Ankyrin Repeat Domain-containing Protein Research Articles

Roles Played by DOCK11, a Guanine Nucleotide Exchange Factor, in HBV Entry and Persistence in Hepatocytes

HBV infection is challenging to cure due to the persistence of viral covalently closed circular viral DNA (cccDNA). The dedicator of cytokinesis 11 (DOCK11) is recognized as a guanine nucleotide exchange factor (GEF) for CDC42 that has been reported to be required for HBV persistence. DOCK11 is expressed in both the cytoplasm and nucleus of human hepatocytes and is functionally associated with retrograde trafficking proteins Arf-GAP with GTPase domain, ankyrin repeat, and pleckstrin homology domain-containing protein 2 (AGAP2), and ADP-ribosylation factor 1 (ARF1), together with the HBV capsid, in the trans-Golgi network (TGN). This opens an alternative retrograde trafficking route for HBV from early endosomes (EEs) to the TGN and then to the endoplasmic reticulum (ER), thereby avoiding lysosomal degradation. DOCK11 also facilitates the association of cccDNA with H3K4me3 and RNA Pol II for activating cccDNA transcription. In addition, DOCK11 plays a crucial role in the host DNA repair system, being essential for cccDNA synthesis. This function can be inhibited by 10M-D42AN, a novel DOCK11-binding peptide, leading to the suppression of HBV replication both in vitro and in vivo. Treatment with a combination of 10M-D42AN and entecavir may represent a promising therapeutic strategy for patients with chronic hepatitis B (CHB). Consequently, DOCK11 may be seen as a potential candidate molecule in the development of molecularly targeted drugs against CHB.

Nuclear AGO2 promotes myocardial remodeling by activating ANKRD1 transcription in failing hearts

Heart failure (HF) is manifested by transcriptional and post-transcriptional reprogramming of critical genes. Multiple studies have revealed that microRNAs could translocate into subcellular organelles such as nucleus to modify gene expression. However, the functional property of subcellular Argonaute2 (AGO2), the core member of the microRNA machinery, has remained elusive in HF. AGO2 was found to be localized in both cytoplasm and nucleus of cardiomyocytes, and robustly increased in failing hearts of patients and animal models. We demonstrated nuclear AGO2 rather than cytosolic AGO2 overexpression by recombinant adeno-associated virus (serotype 9) with cardiomyocyte-specific troponin T promoter exacerbated the cardiac dysfunction in transverse aortic constriction (TAC)-operated mice. Mechanistically, nuclear AGO2 activates the transcription of ANKRD1, encoding Ankyrin repeat domain-containing protein 1 (ANKRD1), which also has a dual function in the cytoplasm as part of I-band of the sarcomere and in the nucleus as a transcriptional cofactor. Overexpression of nuclear ANKRD1 recaptured some key features of cardiac remodeling by inducing pathological MYH7 activation, whereas cytosolic ANKRD1 seemed cardio-protective. For clinical practice, we found ivermectin, an anti-parasite drug, and ANPep, a ANKRD1 nuclear location signal mimetic peptide, were able to prevent ANKRD1 nuclear import, resulting in the improvement of cardiac performance in TAC-induced HF.

Functional investigation of a novel ANKRD11 frameshift variant identified in a Chinese family with KBG syndrome

KBG syndrome is a rare autosomal dominant condition characterized by multisystem developmental disorder, primarily caused by loss-of-function variants in ankyrin repeat domain-containing protein 11 (ANKRD11). Approximately 80 % of ANKRD11 variants associated with KBG syndrome, are frameshift and nonsense variants. Current insight into the pathogenesis of KBG syndrome resulting from ANKRD11 truncating variants remains limited. Here, we presented two members from a non-consanguineous Chinese pedigree both exhibiting characteristics fitting the KBG syndrome-associated phenotypic spectrum. Whole-exome sequencing identified a novel heterozygous frameshift variant in ANKRD11 (NM_013275.6, c.2280_2281delGT, p.Y761Qfs*20) in the proband. Sanger sequencing confirmed that the variant was inherited from her mother and co-segregated with KBG syndrome phenotype. In vitro functional assays revealed that the frameshift variant escaped nonsense-mediated mRNA decay, and resulting in a truncated protein with significantly increased expression levels compared to full-length ANKRD11. Immunofluorescence results demonstrated that truncated protein was predominantly expressed in the nucleus of HEK293 cells, while wild-type ANKRD11 was equally distributed in both the nucleus and cytoplasm. Moreover, the truncated protein significantly reduced CDKN1A/P21-promoter luciferase activity in comparison to wild-type ANKRD11 protein, as well as a remarkably decrease in the endogenous CDKN1A/P21 mRNA level in HEK293 cells. These findings suggest a loss of transcriptional activation function and potentially a dominant-negative mechanism. Overall, our study expands the mutational spectrum of ANKRD11 gene and provides new insights into the pathogenic mechanism of KBG syndrome caused by ANKRD11 truncating variants.

