A green and highly sensitive microwell spectrofluorimetric method with high throughput for the determination of pemigatinib based on dual fluorescence enhancement by photoinduced electron transfer blocking and micellization: Application to the analysis of tablets, content uniformity testing, and human plasma.

Pemigatinib (PGT) is a recently FDA-approved small molecule kinase inhibitor used for the treatment of relapsed or refractory myeloid/lymphoid neoplasms in adults. This study introduces the development of a first microwell spectrofluorimetric method (MW-SFM) for quantifying PGT in FDA-approved tablets and plasma samples. The method utilized the enhancement of PGT's weak native fluorescence by blocking photoinduced electron transfer (PET) and micellization with sodium lauryl sulfate (SLS). The MW-SFM was performed in 96-microwell plates, and fluorescence signals were measured using a fluorescence microplate reader with excitation at 290 nm and emission at 350 nm. The method exhibited a linear range of 2-250 ng mL-1, and a limit of quantitation was 6.5 ng mL-1. The accuracy and precision of the method were confirmed with recovery rates ranging from 96.5% to 102.8% and relative standard deviations of 1.52% to 3.51%. The MW-SFM successfully analyzed Pemazyre® tablets, assessed content uniformity, and analyzed PGT-spiked human plasma samples. The greenness of the MW-SFM was verified using three different metric tools. In conclusion, the proposed MW-SFM is a valuable tool in supporting quality assessment of dosage forms, conducting pharmacokinetic studies, and monitoring therapeutic outcomes.

Insight into the interaction of quinizarin with SDS micelles - effects of additives.

Association behavior between quinizarin (1,4-dihydroxyanthraquinone), an analogue of the chromophore of anthracycline anticancer drugs and sodium dodecyl sulfate (SDS) micelles in the presence of glucose, NaCl and urea additives was studied using absorption spectroscopy and conductometric techniques. The spectral results indicate an increase of binding constant and partition coefficient values in the presence of glucose and NaCl whereas the addition of urea leads to a decrease of binding strength and quinizarin partitioning into SDS micelles. Thus, the rise of NaCl and glucose concentrations is favorable for the quinizarin distribution into SDS micelles. From electrical conductivity measurements it was found that the critical micelle concentration (CMC) of SDS/quinizarin system decreases by adding NaCl and glucose whereas urea has not influence on the micelization process at the concentrations used in the present study. Since biologically compounds like glucose, NaCl and urea are found in the human body, the attained outcomes can be important in finding of effective drug delivery systems.

Production, characterization, and application of Pseudoxanthomonas taiwanensis biosurfactant: a green chemical for microbial enhanced oil recovery (MEOR).

Biosurfactants, as microbial bioproducts, have significant potential in the field of microbial enhanced oil recovery (MEOR). Biosurfactants are microbial bioproducts with the potential to reduce the interfacial tension (IFT) between crude oil and water, thus enhancing oil recovery. This study aims to investigate the production and characterization of biosurfactants and evaluate their effectiveness in increasing oil recovery. Pseudoxanthomonas taiwanensis was cultured on SMSS medium to produce biosurfactants. Crude oil was found to be the most effective carbon source for biosurfactant production. The biosurfactants exhibited comparable activity to sodium dodecyl sulfate (SDS) at a concentration of 400 ppm in reducing IFT. It was characterized as glycolipids, showing stability in emulsions at high temperatures (up to 120 °C), pH levels ranging from 3 to 9, and NaCl concentrations up to 10% (w/v). Response surface methodology revealed the optimized conditions for the most stable biosurfactants (pH 7, temperature of 40 °C, and salinity of 2%), resulting in an EI24 value of 64.45%. Experimental evaluations included sand pack column and core flooding studies, which demonstrated additional oil recovery of 36.04% and 12.92%, respectively. These results indicate the potential application of P. taiwanensis biosurfactants as sustainable and environmentally friendly approaches to enhance oil recovery in MEOR processes.

Selection of lubricant type and concentration for orodispersible tablets.

