Lara Strittmatter

Arabidopsis Separase Functions beyond the Removal of Sister Chromatid Cohesion during Meiosis

Separase is a capase family protease that is required for the release of sister chromatid cohesion during meiosis and mitosis. Proteolytic cleavage of the α-kleisin subunit of the cohesin complex at the metaphase-to-anaphase transition is essential for the proper segregation of chromosomes. In addition to its highly conserved role in cleaving the α-kleisin subunit, separase appears to have acquired additional diverse activities in some organisms, including involvement in mitotic and meiotic anaphase spindle assembly and elongation, interphase spindle pole body positioning, and epithelial cell reorganization. Results from the characterization of Arabidopsis (Arabidopsis thaliana) separase (ESP) demonstrated that meiotic expression of ESP RNA interference blocked the proper removal of cohesin from chromosomes and resulted in the presence of a mixture of fragmented chromosomes and intact bivalents. The presence of large numbers of intact bivalents raised the possibility that separase may also have multiple roles in Arabidopsis. In this report, we show that meiotic expression of ESP RNA interference blocks the removal of cohesin during both meiosis I and II, results in alterations in nonhomologous centromere association, disrupts the radial microtubule system after telophase II, and affects the proper establishment of nuclear cytoplasmic domains, resulting in the formation of multinucleate microspores.

Comparative microsporangium development in male‐fertile and male‐sterile flowers of Consolea (Cactaceae): When and how does pollen abortion occur

Anther development, microsporogenesis, and microgametogenesis were studied using both light and TEM microscopy in the six accessible subdioecious/cryptically dioecious species of Consolea (Cactaceae). Anther wall development, microsporogenesis, and microgametogenesis are uniform in staminate flowers of all six species, and are typical for Cactaceae. Breakdown of microsporogenesis in male‐sterile anthers occurs early, at the onset of meiosis, and results in anthers bearing no pollen grains. The abortive process follows a common pattern in all investigated species. The tapetum is the first layer to deviate from normal male‐fertile anther development. Tapetal cells in male‐sterile anthers elongate at an early stage and have abundant rER with atypical configurations. Ultimately, the tapetum becomes hypertrophied and non‐functional. Male‐sterility in pistillate flowers appears to be directly related to these anomalies. In addition, other anther layers and tissues are affected, and normal patterns of programmed cell death (PCD) are disrupted. The relationship between these patterns and the pattern of PCD in normal male‐fertile anthers is discussed. We hypothesize a single origin for the cryptically dioecious/subdioecious breeding system of Consolea based on the uniformity of the anthers abortive processes in pistillate flowers.

Quantitative analysis of high-resolution, contrast-enhanced, cone-beam CT for the detection of intracranial in-stent hyperplasia

Background Intracranial in-stent hyperplasia is a stroke-associated complication that requires routine surveillance. Objective To compare the results of in vivo experiments to determine the accuracy and precision of in-stent hyperplasia measurements obtained with modified C-arm contrast-enhanced, cone-beam CT (CE-CBCT) imaging with those obtained by ‘gold standard’ histomorphometry. Additionally, to carry out clinical analyses comparing this CE-CBCT protocol with digital subtraction angiography (DSA). Methods A non-binned CE-CBCT protocol (VasoCT) was used that acquires x-ray images with a small field-of-view and applies a full-scale reconstruction algorithm providing high-resolution three-dimensional (3D) imaging with 100 µm isotropic voxels. In an vivo porcine model, VasoCT cross-sectional area measurements were compared with gold standard vessel histology. VasoCT and DSA were used to calculate in-stent stenosis in 23 imaging studies. Results Porcine VasoCT cross-sectional stent, lumen, and in-stent hyperplasia areas strongly correlated with histological measurements (r2=0.97, 0.93, 0.90; slope=1.14, 1.07, and 0.76, respectively; p<0.0001). Clinical VasoCT percentage stenosis correlated well with DSA percentage stenosis (r2=0.84; slope=0.76), and the two techniques were free of consistent bias (Bland–Altman, bias=3.29%; 95% CI −14.75% to 21.33%). An illustrative clinical case demonstrated the advantages of VasoCT, including 3D capability and non-invasive IV contrast administration, for detection of in-stent hyperplasia. Conclusions C-arm VasoCT is a high-resolution 3D capable imaging technique that has been validated in an animal model for measurement of in-stent tissue growth. Successful clinical implementation of the protocol was performed in a small case series.

