Retrograde Tracing with Recombinant Rabies Virus Reveals Correlations Between Projection Targets and Dendritic Architecture in Layer 5 of Mouse Barrel Cortex

A recombinant rabies virus was used as a retrograde tracer to allow complete filling of the axonal and dendritic arbors of identified projection neurons in layer 5 of mouse primary somatosensory cortex (S1) in vivo. Previous studies have distinguished three types of layer 5 pyramids in S1: tall-tufted, tall-simple, and short. Layer 5 pyramidal neurons were retrogradely labeled from several known targets: contralateral S1, superior colliculus, and thalamus. The complete dendritic arbors of labeled cells were reconstructed to allow for unambiguous classification of cell type. We confirmed that the tall-tufted pyramids project to the superior colliculus and thalamus and that short layer 5 pyramidal neurons project to contralateral cortex, as previously described. We found that tall-simple pyramidal neurons contribute to corticocortical connections. Axonal reconstructions show that corticocortical projection neurons have a large superficial axonal arborization locally, while the subcortically projecting neurons limit axonal arbors to the deep layers. Furthermore, reconstructions of local axons suggest that tall-simple cell axons have extensive lateral spread while those of the short pyramids are more columnar. These differences were revealed by the ability to completely label dendritic and axonal arbors in vivo and have not been apparent in previous studies using labeling in brain slices

The genetic regulation of sex-specific motorneurons by the doublesex gene in Drosophila melanogaster and the genetic characterization of an interaction with the sex determination hierarchy

The remodeling of the central nervous system (CNS) during metamorphosis in Drosophila melanogaster is a prime model system in which to study the genetic control of the sexual dimorphisms in the abdominal ganglion of the CNS. I have been using a P[tau-lacZ] enhancer trap line, 4.078, to label a segmentally repeated subset of abdominal motorneurons in order to assess the function of the sex determination hierarchy in controlling sex-specific development of the adult nervous system. In both the male and female larva there are 8 sets of these labeled abdominal motorneurons but only six sets in males and five sets in females survive in the adult. When this P[tau-lacZ] reporter construct is placed into a doublesex (dsx) mutant background, all 8 sets of these labeled abdominal motorneurons survive in both male and female adults. These results strongly suggest that dsx plays a role in the sex-specific survival of larval neurons that have functions in the adult. During the construction of mutant strains containing the sex determining genes transformer (tra) and transformer-2 (tra2), a genetic interactor was discovered in the P[tau-lacZ] 4.078 line. Female flies heterozygous for either tra or tra-2 alleles and the P[tau-lacZ] 4.078 developed with masculinized external and internal sex-specific structures. The external sex-specific structures, such as the genitalia, and ventral muscles are dependent on dsx gene function and a dorsal sex-specific muscle is dependent on fruitless (fru) gene function. From standard genetic crosses, I have characterized and demonstrated that the genetic interaction is linked to the P-element insertion site, which maps to the 85-87 region on the right arm of the third chromosome. By genetic analysis, this new genetic interactor appears to interfere with the tra and tra2 regulated female specific functions of both dsx and fru, potentially by reducing the female-specific splicing of the primary transcripts of the genes dsx and fru. To test the possibility that this newly described genetic interactor was allelic to a known gene, B52, that maps to the same region of the chromosome and alters dsx splicing, complementation tests were conducted which showed that the P[tau-lacZ] is not allelic B52. Additional phenotypes were observed in the crosses that first detected the interaction, suggesting that this newly described locus may affect other gene functions as well. Among the phenotypes observed were XX intersexes, male-female gynandromorphs (XX//XO mosaics), and non-disjunction events evident as XO males and XXY females. This new locus may represent a new member of the family of genes that influence regulated splicing events

Primary cilia respond to fluid shear stress and mediate flow-induced calcium deposition in osteoblasts