Lubricants are essential for most tablet formulations as they assist powder flow, prevent adhesion to tableting tools and facilitate tablet ejection. Magnesium stearate (MgSt) is an effective lubricant but may compromise tablet strength and disintegratability. In the design of orodispersible tablets, tablet strength and disintegratability are critical attributes of the dosage form. Hence, this study aimed to conduct an in-depth comparative study of MgSt with alternative lubricants, namely sodium lauryl sulphate (SLS), stearic acid (SA) and hydrogenated castor oil (HCO), for their effects on the tableting process as well as tablet properties. Powder blends were prepared with lactose, sodium starch glycolate or crospovidone as the disintegrant, and a lubricant at different concentrations. Angle of repose was determined for the mixtures. Comparative evaluation was carried out based on the ejection force, tensile strength, liquid penetration and disintegratability of the tablets produced. As the lubricant concentration increased, powder flow and tablet ejection improved. The lubrication efficiency generally decreased as follows: MgSt > HCO > SA > SLS. Despite its superior lubrication efficacy, MgSt is the only lubricant of four evaluated that reduced tablet tensile strength. Tablet disintegration time was strongly determined by tensile strength and liquid penetration, which were in turn affected by the lubricant type and concentration. All the above factors should be taken into consideration when deciding the type and concentration of lubricant for an orodispersible tablet formulation.

Morphological aspects and the effectiveness of photodynamic inactivation against Rhizopus oryzae in different life cycles.

Mucormycosis is an extremely aggressive fungal disease with a high mortality rate, especially in people with compromised immune systems. Most cases of mucormycosis are caused by the fungus Rhizopus oryzae. The treatments used are based on high doses of antifungals, associated with surgical resections, when it is possible. However, even with this aggressive treatment, the estimated attributable mortality rate is high. There is therefore a need to develop adjuvant treatments. Photodynamic Inactivation (PDI) may be an auxiliary therapeutic option for mucormycosis. Due to the lack of reports in the literature on the morphology and photodynamic inactivation of R. oryzae, characterization of the fungus using Confocal Microscopy and Transmission Electron Microscopy, and different protocols using Photodithazine® (PDZ), a chlorin e6 compound, as a photosensitizer, were performed. The fungus growth rate under different concentrations and incubation times of the photosensitizer and its association with the surfactant Sodium Dodecyl Sulphate (SDS) was evaluated. For the hyphae, both in the light and dark phases, in the protocols using only PDZ, no effective photodynamic response was observed. Meanwhile with the combination of SDS 0.05% and PDZ, inhibition growth rates of 98% and 72% were achieved for the white and black phase, respectively. In the conidia phase, only a 1.7 log10 reduction of the infective spores was observed. High concentration of melanin and the complex and resistant structures, especially at the black phase, results in a high limitation of the PDI inactivation response. The combined use of the SDS resulted in an improved response, when compared to the one obtained with the amphotericin B treatment.

Assessing the precision of a detergent-assisted cartridge precipitation workflow for non-targeted quantitative proteomics.

Detergent-based workflows incorporating sodium dodecyl sulfate (SDS) necessitate additional steps for detergent removal ahead of mass spectrometry (MS). These steps may lead to variable protein recovery, inconsistent enzyme digestion efficiency, and unreliable MS signals. To validate a detergent-based workflow for quantitative proteomics, we herein evaluate the precision of a bottom-up sample preparation strategy incorporating cartridge-based protein precipitation with organic solvent to deplete SDS. The variance of data-independent acquisition (SWATH-MS) data was isolated from sample preparation error by modelling the variance as a function of peptide signal intensity. Our SDS-assisted cartridge workflow yield a coefficient of variance (CV) of 13%-14%. By comparison, conventional (detergent-free) in-solution digestion increased the CV to 50%; in-gel digestion provided lower CVs between 14% and 20%. By filtering peptides predicting to display lower precision, we further enhance the validity of data in global comparative proteomics. These results demonstrate the detergent-based precipitation workflow is a reliable approach for in depth, label-free quantitative proteome analysis.

Solvent-Induced Lag Phase during the Formation of Lysozyme Amyloid Fibrils Triggered by Sodium Dodecyl Sulfate: Biophysical Experimental and In Silico Study of Solvent Effects.

