Biocellulose-based hydrogel dressing as a strategy for the management of chronic arterial wounds.

PURPOSE: To evaluate using a biocellulose-based hydrogel as an adjuvant in the healing process of arterial ulcers. METHODS: A prospective single group quasi-experimental study was carried out with chronic lower limb arterial ulcer patients. These patients received biocellulose-based hydrogel dressings and outpatient guidance on dressing and periodic reassessments. The primary outcomes were the ulcer-healing rate and product safety, which were assessed by ulcer area measured in photographic records of pre-treatment and posttreatment after 7, 30, and 60 days. Secondary outcomes were related to clinical assessment by the quality-of-life scores (SF-36 and EQ-5D) and pain, evaluated by the visual analogue scale (VAS). RESULTS: Seventeen participants were included, and one of them was excluded. Six patients (37%) had complete wound healing, and all patients had a significant reduction in the ulcer area during follow-up (233.6mm2 versus 2.7mm2) and reduction on the score PUSH 3.0 (p < 0.0001). The analysis of the SF-36 and EQ-5D questionnaires showed a statistically significant improvement in almost all parameters analyzed and with a reduction of pain assessed by the VAS. CONCLUSIONS: The biocellulose-based hydrogel was safe and showed a good perspective to promoting the necessary conditions to facilitate partial or complete healing of chronic arterial ulcers within a 60-day follow-up. Quality of life and pain were positively affected by the treatment.

Nonclinical Evaluation of Single-Mutant E. coli Asparaginases Obtained by Double-Mutant Deconvolution: Improving Toxicological, Immune and Inflammatory Responses.

Acute lymphoblastic leukaemia is currently treated with bacterial L-asparaginase; however, its side effects raise the need for the development of improved and efficient novel enzymes. Previously, we obtained low anti-asparaginase antibody production and high serum enzyme half-life in mice treated with the P40S/S206C mutant; however, its specific activity was significantly reduced. Thus, our aim was to test single mutants, S206C and P40S, through in vitro and in vivo assays. Our results showed that the drop in specific activity was caused by P40S substitution. In addition, our single mutants were highly stable in biological environment simulation, unlike the double-mutant P40S/S206C. The in vitro cell viability assay demonstrated that mutant enzymes have a higher cytotoxic effect than WT on T-cell-derived ALL and on some solid cancer cell lines. The in vivo assays were performed in mice to identify toxicological effects, to evoke immunological responses and to study the enzymes' pharmacokinetics. From these tests, none of the enzymes was toxic; however, S206C elicited lower physiological changes and immune/allergenic responses. In relation to the pharmacokinetic profile, S206C exhibited twofold higher activity than WT and P40S two hours after injection. In conclusion, we present bioengineered E. coli asparaginases with high specific enzyme activity and fewer side effects.

Exploring the potential of asparagine restriction in solid cancer treatment: recent discoveries, therapeutic implications, and challenges.

Asparagine is a non-essential amino acid crucial for protein biosynthesis and function, and therefore cell maintenance and growth. Furthermore, this amino acid has an important role in regulating several metabolic pathways, such as tricarboxylic acid cycle and the urea cycle. When compared to normal cells, tumor cells typically present a higher demand for asparagine, making it a compelling target for therapy. In this review article, we investigate different facets of asparagine bioavailability intricate role in malignant tumors raised from solid organs. We take a comprehensive look at asparagine synthetase expression and regulation in cancer, including the impact on tumor growth and metastasis. Moreover, we explore asparagine depletion through L-asparaginase as a potential therapeutic method for aggressive solid tumors, approaching different formulations of the enzyme and combinatory therapies. In summary, here we delve into studies about endogenous and exogenous asparagine availability in solid cancers, analyzing therapeutic implications and future challenges.

Bacterial cellulose nanocrystals obtained through enzymatic and acidic routes: A comparative study of their main properties and in vitro biological responses.