Bone turnover in vivo is regulated by mechanical forces such as shear stress originating from interstitial oscillatory fluid flow (OFF), and bone cells in vitro respond to mechanical loading. However, the mechanisms by which bone cells sense mechanical forces, resulting in increased mineral deposition, are not well understood. The aim of this study was to investigate the role of the primary cilium in mechanosensing by osteoblasts. MLO-A5 murine osteoblasts were cultured in monolayer and subjected to two different OFF regimens: 5 short (2 h daily) bouts of OFF followed by morphological analysis of primary cilia; or exposure to chloral hydrate to damage or remove primary cilia and 2 short bouts (2 h on consecutive days) of OFF. Primary cilia were shorter and there were fewer cilia per cell after exposure to periods of OFF compared with static controls. Damage or removal of primary cilia inhibited OFF-induced PGE2 release into the medium and mineral deposition, assayed by Alizarin red staining. We conclude that primary cilia are important mediators of OFF-induced mineral deposition, which has relevance for the design of bone tissue engineering strategies and may inform clinical treatments of bone disorders causes by load-deficiency.—Delaine-Smith, R. M., Sittichokechaiwut, A., Reilly, G. C. Primary cilia respond to fluid shear stress and mediate flow-induced calcium deposition in osteoblasts

RORα Coordinates Reciprocal Signaling in Cerebellar Development through Sonic hedgehog and Calcium-Dependent Pathways

AbstractThe cerebellum provides an excellent system for understanding how afferent and target neurons coordinate sequential intercellular signals and cell-autonomous genetic programs in development. Mutations in the orphan nuclear receptor RORα block Purkinje cell differentiation with a secondary loss of afferent granule cells. We show that early transcriptional targets of RORα include both mitogenic signals for afferent progenitors and signal transduction genes required to process their subsequent synaptic input. RORα acts through recruitment of gene-specific sets of transcriptional cofactors, including β-catenin, p300, and Tip60, but appears independent of CBP. One target promoter is Sonic hedgehog, and recombinant Sonic hedgehog restores granule precursor proliferation in RORα-deficient cerebellum. Our results suggest a link between RORα and β-catenin pathways, confirm that a nuclear receptor employs distinct coactivator complexes at different target genes, and provide a logic for early RORα expression in coordinating expression of genes required for reciprocal signals in cerebellar development

Preconditioning of mesenchymal stromal cells with low-intensity ultrasound: influence on chondrogenesis and directed SOX9 signaling pathways

Background: Continuous low-intensity ultrasound (cLIUS) facilitates the chondrogenic differentiation of human mesenchymal stromal cells (MSCs) in the absence of exogenously added transforming growth factor-beta (TGFβ) by upregulating the expression of transcription factor SOX9, a master regulator of chondrogenesis. The present study evaluated the molecular events associated with the signaling pathways impacting SOX9 gene and protein expression under cLIUS. Methods: Human bone marrow-derived MSCs were exposed to cLIUS stimulation at 14 kPa (5 MHz, 2.5 Vpp) for 5 min. The gene and protein expression of SOX9 was evaluated. The specificity of SOX9 upregulation under cLIUS was determined by treating the MSCs with small molecule inhibitors of select signaling molecules, followed by cLIUS treatment. Signaling events regulating SOX9 expression under cLIUS were analyzed by gene expression, immunofluorescence staining, and western blotting. Results: cLIUS upregulated the gene expression of SOX9 and enhanced the nuclear localization of SOX9 protein when compared to non-cLIUS-stimulated control. cLIUS was noted to enhance the phosphorylation of the signaling molecule ERK1/2. Inhibition of MEK/ERK1/2 by PD98059 resulted in the effective abrogation of cLIUS-induced SOX9 expression, indicating that cLIUS-induced SOX9 upregulation was dependent on the phosphorylation of ERK1/2. Inhibition of integrin and TRPV4, the upstream cell-surface effectors of ERK1/2, did not inhibit the phosphorylation of ERK1/2 and therefore did not abrogate cLIUS-induced SOX9 expression, thereby suggesting the involvement of other mechanoreceptors. Consequently, the effect of cLIUS on the actin cytoskeleton, a mechanosensitive receptor regulating SOX9, was evaluated. Diffused and disrupted actin fibers observed in MSCs under cLIUS closely resembled actin disruption by treatment with cytoskeletal drug Y27632, which is known to increase the gene expression of SOX9. The upregulation of SOX9 under cLIUS was, therefore, related to cLIUS-induced actin reorganization. SOX9 upregulation induced by actin reorganization was also found to be dependent on the phosphorylation of ERK1/2. Conclusions: Collectively, preconditioning of MSCs by cLIUS resulted in the nuclear localization of SOX9, phosphorylation of ERK1/2 and disruption of actin filaments, and the expression of SOX9 was dependent on the phosphorylation of ERK1/2 under cLIUS

