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.

Systematic investigation of factors, such as the impact of emulsifiers, which influence the measurement of skin barrier integrity by in-vitro trans-epidermal water loss (TEWL).

Trans-epidermal water loss (TEWL) has been the most widely used method to assess the integrity of the skin barrier and evaluate the irritation potential or the protective properties of topical products for many years. It detects the amount of water that diffuses across the stratum corneum (SC) to the external environment. As one of the most important functions of the skin is to keep water inside the body, an increase in TEWL is used to indicate the skin's impaired barrier function. So far, a variety of commercial instruments are available to measure the TEWL. Their applications mainly focus on the in-vivo TEWL measurements for dermatological examinations or formulation development. Recently, an in-vitro TEWL probe has also been commercially released enabling preliminary tests with excised skin samples. In our study, we first aimed to optimize the experimental procedures for detecting the in-vitro TEWL of porcine skin. Secondly, different kinds of emulsifiers were applied to the skin, including polyethylene glycol-containing emulsifiers (PEG-ylated emulsifiers), sorbitan esters, cholesterol, and lecithin. Sodium lauryl sulfate (SLS) was used as a positive control, and water as a negative control. Based on the findings, we established a protocol for accurately measuring the in-vitro TEWL values, emphasizing that the temperature of the skin sample should be constantly maintained at 32℃. Subsequently, the influences of emulsifiers on the in-vitro TEWL were analyzed. They indicated a significant skin barrier impairment of PEG-20 cetyl ether, PEG-20 stearyl ether, and SLS on in-vitro skin. Furthermore, we interestingly found that there consistently was an alteration of the TEWL values, even after the application of water to the skin. Our findings are of special interest, as the European Medicines Agency (EMA) recommends the use of in-vitro TEWL to determine skin barrier intactness during Franz cell experiments. Thus, this study provides a validated protocol for measuring the in-vitro TEWL and elucidates the impact of emulsifiers on the skin barrier. It also improves the understanding of tolerable variations of in-vitro TEWL and offers recommendations for its use in research.

Enhanced Transdermal Delivery of N-Acetylcysteine and 4-Phenylbutyric Acid for Potential Use as Antidotes to Lewisite.

Lewisite is a highly toxic chemical warfare agent that leads to cutaneous and systemic damage. N-acetylcysteine (NAC) and 4-phenylbutryic acid (4-PBA) are two novel antidotes developed to treat toxicity caused by lewisite and similar arsenicals. Our in vivo studies demonstrated safety and effectiveness of these agents against skin injury caused by surrogate lewisite (Phenylarsine oxide) proving their potential for the treatment of lewisite injury. We further focused on exploring various enhancement strategies for an enhanced delivery of these agents via skin. NAC did not permeate passively from propylene glycol (PG). Iontophoresis as a physical enhancement technique and chemical enhancers were investigated for transdermal delivery of NAC. Application of cathodal and anodal iontophoresis with the current density of 0.2 mA/cm2 for 4 h followed by passive diffusion till 24 h significantly enhanced the delivery of NAC with a total delivery of 65.16 ± 1.95 µg/cm2 and 87.23 ± 7.02 µg/cm2, respectively. Amongst chemical enhancers, screened oleic acid, oleyl alcohol, sodium lauryl ether sulfate, and dimethyl sulfoxide (DMSO) showed significantly enhanced delivery of NAC with DMSO showing highest delivery of 28,370.2 ± 2355.4 µg/cm2 in 24 h. Furthermore, 4-PBA permeated passively from PG with total delivery of 1745.8 ± 443.5 µg/cm2 in 24 h. Amongst the chemical enhancers screened for 4-PBA, oleic acid, oleyl alcohol, and isopropyl myristate showed significantly enhanced delivery with isopropyl myristate showing highest total delivery of 17,788.7 ± 790.2 µg/cm2. These studies demonstrate feasibility of delivering these antidotes via skin and will aid in selection of excipients for the development of topical/transdermal delivery systems of these agents.

Phosphorylation of EIF2S1 (eukaryotic translation initiation factor 2 subunit alpha) is indispensable for nuclear translocation of TFEB and TFE3 during ER stress.

