bims-bicyki 2021-12-26 papers

bims-bicyki

Biomed News

on Bicaudal-C1 and interactors in cystic kidney disease

Issue of 2021–12–26
23 papers selected by
Céline Gagnieux, École Polytechnique Fédérale de Lausanne

The association of glucagon with disease severity and progression in patients with autosomal dominant polycystic kidney disease: an observational cohort study.
Recent Discoveries in Epigenetic Modifications of Polycystic Kidney Disease.
Interdependent Regulation of Polycystin Expression Influences Starvation-Induced Autophagy and Cell Death.
Autophagy Promotes Hepatic Cystogenesis in Polycystic Liver Disease via Depletion of Cholangiocyte Ciliogenic Proteins.
Aurora A and AKT Kinase Signaling Associated with Primary Cilia.
Primary Cilia Structure Is Prolonged in Enteric Neurons of 5xFAD Alzheimer's Disease Model Mice.
Incoercible Vomiting in a Polycystic (ADPKD) Patient on Peritoneal Dialysis.
Hepatocyte Nuclear Factor 1 Beta Mutation-associated Newborn Onset of Glomerulocystic Kidney Disease: A Case Presentation.
Multiomic identification of factors associated with progression to cystic kidney disease in mice with nephron Ift88 disruption.
Retinal ciliopathies through the lens of Bardet-Biedl Syndrome: Past, present and future.
Lysophosphatidic Acid and Several Neurotransmitters Converge on Rho-Kinase 2 Signaling to Manage Motoneuron Excitability.
Efficient and Cost Effective Electroporation Method to Study Primary Cilium-dependent Signaling Pathways in the Granule Cell Precursor.
Temporospatial regulation of intraflagellar transport is required for the endochondral ossification in mice.
Sishen Pill Maintained Colonic Mucosal Barrier Integrity to Treat Ulcerative Colitis via Rho/ROCK Signaling Pathway.
Essential Role of Rho-Associated Kinase in ABO Immune Complex-Mediated Endothelial Barrier Disruption.
Adaptive Lipid Immiscibility and Membrane Remodeling Are Active Functional Determinants of Primary Ciliogenesis.
Prostaglandin signaling in ciliogenesis and development.
Tubulin post-translational modifications in protists - Tiny models for solving big questions.
CCDC40 mutation as a cause of infertility in a Chinese family with primary ciliary dyskinesia.
Comparison of the airway microbiota in children with chronic suppurative lung disease.
Microgravity and space radiation inhibit autophagy in human capillary endothelial cells, through either opposite or synergistic effects on specific molecular pathways.
Respiratory muscle strength, exercise capacity and physical activity in patients with primary ciliary dyskinesia: A cross-sectional study.
Genome-Wide RNAi Screening Identifies Novel Pathways/Genes Involved in Oxidative Stress and Repurposable Drugs to Preserve Cystic Fibrosis Airway Epithelial Cell Integrity.

Clin Kidney J. 2021 Dec;14(12): 2582-2590

The association of glucagon with disease severity and progression in patients with autosomal dominant polycystic kidney disease: an observational cohort study.

Martine G E Knol, Bart J Kramers, Ron T Gansevoort, Maatje D A van Gastel.

   Background: Mammalian target of rapamycin (mTOR) inhibitors and ketogenesis have been shown to ameliorate disease progression in experimental autosomal dominant polycystic kidney disease (ADPKD). Glucagon is known to lower mTOR activity and stimulate ketogenesis. We hypothesized that in ADPKD patients, higher endogenous glucagon is associated with less disease severity and progression.
Methods: Data were analysed from 664 Dutch ADPKD patients participating in the Developing Intervention Strategies to Halt Progression of ADPKD observational cohort, including patients >18 years of age with an estimated glomerular filtration rate (eGFR) ≥15 mL/min/1.73 m2 and excluding patients with concomitant diseases or medication use that may impact the natural course of ADPKD. The association between glucagon and disease severity and progression was tested using multivariate linear regression and mixed modelling, respectively.
Results: The median glucagon concentration was 5.0 pmol/L [interquartile range (IQR) 3.4-7.2) and differed significantly between females and males [4.3 pmol/L (IQR 2.9-6.0) and 6.6 (4.5-9.5), P < 0.001, respectively]. Intrasubject stability of glucagon in 30 patients showed a strong correlation (Pearson's correlation coefficient 0.893; P < 0.001). Moreover, glucagon showed significant associations with known determinants (sex, body mass index and copeptin; all P < 0.01) and known downstream effects (glucose, haemoglobin A1c and cholesterol; all P < 0.05), suggesting that glucagon was measured reliably. Cross-sectionally, glucagon was associated with eGFR and height-adjusted total kidney volume, but in the opposite direction of our hypothesis, and these lost significance after adjustment for confounders. Glucagon was not associated with an annual decline in kidney function or growth in kidney volume.
Conclusions: These data do not provide evidence for a role of endogenous glucagon as a protective hormone in ADPKD. Intervention studies are needed to determine the relation between glucagon and ADPKD.

Keywords:  ADPKD; PKD; TKV; eGFR; estimated glomerular filtration rate; glucagon; polycystic kidney disease; total kidney volume

DOI:  https://doi.org/10.1093/ckj/sfab112
Int J Mol Sci. 2021 Dec 11. pii: 13327. [Epub ahead of print]22(24):

Recent Discoveries in Epigenetic Modifications of Polycystic Kidney Disease.

Sarah A Bowden, Euan J Rodger, Aniruddha Chatterjee, Michael R Eccles, Cherie Stayner.

  Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a heritable renal disease that results in end-stage kidney disease, due to the uncontrolled bilateral growth of cysts throughout the kidneys. While it is known that a mutation within a PKD-causing gene is required for the development of ADPKD, the underlying mechanism(s) causing cystogenesis and progression of the disease are not well understood. Limited therapeutic options are currently available to slow the rate of cystic growth. Epigenetic modifications, including DNA methylation, are known to be altered in neoplasia, and several FDA-approved therapeutics target these disease-specific changes. As there are many similarities between ADPKD and neoplasia, we (and others) have postulated that ADPKD kidneys contain alterations to their epigenetic landscape that could be exploited for future therapeutic discovery. Here we summarise the current understanding of epigenetic changes that are associated with ADPKD, with a particular focus on the burgeoning field of ADPKD-specific alterations in DNA methylation.

Keywords:  ADPKD; DNA methylation; PKD1; cystogenesis; epigenetics; epigenome; inherited kidney disease; polycystic kidney disease

DOI:  https://doi.org/10.3390/ijms222413327
Int J Mol Sci. 2021 Dec 16. pii: 13511. [Epub ahead of print]22(24):

Interdependent Regulation of Polycystin Expression Influences Starvation-Induced Autophagy and Cell Death.

Jean-Paul Decuypere, Dorien Van Giel, Peter Janssens, Ke Dong, Stefan Somlo, Yiqiang Cai, Djalila Mekahli, Rudi Vennekens.

  Autosomal dominant polycystic kidney disease (ADPKD) is mainly caused by deficiency of polycystin-1 (PC1) or polycystin-2 (PC2). Altered autophagy has recently been implicated in ADPKD progression, but its exact regulation by PC1 and PC2 remains unclear. We therefore investigated cell death and survival during nutritional stress in mouse inner medullary collecting duct cells (mIMCDs), either wild-type (WT) or lacking PC1 (PC1KO) or PC2 (PC2KO), and human urine-derived proximal tubular epithelial cells (PTEC) from early-stage ADPKD patients with PC1 mutations versus healthy individuals. Basal autophagy was enhanced in PC1-deficient cells. Similarly, following starvation, autophagy was enhanced and cell death reduced when PC1 was reduced. Autophagy inhibition reduced cell death resistance in PC1KO mIMCDs to the WT level, implying that PC1 promotes autophagic cell survival. Although PC2 expression was increased in PC1KO mIMCDs, PC2 knockdown did not result in reduced autophagy. PC2KO mIMCDs displayed lower basal autophagy, but more autophagy and less cell death following chronic starvation. This could be reversed by overexpression of PC1 in PC2KO. Together, these findings indicate that PC1 levels are partially coupled to PC2 expression, and determine the transition from renal cell survival to death, leading to enhanced survival of ADPKD cells during nutritional stress.

Keywords:  ADPKD; autophagy; autosomal dominant polycystic kidney disease; cell death; nutrient stress; polycystins

DOI:  https://doi.org/10.3390/ijms222413511
Hepatology. 2021 Dec 23.

Autophagy Promotes Hepatic Cystogenesis in Polycystic Liver Disease via Depletion of Cholangiocyte Ciliogenic Proteins.

Anatoliy I Masyuk, Tatyana V Masyuk, Christy E Trussoni, Nicholas E Pirius, Nicholas F LaRusso.

   BACKGROUNDS & AIMS: Polycystic liver disease (PLD) is characterized by defective cholangiocyte cilia that regulate progressive growth of hepatic cysts. Because formation of primary cilia is influenced by autophagy through degradation of proteins involved in ciliogenesis, we hypothesized that ciliary defects in PLD cholangiocytes (PLDC) originate from autophagy-mediated depletion of ciliogenic proteins, ARL3 and ARL13B, and ARL-dependent mislocation of a ciliary-localized bile acid receptor, TGR5, activation of which enhances hepatic cystogenesis. The aims here were to determine: i) if ciliogenesis is impaired in PLDC, is associated with increased autophagy, and involves autophagy-mediated depletion of ARL3 and ARL13B; ii) if depletion of ARL3 and ARL13B in PLDC cilia impacts ciliary localization of TGR5; and iii) if pharmacological inhibition of autophagy reestablishes cholangiocyte cilia, ciliary localization of ARL3, ARL3B and TGR5, and reduces hepatic cystogenesis.
APPROACH & RESULTS: By using liver tissue from healthy individuals and patients with PLD, in vitro and in vivo models of PLD, and in vitro models of ciliogenesis, we demonstrated that in PLDC: ciliogenesis is impaired; autophagy is enhanced; ARL3 and ARL13B are ubiquitinated by HDAC6, depleted in cilia and present in autophagosomes; depletion of ARL3 and ARL13B impacts ciliary localization of TGR5; and pharmacologic inhibition of autophagy with mefloquine and verteporfin reestablishes cholangiocyte cilia, ciliary localization of ARL3, ARL13B and TGR5, and reduces hepatic cystogenesis.
CONCLUSION: The intersection between autophagy, defective cholangiocyte cilia, and enhanced hepatic cystogenesis contributes to PLD progression and can be considered a novel target for therapeutic interventions.

Keywords:  Polycystic liver disease; cholangiocytes; ciliogenesis; primary cilia

DOI:  https://doi.org/10.1002/hep.32298
Cells. 2021 Dec 20. pii: 3602. [Epub ahead of print]10(12):

Aurora A and AKT Kinase Signaling Associated with Primary Cilia.

Yuhei Nishimura, Daishi Yamakawa, Takashi Shiromizu, Masaki Inagaki.