Genetic link between renal birth defects and congenital heart disease

Structural birth defects in the kidney and urinary tract are observed in 0.5% of live births and are a major cause of end-stage renal disease, but their genetic aetiology is not well understood. Here we analyse 135 lines of mice identified in large-scale mouse mutagenesis screen and show that 29% of mutations causing congenital heart disease (CHD) also cause renal anomalies. The renal anomalies included duplex and multiplex kidneys, renal agenesis, hydronephrosis and cystic kidney disease. To assess the clinical relevance of these findings, we examined patients with CHD and observed a 30% co-occurrence of renal anomalies of a similar spectrum. Together, these findings demonstrate a common shared genetic aetiology for CHD and renal anomalies, indicating that CHD patients are at increased risk for complications from renal anomalies. This collection of mutant mouse models provides a resource for further studies to elucidate the developmental link between renal anomalies and CHD.

CFAP54 is required for proper ciliary motility and assembly of the central pair apparatus in mice.

Assembly of the C1d projection of the central microtubule pair apparatus in mammalian motile cilia requires the ciliary protein CFAP54. Loss of the C1d projection in mice lacking CFAP54 impairs ciliary motility and cilia-driven fluid flow and results in a primary ciliary dyskinesia phenotype.

In situ tissue engineering: endothelial growth patterns as a function of flow diverter design

Background Vascular remodeling in response to implantation of a tissue engineering scaffold such as a flow diverter (FD) leads to the cure of intracranial aneurysms. We hypothesize that the vascular response is dependent on FD design, and CD34+ progenitor cells play an important role in the endothelialization of the implant. Methods Sixteen rabbit aneurysms were randomly treated with two different single-layer braided FDs made of cobalt–chrome alloys. The FD-48 and FD-72 devices had 48 and 72 wires, respectively. Aneurysm occlusion rate was assessed during the final digital subtraction angiogram at 10, 20, 30, and 60 days (n=2 per device per time point). Implanted vessels were analyzed with scanning electron microscopy for tissue coverage, endothelialization, and immuno-gold labeling for CD34+ cells. Results Complete aneurysm occlusion rates were similar between the devices; however, complete or near complete occlusion was more frequently observed in aneurysms with neck ≤4.2 mm (p=0.008). Total tissue coverage at 10 days over the surface of the FD-48 and FD-72 devices was 56.4±11.6% and 76.6±3.6%, respectively. Endothelial cell growth over the surface was time-dependent for the FD-72 device (Spearmans r=0.86, p=0.013) but not for the FD-48 device (Spearmans r=−0.59, p=0.094). The endothelialization score was marginally correlated with the distance from the aneurysm neck for the FD-48 device (Spearmans r=1, p=0.083) but not for the FD-72 device (Spearmans r=0.8, p=0.33). CD34+ cells were present along the entirety of both devices at all time points. Conclusions This study gives preliminary evidence that temporal and spatial endothelialization is dependent on FD design. Circulating CD34+ progenitor cells contribute to endothelialization throughout the healing process.

Loss of Arf4 causes severe degeneration of the exocrine pancreas but not cystic kidney disease or retinal degeneration

Arf4 is proposed to be a critical regulator of membrane protein trafficking in early secretory pathway. More recently, Arf4 was also implicated in regulating ciliary trafficking, however, this has not been comprehensively tested in vivo. To directly address Arf4’s role in ciliary transport, we deleted Arf4 specifically in either rod photoreceptor cells, kidney, or globally during the early postnatal period. Arf4 deletion in photoreceptors did not cause protein mislocalization or retinal degeneration, as expected if Arf4 played a role in protein transport to the ciliary outer segment. Likewise, Arf4 deletion in kidney did not cause cystic disease, as expected if Arf4 were involved in general ciliary trafficking. In contrast, global Arf4 deletion in the early postnatal period resulted in growth restriction, severe pancreatic degeneration and early death. These findings are consistent with Arf4 playing a critical role in endomembrane trafficking, particularly in the pancreas, but not in ciliary function.

Erratum: Genetic link between renal birth defects and congenital heart disease.