There are diverse links between macroautophagy/autophagy pathways and unfolded protein response (UPR) pathways under endoplasmic reticulum (ER) stress conditions to restore ER homeostasis. Phosphorylation of EIF2S1/eIF2α is an important mechanism that can regulate all three UPR pathways through transcriptional and translational reprogramming to maintain cellular homeostasis and overcome cellular stresses. In this study, to investigate the roles of EIF2S1 phosphorylation in regulation of autophagy during ER stress, we used EIF2S1 phosphorylation-deficient (A/A) cells in which residue 51 was mutated from serine to alanine. A/A cells exhibited defects in several steps of autophagic processes (such as autophagosome and autolysosome formation) that are regulated by the transcriptional activities of the autophagy master transcription factors TFEB and TFE3 under ER stress conditions. EIF2S1 phosphorylation was required for nuclear translocation of TFEB and TFE3 during ER stress. In addition, EIF2AK3/PERK, PPP3/calcineurin-mediated dephosphorylation of TFEB and TFE3, and YWHA/14-3-3 dissociation were required for their nuclear translocation, but were insufficient to induce their nuclear retention during ER stress. Overexpression of the activated ATF6/ATF6α form, XBP1s, and ATF4 differentially rescued defects of TFEB and TFE3 nuclear translocation in A/A cells during ER stress. Consequently, overexpression of the activated ATF6 or TFEB form more efficiently rescued autophagic defects, although XBP1s and ATF4 also displayed an ability to restore autophagy in A/A cells during ER stress. Our results suggest that EIF2S1 phosphorylation is important for autophagy and UPR pathways, to restore ER homeostasis and reveal how EIF2S1 phosphorylation connects UPR pathways to autophagy.Abbreviations: A/A: EIF2S1 phosphorylation-deficient; ACTB: actin beta; Ad-: adenovirus-; ATF6: activating transcription factor 6; ATZ: SERPINA1/α1-antitrypsin with an E342K (Z) mutation; Baf A1: bafilomycin A1; BSA: bovine serum albumin; CDK4: cyclin dependent kinase 4; CDK6: cyclin dependent kinase 6; CHX: cycloheximide; CLEAR: coordinated lysosomal expression and regulation; Co-IP: coimmunoprecipitation; CTSB: cathepsin B; CTSD: cathepsin D; CTSL: cathepsin L; DAPI: 4',6-diamidino-2-phenylindole dihydrochloride; DMEM: Dulbecco's modified Eagle's medium; DMSO: dimethyl sulfoxide; DTT: dithiothreitol; EBSS: Earle's Balanced Salt Solution; EGFP: enhanced green fluorescent protein; EIF2S1/eIF2α: eukaryotic translation initiation factor 2 subunit alpha; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; ERAD: endoplasmic reticulum-associated degradation; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; FBS: fetal bovine serum; gRNA: guide RNA; GSK3B/GSK3ß: glycogen synthase kinase 3 beta; HA: hemagglutinin; Hep: immortalized hepatocyte; IF: immunofluorescence; IRES: internal ribosome entry site; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LMB: leptomycin B; LPS: lipopolysaccharide; MAP1LC3A/B/LC3A/B: microtubule associated protein 1 light chain 3 alpha/beta; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEFs: mouse embryonic fibroblasts; MFI: mean fluorescence intensity; MTORC1: mechanistic target of rapamycin kinase complex 1; NES: nuclear export signal; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; OE: overexpression; PBS: phosphate-buffered saline; PLA: proximity ligation assay; PPP3/calcineurin: protein phosphatase 3; PTM: post-translational modification; SDS: sodium dodecyl sulfate; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEM: standard error of the mean; TEM: transmission electron microscopy; TFE3: transcription factor E3; TFEB: transcription factor EB; TFs: transcription factors; Tg: thapsigargin; Tm: tunicamycin; UPR: unfolded protein response; WB: western blot; WT: wild-type; Xbp1s: spliced Xbp1; XPO1/CRM1: exportin 1.

Distribution of Glycolipids in the Plasma Membrane Monitored by Specific Probes in Combination with Sodium Dodecyl Sulfate-Digested Freeze-Fracture Replica Labeling (SDS-FRL).

Glycolipids are mainly distributed in the outer leaflet of the plasma membrane and are involved in cellular signaling by modulating the activity of cell surface receptor proteins. Glycolipids themselves also work as cell surface receptors of bacterial toxins. Anti-glycolipid antibodies are associated with various pathological conditions. The cellular distribution of glycolipids has been studied using specific toxins or antibodies. However, these proteins are multivalent and thus potentially induce the artificial aggregation of glycolipids. Since chemical fixative such as paraformaldehyde does not fix glycolipids, an alternative methodology is required to localize glycolipids with multivalent probes. Sodium dodecyl sulfate-digested freeze-fracture replica labeling (SDS-FRL) physically fixes glycolipids on the cast after quick freezing. Thus, SDS-FRL provides the opportunity to observe the natural distribution of glycolipids using multivalent probes. Here, we describe the application of SDS-FRL on the cell surface distribution of phosphatidylglucoside.

