Decellularized tissue exhibits large differences of extracellular matrix properties dependent on decellularization method: novel insights from a standardized characterization on skeletal muscle.

Decellularized matrices are an attractive choice of scaffold in regenerative medicine as they can provide the necessary extracellular matrix (ECM) components, signals and mechanical properties. Various detergent-based protocols have already been proposed for decellularization of skeletal muscle tissue. However, a proper comparison is difficult due to differences in species, muscle origin and sample sizes. Moreover, a thorough evaluation of the remaining acellular matrix is often lacking. We compared an in-house developed decellularization protocol to four previously published methods in a standardized manner. Porcine skeletal muscle samples with uniform thickness were subjected to in-depth histological, ultrastructural, biochemical and biomechanical analysis. In addition, 2D and three-dimensional cytocompatibility experiments were performed. We found that the decellularization methods had a differential effect on the properties of the resulting acellular matrices. Sodium deoxycholate combined with deoxyribonuclease I was not an effective method for decellularizing thick skeletal muscle tissue. Triton X-100 in combination with trypsin, on the other hand, removed nuclear material but not cytoplasmic proteins at low concentrations. Moreover, it led to significant alterations in the biomechanical properties. Finally, sodium dodecyl sulphate (SDS) seemed most promising, resulting in a drastic decrease in DNA content without major effects on the ECM composition and biomechanical properties. Moreover, cell attachment and metabolic activity were also found to be the highest on samples decellularized with SDS. Through a newly proposed standardized analysis, we provide a comprehensive understanding of the impact of different decellularizing agents on the structure and composition of skeletal muscle. Evaluation of nuclear content as well as ECM composition, biomechanical properties and cell growth are important parameters to assess. SDS comes forward as a detergent with the best balance between all measured parameters and holds the most promise for decellularization of skeletal muscle tissue.

The effect of electrical stimulation on skin vulnerability to irritants.

PURPOSE: Electrical stimulation (ES) is a widely used technique in the medical field for various purposes. The effect of ES on several skin properties has been investigated; however, its effect on skin vulnerability to irritants remains unknown. This study aimed to investigate the effects of ES application on skin vulnerability to external irritants. MATERIALS AND METHODS: An experimental study on 12 healthy male subjects (Mean ± SD, 22.9 ± 3.6 years) who completed the study. The subjects were free of skin abnormalities in the volar aspect of both forearms. Three areas were allocated to each forearm and marked as areas 1, 2, and A in the treated forearm, and areas 3, 4, and B in the control forearm. ES was applied to the volar aspect of the treated forearm for 30 min three times a week, for 2 weeks. The effect of ES on skin vulnerability was investigated using 5% and 0.5% sodium lauryl sulfate (SLS) patches applied to both treated and control forearms. The skin response to irritants was evaluated using transepidermal water loss (TEWL) and a visual erythema score 24 h after patch removal. RESULTS: Compared to the control forearm, ES increased skin permeability and erythema in response to external irritants (SLS), as measured by the visual analog score (Z = 2.75, p = 0.006) and TEWL (p < 0.05), respectively. CONCLUSIONS: ES escalates skin reactions to low concentrations of irritant substances, such as SLS, in the area between the two electrodes. This emphasizes the use of this substance, and similar irritants should be avoided in areas treated with ES.

Susceptibility of Cu(I) transport ATPases to sodium dodecyl sulfate. Relevance of the composition of the micellar phase.

Sodium dodecyl sulfate (SDS) is a well-known protein denaturing agent. A less known property of this detergent is that it can activate or inactivate some enzymes at sub-denaturing concentrations. In this work we explore the effect of SDS on the ATPase activity of a hyper-thermophilic and a mesophilic Cu(I) ATPases reconstituted in mixed micelles of phospholipids and a non-denaturing detergent. An iterative procedure was used to evaluate the partition of SDS between the aqueous and the micellar phases, allowing to determine the composition of micelles prepared from phospholipid/detergent mixtures. The incubation of enzymes with SDS in the presence of different amounts of phospholipids reveals that higher SDS concentrations are required to obtain the same degree of inactivation when the initial concentration of phospholipids is increased. Remarkably, we found that, if represented as a function of the mole fraction of SDS in the micelle, the degree of inactivation obtained at different amounts of amphiphiles converges to a single inactivation curve. To interpret this result, we propose a simple model involving active and inactive enzyme molecules in equilibrium. This model allowed us to estimate the Gibbs free energy change for the inactivation process and its derivative with respect to the mole fraction of SDS in the micellar phase, the latter being a measure of the susceptibility of the enzyme to SDS. Our results showed that the inactivation free energy changes are similar for both proteins. Conversely, susceptibility to SDS is significantly lower for the hyperthermophilic ATPase, suggesting an inverse relation between thermophilicity and susceptibility to SDS.

