Development of an experimental system to investigate the interaction between the Helicoverpa armigera stunt virus capsid protein and viral RNA

Tetraviruses are entomopathogenic viruses that propagate solely in lepidopteran hosts. Viruses of this group possess non-enveloped 38- to 40-nm capsids arranged in T = 4 surface symmetry. The viral genome consists of one or two single stranded positive sense RNA strands, which define the two genera of this family, the monopartite betatetraviruses and the bipartite omegatetraviruses. Two extensively studied members of the tetraviruses are the omegatetraviruses, Helicoverpa armigera stunt virus (HaSV) and the closely related Nudaurelia capensis ω virus (NωV). The larger genomic strand of HaSV (RNA1) encodes the viral replicase, while the other (RNA2) encodes the 71-kDa capsid precursor protein (p71). The pro-capsid is assembled from 240 copies of p71, which undergo a maturation auto-catalytic cleavage into the 64-kDa (p64) capsid protein and a 7-kDa peptide (p7) forming the capsid shell. The mechanism for the recognition and packaging of the viral genome is poorly understood for these viruses. The principle objective of the research described in this study was to develop in vitro and in vivo experimental systems to investigate interactions between the N terminal domain of HaSV p71 and viral RNAs. More specifically, the two positively charged clusters of predominantly arginine residues that are conserved amongst tetraviruses and the structurally analologous nodaviruses capsid protomers’ N terminal domains were investigated. An in vitro RNA-protein “pull down” system was developed using the rapid protein purification technique of the IMPACTTM-CN system. The coding sequence of the N terminal domain of p71 was fused to that of a chitin binding affinity tag (intein). This fusion protein was used as protein bait for the viral RNA. It was proposed that if RNA interacted with the fusion protein, it would be pulled down by the mass of affinity matrix and be precipitated and fluoresce when analysed by agarose gel electrophoresis using ethidium bromide. Despite optimisation of the in vitro assay, results were affected by the interaction between the intein-tag and nucleic acids, the state of the expressed fusion protein (in particular self-cleavage) and the excessive fluorescence present on the gels. The ADH2-GAPDH yeast expression system was used to investigate the in vivo assembly of p71 containing deletions of either one or both clusters within N terminal domain. It was found that all p71 mutants were expressed with the exception of the mutant containing a deletion of the second cluster. The reasons for this still require further investigation. The expressed p71 mutants were not processed into p64 and were degraded in vivo. In addition, an experimental attempt to purify assembled p71 mutant VLPs was unsuccessful. The assembly defect of p71 mutants emphasised the significance of the clusters, which are possibly required for interaction with viral RNAs for efficient VLP assembly. The results of this study suggest that an alternative tag or in vitro RNA-protein interaction assay be used. In addition, further experiments are required to investigate whether the co-expression of full length viral RNAs are required to rescue the in vivo assembly defect of p71 mutants into VLPs

