Bidirectional multiciliated cell extrusion is controlled by Notch-driven basal extrusion and Piezo1-driven apical extrusion.

Xenopus embryos are covered with a complex epithelium containing numerous multiciliated cells (MCCs). During late-stage development, there is a dramatic remodeling of the epithelium that involves the complete loss of MCCs. Cell extrusion is a well-characterized process for driving cell loss while maintaining epithelial barrier function. Normal cell extrusion is typically unidirectional, whereas bidirectional extrusion is often associated with disease (e.g. cancer). We describe two distinct mechanisms for MCC extrusion, a basal extrusion driven by Notch signaling and an apical extrusion driven by Piezo1. Early in the process there is a strong bias towards basal extrusion, but as development continues there is a shift towards apical extrusion. Importantly, response to the Notch signal is age dependent and governed by the maintenance of the MCC transcriptional program such that extension of this program is protective against cell loss. In contrast, later apical extrusion is regulated by Piezo1, such that premature activation of Piezo1 leads to early extrusion while blocking Piezo1 leads to MCC maintenance. Distinct mechanisms for MCC loss underlie the importance of their removal during epithelial remodeling.

A role for Cep70 in centriole amplification in multiciliated cells.

Centriole amplification in multiciliated cells occurs in a pseudo-cell cycle regulated process that typically utilizes a poorly characterized molecularly dense structure called the deuterosome. We identified the centrosomal protein Cep70 as a novel deuterosome-associated protein that forms a complex with other deuterosome proteins, CCDC78 and Deup1. Cep70 dynamically associates with deuterosomes during centriole amplification in the ciliated epithelia of Xenopus embryos. Cep70 is not found in nascent deuterosomes prior to amplification. However, it becomes localized at deuterosomes at the onset of centriole biogenesis and remains there after the completion of centriole amplification. Deuterosome localization requires a conserved C-terminal "Cep70" motif. Depletion of Cep70 using morpholino oligos or CRISPR/Cas9 editing in F0 embryos leads to a severe decrease in centriole formation in both endogenous MCCs, as well as ectopically induced MCCs. Consistent with a decrease in centrioles, endogenous MCCs have defects in the process of radial intercalation. We propose that Cep70 represents a novel regulator of centriole biogenesis in MCCs.

Integrating biopharmaceutics risk assessment and in vivo absorption model in formulation development of BCS class I drug using the QbD approach.

OBJECTIVE: Clinically relevant critical quality attributes (CQA's) were identified for the development of generic drug products containing fluconazole and potential design spaces relevant to the clinical application of the drug candidate was explored. SIGNIFICANCE: A simplified scoring system for the biopharmaceutics risk assessment roadmap (BioRAM) is proposed to guide product development. METHODS: Factorial design of experiments was employed to study the effect of formulation and process variables on CQA's. The in vivo model was developed for predicting the fraction of drug absorbed and to identify the effect of formulation components on drug absorption. RESULTS: BioRAM yielded low scores for fluconazole absorption with respect to severity (risks of sub and supra-bioavailable drug products), probability of incidence of bioinequivalent results and capacity of detection. The results demonstrated that dissolution was highly influenced by the active pharmaceutical ingredient (API) polymorphism and the ratio of diluents. Process variables (mixing time, lubricant concentration, lubrication time and filling speed) did not impact the clinical outcome of the formulation with respect to dissolution and content uniformity. CONCLUSIONS: Understanding the clinical implications of the adopted formulation approach led to the construction of purposeful design space and control strategy.

EB-SUN, a New Microtubule Plus-End Tracking Protein in Drosophila.

