A novel non-viral delivery method that enables efficient engineering of primary human T cells for ex vivo cell therapy applications.

BACKGROUND AIMS: Next-generation immune cell therapy products will require complex modifications using engineering technologies that can maintain high levels of cell functionality. Non-viral engineering methods have the potential to address limitations associated with viral vectors. However, while electroporation is the most widely used non-viral modality, concerns about its effects on cell functionality have led to the exploration of alternative approaches. Here the authors have examined the suitability of the Solupore non-viral delivery system for engineering primary human T cells for cell therapy applications. METHODS: The Solupore system was used to deliver messenger RNA (mRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) guide RNA ribonucleoprotein (RNP) cargos to T cells, and efficiency was measured by flow cytometry. Cell perturbation was assessed by immune gene expression profiling, including an electroporation comparator. In vitro and in vivo cytotoxicity of chimeric antigen receptor (CAR) T cells generated using the Solupore system was evaluated using a real-time cellular impedance assay and a Raji-luciferase mouse tumor model, respectively. RESULTS: Efficient transfection was demonstrated through delivery of mRNA and CRISPR CAS9 RNP cargos individually, simultaneously and sequentially using the Solupore system while consistently maintaining high levels of cell viability. Gene expression profiling revealed minimal alteration in immune gene expression, demonstrating the low level of perturbation experienced by the cells during this transfection process. By contrast, electroporation resulted in substantial changes in immune gene expression in T cells. CAR T cells generated using the Solupore system exhibited efficient cytotoxicity against target cancer cells in vitro and in vivo. CONCLUSIONS: The Solupore system is a non-viral means of simply, rapidly and efficiently delivering cargos to primary human immune cells with retention of high cell viability and functionality.

Delivery of nucleic acids to ex vivo porcine airways using electrospray.

AIM OF THE STUDY: Nucleic acid-based therapies have the potential to provide clinically meaningful benefit across a wide spectrum of lung disease. However, in vivo delivery remains a challenge. Here we examined the feasibility of using electrospray to deliver nucleic acids to both porcine tracheal tissue sections and whole lung ex vivo. MATERIALS AND METHODS: The effect of electrospray solution, emitter gauge, flow rate and voltage on plasmid DNA integrity was examined by analyzing supercoiled:open circle structure ratio by gel electrophoresis. Optimal parameters were used to deliver luciferase DNA and mRNA and siRNA-FITC to tracheal tissue sections. Luciferase mRNA was delivered to whole porcine lungs ex vivo using a catheter and bronchoscope system. Luciferase activity and fluorescence were analyzed by luminometry and microscopy respectively. RESULTS: The incidence of DNA plasmid nicking was greatest in a low salt solution without ethanol compared with 1% and 20% ethanol with salt. From a range of emitters tested, a 32 gauge emitter produced the best supercoiled:open circle structure ratio, likely because less voltage was required to produce a stable electrospray with this emitter. Lower flow rates also showed a trend towards reduced DNA nicking. GFP DNA electrosprayed at 5 kV and 6 kV resulted in lower levels of GFP expression in A549 lung cells following lipofection compared with 3 kV and 4 kV. Optimised parameters of 20% ethanol solution, 32 gauge emitter, low flow rates and voltages of 3-5 kV, nucleic acid molecules were successful for delivery of luciferase DNA and mRNA as well as siRNA-FITC to porcine tracheal tissue sections and for delivery of luciferase mRNA to whole porcine lungs via bronchoscope. CONCLUSIONS: We report ex vivo delivery of nucleic acids to porcine lung tissue via electrospray and bronchoscopic electrospray delivery of nucleic acid to an ex vivo porcine lung model.

Vector-free intracellular delivery by reversible permeabilization.

