The Dynamics and Regulation of Mesenchymal Cell Fusion in the Sea Urchin Embryo

AbstractCell–cell fusion occurs in a wide variety of developmental contexts, yet the mechanisms involved are just beginning to be elucidated. In the sea urchin embryo, primary mesenchyme cells (PMCs) fuse to form syncytial filopodial cables within which skeletal spicules are deposited. Taking advantage of the optical transparency and ease of micromanipulation of sea urchin embryos, we have developed methods for directly observing the dynamics of PMC fusionin vivo.A fraction of the PMCs was labeled with fluorescent dextran and transfer of the dye to unlabeled PMCs was followed by time-lapse, fluorescence microscopy. Fusion was first detected about 2 h after PMCs began to migrate within the blastocoel. Fusion proceeded in parallel with the assembly of the PMC ring pattern and was complete by the early gastrula stage. The formation of a single, extensive PMC syncytium was confirmed by DiI labeling of fixed embryos. When single micromeres were isolated and cultured in unsupplemented seawater, they divided and their progeny underwent fusion. This shows that the capacity to fuse is autonomously programmed in the micromere–PMC lineage by the 16-cell stage. PMC transplantations at late embryonic stages revealed that these cells remain fusion-competent long after their fusion is complete. At late stages, other mesenchyme cells (blastocoelar cells) are also present within the blastocoel and are migrating and fusing with one another. Fusion-competent blastocoelar cells and PMCs come into contact but do not fuse with one another, indicating that these two cell types fuse by distinct mechanisms. When secondary mesenchyme cells convert to a skeletogenic fate they alter their fusogenic properties and join the PMC syncytium, as shown by transfer of fluorescent dextran. Our analysis has provided a detailed picture of the cellular basis and regulation of mesodermal cell fusion and has important implications regarding molecular mechanisms that underlie fusion

Molecular characterization of microbiota in cerebrospinal fluid from patients with CSF shunt infections using whole genome amplification followed by shotgun sequencing

Understanding the etiology of cerebrospinal fluid (CSF) shunt infections and reinfections requires detailed characterization of associated microorganisms. Traditionally, identification of bacteria present in the CSF has relied on culture methods, but recent studies have used high throughput sequencing of 16S rRNA genes. Here we evaluated the method of shotgun DNA sequencing for its potential to provide additional genomic information. CSF samples were collected from 3 patients near the beginning and end of each of 2 infection episodes. Extracted total DNA was sequenced by: (1) whole genome amplification followed by shotgun sequencing (WGA) and (2) high-throughput sequencing of the 16S rRNA V4 region (16S). Taxonomic assignments of sequences from WGA and 16S were compared with one another and with conventional microbiological cultures. While classification of bacteria was consistent among the 3 approaches, WGA provided additional insights into sample microbiological composition, such as showing relative abundances of microbial versus human DNA, identifying samples of questionable quality, and detecting significant viral load in some samples. One sample yielded sufficient non-human reads to allow assembly of a high-qualit

Lower levels of Th1 and Th2 cytokines in cerebrospinal fluid (CSF) at the time of initial CSF shunt placement in children are associated with subsequent shunt revision surgeries