Effect of Drinking Water Salt Content on the Interaction between Surfactants and Bacteria.

Sodium dodecyl sulfate (SDS) is a common surfactant used in various hygienic products. Its interactions with bacteria were studied previously, but the three-way interaction between surfactants, bacteria, and dissolved salts in the context of bacterial adhesion has not been studied. Here, we examined the combined effects of SDS (at concentrations typical of everyday hygienic activities) and salts, sodium chloride, and calcium chloride (at concentrations typically found in tap water) on the adhesion behavior of the common opportunistic pathogen Pseudomonas aeruginosa. We found that bacterial adhesion in the absence of SDS was dependent on the cation concentration rather than the total ionic strength and that combined treatment with several millimolar NaCl and SDS can increase bacterial adhesion. The addition of low concentrations of SDS (2 mM) to tens to hundreds millimolar concentrations of NaCl, typical of systems that suffer seawater incursion, reduced bacterial adhesion dramatically. Combined treatment with Ca+2 (in concentrations typical of those found in hard water) and SDS produced a small increase in total adhesion but a dramatic increase in the strength of adhesion. We conclude that the type and concentration of salts in water can have a considerable effect on the efficacy of soap in reducing bacterial adhesion and should be taken under consideration in critical applications. IMPORTANCE Surface-adhering bacteria are a reoccurring problem in many settings, including households, municipal water systems, food production facilities, and hospitals. Surfactants, and specifically sodium dodecyl sulfate (also known as SDS/SLS), are commonly used to remove bacterial contamination, but data regarding the interaction of SDS with bacteria and especially the effects of water-dissolved salts on this interaction are lacking. Here, we show that calcium and sodium ions can dramatically affect the efficacy of SDS on bacterial adhesion behavior and conclude that salt concentrations and ion species in the water supply should be considered in SDS applications.

Enhancing antimicrobial photodynamic therapy with phenothiazinium dyes and sodium dodecyl sulfate against Candida albicans at various growth stages.

BACKGROUND: The eradication of C. albicans is difficult due to the organization of the yeast in biofilms. Photodynamic therapy (PDT) has been proposed as an alternative to antifungals. Phenothiazinium dyes, e.g. methylene blue (MB), have been proposed as photosensitizing agents (PS), and their association with sodium dodecyl sulfate (SDS) has recently been shown to improve the effectiveness of PDT in planktonic culture. In this sense, the objective of this work was to evaluate the effect of PDT with phenothiazinium dyes associated to SDS in biofilms at the different stages of growth. METHODS: Experiments were carried out to evaluate the effects of PDT on biofilm formation and on established biofilms of C. albicans ATCC 10231. Samples were exposed to PS 50 mg/L (MB, Azure A - AA, Azure B - AB and dimethyl methylene blue - DMMB) dissolved in water or 0.25% SDS, for 5 min in the dark. After irradiation at 660 nm, 37.3mW/cm2 for 27 min, 60.4J/cm2 colony forming units count assay (CFU/mL) was performed. One or two irradiations were applied. Statistical methods were used to assess effectiveness. RESULTS: PSs showed low toxicity in the dark. An application of PDT irradiation was not able to reduce the CFU/mL both in mature biofilms (24h) and in biofilms in the dispersion phase (48h), only in the adherence phase did PDT prevent the formation of biofilms. With two successive applications of PDT irradiation in the dispersion phase, PDT with MB, AA, and DMMB completely inactivated C. albicans. The similar was not observed with mature biofilms. CONCLUSIONS: Different stages of biofilm growth respond differently to PDT, with the greatest inhibitory effect found in the adhesion stage. Mature and dispersed biofilms are less susceptible to PDT. The use of two successive applications of PDT with PSs associated with SDS may be a useful approach to inactivate C. albicans biofilms.

The combination of methylene blue and sodium dodecyl sulfate enhances the antimicrobial photodynamic therapy of Candida albicans at lower light parameters.