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.

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.

High-level ertapenem resistance in Klebsiella pneumoniae is due to RamA downregulation of ompK35 through micF.

An ertapenem-resistant Klebsiella pneumoniae clinical isolate (KP20) without carbapenemase and negative for the efflux pump inhibition test was resistant to ertapenem at a high level [minimum inhibitory concentration (MIC) = 64 mg/L] but susceptible to meropenem and imipenem. Second-generation sequencing was performed and a termination mutation was found in ramR. Complementation of ramR in KP20 reduced the ertapenem MIC by 128 times (from 64 mg/L to 0.5 mg/L). Overexpression of ramA and loss of OmpK35 were discovered in strain KP20 by quantitative reverse transcription PCR (RT-qPCR) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), respectively. Furthermore, ramA deletion in strain KP20 resulted in a 128-fold decrease in the MIC of ertapenem (from 64 mg/L to 0.5 mg/L), and expression of OmpK35 was observed in KP20ΔramA by SDS-PAGE. Complementation of ramA in KP20ΔramA led to a 45.45-fold downregulation of ompK35. Complementation of ompK35 in KP20 could restore susceptibility to ertapenem (MIC reduced from 64 mg/L to 0.25 mg/L). Furthermore, results of the electrophoretic mobility shift assay showed that RamA could bind to the promoter of micF. These results showed that the termination mutation in ramR resulted in overexpression of ramA causing loss of OmpK35 expression through upregulation of micF, revealing the mechanism of ertapenem resistance only in K. pneumoniae.

How do surfactants unfold and refold proteins?

Although the anionic surfactant sodium dodecyl sulfate, SDS, has been used for more than half a century as a versatile and efficient protein denaturant for protein separation and size estimation, there is still controversy about its mode of interaction with proteins. The term "rod-like" structures for the complexes that form between SDS and protein, originally introduced by Tanford, is not sufficiently descriptive and does not distinguish between the two current vying models, namely protein-decorated micelles a.k.a. the core-shell model (in which denatured protein covers the surface of micelles) versus beads-on-a-string model (where unfolded proteins are surrounded by surfactant micelles). Thanks to a combination of structural, kinetic and computational work particularly within the last 5-10 years, it is now possible to rule decisively in favor of the core-shell model. This is supported unambiguously by a combination of calorimetric and small-angle X-ray scattering (SAXS) techniques and confirmed by increasingly sophisticated molecular dynamics simulations. Depending on the SDS:protein ratio and the protein molecular mass, the formed structures can range from multiple partly unfolded protein molecules surrounding a single shared micelle to a single polypeptide chain decorating multiple micelles. We also have much new insight into how this species forms. It is preceded by the binding of small numbers of SDS molecules which subsequently grow by accretion. Time-resolved SAXS analysis reveals an asymmetric attack by SDS micelles followed by distribution of the increasingly unfolded protein around the micelle. The compactness of the protein chain continues to evolve at higher SDS concentrations according to single-molecule studies, though the protein remains completely denatured on the tertiary structural level. SDS denaturation can be reversed by addition of nonionic surfactants that absorb SDS forming mixed micelles, leaving the protein free to refold. Refolding can occur in parallel tracks if only a fraction of the protein is initially stripped of SDS. SDS unfolding is nearly always reversible unless carried out at low pH, where charge neutralization can lead to superclusters of protein-surfactant complexes. With the general mechanism of SDS denaturation now firmly established, it largely remains to explore how other ionic surfactants (including biosurfactants) may diverge from this path.

A cell wall-anchored glycoprotein confers resistance to cation stress in Actinomyces oris biofilms.