Amyloid aggregates arise from either the partial or complete loss of the native protein structure or the inability of proteins to attain their native conformation. These aggregates have been linked to several diseases, including Alzheimer's, Parkinson's, and lysozyme amyloidosis. A comprehensive dataset was recently reported, demonstrating the critical role of the protein's surrounding environment in amyloid formation. In this study, we investigated the formation of lysozyme amyloid fibrils induced by sodium dodecyl sulfate (SDS) and the effect of solvents in the medium. Experimental data obtained through fluorescence spectroscopy revealed a notable lag phase in amyloid formation when acetone solution was present. This finding suggested that the presence of acetone in the reaction medium created an unfavorable microenvironment for amyloid fibril formation and impeded the organization of the denatured protein into the fibril form. The in silico data provided insights into the molecular mechanism of the interaction between acetone molecules and the lysozyme protofibril, once acetone presented the best experimental results. It was observed that the lysozyme protofibril became highly unstable in the presence of acetone, leading to the complete loss of its ß-sheet conformation and resulting in an open structure. Furthermore, the solvation layer of the protofibril in acetone solution was significantly reduced compared to that in other solvents, resulting in fewer hydrogen bonds. Consequently, the presence of acetone facilitated the exposure of the hydrophobic portion of the protofibril, precluding the amyloid fibril formation. In summary, our study underscores the pivotal role the surrounding environment plays in influencing amyloid formation.

Monitoring of low-molecular-weight protein aggregation by CE-SDS as a complementary method to SE-HPLC.

Capillary electrophoresis with sodium dodecyl sulfate (CE-SDS) has long been proven to have excellent performance in the analysis and characterization of therapeutic proteins. However, it is rarely used for the detection of low-molecular-weight proteins or peptides. Our research has proved the ability of CE-SDS to characterize the purity of low-molecular-weight proteins (i.e., <10 kDa) and even polypeptides. In this article, insulin glargine was used as a model protein, and CE-SDS was used to analyze the samples damaged by heating and light exposure. The monomers, dimers, and trimers of insulin glargine were effectively separated, and the results of the mass spectrometry also confirmed the existence of two kinds of insulin aggregates. For comparison, the size-exclusion high-performance liquid chromatography (SE-HPLC) only showed a single aggregate peak. In addition, the denaturation conditions caused only the covalent aggregates to appear in the CE-SDS analysis. These advantages also make CE-SDS an excellent supplementary technology to the traditional SE-HPLC, providing biopharmaceutical analysts with more information.

Boosting the Separation of Adeno-Associated Virus Capsid Proteins by Liquid Chromatography and Capillary Electrophoresis Approaches.

The purity of the three capsid proteins that make up recombinant adeno-associated virus (rAAV) is considered a critical quality attribute of gene therapy products. As such, there is a clear need to develop separation methods capable of rapidly characterizing these three viral proteins (VPs). In this study, the potential benefits and limitations of different electrophoretic and chromatographic methods were evaluated, including capillary electrophoresis-sodium dodecyl sulfate (CE-SDS), reversed phase liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC), and hydrophobic interaction chromatography (HIC), for the analysis of VPs obtained from different serotypes (i.e., AAV2, AAV5, AAV8, and AAV9). CE-SDS is considered to be the reference method and provides a suitable separation of VP1-3 proteins using generic conditions and laser induced fluorescence detection. However, the characterization of post-translational modifications (i.e., phosphorylation, oxidation) remains difficult, and species identification is almost impossible due to the lack of compatibility between CE-SDS and mass spectrometry (MS). In contrast, RPLC and HILIC were found to be less generic than CE-SDS and require tedious optimization of the gradient conditions for each AAV serotype. However, these two chromatographic approaches are inherently compatible with MS, and were shown to be particularly sensitive in detecting capsid protein variants resulting from different post-translational modifications. Finally, despite being non-denaturing, HIC offers disappointing performance for viral capsid proteins characterization.

Characterization and exploration of an artifact in the reducing capillary electrophoresis-sodium dodecyl sulfate analysis of the 'me-too' drug zuberitamab related to rituximab.

