ARMC4 Mutations Cause Primary Ciliary Dyskinesia with Randomization of Left/Right Body Asymmetry

The motive forces for ciliary movement are generated by large multiprotein complexes referred to as outer dynein arms (ODAs), which are preassembled in the cytoplasm prior to transport to the ciliary axonemal compartment. In humans, defects in structural components, docking complexes, or cytoplasmic assembly factors can cause primary ciliary dyskinesia (PCD), a disorder characterized by chronic airway disease and defects in laterality. By using combined high resolution copy-number variant and mutation analysis, we identified ARMC4 mutations in twelve PCD individuals whose cells showed reduced numbers of ODAs and severely impaired ciliary beating. Transient suppression in zebrafish and analysis of an ENU mouse mutant confirmed in both model organisms that ARMC4 is critical for left-right patterning. We demonstrate that ARMC4 is an axonemal protein that is necessary for proper targeting and anchoring of ODAs

Mutations involving the SRY-related gene SOX8 are associated with a spectrum of human reproductive anomalies.

© The Author(s) 2018. Published by Oxford University Press. All rights reserved. SOX8 is an HMG-box transcription factor closely related to SRY and SOX9. Deletion of the gene encoding Sox8 in mice causes reproductive dysfunction but the role of SOX8 in humans is unknown. Here, we show that SOX8 is expressed in the somatic cells of the early developing gonad in the human and influences human sex determination. We identified two individuals with 46, XY disorders/differences in sex development (DSD) and chromosomal rearrangements encompassing the SOX8 locus and a third individual with 46, XY DSD and a missense mutation in the HMG-box of SOX8. In vitro functional assays indicate that this mutation alters the biological activity of the protein. As an emerging body of evidence suggests that DSDs and infertility can have common etiologies, we also analysed SOX8 in a cohort of infertile men (n=274) and two independent cohorts of women with primary ovarian insufficiency (POI; n=153 and n=104). SOX8 mutations were found at increased frequency in oligozoospermic men (3.5%; P < 0.05) and POI (5.06%; P=4.5×10 -5 ) as compared with fertile/normospermic control populations (0.74%). The mutant proteins identified altered SOX8 biological activity as compared with the wild-type protein. These data demonstrate that SOX8 plays an important role in human reproduction and SOX8 mutations contribute to a spectrum of phenotypes including 46, XY DSD, male infertility and 46, XX POI.Link_to_subscribed_fulltex

Pediatric Chronic Sclerosing Sialadenitis: Kuttner Tumor

Chronic sclerosing sialadenitis is an uncommon cause of salivary gland enlargement mainly occurring in the fifth and seventh decade of life. In the Western population, chronic sclerosing sialadenitis has been characterized as an IgG4-related disease. Although rare, this lesion occurs in children. To increase awareness about this entity in the pediatric age group, we report the case of an 11-year-old boy with a hard, 4.0-cm circumscribed mass in the right submandibular gland. Histologically there was marked distortion of the gland architecture by a dense lymphocytic infiltrate and extensive fibrosis with septa that crossed and distorted the gland, leaving atrophic acini and dilated, irregular ducts. The lymphoid infiltrate formed multiple follicles with active germinal centers, numerous plasma cells, and areas with diffuse arrangement. Immunophenotyping showed abundant CD20- and CD3-positive lymphocytes; cytokeratin AE1/AE3 highlighted the distorted architectural pattern; IgG staining showed large numbers of positive cells infiltrating the interstitium and surrounding the atrophic acini and ducts. IgG4 staining revealed a large proportion of positive infiltrating elements. Kuttner tumor belongs to the group of IgG4-related sclerosing diseases. The differential diagnosis includes pleomorphic adenoma and other salivary gland neoplasms. Its recognition in children is important clinically because this entity is amenable to steroid treatment, and additional work up and follow up is warranted to stave off other IgG4-related diseases/complications

Role of the Beta Catenin Destruction Complex in Mediating Chemotherapy-Induced Senescence-Associated Secretory Phenotype