  Dysregulation of kinase signaling is associated with various pathological conditions, including cancer, inflammation, and autoimmunity; consequently, the kinases involved have become major therapeutic targets. While kinase signaling pathways play crucial roles in multiple cellular processes, the precise manner in which their dysregulation contributes to disease is dependent on the context; for example, the cell/tissue type or subcellular localization of the kinase or substrate. Thus, context-selective targeting of dysregulated kinases may serve to increase the therapeutic specificity while reducing off-target adverse effects. Primary cilia are antenna-like structures that extend from the plasma membrane and function by detecting extracellular cues and transducing signals into the cell. Cilia formation and signaling are dynamically regulated through context-dependent mechanisms; as such, dysregulation of primary cilia contributes to disease in a variety of ways. Here, we review the involvement of primary cilia-associated signaling through aurora A and AKT kinases with respect to cancer, obesity, and other ciliopathies.

Keywords:  AKT kinase; aurora kinase A; cancer; ciliopathy; differentiation; lipid raft; obesity; primary cilium; proliferation; trichoplein

DOI:  https://doi.org/10.3390/cells10123602
Int J Mol Sci. 2021 Dec 17. pii: 13564. [Epub ahead of print]22(24):

Primary Cilia Structure Is Prolonged in Enteric Neurons of 5xFAD Alzheimer's Disease Model Mice.

Vu Thu Thuy Nguyen, Lena Brücker, Ann-Kathrin Volz, Julia C Baumgärtner, Malena Dos Santos Guilherme, Francesco Valeri, Helen May-Simera, Kristina Endres.

  Neurodegenerative diseases such as Alzheimer's disease (AD) have long been acknowledged as mere disorders of the central nervous system (CNS). However, in recent years the gut with its autonomous nervous system and the multitude of microbial commensals has come into focus. Changes in gut properties have been described in patients and animal disease models such as altered enzyme secretion or architecture of the enteric nervous system. The underlying cellular mechanisms have so far only been poorly investigated. An important organelle for integrating potentially toxic signals such as the AD characteristic A-beta peptide is the primary cilium. This microtubule-based signaling organelle regulates numerous cellular processes. Even though the role of primary cilia in a variety of developmental and disease processes has recently been recognized, the contribution of defective ciliary signaling to neurodegenerative diseases such as AD, however, has not been investigated in detail so far. The AD mouse model 5xFAD was used to analyze possible changes in gut functionality by organ bath measurement of peristalsis movement. Subsequently, we cultured primary enteric neurons from mutant mice and wild type littermate controls and assessed for cellular pathomechanisms. Neurite mass was quantified within transwell culturing experiments. Using a combination of different markers for the primary cilium, cilia number and length were determined using fluorescence microscopy. 5xFAD mice showed altered gut anatomy, motility, and neurite mass of enteric neurons. Moreover, primary cilia could be demonstrated on the surface of enteric neurons and exhibited an elongated phenotype in 5xFAD mice. In parallel, we observed reduced β-Catenin expression, a key signaling molecule that regulates Wnt signaling, which is regulated in part via ciliary associated mechanisms. Both results could be recapitulated via in vitro treatments of enteric neurons from wild type mice with A-beta. So far, only a few reports on the probable role of primary cilia in AD can be found. Here, we reveal for the first time an architectural altered phenotype of primary cilia in the enteric nervous system of AD model mice, elicited potentially by neurotoxic A-beta. Potential changes on the sub-organelle level-also in CNS-derived neurons-require further investigations.

Keywords:  Alzheimer’s disease (AD); enteric nervous system (ENS); ganglia; neurites; primary cilia

DOI:  https://doi.org/10.3390/ijms222413564
Case Rep Nephrol Dial. 2021 Sep-Dec;11(3):11(3): 321-326

Incoercible Vomiting in a Polycystic (ADPKD) Patient on Peritoneal Dialysis.

Victor Burguera Vion, R Haridian Sosa Barrios, Maria Delgado Yagüe, Milagros Fernández Lucas, Maite E Rivera Gorrín.

  Although gastrointestinal symptoms are not uncommon in PD patients due to several causes, such as infusion volume with early satiety, constipation, or peritonitis, sometimes the differential diagnosis is more challenging for nephrologists. We present the case of a woman with end-stage renal disease due to autosomal dominant polycystic kidney disease on PD who presented with swollen legs and incoercible vomiting. After ruling out constipation and infection, an abdominal CT was done, revealing extrinsic compression of the intrahepatic inferior cava vein (ICV) and massive venous thrombosis from ICV to bilateral iliofemoral deep veins. In addition, CT also showed displacement and extrinsic compression of the stomach, pylorus, and duodenum due to an enlarged liver cyst. Percutaneous drainage and sclerosis of the cyst compressing the stomach was performed, anticoagulation was started, and the patient clinically improved with complete resolution of symptoms.

Keywords:  Autosomal dominant polycystic kidney disease; Peritoneal dialysis; Pyloric stenosis; Vomiting

DOI:  https://doi.org/10.1159/000520020
Medeni Med J. 2021 Dec 19. 36(4): 352-355

Hepatocyte Nuclear Factor 1 Beta Mutation-associated Newborn Onset of Glomerulocystic Kidney Disease: A Case Presentation.

Nilufer Goknar, Melda Ekici Avci, Diana Uckardes, Emre Kelesoglu, Kubra Tekkus Ermis, Cengiz Candan.

  Mutations in hepatocyte nuclear factor-1 beta (HNF1B) are the most commonly identified genetic cause of renal malformations. Heterozygous mutations are associated with renal cysts and diabetes syndrome. Various renal developmental abnormalities and maturity-onset diabetes of the young could be the presenting factors of these mutations. A 10-year-old boy who was evaluated for bilateral cystic kidneys and chronic kidney disease from the newborn period was diagnosed with HNF1B-related glomerulocystic disease by DNA sequencing. The differential diagnosis of autosomal dominant polycystic kidney disease was a diagnostic pitfall. The genetic screening of the family revealed his mother, sister, and brother to have the same mutation. Therefore, genetic diagnosis and counseling are important for cystic kidney diseases not only for formulating the diagnosis and early management plan but also for the diagnosis of potential asymptomatic cases in the family.