For monoclonal antibody (mAb) drugs, the 'me-too' drug is a pharmacologically active compound that is structurally similar to the first-in-class drugs, acting on the same target and is used for the same therapeutic purposes, but it may differ in drug-drug interactions and adverse drug reactions. Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) has been widely used for quality evaluation of mAb drugs. The properties of the detected substances can interfere with the credibility and accuracy of the method. In the routine comparison analysis for both innovator rituximab and 'me-too' drug zuberitamab samples, an uncommon artifact related to the heavy chain (HC) of zuberitamab was observed in reducing CE-SDS and interfered with our identification of the purity of samples. In this work, the overall hydrophobicity of the HCs of rituximab, zuberitamab, and several other common mAbs was characterized and determined by reversed-phase high-performance liquid chromatography. Additionally, the local hydrophobicity and surface charge were compared using Expasy ProtScale and PyMOL software simulations. We concluded that noncovalent protein aggregation can be related to strong hydrophobicity and low electrostatic repulsion of local amino acid regions, which complicates drug quality control. These findings shed light on the relationship between protein aggregation and the local hydrophobicity region, and broaden the way to analyze the detection 'artifacts' in reducing CE-SDS studies of therapeutic proteins.

Effect of (bio)surfactant type and concentration on the gas-liquid equilibrium partitioning of hydrophobic volatile organic compounds.

The biological removal of hydrophobic volatile organic compounds (VOCs) is limited by their low water solubility and, therefore, low bioavailability. The addition of surfactants is a promising strategy, but to gain understanding and broaden its applicability, its effect on the solubility of hydrophobic VOCs should be investigated. This study evaluates the effect of 2 synthetic surfactants (sodium dodecyl sulfate (SDS) and Tween 80) and 3 biological surfactants (surfactin, rhamnolipid and saponin) on the gas-to-liquid equilibrium partitioning coefficient (KGL) of 7 hydrophobic VOCs at different critical micelle concentrations (CMC). For all VOCs, a decrease in their KGL was observed when a (bio)surfactant was added at 1 and 3 CMC. The highest decrease in KGL (71 - 96 %) was observed for all compounds when SDS was added at 3 CMC, whereas the smallest effect was noticed when Tween 80 or surfactin (5.1 - 75 %) were added at both concentrations. The results are explained in terms of the (bio)surfactant and VOC physical-chemical properties (e.g. CMC and polarity). This is the first study evaluating the effect of biological surfactants on KGL. These fundamental data are essential to improve the design and modeling of air treatment systems using (bio)surfactants.

Ultra-low protein residue of chitosan by one step H2O2 and sodium dodecyl sulfate treatment.

Residual protein in chitosan-based biomaterials may cause inflammation, allergy, and immune rejection after surgery, impeding their clinical application. Facile production of chitosan with ultra-low protein content (residual protein <0.2 %) is yet to be addressed. Herein, we proposed a one-step method for preparing chitosan with residue protein content <0.2 % by using hydrogen peroxide and sodium dodecyl sulfate, which is simple, time-saving, cost-effective, and acid/alkali-free. Notably, the molecular weight of chitosan can be reduced simultaneously. The effects of experimental parameters (i.e. hydrogen peroxide concentration (0.01 %-1 %), SDS concentration (5 %-20 %), and reaction temperature (50 °C-70 °C)) on the protein removal and molecular weight decrease were systematically analyzed by response surface methodology. The results show that temperature and H2O2 concentration are the main parameters affecting the deproteinization of chitosan. Further characterizations on the resulting ultra-low protein residue chitosan revealed unchanged chemical structure, enhanced crystallinity, and reduced thermal stability. The proposed one-step deproteination method may have great potential for industrial mass production of ultra-low protein residue chitosan.

Base-triggerable lauryl sarcosinate-dodecyl sulfate catanionic liposomes: structure, biophysical characterization, and drug entrapment/release studies.

Equimolar mixtures of oppositely charged single-chain amphiphiles form a variety of phases, including vesicles. Such catanionic mixed lipid systems show high stability and exhibit versatile physicochemical properties. In the present study we have investigated the aggregation behaviour of lauryl sarcosinate hydrochloride (LS·HCl) in aqueous dispersion as well as its interaction with the anionic surfactant sodium dodecyl sulfate (SDS). The CMC of LS·HCl was estimated to be ∼5 mM by isothermal titration calorimetry (ITC) and fluorescence spectroscopy using pyrene as the fluorescent probe. Turbidimetric and ITC studies on the interaction of LS·HCl with SDS demonstrated that the two surfactants form an equimolar catanionic complex. The crystal structure of the lauryl sarcosinate-dodecyl sulfate (LS-DS) complex revealed that the complex is stabilized by classical N-H⋯O as well as C-H⋯O hydrogen bonds, besides the electrostatic attraction between LS (cation) and DS (anion) and dispersion interactions between the hydrocarbon chains. Differential scanning calorimetry studies revealed that the phase transition of the equimolar LS-DS complex is significantly reduced compared to the analogous LG-DS and LA-DS complexes in the fully hydrated state. Dynamic light scattering, atomic force microscopy and transmission electron microscopy studies demonstrated that the LS-DS catanionic complex forms stable medium-sized vesicles (diameter of ∼300-500 nm). In vitro studies with 5-fluorouracil and rhodamine 6G showed efficient entrapment and release of these two anti-cancer drugs in the physiologically relevant pH range of 6.0-8.0, but with contrasting pH dependences. These observations indicate that LS-DS catanionic vesicles may find application in designing drug delivery systems.

