Ionic Detergents Research Articles

Characteristic study of Candida rugosa lipase immobilized on lignocellulosic wastes: effect of support material.

For the first time is reported the comparison of solid biocatalysts derived from Candida rugosa lipase (CRL) immobilized on different lignocellulosic wastes (rice husk, brewer's spent grain, hemp tea waste, green tea waste, vine bark, and spent coffee grounds) focusing on the characterization of these materials and their impact on the lipase-support interaction. The wastes were subjected to meticulous characterization by ATR-FTIR, BET, and SEM analysis, besides lignin content and hydrophobicity determination. Investigating parameters influencing immobilization performance revealed the importance of morphology, textural properties, and hydrophobic interactions revealed the importance of morphology, textural properties and especially hydrophobic interactions which resulted in positive correlations between surface hydrophobicity and lipase immobilization efficiency. Hemp tea waste and spent coffee grounds demonstrated superior immobilization performances (7.20 U/g and 8.74 U/g immobilized activity, 102.3% and 33.5% efficiency, 13.4% and 15.4% recovery, respectively). Moreover, they demonstrated good temporal stability (100% and 92% residual activity after 120days, respectively) and retained 100% of their immobilized activity after five reuses in the hydrolysis of p-nitrophenyl palmitate in hexane. In addition, the study of enzymatic desorption caused by ionic strength and detergent treatments indicated mixed hydrophobic and electrostatic interactions in rice husk, vine bark, and spent coffee grounds supports, while hemp tea waste and green tea waste were dominated by hydrophobic interactions.

Synthesis of Asymmetric Ionic Hybrid Detergents enables Micelles with Scalable Properties including Cell Compatibility.

Ionic detergents enable applications and cause harm in biospheres due to cell toxicity. The utility of covalent combinations between ionic and non-ionic detergent headgroups in modulating cell toxicity remains speculative due to the yet rarely explored synthesis. We close this gap and establish the modular synthesis of ionic/non-ionic hybrid detergents. We restructure a combinatorial methallyl dichloride one-pot coupling into a two-step coupling, which reduces by-products, improves product yields, and enables the gram-scale preparation of asymmetric, cationic/non-ionic and anionic/non-ionic hybrid detergents. Our modular synthesis delivers new modalities for the design of ionic detergents, including an unprecedented scaling of properties that determine applications, such as charge, critical micelle concentration, solubilizing properties, hard water tolerance, and cell compatibility. We uncover that shielding the charge in ionic headgroups can switch the detergent species that is toxic to cells from monomers to mixtures of monomers and micellar assemblies. Establishing the chemistry of ionic/non-ionic hybrid detergents provides a missing evolutionary link in the structural comparison of ionic and non-ionic detergents, enables an easy synthesis access to yet unexplored chemical spaces of asymmetric hybrid materials, and delivers new modalities for designing the toxicity of supramolecular nanomaterials.

Optimization of Enzymatic and Chemical Decellularization of Native Porcine Heart Valves for the Generation of Decellularized Xenografts.

One of the most important medical interventions for individuals with heart valvular disease is heart valve replacement, which is not without substantial challenges, particularly for pediatric patients. Due to their biological properties and biocompatibility, natural tissue-originated scaffolds derived from human or animal sources are one type of scaffold that is widely used in tissue engineering. However, they are known for their high potential for immunogenicity. Being free of cells and genetic material, decellularized xenografts, consequently, have low immunogenicity and, thus, are expected to be tolerated by the recipient's immune system. The scaffold ultrastructure and ECM composition can be affected by cell removal agents. Therefore, applying an appropriate method that preserves intact the structure of the ECM plays a critical role in the final result. So far, there has not been an effective decellularization technique that preserves the integrity of the heart valve's ultrastructure while securing the least amount of genetic material left. This study demonstrates a new protocol with untraceable cells and residual DNA, thereby maximally reducing any chance of immunogenicity. The mechanical and biochemical properties of the ECM resemble those of native heart valves. Results from this study strongly indicate that different critical factors, such as ionic detergent omission, the substitution of Triton X-100 with Tergitol, and using a lower concentration of trypsin and a higher concentration of DNase and RNase, play a significant role in maintaining intact the ultrastructure and function of the ECM.