Keywords:  Hyperechogenic kidneys; chronic kidney disease; cystic kidney disease; infant

DOI:  https://doi.org/10.4274/MMJ.galenos.2021.02686
Am J Physiol Renal Physiol. 2021 Dec 20.

Multiomic identification of factors associated with progression to cystic kidney disease in mice with nephron Ift88 disruption.

Chunyan Hu, Katherine Beebe, Edgar J Hernandez, Jose M Lazaro-Guevara, Monica P Revelo, Yufeng Huang, J Alan Maschek, James E Cox, Donald E Kohan.

  Ift88 gene mutations cause primary cilia loss and polycystic kidney disease (PKD) in mice. Nephron Ift88 knockout (KO) at 2 months postnatal does not affect renal histology at 4 months postnatal and causes PKD only in males by 11 months postnatal. To identify factors associated with PKD development, kidneys from 4-month-old male and female control and Ift88 KO mice underwent transcriptomic, proteomic, western, metabolomic and lipidomic analysis. mRNAs involved in extracellular matrix (ECM) synthesis and degradation were selectively upregulated in male KO mice. Proteomic analysis was insufficiently sensitive to detect most ECM components, while western analysis paradoxically revealed reduced fibronectin and collagen I in male KO mice. Only male KO mice upregulated mRNAs encoding fibrinogen subunits and receptors for VEGF and PDGF; Per2, Per3 and Nrld2 clock mRNAs were selectively decreased in male KO mice. Proteomic, metabolomic and lipidomic analysis detected a relative (vs same sex control) decrease in factors involved in fatty acid ß-oxidation in female KO, while increased or unchanged levels in male KO, mice including medium chain acyl-CoA dehydrogenase, 3-hydroxybutyrate, and acylcarnitine. Three putative mRNA biomarkers of cystogenesis in male Ift88 KO mice (similar control levels between sexes and uniquely altered by KO in males) were identified, including high levels (Fga and Sdf2l1) and low levels (Banp) in male KO mice. These findings suggest that relative alterations in renal ECM metabolism, fatty acid ß-oxidation, and other pathways precede cystogenesis in Ift88 KO mice. In addition, potential novel biomarkers of cystogenesis in Ift88 KO mice have been identified.

Keywords:  cilia; cysts; nephron; predictor; sex

DOI:  https://doi.org/10.1152/ajprenal.00409.2021
Prog Retin Eye Res. 2021 Dec 17. pii: S1350-9462(21)00096-3. [Epub ahead of print] 101035

Retinal ciliopathies through the lens of Bardet-Biedl Syndrome: Past, present and future.

Bharatendu Chandra, Moon Ley Tung, Ying Hsu, Todd Scheetz, Val C Sheffield.

  The primary cilium is a highly specialized and evolutionary conserved organelle in eukaryotes that plays a significant role in cell signaling and trafficking. Over the past few decades tremendous progress has been made in understanding the physiology of cilia and the underlying pathomechanisms of various ciliopathies. Syndromic ciliopathies consist of a group of disorders caused by ciliary dysfunction or abnormal ciliogenesis. These disorders have multiorgan involvement in addition to retinal degeneration underscoring the ubiquitous distribution of primary cilia in different cell types. Genotype-phenotype correlation is often challenging due to the allelic heterogeneity and pleiotropy of these disorders. In this review, we discuss the clinical and genetic features of syndromic ciliopathies with a focus on Bardet-Biedl syndrome (BBS) as a representative disorder. We discuss the structure and function of primary cilia and their role in retinal photoreceptors. We describe the progress made thus far in understanding the functional and genetic characterization including expression quantitative trait locus (eQTL) analysis of BBS genes. In the future directions section, we discuss the emerging technologies, such as gene therapy, as well as anticipated challenges and their implications in therapeutic development for ciliopathies.

Keywords:  Bardet-biedl syndrome; Cilia; Ciliopathies; Retinopathy; eQTL

DOI:  https://doi.org/10.1016/j.preteyeres.2021.101035
Front Mol Neurosci. 2021 ;14 788039

Lysophosphatidic Acid and Several Neurotransmitters Converge on Rho-Kinase 2 Signaling to Manage Motoneuron Excitability.

Victoria García-Morales, Ángela Gento-Caro, Federico Portillo, Fernando Montero, David González-Forero, Bernardo Moreno-López.

  Intrinsic membrane excitability (IME) sets up neuronal responsiveness to synaptic drive. Several neurotransmitters and neuromodulators, acting through G-protein-coupled receptors (GPCRs), fine-tune motoneuron (MN) IME by modulating background K+ channels TASK1. However, intracellular partners linking GPCRs to TASK1 modulation are not yet well-known. We hypothesized that isoform 2 of rho-kinase (ROCK2), acting as downstream GPCRs, mediates adjustment of MN IME via TASK1. Electrophysiological recordings were performed in hypoglossal MNs (HMNs) obtained from adult and neonatal rats, neonatal knockout mice for TASK1 (task1 -/-) and TASK3 (task3 -/-, the another highly expressed TASK subunit in MNs), and primary cultures of embryonic spinal cord MNs (SMNs). Small-interfering RNA (siRNA) technology was also used to knockdown either ROCK1 or ROCK2. Furthermore, ROCK activity assays were performed to evaluate the ability of various physiological GPCR ligands to stimulate ROCK. Microiontophoretically applied H1152, a ROCK inhibitor, and siRNA-induced ROCK2 knockdown both depressed AMPAergic, inspiratory-related discharge activity of adult HMNs in vivo, which mainly express the ROCK2 isoform. In brainstem slices, intracellular constitutively active ROCK2 (aROCK2) led to H1152-sensitive HMN hyper-excitability. The aROCK2 inhibited pH-sensitive and TASK1-mediated currents in SMNs. Conclusively, aROCK2 increased IME in task3 -/-, but not in task1 -/- HMNs. MN IME was also augmented by the physiological neuromodulator lysophosphatidic acid (LPA) through a mechanism entailing Gαi/o-protein stimulation, ROCK2, but not ROCK1, activity and TASK1 inhibition. Finally, two neurotransmitters, TRH, and 5-HT, which are both known to increase MN IME by TASK1 inhibition, stimulated ROCK2, and depressed background resting currents via Gαq/ROCK2 signaling. These outcomes suggest that LPA and several neurotransmitters impact MN IME via Gαi/o/Gαq-protein-coupled receptors, downstream ROCK2 activation, and subsequent inhibition of TASK1 channels.