<div><p>Cellular senescence is considered as a tumor suppressive mechanism. Recent evidence indicates however that senescent cells secrete various growth factors and cytokines, some of which may paradoxically promote cancer progression. This phenomenon termed senescence-associated secretory phenotype (SASP) must be inhibited in order for anti-proliferative agents to be effective. The present study was designed to determine whether the β-catenin destruction complex (BCDC), known to integrate the action of various growth factors and cytokines, would represent a suitable target to inhibit the activity of SASP components. For this, we carried out experiments to determine the effect of drug-induced senescence on secretion of SASP, β-catenin transactivation, and the relationship between these processes. Moreover, genetic and pharmacological approaches were used to define the implication of BCDC in mediating the effects of SASP components on cell migration and resistance to drugs. The findings indicate that drug-induced senescence was associated with expression of various Wnt ligands in addition to previously known SASP components. Beta catenin transactivation and expression of genes implicated in epithelial-mesenchymal transition (EMT) also increased in response to drug-induced SASP. These effects were prevented by Pyrvinium, a recently described activator of BCDC. Pyrvinium also suppressed the effects of SASP on cell migration and resistance to doxorubicin. Together, these findings provide insights on the potential role of BCDC in mediating the effects of drug-induced SASP on cancer cell invasion and resistance to therapy, and suggest that targeting this pathway may represent an effective approach to enhance the activity of current and prospective anti-cancer therapeutics.</p> </div

Wnt signaling activation and expression of EMT markers in association with SASP.

<p><u>Panel A.</u> β-catenin transactivation, measured by the Super-Topflash luciferase reporter system (STF) in three melanoma cell lines exposed or not to increased concentrations of doxorubicin. The FopFlash construct containing mutated TCF binding sites was used to define the specificity of doxorubicin’s action. <u>Panel B</u>. Effect of conditioned media from cells pre-treated with increasing concentrations of doxorubicin on TopFlash and FopFlash activities in naïve cells (not previously exposed to the drug). <u>Panel C.</u> Effect of individual SASP components on β-catenin transactivation determined by measure of STF activity. Data in panels A, B and C represents the average of three determinations ±SE. Statistical significance is shown for drug-treated cells versus control (*p<0.05, **p<0.001). <u>Panel D.</u> Western blots showing expression of EMT genes in WM 115 cells incubated with conditioned medium from their senescent counterparts. β-actin is used as a loading control. β-cat (c): β catenin in the cytoplasm, β-cat (N): β-catenin in the nucleus.</p

Effect of pyrvinium on SASP-induced expression of mdr1 gene and resistance to doxorubicin.

<p><u>Panel A.</u> Pyrvinium was added at the indicated concentrations to 293 cells transfected with the PGL3-mdr1-luciferase (mdr1-Luc) and then incubated with CM from those treated with senescence inducing concentration of doxorubicin. After 24 h, the luciferase activity was measured and plotted as a ratio of luminescence measured in cells transfected with the mdr1-Luc versus those transfected with the PGL3-luciferase. <u>Panel B.</u> Effect of pyrvinium on rhodamine 123 uptake (1 hr) and efflux (2 hr). <u>Panel C and D</u>. Effect of pyrvinium (PYR) on cell viability in the melanoma cell line 266, determined morphologically and by MTT assay respectively. The data in panels A and D represent average ± SE of three determinations (*p<0.05, **p<00.1).</p

Role of GSK3-associated β-catenin degradation complex in mediating β-catenin transactivation by SASP.

<p><u>Panel A.</u> Effect of enhanced GSK 3β activity on β-catenin integrity. 293 cells were transfected with a constitutively active form of GSK 3β (GSK3-S9), and expression levels of this enzyme and as well as β-catenin determined by western blot. β-actin is used here as a loading control. <u>Panel B.</u> RT-PCR analysis showing expression levels of GSK 3β, β-catenin and GAPDH in transfected and non-transfected cells as described in panel A. <u>Panel C.</u> Effect of GSK 3β activation on SASP induced β-catenin transactivation. GSK3-S9 transfected and non-transfected cells were incubated in the absence or the presence of CM from those treated with senescence inducing concentration of doxorubicin. STF activity was measured after 24 h. The data represents the average of three determinations ±SE (**p<0.001).</p

Targeting BCDC inhibits β-catenin transactivation, EMT and cell migration.