Actinomyces oris plays an important role in oral biofilm development. Like many gram-positive bacteria, A. oris produces a sizable number of surface proteins that are anchored to bacterial peptidoglycan by a conserved transpeptidase named the housekeeping sortase SrtA; however, the biological role of many A. oris surface proteins in biofilm formation is largely unknown. Here, we report that the glycoprotein GspA-a genetic suppressor of srtA deletion lethality-not only promotes biofilm formation but also maintains cell membrane integrity under cation stress. In comparison to wild-type cells, under elevated concentrations of mono- and divalent cations the formation of mono- and multi-species biofilms by mutant cells devoid of gspA was significantly diminished, although planktonic growth of both cell types in the presence of cations was indistinguishable. Because gspA overexpression is lethal to cells lacking gspA and srtA, we performed a genetic screen to identify GspA determinants involving cell viability. DNA sequencing and biochemical characterizations of viable clones revealed that mutations of two critical cysteine residues and a serine residue severely affected GspA glycosylation and biofilm formation. Furthermore, mutant cells lacking gspA were markedly sensitive to sodium dodecyl sulfate, a detergent that solubilizes the cytoplasmic membranes, suggesting the cell envelope of the gspA mutant was altered. Consistent with this observation, the gspA mutant exhibited increased membrane permeability, independent of GspA glycosylation, compared to the wild-type strain. Altogether, the results support the notion that the cell wall-anchored glycoprotein GspA provides a defense mechanism against cation stress in biofilm development promoted by A. oris.

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.

Conformation and membrane interaction studies of the potent antimicrobial and anticancer peptide palustrin-Ca.

Palustrin-Ca (GFLDIIKDTGKEFAVKILNNLKCKLAGGCPP) is a host defence peptide with potent antimicrobial and anticancer activities, first isolated from the skin of the American bullfrog Lithobates catesbeianus. The peptide is 31 amino acid residues long, cationic and amphipathic. Two-dimensional NMR spectroscopy was employed to characterise its three-dimensional structure in a 50/50% water/2,2,2-trifluoroethanol-[Formula: see text] mixture. The structure is defined by an [Formula: see text]-helix that spans between Ile[Formula: see text]-Ala[Formula: see text], and a cyclic disulfide-bridged domain at the C-terminal end of the peptide sequence, between residues 23 and 29. A molecular dynamics simulation was employed to model the peptide's interactions with sodium dodecyl sulfate micelles, a widely used bacterial membrane-mimicking environment. Throughout the simulation, the peptide was found to maintain its [Formula: see text]-helical conformation between residues Ile[Formula: see text]-Ala[Formula: see text], while adopting a position parallel to the surface to micelle, which is energetically-favourable due to many hydrophobic and electrostatic contacts with the micelle.

Adaptation of Pseudomonas helmanticensis to fat hydrolysates and SDS: fatty acid response and aggregate formation.

An essential part of designing any biotechnological process is examination of the physiological state of producer cells in different phases of cultivation. The main marker of a bacterial cell's state is its fatty acid (FA) profile, reflecting membrane lipid composition. Consideration of FA composition enables assessment of bacterial responses to cultivation conditions and helps biotechnologists understand the most significant factors impacting cellular metabolism. In this work, soil SDS-degrading Pseudomonas helmanticensis was studied at the fatty acid profile level, including analysis of rearrangement between planktonic and aggregated forms. The set of substrates included fat hydrolysates, SDS, and their mixtures with glucose. Such media are useful in bioplastic production since they can help incrementally lower overall costs. Conventional gas chromatography-mass spectrometry was used for FA analysis. Acridine orange-stained aggregates were observed by epifluorescence microscopy. The bacterium was shown to change fatty acid composition in the presence of hydrolyzed fats or SDS. These changes seem to be driven by the depletion of metabolizable substrates in the culture medium. Cell aggregation has also been found to be a defense strategy, particularly with anionic surfactant (SDS) exposure. It was shown that simple fluidity indices (such as saturated/unsaturated FA ratios) do not always sufficiently characterize a cell's physiological state, and morphological examination is essential in cases where complex carbon sources are used.

Microsecond Dynamics During the Binding-induced Folding of an Intrinsically Disordered Protein.