Surface stabilization determines macrophage uptake, cytotoxicity, and bioactivity of curcumin nanocrystals.

Pharmaceutical nanocrystals represent a promising new formulation that combines the benefits of bulk crystalline materials and colloidal nanoparticles. To be applied in vivo, nanocrystals must meet several criteria, namely colloidal stability in physiological media, non-toxicity to healthy cells, avoidance of macrophage clearance, and bioactivity in the target tissue. In the present work, curcumin, a naturally occurring poorly water-soluble molecule with a broad spectrum of bioactivity has been considered a candidate substance for preparing pharmaceutical nanocrystals. Curcumin nanocrystals in the size range of 40-90 nm were prepared by wet milling using the following combination of steric and ionic stabilizers: Tween 80, sodium dodecyl sulfate, Poloxamer 188, hydroxypropyl methylcellulose, phospholipids (with and without polyethylene glycol), and their combination. Nanocrystals stabilized by a combination of phospholipids enriched with polyethylene glycol proved to be the most successful in all evaluated criteria; they were colloidally stable in all media, exhibited low macrophage clearance, and proved non-toxic to healthy cells. This curcumin nanoformulation also exhibited outstanding anticancer potential comparable to commercially used cytostatics (IC50 = 73 µM; 24 h, HT-29 colorectal carcinoma cell line) which represents an improvement of several orders of magnitude when compared to previously studied curcumin formulations. This work shows that the preparation of phospholipid-stabilized nanocrystals allows for the conversion of poorly soluble compounds into a highly effective "solution-like" drug delivery system at pharmaceutically relevant drug concentrations.

Poly(ethylene glycol)-Based Surfactant Reduces the Conformational Change of Adsorbed Proteins on Nanoparticles.

When in contact with a biological medium, the surfaces of nanoparticles are usually covered by proteins. In this regard, it was found that poly(ethylene glycol) (PEG) promotes the "stealth effect". This implies a reduction of unspecific protein adsorption and cellular uptake. Although information about the PEG-protein interaction was reported, more accurate and sophisticated structure and dynamics analyses are needed to understand the interaction processes in detail. This work studies the PEG-protein interaction using model nanoparticles stabilized either by the PEG-based surfactant Lutensol AT50 or sodium dodecyl sulfate. The interaction with human serum albumin was studied using neutron scattering techniques. The parameters obtained by small-angle neutron scattering yielded information about the adsorbed protein layer thickness. Protein structure changes were detected via differential scanning fluorimetry and elastic neutron scattering. This combination gives a better insight into the PEG-protein interaction, contributing to the design of nanomaterials for medical applications.

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.

Molecular Mechanism of Interaction of Curcumin with BSA, Surfactants and Live E. Coli Cell Membrane Revealed by Fluorescence Spectroscopy and Confocal Microscopy.

Clinical trials on the therapeutic effect of curcumin have proven to be highly effective against many diseases including cancer and Alzheimer's. However, the molecular mechanism of interaction of curcumin with protein and live cell membrane is poorly understood. Here, we report the mechanism of interaction of curcumin with bovine serum albumin (BSA) and live E. coli cell membrane in the presence of organized assemblies of sodium dodecyl sulfate (SDS) and cetrimonium bromide (CTAB) by fluorescence spectroscopy, laser-scanning confocal microscopy, and computation. Enhanced binding constant, blue-shifted emission spectra, and imaging of heterogeneous FRET on live bacteria cell membrane strongly indicate the complex formation of curcumin with strong hydrophobic interaction, which is further validated by computation. Finally, our results may shed light on the efficient strategy of applications of curcumin as a natural therapeutic lead in clinical trials against many life-threatening diseases.