Microtubule (MT) regulation is essential for oocyte development. In Drosophila, MT stability, polarity, abundance, and orientation undergo dynamic changes across developmental stages. In our effort to identify novel microtubule-associated proteins (MAPs) that regulate MTs in the Drosophila ovary, we identified a previously uncharacterized gene, CG18190, encoding a novel MT end-binding (EB) protein, which we propose to name EB-SUN. We show that EB-SUN colocalizes with EB1 at growing microtubule plus-ends in Drosophila S2 cells. Tissue-specific and developmental expression profiles from Paralog Explorer reveal that EB-SUN is predominantly expressed in the ovary and early embryos, while EB1 is ubiquitously expressed. Furthermore, as early as oocyte determination, EB-SUN comets are highly concentrated in oocytes during oogenesis. EB-SUN knockout (KO) results in a decrease in MT density at the onset of mid-oogenesis (Stage 7) and delays oocyte growth during late mid-oogenesis (Stage 9). Combining EB-SUN KO with EB1 knockdown (KD) in germ cells significantly further reduced MT density at Stage 7. Notably, all eggs from EB-SUN KO/EB1 KD females fail to hatch, unlike single gene depletion, suggesting a functional redundancy between these two EB proteins during embryogenesis. Our findings indicate that EB-SUN and EB1 play distinct roles during early embryogenesis.

EB-SUN, a New Microtubule Plus-End Tracking Protein in Drosophila.

Microtubule (MT) regulation is essential for oocyte development. In Drosophila, MT stability, polarity, abundance, and orientation undergo dynamic changes across developmental stages. In our effort to identify novel microtubule-associated proteins (MAPs) that regulate MTs in the Drosophila ovary, we identified a previously uncharacterized gene, CG18190, encoding a novel MT end-binding (EB) protein, which we propose to name EB-SUN. We show that EB-SUN colocalizes with EB1 at growing microtubule plus-ends in Drosophila S2 cells. Tissue-specific and developmental expression profiles from Paralog Explorer reveal that EB-SUN is predominantly expressed in the ovary and early embryos, while EB1 is ubiquitously expressed. Furthermore, as early as oocyte determination, EB-SUN comets are highly concentrated in oocytes during oogenesis. EB-SUN knockout (KO) results in a decrease in MT density at the onset of mid-oogenesis (Stage 7) and delays oocyte growth during late mid-oogenesis (Stage 9). Combining EB-SUN KO with EB1 knockdown (KD) in germ cells significantly further reduced MT density at Stage 7. Hatching assays of single protein depletion reveal distinct roles for EB-SUN and EB1 in early embryogenesis, likely due to differences in expression and binding partners. Notably, all eggs from EB-SUN KO/EB1 KD females fail to hatch, suggesting partial redundancy between these proteins. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].

Bidirectional multiciliated cell extrusion is controlled by Notch driven basal extrusion and Piezo 1 driven apical extrusion.

Xenopus embryos are covered with a complex epithelium containing numerous multiciliated cells (MCCs). During late stage development there is a dramatic remodeling of the epithelium that involves the complete loss of MCCs. Cell extrusion is a well-characterized process for driving cell loss while maintaining epithelial barrier function. Normal cell extrusion is typically unidirectional whereas bidirectional extrusion is often associated with disease (e.g. cancer). We describe two distinct mechanisms for MCC extrusion, a basal extrusion driven by Notch signaling and an apical extrusion driven by Piezo1. Early in the process there is a strong bias towards basal extrusion, but as development continues there is a shift towards apical extrusion. Importantly, receptivity to the Notch signal is age-dependent and governed by the maintenance of the MCC transcriptional program such that extension of this program is protective against cell loss. In contrast, later apical extrusion is regulated by Piezo 1 such that premature activation of Piezo 1 leads to early extrusion while blocking Piezo 1 leads to MCC maintenance. Distinct mechansms for MCC loss underlie the importance of their removal during epithelial remodeling. Summay Statement: Cell extrusion typically occurs unidirectionally. We have identified a single population of multiciliated cells that extrudes bidirectionally: Notch-driven basal extrusion and Piezo 1-mediated apical extrusion.

Tubulin acetylation promotes penetrative capacity of cells undergoing radial intercalation.

