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The X-linked geneRlimplays major roles in female mouse development and reproduction, where it is crucial for the maintenance of imprinted X chromosome inactivation in extraembryonic tissues of embryos. However, while females carrying a systemicRlimknockout (KO) die around implantation, maleRlimKO mice appear healthy and are fertile. Here, we report an important role forRlimin testis where it is highly expressed in post-meiotic round spermatids as well as in Sertoli cells. Systemic deletion of theRlimgene results in lower numbers of mature sperm that contains excess cytoplasm, leading to decreased sperm motility and in vitro fertilization rates. Targeting the conditionalRlimcKO specifically to the spermatogenic cell lineage largely recapitulates this phenotype. These results reveal functions ofRlimin male reproduction specifically in round spermatids during spermiogenesis.

As organisms develop, individual cells generate mitochondria to fulfill physiological requirements. However, it remains unknown how mitochondrial network expansion is scaled to cell growth. The mitochondrial unfolded protein response (UPRmt) is a signaling pathway mediated by the transcription factor ATFS-1 which harbors a mitochondrial targeting sequence (MTS). Here, using the model organism Caenorhabditis elegans we demonstrate that ATFS-1 mediates an adaptable mitochondrial network expansion program that is active throughout normal development. Mitochondrial network expansion requires the relatively inefficient MTS in ATFS-1, which allows the transcription factor to be responsive to parameters that impact protein import capacity of the mitochondrial network. Increasing the strength of the ATFS-1 MTS impairs UPRmt activity by increasing accumulation within mitochondria. Manipulations of TORC1 activity increase or decrease ATFS-1 activity in a manner that correlates with protein synthesis. Lastly, expression of mitochondrial-targeted GFP is sufficient to expand the muscle cell mitochondrial network in an ATFS-1-dependent manner. We propose that mitochondrial network expansion during development is an emergent property of the synthesis of highly expressed mitochondrial proteins that exclude ATFS-1 from mitochondrial import, causing UPRmt activation., The mitochondrial network expands to accommodate cell growth, but how scaling occurs is unclear. Here, the authors show in C. elegans that ATFS-1 mitochondrial import is reduced when mitochondrial proteins are highly expressed, activating the unfolded protein response and causing expansion.

The X-linked gene Rlim plays major roles in female mouse development and reproduction, where it is crucial for the maintenance of imprinted X chromosome inactivation in extraembryonic tissues of embryos. However, while females carrying a systemic Rlim knockout (KO) die around implantation, male Rlim KO mice appear healthy and are fertile. Here, we report an important role for Rlim in testis where it is highly expressed in post-meiotic round spermatids as well as in Sertoli cells. Systemic deletion of the Rlim gene results in lower numbers of mature sperm that contains excess cytoplasm, leading to decreased sperm motility and in vitro fertilization rates. Targeting the conditional Rlim cKO specifically to the spermatogenic cell lineage largely recapitulates this phenotype. These results reveal functions of Rlim in male reproduction specifically in round spermatids during spermiogenesis.

SummaryThe X-linked geneRlimplays major roles in female mouse development and reproduction, where it is crucial for the maintenance of imprinted X chromosome inactivation in extraembryonic tissues of embryos. However, while females carrying a systemicRlimknockout (KO) die around implantation, maleRlimKO mice appear healthy and are fertile, raising questions as to the pressures drivingRlimgene selection during evolution. Here we report an important role forRlimin testis where it is highly expressed in post-meiotic round spermatids as well as in Sertoli cells. Systemic deletion of theRlimgene leads to lower numbers of mature sperm that contains excess cytoplasm, leading to decreased sperm motility andin vitrofertilization rates. Targeting the conditionalRlimcKO specifically to the spermatogenic cell lineage largely recapitulates this phenotype. These results reveal functions ofRlimin male reproduction specifically in round spermatids during spermiogenesis with likely evolutionary implications.

