In vivo imaging and quantitative analysis of leukocyte directional migration and polarization in inflamed tissue

Directional migration of transmigrated leukocytes to the site of injury is a central event in the inflammatory response. Here, we present an in vivo chemotaxis assay enabling the visualization and quantitative analysis of subtype-specific directional motility and polarization of leukocytes in their natural 3D microenvironment. Our technique comprises the combination of i) semi-automated in situ microinjection of chemoattractants or bacteria as local chemotactic stimulus, ii) in vivo near-infrared reflected-light oblique transillumination (RLOT) microscopy for the visualization of leukocyte motility and morphology, and iii) in vivo fluorescence microscopy for the visualization of different leukocyte subpopulations or fluorescence-labeled bacteria. Leukocyte motility parameters are quantified off-line in digitized video sequences using computer-assisted single cell tracking. Here, we show that perivenular microinjection of chemoattractants [macrophage inflammatory protein-1alpha (MIP-1alpha/Ccl3), platelet-activating factor (PAF)] or E. coli into the murine cremaster muscle induces target-oriented intravascular adhesion and transmigration as well as polarization and directional interstitial migration of leukocytes towards the locally administered stimuli. Moreover, we describe a crucial role of Rho kinase for the regulation of directional motility and polarization of transmigrated leukocytes in vivo. Finally, combining in vivo RLOT and fluorescence microscopy in Cx3CR1(gfp/gfp) mice (mice exhibiting green fluorescent protein-labeled monocytes), we are able to demonstrate differences in the migratory behavior of monocytes and neutrophils.Taken together, we propose a novel approach for investigating the mechanisms and spatiotemporal dynamics of subtype-specific motility and polarization of leukocytes during their directional interstitial migration in vivo

Reliable assessment of telomere maintenance mechanisms in neuroblastoma

BACKGROUND: Telomere maintenance mechanisms (TMM) are a hallmark of high-risk neuroblastoma, and are conferred by activation of telomerase or alternative lengthening of telomeres (ALT). However, detection of TMM is not yet part of the clinical routine, and consensus on TMM detection, especially on ALT assessment, remains to be achieved. METHODS: Whole genome sequencing (WGS) data of 68 primary neuroblastoma samples were analyzed. Telomere length was calculated from WGS data or by telomere restriction fragment analysis (n = 39). ALT was assessed by C-circle assay (CCA, n = 67) and detection of ALT-associated PML nuclear bodies (APB) by combined fluorescence in situ hybridization and immunofluorescence staining (n = 68). RNA sequencing was performed (n = 64) to determine expression of TERT and telomeric long non-coding RNA (TERRA). Telomerase activity was examined by telomerase repeat amplification protocol (TRAP, n = 15). RESULTS: Tumors were considered as telomerase-positive if they harbored a TERT rearrangement, MYCN amplification or high TERT expression (45.6%, 31/68), and ALT-positive if they were positive for APB and CCA (19.1%, 13/68). If all these markers were absent, tumors were considered TMM-negative (25.0%, 17/68). According to these criteria, the majority of samples were classified unambiguously (89.7%, 61/68). Assessment of additional ALT-associated parameters clarified the TMM status of the remaining seven cases with high likelihood: ALT-positive tumors had higher TERRA expression, longer telomeres, more telomere insertions, a characteristic pattern of telomere variant repeats, and were associated with ATRX mutations. CONCLUSIONS: We here propose a workflow to reliably detect TMM in neuroblastoma. We show that unambiguous classification is feasible following a stepwise approach that determines both, activation of telomerase and ALT. The workflow proposed in this study can be used in clinical routine and provides a framework to systematically and reliably determine telomere maintenance mechanisms for risk stratification and treatment allocation of neuroblastoma patients

Characterisation of proteins in excretory/secretory products collected from salmon lice, Lepeophtheirus salmonis

Background  The salmon louse, Lepeophtheirus salmonis, is an ectoparasitic copepod which feeds on the mucus, skin and blood of salmonid fish species. The parasite can persist on the surface of the fish without any effective control being exerted by the host immune system. Other ectoparasitic invertebrates produce compounds in their saliva, excretions and/or secretions which modulate the host immune responses allowing them to remain on or in the host during development. Similarly, compounds are produced in secretions of L. salmonis which are thought to be responsible for immunomodulation of the host responses as well as other aspects of crucial host-parasite interactions.  Methods  In this study we have identified and characterised the proteins in the excretory/secretory (E/S) products of L. salmonis using LC-ESI-MS/MS.  Results  In total 187 individual proteins were identified in the E/S collected from adult lice and pre-adult sea lice. Fifty-three proteins, including 13 serine-type endopeptidases, 1 peroxidase and 5 vitellogenin-like proteins were common to both adult and pre-adult E/S products. One hundred and seven proteins were identified in the adult E/S but not in the pre-adult E/S and these included serine and cysteine-type endopeptidases, vitellogenins, sphingomyelinase and calreticulin. A total of 27 proteins were identified in pre-adult E/S products but not in adult E/S.  Conclusions  The assigned functions of these E/S products and the potential roles they play in host-parasite interaction is discussed