Cellulose nanocrystals (CNCs) are crystalline domains isolated from cellulosic fibers. They have been utilized in a wide range of applications, such as reinforcing fillers, antibacterial agents and manufacturing of biosensors. Whitin this context, the aim of this work was to obtain and analyze CNCs extracted from bacterial nanocellulose (BNC) using two distinct methods combined with milling pre-treatment: an acidic hydrolysis using 64 % sulfuric acid and an enzymatic hydrolysis using a commercial cellulase enzyme mixture. The CNCs obtained from the enzymatic route (e-CNCs) were observed to be spherical nanoparticles with diameter of 56 ± 11 nm. In contrast, the CNCs from the acid hydrolysis (a-CNCs) appeared as needle-shaped nanoparticles with a high aspect ratio with lengths/widths of 158 ± 64 nm/11 ± 2 nm. The surface zeta potential (ZP) of the a-CNCs was -30,8 mV, whereas the e-CNCs has a potential of +2.70 ± 3.32 mV, indicating that a-CNCs consisted of negatively charged particles with higher stability in solution. Although the acidic route resulted in nanocrystals with a slightly higher crystallinity index compared to the enzymatic route, e-CNCs was found to be more thermally stable than BNC and a-CNCs. Here, we also confirmed the safety of a-CNCs and e-CNCs using L929 cell line. Lastly, this article describes two different CNCs synthesis approaches that leads to the formation of nanoparticles with different dimensions, morphology and unique physicochemical properties. To the best of our knowledge, this is the first study to yield spherical nanoparticles as a result of BNC enzymatic treatment.

Membranes composed of poly(lactic acid)/poly(ethylene glycol) and Ora-pro-nóbis (Pereskia aculeata Miller) extract for dressing applications.

Wounds are considered one of the most critical medical conditions that must be managed appropriately due to the psychological and physical stress they cause for patients, as well as creating a substantial financial burden on patients and global healthcare systems. Nowadays, there is a growing interest in developing nanofiber mats loaded with varying plant extracts to meet the urgent need for advanced wound ressings. This study investigated the development and characterization of poly(lactic acid) (PLA)/ poly(ethylene glycol) (PEG) nanofiber membranes incorporated with Ora-pro-nóbis (OPN; 12.5, 25, and 50 % w/w) by the solution-blow-spinning (SBS) technique. The PLA/PEG and PLA/PEG/OPN nanofiber membranes were characterized by scanning electron microscopy (SEM), thermal properties (TGA and DSC), Fourier transform infrared spectroscopy (FTIR), contact angle measurements and water vapor permeability (WVTR). In addition, the mats were analyzed for swelling properties in vitro cell viability, and fibroblast adhesion (L-929) tests. SEM images showed that smooth and continuous PLA/PEG and PLA/PEG/OPN nanofibers were obtained with a diameter distribution ranging from 171 to 1533 nm. The PLA/PEG and PLA/PEG/OPN nanofiber membranes showed moderate hydrophobicity (~109-120°), possibly preventing secondary injuries during dressing removal. Besides that, PLA/PEG/OPN nanofibers exhibited adequate WVTR, meeting wound healing requirements. Notably, the presence of OPN gave the PLA/PEG membranes better mechanical properties, increasing their tensile strength (TS) from 3.4 MPa (PLA/PEG) to 5.3 MPa (PLA/PEG/OPN), as well as excellent antioxidant properties (Antioxidant activity with approximately 45 % oxidation inhibition). Therefore, the nanofiber mats based on PLA/PEG, especially those incorporated with OPN, are promising options for use as antioxidant dressings to aid skin healing.

3D Filaments Based on Polyhydroxy Butyrate-Micronized Bacterial Cellulose for Tissue Engineering Applications.

In this work, scaffolds based on poly(hydroxybutyrate) (PHB) and micronized bacterial cellulose (BC) were produced through 3D printing. Filaments for the printing were obtained by varying the percentage of micronized BC (0.25, 0.50, 1.00, and 2.00%) inserted in relation to the PHB matrix. Despite the varying concentrations of BC, the biocomposite filaments predominantly contained PHB functional groups, as Fourier transform infrared spectroscopy (FTIR) demonstrated. Thermogravimetric analyses (i.e., TG and DTG) of the filaments showed that the peak temperature (Tpeak) of PHB degradation decreased as the concentration of BC increased, with the lowest being 248 °C, referring to the biocomposite filament PHB/2.0% BC, which has the highest concentration of BC. Although there was a variation in the thermal behavior of the filaments, it was not significant enough to make printing impossible, considering that the PHB melting temperature was 170 °C. Biological assays indicated the non-cytotoxicity of scaffolds and the provision of cell anchorage sites. The results obtained in this research open up new paths for the application of this innovation in tissue engineering.

Combining Polymer and Cyclodextrin Strategy for Drug Release of Sulfadiazine from Electrospun Fibers.

