In-Depth Comparison of Matrigel Dissolving Methods on Proteomic Profiling of Organoids.

Patient-derived organoids recently emerged as promising ex vivo 3D culture models recapitulating histological and molecular characteristics of original tissues, thus proteomic profiling of organoids could be valuable for function investigation and clinical translation. However, organoids are usually cultured in murine Matrigel (served as scaffolds and matrix), which brings an issue to separate organoids from Matrigel. Because of the complex compositions of Matrigel and thousands of identical peptides shared between Matrigel and organoids, insufficiently dissolved Matrigel could influence proteomic analysis of organoids in multiple ways. Thus, how to dissolve Matrigel matrix and recovery organoid cells efficiently is vital for sample preparation. Here, we comprehensively compared three popular Matrigel dissolving methods (cell recovery solution, dispase, and PBS-EDTA buffer) and investigated the effect of undissolved Matrigel proteins on proteomic profiles of organoids. By integrative analysis of label-free proteomes of Matrigel and stable isotope labeling by amino acids in cell culture proteomes of organoids collected by three methods, respectively, we found that dispase showed an optimal efficiency, with the highest peptide yield and the highest incorporation ratio of stable isotope labeling by amino acids in cell culture labels (97.1%), as well as with the least potential Matrigel contaminants. To help analysis of proteomic profiles of organoids collected by the other two methods, we identified 312 high-confidence Matrigel contaminants, which could be filtered out to attenuate Matrigel interference with minimal loss of biological information. Together, our study identifies bioinformatics and experimental approaches to eliminate interference of Matrigel contaminants efficiently, which will be valuable for basic and translational proteomic research using organoid models.

Octenidine Versus Dispase Gels for Wound Healing After Cryosurgery Treatment in Patients with Basal Cell Carcinoma.

For a specific group of patients with basal cell carcinoma (small, low risk), cryosurgery could be the suggested treatment, which results in the formation of an ulcer in the lesion area. The proteolytic enzymes' contribution to the wound healing is an ongoing research goal. Preclinical animal experiments in the Laboratory of the Pharmaceutical Technology Department of the National and Kapodistrian University of Athens have showed that a dose of 5 U/mL of dispase gel after the formation of tissue rashes, significantly promoted wound healing. Herein, a feasibility study in 16 patients enrolled by the First Department of Dermatology of Andreas Syggros Hospital was designed: 5 U/mL of dispase gel (once every 3 days) versus a drug reference containing octenidine (daily administration). The evaluation of the healing effect, safety, and tolerance was done on days 1 (cryosurgery), 2, 7, 21, and 60. The study end point was considered either the ulcer complete healing or the eighth week since treatment initiation. Wound healing was faster with dispase gel and hemoglobin reduced rapidly after the seventh day. Yet, hydration was higher in the control group. Our non-parametric analysis provides evidence that the dispase gel shows faster healing compared to the reference drug, in humans, meriting further investigation in larger human sample sizes before massive production of the product.

Effect of aflibercept on proliferative vitreoretinopathy: Proteomic analysis in an experimental animal model.

PURPOSE: The aim of this study was to monitor inflammatory, proliferative and progressive effects of proliferative vitreoretinopathy (PVR) and aflibercept treatment in dispase induced PVR rat model by proteomic analysis. MATERIAL AND METHODS: A total of 35 male Long Evans pigmented rats were divided into three groups, namely, PVR (dispase+saline), PVR+aflibercept (dispase+aflibercept) and control. The PVR group received 2 µl of 0.03 IU/µl dispase and 2 µl saline, the PVR+aflibercept group received 2 µl of 0.03 IU/µl and 2 µl of 40 mg/ml aflibercept at the first day of the experiment. At the end of the 6th week all retina and vitreous specimens were collected by evisceration and transferred to the proteomics laboratory for analysis. Proteomic analysis by 2D gel electrophoresis coupled with MALDI-TOF/TOF was performed. RESULTS: In the PVR and PVR+aflibercept group 16 different proteins that were identified to be differentially regulated in comparison to the control group. In the PVR+aflibercept group, ENO1, ENO2, LDH-B, PEBP-1 and GS levels were higher than the PVR group. In addition, the association of proteins such as UCHL, PEBP1, PDHB and ENO1 with PVR has been demonstrated for the first time. CONCLUSION: STRING analysis elucidated the functional protein-protein interaction among the differentially regulated proteins and highlighted that those proteins mainly played roles in carbon and nucleotide metabolisms. Functional analysis of the differentially regulated proteins indicated the presence of inflammation, gliosis and retinal damage in the PVR group. Aflibercept treatment had pronounced effect on prevention of inflammation and retinal damage while causing a slight increase in gliosis. However, aflibercept treatment was not effective enough to normalize the levels of differentially regulated proteins of the PVR group. Therefore, we predict that the treatment dose of aflibercept used in this study was below of its ideal concentration and should be increased in the future studies. The differential regulation of these structural proteins in this study should shed some light to the mechanism of glial wound formation in the retina and guide future treatment modalities.

