Ionic Detergents Research Articles

Polymers for the stabilization and delivery of proteins topically and per os to the insect hemocoel through conjugation with aliphatic polyethylene glycol

Co-feeding of aliphatic polyethylene glycol (PEG), phospholipase A2, anionic and ionic detergents, and amphipathic glycoside with bovine serum albumin (BSA) as a model protein to fourth stadium tobacco budworms, Heliothis virescens, did not affect the levels of BSA in the hemolymph. Covalent conjugation of small proteins like the decapeptide trypsin modulating oostatic factor (TMOF) to polyethylene glycol was previously shown to protect the peptide from protease attack and enhance its accumulation in the insect hemocoel. Whether this polymer chemistry could do the same for larger proteins was examined. The chemistry for the synthesis of polydispersed aliphatic PEG350-insulin and monodispersed aliphatic PEG333-insulin are described herein. Insulin was used for this synthesis and not BSA to better control conjugation among the available free amine groups. When PEGylated insulin or free insulin were fed in artificial diet to fifth stadium budworms, greater concentrations of insulin using the PEGylated variants were found in the hemolymph than when free insulin was used (a 6.7 and 7.3-fold increase for the PEG350 and PEG333 conjugates, respectively). When insulin is topically applied to the dorsum of H. virescens, no insulin is found in the hemolymph. However, after topical application of the PEGylated insulins, PEG350-insulin and PEG333-insulin were detected in the hemolymph. After injections of insulin into the hemocoel of fourth stadium H. virescens, insulin is completely cleared from the hemolymph in 120min. In comparison, PEG350-insulin and PEG333-insulin were present in the hemolymph for 300 and 240min after injection, respectively, translating to a 3.3 and 2.7-fold increase in the length of time insulin remains in the hemolymph after injection.

Latent NOTCH3 epitopes unmasked in CADASIL and regulated by protein redox state

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy CADASIL is caused by more than a hundred NOTCH3 mutations. Virtually all encoded mutant proteins contain an odd number of cysteines. As such, structural changes in NOTCH3 may be the primary molecular abnormality in CADASIL. Thus, we sought evidence for structurally altered NOTCH3 protein in CADASIL tissue. Four antibodies were raised in rabbits against two non-overlapping N-terminal NOTCH3 sequences. These reagents were used in immunohistochemical experiments to detect epitopes in post-mortem CADASIL brains (n=8), control brains, and cells overexpressing NOTCH3. To determine the biochemical nature of NOTCH3 epitopes, we used these antibodies to probe pure NOTCH3–Fc fusion proteins treated with acid, urea, guanidinium, ionic detergents, acrylamide, and thiol- and phosphorus-based reductants. All antibodies avidly stained arteries in 8 of 8 CADASIL brain samples. The most prominent staining was in degenerating media of leptomeningeal arteries and sclerotic penetrating vessels. Normal appearing vessels from control brains were not reactive. Antibodies did not react with cultured cells overexpressing NOTCH3 or with purified NOTCH3–Fc protein. Furthermore, treatment of pure protein with acid, chaotropic denaturants, alkylators, and detergents failed to unmask N-terminal NOTCH3 epitopes. Antibodies, however, recognized novel N-terminal epitopes in purified NOTCH3–Fc protein treated with three different reductants (DTT, beta-mercaptoethanol, and TCEP). We conclude that CADASIL arteries feature latent N-terminal NOTCH3 epitopes, suggesting the first evidence in vivo of NOTCH3 structural alterations.

Corneal decellularisation: recycling tissue for transplantation

AbstractPurpose There is a clinical need for reliable, reproducible biomimetic corneas. Decellularised tissues are advantageous compared to synthetic/semi‐synthetic tissues in that the native matrix ultrastructure and intrinsic cues including growth factors, cytokines and glycosaminoglycans (GAGs) may be retained. However, there is currently no reliable, standardised human corneal decellularisation method. Here, we provide a systematic study of commonly used decellularisation methods and assess their appropriateness for corneal applications.Methods Eye‐bank tissue unsuitable for transplantation was used to test decellularisation methods: Dispase removal of the epi‐ and endothelium; Mechanical agitation; Hypertonic NaCl; Ionic detergent (SDS); Non‐ionic detergent (Triton‐X100); all followed by nuclease treatment. Removal of cellular material, preservation of transparency, retention of corneal architecture and GAGs was assessed via histological, immunofluorescence and quantitative analysis. Potential cytotoxicity in vitro of the treated tissue was also assessed.Results No decellularisation technique investigated successfully removed 100% of cellular components. The techniques which had the least residual DNA, SDS and Triton‐X100, were most structurally compromised with reduced GAG content. Dispase treated, NaCl and mechanically agitated corneas had better preservation of structure, transparency and GAGs, but had higher residual DNA.Conclusion The ability to reprocess and regenerate tissues deemed “unsuitable” for transplantation allows us to salvage valuable tissue. However, in order to progress, we may need to take a step back to establish a “decellularisation” criterion; which should balance effective removal of immune reactive material with maintenance of tissue functionality.