ANKRD22 aggravates sepsis-induced ARDS and promotes pulmonary M1 macrophage polarization

Acute respiratory distress syndrome (ARDS) is independently associated with a poor prognosis in patients with sepsis. Macrophage M1 polarization plays an instrumental role in this process. Therefore, the exploration of key molecules affecting acute lung injury and macrophage M1 polarization may provide therapeutic targets for the treatment of septic ARDS. Here, we identified that elevated levels of Ankyrin repeat domain-containing protein 22 (ANKRD22) were associated with poor prognosis and more pronounced M1 macrophage polarization in septic patients by analyzing high-throughput data. ANKRD22 expression was also significantly upregulated in the alveolar lavage fluid, peripheral blood, and lung tissue of septic ARDS model mice. Knockdown of ANKRD22 significantly attenuated acute lung injury in mice with sepsis-induced ARDS and reduced the M1 polarization of lung macrophages. Furthermore, deletion of ANKRD22 in macrophages inhibited M1 macrophage polarization and reduced levels of phosphorylated IRF3 and intracellular interferon regulatory factor 3 (IRF3) expression, while re-expression of ANKRD22 reversed these changes. Further experiments revealed that ANKRD22 promotes IRF3 activation by binding to mitochondrial antiviral-signaling protein (MAVS). In conclusion, these findings suggest that ANKRD22 promotes the M1 polarization of lung macrophages and exacerbates sepsis-induced ARDS.

ANKS1A-Deficiency Aberrantly Increases the Entry of the Protein Transport Machinery into the Ependymal Cilia.

In this study, we examine whether a change in the protein levels for FOP in Ankyrin repeat and SAM domain-containing protein 1A (ANKS1A)-deficient ependymal cells affects the intraflagellar transport (IFT) protein transport system in the multicilia. Three distinct abnormalities are observed in the multicilia of ANKS1A-deficient ependymal cells. First, there were a greater number of IFT88-positive trains along the cilia from ANKS1A deficiency. The results are similar to each isolated cilium as well. Second, each isolated cilium contains a significant increase in the number of extracellular vesicles (ECVs) due to the lack of ANKS1A. Third, Van Gogh-like 2 (Vangl2), a ciliary membrane protein, is abundantly detected along the cilia and in the ECVs attached to them for ANKS1A-deficient cells. We also use primary ependymal culture systems to obtain the ECVs released from the multicilia. Consequently, we find that ECVs from ANKS1A-deficient cells contain more IFT machinery and Vangl2. These results indicate that ANKS1A deficiency increases the entry of the protein transport machinery into the multicilia and as a result of these abnormal protein transports, excessive ECVs form along the cilia. We conclude that ependymal cells make use of the ECV-based disposal system in order to eliminate excessively transported proteins from basal bodies.

Evaluating the Molecular Properties and Function of ANKHD1, and Its Role in Cancer.

Ankyrin repeat and single KH domain-containing protein 1 (ANKHD1) is a large, scaffolding protein composed of two stretches of ankyrin repeat domains that mediate protein-protein interactions and a KH domain that mediates RNA or single-stranded DNA binding. ANKHD1 interacts with proteins in several crucial signalling pathways, including receptor tyrosine kinase, JAK/STAT, mechanosensitive Hippo (YAP/TAZ), and p21. Studies into the role of ANKHD1 in cancer cell lines demonstrate a crucial role in driving uncontrolled cellular proliferation and growth, enhanced tumorigenicity, cell cycle progression through the S phase, and increased epithelial-to-mesenchymal transition. Furthermore, at a clinical level, the increased expression of ANKHD1 has been associated with greater tumour infiltration, increased metastasis, and larger tumours. Elevated ANKHD1 resulted in poorer prognosis, more aggressive growth, and a decrease in patient survival in numerous cancer types. This review aims to gather the current knowledge about ANKHD1 and explore its molecular properties and functions, focusing on the protein's role in cancer at both a cellular and clinical level.