BACKGROUND: The growth of resistant microorganisms has been a challenge for health systems. Antimicrobial Photodynamic Therapy (aPDT) has gained attention due to its effects on resistant strains. Recently, it was shown that the association of methylene blue (MB) and sodium dodecyl sulfate (SDS) are an effective strategy to increase the effect of aPDT; however, it is unknown which are the best light parameters (such as irradiance and radiant exposure, RE), to reach the most effective protocols. This work aimed to evaluate the light parameters, irradiance, and radiant exposure, in aPDT with MB when conveyed in water compared to MB associated with SDS. METHODS: Tests were carried out to quantify the colony-forming units (CFU) of ATCC 10,231 strain of Candida albicans when using MB in different media and with different light parameters: Control (water), SDS (0.25%), MB (20 mg/mL), and the MB/SDS at irradiances of 3.7; 11.2; 18.6, and 26.1 mW/cm2 and varied irradiation times to reach radiant exposures of 4.4; 17.8; 26.7, and 44 J/cm². RESULTS: The results showed that aPDT with MB/SDS had a higher antimicrobial effect than MB when conveyed in water. Furthermore, for the highest irradiance studied (26.1 mW/cm2), CFU decreases exponentially with increasing RE from 4.4 up to 44 J/cm2. Similarly, at a fixed RE, the higher the irradiance used, the higher the antimicrobial effect was observed, except for the lowest RE studied (4.4 J/cm2). CONCLUSIONS: aPDT with MB/SDS had a greater antimicrobial action at the lower light parameters when compared to MB conveyed in water. The authors suggest the use of RE above 18 J/cm2 and irradiance above 26 mW/cm2 since at the mentioned parameters the increase in its value caused a greater antimicrobial effect.

Inactivation of Staphylococcus aureus by Levulinic Acid Plus Sodium Dodecyl Sulfate and their Antibacterial Mechanisms on S. aureus Biofilms by Transcriptomic Analysis.

The combination of levulinic acid (LVA) and sodium dodecyl sulfate (SDS) in recent years has shown a considerable potential to use as an antimicrobial intervention. The objectives of this study were to evaluate the antimicrobial efficacy of the combination against Staphylococcus aureus in both planktonic and biofilm states and to investigate the transcriptional changes in S. aureus biofilms coincubated with sublethal concentrations of LVA and/or SDS. The minimum inhibitory concentrations (MICs) of LVA and SDS determined by the microdilution method were 3.125 and 0.039 mg/mL, respectively. An additive bacteriostatic interaction (fractional inhibitory concentration index = 1) between the two compounds was observed by the checkerboard assay, whereas a synergistic bactericidal activity was displayed by the time-kill assay. The biomass and viable cells in the biofilms were reduced by both antimicrobials either alone or in combination in a dose-dependent manner. Transcriptomics indicated that more differentially expressed (DE) genes were observed in the biofilm treated with SDS (103 up- and 205 downregulated DE genes) and LVA + SDS (187 up and 162 down) than that coincubated with LVA (34 up and 32 down). The SDS and LVA + SDS treatments mainly affected the expression of genes responsible for cell surface proteins, virulence factors, adhesins, and capsular polysaccharides. Both the antibiofilm assay and the transcriptomics indicated that SDS, not LVA, was the major chemical contributing to the antibacterial efficacy of the combination. This study reveals the behavioral responses and protective mechanisms of S. aureus to LVA and SDS applied individually or in combination.

Reduced Biofilm Formation at the Air-Liquid-Solid Interface via Introduction of Surfactants.

Reduced biofilm formation is highly desirable in applications ranging from transportation to separations and healthcare. Biofilms often form at the three-phase interface where air, liquid, and solid coexist due to the close proximity to nutrients and oxygen. Reducing biofilm formation at the triple interface presents challenges because of the conflicting requirements for hydrophobicity at the air-solid interface (for self-cleaning properties) and for hydrophilicity at the liquid-solid interface (for reduced foulant adhesion). Meeting those needs simultaneously likely entails a dynamic surface, capable of shifting the surface energy landscape in response to wetting conditions and thus enabling hydrophobicity in air and hydrophilicity in water. Here, we designed a facile approach to render existing surfaces resistant to biofilm formation at the triple interface. By adding trace amounts (∼0.1 mM) of surfactants, biofilm formation of Pseudomonas aeruginosa (known to form biofilm at the triple interface) was reduced on all surfaces tested, ranging from hydrophilic to hydrophobic, polar to nonpolar. That reduced fouling was not a result of the known antimicrobial effects. Instead, it was attributed to the surface-adsorbed surfactants that dynamically control surface energy at the triple interface. To further understand the effect of surfactant-surface interactions on biofilm reduction, we systematically varied the surfactant charge type and surface properties (surface energy and charge). Electrostatic interactions between surfactants and surfaces were identified as an influential factor when predicting the relative fouling reduction upon introduction of surfactants. Nevertheless, biofilm formation was reduced even on the charge-neutral, fluorinated surface made of poly(1H, 1H, 2H, 2H-perfluorodecyl acrylate) by more than 2-fold simply via adding 0.2 mM dodecyl trimethylammonium chloride or 0.3 mM sodium dodecyl sulfate. Given its robustness, this strategy is broadly applicable for reducing fouling on existing surfaces, which in turn improves the cost-effectiveness of membrane separations and mitigates contaminations and nosocomial infections in healthcare.