This study reports the fabrication of polymeric matrices through electrospinning using polymethyl methacrylate (PMMA) and poly(lactic-co-glycolic acid) (PLGA), biocompatible polymers commonly used in medical systems. These polymers were combined with an antibacterial drug, sulfadiazine sodium salt (SDS) or its supramolecular system formed with hydroxypropyl-ß-cyclodextrin (HPß/CD) at 1:1 molar ratio, aiming to assemble a transdermal drug delivery system. The formation of fibers was confirmed by scanning electron microscopy (SEM), and the fibers' surface properties were analyzed using contact angle and water vapor permeability techniques. Drug release tests and cell viability assays were performed to evaluate the potential toxicity of the material. SEM images demonstrated that the obtained fibers had nanoscale- and micrometer-scale diameters in PLGA and PMMA systems, respectively. The contact angle analyses indicated that, even in the presence of hydrophilic molecules (SDS and HPßCD), PMMA fibers exhibited hydrophobic characteristics, while PLGA fibers exhibited hydrophilic surface properties. These data were also confirmed by water vapor permeability analysis. The drug release profiles demonstrated a greater release of SDS in the PLGA system. Moreover, the presence of HPßCD improved the drug release in both polymeric systems and the cell viability in the PMMA SDS/HPßCD system. In terms of antibacterial activity, all membranes yielded positive outcomes; nevertheless, the PLGA SDS/HPßCD membrane exhibited the most remarkable results, with the lowest microbial load values. Additionally, the pseudo wound healing analysis demonstrated that the PLGA SDS/HPßCD fiber exhibited results similar to the control group. Consequently, these findings exemplify the substantial potential of the obtained materials for use in wound healing applications.

Alginate-Based 3D A549 Cell Culture Model to Study Paracoccidioides Infection.

A three-dimensional (3D) lung aggregate model based on sodium alginate scaffolds was developed to study the interactions between Paracoccidioides brasiliensis (Pb) and lung epithelial cells. The suitability of the 3D aggregate as an infection model was examined using cell viability (cytotoxicity), metabolic activity, and proliferation assays. Several studies exemplify the similarity between 3D cell cultures and living organisms, which can generate complementary data due to the greater complexity observed in these designed models, compared to 2D cell cultures. A 3D cell culture system of human A549 lung cell line plus sodium alginate was used to create the scaffolds that were infected with Pb18. Our results showed low cytotoxicity, evidence of increased cell density (indicative of cell proliferation), and the maintenance of cell viability for seven days. The confocal analysis revealed viable yeast within the 3D scaffold, as demonstrated in the solid BHI Agar medium cultivation. Moreover, when ECM proteins were added to the alginate scaffolds, the number of retrieved fungi was significantly higher. Our results highlight that this 3D model may be promising for in vitro studies of host-pathogen interactions.

Review of Bacterial Nanocellulose as Suitable Substrate for Conformable and Flexible Organic Light-Emitting Diodes.

As the development of nanotechnology progresses, organic electronics have gained momentum in recent years, and the production and rapid development of electronic devices based on organic semiconductors, such as organic light-emitting diodes (OLEDs), organic photovoltaic cells (OPVs), and organic field effect transistors (OFETs), among others, have excelled. Their uses extend to the fabrication of intelligent screens for televisions and portable devices, due to their flexibility and versatility. Lately, great efforts have been reported in the literature to use them in the biomedical field, such as in photodynamic therapy. In tandem, there has been considerable interest in the design of advanced materials originating from natural sources. Bacterial nanocellulose (BNC) is a natural polymer synthesized by many microorganisms, notably by non-pathogenic strains of Komagataeibacter (K. xylinus, K. hansenii, and K. rhaeticus). BNC shows distinct physical and mechanical properties, including its insolubility, rapid biodegradability, tensile strength, elasticity, durability, and nontoxic and nonallergenic features, which make BNC ideal for many areas, including active and intelligent food packaging, sensors, water remediation, drug delivery, wound healing, and as conformable/flexible substrates for application in organic electronics. Here, we review BNC production methods, properties, and applications, focusing on electronic devices, especially OLEDs and flexible OLEDs (FOLEDs). Furthermore, we discuss the future progress of BNC-based flexible substrate nanocomposites.

Multiple Tolerization Subtractive Immunization (MTSI) Protocol: Effects on Mice and Monoclonal Antibody Specificity.