Dissecting capture and twisting of aureolysin and pseudolysin: functional amino acids of the Dispase autolysis-inducing protein.

The Dispase autolysis-inducing protein (DAIP) from Streptomyces mobaraensis attracts M4 metalloproteases, which results in inhibition and autolysis of bacillolysin (BL) and thermolysin (TL). The present study shows that aureolysin (AL) from Staphylococcus aureus and pseudolysin (LasB) from Pseudomonas aeruginosa are likewise impaired by DAIP. Complete inhibition occurred when DAIP significantly exceeded the amount of the target protease. At low DAIP concentrations, AL and BL performed autolysis, while LasB and TL degradation required reductants or detergents that break intramolecular disulfide bonds or change the protein structure. Site directed mutagenesis of DAIP and removal of an exposed protein loop either influenced binding or inhibition of AL and TL but had no effect on LasB and BL. The Y170A and Δ239-248 variants had completely lost affinity for TL and AL. The exchange of Asn-275 also impaired the interaction of DAIP with AL. In contrast, DAIP Phe-297 substitution abolished inhibition and autolysis of both target proteases but still allowed complex formation. Our results give rise to the conclusion that other, yet unknown DAIP amino acids inactivate LasB and BL. Obviously, various bacteria in the same habitat caused Streptomyces mobaraensis to continuously optimize DAIP in inactivating the tackling metalloproteases.

Specificity-directed design of a FRET-quenched heptapeptide for assaying thermolysin-like proteases.

Thermolysin (TL) is an industrially important zinc endopeptidase, and the prototype of the M4 family of metallopeptidases. The catalytic function of TL and its relatives is typically assessed using chromogenic or more sensitive fluorescent peptides, with the latter substrates relying on Förster resonance energy transfer (FRET). Here, we demonstrate that a FRET-quenched heptapeptide designed on the basis of the enzyme's substrate specificity (Dabcyl-FKFLGKE-EDANS) is efficiently cleaved by TL and dispase (a TL-like protease) in between the Phe3 and Leu4 residues. The specificity constants (determined at pH 7.4 and 25 °C) for TL and dispase (3.6 × 106 M-1 s-1 and 4.6 × 106 M-1 s-1, respectively) were found to be amongst the highest documented for any TL substrate. Maximal peptide cleavage rates were achieved at pH 6.5 and a temperature of 65 °C. In view of the sensitivity of the assay, concentrations as low as 10 pM TL could be detected. Furthermore, the rate of hydrolysis of Dabcyl-FKFLGKE-EDANS was slow or immeasurable with some other unrelated metallo-, serine- and cysteine proteases, suggesting that the peptide has the potential to serve as a selective substrate for TL and TL-like proteases.

The effect of infliximab and octreotide on cytokine levels experimental proliferative vitreoretinopathy.