Associated and intermediate factors between genetic variants of the dopaminergic D2 receptor gene and harmful alcohol use in young adults.

Dopamine receptor D2 (DRD2) and ankyrin repeat and kinase domain-containing protein 1 (ANKK1) genes have received considerable attention for their involvement in alcohol use disorder (AUD), but many questions remain on their exact role. We conducted a population-based case-control and genetic association study in a large sample of young adults. Our aim was to assess the association between DRD2 and ANKK1 single nucleotide polymorphisms (SNPs) and harmful alcohol use, disentangling associated and possible intermediate factors. A total of 1841 college students from the French region Champagne-Ardennes, aged between 18 and 21 years and who reported at least one lifetime alcohol consumption, were included in this study. Allele frequencies were analysed according to harmful alcohol use (assessed through the Alcohol Use Disorder Identification Test [AUDIT] questionnaire). Different substance use disorders, including nicotine and cannabis dependences, were also assessed through questionnaires, as was a list of potential associated factors (e.g., major depressive episode, conduct disorder, attention-deficit/hyperactivity disorder [ADHD], school failure, sugar consumption, sexual trauma, parents' use of alcohol, tobacco or cannabis). We found that DRD2 rs1800498 was associated with harmful alcohol use. Many factors were detected, but a global path analysis revealed that DRD2 rs1800498 had a significant direct effect on harmful alcohol use and that early age at first alcohol consumption and depressive symptoms moderated this effect. This study suggests an interplay between harmful alcohol use, DRD2 genotypes and other risk factors that, with a full understanding, could be useful for preventive purposes.

Dendritic Spine in Autism Genetics: Whole-Exome Sequencing Identifying De Novo Variant of CTTNBP2 in a Quad Family Affected by Autism Spectrum Disorder.

Autism spectrum disorder (ASD) affects around 1% of children with no effective blood test or cure. Recent studies have suggested that these are neurological disorders with a strong genetic basis and that they are associated with the abnormal formation of dendritic spines. Chromosome microarray (CMA) together with high-throughput sequencing technology has been used as a powerful tool to identify new candidate genes for ASD. In the present study, CMA was first used to scan for genome-wide copy number variants in a proband, and no clinically significant copy number variants were found. Whole-exome sequencing (WES) was used further for genetic testing of the whole quad family affected by ASD, including the proband, his non-autistic sister, and his parents. Sanger sequencing and MassARRAY-based validation were used to identify and confirm variants associated with ASD. WES yielded a 151-fold coverage depth for each sample. A total of 98.65% of the targeted whole-exome region was covered at >20-fold depth. A de novo variant in CTTNBP2, p.M115T, was identified. The CTTNBP2 gene belongs to a family of ankyrin repeat domain-containing proteins associated with dendritic spine formation. Although CTTNBP2 has been associated with ASD, limited studies have been developed to identify clinically relevant de novo mutations of CTTNBP2 in children with ASD; family-based WES successfully identified a clinically relevant mutation in the CTTNBP2 gene in a quad family affected by ASD. Considering the neuron-specific expression of CTTNBP2 and its role in dendritic spine formation, our results suggest a correlation between the CTTNBP2 mutation and ASD, providing genetic evidence for ASD spine pathology. Although the present study is currently insufficient to support the assertion that the de novo mutation M115T in CTTNBP2 directly causes the autism phenotype, our study provides support for the assertion that this mutation is a candidate clinically relevant variant in autism.