Despite advances in intracellular delivery technologies, efficient methods are still required that are vector-free, can address a wide range of cargo types and can be applied to cells that are difficult to transfect whilst maintaining cell viability. We have developed a novel vector-free method that uses reversible permeabilization to achieve rapid intracellular delivery of cargos with varying composition, properties and size. A permeabilizing delivery solution was developed that contains a low level of ethanol as the permeabilizing agent. Reversal of cell permeabilization is achieved by temporally and volumetrically controlling the contact of the target cells with this solution. Cells are seeded in conventional multi-well plates. Following removal of the supernatant, the cargo is mixed with the delivery solution and applied directly to the cells using an atomizer. After a short incubation period, permeabilization is halted by incubating the cells in a phosphate buffer saline solution that dilutes the ethanol and is non-toxic to the permeabilized cells. Normal culture medium is then added. The procedure lasts less than 5 min. With this method, proteins, mRNA, plasmid DNA and other molecules have been delivered to a variety of cell types, including primary cells, with low toxicity and cargo functionality has been confirmed in proof-of-principle studies. Co-delivery of different cargo types has also been demonstrated. Importantly, delivery occurs by diffusion directly into the cytoplasm in an endocytic-independent manner. Unlike some other vector-free methods, adherent cells are addressed in situ without the need for detachment from their substratum. The method has also been adapted to address suspension cells. This delivery method is gentle yet highly reproducible, compatible with high throughput and automated cell-based assays and has the potential to enable a broad range of research, drug discovery and clinical applications.

The development of a tissue-engineered tracheobronchial epithelial model using a bilayered collagen-hyaluronate scaffold.

Today, chronic respiratory disease is one of the leading causes of mortality globally. Epithelial dysfunction can play a central role in its pathophysiology. The development of physiologically-representative in vitro model systems using tissue-engineered constructs might improve our understanding of epithelial tissue and disease. This study sought to engineer a bilayered collagen-hyaluronate (CHyA-B) scaffold for the development of a physiologically-representative 3D in vitro tracheobronchial epithelial co-culture model. CHyA-B scaffolds were fabricated by integrating a thin film top-layer into a porous sub-layer with lyophilisation. The film layer firmly connected to the sub-layer with delamination occurring at stresses of 12-15 kPa. Crosslinked scaffolds had a compressive modulus of 1.9 kPa and mean pore diameters of 70 µm and 80 µm, depending on the freezing temperature. Histological analysis showed that the Calu-3 bronchial epithelial cell line attached and grew on CHyA-B with adoption of an epithelial monolayer on the film layer. Immunofluorescence and qRT-PCR studies demonstrated that the CHyA-B scaffolds facilitated Calu-3 cell differentiation, with enhanced mucin expression, increased ciliation and the formation of intercellular tight junctions. Co-culture of Calu-3 cells with Wi38 lung fibroblasts was achieved on the scaffold to create a submucosal tissue analogue of the upper respiratory tract, validating CHyA-B as a platform to support co-culture and cellular organisation reminiscent of in vivo tissue architecture. In summary, this study has demonstrated that CHyA-B is a promising tool for the development of novel 3D tracheobronchial co-culture in vitro models with the potential to unravel new pathways in drug discovery and drug delivery.

SMAD Signaling in the Airways of Healthy Rhesus Macaques versus Rhesus Macaques with Asthma Highlights a Relationship Between Inflammation and Bone Morphogenetic Proteins.