Purpose: To investigate the efficiency of intravitreal octreotide, which has previously been shown to have benefits in the treatment of proliferative vitreoretinopathy (PVR), and intravitreal infliximab as a novel option in an experimental dispase-induced PVR model.Methods: A total of 28 pigmented guinea pigs were divided into four groups, and each group consisted of seven subjects. Group 1 (control) was treated with a 0.2 mL saline solution intravitreally from 1.5 mm behind the limbus. Group 2 (sham) was treated with 0.07 IU/0.1 mL dispase 0.1 mL saline solution using the same method. Group 3(infliximab) received 0.07 IU/0.1 mL dispase and 1 mg/0.1 mL infliximab, and group 4(octreotide) was treated with 0.07 IU/0.1 mL dispase and 1 mg/0.1 mL octreotide. An intravitreal injection of infliximab and octreotide was administered to groups 3 and 4 two times during the experiment. The subjects were held for a 10-week period to await for the formation of PVR. At the end of ten weeks, the eyes were enucleated, and tumour necrosis factor-alpha (TNF-α), interleukin 1(IL-1), interleukin 6 (IL-6), transforming growth factor (TGF-ß), and platelet-derived growth factor (PDGF) and levels in homogenised retina tissue were measured using the enzyme linked-immuno-sorbent assay (ELISA) method.Results: Retinal TNF-α, IL-1, IL-6, and PDGF levels had significantly decreased in treatment groups compared to the sham group (p < 0.05). The decrease in the level of TGF-ß was not statistically significant between the treatment and the sham groups (p > 0.05).Conclusions: Intravitreal infliximab can inhibit the development of PVR and reduce levels of cytokine, which plays an essential role in the pathogenesis of PVR. The results of our study suggest that it may be possible to identify the ideal adjuvant pharmacological drugs that are effective in preventing PVR.

An improved method for primary culture of normal cervical epithelial cells and establishment of cell model in vitro with HPV-16 E6 gene by lentivirus.

Normal human cervical epitheliums infected with HPVs gene in vitro are underlying molecular models to investigate physiological mechanisms of cervical epithelia and cervical disease. The current study aimed to establish a modified culture method for cervical epithelium and explore the feasibility of transfection with HPV-16 E6 gene mediated by lentivirus in primary cervical cells. The cells were dissociated enzymatically using Dispase II combined with 0.25% Trypsin-0.01% ethylenediamine tetracetic acid (EDTA) or Collagenase I. The detached effectiveness of Dispase II at different times was compared. Isolated cells were cultured and subcultured in modified keratinocyte serum-free medium (K-SFM) supplemented with 5% fetal bovine serum (FBS) or K-SFM alone. Cytokeratin was used as the identification of cervical epitheliums. Proliferative capacity and growth curve of cervical epitheliums were evaluated by cell counting kit-8 (CCK-8). The cells at passages 3 were used to infect with HPV-16 E6 gene by lentivirus. The expression of green fluorescent protein (GFP) presented in the infected cells was observed via fluorescence microscopy and the levels of E6 mRNA were detected by quantitative real-time PCR (qRT-PCR). The results indicate that cervical epithelial cells can be isolated successfully by Dispase II combined with 0.25% Trypsin-0.01% EDTA method for 20 hr and maintained for five or six passages in K-SFM medium with 5% FBS. The present study proposed a brief and high-yield protocol for isolation and culture of human cervical epitheliums. Moreover, an infected cell model with HPV-16 E6 gene mediated by lentivirus was established which can do duty for studies in vitro on the carcinogenic mechanism of HR-HPVs.

Structure of the Dispase Autolysis-inducing Protein from Streptomyces mobaraensis and Glutamine Cross-linking Sites for Transglutaminase.

Transglutaminase from Streptomyces mobaraensis (MTG) is an important enzyme for cross-linking and modifying proteins. An intrinsic substrate of MTG is the dispase autolysis-inducing protein (DAIP). The amino acid sequence of DAIP contains 5 potential glutamines and 10 lysines for MTG-mediated cross-linking. The aim of the study was to determine the structure and glutamine cross-linking sites of the first physiological MTG substrate. A production procedure was established in Escherichia coli BL21 (DE3) to obtain high yields of recombinant DAIP. DAIP variants were prepared by replacing four of five glutamines for asparagines in various combinations via site-directed mutagenesis. Incorporation of biotin cadaverine revealed a preference of MTG for the DAIP glutamines in the order of Gln-39 ≫ Gln-298 > Gln-345 ∼ Gln-65 ≫ Gln-144. In the structure of DAIP the preferred glutamines do cluster at the top of the seven-bladed ß-propeller. This suggests a targeted cross-linking of DAIP by MTG that may occur after self-assembly in the bacterial cell wall. Based on our biochemical and structural data of the first physiological MTG substrate, we further provide novel insight into determinants of MTG-mediated modification, specificity, and efficiency.

