Signaling pathways in cell models of Fabry disease nephropathy

Chronic Kidney Disease is a leading cause of morbidity, impaired quality of life and premature death in patients with Fabry disease, being of major public health significance. At the cellular level, besides within lysosomes, glycosphingolipids that accumulate in Fabry disease due to alpha-galactosidase A (α-gal A) deficiency localize to membrane microdomains, which play crucial roles in protein clustering, membrane trafficking, and especially cell signaling. The mechanisms by which increased levels of these glycosphingolipids and consequent changes in microdomain dynamics and lysosomal dysfunction all result in cellular and organ injury are not well understood. To effectively study Fabry disease disease mechanisms at the cellular level, I first established and characterized an epithelial kidney cell model of Fabry disease in Madin-Darby canine kidney (MDCK) cells using small interfering RNA (siRNA). I then examined protein dynamics at the plasma membrane of a model raft-associated protein, GFP-GPI, in this model system. Number and Brightness Analysis in live cells showed a significant increase in the oligomeric size of antibody-induced clusters in α-gal A silenced cells compared to control cells (5.08 ± 0.45 vs 2.74 ± 0.24, respectively). To explore possible consequences of these findings in signaling pathways that are relevant to human disease, I first generated human kidney cell models of Fabry disease in immortalized podocytes and tubule epithelial cells (HK-2) applying the genome editing technique of clustered, regularly interspaced, short palindromic repeats and associated endonuclease 9 from S. pyogenes (CRISPR/Cas9). I compared abundance and phosphorylation of relevant signaling proteins through a high-throughput phosphorylation profiling for Fabry disease and control immortalized human podocytes. Fabry disease podocytes showed significant changes in total protein abundance and/or phosphorylation in 59 proteins. Pathway analysis predicted differential signaling of several canonical pathways in Fabry disease podocytes. These studies provided for the first time an understanding of raft protein dynamics and signaling in kidney cells deficient for α-gal A, potentially opening new avenues for biomarker discovery and drug development for Fabry disease nephropathy

Low Threshold Two-Dimensional Annular Bragg Lasers

Lasing at telecommunication wavelengths from annular resonators employing radial Bragg reflectors is demonstrated at room temperature under pulsed optical pumping. Sub milliwatt pump threshold levels are observed for resonators with 0.5-1.5 wavelengths wide defects of radii 7-8 mm. The quality factors of the resonator modal fields are estimated to be on the order of a few thousands. The electromagnetic field is shown to be guided by the defect. Good agreement is found between the measured and calculated spectrum.Comment: 8 pages, 4 figure

Photonic crystals for light-emitting devices

Photonic crystals or photonic bandgap (PBG) structures promise to revolutionize optoelectronics by making anew class of highly efficient, low noise light emitters possible. We present data to show that their properties, in particular 2D systems, have now been fully characterized in the relevant semiconductor material system and at near-IR wavelengths, so effort can be redirected towards making active light emitters. As a first example, we present a semiconductor laser with one output mirror designed according to PBG principles. From threshold and efficiency data, we derive a reflectivity of 95 +/- 10 percent for this mirror, which underlines the viability of the PBG approach for practical devices. In order to realize the full potential of photonic crystal light emitters, however, important material issues need to be considered. Non- radiative recombination, for example, is a big problem when the photonic crystal is an integral part of the active region because of the relatively large areas of exposed surface. Several possible solutions to this problem are presented

Photonic crystals for light-emitting devices

Photonic crystals or photonic bandgap (PBG) structures promise to revolutionize optoelectronics by making anew class of highly efficient, low noise light emitters possible. We present data to show that their properties, in particular 2D systems, have now been fully characterized in the relevant semiconductor material system and at near-IR wavelengths, so effort can be redirected towards making active light emitters. As a first example, we present a semiconductor laser with one output mirror designed according to PBG principles. From threshold and efficiency data, we derive a reflectivity of 95 +/- 10 percent for this mirror, which underlines the viability of the PBG approach for practical devices. In order to realize the full potential of photonic crystal light emitters, however, important material issues need to be considered. Non- radiative recombination, for example, is a big problem when the photonic crystal is an integral part of the active region because of the relatively large areas of exposed surface. Several possible solutions to this problem are presented

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

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

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

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