As organisms develop, individual cells generate mitochondria to fulfill physiologic requirements. However, it remains unknown how mitochondrial network expansion is scaled to cell growth and impacted by environmental cues. The mitochondrial unfolded protein response (UPRmt) is a signaling pathway mediated by the transcription factor ATFS-1 which harbors a mitochondrial targeting sequence (MTS)1. Here, we demonstrate that ATFS-1 mediates an adaptable mitochondrial expansion program that is active throughout normal development. Developmental mitochondrial network expansion required the relatively inefficient MTS2 in ATFS-1, which allowed the transcription factor to be responsive to parameters that impact protein import capacity of the entire mitochondrial network. Increasing the strength of the ATFS-1 MTS impaired UPRmt activity throughout development due to increased accumulation within mitochondria. The insulin-like signaling-TORC13 and AMPK pathways affected UPRmt activation4,5 in a manner that correlated with protein synthesis. Manipulation to increase protein synthesis caused UPRmt activation. Alternatively, S6 kinase inhibition had the opposite effect due to increased mitochondrial accumulation of ATFS-1. However, ATFS-1 with a dysfunctional MTS6 constitutively increased UPRmt activity independent of TORC1 function. Lastly, expression of a single protein with a strong MTS, was sufficient to expand the muscle cell mitochondrial network in an ATFS-1-dependent manner. We propose that mitochondrial network expansion during development is an emergent property of the synthesis of highly expressed mitochondrial proteins that exclude ATFS-1 from mitochondrial import, causing UPRmt activation. Mitochondrial network expansion is attenuated once ATFS-1 can be imported.

Argonaute proteins use nucleic acid guides to protect organisms against transposons and viruses. In the eubacterium Thermus thermophilus, the DNA-guided Argonaute TtAgo defends against transformation by DNA plasmids. Here, we report that TtAgo also participates in DNA replication. TtAgo binds small DNA guides derived from the chromosomal region where replication terminates and associates with proteins known to act in DNA replication. T. thermophilus deploys a single type II topoisomerase, gyrase. When gyrase is inhibited, T. thermophilus relies on TtAgo to complete replication of its circular genome; loss of both gyrase and TtAgo activity produces long filaments that fail to separate into individual bacteria. We propose that the primary role of TtAgo is to help T. thermophilus disentangle the catenated circular chromosomes made by DNA replication.One Sentence SummaryThe DNA-guided Argonaute protein of Thermus thermophilus helps separate daughter chromosomes at the end of DNA replication.