A non-aggressive, highly efficient, enzymatic method for dissociation of human brain-tumors and brain-tissues to viable single-cells.

BACKGROUND: Conducting research on the molecular biology, immunology, and physiology of brain tumors (BTs) and primary brain tissues requires the use of viably dissociated single cells. Inadequate methods for tissue dissociation generate considerable loss in the quantity of single cells produced and in the produced cells' viability. Improper dissociation may also demote the quality of data attained in functional and molecular assays due to the presence of large quantities cellular debris containing immune-activatory danger associated molecular patterns, and due to the increased quantities of degraded proteins and RNA. RESULTS: Over 40 resected BTs and non-tumorous brain tissue samples were dissociated into single cells by mechanical dissociation or by mechanical and enzymatic dissociation. The quality of dissociation was compared for all frequently used dissociation enzymes (collagenase, DNase, hyaluronidase, papain, dispase) and for neutral protease (NP) from Clostridium histolyticum. Single-cell-dissociated cell mixtures were evaluated for cellular viability and for the cell-mixture dissociation quality. Dissociation quality was graded by the quantity of subcellular debris, non-dissociated cell clumps, and DNA released from dead cells. Of all enzymes or enzyme combinations examined, NP (an enzyme previously not evaluated on brain tissues) produced dissociated cell mixtures with the highest mean cellular viability: 93 % in gliomas, 85 % in brain metastases, and 89 % in non-tumorous brain tissue. NP also produced cell mixtures with significantly less cellular debris than other enzymes tested. Dissociation using NP was non-aggressive over time-no changes in cell viability or dissociation quality were found when comparing 2-h dissociation at 37 °C to overnight dissociation at ambient temperature. CONCLUSIONS: The use of NP allows for the most effective dissociation of viable single cells from human BTs or brain tissue. Its non-aggressive dissociative capacity may enable ambient-temperature shipping of tumor pieces in multi-center clinical trials, meanwhile being dissociated. As clinical grade NP is commercially available it can be easily integrated into cell-therapy clinical trials in neuro-oncology. The high quality viable cells produced may enable investigators to conduct more consistent research by avoiding the experimental artifacts associated with the presence dead cells or cellular debris.

Presence of native limbal stromal cells increases the expansion efficiency of limbal stem/progenitor cells in culture.

Niche factors are important in the maintenance and regulation of stem cells. Limbal stromal cells are potentially a component of limbal stem cell (LSC) niche. We investigated the role of the limbal stromal cells in the ex vivo expansion of limbal stem/progenitor cells. Limbal epithelial cells were cultured as single-cell suspension and cell clusters from dispase II or collagenase A (ColA), or tissue explant. ColA isolated limbal stromal cells along with limbal epithelial cells. In the presence of limbal stromal cells, a higher absolute number of p63α(bright) cells (p < 0.05) and a higher proportion of K14 positive epithelial cells were obtained from both ColA and explant tissue cultures. Expansion of the stem/progenitor population from dispase isolation was more efficient in the form of cell clusters than single cell suspension based on the absolute number of p63α(bright) cells. Expansion of the stem cell population is similar in the single cell and cell cluster cultures that are derived from ColA isolation. Our finding suggests that limbal stromal cells and an intact cell-cell contact help to maintain LSCs in an undifferentiated state in vitro during expansion.

Human Skin Drug Metabolism: Relationships between Methyl Salicylate Metabolism and Esterase Activities in IVPT Skin Membranes.