Bone morphogenetic protein (BMP) signaling is important for correct lung morphogenesis, and there is evidence of BMP signaling reactivation in lung diseases. However, little is known about BMP signaling patterns in healthy airway homeostasis and inflammatory airway disease and during epithelial repair. In this study, a rhesus macaque (Macaca mulatta) model of allergic airway disease was used to investigate BMP signaling throughout the airways in health, disease, and regeneration. Stereologic quantification of immunofluorescent images was used to determine the expression of BMP receptor (BMPR) Ia and phosphorylated SMAD (pSMAD) 1/5/8 in the airway epithelium. A pSMAD 1/5/8 expression gradient was found along the airways of healthy juvenile rhesus macaques (n = 3, P < 0.005). Membrane-localized BMPRIa expression was also present in the epithelium of the healthy animals. After exposure to house dust mite allergen and ozone, significant down-regulation of nuclear pSMAD 1/5/8 occurs in the epithelium. When the animals were provided with a recovery period in filtered air, proliferating cell nuclear antigen, pSMAD 1/5/8, and membrane-localized BMPRIa expression were significantly increased in the epithelium of conducting airways (P < 0.005). Furthermore, in the asthmatic airways, altered BMPRIa localization was evident. Because of the elevated eosinophil presence in these airways, we investigated the effect of eosinophil-derived proteins on BMPRIa trafficking in epithelial cells. Eosinophil-derived proteins (eosinophil-derived neurotoxin, eosinophil peroxidase, and major basic protein) induced transient nuclear translocation of membrane-bound BMPRIa. This work mapping SMAD signaling in the airways of nonhuman primates highlights a potential mechanistic relationship between inflammatory mediators and BMP signaling and provides evidence that basal expression of the BMP signaling pathway may be important for maintaining healthy airways.

Respiratory Tissue Engineering: Current Status and Opportunities for the Future.

Currently, lung disease and major airway trauma constitute a major global healthcare burden with limited treatment options. Airway diseases such as chronic obstructive pulmonary disease and cystic fibrosis have been identified as the fifth highest cause of mortality worldwide and are estimated to rise to fourth place by 2030. Alternate approaches and therapeutic modalities are urgently needed to improve clinical outcomes for chronic lung disease. This can be achieved through tissue engineering of the respiratory tract. Interest is growing in the use of airway tissue-engineered constructs as both a research tool, to further our understanding of airway pathology, validate new drugs, and pave the way for novel drug therapies, and also as regenerative medical devices or as an alternative to transplant tissue. This review provides a concise summary of the field of respiratory tissue engineering to date. An initial overview of airway anatomy and physiology is given, followed by a description of the stem cell populations and signaling processes involved in parenchymal healing and tissue repair. We then focus on the different biomaterials and tissue-engineered systems employed in upper and lower respiratory tract engineering and give a final perspective of the opportunities and challenges facing the field of respiratory tissue engineering.

Anatomy and bronchoscopy of the porcine lung. A model for translational respiratory medicine.

The porcine model has contributed significantly to biomedical research over many decades. The similar size and anatomy of pig and human organs make this model particularly beneficial for translational research in areas such as medical device development, therapeutics and xenotransplantation. In recent years, a major limitation with the porcine model was overcome with the successful generation of gene-targeted pigs and the publication of the pig genome. As a result, the role of this model is likely to become even more important. For the respiratory medicine field, the similarities between pig and human lungs give the porcine model particular potential for advancing translational medicine. An increasing number of lung conditions are being studied and modeled in the pig. Genetically modified porcine models of cystic fibrosis have been generated that, unlike mouse models, develop lung disease similar to human cystic fibrosis. However, the scientific literature relating specifically to porcine lung anatomy and airway histology is limited and is largely restricted to veterinary literature and textbooks. Furthermore, methods for in vivo lung procedures in the pig are rarely described. The aims of this review are to collate the disparate literature on porcine lung anatomy, histology, and microbiology; to provide a comparison with the human lung; and to describe appropriate bronchoscopy procedures for the pig lungs to aid clinical researchers working in the area of translational respiratory medicine using the porcine model.

Regulation of epithelial to mesenchymal transition by bone morphogenetic proteins.