Decellularized human amniotic membrane scaffolds: influence on the biological behavior of dental pulp stem cells

ObjectiveThe objective of this study was to assess the characterization of human acellular amniotic membrane (HAAM) using various decellularization methods and their impact on the proliferation and differentiation of human dental pulp stem cells (DPSCs). The goal was to identify scaffold materials that are better suited for pulp regeneration.MethodsSix different decellularization methods were used to generate the amniotic membranes. The characteristics of these scaffolds were examined through hematoxylin and eosin (H&E) staining, scanning electron microscopy (SEM), and immunohistofluorescence staining (IHF). The DPSCs were isolated, cultured, and their capacity for multidirectional differentiation was verified. The third generation (P3) DPSCs, were then combined with HAAM to form the decellularized amniotic scaffold-dental pulp stem cell complex (HAAM-DPSCs complex). Subsequently, the osteogenic capacity of the HAAM-DPSCs complex was evaluated using CCK8 assay, live-dead cell staining, alizarin red and alkaline phosphatase staining, and real-time quantitative PCR (RT-PCR).ResultsOut of the assessed decellularization methods, the freeze-thaw + DNase method and the use of ionic detergent (CHAPS) showed minimal changes in structure after decellularization, making it the most effective method. The HAAM-DPSCs complexes produced using this method demonstrated enhanced biological properties, as indicated by CCK8, alizarin red, alkaline phosphatase staining, and RT-PCR.ConclusionThe HAAM prepared using the freeze-thaw + DNase method and CHAPS methods exhibited improved surface characteristics and significantly enhanced the proliferation and differentiation capacity of DPSCs when applied to them. The findings, therefore demonstrate the capacity for enhanced pulp regeneration therapy.

Adsorption features of reduced aminated supports modified with glutaraldehyde: Understanding the heterofunctional features of these supports

Immobilization of enzymes on aminated supports using the glutaraldehyde chemistry may involve three different interactions, cationic, hydrophobic, and covalent interactions. To try to understand the impact this heterofunctionality, we study the physical adsorption of the beta-galactosidase from Aspergillus niger, on aminated supports (MANAE) and aminated supports with one (MANAE-GLU) or two molecules of glutaraldehyde (MANAE-GLU-GLU). To eliminate the chemical reactivity of the glutaraldehyde, the supports were reduced using sodium borohydride. After enzyme adsorption, the release of the enzyme from the supports using different NaCl concentrations, Triton X100, ionic detergents (SDS and CTAB), or different temperatures (4 °C to 55 °C) was studied. Using MANAE support, at 0.3 M NaCl almost all the immobilized enzyme was released. Using MANAE-GLU, 0.3 M, and 0.6 M NaCl similar results were obtained. However, incubation at 1 M or 2 M NaCl, many enzyme molecules were not released from the support. For the MANAE-GLU-GLU support, none of the tested concentrations of NaCl was sufficient to release all enzyme bound to the support. Only using high temperatures, 0.6 M NaCl, and 1 % CTAB or SDS, could the totality of the proteins be released from the support. The results shown in this paper confirm the heterofunctional character of aminated supports modified with glutaraldehyde.

An update on a technical method of cartilage decellularization: (a physical-based protocol)

Introduction: Despite advances in orthopedic surgery, the lack of effective conventional treatment for cartilage defects has led to research in cartilage tissue engineering. One of the interesting topics is the use of decellularized extracellular matrix (ECM) as a suitable natural scaffold that supports the growth and function of cells cultured in it. A concern with decellularization protocols, especially those with high detergent concentrations, is the disruption of native ECM, which has deleterious effects on subsequent scaffold recellularization. Therefore, this study focused on optimizing cartilage decellularization by physical methods without the use of ionic detergents. Methods: The bovine tracheal cartilage fragments were decellularized by a combination of 8 cycles of freeze-thaw and ultrasound techniques. Then, the tissues were immersed and shaken in 0.25% trypsin for 24 hours. Efficient cell removal and preservation of ECM were confirmed by histological and cytocompatibility assessments. The in-vivo studies were performed to evaluate the biocompatibility and bioactivity of the scaffold. Results: The histological assessments indicated the appropriate cytocompatibility and the fibroblast cell culture study demonstrated that cells were able to proliferate and migrate on the decellularized cartilage. In-vivo evaluation also showed a reduced adverse immune response, including leukocyte infiltration into the ECM. Conclusions: These results suggest that a cartilage scaffold created using a physical decellularization protocol that efficiently removes cells while preserving the native ECM can be a suitable scaffold for cartilage reconstruction. The main advantage of this protocol is the absence of potentially toxic chemicals in the tissues.