The presence of esterase enzymes in human skin and their role in drug metabolism has been reported, but their distribution in the various skin layers and the relative contributions of those layers to metabolism is poorly defined. To gain further insight into esterase distribution, we performed in vitro skin permeation of a commercial 28.3% methyl salicylate (MeSA) cream (Metsal™) in Franz diffusion cells, using a range of human skin membranes, all from the same donor. The membranes were viable epidermis separated by a dispase II enzymatic method, heat separated epidermis, dermatomed skin, and dermis separated by a dispase II enzymatic method. Methyl salicylate and its metabolite, salicylic acid (SA), were measured by high-performance liquid chromatography. Alpha naphthyl acetate and Hematoxylin and Eosin staining provided qualitative estimations of esterase distribution in these membranes. The permeation of methyl salicylate after 24 h was similar across all membranes. Salicylic acid formation and permeation were found to be similar in dermatomed skin and dermis, suggesting dermal esterase activity. These results were supported by the staining studies, which showed strong esterase activity in the dermal-epidermal junction region of the dermis. In contrast with high staining of esterase activity in the stratum corneum and viable epidermis, minimal stained and functional esterase activity was found in heat-separated and dispase II-prepared epidermal membranes. The results are consistent with dispase II digesting hemidesmosomes, penetrating the epidermis, and affecting epidermal esterases but not those in the dermis. Accordingly, whilst the resulting dispase II-generated dermal membranes may be used for in vitro permeation tests (IVPT) involving esterase-based metabolic studies, the dispase II-generated epidermal membranes are not suitable for this purpose.

Whole-Mount Preparation and Microscopic Analysis of Epidermis.

The epidermis is a stratified epithelium. Compared to that for monolayered epithelia, understanding of the cell biology of stratified epithelia lags far behind. The major reason for this is the limitation of methods to reproduce the epidermis in vitro using cultured keratinocytes: for example, cultured keratinocyte cell sheets lack Langerhans cells, melanocytes, nerves, sweat ducts, and hair follicles. One current way to overcome this limitation is to observe the epidermis in vivo via whole-mount staining and three-dimensional imaging. Here, we describe how to prepare epidermal sheets from skin and how to immunostain and observe them in whole mount. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of mouse epidermal sheets by the ammonium thiocyanate method Alternate Protocol: Preparation of mouse epidermal sheets by the dispase method Basic Protocol 2: Preparation of human epidermal sheets by the dispase method Basic Protocol 3: Whole-mount immunostaining of epidermis.

A simple method to isolate term trophoblasts and maintain them in extended culture.

INTRODUCTION: Primary trophoblast cultures obtained from term placentae are an important research tool. Term trophoblasts, while isolated as mononuclear cells, spontaneously fuse to form multinucleated syncytial clusters. Since term trophoblast cells do not replicate in vitro, contaminating cells can overgrow the culture limiting the lifespan of primary trophoblast cultures to about seven days. We aimed to develop a method that would allow the prolonged culture of term trophoblasts. METHODS: Trophoblasts were isolated from term placentae, following vaginal or cesarean section delivery, using either trypsin/DNase or dispase/DNase to digest the tissue. Purity of the trophoblasts was confirmed using flow cytometry prior to plating and during culture using immunocytochemistry. Cell death was examined with propidium iodide and trophoblast fusion monitored using PKH67 membrane stain. RESULTS: Digestion of term villous tissue with dispase/DNase resulted in the release of significantly more trophoblasts than digestion with trypsin/DNase (n = 8, p = 0.0051). Viability of the trophoblasts was unaffected by enzyme choice. The use of Advanced DMEM/F12 supplemented with 2% fetal bovine serum allowed culture of the trophoblasts with minimal cell death or contamination for 30 days. Despite prolonged culture over half of the trophoblasts remained mononuclear. DISCUSSION: We report a simple, optimized method to isolate and culture trophoblasts from term placentae for prolonged periods without substantial contamination with other cell types. Consistent with previous findings, trophoblasts cultured using our method were able to syncytialise, forming multi-nucleated syncytia. This extended growth time allows long term in vitro experimentation to further understand the nature of trophoblasts.

Simple Method for Establishing Primary Leporidae Skin Fibroblast Cultures.