<p><u>Panel A. </u>Effect of direct and indirect activators of the BCDC on β-catenin transactivation as measured by STF activity. STF transfected cells were pre-treated with the indicated effectors for 2 hrs and then exposed to CM from those pre-treated with senescence-inducing doxorubicin concentration for an additional 24 h, followed by measure of STF activity. LY: PI3 kinase inhibitor. (LY294002), PD: MAP kinase inhibitor (PD98059), Y27: Rho kinase inhibitor (Y27632), RAP: mTOR inhibitor (rapamycin). XAV: Tankyrase inhibitor (XAV939), and PYR: casein kinase 1 activator (Pyrvinium). <u>Panel B</u>. Effect of Pyrvinium (PY) on Wnt3a- mediated activation of TopFlash. Cells were exposed for 24 hours to Wnt 3a at 10 or 50 ng/ml (Wnt-10 and Wnt-50 respectively), in the absence or the presence of pyrvinium (PYR at 500 nM). Data in panels A and B represents average of three determinations ±SE. Statistical significance is shown in panel A for drug-treated cells versus control, and in panel B between Wnt-50+PYR compared to Wnt-50-treated cells (*p<0.05, **p<0.001). <u>Panels C and D</u>. Effect of cellular exposure to PYR for 24 h on the expression of Zeb1 at the protein (Western blot) and the mRNA level (RT-PCR). <u>Panel E.</u> Effect of PYR (500 nM) on cell migration as determined by the monolayer scratch assay described in the method section.</p

Approaches to Research Determination of Late Acute Cellular Rejection in Pediatric Liver Transplant Recipients

A central pathology or site reading of biopsy slides is used in liver transplant clinical trials to determine rejection. We evaluated interrater reliability of readings of "rejection or not" using digitized slides from the Medication Adherence in Children who had a Liver Transplant (MALT) study. Four masked experienced pathologists read the digitized slides and then reread them after a study-specific histologic endpoint development program. Agreement was expressed throughout as a Kappa or Fleiss Kappa statistic (ҡ). A ҡ&nbsp;&gt;&nbsp;0.6 was predefined as desirable. Readings were correlated with immunosuppressant adherence (the Medication Level Variability Index, [MLVI]), and maximal liver enzyme levels during the study period. Interrater agreement between site and central review in MALT, and between 4 pathologists later on, was low (ҡ&nbsp;=&nbsp;0.44, Fleiss ҡ&nbsp;=&nbsp;0.41, respectively). Following the endpoint development program, agreement improved and became acceptable (ҡ&nbsp;=&nbsp;0.71). The final reading was better-aligned with maximal gamma-glutamyl transferase levels and MLVI as compared with the original central reading. We found substantial disagreement between experienced pathologists reading the same slides. A unique study-specific procedure improved interrater reliability to the point it was acceptable. Such a procedure may be indicated to increase reliability of histopathologic determinations in future research, and perhaps also clinically

Identification of Intestinal Ion Transport Defects in Microvillus Inclusion Disease

Loss of function mutations in the actin motor Myosin Vb (Myo5b) lead to Microvillus Inclusion Disease (MVID) and death in newborns and children. MVID results in secretory diarrhea (SD), brush border (BB) defects, villus atrophy and microvillus inclusions (MVIs) in enterocytes. How loss of Myo5b results in increased stool loss of chloride (Cl(-)) and sodium (Na(+)) is unknown. The current study used Myo5b loss of function human MVID intestine, polarized intestinal cell models of secretory crypt (T84) and villus resembling (CaCo2BBe, C2BBe) enterocytes lacking Myo5b in conjunction with immunofluorescence confocal gSTED imaging, immunohistochemical staining, TEM, shRNA silencing, immunoblots, and electrophysiological approaches to examine the distribution, expression and function of the major BB ion transporters NHE3 (Na(+)), CFTR (Cl(-)) and SLC26A3 (DRA) (Cl(-) /HCO3(-)) that control intestinal fluid transport. We hypothesized that enterocyte maturation defects lead villus atrophy with immature secretory crypt like enterocytes in the MVID epithelium. We investigated the role of Myo5b in enterocyte maturation. NHE3 and DRA localization and function were markedly reduced on the BBM of human MVID enterocytes and Myo5bKD C2BBe cells, while CFTR localization was preserved. Forskolin-stimulated CFTR ion transport in Myo5bKD T84 cells resembled that of control. Loss of Myo5b led to YAP1 nuclear retention, retarded enterocyte maturation and a crypt-like phenotype. We conclude that preservation of functional CFTR in immature enterocytes, reduced functional expression of NHE3 and DRA contribute to Cl(-) and Na+ stool loss in MVID diarrhea