Characterization of goat fiber physical properties from indigenous Ethiopian goat breeds

This study aimed to characterize the physical properties of goat fibers from some indigenous Ethiopian goat breeds. Ethiopia has different indigenous goat breeds, which are the livelihood of Ethiopian farmers and are mainly used as a source of meat, milk, fiber, hides, etc. The fiber samples were collected from Abergelle, Central Highland, and Western Lowland indigenous Ethiopian goat breeds. After shearing, scouring was applied for the removal of some impurities using ionic and non-ionic detergents and chemicals. In this study, fiber length, fineness, tensile strength and elongation, and fiber diameter were evaluated using scientific test-standard methods. The study results showed that the average tensile strengths of goat fibers obtained from Abergelle, Central Highland, and Western Lowland were 13.38, 13.31, and 13.26 cN/Tex, respectively. Similarly, the elongation percentage values were found between 22% and 24% and 32% and 34% in dry and wet conditions, respectively. The moisture regain and moisture content values ranged from 14.04% to 14.10% and 12.61% to 12.68%, respectively. The fiber length values were between 38 and 43 mm, with a diameter ranging from 18 to 21 μm. Even though there was a slight difference in some values of physical properties, the fibers obtained from each goat breed have satisfactory mechanical properties: good strength, fineness, elongation, moisture regain, and low density. Generally, this study proved that the goat fiber obtained from some indigenous Ethiopian goat breeds is a potential source of raw material for textile manufacturers and researchers.

Descelularización de ovario de rata: comparación de diferentes protocolos de generación de andamios para aplicaciones en bioingeniería

Ovarian failure, hormonal disorders, and side effects of cancer treatments are some underlying causes that can affect a woman’s ability to conceive. Given this concern, different preventive options arise to preserve fertility such as cryopreservation of embryos, oocytes, and ovarian tissue; however, they have significant limitations. Currently, bioengineering allows the adoption of different strategies against reproductive disorders and that is why this study evaluates the first step to manufacture in vitro decellularized ovarian bioscaffolds based on the specific ECM of ovarian tissue. To this end, complete rat ovaries have been used that have been subjected to four decellularization protocols that combine different mechanical-chemical-enzymatic treatments in order to eliminate cell nuclei, while maintaining the macro and microstructure of the native tissue. The results of this study, of a histological and histochemical nature, have not revealed significant data regarding the elimination of cell nuclei. However, a novel protocol was identified that has generously removed some of the cellular material while preserving a good ECM structure. All this supports the idea of ​​the importance of the selection of decellularizing agents and their respective concentrations; with special emphasis on the concentration of the strongest ionic detergents such as SDS and the effect it causes on the samples. In addition, what has already been revealed in recent studies has been confirmed, which is that the ovarian scaffolds generated with SDC have been able to better preserve the extracellular composition, which could be beneficial for recellularization and other applications. These generated bioscaffolds form a promising basis for future experiments where the objective should be to optimize the protocols and conduct additional studies.

Self-Cleavage of Human Chloride Channel Accessory 2 Causes a Conformational Shift That Depends on Membrane Anchorage and Is Required for Its Regulation of Store-Operated Calcium Entry.

Human CLCA2 regulates store-operated calcium entry (SOCE) by interacting with Orai1 and STIM1. It is expressed as a 943aa type I transmembrane protein that is cleaved at amino acid 708 to produce a diffusible 100 kDa product. The N-terminal ectodomain contains a hydrolase-like subdomain with a conserved HEXXH zinc-binding motif that is proposed to cleave the precursor autoproteolytically. Here, we tested this hypothesis and its link to SOCE. We first studied the conditions for autocleavage in isolated membranes and then in a purified protein system. Cleavage was zinc-dependent and abolished by mutation of the E in the HEXXH motif to Q, E165Q. Cleavage efficiency increased with CLCA2 concentration, implying that it occurs in trans. Accordingly, the E165Q mutant was cleaved by co-transfected wildtype CLCA2. Moreover, CLCA2 precursors with different epitope tags co-immunoprecipitated. In a membrane-free system utilizing immunopurified protease and target, no cleavage occurred unless the target was first denatured, implying that membranes provide essential structural or conformational cues. Unexpectedly, cleavage caused a conformational shift: an N-terminal antibody that immunoprecipitated the precursor failed to precipitate the N-terminal product unless the product was first denatured with an ionic detergent. The E165Q mutation abolished the stimulation of SOCE caused by wildtype CLCA2, establishing that the metalloprotease activity is required for this regulatory function.