Methods for Improving Anaerobic Lignocellulosic Substrates Degradation for Enhanced Biogas Production

A successful biogas production process depends upon adequate hydrolysis of macromolecules in the substrate and stable further conversion. The complex and rigid structure of cellulosic, hemicellulosic and lignin chain is preventing lignocellulosic biomass to reach efficient hydrolysis, therefore pretreatment of a substrate is needed for higher biogas and methane yields. There are several different physical and chemical methods of pretreatments available which include the usage of acids, alkalis, organic solvents, ionic detergents, steam, high pressure, grinding, ultrasound, and microwave irradiation. Physico-chemical pretreatments act rapidly on lignocellulose but their upscaling is very expensive in industry. Many studies have been made in finding the best combination of different pretreatment methods and also new biological techniques which could make lignocellulose pretreatment cheaper and environmentally more friendly. Using natural abilities of different fungi, bacteria or yeast to degrade lignocellulose simplifies the whole process. Also cocktails of biotechnologically produced enzymes are effective in degrading lignocellulose.

Posttranslational regulation of the bile salt export pump

Bile plays an essential role in the nutrient uptake of the human body. It consists mostly of amphipathic bile salts, phospholipids and cholesterol in mixed micelles. The bile solubilizes lipophilic nutrients, such as lipids and fat-soluble vitamins, and facilitates their uptake. The formation of bile depends on the activity of various ATP-binding cassette (ABC) transporters localized in the apical membrane of hepatocytes. The main components of bile, phosphatidylcholine, cholesterol and bile salts, are transported by multidrug resistance protein 3 (MDR3, ABCB4), ABCG5/G8 and the bile salt export pump (BSEP, ABCB11), respectively. BSEP, as the transporter of the main solute in bile, is considered to be the primary driving force of bile formation. BSEP belongs to the family of ABC transporters, which constitute one of the largest protein families. They are primary active transporters with a common functional unit: two nucleotide–binding domains and two transmembrane domains. The nucleotide–binding domains fuel the transport by hydrolyzing ATP, while the transmembrane domains provide the translocation pathway over the membrane and are thought to be responsible for substrate recognition. Eukaryotic ABC transporters are usually encoded by one gene as in the case of BSEP (full-size transporters) or by two genes coding for one nucleotide-binding domain and one transmembrane domain each. In the latter case the transporter is called a half-size transporter and the two polypeptides homo- or heterodimerize to form a functional unit. An example for such a half-size transporter is ABCG5/G8. Mutations in ABC transporters can lead to severe diseases. For example an impairment of targeting or activity in BSEP leads to the accumulation of bile salts in the hepatocytes. Bile salts, in addition to their detergent function, can furthermore act as signaling molecules and thereby cause different forms of cholestasis, such as progressive familial intrahepatic cholestasis type 2 (PFIC2) or benign recurrent intrahepatic cholestasis type 2 (BRIC2). An additional influence that can affect the development of diseases is the post-translational regulation of ABC transporter functionality. The regulation can occur directly in the form of chemical modifications or can be mediated for example by protein-protein interactions. For several ABC transporters of the apical hepatocyte membrane an influence of protein-protein interactions on targeting, turnover or activity could be shown. MRP2 for example interacts with NHERF-1, a scaffold protein that is responsible for linking its interacting partners to the cytoskeleton. It has furthermore been reported that the interaction of MRP2 with NHERF-1 influences the apical membrane expression of MRP2. For example, NHERF-1 knock–out mice show reduced abundance of MRP2 in the membrane [1]. For BSEP two interaction partners are known to date. One of them is the AP2 adaptor related protein complex, which is involved in internalization of BSEP [2]. To study ABC transporters in their isolated form a heterologous overexpression system is required to obtain sufficient amounts of protein. Initially, toxicity of the BSEP cDNA for Escherichia coli was an obstacle. Therefore a Saccharomyces cerevisiae based cloning technique has been developed, which is independent of E. coli[3]. Subsequently, with the optimized cloning procedure an overexpression of BSEP could be established in the methylotrophic yeast Pichia pastoris. With the help of a GFP-fusion protein BSEP could be traced to the P. pastoris plasma membrane. Additionally, sucrose density centrifugation experiments confirmed the correct localization of BSEP. A subsequent detergent screen led to the identification of detergents suitable for BSEP solubilization. The transporter appears to be in a detergent-resistant environment as only the lipid–like, zwitter–ionic detergents of the Fos–choline and Cyclofos series, comparatively “harsh” detergents, could efficiently extract BSEP. Finally, a purification protocol was established to obtain BSEP in a detergent-solubilized form. With a dual–affinity tag tandem affinity purification could be employed to obtain the ABC transporter with approximately 75% purity [4]. In summary, the optimized cloning strategy for BSEP cDNA in S. cerevisiae as well as the efficient expression and purification protocols enable us to investigate the interaction of BSEP with other proteins in vitro.