Automated Decellularization of the Rodent Epigastric Free Flap: A Comparison of Sodium Dodecyl Sulfate-Based Protocols.

BACKGROUND: Free tissue transfer to cover complex wounds with exposed critical structures results in donor-site morbidity. Perfusion decellularization and recellularization of vascularized composite tissues is an active area of research to fabricate complex constructs without a donor site. Sodium dodecyl sulfate (SDS)-based protocols remain the predominant choice for decellularization despite the deleterious effects on tissue ultrastructure and capillary networks. We aimed to develop an automated decellularization process and compare different SDS perfusion times to optimize the protocol. METHODS: A three-dimensional-printed closed-system bioreactor capable of continuously perfusing fluid through the vasculature was used for decellularization. The artery and vein of rat epigastric fasciocutaneous free flaps were cannulated and connected to the bioreactor. Protocols had varying durations of 1% SDS solution (3, 5, and 10 days) followed by 1 day of 1% Triton X-100 and 1 day of 1x phosphate-buffered saline. The residual DNA was quantified. Microarchitecture of the constructs was assessed with histology, and the vascular network was visualized for qualitative assessment. RESULTS: The structural integrity and the microarchitecture of the extracellular matrix was preserved in the 3- and 5-day SDS perfusion groups; however, the subcutaneous tissue of the 10-day protocol lost its structure. Collagen and elastin structures of the pedicle vessels were not compromised by the decellularization process. Five-day SDS exposure group had the least residual DNA content (p < 0.001). Across all protocols, skin consistently had twice as much residual DNA over the subcutaneous tissues. CONCLUSION: A compact and integrated bioreactor can automate decellularization of free flaps to bioengineer regenerative constructs for future use in reconstruction of complex defects. A decellularization protocol with 5 days of 1% SDS exposure was the most successful to keep the residual DNA content at a minimum while preserving the structural integrity of the tissues.

Influence of permeability enhancers on the paracellular permeability of metformin hydrochloride and furosemide across Caco-2 cells.

Permeability enhancers can affect absorption of paracellularly transported drugs. This study aims to evaluate effects of permeability enhancers (chitosan, methyl-ß -cyclodextrin, sodium caprate, sodium lauryl sulfate, etc.) on the permeability of paracellularly absorbed furosemide and metformin hydrochloride. Methyl thiazole tetrazolium bromide test was carried out to determine the drug concentrations in permeability study. Trans-epithelial electrical resistance (TEER) values determined to assess the integrity of tight junctions. Permeability enhancers were applied at different concentrations alone, in dual/triple combinations. Permeability was determined using human colorectal adenocarcinoma (Caco-2) cells (TEER > 400 Ω·cm2). Permeability enhancers have no significant effect (<2-fold; p > 0.05) on the permeability of furosemide (1.80 × 10-5 ± 4.55 × 10-7 cm/s); however, metformin permeability (1.36 × 10-5 ± 1.25 × 10-6 cm/s) increased significantly (p < 0.05) with 0.3% and 0.5% (w/v) chitosan (2.0- and 2.7-fold, respectively), 1% methyl-ß -cyclodextrin (w/v) (3.5-fold), 10 and 20 µmol/L sodium caprate (2.2- and 2.8-fold, respectively), and 0.012% sodium lauryl sulfate (w/v) (1.9-fold). Furosemide permeability increased significantly (p < 0.05) with chitosan-sodium lauryl sulfate combination (1.7-fold), and all triple combinations (1.4- to 1.9-fold). Chitosan containing dual/triple combinations resulted in significant increase (p < 0.05) in metformin permeability (1.7 to 2.8-fold). All results indicated that absorption of furosemide and metformin can be improved by the combination of permeability enhancers. Therefore, it can be evaluated for the formulation of development strategies containing furosemide and metformin by the pharmaceutical industry.

Antifungal and antibiofilm activities of bee venom loaded on chitosan nanoparticles: a novel approach for combating fungal human pathogens.