Impact of various detergent-based immersion and perfusion decellularization strategies on the novel caprine pancreas derived extracellular matrix scaffold.

Limited availability of the organs donors has facilitated the establishment of xenogeneic organ sources for transplantation. Numerous studies have decellularized several organs and assessed their implantability in order to provide such organs. Among all the decellularized organs studies for xenotransplantation, the pancreas has garnered very limited amount of research. The presently offered alternatives for pancreas transplantation are unable to liberate patients from donor dependence. The rat and mice pancreas are not of an accurate size for transplantation but can only be used for in-vitro studies mimicking in-vivo immune response in humans, while the porcine pancreas can cause zoonotic diseases as it carries porcine endogenous retrovirus (PERV- A/B/C). Therefore, we propose caprine pancreas as a substitute for these organs, which not only reduces donor dependence but also poses no risk of zoonosis. Upon decellularization the extracellular matrix (ECM) of different tissues responds differently to the detergents used for decellularization at physical and physiological level; this necessitates a comprehensive analysis of each tissue independently. This study investigates the impact of decellularization by ionic (SDS and SDC), non-ionic (Triton X-100 and Tween-20), and zwitterionic detergents (CHAPS). All these five detergents have been used to decellularize caprine pancreas via immersion (ID) and perfusion (PD) set-up. In this study, an extensive comparison of these two configurations (ID and PD) with regard to each detergent has been conducted. The final obtained scaffold with each set-up has been evaluated for the left-over cytosolic content, ECM components like sGAG, collagen, and fibronectin were estimated via Prussian blue and Immunohistochemical staining respectively, and finally for the tensile strength and antimicrobial activity. All the detergents performed consistently superior in PD than in ID. Conclusively, PD with SDS, SDC, and TX-100 successfully decellularizes caprine pancreatic tissue while retaining ECM architecture and mechanical properties. This research demonstrates the viability of caprine pancreatic tissue as a substitute scaffold for porcine organs and provides optimal decellularization protocol for this xenogeneic tissue. This research aims to establish a foundation for further investigations into potential regenerative strategies using this ECM in combination with other factors.

Conformations of Human Immunodeficiency Virus Envelope Glycoproteins in Detergents and Styrene-Maleic Acid Lipid Particles.

The mature human immunodeficiency virus (HIV) envelope glycoprotein (Env) trimer, which consists of noncovalently associated gp120 exterior and gp41 transmembrane subunits, mediates virus entry into cells. The pretriggered (State-1) Env conformation is the major target for broadly neutralizing antibodies (bNAbs), whereas receptor-induced downstream Env conformations elicit immunodominant, poorly neutralizing antibody (pNAb) responses. To examine the contribution of membrane anchorage to the maintenance of the metastable pretriggered Env conformation, we compared wild-type and State-1-stabilized Envs solubilized in detergents or in styrene-maleic acid (SMA) copolymers. SMA directly incorporates membrane lipids and resident membrane proteins into lipid nanoparticles (styrene-maleic acid lipid particles [SMALPs]). The integrity of the Env trimer in SMALPs was maintained at both 4°C and room temperature. In contrast, Envs solubilized in Cymal-5, a nonionic detergent, were unstable at room temperature, although their stability was improved at 4°C and/or after incubation with the entry inhibitor BMS-806. Envs solubilized in ionic detergents were relatively unstable at either temperature. Comparison of Envs solubilized in Cymal-5 and SMA at 4°C revealed subtle differences in bNAb binding to the gp41 membrane-proximal external region, consistent with these distinct modes of Env solubilization. Otherwise, the antigenicity of the Cymal-5- and SMA-solubilized Envs was remarkably similar, both in the absence and in the presence of BMS-806. However, both solubilized Envs were recognized differently from the mature membrane Env by specific bNAbs and pNAbs. Thus, detergent-based and detergent-free solubilization at 4°C alters the pretriggered membrane Env conformation in consistent ways, suggesting that Env assumes default conformations when its association with the membrane is disrupted. IMPORTANCE The human immunodeficiency virus (HIV) envelope glycoproteins (Envs) in the viral membrane mediate virus entry into the host cell and are targeted by neutralizing antibodies elicited by natural infection or vaccines. Detailed studies of membrane proteins rely on purification procedures that allow the proteins to maintain their natural conformation. In this study, we show that a styrene-maleic acid (SMA) copolymer can extract HIV-1 Env from a membrane without the use of detergents. The Env in SMA is more stable at room temperature than Env in detergents. The purified Env in SMA maintains many but not all of the characteristics expected of the natural membrane Env. Our results underscore the importance of the membrane environment to the native conformation of HIV-1 Env. Purification methods that bypass the need for detergents could be useful tools for future studies of HIV-1 Env structure and its interaction with receptors and antibodies.