Monoclonal antibodies (mAbs) have been a valuable tool to elucidate several biological processes, such as stem cell differentiation and cancer, and contributed to virtually all areas of biomedical sciences. Yet, it remains a challenge to obtain mAbs specific to poorly expressed epitopes, or to epitopes that are actually involved in important biological phenomena, such as cell differentiation and metastasis. Drug-induced subtractive immunization, and recently the multiple tolerization subtractive immunization (MTSI) technique, reported by our group, have the potential to level up the field, as they direct the host´s immune response towards these epitopes. However, due to cyclophosphamide (CY) treatment, high mice mortality can be observed, and only a few data are available on how these techniques affect the immune system of mice. Tolerogen and immunogen cells, RWPE-1 and PC-3 cells, respectively, were individually seeded at 2 × 104 cells/cm2, and then adjusted to 2 × 106 cells per mouse before immunization, which was conducted in a subtractive approach (MTSI) with CY. Immunosuppression of mice was recorded via total white blood counting, as well the reactivity of circulating polyclonal antibodies (pAbs). General parameters, including weight, physical appearance, and behavior on mice subjected to three different concentrations of CY were recorded. mAbs were obtained using classical hybridoma techniques, using the spleen of immunized mice. After purification, antibodies were characterized by Western blotting, and Indirect immunofluorescence. In conclusion, all CY dosage were efficient in creating an immunosuppression state, but only the 100 mg/kg body weight was feasible, as the others resulted in extensive mice mortality. pAbs obtained in the peripheral blood of mice showed more reactivity towards tumor cells. MAbs 2-7A50 and 2-5C11 recognized antigens from tumor cells, but not from their non-tumor counterparts, as shown in western blotting and immunofluorescence assays. MTSI technique was successful in generating mAbs that recognize tumor-specific antigens.

Biophysical Analysis of Schistosoma mansoni Septins.

Septins are dynamic filament-forming proteins that are recognized as important components of the cytoskeleton and are involved in numerous functions inside the cells, such as cytokinesis, exocytosis, and ciliogenesis and even in defense against pathogenic bacteria. Despite being highly conserved in eukaryotes, there is scarce literature on the role of septins in organisms other than humans and yeast. Therefore, septins from Schistosoma mansoni represent an interesting model to study an unexplored branch of this protein family. Here we described standard protocols for recombinant production and initial characterization of septins from S. mansoni. Septins are notably difficult to purify, mostly due to their tendency to assemble into filaments. Therefore, specific protocols to stabilize these proteins have been developed. In this chapter, we systematically describe protocols to clone, express, and purify schistosome septins. We also describe the use of circular dichroism to assess the folding and stability of septins and use of chromatography to characterize their oligomeric state, bound guanine nucleotide, and GTP hydrolysis. We expect that these protocols may help researchers involved in the study of schistosome septins as well as assist to establish protocols for septins from other organisms.

Multiple Tolerization Reduces Antibody Binding Against Tolerogen Cells: Implications for the Production of Monoclonal Antibodies.

We report an immunization technique that can update the production of monoclonal antibodies (mAbs): the multiple tolerization subtractive immunization (MTSI). A total of 10 BALB/C mice were used. Animals in group 1 received one inoculation of RWPE-1 cells (nontumoral), followed by cyclophosphamide, and then received serial inoculations of nonirradiated PC3 cells (tumoral). Animals in group 2 received our MTSI protocol, as follows: one inoculation of RWPE-1 cells, followed by cyclophosphamide (Cy). This whole tolerization step was repeated three other times, with 14-day intervals between the last Cy exposure and the next RWPE-1 cell inoculation. Finally, the animals received the same nonirradiated PC3 cell exposure as group 1. Blood was taken from each animal, and their polyclonal sera individually tested against the nontumoral RWPE-1 cells in flow cytometry. We found out that, after the MTSI was employed, the serum of the immunized animals, in group 2, contained considerably less antibodies that reacted against the tolerogenic cells, compared with the serum of the animals that underwent regular subtractive immunization. We showed that, by repeating the tolerization cycles, the polyclonal antibodies produced by mice have a reduced specificity toward common/immunodominant epitopes present at nontumoral cells, and thus this technique can be readily used by others in studies involving murine mAb protocols.

Enhanced immunization techniques to obtain highly specific monoclonal antibodies.

Despite fast advances in genomics and proteomics, monoclonal antibodies (mAbs) are still a valuable tool for areas such as the evolution of basic research in stem cells and cancer, for immunophenotyping cell populations, diagnosing and prognosis of diseases, and for immunotherapy. To summarize different subtractive immunization approaches successfully used for the production of highly specific antibodies, we identified scientific articles in NCBI PubMed using the following search terms: subtractive immunization, monoclonal antibody, tolerization, neonatal, high-zone tolerance, masking immunization. Patent records were also consulted. From the list of results, we included all available reports, from 1985 to present, that used any enhanced immunization technique to produce either polyclonal or monoclonal antibodies. Our examination yielded direct evidence that these enhanced immunization techniques are efficient in obtaining specific antibodies to rare epitopes, with different applications, such as to identify food contaminants or tumor cells.