Nature Communications 7 Article number:1110310.1038/ncomms11103 (2016); Published March222016; Updated June082016 In Fig. 6 of this Article, defects in the tectonic complex were inadvertently labelled during the production process as causing postnatal, rather than prenatal cysts. The correct version of the figure appears below. Figure 6

Quantitative In Vivo Evaluation of Neointimal Hyperplasia Using High-Resolution Contrast-Enhanced Cone-Beam Computed Tomography

Intracranial atherosclerotic disease (ICAD) is the most common cause of stroke throughout the world. Patients with severe arterial stenosis and a recent stroke or transient ischemic attack are at high risk of recurring stroke. In an attempt to improve the clinical outcome of patients with severe, symptomatic ICAD, percutaneous transluminal angioplasty and intracranial stenting have become treatment options over the last years. Follow-up imaging of patients with ICAD that underwent intracranial stenting is generally done using 2D digitally subtracted angiography. Since often the vessel path is curved and the neointimal hyperplasia (NH) is eccentric, in-stent restenosis (ISR) may be under- or overestimated using 2D techniques.Copyright

P-016 Quantitative Assessment of Stent Induced Neointimal Hyperplasia with Contrast Enhanced Cone-Beam CT: In Vivo Validation with Histomorphometry

Introduction Intracranial stenting is an effective therapy for specific cerebrovascular disorders including treatment-resistant atherosclerosis, cerebral aneurysms, and arterial dissections. However, in-stent tissue growth (neointimal hyperplasia (NIH) and/or in-stent restenosis (ISR)), is a significant long-term complication that necessitates routine surveillance. Catheter-based digital subtraction angiography, (DSA), is the current imaging standard for NIH/ISR detection; however, DSA is invasive and relies on 2D vascular representations that may over- or underestimate asymmetric tissue growth and consequentially, confuse clinical management decisions. A less invasive 3D capable, cross-sectional imaging technique with resolution to detect NIH/ISR, could circumvent these limitations, better inform clinicians, and improve patient care. Herein, contrast-enhanced C-arm Cone-Beam Computed Tomography, (CE-CBCT), recently optimised for high resolution 3D stent imaging by reducing the field-of-view during acquisition and performing full-scale reconstruction1, was quantitatively compared to vessel histology in a porcine model of in-stent NIH to validate the CE-CBCT approach. Materials and Methods All experiments were approved by our IACUC. The following was performed to model in-stent NIH: - 3 days, adult pig started on daily aspirin; day 0, pig anaesthetised, arterial access obtained, 4 arterial areas identified, damaged via a cutting balloon, and stented (Neuroform), pig recovered alive and returned to the animal facility; day 42, daily aspirin stopped; day 49, pig anaesthetised, CE-CBCT data acquired, animal sacrificed and perfused, stented vessels explanted, embedded in resin, sectioned and stained for analysis. Image J was used to quantify stent and luminal area from CE-CBCT and histological cross-sections that were spatially matched to best approximation; the measurements were compared with statistical software (Prism). Results Stent struts, lumen, and in-stent growth were clearly visualised and easily demarcated for quantitative analysis in both CE-CBCT and histological cross-sections (fig. 1a). CE-CBCT stent, lumen, and in-stent tissue growth calculated areas closely correlated with corresponding histological measurements (r2 = 0.96, 0.84, 0.87, respectively; fig. 1b). However, CE-CBCT was found to consistently overestimate lumen area relative to histology, which resulted in a lower Pearson’s r2 value and a non-zero intercept in the latter two measurements. Conclusion CE-CBCT quantification of in-stent tissue growth correlates well with histology in a porcine model and may be an important new clinical tool for post-stent vascular surveillance. Further evaluation of intravenous CE-CBCT as a non-invasive alternative to DSA in post-stent cerebrovascular patients is ongoing. Disclosures T. Flood: None. I. van der Bom: None. L. Strittmatter: None. G. Hendricks: None. A. Puri: None. A. Wakhloo: 1; C; Philips Healthcare. M. Gounis: 1; C; Philips Healthcare. References 1. Patel et al. AJNR 2011;32 (1):137–144. 2. Psychogios et al. Investigative Radiology;2013:48 (2);98–103. 3. Psychogios et al. AJNR 2010;31 (10):1886–91. Abstract P-016 Figure 1