The prevalence of opportunistic human fungal pathogens is increasing worldwide, and antimicrobial resistance is one of the greatest medical challenges the world faces. Therefore, this study aimed to develop a novel agent to control fungal pathogens. The honeybee products (honey, royal jelly, propolis, bee bread, and bee venom) were screened against unicellular fungal (UCF) pathogens (Cryptococcus neoformans, Kodamaea ohmeri, and Candida albicans) and the bee venom was only exhibited an inhibitory effect against them. The protein contents of crude bee venom were separated using the gel filtration technique into eight fractions which were visualized on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to confirm the presence of five bands with molecular weights of 65, 43, 21, 15, and 3 KDa. Bee venom (BV) of Apis mellifera loaded chitosan nanoparticles were prepared by the ionotropic gelation method. The encapsulation efficiency%, average size, zeta potentials, and surface appearance by Transmission electron microscope (TEM) were evaluated for the prepared nanoparticles. The minimum inhibitory concentration (MIC) of crude BV and BV loaded chitosan nanoparticles (BV-CNPs) was evaluated against the offer mentioned UCF where the MIC values of crude BV were 6.25, 3.12 & 6.25 while MIC values in the case of BV-CNPs were decreased to 3.12, 3.12 & 1.56 mg/ml against C. neoformans, K. ohmeri and C. albicans, respectively. Also, the results showed that BV-CNPs suppressed the biofilm formation as well as yeast to hyphal transition formed by the examined UCF. These results revealed that BV-CNPs are a promising natural compound for fungal pathogens treatment.

Cardiac-derived extracellular matrix: A decellularization protocol for heart regeneration.

Extracellular matrix (ECM) is a fundamental component of the heart, guiding vital cellular processes during organ homeostasis. Most cardiovascular diseases lead to a remarkable remodeling of the ECM, accompanied by the formation of a fibrotic tissue that heavily compromises the heart function. Effective therapies for managing fibrosis and promoting physiological ECM repair are not yet available. The production of a decellularized extracellular matrix (d-ECM) serving as a three-dimensional and bioactive scaffold able to modulate cellular behavior and activities is considered crucial to achieve a successful regeneration. The protocol represents a step-by-step method to obtain a decellularized cardiac matrix through the combination of sodium dodecyl sulphate (SDS) and Triton X-100. Briefly, cardiac samples obtained from left ventricles of explanted, pathological human hearts were dissected and washed to remove residual body fluids. Samples were then snap-frozen and sliced by a cryostat into 350 µm thick sections. The sections obtained were decellularized using a solution containing 1% Triton X-100 and 1% SDS in combination, for 24 hours, until observing the color change from brownish-red to translucent-white. As a result, the protocol shows efficiency in preserving ECM architecture and protein composition during the whole process, suggesting that it is worthwhile, highly reproducible and produces a well- preserved decellularized extracellular matrix from cardiac samples. Notwithstanding, some limitations need to be addressed, such as the risk for microbial contamination and the unpredictable trend of the protocol when applied to decellularize samples other than myocardium, vessels, or skin. These issues require antibiotics mixture supplement during the procedure followed by UV sterilization, and appropriate adjustments for a tissue-specific utilization, respectively. The protocol is intended to produce a cardiac d-ECM for cell settlement, representing the ideal scaffold for tissue engineering purposes.

Combinatorial treatment of brain samples from sheep with scrapie using sodium percarbonate, sodium dodecyl sulfate, and proteinase K increases survival time in inoculated susceptible sheep.

The agent of scrapie is resistant to most chemical and physical methods of inactivation. Prions bind to soils, metals, and various materials and persist in the environment confounding the control of prion diseases. Most methods of prion inactivation require severe conditions such as prolong exposure to sodium hypochlorite or autoclaving, which may not be suitable for field conditions. We evaluated the efficacy of a combinatorial approach to inactivation of US scrapie strain x124 under the mild conditions of treating scrapie-affected brain homogenate with sodium percarbonate (SPC), sodium dodecyl sulfate (SDS), or in combination followed by proteinase K (PK) digestion at room temperature. Western blot analysis of treated brain homogenate demonstrates partial reduction in PrPSc immunoreactivity. Genetically susceptible VRQ/ARQ Suffolk sheep were oronasally inoculated with 1 g of SPC (n = 1), SDS (n = 2), SDS + PK (n = 2), and SPC + SDS + PK (n = 4) treated brain homogenate. Sheep were assessed daily for clinical signs, euthanized at the development of clinical disease, and tissues were assessed for accumulation of PrPSc. Scrapie status in all sheep was determined by western blot, enzyme immunoassay, and immunohistochemistry. Mean incubation periods (IPs) for SPC (11.9 months, 0% survival), SDS (12.6 months, 0% survival), SDS + PK (14.0 months, 0% survival), and SPC + SDS + PK (12.5 months, 25% survival) were increased compared to positive control sheep (n = 2, 10.7 months, 0% survival) by 1.2, 1.9, 3.3, and 1.8 months, respectively. Treatment did not influence PrPSc accumulation and distribution at the clinical stage of disease. Differences in mean IPs and survival indicates partial but not complete reduction in scrapie infectivity.