Introduction Advances in pump design make aspiration patterns programmable, producing an infinite combination of cyclical aspiration profiles for clot removal during treatment of acute ischemic stroke (AIS). However, little is known about how clots respond to different aspiration patterns. The goal of this study is to assess the structural and mechanical changes of the clot under aspiration. It is our hypothesis that a decreased fibrin pore size correlates to a higher aspiration efficacy. Materials and methods A fibrin-rich bovine clot was used to create an MCA occlusion in a patient-specific silicone model and underwent thromboaspiration via a 6F Sofia Plus catheter (MicroVention, Tustin, CA). Three sets of the retrieved clots were prepared for scanning electron microscopy (SEM) analysis, including clots lodged at the MCA without aspiration (control) as well as clots subjected to static (28 mmHg) or cyclic aspiration (amplitude: 18 to 27 mmHg) for 2 minutes (CLEARTM Aspiration System, Insera Therapeutics, LLC, Sacramento, CA). All the clot samples were fixed, dehydrated and frozen fractured. Surfaces of the fractured clots were imaged at 20 Kx at four different locations to obtain pore-size estimates of fibrin networks using ImageJ. Dynamic mechanical analysis (DMA) was conducted to understand the viscoelastic behavior of the clots obtained from patients with AIS under cyclic loading conditions. Results The fibrin-rich clot was corked in the distal tip of the aspiration catheter during clot retrieval. The average clot diameter measured proximally was 3.5 mm, 2.3 mm and 1.8 mm for the control, static and cyclic groups, respectively. Mechanical loading-unloading-reloading on the clot resulted in clot deformations; however, the changes in the clot dimension were less pronounced as the mechanical stimulus continued during the DMA tests. Clot deformation during aspiration was accompanied by a reduction in the average pore size in the fibrin-rich clot. The figure 1 demonstrates that the fibrin pore size measured from the proximal end of the clot was significantly reduced after aspiration when compared to the control clot (control: 857µm2, cyclic: 434µm2, static: 420 µm2). No significant differences in fibrin pore size were observed between the cyclic and static groups; however, it should be noted that the smallest pore size measurement was found in the cyclic group. Conclusion Applying cyclic or static aspiration to the fibrin-rich clot forms a denser fibrin mesh and results in clot stiffening. A comprehensive study is ongoing to study the structural changes of different clot models under aspiration at various frequencies and amplitudes. Disclosures M. Marosfoi: 2; C; Stryker Neurovascular and InNeuroCo Inc. L. Strittmatter: None. R. Arslanian: None. C. Raskett: None. M. Gounis: 1; C; the National Institutes of Health, Israel Binational Science Foundation, Anaconda, Cerenovus, Cook Medical, Gentuity, Imperative Care, InNeuroCo, Magneto, Microvention, Medtronic Neurovascular, MIVI Neurosciences, Neuravi, Neurogami, Philips Healthcare, Rapid Medical, Route 92 Medical, Stryker Neurovascular, Syntheon, and the Wyss Institute. 2; C; Cerenovus, Imperative Care, MIVI Neurosciences, phenox, Route 92 Medical, Stryker Neurovascular. 4; C; Imperative Care and Neurogami. A. Puri: 1; C; Medtronic Neurovascular and Stryker Neurovascular. 2; C; Medtronic Neurovascular and Stryker Neurovascular. F. Massari: None. K. De Macedo Rodrigues: None. J. Singh: None. J. Chueh: 2; C; Stryker Neurovascular and InNeuroCo Inc.

Summary In many eukaryotes, Argonaute proteins, guided by short RNA sequences, defend cells against transposons and viruses. In the eubacterium Thermus thermophilus, the DNA-guided Argonaute TtAgo defends against transformation by DNA plasmids. Here, we report that TtAgo also participates in DNA replication. In vivo, TtAgo binds 15- to 18-nt DNA guides derived from the chromosomal region where replication terminates and associates with proteins known to act in DNA replication. When gyrase, the sole T. thermophilus type II topoisomerase, is inhibited, TtAgo allows the bacterium to finish replicating its circular genome. In contrast, loss of gyrase and TtAgo activity slows growth and produces long sausage-like filaments in which the individual bacteria are linked by DNA. Finally, wild-type T. thermophilus outcompetes an otherwise isogenic strain lacking TtAgo. We propose that the primary role of TtAgo is to help T. thermophilus disentangle the catenated circular chromosomes generated by DNA replication.

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., Motile cilia and flagella play critical roles in fluid clearance and cell motility, and dysfunction commonly results in the pediatric syndrome primary ciliary dyskinesia (PCD). CFAP221, also known as PCDP1, is required for ciliary and flagellar function in mice and Chlamydomonas reinhardtii, where it localizes to the C1d projection of the central microtubule apparatus and functions in a complex that regulates flagellar motility in a calcium-dependent manner. We demonstrate that the genes encoding the mouse homologues of the other C. reinhardtii C1d complex members are primarily expressed in motile ciliated tissues, suggesting a conserved function in mammalian motile cilia. The requirement for one of these C1d complex members, CFAP54, was identified in a mouse line with a gene-trapped allele. Homozygous mice have PCD characterized by hydrocephalus, male infertility, and mucus accumulation. The infertility results from defects in spermatogenesis. Motile cilia have a structural defect in the C1d projection, indicating that the C1d assembly mechanism requires CFAP54. This structural defect results in decreased ciliary beat frequency and perturbed cilia-driven flow. This study identifies a critical role for CFAP54 in proper assembly and function of mammalian cilia and flagella and establishes the gene-trapped allele as a new model of PCD.

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

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

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.