Conformational changes induced by detergents during the refolding of chemically denatured cysteine protease ppEhCP-B9 from Entamoeba histolytica

EhCP-B9, a cysteine protease (CP) involved in Entamoeba histolytica virulence, is a potential target for disease diagnosis and drug design. After purification from inclusion bodies produced in Escherichia coli, the recombinant EhCP-B9 precursor (ppEhCP-B9) can be refolded using detergents as artificial chaperones. However, the conformational changes that occur during ppEhCP-B9 refolding remain unknown. Here, we comprehensively describe conformational changes of ppEhCP-B9 that are induced by various chemical detergents acting as chaperones, including non-ionic, zwitterionic, cationic and anionic surfactants. We monitored the effect of detergent concentration and incubation time on the secondary and tertiary structures of ppEhCP-B9 using fluorescence and circular dichroism (CD) spectroscopy. In the presence of non-ionic and zwitterionic detergents, ppEhCP-B9 adopted a β-enriched structure (ppEhCP-B9β1) without proteolytic activity at all detergent concentrations and incubation times evaluated. ppEhCP-B9 also exhibits a β-rich structure in low concentrations of ionic detergents, but at concentrations above the critical micelle concentration (CMC), the protein acquires an α+β structure, similar to that of papain but without proteolytic activity (ppEhCP-B9α+β1). Interestingly, only within a narrow range of experimental conditions in which SDS concentrations were below the CMC, ppEhCP-B9 refolded into a β-sheet rich structure (ppEhCP-B9β2) that slowly transforms into a different type of α+β conformation that exhibited proteolytic activity (ppEhCP-B9α+β2) suggesting that enzymatic activity is gained as slow transformation occurs.

Stem Cells Accumulate on a Decellularized Arterial Xenograft In Vivo

Surgical interest in complete arterial revascularization is expanding, but some patients lack suitable conduits for this goal. The field of stem cell biology is rapidly expanding and, together with the concepts of tissue engineering, offers the promise of growing autologous grafts in the laboratory. We aim to develop a model using human arteries as vascular grafts in a murine model and to assess the cellular accumulation on these grafts. Human arterial samples were collected and decellularized using an ionic detergent. These vessel scaffolds were then used as grafts in an invivo mouse model, and the cellular accumulation on them was examined histologically and by cell culture with assessment of their physiologic properties. Left internal mammary artery branches werefully decellularized and successfully implanted intoa murine model. Grafts were repopulated by cells expressing stem cell markers cluster of differentiation 34 and stage-specific embryonic antigen, and subsequently expressed markers of mature endothelial and smooth muscle cells (cluster of differentiation 31, calponin, and myosin heavy chain). The migratory capacity of the cultured cells was significantly higher than that of mouse smooth muscle cells (p < 0.001). We describe the successful use of human arteries in a murine graft model, allowing the study of repopulation. Decellularized grafts are repopulated by cells expressing stem cell markers and subsequently express smooth muscle and endothelial cell markers. This model has the potential to be used for further development of laboratory-grown vascular grafts.