OBJECTIVE: We compare cytokine profiles at the time of initial CSF shunt placement between children who required no subsequent shunt revision surgeries and children requiring repeated CSF shunt revision surgeries for CSF shunt failure. We also describe the cytokine profiles across surgical episodes for children who undergo multiple subsequent revision surgeries. METHODS: This pilot study was nested within an ongoing prospective multicenter study collecting CSF samples and clinical data at the time of CSF shunt surgeries since August 2014. We selected cases where CSF was available for children who underwent an initial CSF shunt placement and had no subsequent shunt revision surgeries during \u3e=24 months of follow-up (n = 7); as well as children who underwent an initial CSF shunt placement and then required repeated CSF shunt revision surgeries (n = 3). Levels of 92 human cytokines were measured using the Olink immunoassay and 41 human cytokines were measured using Luminex based bead array on CSF obtained at the time of each child\u27s initial CSF shunt placement and were displayed in heat maps. RESULTS: Qualitatively similar profiles for the majority of cytokines were observed among the patients in each group in both Olink and Luminex assays. Lower levels of MCP-3, CASP-8, CD5, CXCL9, CXCL11, eotaxin, IFN-γ, IL-13, IP-10, and OSM at the time of initial surgery were noted in the children who went on to require multiple surgeries. Pro- and anti-inflammatory cytokines were selected a priori and shown across subsequent revision surgeries for the 3 patients. Cytokine patterns differed between patients, but within a given patient pro-inflammatory and anti-inflammatory cytokines acted in a parallel fashion, with the exception of IL-4. CONCLUSIONS: Heat maps of cytokine levels at the time of initial CSF shunt placement for each child undergoing only a single initial CSF shunt placement and for each child undergoing repeat CSF shunt revision surgeries demonstrated qualitatively similar profiles for the majority of cytokines. Lower levels of MCP-3, CASP-8, CD5, CXCL9, CXCL11, eotaxin, IFN-γ, IL-13, IP-10, and OSM at the time of initial surgery were noted in the children who went on to require multiple surgeries. Better stratification by patient age, etiology, and mechanism of failure is needed to develop a deeper understanding of the mechanism of inflammation in the development of hydrocephalus and response to shunting in children

Mesenchymal cell fusion in the sea urchin embryo.

Mesenchymal cells of the sea urchin embryo provide a valuable experimental model for the analysis of cell-cell fusion in vivo. The unsurpassed optical transparency of the sea urchin embryo facilitates analysis of cell fusion in vivo using fluorescent markers and time-lapse three-dimensional imaging. Two populations of mesodermal cells engage in homotypic cell-cell fusion during gastrulation: primary mesenchyme cells and blastocoelar cells. In this chapter, we describe methods for studying the dynamics of cell fusion in living embryos. These methods have been used to analyze the fusion of primary mesenchyme cells and are also applicable to blastocoelar cell fusion. Although the molecular basis of cell fusion in the sea urchin has not been investigated, tools have recently become available that highlight the potential of this experimental model for integrating dynamic morphogenetic behaviors with underlying molecular mechanisms.</p

Characterization of cerebrospinal fluid (CSF) microbiota from patients with CSF shunt infection and reinfection using high throughput sequencing of 16S ribosomal RNAgenes.

BackgroundNearly 20% of patients with cerebrospinal fluid (CSF) shunt infection develop reinfection. It is unclear whether reinfections are caused by an organism previously present or are independent infection events.ObjectiveWe used bacterial culture and high throughput sequencing (HTS) of 16S ribosomal RNA (rRNA) genes to identify bacteria present in serial CSF samples obtained from children who failed CSF shunt infection treatment. We hypothesized that organisms that persist in CSF despite treatment would be detected upon reinfection.Design/methodsSerial CSF samples were obtained from 6 patients, 5 with 2 infections and 1 with 3 infections; the study was limited to those for which CSF samples were available from the end of infection and beginning of reinfection. Amplicons of the 16S rRNA gene V4 region were sequenced. Taxonomic assignments of V4 sequences were compared with bacterial species identified in culture.ResultsSeven infection dyads averaging 13.5 samples per infection were analyzed. A median of 8 taxa [interquartile range (IQR) 5-10] were observed in the first samples from reinfection using HTS. Conventional culture correlated with high abundance of an organism by HTS in all but 1 infection. In 6 of 7 infection dyads, organisms identified by culture at reinfection were detected by HTS of culture-negative samples at the end of the previous infection. The median Chao-Jaccard abundance-based similarity index for matched infection pairs at end of infection and beginning of reinfection was 0.57 (IQR 0.07-0.87) compared to that for unmatched pairs of 0.40 (IQR 0.10-0.60) [p = 0.46].Conclusion(s)HTS results were generally consistent with culture-based methods in CSF shunt infection and reinfection, and may detect organisms missed by culture at the end of infection treatment but detected by culture at reinfection. However, the CSF microbiota did not correlate more closely within patients at the end of infection and beginning of reinfection than between any two unrelated infections. We cannot reject the hypothesis that sequential infections were independent