35 research outputs found

    The Tatton-Brown-Rahman Syndrome: A clinical study of 55 individuals with de novo constitutive DNMT3A variants.

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    Tatton-Brown-Rahman syndrome (TBRS; OMIM 615879), also known as the DNMT3A-overgrowth syndrome, is an overgrowth intellectual disability syndrome first described in 2014 with a report of 13 individuals with constitutive heterozygous DNMT3A variants. Here we have undertaken a detailed clinical study of 55 individuals with de novoDNMT3A variants, including the 13 previously reported individuals. An intellectual disability and overgrowth were reported in >80% of individuals with TBRS and were designated major clinical associations. Additional frequent clinical associations (reported in 20-80% individuals) included an evolving facial appearance with low-set, heavy, horizontal eyebrows and prominent upper central incisors; joint hypermobility (74%); obesity (weight ³2SD, 67%); hypotonia (54%); behavioural/psychiatric issues (most frequently autistic spectrum disorder, 51%); kyphoscoliosis (33%) and afebrile seizures (22%). One individual was diagnosed with acute myeloid leukaemia in teenage years. Based upon the results from this study, we present our current management for individuals with TBRS

    VAMP7 modulates ciliary biogenesis in kidney cells

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    Epithelial cells elaborate specialized domains that have distinct protein and lipid compositions, including the apical and basolateral surfaces and primary cilia. Maintaining the identity of these domains is required for proper cell function, and requires the efficient and selective SNARE-mediated fusion of vesicles containing newly synthesized and recycling proteins with the proper target membrane. Multiple pathways exist to deliver newly synthesized proteins to the apical surface of kidney cells, and the post-Golgi SNAREs, or VAMPs, involved in these distinct pathways have not been identified. VAMP7 has been implicated in apical protein delivery in other cell types, and we hypothesized that this SNARE would have differential effects on the trafficking of apical proteins known to take distinct routes to the apical surface in kidney cells. VAMP7 expressed in polarized Madin Darby canine kidney cells colocalized primarily with LAMP2-positive compartments, and siRNA-mediated knockdown modulated lysosome size, consistent with the known function of VAMP7 in lysosomal delivery. Surprisingly, VAMP7 knockdown had no effect on apical delivery of numerous cargoes tested, but did decrease the length and frequency of primary cilia. Additionally, VAMP7 knockdown disrupted cystogenesis in cells grown in a three-dimensional basement membrane matrix. The effects of VAMP7 depletion on ciliogenesis and cystogenesis are not directly linked to the disruption of lysosomal function, as cilia lengths and cyst morphology were unaffected in an MDCK lysosomal storage disorder model. Together, our data suggest that VAMP7 plays an essential role in ciliogenesis and lumen formation. To our knowledge, this is the first study implicating an R-SNARE in ciliogenesis and cystogenesis. © 2014 Szalinski et al

    Using enhanced number and brightness to measure protein oligomerization dynamics in live cells

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    Protein dimerization and oligomerization are essential to most cellular functions, yet measurement of the size of these oligomers in live cells, especially when their size changes over time and space, remains a challenge. A commonly used approach for studying protein aggregates in cells is number and brightness (N&B), a fluorescence microscopy method that is capable of measuring the apparent average number of molecules and their oligomerization (brightness) in each pixel from a series of fluorescence microscopy images. We have recently expanded this approach in order to allow resampling of the raw data to resolve the statistical weighting of coexisting species within each pixel. This feature makes enhanced N&B (eN&B) optimal for capturing the temporal aspects of protein oligomerization when a distribution of oligomers shifts toward a larger central size over time. In this protocol, we demonstrate the application of eN&B by quantifying receptor clustering dynamics using electron-multiplying charge-coupled device (EMCCD)-based total internal reflection microscopy (TIRF) imaging. TIRF provides a superior signal-to-noise ratio, but we also provide guidelines for implementing eN&B in confocal microscopes. For each time point, eN&B requires the acquisition of 200 frames, and it takes a few seconds up to 2 min to complete a single time point. We provide an eN&B (and standard N&B) MATLAB software package amenable to any standard confocal or TIRF microscope. The software requires a high-RAM computer (64 Gb) to run and includes a photobleaching detrending algorithm, which allows extension of the live imaging for more than an hour

    Complete photonic bandgaps in 12-fold symmetric quasicrystals

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    Photonic crystals are attracting current interest for a variety of reasons, such as their ability to inhibit the spontaneous emission of light. This and related properties arise from the formation of photonic bandgaps, whereby multiple scattering of photons by lattices of periodically varying refractive indices acts to prevent the propagation of electromagnetic waves having certain wavelengths. One route to forming photonic crystals is to etch two-dimensional periodic lattices of vertical air holes into dielectric slab waveguides. Such structures can show complete photonic bandgaps, but only for large-diameter air holes in materials of high refractive index (such as gallium arsenide, n = 3.69), which unfortunately leads to significantly reduced optical transmission when combined with optical fibres of low refractive index. It has been suggested that quasicrystalline (rather than periodic) lattices can also possess photonic bandgaps. Here we demonstrate this concept experimentally and show that it enables complete photonic bandgaps—non-directional and for any polarization—to be realized with small air holes in silicon nitride (n = 2.02), and even glass (n = 1.45). These properties make photonic quasicrystals promising for application in a range of optical devices

    VAMP7 knockdown and overexpression alters lysosome volume.