Keywords:  background potassium channels; intrinsic membrane excitability; lysophosphatidic acid (LPA); serotonin; thyrotropin-releasing hormone (TRH)

DOI:  https://doi.org/10.3389/fnmol.2021.788039
J Vis Exp. 2021 Nov 30.

Efficient and Cost Effective Electroporation Method to Study Primary Cilium-dependent Signaling Pathways in the Granule Cell Precursor.

Jason Cho Wai Lo, Wee Lin Wong, Catherine Hong Huan Hor.

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In the granule cell precursor (GCP), the primary cilium acts as a pivotal signaling center that orchestrates precursor cell proliferation by modulating the Hh signaling pathway. The investigation of primary cilium-dependent Hh signaling machinery is facilitated by in vitro genetic manipulation of the pathway components to visualize their dynamic localization to the primary cilium. However, transfection of transgenes in the primary cultures of GCPs using the currently known electroporation methods is generally costly and often results in low cell viability and undesirable transfection efficiency. This paper introduces an efficient, cost-effective, and simple electroporation protocol that demonstrates a high transfection efficiency of ~80-90% and optimal cell viability. This is a simple, reproducible, and efficient genetic modification method that is applicable to the study of the primary cilium-dependent Hedgehog signaling pathway in primary GCP cultures.

DOI: https://doi.org/10.3791/63283
Dev Biol. 2021 Dec 17. pii: S0012-1606(21)00249-9. [Epub ahead of print]

Temporospatial regulation of intraflagellar transport is required for the endochondral ossification in mice.

Hiroyuki Yamaguchi, Megumi Kitami, Karin H Uchima Koecklin, Li He, Jianbo Wang, William R Lagor, Daniel S Perrien, Yoshihiro Komatsu.

  Ciliogenic components, such as the family of intraflagellar transport (IFT) proteins, are recognized to play key roles in endochondral ossification, a critical process to form most bones. However, the unique functions and roles of each IFT during endochondral ossification remain unclear. Here, we show that IFT20 is required for endochondral ossification in mice. Utilizing osteo-chondrocyte lineage-specific Cre mice (Prx1-Cre and Col2-Cre), we deleted Ift20 to examine its function. Although chondrocyte-specific Ift20 deletion with Col2-Cre mice did not cause any overt skeletal defects, mesoderm-specific Ift20 deletion using Prx1-Cre (Ift20:Prx1-Cre) mice resulted in shortened limb outgrowth. Primary cilia were absent on chondrocytes of Ift20:Prx1-Cre mice, and ciliary-mediated Hedgehog signaling was attenuated in Ift20:Prx1-Cre mice. Interestingly, loss of Ift20 also increased Fgf18 expression in the perichondrium that sustained Sox9 expression, thus preventing endochondral ossification. Inhibition of enhanced phospho-ERK1/2 activation partially rescued defective chondrogenesis in Ift20 mutant cells, supporting an important role for FGF signaling. Our findings demonstrate that IFT20 is a critical regulator of temporospatial FGF signaling that is required for endochondral ossification.

Keywords:  Cartilage; Cilia; FGF; Hedgehog; Intraflagellar transport

DOI:  https://doi.org/10.1016/j.ydbio.2021.12.004
Evid Based Complement Alternat Med. 2021 ;2021 5536679

Sishen Pill Maintained Colonic Mucosal Barrier Integrity to Treat Ulcerative Colitis via Rho/ROCK Signaling Pathway.

Xiao-Yun Zhang, Hai-Mei Zhao, Yi Liu, Xiu-Yun Lu, Yan-Zhen Li, Qi-Hong Pan, Hai-Yan Wang, Wei Ge, Duan-Yong Liu.

Sishen Pill (SSP) is a classical prescription of traditional Chinese medicine and often used to treat gastrointestinal diseases, including ulcerative colitis (UC). However, its mechanism is still unclear. We aimed to determine the mechanism of SSP in the treatment of UC by investigating if it maintains the integrity of the intestinal mucosal barrier via the Rho A/Rho kinase (ROCK) signaling pathway. Administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) successfully induced chronic UC in rats, while the treatment effect of SSP was evaluated by body weight change, colonic length, colonic weight, colonic weight index, histological injury score, and pathological injury score after colitis rats were treated for 7 days. TNF-α and IL-1β levels were analyzed by ELISA, and the proteins of PI3K/Akt and RhoA/ROCK signaling pathway and junction proteins expression were measured by western blotting assay, and the distribution of Claudin 5 was shown by immunofluorescence. SSP significantly improved the clinical symptoms of colitis in rats and reduced the expression of p-RhoA, ROCK1, PI3K, and Akt in the colon mucosa, while it increased the expression of p-Rac and related proteins (Claudin-5, JAM1, VE-cadherin, and Connexin 43). In addition, SSP increased p-AMPKα and PTEN proteins expression, decreased Notch1 level, and hinted that activation of the PI3K/Akt signaling pathway was inhibited. In conclusion, SSP effectively treated chronic colitis induced by TNBS, which may have been achieved by inhibiting PI3K/Akt signal to suppress activation of the Rho/ROCK signaling pathway to finally maintain the integrity of the intestinal mucosal barrier.