Establishment of decellularized extracellular matrix scaffold derived from caprine pancreas as a novel alternative template over porcine pancreatic scaffold for prospective biomedical application.

In this study, the caprine pancreas has been presented as an alternative to the porcine organ for pancreatic xenotransplantation with lesser risk factors. The obtained caprine pancreas underwent a systematic cycle of detergent perfusion for decellularization. It was perfused using anionic (0.5% w/v sodium dodecyl sulfate) as well as non-ionic (0.1% v/v triton X-100, t-octyl phenoxy polyethoxy ethanol) detergents and washed intermittently with 1XPBS supplemented with 0.1% v/v antibiotic and nucleases in a gravitation-driven set-up. After 48 h, a white decellularized pancreas was obtained, and its extracellular matrix (ECM) content was examined for scaffold-like properties. The ECM content was assessed for removal of cellular content, and nuclear material was evaluated with temporal H&E staining. Quantified DNA was found to be present in a negligible amount in the resultant decellularized pancreas tissue (DPT), thus prohibiting it from triggering any immunogenicity. Collagen and fibronectin were confirmed to be preserved upon trichrome and immunohistochemical staining, respectively. SEM and AFM images reveal interconnected collagen fibril networks in the DPT, confirming that collagen was unaffected. sGAG was visualized using Prussian blue staining and quantified with DMMB assay, where DPT has effectively retained this ECM component. Uniaxial tensile analysis revealed that DPT possesses better elasticity than NPT (native pancreatic tissue). Physical parameters like tensile strength, stiffness, biodegradation, and swelling index were retained in the DPT with negligible loss. The cytocompatibility analysis of DPT has shown no cytotoxic effect for up to 72 h on normal insulin-producing cells (MIN-6) and cancerous glioblastoma (LN229) cells in vitro. The scaffold was recellularized using isolated mouse islets, which have established in vitro cell proliferation for up to 9 days. The scaffold received at the end of the decellularization cycle was found to be non-toxic to the cells, retained biological and physical properties of the native ECM, suitable for recellularization, and can be used as a safer and better alternative as a transplantable organ from a xenogeneic source.

A critical role of calcineurin in stress responses, hyphal formation, and virulence of the pathogenic fungus Trichosporon asahii.

Trichosporon asahii is a conditional pathogenic fungus that causes severe and sometimes fatal infections in immunocompromised patients. While calcineurin, an essential component of a calcium-dependent signaling pathway, is known to regulate stress resistance and virulence of some pathogenic fungi, its role in T. asahii has not been investigated. Here, we demonstrated that calcineurin gene-deficient T. asahii mutants are sensitive to high temperature as well as cell-membrane and cell-wall stress, and exhibit decreased hyphal formation and virulence against silkworms. Growth of T. asahii mutants deficient in genes encoding subunits of calcineurin, cna1 and cnb1, was delayed at 40 °C. The cna1 and cnb1 gene-deficient mutants also showed sensitivity to sodium dodecyl sulfate, Congo red, dithiothreitol, and tunicamycin. On the other hand, these mutants exhibited no sensitivity to caffeine, sorbitol, monensin, CaCl2, LiCl, NaCl, amphotericin B, fluconazole, or voriconazole. The ratio of hyphal formation in the cna1 and cnb1 gene-deficient mutants was decreased. Moreover, the virulence of the cna1 and cnb1 gene-deficient mutants against silkworms was attenuated. These phenotypes were restored by re-introducing each respective gene into the gene-deficient mutants. Our findings suggest that calcineurin has a role in regulating the cellular stress response and virulence of T. asahii.

Guaiacol augments quorum quenching potential of ciprofloxacin against Pseudomonas aeruginosa.