Epithelial to mesenchymal transition (EMT) is a process in which fully differentiated epithelial cells lose many of their epithelial characteristics and adopt features typical of mesenchymal cells, thus allowing cells to become migratory and invasive. EMT is a critical process in development and its role in cancer and fibrosis is becoming increasingly recognised. It is also becoming apparent that EMT is not just restricted to embryonic development and disease in adults, but in fact may be an important process for the maintenance and regeneration of adult tissue architecture. While transforming growth factor-ß (TGF-ß) is considered a prototypic inducer of EMT, relatively little is known about other signalling molecules that regulate EMT. Bone morphogenic proteins (BMPs) are members of the TGF-ß superfamily and 20 different human BMPs have been identified. Originally named for their effects on bone, these proteins are now considered to be key morphogenetic signals that orchestrate tissue architecture throughout the body. BMP2, -4 and -7 are the best studied to date. There are disparate reports of the roles of BMPs in EMT during development, cancer and fibrosis. Here, we present an overview of this literature as well as the emerging role of EMT in tissue regeneration and the involvement of BMPs in regulating this process.

IFN-γ stimulated human umbilical-tissue-derived cells potently suppress NK activation and resist NK-mediated cytotoxicity in vitro.

Umbilical cord tissue represents a unique source of cells with potential for cell therapy applications for multiple diseases. Human umbilical tissue-derived cells (hUTC) are a developmentally early stage, homogenous population of cells that are HLA-ABC dim, HLA-DR negative, and lack expression of co-stimulatory molecules in the unactivated state. The lack of HLA-DR and co-stimulatory molecule expression on unactivated hUTC may account for their reduced immunogenicity, facilitating their use in allogeneic settings. However, such approaches could be confounded by host innate cells such as natural killer (NK) cells. Here, we evaluate in vitro NK cell interactions with hUTC and compare them with human mesenchymal stem cells (MSC). Our investigations show that hUTC suppress NK activation, through prostaglandin-E2 secretion in a contact-independent manner. Prestimulation of hUTC or human MSC with interferon gamma (IFN-γ) induced expression of the tryptophan degrading enzyme indoleamine 2, 3 dioxygenase, facilitating enhanced suppression. However, resting NK cells of different killer immunoglobulin-like receptor haplotypes did not kill hUTC or MSC; only activated NK cells had the ability to kill nonstimulated hUTC and, to a lesser extent, MSC. The cell killing process involved signaling through the NKG2D receptor and the perforin/granzyme pathway; this was supported by CD54 (ICAM-1) expression by hUTC. IFN-γ-stimulated hUTC or hMSC were less susceptible to NK killing; in this case, protection was associated with elevated HLA-ABC expression. These data delineate the different mechanisms in a two-way interaction between NK cells and two distinct cell therapies, hUTC or hMSC, and how these interactions may influence their clinical applications.

Bone morphogenetic proteins enhance an epithelial-mesenchymal transition in normal airway epithelial cells during restitution of a disrupted epithelium.

BACKGROUND: Mechanisms of airway repair are poorly understood. It has been proposed that, following injury, progenitor populations such as club cells (Clara) become undifferentiated, proliferate and re-differentiate to re-epithelialise the airway. The exact phenotype of such cells during repair is unknown however. We hypothesised that airway epithelial cells (AECs) undergo some degree of epithelial-mesenchymal transition (EMT) in order to migrate over a denuded airway and effect re-epithelialisation. Furthermore, based on our previous findings that BMP signalling is an early event in AECs following injury in vivo and that BMP4 down-regulates E-cadherin expression and enhances migration in AECs in vitro, we hypothesised that BMPs could play a role in inducing such a phenotypic switch. METHODS: Normal AECs were isolated from mouse lungs and analysed in a model of a disrupted epithelium. EMT marker expression and BMP signalling were examined by immunofluorescence, Western blotting and RT-PCR. RESULTS: Following generation of a wound area, AECs at the wound edge migrated and acquired a mesenchymal-like morphology. E-cadherin expression was reduced in migrating cells while vimentin and α-smooth muscle actin (α-SMA) expression was increased. Re-expression of membrane E-cadherin was subsequently observed in some cells in the wound area following re-establishment of the monolayer. A transient increase in the incidence of nuclear phosphorylated Smad1/5/8 was observed in migrating cells compared with confluent cells, indicating active BMP signalling during migration. BMP antagonists noggin and gremlin inhibited cell migration, confirming the involvement of BMP signalling in migration and indicating autocrine signalling, possibly involving BMP7 or BMP4 which were expressed in AECs. Exogenous BMP2, BMP4 and BMP7 induced a mesenchymal-like morphology in AECs, enhanced the rate of cell migration and increased α-SMA protein expression in AECs. CONCLUSIONS: Following disruption of an intact epithelium, migrating AECs at the wound edge acquire an EMT-like phenotype involving altered expression of E-cadherin, vimentin and α-SMA. BMP signalling is involved in AEC migration and is likely to mediate the switch towards an EMT-like phenotype by altering protein expression to facilitate cell migration and wound closure. We propose therefore that acquisition of an EMT-like phenotype by AECs is a normal aspect of wound repair. Furthermore, we suggest that diseases involving fibrosis may arise because the EMT phase of repair is prolonged by chronic injury/inflammation, rather than being caused by it, as is the current paradigm.