Identification of novel cold-adapted nitrilase superfamily enzymes

Philosophiae Doctor - PhDIn bacteria, nitrile hydratases and enzymes of nitrilase and signature amidase superfamilies hydrolyse nitriles and amides to their corresponding carboxylic acids releasing ammonia. Bacteria expressing these enzymes are typically isolated where a sole nitrogen and/or carbon source is used to support their growth. The majority of characterised enzymes of industrial potential have been identified for their stabilities at elevated temperatures. To date, no reports of such enzymes have been isolated from cold adapted bacteria.In this study, an extensive screening program of cold-active microbial isolates for enzymes of this group led to the selection and detailed characterisation of an aliphatic amidase from Nesterenkonia.Nesterenkonia AN1, a new psychrotrophic isolate of the genus, was isolated from soil samples collected from the Miers Valley, Antarctica. AN1 showed significant 16S rRNA sequence identity to known members of the genera, but this is the only strain that had optimal growth at approximately 21oC. AN1, similar to known members, is an obligately alkaliphilic (pH 9-10) and halotolerant (Na+ 0- 15% (w/v)) strain.The genome of Nesterenkonia AN1, sequenced in-house, revealed two ORFs encoding putative nitrilases, referred to as Nit1 and Nit2. Based on analysis of their deduced protein sequences, both belonged to the nitrilase superfamily. Both sequences showed conserved catalytic residues (EKEC), glycine residues and contained the characteristic áââá monomer fold. Homology modelling using known structures suggested that both genes could encode N-carbamoyl D-amino acid amidohydrolases, although neither showed conserved residues implicated in the hydrolysis of carbamoyls.Nit1 and Nit2 were expressed in Escherichia coli BL21 (DE3) pLysS as Cterminal and N-terminal hexahistidine tagged fusion proteins, and purified using Ni-chelation chromatography. Nit1 showed no activity towards nitrile, amide and carbamoyl substrates. This protein, unlike members of the multimeric enzymes of the nitrilase superfamily, was a monomer ~30 kDa protein. It is possible that the C-terminal hexahistidine tag might have prevented Nit1 from forming multimeric proteins.Nit2 showed substrate specificity similar to known aliphatic amidases with a preference for small amides. Nit2 had maximal activity at 30oC and between pH 6.5 and 7.5, properties compatible with its cold-adapted alkaliphilic origins. In addition, the enzyme was irreversibly inactivated at temperatures above 30oC and had a half-life of approximately 7 mins at 60oC. The crystal structure of Nit2 was solved to 1.66 Å. It revealed a ~45.5 kDa dimer, composed of two tightly bound ~30 kDa monomers. These monomers associated along the A surface forming a áââá-áââá sandwich architecture that is conserved in known structures of the nitrilase superfamily.Nit2 is distinct from known aliphatic amidases in both its structure and enzymic activity: the enzyme did not possess an extended C-terminal region; is active in dimeric form; has high affinity for 3C amides rather than 2C amides; and has a low overall catalytic rate. The short C-terminal region of Nit2 may have contributed to the low stability of the enzyme at elevated temperatures. A dendrogram composed of protein sequences of members of the nitrilase superfamily and Nit2 further supported evidence that this aliphatic amidase falls within a distinct group of enzymes.This is the first report of the enzymic characterisation and structural analysis of an aliphatic amidase from a psychrotolerant, alkaliphilic and halotolerant extremophile

Defining stem cell dynamics and migration during wound healing in mouse skin epidermis.

Wound healing is essential to repair the skin after injury. In the epidermis, distinct stem cells (SCs) populations contribute to wound healing. However, how SCs balance proliferation, differentiation and migration to repair a wound remains poorly understood. Here, we show the cellular and molecular mechanisms that regulate wound healing in mouse tail epidermis. Using a combination of proliferation kinetics experiments and molecular profiling, we identify the gene signatures associated with proliferation, differentiation and migration in different regions surrounding the wound. Functional experiments show that SC proliferation, migration and differentiation can be uncoupled during wound healing. Lineage tracing and quantitative clonal analysis reveal that, following wounding, progenitors divide more rapidly, but conserve their homoeostatic mode of division, leading to their rapid depletion, whereas SCs become active, giving rise to new progenitors that expand and repair the wound. These results have important implications for tissue regeneration, acute and chronic wound disorders.This work was supported by the FNRS, TELEVIE, the PAI programme, a research grant from the Fondation contre le Cancer, the ULB fondation, the foundation Bettencourt Schueller, the foundation Baillet Latour and a consolidator grant the European Research Council (ERC-EXPAND)

Defining the clonal dynamics leading to mouse skin tumour initiation.

The changes in cell dynamics after oncogenic mutation that lead to the development of tumours are currently unknown. Here, using skin epidermis as a model, we assessed the effect of oncogenic hedgehog signalling in distinct cell populations and their capacity to induce basal cell carcinoma, the most frequent cancer in humans. We found that only stem cells, and not progenitors, initiated tumour formation upon oncogenic hedgehog signalling. This difference was due to the hierarchical organization of tumour growth in oncogene-targeted stem cells, characterized by an increase in symmetric self-renewing divisions and a higher p53-dependent resistance to apoptosis, leading to rapid clonal expansion and progression into invasive tumours. Our work reveals that the capacity of oncogene-targeted cells to induce tumour formation is dependent not only on their long-term survival and expansion, but also on the specific clonal dynamics of the cancer cell of origin.C.B. is an investigator of WELBIO. A.S-D. and JC.L. are supported by a fellowship of the FNRS and FRIA respectively. B.D.S. and E.H. are supported by the Wellcome Trust (grant number 098357/Z/12/Z and 110326/Z/15/Z). EH is supported by a fellowship from Trinity College, Cambridge. This work was supported by the FNRS, the IUAP program, the Fondation contre le Cancer, the ULB fondation, the foundation Bettencourt Schueller, the foundation Baillet Latour, a consolidator grant of the European Research Council.This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nature1906