Fluorescence of pyrene in inhomogeneous media containing silver nanoparticles

Pyrene fluorescence in inhomogeneous media based on ionic detergents containing silver nanoparticles with different morphologies is investigated. An increase in pyrene monomer emissions in the spectral range of 400–500 nm is observed, due to the resonance between electronic transitions in pyrene molecules in that region and the plasmonic oscillations of silver nanoparticles.

Kinetics and thermodynamics of membrane protein folding.

Understanding protein folding has been one of the great challenges in biochemistry and molecular biophysics. Over the past 50 years, many thermodynamic and kinetic studies have been performed addressing the stability of globular proteins. In comparison, advances in the membrane protein folding field lag far behind. Although membrane proteins constitute about a third of the proteins encoded in known genomes, stability studies on membrane proteins have been impaired due to experimental limitations. Furthermore, no systematic experimental strategies are available for folding these biomolecules in vitro. Common denaturing agents such as chaotropes usually do not work on helical membrane proteins, and ionic detergents have been successful denaturants only in few cases. Refolding a membrane protein seems to be a craftsman work, which is relatively straightforward for transmembrane β-barrel proteins but challenging for α-helical membrane proteins. Additional complexities emerge in multidomain membrane proteins, data interpretation being one of the most critical. In this review, we will describe some recent efforts in understanding the folding mechanism of membrane proteins that have been reversibly refolded allowing both thermodynamic and kinetic analysis. This information will be discussed in the context of current paradigms in the protein folding field.

Cellular Localization, Invasion, and Turnover Are Differently Influenced by Healthy and Tumor-Derived Extracellular Matrix

The interplay between tumor cells and the microenvironment has been recognized as one of the hallmarks of cancer biology. To assess the role of extracellular matrix (ECM) in the modulation of tissue homeostasis and tumorigenesis, we developed a protocol for the purification of tissue-derived ECM using mucosae from healthy human colon, perilesional area, and colorectal carcinoma (CRC). Matched specimens were collected from the left colon of patients undergoing CRC resection surgery. ECMs were obtained from tissues that were decellularized with hypotonic solutions containing ionic and nonionic detergents, hypertonic solution, and endonuclease in the absence of denaturing agents. Mucosae-derived ECMs maintained distribution and localization of proteins and glycoproteins typical of the original tissues, and showed different three-dimensional (3D) structures among normal versus perilesional and tumor-derived stroma. The three types of ECM differentially regulated the localization and organization of seeded monocytes and cancer cells that were located and organized as in the original tissue. Specifically, healthy, perilesional, and CRC-derived ECMs sustained differentiation and polarization of cancer epithelial cells. In addition, healthy, but not perilesional and CRC-derived ECM constrained invasion of cancer cells. All three ECMs sustained turnover between cell proliferation and death up to 40 days of culture, although each ECM showed different ability in supporting cell proliferation, with tumor>perilesional>healthy-derived ECMs. Healthy-, perilesional- and CRC-derived ECM differently modulated cell homeostasis, spreading in the stroma and turnover between proliferation and death, and equally supported differentiation and polarization of cancer epithelial cells, thus highlighting the contribution of different ECMs modulating some features of tissue homeostasis and tumorigenesis. Moreover, these ECMs provide competent scaffolds useful to assess efficacy of antitumor drugs in a 3D setting that more closely recapitulates the native microenvironment. Further, ECM-based scaffolds may also be beneficial for future studies seeking prognostic and diagnostic stromal markers and targets for antineoplastic drugs.

Expression of a Deschampsia antarctica Desv. Polypeptide with Lipase Activity in a Pichia pastoris Vector

The current study isolated and characterized the Lip3F9 polypeptide sequence of Deschampsia antarctica Desv. (GeneBank Accession Number JX846628), which was found to be comprised of 291 base pairs and was, moreover, expressed in Pichia pastoris X-33 cells. The enzyme was secreted after 24 h of P. pastoris culture incubation and through induction with methanol. The expressed protein showed maximum lipase activity (35 U/L) with an optimal temperature of 37 °C. The lipase-expressed enzyme lost 50% of its specific activity at 25 °C, a behavior characteristic of a psychrotolerant enzyme. Recombinant enzyme activity was measured in the presence of ionic and non-ionic detergents, and a decrease in enzyme activity was detected for all concentrations of ionic and non-ionic detergents assessed.