Deconstruction of a Metastatic Tumor Microenvironment Reveals a Common Matrix Response in Human Cancers.

We have profiled, for the first time, an evolving human metastatic microenvironment by measuring gene expression, matrisome proteomics, cytokine and chemokine levels, cellularity, extracellular matrix organization, and biomechanical properties, all on the same sample. Using biopsies of high-grade serous ovarian cancer metastases that ranged from minimal to extensive disease, we show how nonmalignant cell densities and cytokine networks evolve with disease progression. Multivariate integration of the different components allowed us to define, for the first time, gene and protein profiles that predict extent of disease and tissue stiffness, while also revealing the complexity and dynamic nature of matrisome remodeling during development of metastases. Although we studied a single metastatic site from one human malignancy, a pattern of expression of 22 matrisome genes distinguished patients with a shorter overall survival in ovarian and 12 other primary solid cancers, suggesting that there may be a common matrix response to human cancer.Significance: Conducting multilevel analysis with data integration on biopsies with a range of disease involvement identifies important features of the evolving tumor microenvironment. The data suggest that despite the large spectrum of genomic alterations, some human malignancies may have a common and potentially targetable matrix response that influences the course of disease. Cancer Discov; 8(3); 304-19. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 253

Archaeology, Architecture, and the Postcolonial Critique

Peer reviewe

BMJ Open

PURPOSE: The currently ongoing Epidemiological Strategy and Medical Economics (ESME) research programme aims at centralising real-life data on oncology care for epidemiological research purposes. We draw on results from the metastatic breast cancer (MBC) cohort to illustrate the methodology used for data collection in the ESME research programme. PARTICIPANTS: All consecutive >/=18 years patients with MBC treatment initiated between 2008 and 2014 in one of the 18 French Comprehensive Cancer Centres were selected. Diagnostic, therapeutic and follow-up data (demographics, primary tumour, metastatic disease, treatment patterns and vital status) were collected through the course of the disease. Data collection is updated annually. FINDING TO DATE: With a recruitment target of 30 000 patients with MBC by 2019, we currently screened a total of 45 329 patients, and >16 700 patients with a metastatic disease treatment initiated after 2008 have been selected. 20.7% of patients had an hormone receptor (HR)-negative MBC, 73.7% had a HER2-negative MBC and 13.9% were classified as triple-negative BC (ie, HER2 and HR status both negative). Median follow-up duration from MBC diagnosis was 48.55 months for the whole cohort. FUTURE PLANS: These real-world data will help standardise the management of MBC and improve patient care. A dozen of ancillary research projects have been conducted and some of them are already accepted for publication or ready to be issued. The ESME research programme is expanding to ovarian cancer and advanced/metastatic lung cancer. Our ultimate goal is to achieve a continuous link to the data of the cohort to the French national Health Data System for centralising data on healthcare reimbursement (drugs, medical procedures), inpatient/outpatient stays and visits in primary/secondary care settings. TRIAL REGISTRATION NUMBER: NCT03275311; Pre-results