Effect of in situ modification of bacterial cellulose with carboxymethylcellulose on its nano/microstructure and methotrexate release properties.

Bacterial cellulose/carboxymethylcelullose (BC/CMC) biocomposites with different DS-CMC (DS from 0.7 to 1.2) were developed in order to evaluate their impact as a drug delivery system. Biocomposites were loaded with methotrexate (MTX) as an alternative for the topical treatment of psoriasis. Scanning electron microscopy and atomic force microscopy showed that the CMC coated the cellulose nanofibers, leading to the decrease of the elastic modulus as the DS of CMC increased. BC/CMC0.9 exhibited the lower liquid uptake (up to 11 times lower), suggesting that the more linear structure of the intermediate substitute CMC grade (0.9) was able to interact more strongly with BC, resulting in a denser structure. All samples showed a typical burst release effect in the first 15min of test, however the BC/CMC0.9 biocomposite promoted a slight lowering of MTX release rates, suggesting that the DS of CMC can be considered the key factor to modulate the BC properties.

Adaptación y validación en población mexicana del Cuestionario de personalidad tipo C (PCTC) / Adaptation and validation of the Mexican population type C personality questionnaire (PCTC)

Resumen El objetivo de este trabajo es la adaptación y validación del Cuestionario de personalidad tipo C, elaborado por López, Ramírez y Esteve & Anarte (1998, 2002) a una población mexicana. Se realizó una adaptación lingüística del Cuestionario, y se aplicó a una muestra de 391 personas en las ciudades de Chihuahua y Xalapa, México. El análisis factorial llevó a eliminar seis de los reactivos de la escala original. El resto de los reactivos se agrupan en cuatro factores. El análisis factorial confirmatorio indica que los factores fundamentales del constructo de personalidad tipo C en población mexicana son los de comprensión, no expresión emocional y necesidad de armonía, aunque en la muestra de la ciudad de Xalapa, este último factor no fue fundamental. La confiabilidad total de la escala por el método alfa de Cronbach fue de 0.757, y en las cuatro subescalas osciló entre 0.645 y 0.842.

Abstract The aim of this work was the adaptation and validation of the Type-C Personality scale C, by Lopez, Ramirez and Esteve & Anarte (1998, 2002). Linguistic adaptation of the questionnaire was carried out with a sample of 391 people in the cities of Chihuahua and Xalapa, México. Factor analysis led to eliminate six items from the original scale. The remaining items were grouped in four factors. Confirmatory factor analysis indicates that the fundamental factors of type C personality construct are understanding, emotional inexpressiveness, and need for harmony. Nevertheless, need for harmony was not fundamental in the sample from Xalapa. The overall reliability (Cronbach's alpha) of the scale was 757 and for the four subscales ranged from 0.645 to 0.842.

Biophysical dissection of schistosome septins: Insights into oligomerization and membrane binding.

Septins are GTP-binding proteins that are highly conserved among eukaryotes and which are usually membrane-associated. They have been linked to several critical cellular functions such as exocytosis and ciliogenesis, but little mechanistic detail is known. Their assembly into filaments and membrane binding properties are incompletely understood and that is specially so for non-human septins where such information would offer therapeutic potential. In this study we use Schistosoma mansoni, exhibiting just four septin genes, as a simpler model for characterizing the septin structure and organization. We show that the biochemical and biophysical proprieties of its SmSEPT5 and SmSEPT10 septins are consistent with their human counterparts of subgroups SEPT2 and SEPT6, respectively. By succeeding to isolate stable constructs comprising distinct domains of SmSEPT5 and SmSEPT10 we were able to infer the influence of terminal interfaces in the oligomerization and membrane binding properties. For example, both proteins tended to form oligomers interacting by the N- and C-terminal interfaces in a nucleotide independent fashion but form heterodimers via the G interface, which are nucleotide dependent. Furthermore, we report for the first time that it is the C-terminus of SmSETP10, rather than the N-terminal polybasic region found in other septins, that mediates its binding to liposomes. Upon binding we observe formation of discrete lipo-protein clusters and higher order septin structures, making our system an exciting model to study interactions of septins with biological membranes.