Gel electrophoresis/electroelution sorting fractionator combined with filter-aided sample preparation for deep proteomic analysis.

Sample preparation and protein fractionation are important issues for proteomic studies. Protein extraction procedures strongly affect the performance of fractionation methods by provoking protein dispersion in several fractions. The most notable exception is the gel-based electrophoretic protein fractionation due to its resolution and effectiveness of sodium dodecyl sulfate as a solubilizing agent, while its main limitation lies in the poor recovery of the gel-trapped proteins. We created a fractionator device to separate complex mixture of proteins and peptides that is based on the continuous gel electrophoresis/electroelution sorting of these molecules. In an unsupervised process, complex mixtures of proteins or peptides are fractionated into the gel while separated fractions are simultaneously and sequentially electroeluted to the solution containing wells. The performance of the device was studied for protein fractionation in terms of reproducibility, protein recovery, and loading capacity. In a setup free of sodium dodecyl sulfate, complex peptide mixtures can also be fractionated. More than 11,700 proteins were identified in the whole-cell lysate of the CaSki cell line by using the fractionator combined with the filter-aided sample preparation method and mass spectrometry analysis. Fractionator-based proteome characterization increased 1.7-fold the number of identified proteins compared to the unfractionated sample analysis.

Decellularization with triton X-100 provides a suitable model for human kidney bioengineering using human mesenchymal stem cells.

AIMS: Regeneration of discarded human kidneys has been considered as an ideal approach to overcome organ shortage for the end-stage renal diseases (ESRDs). The aim of this study was to develop an effective method for preparation of kidney scaffolds that retain the matrix structure required for proliferation and importantly, differentiation of human adipose-derived mesenchymal stem cells (hAd-MSCs) into renal cells. MAIN METHODS: We first compared two different methods using triton X-100 and sodium dodecyl sulfate (SDS) for human kidney decellularization; followed by characterization of the prepared human renal extracellular matrix (ECM) scaffolds. Then, hAd-MSCs were seeded on the scaffolds and cultured for up to 3 weeks. Next, viability, proliferation, and migration of seeded hAd-MSCs underwent histological and scanning electron microscopy (SEM) assessments. Moreover, differentiation of hAd-MSCs into kidney-specific cell types was examined using immunohistochemistry (IHC) staining and qRT-PCR. KEY FINDINGS: Our results indicated that triton X-100 was a more effective detergent for decellularization of human kidneys compared with SDS. Moreover, attachment and proliferation of hAd-MSCs within the recellularized human kidney scaffolds, were confirmed. Seeded cells expressed epithelial and endothelial differentiation markers, and qRT-PCR results indicated increased expression of platelet and endothelial cell adhesion molecule 1 (PECAM-1), paired box 2 (PAX2), and E-cadherine (E-CDH) as markers of differentiation into epithelial and endothelial cells. SIGNIFICANCE: These observations indicate the effectiveness of decellularization with triton X-100 to generate suitable human ECM renal scaffolds, which supported adhesion and proliferation of hAd-MSCs and could induce their differentiation towards a renal lineage.

Biochemical Characterisation of Human Transglutaminase 4.

Transglutaminases are protein-modifying enzymes involved in physiological and pathological processes with potent therapeutic possibilities. Human TG4, also called prostate transglutaminase, is involved in the development of autoimmune and tumour diseases. Although rodent TG4 is well characterised, biochemical characteristics of human TG4 that could help th e understanding of its way of action are not published. First, we analysed proteomics databases and found that TG4 protein is present in human tissues beyond the prostate. Then, we studied in vitro the transamidase activity of human TG4 and its regulation using the microtitre plate method. Human TG4 has low transamidase activity which prefers slightly acidic pH and a reducing environment. It is enhanced by submicellar concentrations of SDS suggesting that membrane proximity is an important regulatory event. Human TG4 does not bind GTP as tested by GTP-agarose and BODIPY-FL-GTPγS binding, and its proteolytic activation by dispase or when expressed in AD-293 cells was not observed either. We identified several potential human TG4 glutamine donor substrates in the AD-293 cell extract by biotin-pentylamine incorporation and mass spectrometry. Several of these potential substrates are involved in cell-cell interaction, adhesion and proliferation, suggesting that human TG4 could become an anticancer therapeutic target.