Olfactomedin III expression contributes to anoikis-resistance in clonal variants of a human lung squamous carcinoma cell line.

Three clonal subpopulations of DLKP, a poorly differentiated squamous lung carcinoma cell line, display striking differences in ability to survive in suspension (anoikis resistance). DLKP-SQ is anoikis resistant (7.5% anoikis at 24 h). In contrast, DLKP-M and DLKP-I are sensitive to anoikis (49.2% and 42.6% respectively). DLKP-I shows increased apoptosis consistently over all time points tested while DLKP-M appear to slow down metabolically and perhaps delays onset of anoikis by undergoing autophagy. Expression microarray analysis identified pronounced differential expression of Olfactomedin 3 (OLFM3) between the clones. High expression of OLFM3 was confirmed at the RNA level by qRT-PCR in DLKP-SQ and at the protein level by Western blotting (within the cell and secreted). Little or no OLFM3 was detected in the other two clones (DLKP-M and DLKP-I). Following siRNA knockdown of OLFM3 in DLKP-SQ, anoikis was increased 2.8-fold to 21% which was intermediate between the anoikis levels in DLKP-SQ and DLKP-M or DLKP-I. This knockdown correlated with increased apoptosis in suspension but not in attached culture conditions. Addition of recombinant OLFM3 reduced anoikis in DLKP-I. This is the first instance of OLFM3 being linked with anoikis resistance in a human cancer cell line.

Bone morphogenetic protein signalling in airway epithelial cells during regeneration.

Mechanisms of lung regeneration after injury remain poorly understood. Bone morphogenetic protein 4 (BMP4) is critical for lung morphogenesis and regulates differentiation of the airway epithelium during development, although its mechanism of action is unknown. The role of BMPs in adult lungs is unclear. We hypothesised that BMP signalling is involved in regeneration of damaged adult airways after injury. Our aims were to characterise the regeneration process in 1-nitronaphthalene (1-NN) injured airways, to determine if and when BMP signalling is activated during this process and investigate the effects of BMP4 on normal adult airway epithelial cells (AECs). Rats were injected with 50 mg/kg 1-NN and protein expression in AECs was examined by Western blotting of lung lysis lavage, and by immunofluorescence, at 6, 24, 48 and 96 h post injection. Expression of signalling molecules p-ERK-1, p-ERK-2 and p-Smad1/5/8 in AECs peaked at 6 h post injection, coincident with maximal inflammation and prior to airway denudation which occurred at 24 h. While airways were re-epithelialised by 48h, AEC proliferation peaked later at 96 h post 1-NN injection. In vitro, BMP4 induced a mesenchymal-like morphology in normal AECs, downregulated E-cadherin expression and increased migration in a wound closure assay. Thus, following acute injury, increased BMP signalling in AECs coincides with inflammation and precedes airway denudation and re-epithelialisation. Our data indicate that, similar to its role in controlling tissue architecture during development, BMP signalling regulates regeneration of the airways following acute injury, involving downregulation of E-cadherin and induction of migration in AECs.

Regulation of surfactant protein B gene expression in bone marrow-derived cells.