#4868 MASS-SPECTROMETRY ANALYSIS OF CONGOPHILIC URINE AND ITS DETERGENT RESISTANT FRACTION IN PATIENTS WITH AMYLOID AND NON-AMYLOID NEPHROPATHIES

Abstract Background and Aims Being a well-known intrinsic property of amyloid, сongophilia was recently demonstrated in urine of patients with proteinuria of different etiology including renal amyloidosis (RA) and non-amyloid nephropathies (NANP) [1]. Urine proteins (UPs) responsible for congophilia in RA and NANP are supposed to have another property of amyloid such as resistance to ionic detergents and present in urine as detergent-resistant aggregates (DRA). In the pilot study we performed mass-spectrometry (MS) analysis of congophilic urine samples and its detergent-resistant fraction in patients with RA and NANP to investigate UPs and their specificity to the particular kidney disease. Method We collected first morning void urine samples from patients with RA (n = 4) and NANP (n = 4). Urine congophilia was assessed by Congo red Dot test as described previously [1]. To analyze bulk urine (BU) proteins, 30 μl of supernatant was precipitated with 80% acetone and boiled in 2% sodium dodecyl sulfate (SDS) for 30 minutes. Then 5 μg of UPs was separated in 10% polyacrylamide gel electrophoresis (PAGE) followed by Coomassie blue staining. The ultracentrifugation of 0.5-3 ml of urine at 300,000x g for 16h following by the treatment of precipitate with 3% sarcosyl in phosphate-buffered saline (PBS) for 10 min with subsequent washing of resistant aggregates in PBS were applied to obtain DRA. After boiling in SDS 8 μl of the sample was separated in 10% PAGE. UPs concentration in the DRA was estimated by densitometry regarding to 2 μg of bovine serum albumin. Then 6 μg of UPs was digested with trypsin, followed by purification on silicate (CDS Empore™ C18 Extraction Disks). Prepared samples of BU and DRA were analyzed by electrospray ionization tandem MS. We used MsFragger software to obtain lists of UPs for each sample. Most representative UPs in the sample were selected by the unique spectral count (USC). We considered the protein as having diagnosis-specific potential (DSP) if it was present in every sample of the particular disease group, i.e. AA, AL, NANP in BU or DRA, and was absent in any sample of another disease group. Results The patients had following characteristics: age 51±13 years, 3 male/ 5 female, eGFR = 45 (19; 95) ml/min/1.73 m2; 24 h proteinuria = 6.5 (4.5; 7.9) g. In PAGE analysis, BU proteins appeared to be similar in all 8 samples with 2 major bends in 70 kDa and 50 kDa regions, corresponding to albumin and immunoglobulin (Ig) heavy chains, respectively (Fig. 1A). The amount of protein in DRA was small and comprised 1.02 (0.71; 1.61) % of the total protein in the sample (4.7 (2.3; 5,1) g/l). Compared with BU PAGE analysis of DRA proteins revealed other bends with trace albumin bend, predominance of 45 kDA region bend and more apparent 30 kDa bend (Ig light chains) in the majority of patients (Fig. 1B). Results of MS analysis are shown on the Figure 2. There were more DSP revealed in DRA vs BU: 14 vs 5, 46 vs 2 and 4 vs 2 UPs in samples of AL, AA and NANP, respectively. When compare by a particular disease group, there were no DSP found either in BU or DRA in RA samples. One protein (aminopeptidase N) and 3 proteins (isoform 3 of unconventional myosin-LC, protein S100-A8, elongation factor 1-α 1) were detected as DSP in BU and DRA in AL, respectively. Alpha-1-acid glycoprotein 1 was only DSP for AA in DRA as well as serum paraoxonase 1 for NANP in BU. Venn-diagrams of shared and divergent UPs are present on the Figures 2D-G. Conclusion Although DRA represented a small portion of UPs, its composition significantly differed from BU and could contain more specific disease markers that makes urine detergent-resistant fraction promising for the future research. Understanding the role of DRA proteins in the pathogenesis of amyloid and non-amyloid renal disease and their diagnostic utility requires further studies.