DOI: https://doi.org/10.1155/2021/5536679
Biomedicines. 2021 Dec 07. pii: 1851. [Epub ahead of print]9(12):

Essential Role of Rho-Associated Kinase in ABO Immune Complex-Mediated Endothelial Barrier Disruption.

Hannah L McRae, Michelle Warren Millar, Spencer A Slavin, Neil Blumberg, Arshad Rahman, Majed A Refaai.

  ABO immune complexes (ABO-IC) formed by ABO-incompatible antigen-antibody interaction are associated with hemolysis and platelet destruction in patients transfused with ABO-nonidentical blood products. However, the effects of ABO-IC on endothelial cells (EC) are unclear. ABO-IC were formed in vitro from normal donor-derived plasma and serum. Human pulmonary artery EC (HPAEC) were cultured and treated with media, ABO-identical and -non-identical plasma, and ABO-IC. EC barrier integrity was evaluated using transendothelial electrical resistance (TEER), scanning electron microscopy (SEM), vascular endothelial (VE)-cadherin and phalloidin staining, and Rho-associated Kinase (ROCK) inhibitor treatment. TEER revealed significant/irreversible barrier disruption within 1-2 h of exposure to ABO non-identical plasma and ABO-IC; this occurred independently of EC ABO type. Treatment with ABO-IC resulted in decreased VE-cadherin staining and increased phalloidin staining in a time-dependent manner, suggesting that the resultant increased EC barrier permeability is secondary to actin stress fiber formation and loss of cell surface VE-cadherin. Inhibition of ROCK was effective in protecting against IC-induced barrier disruption even two hours after ABO-IC exposure. ABO-IC causes increased EC barrier permeability by decreasing cell surface VE-cadherin and promoting stress fiber formation, which is preventable by inhibiting ROCK activation to protect against EC contraction and gap formation.

Keywords:  ABO blood groups; endothelial cells; immune complexes; transfusion; vascular biology

DOI:  https://doi.org/10.3390/biomedicines9121851
Small Methods. 2021 Feb;5(2): e2000711

Adaptive Lipid Immiscibility and Membrane Remodeling Are Active Functional Determinants of Primary Ciliogenesis.

Miguel Bernabé-Rubio, Minerva Bosch-Fortea, Esther García, Jorge Bernardino de la Serna, Miguel A Alonso.

  Lipid liquid-liquid immiscibility and its consequent lateral heterogeneity have been observed under thermodynamic equilibrium in model and native membranes. However, cholesterol-rich membrane domains, sometimes referred to as lipid rafts, are difficult to observe spatiotemporally in live cells. Despite their importance in many biological processes, robust evidence for their existence remains elusive. This is mainly due to the difficulty in simultaneously determining their chemical composition and physicochemical nature, whilst spatiotemporally resolving their nanodomain lifetime and molecular dynamics. In this study, a bespoke method based on super-resolution stimulated emission depletion (STED) microscopy and raster imaging correlation spectroscopy (RICS) is used to overcome this issue. This methodology, laser interleaved confocal RICS and STED-RICS (LICSR), enables simultaneous tracking of lipid lateral packing and dynamics at the nanoscale. Previous work indicated that, in polarized epithelial cells, the midbody remnant licenses primary cilium formation through an unidentified mechanism. LICSR shows that lipid immiscibility and its adaptive collective nanoscale self-assembly are crucial for the midbody remnant to supply condensed membranes to the centrosome for the biogenesis of the ciliary membrane. Hence, this work poses a breakthrough in the field of lipid biology by providing compelling evidence of a functional role for liquid ordered-like membranes in primary ciliogenesis.

Keywords:  cholesterol-rich nanodomains; ciliary membranes; lipid immiscibility; membrane dynamics; midbody remnant; primary cilium; stimulated emission depletion nanoscopy

DOI:  https://doi.org/10.1002/smtd.202000711
J Cell Physiol. 2021 Dec 20.

Prostaglandin signaling in ciliogenesis and development.

Daqing Jin, Tao P Zhong.

  Prostaglandin (PG) signaling regulates a wide variety of physiological and pathological processes, including body temperature, cardiovascular homeostasis, reproduction, and inflammation. Recent studies have revealed that PGs play pivotal roles in embryo development, ciliogenesis, and organ formation. Prostaglandin E2 (PGE2) and its receptor EP4 modulate ciliogenesis by increasing the anterograde intraflagellar transport. Many G-protein-coupled receptors (GPCRs) including EP4 are localized in cilia for modulating cAMP signaling under various conditions. During development, PGE2 signaling regulates embryogenesis, hepatocyte differentiation, hematopoiesis, and kidney formation. Prostaglandins are also essential for skeletal muscle repair. This review outlines recent advances in understanding the functions and mechanisms of prostaglandin signaling in ciliogenesis, embryo development, and organ formation.

Keywords:  ciliogenesis; development; prostaglandin; repair

DOI:  https://doi.org/10.1002/jcp.30659
Semin Cell Dev Biol. 2021 Dec 15. pii: S1084-9521(21)00312-8. [Epub ahead of print]

Tubulin post-translational modifications in protists - Tiny models for solving big questions.

Ewa Joachimiak, Dorota Wloga.

