film with immobilized Cramoll 1,4. Macroscopic evaluations were performed by clinical observations and area measurements, and microscopic analysis by histological criteria. Epithelial cell proliferation and differentiation was immunohistochemically assessed using CK14 and PCNA. The presence of CO peaks in the FTIR spectrum confirmed the immobilization of Cramoll 1,4 in the film, while the residual HA confirmed the stability of the lectin after immobilization with 90.94% of the initial HA. The films presented non-cytotoxicity and cell viability exceeding 80%. All of the animals presented re-epithelization around 10days, furthermore test 2 group showed a diffuse response at the stromal tissue and the basal layer associated with wounds completely closed with 11days of experiment. The results suggest a promising use of the films as topical wound curatives.

Combined therapy using low level laser and chitosan-policaju hydrogel for wound healing.

We have evaluated the effect of POLI-CHI hydrogel based on policaju (POLI) from cashew tree (Anacardium occidentale L.) gum and chitosan (CHI), associated or not with Low level laser therapy (LLLT), in wound healing. Sixty male Wistar rats were assigned into four groups: POLI-CHI hydrogel (H); LLLT (L); POLI-CHI with LLLT (HL) and saline control (C). Macroscopic evaluations were carried out using clinical observations and area measurements, as well as microscopic analysis by histological criteria. H and HL presented more esthetical scar tissue and larger wound contraction compared to C. Histopathological analyzes showed: stronger presence of fibrin-leukocyte crust in L and HL at day 3; stronger collagen presence in H, L and HL; weak presence of focal necrosis at 7 and 14days in H; weak neutrophilic exudate in H, L and HL; regression of the vascular neoformation at 7days in H, and modulation of the same in L and HL. These results demonstrated that POLI-CHI contributed to more efficient healing process and modulation of the inflammation; furthermore, the combined use with LLLT subtle potentiated this process.

Can Sophorolipids prevent biofilm formation on silicone catheter tubes?

Given the impact of biofilms in health care environment and the increasing antibiotic resistance and/or tolerance, new strategies for preventing that occurrence in medical devices are obligatory. Thus, biomaterials surface functionalization with active compounds can be a valuable approach. In the present study the ability of the biosurfactants sophorolipids to prevent biofilms formation on silicone rubber aimed for medical catheters was investigated. Sophorolipids produced by Starmerella bombicola, identified by HPLC-MS/MS were used to cover silicone and surface characterization was evaluated through contact angle measurements and FTIR-ATR. Results revealed that sophorolipids presence on silicone surface decreased the hydrophobicity of the material and biofilm formation of Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922. Antibiofilm activity was evaluated through different methods and was more pronounced against S. aureus. Furthermore, biocompatibility of silicone specimens with HaCaT cells was also obtained. From this study it was possible to conclude that sophorolipids seem to be a favourable approach for coating silicone catheters. Such compounds may represent a novel source of antibiofilm agents for technological development passing through strategies of permanent functionalization of surfaces.

Selective recovery of acidic and lactonic sophorolipids from culture broths towards the improvement of their therapeutic potential.

Sophorolipids (SLs) were produced by Starmerella bombicola. The separation and purification of SLs are a complex process, since they are produced as a mixture of compounds with few structural differences. Solvent extraction is commonly used in downstream processing. In this work, an environmental friendly approach was developed for SLs recovery and purification, based on neutral polymeric sorbents, Amberlite XAD16NTM, XAD18TM, and XAD1600NTM. In batch microassays, key parameters of sorption/desorption process (e.g., contact time, temperature, sorbents, and SLs concentrations) were optimized for separation of acidic and lactonic SLs. Sorption equilibrium was reached after 2-3 h, for all the sorbents tested. Among them XAD1600NTM showed a higher sorption capacity (q max 230 mg g-1), a higher removal (≈100 %) of acidic and lactonic SLs [1 and 2.5 % (w/v)], and the best selectivity. Methanol, ethanol, and acetone were suitable for SLs elution. A selective desorption of SLs was attained with acetonitrile aqueous solutions (v/v): (1) 25 % led to 88.3 % of acidic SLs and (2) 55 % followed by methanol solution (100 %) led to 93.2 % of purified lactonic SLs. This achievement was particularly important regarding SLs potential therapeutic applications, since acidic and lactonic SLs show different biologic activities. In fact, acid SLs show higher virucidal and pro-inflammatory cytokine activity, while lactonic SLs show stronger spermicidal and anti-cancer activity.