AIM: The present study aims to investigate the antimicrobial as well as antivirulence potential and the principle mechanism of action of guaiacol against Pseudomonas aeruginosa. METHODS AND RESULTS: Quorum sensing inhibition and membrane disruption studies were performed to check the effect of guaiacol on the virulence of P. aeruginosa. Production of various virulence factors and biofilm formation was studied at a sub-MIC concentration of guaiacol alone (1/8 MIC) and in combination with ciprofloxacin (1/2 FIC). Guaiacol exhibited synergistic interactions with ciprofloxacin and further reduced the production of all virulence factors and biofilm formation. Using crystal violet (CV) assay and quantification of exopolysaccharide, we observed weak biofilm formation, together with reduced motilities at sub-MIC, which was further visualized by confocal laser microscopy and Field Emission Scanning Electron Microscopy. The antibacterial activity of guaiacol against P. aeruginosa upon 2 × MIC exposure coincided with enhanced membrane permeability leading to disruption and release of cellular material as quantified by CV uptake assay and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results demonstrated that sub-MICs of guaiacol in combination with ciprofloxacin can act as a potent alternate compound for attenuation of quorum sensing in P. aeruginosa. CONCLUSION: The study reports that guaiacol in combination with ciprofloxacin at 1/2 FIC significantly compromised the bacterial growth and motilities alongside inducing quorum quenching potential. This was accompanied by inhibition of biofilm which subsequently decreased EPS production at sub-MIC concentration. Furthermore, guaiacol in combination displayed a severe detrimental effect on bacterial membrane disruption, thereby enhancing cellular material release. NOVELTY IMPACT STATEMENT: For the first time, the potential of guaiacol in combination with ciprofloxacin in attenuation of virulence factors, and biofilm formation in Pseudomonas aeruginosa was described. Results corroborate how plant bioactive in synergism with antibiotics can act as an alternate treatment regime to tackle the menace of drug resistance.

Sodium dodecyl sulphate, N-octyl ß-D glucopyranoside and 4-methoxy phenyl ß-D glucopyranoside effect on post-thaw sperm motion and viability traits of Murrah buffalo (Bubalus bubalis) bulls.

Sodium Dodecyl Sulphate (SDS), N-Octyl ß-D Glucopyranoside (NOG), 4-Methoxy Phenyl ß-D Glucopyranoside (4-MPG) as ice recrystallization inhibitors were added to Tris Egg Yolk Glycerol (TEYG) semen extender for cryopreservation of semen of buffalo bulls. Post-thaw sperm motion and viability traits were evaluated. Pilot study involved six semen ejaculates (2 ejaculates/bull, from three bulls); second experiment was conducted using twenty seven semen ejaculates (9 ejaculates/bull, from 3 bulls) and in third experiment three semen ejaculates (one bull) were used. Eight concentrations of SDS (2, 1, 0.5, 0.25, 0.15, 0.125, 0.0625 and 0.0312%), twelve concentrations of NOG (33, 22, 11, 5.5, 2.5, 0.75, 0.5, 0.25, 0.125, 0.0625, 0.03125 and 0.0156 mM), and, eleven concentrations of 4-MPG (220, 165, 110, 55, 50, 25, 12.5, 6.25, 3.125, 1.56 and 0.78 mM) were supplemented in TEYG semen extender to evaluate the post-thaw sperm motility and viability traits. Computer Assisted Sperm Analysis (CASA) was used to measure the kinetic and functional parameters for sperm motion traits, Hypo Osmotic Swelling Test (HOST) for sperm plasma membrane integrity, Eosin Nigrosin staining for viability and Rose Bengal staining for sperm abnormalities for all the experiments except for pilot study where only Total Motility (TM) and Rapid Progressive Motility (RP) were evaluated. Three freezing protocols; i) Normal P24 (freezing rate of -30 °C min-1 from 4 °C to -15 °C; -40 °C min-1 from -15 °C to -60 °C; and -50 °C min-1 from -60 °C to -140 °C; and then plunged in liquid Nitrogen at -196 °C); ii) Moderate P25 (freezing rate of -30 °C min-1 from 4 °C to -15 °C; -50 °C min-1 from -15 °C to -60 °C; and -50 °C min-1 from -60 °C to -140 °C; and then plunged in liquid Nitrogen at -196 °C); and iii) Rapid P26 (freezing rate of -30 °C min-1 from 4 °C to -15 °C; -60 °C min-1 from -15 °C to -60 °C; and -50 °C min-1 from -60 °C to -140 °C; and then plunged in liquid Nitrogen at -196 °C) were evaluated using SDS 0.125% in TEYG semen extender. SDS ≤0.125%, NOG ≤0.0625 mM and 4-MPG ≤ 3.125 mM in TEYG buffalo semen extender improved significantly (p < .05) the kinetic and functional parameters as compared to the other Ice Recrystallization Inhibitors (IRIs) concentrations used for cryopreservation of buffalo bull semen in the pilot study. SDS 0.125% supplementation was the best IRI among all which resulted in improved kinetic and functional parameters of bull semen in second experiment. Conclusion was drawn that buffalo bull semen cryopreservation using sodium dodecyl sulphate, 0.125% as IRI in TEYG semen extender along with freezing protocol P 25 revealed optimum kinetic and functional parameters for post-thaw spermatozoa.