  Protists are an exceptionally diverse group of mostly single-celled eukaryotes. The organization of the microtubular cytoskeleton in protists from various evolutionary lineages has different levels of sophistication, from a network of microtubules (MTs) supporting intracellular trafficking as in Dictyostelium, to complex structures such as basal bodies and cilia/flagella enabling cell motility, and lineage-specific adaptations such as the ventral disc in Giardia. MTs building these diverse structures have specific properties partly due to the presence of tubulin post-translational modifications (PTMs). Among them there are highly evolutionarily conserved PTMs: acetylation, detyrosination, (poly)glutamylation and (poly)glycylation. In some protists also less common tubulin PTMs were identified, including phosphorylation, methylation, Δ2-, Δ5- of α-tubulin, polyubiquitination, sumoylation, or S-palmitoylation. Not surprisingly, several single-celled organisms become models to study tubulin PTMs, including their effect on MT properties and discovery of the modifying enzymes. Here, we briefly summarize the current knowledge on tubulin PTMs in unicellular eukaryotes and highlight key findings in protists as model organisms.

Keywords:  Cilia; Flagella; Microtubule; Post-translational modifications; Protists; Tubulin

DOI:  https://doi.org/10.1016/j.semcdb.2021.12.004
Medicine (Baltimore). 2021 Dec 23. 100(51): e28275

CCDC40 mutation as a cause of infertility in a Chinese family with primary ciliary dyskinesia.

Li Liu, Kechong Zhou, Yuxuan Song, Xiaoqiang Liu.

TRIAL DESIGN: Primary ciliary dyskinesia (PCD) is a genetical disease that inherited in an autosomal-recessive way. Its clinical manifestations (such as male infertility) are mainly caused by defects of motion-related cilia that encoded by mutated genes. Although some mutation has been verified, a number of mutations of PCD remain elusive. The main purpose of this study is to identify mutant genes in a Chinese family with PCD, and to verify the safety and effectiveness of intracytoplasmic sperm injection (ICSI) of infertility caused by PCD.
METHODS: Imaging examination was used to exclude pulmonary inflammation and visceral translocation. Semen analysis was used to assess the quality of the proband's sperm. Transmission electron microscopy (TEM) was conducted to assess the ultrastructure of flagella and cilia. Targeted next generation sequencing and Sanger sequencing and qPCR (real-time quantitative polymerase chain reaction detecting system) were applied to identified mutation of Chinese Family suspected of having PCD. Viable sperm were selected by hypo-osmotic swelling test (HOST) for ICSI.
RESULTS: We report 2 novel mutations in CCDC40 gene (c.1259delA and EX17_20 deletion) resulted in immobility of sperm and infertility of the proband. These mutations were confirmed in the proband's sister (heterozygous) and his parents (recessive carrier) by Sanger sequencing and qPCR. All the spermatozoa from the proband were immotile. Ultrastructural defects were found in flagella and cilia of proband and his sister. Viable sperms were selected by HOST for ICSI and fertilized 9 of 21 eggs. Two frozen embryos were transplanted and a healthy 3500 g boy was delivered at 40 + 4 weeks' gestation. And then, we summarized the genes related to PCD and the mutant sites of CCDC40 gene.
CONCLUSION: We reported 2 novel mutants in CCDC40 gene (c.1259delA and EX17_20 deletion), which could be candidates for genetic diagnosis in PCD patients. The combination of targeted next generation sequencing and Sanger sequencing may be a useful tool to diagnose PCD. ICSI is a considerable method in treatment of infertility caused by PCD.

DOI: https://doi.org/10.1097/MD.0000000000028275
BMJ Open Respir Res. 2021 Dec;pii: e001106. [Epub ahead of print]8(1):

Comparison of the airway microbiota in children with chronic suppurative lung disease.

Bushra Ahmed, Michael J Cox, Leah Cuthbertson, Phillip James, Laura Gardner, William Cookson, Jane Davies, Miriam Moffatt, Andrew Bush.

   RATIONALE: The airway microbiota is important in chronic suppurative lung diseases, such as primary ciliary dyskinesia (PCD) and cystic fibrosis (CF). This comparison has not previously been described but is important because difference between the two diseases may relate to the differing prognoses and lead to pathological insights and potentially, new treatments.
OBJECTIVES: To compare the longitudinal development of the airway microbiota in children with PCD to that of CF and relate this to age and clinical status.
METHODS: Sixty-two age-matched children (age range 0.5-17 years) with PCD or CF (n=31 in each group) were recruited prospectively and followed for 1.1 years. Throat swabs or sputum as well as clinical information were collected at routine clinical appointments. 16S rRNA gene sequencing was performed.
MEASUREMENTS AND MAIN RESULTS: The microbiota was highly individual and more diverse in PCD and differed in community composition when compared with CF. While Streptococcus was the most abundant genus in both conditions, Pseudomonas was more abundant in CF with Haemophilus more abundant in PCD (Padj=0.0005). In PCD only, an inverse relationship was seen in the relative abundance of Streptococcus and Haemophilus with age.
CONCLUSIONS: Bacterial community composition differs between children with PCD and those with CF. Pseudomonas is more prevalent in CF and Haemophilus in PCD, at least until infection with Pseudomonas supervenes. Interactions between organisms, particularly members of Haemophilus, Streptococcus and Pseudomonas genera appear important. Study of the interactions between these organisms may lead to new therapies or risk stratification.

Keywords:  cystic fibrosis; paediatric lung disaese; respiratory infection

DOI:  https://doi.org/10.1136/bmjresp-2021-001106
Cell Mol Life Sci. 2021 Dec 22.

Microgravity and space radiation inhibit autophagy in human capillary endothelial cells, through either opposite or synergistic effects on specific molecular pathways.

Ivana Barravecchia, Chiara De Cesari, Mattia Forcato, Francesca Scebba, Olga V Pyankova, Joanna M Bridger, Helen A Foster, Giovanni Signore, Andrea Borghini, Mariagrazia Andreassi, Massimiliano Andreazzoli, Silvio Bicciato, Mario Enrico Pè, Debora Angeloni.