Ovule development, megasporogenesis, and megagametogenesis were studied in six cryptically dioecious species of Consolea. All species showed uniform development typical for the Opuntioideae. Ovule development proceeds acropetally, but shows developmental asynchrony across floral morphs. At anthesis, female morph ovules are functional and available for fertilization, whereas staminate flower ovules are senescing and incapable of being fertilized. In occasional plants of some species, staminate flowers may reach anthesis with a few functional apical ovules capable of seed formation. Such plants are described as inconstant/leaky males. Ovule fertility differences across morphs are interpreted as resulting from heterochronic ovule development and senescence, although variation in embryo sac longevity cannot be ruled out. Significantly, ovule abortion follows a common pattern and timing in staminate flowers of both male morphs in all species. Thus, on the basis of this uniformity, a common origin for the cryptically dioecious breeding system in Consolea is hypothesized. Furthermore, staminate expression in Consolea appears to be controlled by a common, genetically determined heterochronic ovule developmental programme affecting the relative timing of ovule receptivity and flower opening. This is the first report of heterochrony as a mechanism of male sex determination. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 156, 305–326.

Summary α-kleisins are core components of meiotic and mitotic cohesin complexes. Arabidopsis contains genes for four α-kleisin proteins encoded by SYN genes. SYN1, a REC8 ortholog, is essential for meiosis, while SYN2 and SYN4 appear to be SCC1 orthologs and function in mitosis. Our analysis of AtSYN3 shows that it localizes primarily in the nucleolus of both meiotic and mitotic cells. Furthermore, analysis of plants containing an AtSYN3 T-DNA knockout mutation demonstrated that it is essential for megagametogenesis and plays an important role in pollen. These results suggest that SYN3 may not function as part of a typical cohesin complex; rather it may have evolved a specialized role in controlling rDNA structure, transcription or rRNA processing.

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., Using forward genetic screen in fetal mice, Gregory Pazour and colleagues describe mutants affecting kidney/urinary tract development. The authors also show that mutants that cause kidney defects overlaps with those leading to congenital heart defects, thus linking renal anomalies and congenital heart disease.

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...

The breeding system and the embryology of Consolea spinosissima, a tree-like opuntioid endemic to Jamaica, were investigated. Morphological and embryological studies revealed that the species is subdioecious, with three sexual morphs present in the 150 3 120 m plot studied at Hellshire Hills, Jamaica. The female morph has pistillate flowers with open stigma lobes, no pollen grains, and sets fruit. The male morph has cryptic staminate flowers with closed stigma lobes, viable pollen grains, and a nonfunctional gynoecium that does not set seed. The weak hermaphrodite morph has low fruit set and ‘‘perfect’’ flowers that superficially resemble the functionally staminate flowers of the male morph. These perfect flowers reach anthesis with viable pollen grains, with no or only a few functional ovules, and with the style supporting pollen tube growth. Embryological studies showed that the critical stage for sex determination occurs earlier in pistillate than in staminate and perfect flowers. Anthers of pistillate flowers abort prior to microspore tetrad formation, whereas ovules of the staminate and perfect flowers degenerate after the complete maturation of the embryo sac. Based on flower structure and embryological data, we hypothesize that the ancestor of C. spinosissima is/was hermaphroditic.

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., Author summary Primary cilia are sensory organelles found on most cells and contain specific receptors that detect extracellular stimuli. Defects in trafficking receptors to cilia cause a diverse set of diseases called ciliopathies, which include polycystic kidney disease, obesity, cerebral anomalies and retinal degeneration. Based mostly on in vitro studies, the small GTPase Arf4 was thought to be critically important for localizing rhodopsin to the outer segment of photoreceptor cells and cystoproteins to kidney cilia. Here we genetically remove Arf4 from mice in either a tissue specific or time dependent manner. To our surprise, the loss of Arf4 does not cause retinal degeneration or cystic kidney disease. Since ciliary dysfunction causes retinal degeneration and cystic disease, our findings indicate that Arf4 does not play a role in ciliary function. Instead, mice have zymogen granule defects and degeneration of the exocrine pancreas supporting roles for Arf4 in regulating endomembrane trafficking in specific cells.

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 © 2013 by ASME

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.