Purification of VDAC1 from Rat Liver Mitochondria

To make biophysical measurements of functions such as the pore-forming activity of mitochondrial voltage-dependent anion-selective channel protein 1 (VDAC1), it is first necessary to obtain a source of purified VDAC protein. In this protocol, we present a method for obtaining rat liver mitochondria as a source of VDAC1 and then describe two methods, one using a nonionic detergent and the other an ionic detergent, for purifying VDAC1 from the isolated mitochondria. This produces a source of VDAC1 proteins that are suitable for subsequent incorporation into artificially prepared phospholipid bilayers. Furthermore, the isolated mitochondria can be used for assaying the mitochondrial permeability transition pore (MPTP).

High Density Lipoprotein and Apolipoprotein A-I Bind Von Willebrand Factor and Prevent Its Self-Association Into Thick Fibers

Von Willebrand factor (VWF) is a large plasma protein secreted constitutively or upon activation by endothelial cells. On activated endothelium, a portion of the VWF molecules remain attached to the surface, where they self-associate to produce long hyperadhesive strands and fibers capable of capturing platelets and other blood cells if not removed by the plasma metalloprotease ADAMTS13. Failure to clear these fibers underlies the pathophysiology of many microangiopathies, most prominently thrombotic thrombocytopenic purpura, in which accumulation of VWF-platelet aggregates in the microvasculature leads to ischemia and organ failure. VWF self-association also plays a major role in the adsorption of purified VWF to surfaces. We observed that adsorption of VWF to surfaces began with contact and direct binding of VWF to the surface via hydrophobic interactions. After saturating the binding sites on the surface, VWF continued to bind to and associate with the immobilized VWF molecules via hydrophilic VWF-VWF interactions, depositing multiple layers of VWF molecules on the surface until essentially all VWF molecules were depleted from the fluid phase. Using the method of Magnani and coworkers, we eluted VWF molecules that were bound to the surface via VWF-VWF interactions with the ionic detergent SDS, and then eluted VWF molecules that were bound to the surface via VWF-surface interactions with the zwitterion detergent CHAPS. Quantification of the eluted VWF showed that self-association of VWF onto the surface accounted for >80% of the adsorptive loss. Using the disappearance of purified VWF from the fluid phase as a measurement of self-association, we fractionated human plasma by heat and identified that apolipoprotein A-I (ApoAI), the major apolipoprotein component in high density lipoprotein (HDL) particles, which is stable to heat at 100° C, stabilized and prevented VWF surface adsorption by interfering with VWF self-association. Commercial preparations of ApoAI and HDL, prepared without exposure to heat, similarly prevented adsorption of purified VWF to surfaces. Half-maximal stabilization occurred at a molar ratio of eight ApoAI molecules to each VWF subunit. We assessed the role of HDL in VWF self-association that leads to the assembly of ultra-large VWF (ULVWF) strings on the surface of phorbol myristyl acetate-stimulated endothelial cells in flow chambers. We observed that HDL reduced the number and length of platelet-decorated ULVWF strings on the endothelial surface, consistent with the ability of HDL to interfere with VWF self-association and ULVWF assembly. We also studied the role of ApoAI in the recruitment of fluid-phase VWF to hyperadhesive ULVWF fibers in a synthetic microvessel system. We observed that ApoAI directly bound to hyperadhesive transluminal ULVWF fibers under flow, and this binding completely blocked the recruitment of fluid phase VWF molecules to the immobilized ULVWF fibers. These results showed that ApoAI or HDL interacted with hyperadhesive forms of VWF and modified the adhesive properties of the ULVWF strings and fibers. Consistent with its antithrombotic properties, the level of ApoAI in patients with hyperadhesive forms of VWF, such as thrombotic thrombocytopenic purpura and sepsis, was significantly reduced. These results suggest that regulation of VWF self-association may be another mechanism by which HDL protects against cardiovascular disease and extends its protective effects from large arteries to the microvasculature. Disclosures:No relevant conflicts of interest to declare.

Characterization of hydrophobic peptides in the presence of detergent by photoionization mass spectrometry.