Commercial hare and rabbit immortalized cell lines are extremely limited regarding the many species within the lagomorpha order. To overcome this limitation, researchers and technicians must establish primary cell cultures derived from biopsies or embryos. Among all cell types, fibroblasts are plastic and resilient cells, highly convenient for clinical and fundamental research but also for diagnosis, particularly for viral isolation. Here, we describe a fast and cheap method to produce primary fibroblast cell cultures from leporid species, using dispase II, a protease that allows dermal-epidermal separation, followed by a simple enzymatic digestion with trypsin. This method allows for the establishment of an in vitro cell culture system with an excellent viability yield and purity level higher than 85% and enables the maintenance and even immortalization of leporid fibroblastic cells derived from tissues already differentiated.

Dermal-Epidermal Separation by Enzyme.

The skin contains three primary layers: epidermis, dermis, and hypodermis. Separation of epidermal components from dermis (dermal-epidermal separation) is an important basic investigation technique for pharmacology, toxicology, and biology. There are different systems of epidermal separation, including typical methods of chemical, enzyme, heat, etc. Each approach has advantages versus disadvantages, and thus the appropriate method should be chosen for a given research question. Here we described the method of enzyme separation.

E-cadherin loss in RMG-1 cells inhibits cell migration and its regulation by Rho GTPases.

E-cadherin is an adherens junction protein that forms intercellular contacts in epithelial cells. Downregulation of E-cadherin is frequently observed in epithelial tumors and it is a hallmark of epithelial-mesenchymal transition (EMT). However, recent findings suggest that E-cadherin plays a more complex role in certain types of cancers. Previous studies investigating the role of E-cadherin mainly used gene-knockdown systems; therefore, we used the CRISPR/Cas9n system to develop E-cadherin-knockout (EcadKO) ovarian cancer RMG-1 cell to clarify the role of E-cadherin in RMG-1 cells. EcadKO RMG-1 cells demonstrated a complete loss of the adherens junctions and failed to form cell clusters. Cell-extracellular matrix (ECM) interactions were increased in EcadKO RMG-1 cells. Upregulation of integrin beta1 and downregulation of collagen 4 were confirmed. EcadKO RMG-1 cells showed decreased ß-catenin levels and decreased expression of its transcriptional target cyclin D1. Surprisingly, a marked decrease in the migratory ability of EcadKO RMG-1 cells was observed and the cellular response to Rho GTPase inhibitors was diminished. Thus, we demonstrated that E-cadherin in RMG-1 cells is indispensable for ß-catenin expression and ß-catenin mediated transcription and Rho GTPase-regulated directionally persistent cell migration.

Simple method for analyzing the purity of protease-containing samples by acid-treatment SDS-PAGE.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used technique to analyze the purity of a protein. However, it is necessary to denature (via boiling) the samples before subjecting them to electrophoresis. In the case of protease-containing samples, autolysis of the protease can occur, affecting the accuracy of results. In this study, we investigated the methods for analyzing the purity of Dispase I, a thermolysin-like neutral protease. When we analyzed D protease, a neutral metalloprotease component of Dispase I and highly purified Dispase I using the conventional SDS-PAGE method, a large number of bands were detected in both cases. These bands (putative D protease fragments) were assumed to result from autolysis. To inactivate D protease (optimal pH 7-8), 0.05 M sulfuric acid was utilized (pH 0.7-2.5). Using a conventional sample preparation solution, acid-treated Dispase I samples (without boiling) were made, and SDS-PAGE (15% w/v gel) was carried out. Our findings show that autolysis was inhibited under strong acidic conditions, and protein denaturation was achieved by treatment with sulfuric acid and SDS without boiling. Using this modified SDS-PAGE method, the purities of Dispase I and the purified enzyme were determined to be approximately 80% and 98%, respectively. Furthermore, we demonstrated that this method can be applied for the analysis of other samples including non-acidic proteases (e.g., thermolysin, subtilisin, and trypsin) and protease-contaminated samples (a mixed solution of albumin and D protease).

Destructive twisting of neutral metalloproteases: the catalysis mechanism of the Dispase autolysis-inducing protein from Streptomyces mobaraensis DSM 40487.