Amyloid fibrils degradation: the pathway to recovery or aggravation of the disease?

Background: The most obvious manifestation of amyloidoses is the accumulation of amyloid fibrils as plaques in tissues and organs, which always leads to a noticeable deterioration in the patients' condition and is the main marker of the disease. For this reason, early diagnosis of amyloidosis is difficult, and inhibition of fibrillogenesis, when mature amyloids are already accumulated in large quantities, is ineffective. A new direction for amyloidosis treatment is the development of approaches aimed at the degradation of mature amyloid fibrils. In the present work, we investigated possible consequences of amyloid's degradation. Methods: We analyzed the size and morphology of amyloid degradation products by transmission and confocal laser scanning microscopy, their secondary structure and spectral properties of aromatic amino acids, intrinsic chromophore sfGFP, and fibril-bound amyloid-specific probe thioflavin T (ThT) by the absorption, fluorescence and circular dichroism spectroscopy, as well as the cytotoxicity of the formed protein aggregates by MTT-test and their resistance to ionic detergents and boiling by SDS-PAGE. Results: On the example of sfGFP fibrils (model fibrils, structural rearrangements of which can be detected by a specific change in the spectral properties of their chromophore), and pathological Aβ-peptide (Aβ42) fibrils, leading to neuronal death in Alzheimer's disease, the possible mechanisms of amyloids degradation after exposure to factors of different nature (proteins with chaperone and protease activity, denaturant, and ultrasound) was demonstrated. Our study shows that, regardless of the method of fibril degradation, the resulting species retain some amyloid's properties, including cytotoxicity, which may even be higher than that of intact amyloids. Conclusion: The results of our work indicate that the degradation of amyloid fibrils in vivo should be treated with caution since such an approach can lead not to recovery, but to aggravation of the disease.

Effects of Chemical Additives in Refolding Buffer on Recombinant Human BMP-2 Dimerization and the Bioactivity on SaOS-2 Osteoblasts.

Bone morphogenetic protein-2 (BMP-2) is a promising osteogenic agent in tissue engineering. BMP-2 is usually expressed in Escherichia coli owing to the high yield and low cost, but the protein is expressed as inclusion bodies. Thus, the bottleneck for BMP-2 production in E. coli is the refolding process. Here, we explored the effects of the refolding buffer composition on BMP-2 refolding. The BMP-2 inclusion body was solubilized in urea and subjected to refolding by the dilution method. Various additives were investigated to improve the BMP-2 refolding yield. Nonreducing SDS-PAGE showed that BMP-2 dimers, the presumably biologically active form, were detected at approximately 25 kDa. The highest yield of the BMP-2 dimers was observed in the refolding buffer that contained ionic detergents (sarkosyl and cetylpyridinium chloride) followed by zwitterionic and nonionic detergents (NDSB-195, NP-40, and Tween 80). In addition, sugars (glucose, sorbitol, and sucrose) in combination with anionic detergents (sodium dodecyl sulfate and sarkosyl) reduced BMP-2 oligomers and increased the BMP-2 dimer yield. Subsequently, the refolded BMP-2s were tested for their bioactivity using the alkaline phosphatase assay in osteogenic cells (SaOS-2), as well as the luciferase reporter assay and the calcium assays. The refolded BMP-2 showed the activities in the calcium deposition assay and the luciferase reporter assay but not in the alkaline phosphatase activity assay or the intracellular calcium assay even though the dimers were clearly detected. Therefore, the detection of the disulfide-linked dimeric BMP-2 in nonreducing SDS-PAGE is an inadequate proxy for the bioactivity of BMP-2.

A rapid and efficient technique for the isolation of Bacillus genomic DNA using a cocktail of peptidoglycan hydrolases of different type.