Improved thermostable polyvinyl alcohol electrospun nanofibers with entangled naringinase used in a novel mini-packed bed reactor.

Polyvinyl alcohol (PVA) electrospun nanofibers were produced using an electrospinning technique. Key parameters (e.g. collectors, distance from needle tip to collector, among others) that influence the structure and morphology of fibers were optimized. The naringinase entrapped in PVA nanofibers retained over 100% of its initial activity after 212h of operation, at 25°C. Chemical crosslinking with several boronic acids further increased the hydrolysis temperature (up to 85°C) and yielded nanofibers with thermal stability up to 121°C. A mini packed bed reactor (PBR) developed to establish the feasibility for continuous enzymatic operation, ran for 16days at 45°C. Highest naringenin biosynthesis was attained at a flow rate of 10mLh(-1). Highest volumetric (78molL(-1)h(-1)) and specific (26molh(-1)genzyme(-1)) productivities were attained at 30mLh(-1). The activity of NGase in electrospun nanofibers remained constant for almost 16days of operation at 10mLh(-1).

Portuguese family with the co-occurrence of frontotemporal lobar degeneration and neuronal ceroid lipofuscinosis phenotypes due to progranulin gene mutation.

We and others have reported heterozygous progranulin mutations as an important cause of frontotemporal lobar degeneration (FTLD). It has been identified a complete progranulin deficiency because of a homozygous mutation in a sibling pair with neuronal ceroid lipofuscinosis (NCL). Here, we describe the first case of NCL caused by a homozygous progranulin mutation segregating in a family with neuropathological confirmed FTLD. In this FTLD-NCL family, we detail the clinical phenotype, neuropsychological evaluation and imaging data of our proband harboring a homozygous mutation, c.900_901dupGT, with serum progranulin level (<6 ng/mL). Symptoms included rapidly progressive visual deficit, slightly dysarthria, and cerebellar ataxia. The electroretinogram confirmed a severe attenuation of rod and cone responses compatible with retinal dystrophy diagnosis and magnetic resonance imaging showed severe global cerebellar atrophy. In contrast, heterozygous relatives presented behavioral variant of frontotemporal dementia (FTD) and some also developed extrapyramidal features compatible with corticobasal syndrome. Our findings suggest the importance of assessing serum progranulin levels in suspected recessive adult-onset NCL cases. Overall, a more holistic neurologic intervention is needed to guarantee a proper genetic counseling in cases like the present family where two distinct phenotypes are generated according to the individuals' mutation state.

Fluid Flow Regulation of Revascularization and Cellular Organization in a Bioengineered Liver Platform.

OBJECTIVE: Modeling of human liver development, especially cellular organization and the mechanisms underlying it, is fundamental for studying liver organogenesis and congenital diseases, yet there are no reliable models that mimic these processes ex vivo. DESIGN: Using an organ engineering approach and relevant cell lines, we designed a perfusion system that delivers discrete mechanical forces inside an acellular liver extracellular matrix scaffold to study the effects of mechanical stimulation in hepatic tissue organization. RESULTS: We observed a fluid flow rate-dependent response in cell distribution within the liver scaffold. Next, we determined the role of nitric oxide (NO) as a mediator of fluid flow effects on endothelial cells. We observed impairment of both neovascularization and liver tissue organization in the presence of selective inhibition of endothelial NO synthase. Similar results were observed in bioengineered livers grown under static conditions. CONCLUSION: Overall, we were able to unveil the potential central role of discrete mechanical stimulation through the NO pathway in the revascularization and cellular organization of a bioengineered liver. Last, we propose that this organ bioengineering platform can contribute significantly to the identification of physiological mechanisms of liver organogenesis and regeneration and improve our ability to bioengineer livers for transplantation.

Development of novel sophorolipids with improved cytotoxic activity toward MDA-MB-231 breast cancer cells.