While investigating the differentiation potential of bone marrow-derived cells, we previously demonstrated upregulated expression of the lung-related surfactant protein B (SP-B) gene in hematopoietic progenitor cells (HPCs) when they were cocultured with macerated lung tissue. During coculture, HPCs differentiated toward a dendritic-like myeloid cell phenotype (hematopoietic progenitor cell-derived dendritic-like cells [HPC-DCs]). However, immature dendritic cells (iDCs) cocultured under identical conditions did not express SP-B mRNA before or after coculture. We have now further examined the regulation of SP-B expression in HPC-DCs and iDCs. Of the transcription factors involved in SP-B gene expression, neither cell type expressed TTF-1, HNF3alpha, or HNF3beta, but both cell types expressed Sp1 and Sp3. Sp1 binding to the SP-B promoter was investigated in these cells. Three novel Sp1 binding motifs were identified in the mouse SP-B promoter. Using chromatin immunoprecipitation, it was demonstrated that Sp1 was bound to all three sites in HPC-DCs after coculture with lung tissue, but not in iDCs. We hypothesized that although genes from multiple lineages may be active in HPCs, gene silencing events, such as methylation, may subsequently occur to suppress expression of these genes in more mature myeloid cells, such as iDCs. Treatment with the demethylating agent 5-azacytidine resulted in expression of the SP-B gene in iDCs. These data indicate that tissue-specific transcription factors are not required to express the lung-related gene SP-B in hematopoietic progenitor cells. Furthermore, silencing events, such as methylation, may occur to suppress lung-related gene expression as progenitor cells become committed toward more mature hematopoietic cell phenotypes.

A possible role for protein synthesis, extracellular signal-regulated kinase, and brain-derived neurotrophic factor in long-term spatial memory retention in the water maze.

Hippocampal protein synthesis is dependent upon a number of different molecular and cellular mechanisms that act together to make previously labile memories more stable and resistant to disruption. Both brain-derived neurotrophic factor (BDNF) and extracellular signal-regulated kinase (ERK) are known to play an important role in protein synthesis-dependent memory consolidation, via the mitogen-activated protein-kinase (MAP-K) signaling pathway during the transcription phase of protein synthesis. The current study investigates the influence of protein synthesis inhibition (PSI) by cycloheximide on spatial learning and memory. In an initial experiment, the authors utilized two doses of cycloheximide (0.5 mg/kg and 1.0 mg/kg, intraperitoneally) to determine the dose at which long-term (>24 hours) memories are impaired. A second experiment was designed to investigate the effect of PSI on the formation of cue-platform associations in the watermaze, and on BDNF and ERK expression in the hippocampus. At the higher dose (1.0 mg/kg) cycloheximide resulted in impaired retention of the water maze. BDNF and ERK expression was also down-regulated in animals injected with this dose of cycloheximide. Our results demonstrate a role of protein synthesis in spatial memory retention, along with a possible relationship between protein synthesis and hippocampal BDNF/ERK expression.

Comparative evaluation of viral, nonviral and physical methods of gene delivery to normal and transformed lung epithelial cells.

Few studies have directly compared the efficiencies of gene delivery methods that target normal lung cells versus lung tumor cells. We report the first study directly comparing the efficiency and toxicity of viral [adeno-associated virus (AAV2, 5, 6) and lentivirus], nonviral (Effectene, SuperFect and Lipofectamine 2000) and physical [particle-mediated gene transfer (PMGT)] methods of gene delivery in normal mouse lung cells and in mouse adenocarcinoma cells. Lentivirus pseudotyped with the vesicular stomatitis virus glycoprotein was the most efficient gene transfer method for normal mouse airway epithelial cells [25.95 (+/-3.57) %] whereas AAV6 was most efficient for MLE-12 adenocarcinoma cells [68.2 (+/-3.2) %]. PMGT was more efficient in normal mouse airway epithelial cells than AAV5, Lipofectamine 2000 and SuperFect. AAV5 displayed the lowest transfection efficiency at less than 10% in both cell types. PMGT was the only method that resulted in significant toxicity. In summary, for all of the gene delivery methods examined here, lung tumor cells were transfected more easily than normal lung cells. Lipofectamine 2000 is potentially highly selective for lung tumor cells whereas AAV6 and lentivirus vesicular stomatitis virus glycoprotein may be useful for gene delivery strategies that require targeting of both normal and tumor cells.