The paper suggests a rapid and efficient technique for isolation of genomic DNA from the bacteria of the genus Bacillus, which is based on the hydrolysis of cell wall peptidoglycan by a cocktail of peptidoglycan hydrolases of different type (L,D-peptidase and N-acetylmuramidase). The comparing of conventional techniques for the isolation of genomic DNA using: a microwave treatment; a treatment with ionic detergents (SDS, CTAB) or a chaotropic agent (GuSCN); and enzymatic hydrolysis (nonspecific, with proteinase K, or specific, with peptidoglycan hydrolases) conducted on Bacillus megaterium, B. subtilis, B. licheniformis, B. cereus showed that the most effective ones were techniques based on the specific hydrolysis of cell wall peptidoglycan. The highest efficiency of hydrolysis was obtained with an enzyme cocktail consisted of hen egg muramidase (HEWL) and highly active phage-specific L,D-peptidase EndoRB49 revealed a pronounced synergism between the peptidase and the muramidase. The cocktail treatment of Bacillus cells could be reduced to 10min without affecting the yield of nucleic acids. The quality of DNA preparations was assessed using the restriction and PCR assays, as well as agarose gel electrophoresis. Using peptidoglycan hydrolases of different type, which have a good synergy, makes the technique very efficient and perspective for the application when rapid and effective disintegration of cell wall is crucial to avoid adverse effects of macromolecular denaturation.

The Development of Small-Caliber Vascular Grafts Using Human Umbilical Artery: An Evaluation of Methods.

Due to the prevalence of cardiovascular disease in the United States, small-caliber vascular grafts for coronary bypass surgery continue to be in high demand. Human umbilical arteries, an underutilized resource, were decellularized using zwitterionic (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate [CHAPS]) and ionic (sodium dodecyl sulfate [SDS]) detergents and evaluated as potential vascular grafts. Vessels were tested for decellularization efficacy, mechanical integrity, and recellularization potential. Hematoxylin and eosin staining and DNA quantification revealed moderate to successful removal of cells in both conditions. While CHAPS-decellularized vessels displayed collagen structure most similar to intact tissue, both CHAPS- and SDS-decellularized vessels demonstrated burst pressures lower than that of intact tissue. Alcian Blue staining and sulfated glycosaminoglycan (sGAG) quantification indicated the preservation of sGAG content after both decellularization pathways. Both conditions were also capable of recellularization with human umbilical vein endothelial cells, and the use of a basic fibroblast growth factor treatment did not have a significant effect on the density of adhered cells after 5 days. Whole CHAPS-decellularized vessels were successfully recellularized. Additionally, an evaluation of the effects of freeze-thaw cycles was performed. In summary, human umbilical arteries present a promising alternative for small-caliber vascular grafts due to their high availability and ability to be decellularized and recellularized for safe and successful implantation. Impact Statement Coronary heart disease accounts for one of nine deaths in the United States each year. Bypass surgery has been shown to decrease the risk of heart attack; however, many patients do not have a suitable saphenous vein, which is required to redirect blood flow around their blocked arteries. In this study, we evaluate decellularized umbilical artery as a potential small-diameter vascular graft based on its mechanical properties and its recellularization potential.

Development and evaluation of a novel combined perfusion decellularization heart-lung model for tissue engineering of bioartificial heart-lung scaffolds.

Bioengineered transplantable heart-lung scaffolds could be potentially lifesaving in a large number of congenital and acquired cardiothoracic disorders including terminal heart-lung disease. We decellularized heart-lung organ-blocks from rats (n=10) by coronary and tracheal perfusion with ionic detergents in a modified Langendorff circuit. In the present project, we were able to achieve complete decellularization of the heart-lung organ-block. Decellularized heart-lung organ-blocks lacked intracellular components but maintained structure of the cellular walls with collagen and elastic fibers. We present a novel model of combined perfusion and decellularization of heart-lung organ-blocks. This model is the first step on the pathway to creating bioengineered transplantable heart-lung scaffolds. We believe that further development of this technology could provide a life-saving conduit, significantly reducing the risks of heart-lung failure surgery and improving postoperative quality of life.

Human RAD51 Protein Forms Amyloid-like Aggregates In Vitro.