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    <p>MDCK cells were transfected with cDNA encoding GFP-VAMP7 (A) or with control or VAMP7 siRNA (B) and processed for immunofluorescence to detect LAMP2 after four days. (C) Percent change +/− standard error of the mean in lysosome volume normalized to its respective control was quantitated using Imaris software as described in Methods. Compared to cells transfected with control siRNA, cells treated with VAMP7 siRNA had smaller LAMP2-positive structures compared to control. In contrast, VAMP7 overexpression (OE) led to an enlargement of LAMP2-positive structures. A total of ten stacks in two experiments were quantitated Scale bars = 10 µm; (* p<0.01 using two-sample t-test; **p<0.001 using paired t-test; see Methods for details.).</p

    Knockdown of VAMP7 has no effect on apical secretion of lipid-raft associated, raft-independent, and secreted cargo.

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    <p>(A) MDCK cells were transfected with control siRNA or siRNA targeting VAMP7, and knockdown after four days was assessed using RT-PCR. A representative gel is shown. We routinely observed approximately 80% knockdown using this oligonucleotide. (B) Apical delivery kinetics of the lipid raft associated protein HA in MDCK cells transfected with control or VAMP7 siRNA were quantitated using a cell surface trypsinization assay as described in Methods. Results from a representative experiment performed in duplicate are plotted (mean +/− range). Similar results were obtained in three experiments. (C) Apical delivery kinetics of the raft-independent protein endolyn were quantitated using a surface biotinylation assay as described in Methods. The results are representative (mean +/− range of duplicate samples) of two independent experiments. (D) Apical (AP) and basolateral (BL) secretion kinetics of Ensol were measured in control and VAMP7 knockdown cells as described in Methods. A representative experiment (mean +/− range of duplicate samples) is shown. Similar results were obtained in five experiments.</p

    VAMP7 knockdown decreases cilia frequency and cilia length in MDCK cells.

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    <p>(A) MDCK 2001 cells were transfected as indicated with control siRNA, our standard VAMP7 siRNA (VAMP7 #1), a less effective VAMP7 targeting sequence (VAMP7 #2), or VAMP8 siRNA. Cells were cultured for four days on permeable supports, then fixed and processed for indirect immunofluorescence to detect acetylated tubulin and cell nuclei. Representative fields acquired using epifluorescence microscopy are shown. Scale bar = 10 µm. (B, D) The number of cells in multiple fields was determined using DAPI nuclear stain and used to quantify the fraction of cells with a primary cilium in each sample. The average of three experiments is plotted (mean +/− SEM); *p<0.05 compared to control using one-way ANOVA with Bonferroni correction. (B) Control n = 1,907; VAMP7 n = 1,747; VAMP7 #2 n = 1,608 cells. (D) Control n = 1,040, VAMP7 n = 1,169, VAMP8 n = 1,049 cells. (C, E) Cilia lengths in multiple fields were measured using ImageJ software, then sorted by length in ascending order and graphed by percentile (100<sup>th</sup> percentile = longest cilium in each sample). Each point on the graph represents the length of an individual cilium. (C) There was a statistically significant reduction in cilia length in cells transfected with VAMP7 #1 and VAMP7 #2 siRNAs, as assessed by comparing the medians from four experiments (*p<0.05, **p<0.01 using one-way ANOVA with Bonferroni correction). (E) There was a statistically significant reduction in cilia length in VAMP7 depleted cells, as assessed by comparing the medians from three experiments (*p<0.05 using one-way ANOVA with Bonferroni correction).</p

    VAMP isoforms in MDCK cells.

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    <p>(A) mRNA isolated from MDCK cells and converted to cDNA was subjected to reverse transcription PCR using primers designed to detect VAMP isoforms. VAMP1 (218 bp expected product), VAMP2 (101 bp), VAMP3 (462 bp), VAMP4 (244 bp), VAMP5 (216 bp), VAMP7 (261 bp), and VAMP8 (298 bp) were detected. Actin (231 bp) was detected as an additional control to ensure amplification of mRNA and reverse transcriptase was excluded from the indicated sample as a negative control.</p
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