Post-translational modification of tubulin provides differential functions to microtubule networks. Here, we address the role of tubulin acetylation on the penetrative capacity of cells undergoing radial intercalation, which is the process by which cells move apically, insert between outer cells, and join an epithelium. There are opposing forces that regulate intercalation, namely, the restrictive forces of the epithelial barrier versus the penetrative forces of the intercalating cell. Positively and negatively modulating tubulin acetylation in intercalating cells alters the developmental timing such that cells with more acetylation penetrate faster. We find that intercalating cells preferentially penetrate higher-order vertices rather than the more prevalent tricellular vertices. Differential timing in the ability of cells to penetrate different vertices reveals that lower-order vertices represent more restrictive sites of insertion. We shift the accessibility of intercalating cells toward more restrictive junctions by increasing tubulin acetylation, and we provide a geometric-based mathematical model that describes our results.

Functional Architecture of Deleterious Genetic Variants in the Genome of a Wrangel Island Mammoth.

Woolly mammoths were among the most abundant cold-adapted species during the Pleistocene. Their once-large populations went extinct in two waves, an end-Pleistocene extinction of continental populations followed by the mid-Holocene extinction of relict populations on St. Paul Island ∼5,600 years ago and Wrangel Island ∼4,000 years ago. Wrangel Island mammoths experienced an episode of rapid demographic decline coincident with their isolation, leading to a small population, reduced genetic diversity, and the fixation of putatively deleterious alleles, but the functional consequences of these processes are unclear. Here, we show that a Wrangel Island mammoth genome had many putative deleterious mutations that are predicted to cause diverse behavioral and developmental defects. Resurrection and functional characterization of several genes from the Wrangel Island mammoth carrying putatively deleterious substitutions identified both loss and gain of function mutations in genes associated with developmental defects (HYLS1), oligozoospermia and reduced male fertility (NKD1), diabetes (NEUROG3), and the ability to detect floral scents (OR5A1). These data suggest that at least one Wrangel Island mammoth may have suffered adverse consequences from reduced population size and isolation.

CLAMP/Spef1 regulates planar cell polarity signaling and asymmetric microtubule accumulation in the Xenopus ciliated epithelia.

Most epithelial cells polarize along the axis of the tissue, a feature known as planar cell polarity (PCP). The initiation of PCP requires cell-cell signaling via the noncanonical Wnt/PCP pathway. Additionally, changes in the cytoskeleton both facilitate and reflect this polarity. We have identified CLAMP/Spef1 as a novel regulator of PCP signaling. In addition to decorating microtubules (MTs) and the ciliary rootlet, a pool of CLAMP localizes at the apical cell cortex. Depletion of CLAMP leads to the loss of PCP protein asymmetry, defects in cilia polarity, and defects in the angle of cell division. Additionally, depletion of CLAMP leads to a loss of the atypical cadherin-like molecule Celrs2, suggesting that CLAMP facilitates the stabilization of junctional interactions responsible for proper PCP protein localization. Depletion of CLAMP also affects the polarized organization of MTs. We hypothesize that CLAMP facilitates the establishment of cell polarity and promotes the asymmetric accumulation of MTs downstream of the establishment of proper PCP.

3 D Network-Structured Crumpled Graphene/Carbon Nanotube/Polyaniline Composites for Supercapacitors.

Crumpled graphene (CGR) is considered a promising supercapacitor material to achieve high power and energy density because it could overcome the disadvantages of 2 D GR sheets such as aggregation during the electrode fabrication process, reduction of the available surface area, and limitation of the electron and ion transport. Even though CGR shows good results, carbon materials are limited in terms of their capacitance performance. Here, we report highly enhanced supercapacitor materials by fabricating a 3 D composite containing CGR, carbon nanotubes (CNTs), and polyaniline (PANI). The CNTs increased the basal spacing and bridged the defects for electron transfer between the GR sheets in CGR. PANI can enhance the rate of conduction of electrons and offer high pseudocapacitance originating from its redox reactions. The synergistic effect of the CNTs and PANI may also result in a higher electrochemical capacitance and better stability than each individual component as electrode materials for supercapacitors in a two-electrode system. More importantly, the performance of the supercapacitors can be further enhanced by employing 2 D GR as the binder for the composite electrodes, resulting in specific capacitance of 456 F g-1 , rate capability of 89 %, and cyclic stability of 97 % after 1000 cycles.

c21orf59/kurly Controls Both Cilia Motility and Polarization.