Reducing the false positive rate in the non-parametric analysis of molecular coevolution.

BACKGROUND: The strength of selective constraints operating on amino acid sites of proteins has a multifactorial nature. In fact, amino acid sites within proteins coevolve due to their functional and/or structural relationships. Different methods have been developed that attempt to account for the evolutionary dependencies between amino acid sites. Researchers have invested a significant effort to increase the sensitivity of such methods. However, the difficulty in disentangling functional co-dependencies from historical covariation has fuelled the scepticism over their power to detect biologically meaningful results. In addition, the biological parameters connecting linear sequence evolution to structure evolution remain elusive. For these reasons, most of the evolutionary studies aimed at identifying functional dependencies among protein domains have focused on the structural properties of proteins rather than on the information extracted from linear multiple sequence alignments (MSA). Non-parametric methods to detect coevolution have been reported to be especially susceptible to produce false positive results based on the properties of MSAs. However, no formal statistical analysis has been performed to definitively test the differential effects of these properties on the sensitivity of such methods. RESULTS: Here we test the effect that variations on the MSA properties have over the sensitivity of non-parametric methods to detect coevolution. We test the effect that the size of the MSA (number of sequences), mean pairwise amino acid distance per site and the strength of the coevolution signal have on the ability of non-parametric methods to detect coevolution. Our results indicate that all three factors have significant effects on the accuracy of non-parametric methods. Further, introducing statistical filters improves the sensitivity and increases the statistical power of the methods to detect functional coevolution. Statistical analysis of the physico-chemical properties of amino acid sites in the context of the protein structure reveals striking dependencies among amino acid sites. Results indicate a covariation trend in the hydrophobicities and molecular weight characteristics of amino acid sites when analysing a non-redundant set of 8000 protein structures. Using this biological information as filter in coevolutionary analyses minimises the false positive rate of these methods. Application of these filters to three different proteins with known functional domains supports the importance of using biological filters to detect coevolution. CONCLUSION: Coevolutionary analyses using non-parametric methods have proved difficult and highly prone to provide spurious results depending on the properties of MSAs and on the strength of coevolution between amino acid sites. The application of statistical filters to the number of pairs detected as coevolving reduces significantly the number of artifactual results. Analysis of the physico-chemical properties of amino acid sites in the protein structure context reveals their structure-dependent covariation. The application of this known biological information to the analysis of covariation greatly enhances the functional coevolutionary signal and removes historical covariation. Simultaneous use of statistical and biological data is instrumental in the detection of functional amino acid sites dependencies and compensatory changes at the protein level.

BMP4 induces an epithelial-mesenchymal transition-like response in adult airway epithelial cells.

Bone morphogenetic proteins (BMPs) are critical morphogens and play key roles in epithelial-mesenchymal transitions (EMTs) during embryogenesis. BMP4 is required for early mesoderm formation and also regulates morphogenesis and epithelial cell differentiation in developing lungs. While, BMP signalling pathways are activated during lung inflammation in adult mice, the role of BMPs in adult lungs remains unclear. We hypothesised that BMPs are involved in remodelling processes in adult lungs and investigated effects of BMP4 on airway epithelial cells. BEAS-2B cell growth decreased in the presence of BMP4. Cells acquired a mesenchymal-like morphology with downregulation of adherens junction proteins and increased cell motility. Changes in extracellular matrix-related gene expression occurred with BMP4 treatment including upregulation of collagens, fibronectin and tenascin C. We conclude that the activity of BMP4 in EMT during development is recapitulated in adult airway epithelial cells and suggest that this activity may contribute to inflammation and fibrosis in vivo.