RAD51 is a central protein of homologous recombination and DNA repair processes that maintains genome stability and ensures the accurate repair of double-stranded breaks (DSBs). In this work, we assessed amyloid properties of RAD51 in vitro and in the bacterial curli-dependent amyloid generator (C-DAG) system. Resistance to ionic detergents, staining with amyloid-specific dyes, polarized microscopy, transmission electron microscopy (TEM), X-ray diffraction and other methods were used to evaluate the properties and structure of RAD51 aggregates. The purified human RAD51 protein formed detergent-resistant aggregates in vitro that had an unbranched cross-β fibrillar structure, which is typical for amyloids, and were stained with amyloid-specific dyes. Congo-red-stained RAD51 aggregates demonstrated birefringence under polarized light. RAD51 fibrils produced sharp circular X-ray reflections at 4.7 Å and 10 Å, demonstrating that they had a cross-β structure. Cytoplasmic aggregates of RAD51 were observed in cell cultures overexpressing RAD51. We demonstrated that a key protein that maintains genome stability, RAD51, has amyloid properties in vitro and in the C-DAG system and discussed the possible biological relevance of this observation.

Efficacy of Nerve-Derived Hydrogels to Promote Axon Regeneration Is Influenced by the Method of Tissue Decellularization.

Injuries to large peripheral nerves are often associated with tissue defects and require reconstruction using autologous nerve grafts, which have limited availability and result in donor site morbidity. Peripheral nerve-derived hydrogels could potentially supplement or even replace these grafts. In this study, three decellularization protocols based on the ionic detergents sodium dodecyl sulfate (P1) and sodium deoxycholate (P2), or the organic solvent tri-n-butyl phosphate (P3), were used to prepare hydrogels. All protocols resulted in significantly decreased amounts of genomic DNA, but the P2 hydrogel showed the best preservation of extracellular matrix proteins, cytokines, and chemokines, and reduced levels of sulfated glycosaminoglycans. In vitro P1 and P2 hydrogels supported Schwann cell viability, secretion of VEGF, and neurite outgrowth. Surgical repair of a 10 mm-long rat sciatic nerve gap was performed by implantation of tubular polycaprolactone conduits filled with hydrogels followed by analyses using diffusion tensor imaging and immunostaining for neuronal and glial markers. The results demonstrated that the P2 hydrogel considerably increased the number of axons and the distance of regeneration into the distal nerve stump. In summary, the method used to decellularize nerve tissue affects the efficacy of the resulting hydrogels to support regeneration after nerve injury.

Ionic Detergent Under Pressure-Vacuum as an Innovative Strategy to Generate Canine Tracheal Scaffold for Organ Engineering

Decellularized scaffolds applied in tissue engineering offer improvements, supplying the elevated necessity for organs and tissues for replacement. However, obtaining a functional trachea for autotransplantation or allotransplantation is tricky due to the organ anatomical and structural complexity. Most tracheal decellularization protocols are lengthy, expensive, and could damage the tracheal extracellular matrix (ECM) architecture and functionality. Here, we aimed to evaluate the effectiveness of 3 different decellularization protocols combined with chemical and physical methods to obtain acellular canine tracheal scaffolds. Six adult dog tracheas were incised (tracheal segments) resulting in 28 rings for control tissue and 84 rings for decellularization (5–7 mm thick). Subsequently, decellularized tracheal scaffolds were microscopically/macroscopically characterized by histological analysis (Hematoxylin-Eosin, Masson’s trichrome, Picrosirius red, Alcian blue, and Safranin O), immunohistochemistry for ECM components, scanning electron microscopy, and genomic DNA quantification. After decellularization, the tracheal tissue revealed reduced genomic DNA, and maintenance of ECM components preserved (structural proteins, adhesive glycoproteins, glycosaminoglycans and proteoglycans), suggesting ECM integrity and functionality. Comparatively, the combined ionic detergent with high vacuum pressure decellularization protocol revealed superior genomic DNA decrease (13.5 ng/mg) and improvement on glycosaminoglycans and proteoglycans preservation regarding the other decellularized trachea scaffolds and native tissue. Our results indicate that the 3 chemical/physical protocols reduce the decellularization time without ECM proteins damage. Notwithstanding, the use of ionic detergent under vacuum pressure was able to generate an innovative strategy to obtain acellular canine tracheal scaffolds with the highest levels of adhesive proteins that support its potentiality for recellularization and future tissue engineering application.