Two-photon microscopy analysis of leukocyte trafficking and motility

During the last several years, live tissue imaging, in particular using two-photon laser microscopy, has advanced our understanding of leukocyte trafficking mechanisms. Studies using this technique are revealing distinct molecular requirements for leukocyte migration in different tissue environments. Also emerging from the studies are the ingenious infrastructures for leukocyte trafficking, which are produced by stromal cells. This review summarizes the recent imaging studies that provided novel mechanistic insights into in vivo leukocyte migration essential for immunosurveillance

Dynamically Integrating Knowledge in Teams: Transforming Resources into Performance

In knowledge-based environments, teams must develop a systematic approach to integrating knowledge resources throughout the course of projects in order to perform effectively. Yet, many teams fail to do so. Drawing on the resource-based view of the firm, we examine how teams can develop a knowledge-integration capability to dynamically integrate members‘ resources into higher performance. We distinguish among three sets of resources: relational, experiential, and structural, and propose that they differentially influence a team‘s knowledge-integration capability. We test our theoretical framework using data on knowledge workers in professional services, and discuss implications for research and practice

A phylogenetically conserved APETALA2/ETHYLENE RESPONSE FACTOR, ERF12, regulates Arabidopsis floral development

Key message Arabidopsis ETHYLENE RESPONSE FACTOR12 (ERF12), the rice MULTIFLORET SPIKELET1 orthologue pleiotropically affects meristem identity, floral phyllotaxy and organ initiation and is conserved among angiosperms. Reproductive development necessitates the coordinated regulation of meristem identity and maturation and lateral organ initiation via positive and negative regulators and network integrators. We have identified ETHYLENE RESPONSE FACTOR12 (ERF12) as the Arabidopsis orthologue of MULTIFLORET SPIKELET1 (MFS1) in rice. Loss of ERF12 function pleiotropically affects reproductive development, including defective floral phyllotaxy and increased floral organ merosity, especially supernumerary sepals, at incomplete penetrance in the first-formed flowers. Wildtype floral organ number in early formed flowers is labile, demonstrating that floral meristem maturation involves the stabilisation of positional information for organogenesis, as well as appropriate identity. A subset of erf12 phenotypes partly defines a narrow developmental time window, suggesting that ERF12 functions heterochronically to fine-tune stochastic variation in wild type floral number and similar to MFS1, promotes meristem identity. ERF12 expression encircles incipient floral primordia in the inflorescence meristem periphery and is strong throughout the floral meristem and intersepal regions. ERF12 is a putative transcriptional repressor and genetically opposes the function of its relatives DORNRoSCHEN, DORNRoSCHEN-LIKE and PUCHI and converges with the APETALA2 pathway. Phylogenetic analysis suggests that ERF12 is conserved among all eudicots and appeared in angiosperm evolution concomitant with the generation of floral diversity

DORNROSCHEN, DORNROSCHEN-LIKE, and PUCHI redundantly control floral meristem identity and organ initiation in Arabidopsis

The biphasic floral transition in Arabidopsis thaliana involves many redundant intersecting regulatory networks. The related AP2 transcription factors DORNROSCHEN (DRN), DORNROSCHEN-LIKE (DRNL), and PUCHI individually execute well-characterized functions in diverse developmental contexts, including floral development. Here, we show that their combined loss of function leads to synergistic floral phenotypes, including reduced floral merosity in all whorls, which reflects redundant functions of all three genes in organ initiation rather than outgrowth. Additional loss of BLADE-ON-PETIOLE1 (BOP1) and BOP2 functions results in the complete conversion of floral meristems into secondary inflorescence shoots, demonstrating that all five genes define an essential regulatory network for establishing floral meristem identity, and we show that their functions converge to regulate LEAFY expression. Thus, despite their largely discrete spatiotemporal expression domains in the inflorescence meristem and early floral meristem, PUCHI, DRN, and DRNL interdependently contribute to cellular fate decisions. Auxin might represent one potential non-cell-autonomous mediator of their gene functions, because PUCHI, DRN, and DRNL all interact with auxin transport and biosynthesis pathways