Tau is an intrinsically disordered protein implicated in many neurodegenerative diseases. The repeat domain fragment of tau, tau-K18, is known to undergo a disorder to order transition in the presence of lipid micelles and vesicles, in which helices form in each of the repeat domains. Here, the mechanism of helical structure formation, induced by a phospholipid mimetic, sodium dodecyl sulfate (SDS) at sub-micellar concentrations, has been studied using multiple biophysical probes. A study of the conformational dynamics of the disordered state, using photoinduced electron transfer coupled to fluorescence correlation spectroscopy (PET-FCS) has indicated the presence of an intermediate state, I, in equilibrium with the unfolded state, U. The cooperative binding of the ligand (L), SDS, to I has been shown to induce the formation of a compact, helical intermediate (IL5) within the dead time (∼37 µs) of a continuous flow mixer. Quantitative analysis of the PET-FCS data and the ensemble microsecond kinetic data, suggests that the mechanism of induction of helical structure can be described by a U ↔ I ↔ IL5 ↔ FL5 mechanism, in which the final helical state, FL5, forms from IL5 with a time constant of 50-200 µs. Finally, it has been shown that the helical conformation is an aggregation-competent state that can directly form amyloid fibrils.

Stratum Corneum Function: A Structural Study with Dynamic Synchrotron X-ray Diffraction Experiments.

Studies on the effectiveness of substances such as drugs and cosmetics that act on the skin require structural evidence at the molecular level in the stratum corneum to clarify their interaction with intercellular lipid and soft keratin. For this purpose, when applying the substances to the stratum corneum X-ray diffraction experiment is one of the powerful tools. To detect minute structural changes in a stratum corneum sample, using a "solution cell", dynamic synchrotron X-ray diffraction measurements were performed when applying aqueous solution of the substances to the stratum corneum: (1) It was found that a surfactant, sodium dodecyl sulfate, significantly disrupted the long-period lamellar structure. (2) To study the effects of water, structural modifications of the short-period lamellar structure and the soft keratin in corneocytes were measured as a function of time. At the initial water content of 15 wt%, the spacings of the short-period lamellar structure and the soft keratin increased toward those at the water content of 25 wt%, that is a key water content in the stratum corneum. (3) Nanoparticles composed of assembly of amphiphilic molecules are one of the leading pharmaceutical formulations. When the nanoparticles were applied, a new assembly of amphiphilic molecules originated from the nanoparticle appeared. This phenomenon suggests that the formation of the new assembly at the surface of skin is concerned with the release of the drug from the nanoparticles. (4) When ethanol was applied to the stratum corneum, only the liquid state in the intercellular lipid matrix was dissolved. After the removal of ethanol from this stratum corneum, the ordered hydrocarbon-chain packing structures appeared. From this fact we would propose that the liquid state region is the main pathway for hydrophobic drugs with a small molecular weight in connection with the so-called 500 Da rule. Here, not only the technique but also the background to these studies and the characteristic results obtained from these studies are explained.

Energetic and structural effects of the Tanford transition on ligand recognition of bovine ß-lactoglobulin.

Bovine ß-lactoglobulin, an abundant protein in whey, is a promising nanocarrier for peroral administration of drug-like hydrophobic molecules, a process that involves transit through the different acidic conditions of the human digestive tract. Among the several pH-induced conformational rearrangements that this lipocalin undergoes, the Tanford transition is particularly relevant. This transition, which occurs with a midpoint around neutral pH, involves a conformational change of the E-F loop that regulates accessibility to the primary binding site. The effect of this transition on the ligand binding properties of this protein has scarcely been explored. In this study, we carried out an energetic and structural characterization of ß-lactoglobulin molecular recognition at pH values above and below the zone in which the Tanford transition occurs. The combined analysis of crystallographic, calorimetric, and molecular dynamics data sheds new light on the interplay between self-association, ligand binding, and the Tanford pre- and post-transition conformational states, revealing novel aspects underlying the molecular recognition mechanism of this enigmatic lipocalin.

Elevated Skin pH Is Associated With an Increased Permeability to Synthetic Urine.

PURPOSE: The aim of this study was to investigate the permeability of the skin following cleansing activities and its susceptibility to synthetic urine penetration. SUBJECTS AND SETTING: Ten healthy volunteers (aged 22-58 years) participated in the study, which was conducted in a university bioengineering laboratory. METHODS: Tape stripping and sodium lauryl sulfate were used to simulate the physical and chemical irritation exacerbated by frequent cleansing activities, respectively. An untreated site also was selected to evaluate responses of intact skin. Synthetic urine was then applied for a period of 2 hours. Measurements of transepidermal water loss and skin pH were taken at baseline and after each challenge. To quantify the permeability of the skin following exposure, desorption curves of transepidermal water loss were measured and skin surface water loss was calculated. RESULTS: Chemically irritated skin, characterized by increased pH (7.34 ± 0.22), demonstrated an increased permeability to urine, as reflected by a significant increase in mean skin surface water loss (46,209 ± 15,596 g/m2) compared to both the intact (14,631 ± 6164 g/m2) and physically irritated (14,545 ± 4051 g/m2) skin (P = .005 in both cases). In contrast, the differences between the intact and physically irritated skin were not significant (P = .88). CONCLUSION: Permeability of the skin to irritants is influenced by the status of the skin and its acid mantle. These highlight the need to reevaluate the frequency of cleansing activities, along with the choice of product in clinical settings, favoring the use of pH-balanced cleansers.