The Dispase autolysis-inducing protein (DAIP) is produced by Streptomyces mobaraensis to disarm neutral metalloproteases by decomposition. The absence of a catalytic protease domain led to the assumption that the seven-bladed ß-propeller protein DAIP causes structural modifications, thereby triggering autolysis. Determination of protein complexes consisting of DAIP and thermolysin or DAIP and a nonfunctional E138A bacillolysin variant supported this postulation. Protein twisting was indicated by DAIP-mediated inhibition of thermolysin while bacillolysin underwent immediate autolysis under the same conditions. Interestingly, an increase in SYPRO orange fluorescence allowed tracking of the fast degradation process. Similarly rapid autolysis of thermolysin mediated by DAIP was only observed upon the addition of amphiphilic compounds, which probably amplify the induced structural changes. DAIP further caused degradation of FITC-labeled E138A bacillolysin by trypsin, as monitored by a linear decrease in fluorescence polarization. The kinetic model, calculated from the obtained data, suggested a three-step mechanism defined by (a) fast DAIP-metalloprotease complex formation, (b) slower DAIP-mediated protein twisting, and (c) fragmentation. These results were substantiated by crystallized DAIP attached to a C-terminal helix fragment of thermolysin. Structural superposition of the complex with thermolysin is indicative of a conformational change upon binding to DAIP. Importantly, the majority of metalloproteases, also including homologs from various pathogens, are highly conserved at the autolysis-prone peptide bonds, suggesting their susceptibility to DAIP-mediated decomposition, which may offer opportunities for pharmaceutical applications. DATABASES: The atomic coordinates and structure factors (PDB ID: 6FHP) have been deposited in the Protein Data Bank (http://www.pdb.org/). ENZYMES: Aureolysin, EC 3.4.24.29; bacillolysin (Dispase, Gentlyase), EC 3.4.24.28; lasB (elastase), EC 3.4.24.4; subtilisin, EC 3.4.21.62; thermolysin, EC 3.4.24.27; transglutaminase, EC 2.3.2.13; trypsin, EC 3.4.21.4; vibriolysin (hemagglutinin(HA)/protease), EC 3.4.24.25.

Isolation of Murine Alveolar Type II Epithelial Cells.

We have optimized a protocol for isolation of alveolar type II epithelial cells from mouse lung. Lung cell suspensions are prepared by intratracheal instillation of dispase and agarose followed by mechanical disaggregation of the lungs. Alveolar type II epithelial cells are purified from these lung cell suspensions through magnetic-based negative selection using a Biotin-antibody, Streptavidin-MicroBeads system. The purified alveolar type II epithelial cells can be cultured and maintained on fibronectin-coated plates in DMEM with 10% FBS. This protocol enables specific investigation of alveolar type II epithelial cells at molecular and cellular levels and provides an important tool to investigate in vitro the mechanisms underlying lung pathogenesis.

Harvesting epithelial keratinocyte sheets from temperature-responsive dishes preserves basement membrane proteins and improves cell survival in a skin defect model.

Cultured epithelial autograft (CEA) therapy has been used in clinical applications since the 1980s. However, there are some issues related to this treatment that still remain unsolved. Enzymatic treatment is typically used in the collection of epithelial keratinocyte sheets, but it tends to break the adhesion and basement membrane proteins. It is thought that the loss of proteins after enzymatic treatment is responsible for the poor survival of transplanted cell sheets. Our laboratory has developed a temperature-responsive culture dish that does not require enzymatic treatment to harvest the cells. In this study, we compare morphological and survival results from rat epithelial keratinocyte cell sheets harvested by temperature-reducing treatment (TT sheets) against cell sheets harvested by enzymatic (dispase) treatment (DT sheets). TT sheets preserve keratin structure in better conditions and express higher levels of collagen IV and laminin 5 than DT sheets. In order to evaluate cell sheet survival after transplantation, we created an in vivo transplant model. Keratinocyte sheets obtained from GFP-positive animals were transplanted into athymic rats. The survival rate 7 days after transplantation of TT sheet was higher than that of DT sheets. Collagen IV and Laminin 5 expression was observed in the TT sheet transplantation group. These results indicate that the remaining basement membrane proteins are important for initial attachment and cell survival. We believe that the cell sheet harvesting method using temperature-responsive culture dishes provides superior cell survival and can solve one of the roadblocks in CEA therapy. Copyright © 2016 John Wiley & Sons, Ltd.