Sophorolipids (SLs) are glycolipid biosurfactants, produced as a mixture of several compounds by some nonpathogenic yeast. In the current study, separation of individual SLs from mixtures with further evaluation of their surface properties and biologic activity on MDA-MB-321 breast cancer cell line were investigated. SLs were biosynthesized by Starmerella bombicola in a culture media supplemented with borage oil. A reverse-phase flash chromatography method with an automated system coupled with a prepacked cartridge was used to separate and purify the main SLs. Compositional analysis of SLs was performed by high-performance liquid chromatography with electrospray ionization mass spectrometry and tandem mass spectrometry. The following diacetylated lactonic SLs were isolated and purified: C18:0, C18:1, C18:2, and C18:3. The critical micelle concentration (CMC) and surface tension at CMC (γCMC ) of the purified SLs showed an increase with the number of double bonds. High cytotoxic effect against MDA-MB-231 cells was observed with C18:0 and C18:1 lactonic SLs. The cytotoxic effects of C18:3 lactonic SL on cancerous cells were for the first time studied. This cytotoxic effect was considerably higher than the promoted by acidic SLs; however, it induced a lower effect than the previously mentioned SLs, C18:0 and C18:1. To our knowledge, for the first time, C18:1 lactonic SL, in selected concentrations, proved to be able to inhibit MDA-MB-231 cell migration without compromising cell viability and to increase intracellular reactive oxygen species.

Operational stability of naringinase PVA lens-shaped microparticles in batch stirred reactors and mini packed bed reactors-one step closer to industry.

The immobilization of naringinase in PVA lens-shaped particles, a cheap and biocompatible hydrogel was shown to provide an effective biocatalyst for naringin hydrolysis, an appealing reaction in the food and pharmaceutical industries. The present work addresses the operational stability and scale-up of the bioconversion system, in various types of reactors, namely shaken microtiter plates (volume ⩽ 2 mL), batch stirred tank reactors (volume <400 mL) and a packed-bed reactor (PBR, 6.8 mL). Consecutive batch runs were performed with the shaken/stirred vessels, with reproducible and encouraging results, related to operational stability. The PBR was used to establish the feasibility for continuous operation, running continuously for 54 days at 45°C. The biocatalyst activity remained constant for 40 days of continuous operation. The averaged specific productivity was 9.07 mmol h(-1) g enzyme(-1) and the half-life of 48 days.

Microtiter plates versus stirred mini-bioreactors in biocatalysis: a scalable approach.

To place the application of miniaturized vessels as microbioreactors on a firm footing, focus has been given to engineering characterization. Studies on this matter have mostly involved carrier-free biological systems, while support-based systems have been overlooked. The present work aims to contribute to fill in such gap. Thus, it intended to establish a robust scaled down approach to identify and optimize relevant operational conditions of naringin hydrolysis by naringinase in PVA lens-shaped particles. The influence of geometric and dynamic (viz. Reynolds number) parameters was evaluated. Naringin hydrolysis in round, flat bottom MTP proved more effective than in square, pyramidal bottom. The bioconversion at MTP and stirred tank reactors scales showed that, given the 12.5-fold scale difference was in agreement between the bioconversion rates. The external mass transfer resistances were negligible as deduced from Damkohler modulus ≤1. The bioconversion was effectively scaled-up 200-fold from shaken microtiter plates to stirred tank reactors.

Sophorolipids: improvement of the selective production by Starmerella bombicola through the design of nutritional requirements.

Microtiter plates were used as minireactors to study Starmerella bombicola growth and sophorolipid (SL) production. Compositional analysis of SL mixtures by liquid chromatography with electrospray ionization tandem mass spectrometry showed similar results on SLs produced using the laboratory scale (shake flask) and the microscale (24-well microtiter plates (MTP)) approach. MTP suitability on SL production was proven, being this approach, especially advantageous on SL screening. Several hydrophilic carbon sources, hydrophobic co-substrates and nitrogen sources were supplied to culture media, and their influence on SL production was evaluated. The selection of specific hydrophobic co-substrate and nitrogen sources influenced the ratio acidic/lactonic SLs. In fact, it was observed that the production of acidic C18:1 diacetylated hydroxy fatty acid SLs was favoured when culture media was supplied with avocado, argan, sweet almond and jojoba oil or when NaNO(3) was supplied instead of urea. This last case was observed after 144 h of cultivation. A new SL, lactonic C18:3 hydroxy fatty acid diacetylated SL, was detected when borage and onagra oils were used individually as co-substrates. Overall results indicated the potential of the selective production of different and new sophorolipids by Starmerella bombicola based on the selection of carbon and nitrogen sources to culture media.