Methylene blue active substances in plaque of Bacillus subtilis subsp. subtilis and enrichment by supplemental calcium in culture media.

A series of experiments was conducted to identify the molecular species responsible for surface active emulsification (surfactant) bioactivity in Bacillus subtilis subsp. subtilis strain ATCC PTA-125135, and to describe culture conditions to support the enriched production of said bioactivity in cultured plaque of the strain. The assay for methylene blue active substances (MBAS) was found to be suitable for describing surfactant activity, where a solvent-extracted molecular fraction from the biofilm was found to retain surfactant activity and positively quantified as MBAS. Furthermore, an HPLC-refined protein fraction was found to quantify as MBAS with approximately 1·36-fold or greater surfactant activity per mol than sodium dodecyl sulphate, and a proteomic analysis of solvent extracted residues confirmed that biofilm surface layer protein BslA was a primary constituent of extracted residues. Surfactant bioactivity, quantified as MBAS, was enriched in cultured plaque by the supplementation of culture media with calcium chloride or calcium nitrate.

Sodium dodecyl sulfate improved stability and transdermal delivery of salidroside-encapsulated niosomes via effects on zeta potential.

Niosomes are novel carriers that show superior transdermal permeation enhancement but require the addition of charged stabilizers. In this study, niosomes were prepared using Span 40, cholesterol, and sodium dodecyl sulfate (SDS) as stabilizers for transdermal delivery of salidroside. At concentrations of 0.05-0.40% (w/v), SDS significantly increased the zeta potential of the nanovesicles from -18.5 ± 3.2 to -157.0 ± 5.2 mV and improved the stability of the niosomal formulations. Niosomes prepared with a Span 40:cholesterol molar ratio of 4:3 and 0.1% SDS showed good stability and the highest transdermal drug delivery among all tested formulations, with 2.75-fold higher transdermal flux of 20.26 ± 1.05 µg/(cm2·h) than that of aqueous salidroside solution. However, excess SDS increased the negative charge on the vesicle surface and hence repulsion with skin cells, leading to reduced drug entrapment efficiency and cellular uptake of niosomes. Although SDS in the niosomes dose-dependently increased the in vitro cytotoxicity of the formulation in skin cells, HaCaT and CCC-ESF-1 cell viability was ≥ 80% for formulations containing ≤0.1% SDS. No significant irritation was observed on rat skin with once-a-day topical application of the niosomal formulations for 7 consecutive days. Thus, SDS is a promising stabilizer for nanomedicines, including niosomes, for transdermal administration.

Influence of micelles on protein's denaturation.

To evaluate the role of micelles for protein-surfactant interaction, we have studied the binding modes of serum albumin proteins (human (HSA) and rabbit (RSA)) with anionic-surfactant, sodium dodecyl sulfate (SDS) by using UV-visible, fluorescence, circular dichroism, fluorescence lifetime, atomic force microscopy (AFM) techniques. The study performed with three different pHs (below (4.0), at (4.7), and above (7.0) isoelectric point). Hydrocarbon chain of the surfactant, dominant role of hydrophobic forces and electrostatic interactions helped in polar interaction on protein on binding surfaces. The change above and below the critical micelle concentration (CMC) in fluorescence spectra was due to polarity of the microenvironment. The CD spectra different binding aspects as below CMC and above CMC, explain about folding and unfolding in secondary structure. Surfactant's binding induces fluctuations in the microenvironment of aromatic amino acid's residues of both proteins at different pHs. AFM images clarify the structural changes in both proteins (HSA & RSA). AFM images also indicate some different interesting conformational and structural changes in both proteins below/above the CMC of the surfactant. The molecular docking studies indicate the binding energy -4.8 kcal mol-1 and -4.7 kcal mol-1 for HSA-SDS and RSA-SDS, respectively. Structural changes can be seen above and below the CMC.