Screening and Research on Skin Barrier Damage Protective Efficacy of Different Mannosylerythritol Lipids.

Mannosylerythritol lipids (MELs) may prevent skin barrier damage, although their protective mechanisms and active monomeric constituents remain unclear. Here, three MELs were extracted from Candida antarctica cultures containing fermented olive oil then purified using silica gel-based column chromatography and semipreparative HPLC. All three compounds (MEL-A, MEL-B, MEL-C) were well separated and stable, and reliable materials were used for NMR and HRESIMS chemical structure determinations and for assessing MELs' protective effects against skin damage. Notably, MEL-B and MEL-C effectively protected HaCaT cells from UVB-induced damage by upregulating the contents of filaggrin (FLG) and transglutaminase-1 (TGM1), as determined via ELISA. Moreover, MEL-B treatment (20 µg/mL) of UVB-irradiated HaCaT cells led to the upregulation of both the expression of mRNA genes and the key proteins FLG, LOR, and TGM1, which are known to be decreased in damaged skin cells. Additionally, histopathological analysis results revealed a markedly reduced intracellular vacuolation and cell damage, reflecting improved skin function after MEL-B treatment. Furthermore, immunofluorescence results revealed that MEL-B protected EpiKutis® three-dimensional cultured human skin cells from sodium dodecyl sulfate-induced damage by up-regulating FLG, LOR, and TGM1 expression. Accordingly, MELs' protection against skin barrier damage depended on MEL-B monomeric constituent activities, thus highlighting their promise as beneficial ingredients for use in skin-care products.

Effect of ethanol washing on porcine pulmonary artery wall decellularization using sodium dodecyl sulfate.

BACKGROUND: To determine the effectiveness of ethanol (EtOH) washing on porcine pulmonary artery (PA) wall decellularization using sodium dodecyl sulfate (SDS), we compared three different washing methods (phosphate-buffered saline [PBS], pH 9 alkali, and EtOH washing). METHODS: Fresh porcine PA walls were decellularized using 0.5% SDS and 0.5% sodium deoxycholate (SDC). The decellularized tissues were rinsed using three different washing techniques. Histological, biochemical, and mechanical analyses were conducted. Implantation into the subcutaneous tissue of rats and patch implantation into the carotid artery of dogs were performed as preliminary in vivo studies. RESULTS: The decellularization protocol based on SDS and SDC effectively removed the cells. The major extracellular matrix (ECM) structures (collagen, elastic fiber, and glycosaminoglycan) were properly preserved with the 75% EtOH-washing method. Significantly reduced residual SDS content was identified in EtOH-washed tissues compared to that in the other methods. No significant difference in the mechanical strength test was observed between the washing methods, and the EtOH-washing method showed better results in the metabolic activity test compared to the PBS-washing method. In the rat study model, no acute rejection or massive calcification was observed. The in vivo preliminary canine study showed better cell repopulation in the EtOH-washed group. CONCLUSION: EtOH washing of SDS-based decellularized porcine PA wall can reduce the residual SDS content and preserve ECM structures, especially the elastin content, and could also enhance cell repopulation after re-implantation.

A novel method for providing scaffold: Decellularization of parathyroid capsule.

This study aimed to generate a novel biomatrix from the decellularized human parathyroid capsule using different methods and to compare the efficiency of decellularization in the means of cell removal, structural integrity and extracellular matrix preservation. The parathyroid capsules, which were carefully dissected from the parathyroid tissue, were randomly divided into four groups and then decellularized using three different protocols: freeze-thaw only, sodium dodecyl sulphate and Triton X-100 treatments after freeze-thawing. Quantitative DNA analysis, agarose gel electrophoresis, sulphated glycosaminoglycan assay, histological analysis, immunohistochemistry and scanning electron microscopy were used to observe the efficiency of parathyroid capsule decellularization and preservation of extracellular matrix components. Considering all the results, it can be said that only freeze-thawing is not an effective method in parathyroid capsule decellularization. When the tissue was treated with a detergent agent in addition to freeze-thawing, the amount of DNA decreased by 90% while sulphated glycosaminoglycan amount maintained 50% compared to untreated tissue. Comparing the effects of the two detergents on the preservation of extracellular matrix such as collagen and sulphated glycosaminoglycan, it was seen that the integrity of tissues treated with Triton X-100 was preserved more than tissues treated with sodium dodecyl sulphate. It is concluded that Triton X-100 treatment with freeze-thawing is the most suitable and effective method for decellularizing the human parathyroid capsule. The biomatrix obtained with this method can be applied in the transplantation of parathyroid tissue and other endocrine tissue types in the body.