Cell motion predicts human epidermal stemness

Image-based identification of cultured stem cells and noninvasive evaluation of their proliferative capacity advance cell therapy and stem cell research. Here we demonstrate that human keratinocyte stem cells can be identified in situ by analyzing cell motion during their cultivation. Modeling experiments suggested that the clonal type of cultured human clonogenic keratinocytes can be efficiently determined by analysis of early cell movement. Image analysis experiments demonstrated that keratinocyte stem cells indeed display a unique rotational movement that can be identified as early as the two-cell stage colony. We also demonstrate that α6 integrin is required for both rotational and collective cell motion. Our experiments provide, for the first time, strong evidence that cell motion and epidermal stemness are linked. We conclude that early identification of human keratinocyte stem cells by image analysis of cell movement is a valid parameter for quality control of cultured keratinocytes for transplantation

Identification of Lineage-Uncommitted, Long-Lived, Label-Retaining Cells in Healthy Human Esophagus and Stomach, and in Metaplastic Esophagus

Background & Aims The existence of slowly cycling, adult stem cells has been challenged by the identification of actively cycling cells. We investigated the existence of uncommitted, slowly cycling cells by tracking 5-iodo-2'-deoxyuridine (IdU) label-retaining cells (LRCs) in normal esophagus, Barrett's esophagus (BE), esophageal dysplasia, adenocarcinoma, and healthy stomach tissues from patients. Methods Four patients (3 undergoing esophagectomy, 1 undergoing esophageal endoscopic mucosal resection for dysplasia and an esophagectomy for esophageal adenocarcinoma) received intravenous infusion of IdU (200 mg/m2 body surface area; maximum dose, 400 mg) over a 30-minute period; the IdU had a circulation half-life of 8 hours. Tissues were collected at 7, 11, 29, and 67 days after infusion, from regions of healthy esophagus, BE, dysplasia, adenocarcinoma, and healthy stomach; they were analyzed by in situ hybridization, flow cytometry, and immunohistochemical analyses. Results No LRCs were found in dysplasias or adenocarcinomas, but there were significant numbers of LRCs in the base of glands from BE tissue, in the papillae of the basal layer of the esophageal squamous epithelium, and in the neck/isthmus region of healthy stomach. These cells cycled slowly because IdU was retained for at least 67 days and co-labeling with Ki-67 was infrequent. In glands from BE tissues, most cells did not express defensin-5, Muc-2, or chromogranin A, indicating that they were not lineage committed. Some cells labeled for endocrine markers and IdU at 67 days; these cells represented a small population (<0.1%) of epithelial cells at this time point. The epithelial turnover time of the healthy esophageal mucosa was approximately 11 days (twice that of the intestine). Conclusions LRCs of human esophagus and stomach have many features of stem cells (long lived, slow cycling, uncommitted, and multipotent), and can be found in a recognized stem cell niche. Further analyses of these cells, in healthy and metaplastic epithelia, is required

Mechanisms of stretch-mediated skin expansion at single-cell resolution.

The ability of the skin to grow in response to stretching has been exploited in reconstructive surgery1. Although the response of epidermal cells to stretching has been studied in vitro2,3, it remains unclear how mechanical forces affect their behaviour in vivo. Here we develop a mouse model in which the consequences of stretching on skin epidermis can be studied at single-cell resolution. Using a multidisciplinary approach that combines clonal analysis with quantitative modelling and single-cell RNA sequencing, we show that stretching induces skin expansion by creating a transient bias in the renewal activity of epidermal stem cells, while a second subpopulation of basal progenitors remains committed to differentiation. Transcriptional and chromatin profiling identifies how cell states and gene-regulatory networks are modulated by stretching. Using pharmacological inhibitors and mouse mutants, we define the step-by-step mechanisms that control stretch-mediated tissue expansion at single-cell resolution in vivo.Wellcome Trust Royal Societ