Cilia are microtubule-based projections that function in the movement of extracellular fluid. This requires cilia to be: (1) motile and driven by dynein complexes and (2) correctly polarized on the surface of cells, which requires planar cell polarity (PCP). Few factors that regulate both processes have been discovered. We reveal that C21orf59/Kurly (Kur), a cytoplasmic protein with some enrichment at the base of cilia, is needed for motility; zebrafish mutants exhibit characteristic developmental abnormalities and dynein arm defects. kur was also required for proper cilia polarization in the zebrafish kidney and the larval skin of Xenopus laevis. CRISPR/Cas9 coupled with homologous recombination to disrupt the endogenous kur locus in Xenopus resulted in the asymmetric localization of the PCP protein Prickle2 being lost in mutant multiciliated cells. Kur also makes interactions with other PCP components, including Disheveled. This supports a model wherein Kur plays a dual role in cilia motility and polarization.

Cell biology. Reversible centriole depletion with an inhibitor of Polo-like kinase 4.

Centrioles are ancient organelles that build centrosomes, the major microtubule-organizing centers of animal cells. Extra centrosomes are a common feature of cancer cells. To investigate the importance of centrosomes in the proliferation of normal and cancer cells, we developed centrinone, a reversible inhibitor of Polo-like kinase 4 (Plk4), a serine-threonine protein kinase that initiates centriole assembly. Centrinone treatment caused centrosome depletion in human and other vertebrate cells. Centrosome loss irreversibly arrested normal cells in a senescence-like G1 state by a p53-dependent mechanism that was independent of DNA damage, stress, Hippo signaling, extended mitotic duration, or segregation errors. In contrast, cancer cell lines with normal or amplified centrosome numbers could proliferate indefinitely after centrosome loss. Upon centrinone washout, each cancer cell line returned to an intrinsic centrosome number "set point." Thus, cells with cancer-associated mutations fundamentally differ from normal cells in their response to centrosome loss.

Radial intercalation is regulated by the Par complex and the microtubule-stabilizing protein CLAMP/Spef1.

The directed movement of cells is critical for numerous developmental and disease processes. A developmentally reiterated form of migration is radial intercalation; the process by which cells move in a direction orthogonal to the plane of the tissue from an inner layer to an outer layer. We use the radial intercalation of cells into the skin of Xenopus laevis embryos as a model to study directed cell migration within an epithelial tissue. We identify a novel function for both the microtubule-binding protein CLAMP and members of the microtubule-regulating Par complex during intercalation. Specifically, we show that Par3 and aPKC promote the apical positioning of centrioles, whereas CLAMP stabilizes microtubules along the axis of migration. We propose a model in which the Par complex defines the orientation of apical migration during intercalation and in which subcellular localization of CLAMP promotes the establishment of an axis of microtubule stability required for the active migration of cells into the outer epithelium.

Endoglin in amniotic fluid as a risk factor for the subsequent development of bronchopulmonary dysplasia.