5-Bromo-2-deoxyuridine activates DNA damage signalling responses and induces a senescence-like phenotype in p16-null lung cancer cells.

5-Bromo-2-deoxyuridine (BrdU) is a thymidine analogue that is incorporated into replicating DNA. Although originally designed as a chemotherapeutic agent, sublethal concentrations of BrdU have long been known to alter the growth and phenotype of a wide range of cell types. Mechanisms underlying these BrdU-mediated effects remain unknown, however. We have characterized the effects of BrdU on A549 lung cancer cells by examining DNA damage responses, cell cycle effects and phenotypic changes. A549 cells express wild-type p53, but are p16-null. Sublethal concentrations of BrdU evoke a DNA damage response in these cells that involves the activation of Chk1, Chk2 and p53. Increased numbers of enlarged nuclei and multinucleated cells are evident in the treated populations. Cell cycle inhibition occurs, resulting in reduced proliferation and accumulation of cells in the S, G2/M and G0 phases. BrdU induces an early inhibition of p21 expression that coincides with nuclear localization of proliferating cell nuclear antigen. Subsequently, p21 levels increase, whereas proliferating cell nuclear antigen levels decrease compared with control cells. Upregulation of p27 and p57 expression also occurs. By day 7 of exposure to BrdU, treated cells acquire a senescent-like phenotype with an increase in cell size, granularity and beta-galactosidase activity. We conclude that BrdU induces a DNA damage response in A549 cells, which results in reduced proliferation mitotic exit and phenotypic changes that resemble senescence.

Posttranslational truncation of E-cadherin and significance for tumour progression.

Stable intraepithelial adhesion complexes are essential for the maintenance of epithelial integrity. Alterations in these complexes are key events in the development and progression of many diseases. One of the major proteins involved in maintaining epithelial cell-cell adhesion is the cell-adhesion junction protein E-cadherin, a member of the cadherin family of transmembrane adhesion proteins. E-cadherin is involved in many cellular processes including morphogenesis, adhesion, recognition, communication and oncogenesis. Inactivation of its adhesive properties is often a key step in tumour progression and metastasis, leading to its recent description as a tumour suppressor gene. Mutations of the E-cadherin gene CDH1 in gastric and mammary cancers have been well documented and reports of transcriptional repression during tumour progression are increasing. This review examines the role of posttranslational truncation of E-cadherin in cancer cells focusing on implications for tumour progression. The various proteins involved in the directed cleavage of E-cadherin and consequences of these truncations are discussed.

p21(Waf1/Cip1) regulates proliferation and apoptosis in airway epithelial cells and alternative forms have altered binding activities.

p21(Waf1/Cip1) plays central roles in proliferation, differentiation, and apoptosis. Alterations in the expression and subcellular localisation of p21 occur during several lung diseases but the roles of p21 in the lung epithelium are unknown. The effects of p21 on proliferation and apoptosis in mouse airway epithelial cells (AECs) were examined using p21-null mice. AECs isolated from p21-null mice had increased proliferation and apoptotic rates compared to AECs from wild-type mice. Alterations in the subcellular localization of the cell cycle regulatory proteins p27, PCNA, and p53 were also evident in p21(-/-) cells. The nuclear and cytoplasmic forms of p21 present in AECs were also examined. Full-length p21 (20 kDa) was detected in nuclear fractions but a C-terminal truncated form (17 kDa) of p21 was present in cytoplasmic fractions. The binding activities of truncated p21 were altered compared to full-length p21. Although the latter was complexed with PCNA, Cdk2, Cdk4, Cdk6, cyclin D3, and cyclin E, truncated p21 was bound only to Cdk4 and cyclin D3. In conclusion, p21 regulates proliferation and protects against apoptosis in AECs. In addition, different forms of p21 are present in AECs and the subcellular localization of these forms reflects differences in p21 activity.