  Microgravity and space radiation (SR) are two highly influential factors affecting humans in space flight (SF). Many health problems reported by astronauts derive from endothelial dysfunction and impaired homeostasis. Here, we describe the adaptive response of human, capillary endothelial cells to SF. Reference samples on the ground and at 1g onboard permitted discrimination between the contribution of microgravity and SR within the combined responses to SF. Cell softening and reduced motility occurred in SF cells, with a loss of actin stress fibers and a broader distribution of microtubules and intermediate filaments within the cytoplasm than in control cells. Furthermore, in space the number of primary cilia per cell increased and DNA repair mechanisms were found to be activated. Transcriptomics revealed the opposing effects of microgravity from SR for specific molecular pathways: SR, unlike microgravity, stimulated pathways for endothelial activation, such as hypoxia and inflammation, DNA repair and apoptosis, inhibiting autophagic flux and promoting an aged-like phenotype. Conversely, microgravity, unlike SR, activated pathways for metabolism and a pro-proliferative phenotype. Therefore, we suggest microgravity and SR should be considered separately to tailor effective countermeasures to protect astronauts' health.

Keywords:  Cytoskeleton; HMEC-1; Immunofluorescence staining; International Space Station; RNA sequencing; Telomeres

DOI:  https://doi.org/10.1007/s00018-021-04025-z
Respir Med. 2021 Dec 17. pii: S0954-6111(21)00427-3. [Epub ahead of print]191 106719

Respiratory muscle strength, exercise capacity and physical activity in patients with primary ciliary dyskinesia: A cross-sectional study.

Merve Firat, Meral Bosnak-Guclu, Tugba Sismanlar-Eyuboglu, Ayse Tana-Aslan.

   BACKGROUND AND OBJECTIVES: The pathophysiological mechanisms of primary ciliary dyskinesia (PCD) may affect many functions, including respiratory, physical, and health status. This study aimed to compare respiratory muscle strength, inspiratory muscle endurance, muscle strength, exercise capacity, physical activity levels, and quality of life in PCD patients and controls.
METHODS: Twenty-seven patients and 28 controls were included. Respiratory muscle strength (maximal inspiratory (MIP) and maximal expiratory (MEP) pressures), inspiratory muscle endurance (incremental threshold loading test), muscle strength (quadriceps femoris, shoulder abductor, elbow flexor, handgrip), exercise capacity (6-min walk test (6MWT)), physical activity and quality of life (QOL-PCD) were evaluated.
RESULTS: MIP, inspiratory muscle endurance, quadriceps femoris, and handgrip muscle strength, 6MWT distance, total energy expenditure; childrens' (6-12 years) and their parents' physical function, upper, lower respiratory, and hearing symptoms and treatment burden QOL-PCD subscales scores were significantly lower in patients compared with controls (p < 0.05). The 66.7% of patients did not meet the optimal number of steps. MEP, shoulder abductor, and elbow flexor muscle strength, active energy expenditure, physical activity duration, average metabolic equivalents, number of steps, lying time, and sleep duration; childrens' and their parents' other subscales and adolescents' all QOL-PCD subscales scores were similar between groups (p > 0.05).
CONCLUSION: Inspiratory muscle strength and endurance, lower extremity and total muscle strength, exercise capacity, total energy expenditure, and childrens' quality of life are impaired compared to healthy controls. Decreased physical activity level is prevalent in these patients. Effects of pulmonary rehabilitation on these impaired outcomes for PCD patients should be investigated.
TRIAL REGISTRATION: Clinicaltrials.gov: NCT03370029; December 12, 2017.

Keywords:  Exercise tolerance; Physical activity; Primary ciliary dyskinesia; Quality of life; Respiratory muscles

DOI:  https://doi.org/10.1016/j.rmed.2021.106719
Antioxidants (Basel). 2021 Dec 02. pii: 1936. [Epub ahead of print]10(12):

Genome-Wide RNAi Screening Identifies Novel Pathways/Genes Involved in Oxidative Stress and Repurposable Drugs to Preserve Cystic Fibrosis Airway Epithelial Cell Integrity.

Javier Checa, Itziar Martínez-González, Maria Maqueda, Jose Luis Mosquera, Josep M Aran.

  Recurrent infection-inflammation cycles in cystic fibrosis (CF) patients generate a highly oxidative environment, leading to progressive destruction of the airway epithelia. The identification of novel modifier genes involved in oxidative stress susceptibility in the CF airways might contribute to devise new therapeutic approaches. We performed an unbiased genome-wide RNAi screen using a randomized siRNA library to identify oxidative stress modulators in CF airway epithelial cells. We monitored changes in cell viability after a lethal dose of hydrogen peroxide. Local similarity and protein-protein interaction network analyses uncovered siRNA target genes/pathways involved in oxidative stress. Further mining against public drug databases allowed identifying and validating commercially available drugs conferring oxidative stress resistance. Accordingly, a catalog of 167 siRNAs able to confer oxidative stress resistance in CF submucosal gland cells targeted 444 host genes and multiple circuitries involved in oxidative stress. The most significant processes were related to alternative splicing and cell communication, motility, and remodeling (impacting cilia structure/function, and cell guidance complexes). Other relevant pathways included DNA repair and PI3K/AKT/mTOR signaling. The mTOR inhibitor everolimus, the α1-adrenergic receptor antagonist doxazosin, and the Syk inhibitor fostamatinib significantly increased the viability of CF submucosal gland cells under strong oxidative stress pressure. Thus, novel therapeutic strategies to preserve airway cell integrity from the harsh oxidative milieu of CF airways could stem from a deep understanding of the complex consequences of oxidative stress at the molecular level, followed by a rational repurposing of existing "protective" drugs. This approach could also prove useful to other respiratory pathologies.

Keywords:  RNAi screening; airway epithelial cells; cystic fibrosis; data mining; drug databases; oxidative stress

DOI:  https://doi.org/10.3390/antiox10121936