The characterization of membrane proteins is still challenging. The major issue is the high hydrophobicity of membrane proteins that necessitates the use of detergents for their extraction and solubilization. The very poor compatibility of mass spectrometry with detergents remains a tremendous obstacle in studies of membrane proteins. Here, we investigated the potential of atmospheric pressure photoionization (APPI) for mass spectrometry study of membrane proteins. This work was focused on the tetraspanin CD9 and the multidrug transporter BmrA. A set of peptides from CD9, exhibiting a broad range of hydropathicity, was investigated using APPI as compared to electrospray ionization (ESI). Mass spectrometry experiments revealed that the most hydrophobic peptides were hardly ionized by ESI whereas all peptides, including the highly hydrophobic one that corresponds to the full sequence of the first transmembrane domain of CD9, were easily ionized by APPI. The native protein BmrA purified in the presence of the non-ionic detergent beta-D-dodecyl maltoside (DDM) was digested in-solution using trypsin. The resulting peptides were investigated by flow injection analysis of the mixture followed by mass spectrometry. Upon ESI, only detergent ions were detected and the ionic signals from the peptides were totally suppressed. In contrast, APPI allowed many peptides distributed along the sequence of the protein to be detected. Furthermore, the parent ion corresponding to the first transmembrane domain of the protein BmrA was detected under APPI conditions. Careful examination of the APPI mass spectrum revealed a-, b-, c- and y- fragment ions generated by in-source fragmentation. Those fragment ions allowed unambiguous structural characterization of the transmembrane domain. In conclusion, APPI–MS appears as a versatile method allowing the ionization and fragmentation of hydrophobic peptides in the presence of detergent.

A modified protein assay from microgram to low nanogram levels in dilute samples

In this article, we present a modified and improved protein assay that was previously described as “amidoschwarz assay” by Schaffner and Weissmann [13]. Our improved protein assay is user-friendly and 30–40 times more sensitive than the earlier method. The assay was developed into three formats (macro-, micro-, and nanoassay) with trichloroacetic acid (TCA) as protein precipitating agent, measuring up to 96 samples. The macro and micro formats of this assay require a single reagent staining with amido black of protein dots bound to nitrocellulose membrane with lowest protein measurements to 1 and 0.1μg, respectively. On the other hand, the nanoassay, with combination staining of amido black followed by colloidal gold, can extend the detection limit to 2.5ng of protein. Protein concentrations were determined by densitometry and/or spectrophotometry. This assay is compatible with many ionic and non-ionic detergents. This improved protein assay provides an additional choice to researchers in measuring total protein concentration accurately in dilute biological samples as low as 0.125μg/ml prior to their biochemical analysis such as in comparative proteomics.

A Comparison of DNA Extraction Methods using Petunia hybrida Tissues

Extraction of DNA from plant tissue is often problematic, as many plants contain high levels of secondary metabolites that can interfere with downstream applications, such as the PCR. Removal of these secondary metabolites usually requires further purification of the DNA using organic solvents or other toxic substances. In this study, we have compared two methods of DNA purification: the cetyltrimethylammonium bromide (CTAB) method that uses the ionic detergent hexadecyltrimethylammonium bromide and chloroform-isoamyl alcohol and the Edwards method that uses the anionic detergent SDS and isopropyl alcohol. Our results show that the Edwards method works better than the CTAB method for extracting DNA from tissues of Petunia hybrida. For six of the eight tissues, the Edwards method yielded more DNA than the CTAB method. In four of the tissues, this difference was statistically significant, and the Edwards method yielded 27-80% more DNA than the CTAB method. Among the different tissues tested, we found that buds, 4 days before anthesis, had the highest DNA concentrations and that buds and reproductive tissue, in general, yielded higher DNA concentrations than other tissues. In addition, DNA extracted using the Edwards method was more consistently PCR-amplified than that of CTAB-extracted DNA. Based on these results, we recommend using the Edwards method to extract DNA from plant tissues and to use buds and reproductive structures for highest DNA yields.