OBJECTIVE: Cross-talk between inflammation and angiogenesis pathways has been recently reported. The objectives of this study were to: (i) examine whether amniotic fluid (AF) concentrations of soluble endoglin (sEng), a protein with anti-angiogenic properties, change during pregnancy, parturition, or intra-amniotic infection and/or inflammation (IAI); (ii) determine whether an increase in sEng in the AF of patients with preterm labor (PTL) and preterm prelabor rupture of membranes (PROM) is associated with adverse neonatal outcomes; and (iii) investigate potential sources of sEng in AF. STUDY DESIGN: A cross-sectional study was conducted to include patients in the following groups: (i) mid-trimester (n = 20); (ii) PTL with term delivery (n = 95); (iii) PTL leading to preterm delivery with (n = 40) and without IAI (n = 46); (iv) preterm PROM with (n = 37) and without IAI (n = 37); (v) term in labor (n = 48) and not in labor (n = 44). AF concentrations of sEng were determined by enzyme-linked immunosorbent assay. Chorioamniotic membranes, umbilical cord blood, and AF macrophages were examined for the expression of endoglin. RESULTS: (i) Patients with IAI had a higher median AF concentration of sEng than those without IAI (P = 0.02 for PTL and 0.06 for preterm PROM); (ii) AF concentrations of sEng in the 3rd and 4th quartiles were associated with IAI (OR 2.5 and 7.9, respectively); (iii) an AF sEng concentration ≥779.5 pg/mL was associated with bronchopulmonary dysplasia (BPD) (OR 7.9); (iv) endoglin was co-localized with CD14+ macrophages in AF pellets of patients with IAI by immunofluorescence and flow cytometry; and (v) the concentration of sEng in the supernatant was significantly increased after the treatment of macrophages with endotoxin or TNF-α. CONCLUSIONS: Soluble endoglin participates in the host response against IAI. Activated macrophages may be a source of sEng concentrations in the AF of patients with IAI. An increase of sEng in the AF is associated with BPD and adverse neonatal outcomes.

Physical activity and screen time in adolescents and their friends.

BACKGROUND: Little is known about the actual physical activity and screen time behaviors of an adolescent's friends relative to the individual's behavior. PURPOSE: To determine the associations between an adolescent's physical activity and screen time and his/her nominated friends' physical activity and screen time. METHODS: Data were obtained from EAT 2010 (Eating and Activity Among Teens), a large cross-sectional study (n=2126) conducted in 20 middle schools and high schools in Minneapolis/St. Paul MN during the 2009-2010 academic year and analyzed during 2011. Each participant nominated up to six friends from a school roster, and data from those friends were obtained as part of the school-based data collection procedures. Physical activity and screen time were assessed with previously used and validated questionnaires. Generalized estimating equation models, stratified by gender, were used to assess associations between adolescents' physical activity and screen time and their friends' physical activity and screen time. RESULTS: Physical activity for female adolescents was associated with their male and female friends' physical activity, including their male and female best friends (all p<0.05). Male adolescents' physical activity was associated with their female friends' physical activity (p<0.03). Female adolescents' screen time was associated with their male and female friends' screen time (p≤0.03), but not with that of their best friends. Male adolescents' screen time was associated with only their female friends' screen time (p=0.04). CONCLUSIONS: The consistent association between female adolescents' physical activity and their friends' physical activity indicates a need to include peer effects on adolescent female physical activity in future intervention work.

Smooth muscle specific deletion of Ndst1 leads to decreased vessel luminal area and no change in blood pressure in conscious mice.

Heparan sulfate proteoglycans are abundant matrix and membrane molecules. Smooth muscle specific deletion of one heparan sulfate biosynthetic enzyme, N-deacetylase-N-sulfotransferase1 leads to decreased vascular smooth muscle cell proliferation, and vascular wall thickness. We hypothesized that this may lead to changes in blood pressure in conscious mice. Blood pressure was measured via telemetry in SM22αCre(+)Ndst1(-/-)(n = 4) and wild type (n = 8) mice. Aorta and thoracodorsal artery luminal area is significantly smaller in SM22αCre(+)Ndst1(-/-) (n = 4-8, P = 0.02, P = 0.0002) compared to wild type (n = 7) mice. Diurnal differences were observed in both cohorts for systolic, diastolic, mean arterial blood pressure, and heart rate (P < 0.001 from T test). No significant differences were found in the above parameters between the cohorts in either light or dark times using a linear mixed model. In conclusion, deletion of N-deacetylase-N-sulfotransferase1 in smooth muscle did not influence any of the blood pressure parameters measured despite significant decrease in aorta and thoracodorsal artery luminal area.