Multiple Protein Analysis of Formalin-fixed and Paraffin-embedded Tissue Samples with Reverse phase Protein Arrays

Reverse-phase protein arrays (RPPAs) have become an important tool for the sensitive and high-throughput detection of proteins from minute amounts of lysates from cell lines and cryopreserved tissue. The current standard method for tissue preservation in almost all hospitals worldwide is formalin fixation and paraffin embedding, and it would be highly desirable if RPPA could also be applied to formalin-fixed and paraffin embedded (FFPE) tissue. We investigated whether the analysis of FFPE tissue lysates with RPPA would result in biologically meaningful data in two independent studies. In the first study on breast cancer samples, we assessed whether a human epidermal growth factor receptor (HER) 2 score based on immunohistochemistry (IHC) could be reproduced with RPPA. The results showed very good concordance between the IHC and RPPA classifications of HER2 expression. In the second study, we profiled FFPE tumor specimens from patients with adenocarcinoma and squamous cell carcinoma in order to find new markers for differentiating these two subtypes of non-small cell lung cancer. p21-activated kinase 2 could be identified as a new differentiation marker for squamous cell carcinoma. Overall, the results demonstrate the technical feasibility and the merits of RPPA for protein expression profiling in FFPE tissue lysates.

Extraction, purification and application of thermostable and halostable alkaline protease from Bacillus alveayuensis CAS 5 using marine wastes

A marine bacterium Bacillus alveayuensis CAS 5 produced protease when grown at 55°C for 60h in 100ml of basal medium containing 1% SSP (w/v) and purified to 7.77 fold with specific activity of 518.78U/mg. The optimum temperature, pH and NaCl for enzyme activity were 50°C, 9 and 35% respectively. The enzyme was highly stable even at 80°C, pH 12, 35% NaCl and presence of ionic, non-ionic and commercial detergents. The protease was investigated for its application as cleansing additive in blood stain removal. The preset study emphasized that marine wastes can be utilized to generate high value-added products and hidden potential in the production of functional foods.

Abstracts from The Aerosol Society Drug Delivery to the Lungs 22Edinburgh International Conference CentreEdinburgh, Scotland, UKDecember 7–9, 2011

Summary Background: In bronchial challenge testing lung deposition of the stimulus may be poorly controlled due to incorrect use of nebulisers. Furthermore, the need for freshly prepared solutions burdens personnel and budget. In this study we aim to develop a dry powder alternative with higher reproducibility, better stability and lower costs than the current nebulisation test. Methods: Different pharmacological stimuli in the solid state were characterised on physico-chemical properties to determine the optimal conditions for processing and storage and were tested on their potential for dry powder inhalation. Adenosine and methacholine were micronised and dispersed with the Twincer ™ dry powder inhaler (DPI). Laser diffraction technique was applied to measure particle size distributions in the aerosol clouds. Results: Adenosine allows processing under ambient conditions. It was mixed with a lactose carrier in various mass ratios to produce adhesive mixtures and nucleus agglomerates. With three formulations, almost the entire dose range could be delivered with the Twincer ™ DPI. However, the process of preparing nucleus agglomerates might not be suitable for upscaling. Currently, spherical pellets are investigated as a more robust alternative. Methacholine is very hygroscopic and should therefore not be exposed to a high relative humidity. The methacholine particles leaving the Twincer ™ had an appropriate particle size distribution for inhalation. However, inhaler retention was high and should be reduced by controlling processing and storage conditions. Conclusions: Dry powder adenosine and methacholine seem promising alternatives for nebulisation. Methacholine must be handled under dry conditions. The highest doses of adenosine require further fine tuning.

An efficient method for decellularization of the rat liver

Using gradient ionic detergent, we optimized the preparation procedure for the decellularized liver biologic scaffold, and analyzed its immunogenicity and biocompatibility. EDTA, hypotonic alkaline solution, Triton X-100, and gradient sodium dodecyl sulfate (1%, 0.5%, and 0.1%, respectively) were prepared for continuous perfusion through the hepatic vascular system. The decellularization of the liver tissue was performed with the optimized reagent buffer and washing protocol. In addition, the preservation of the original extracellular matrix was observed. To analyze its biocompatibility, the scaffold was embedded in a heterologous animal and the inflammation features, including the surrounding cell infiltration and changes of the scaffold architecture, were detected. The cell-attachment ability was also validated by the perfusion culture of HepG2 cells with the scaffold. By using gradient ionic detergent, we completed the decellularization process in approximately 5 h, which was shorter than >10 hours in previous experiments (p<0.001). The extracellular matrix was kept relatively intact, with no obvious inflammatory cellular infiltration or structural damage in the grafted tissue. The engraftment efficiencies of HepG2 were 86±5% (n=8). The levels of albumin and urea synthesis were significantly superior to the ones in traditional two-dimensional culture. The current new method can be used efficiently for the decellularization of the liver biologic scaffold with satisfying biocomparability for application both in vivo and in vitro.