Hepatitis B Virus Utilizes a Retrograde Trafficking Route via the Trans-Golgi Network to Avoid Lysosomal Degradation

Hepatitis B virus (HBV) infection is difficult to cure owing to the persistence of covalently closed circular viral DNA (cccDNA). We performed single-cell transcriptome analysis of newly established HBV-positive and HBV-negative hepatocellular carcinoma cell lines and found that dedicator of cytokinesis 11 (DOCK11) was crucially involved in HBV persistence. However, the roles of DOCK11 in the HBV lifecycle have not been clarified. The cccDNA levels were measured by Southern blotting and real-time detection polymerase chain reaction in various hepatocytes including PXB cells by using an HBV-infected model. The retrograde trafficking route of HBV capsid was investigated by super-resolution microscopy, proximity ligation assay, and time-lapse analysis. The downstream molecules of DOCK11 and underlying mechanism were examined by liquid chromatography-tandem mass spectrometry, immunoblotting, and enzyme-linked immunosorbent assay. The cccDNA levels were strongly increased by DOCK11 overexpression and repressed by DOCK11 suppression. Interestingly, DOCK11 functionally associated with retrograde trafficking proteins in the trans-Golgi network (TGN), Arf-GAP with GTPase domain, ankyrin repeat, and pleckstrin homology domain-containing protein 2 (AGAP2), and ADP-ribosylation factor 1 (ARF1), together with HBV capsid, to open an alternative retrograde trafficking route for HBV from early endosomes (EEs) to the TGN and then to the endoplasmic reticulum (ER), thereby avoiding lysosomal degradation. Clinically, DOCK11 levels in liver biopsies from patients with chronic hepatitis B were significantly reduced by entecavir treatment, and this reduction correlated with HBV surface antigen levels. HBV uses a retrograde trafficking route via EEs-TGN-ER for infection that is facilitated by DOCK11 and serves to maintain cccDNA. Therefore, DOCK11 is a potential therapeutic target to prevent persistent HBV infection.

ANKHD1 contributes to the malignant phenotype of triple-negative breast cancer cells.

Ankyrin repeat and KH domain-containing protein 1, ANKHD1, has been identified as a regulator of signaling pathways and cellular processes of relevance in carcinogenesis. However, the role of ANKHD1 in breast cancer remains unclear. The aim of the present study was to characterize the expression pattern and involvement of ANKHD1 in the malignant phenotype of breast cancer cell lines and to investigate the clinical relevance of ANKHD1 in a breast cancer context. Gene and protein expressions were assessed in the cell lines by quantitative reverse transcription PCR and Western blot analysis, respectively, and ANKHD1 silencing through siRNA transfection was conducted for further in vitro functional assays. The expression of ANKHD1 was identified in non-tumorigenic breast epithelium and breast cancer cell lines, but differences in cellular localization were found among the neoplasia subtypes. ANKHD1 silencing reduced the viability, clonogenicity, and migration of triple-negative breast cancer (TNBC) cells. Bioinformatics analyses demonstrated that patients with triple-negative basal-like 2 and mesenchymal breast cancer subtypes had high ANKHD1 expression associated with poor recurrence-free survival. Therefore, these data indicate that ANKHD1 relevance in breast cancer varies among its subtypes, indicating the importance of ANKHD1 in TNBC.

Riociguat attenuates the changes in left ventricular proteome and microRNA profile after experimental aortic stenosis in mice.

Development and progression of heart failure involve endothelial and myocardial dysfunction as well as a dysregulation of the NO-sGC-cGMP signalling pathway. Recently, we reported that the sGC stimulator riociguat has beneficial effects on cardiac remodelling and progression of heart failure in response to chronic pressure overload. Here, we examined if these beneficial effects of riociguat were also reflected in alterations of the myocardial proteome and microRNA profiles. Male C57BL/6N mice underwent transverse aortic constriction (TAC) and sham-operated mice served as controls. TAC and sham animals were randomised and treated with either riociguat or vehicle for 5 weeks, starting 3 weeks after surgery, when cardiac hypertrophy was established. Afterwards, we performed mass spectrometric proteome analyses and microRNA sequencing of proteins and RNAs, respectively, isolated from left ventricles (LVs). TAC-induced changes of the LV proteome were significantly reduced by treatment with riociguat. Bioinformatics analyses revealed that riociguat improved TAC-induced cardiovascular disease-related pathways, metabolism and energy production, for example, reversed alterations in the levels of myosin heavy chain 7, cardiac phospholamban and ankyrin repeat domain-containing protein 1. Riociguat also attenuated TAC-induced changes of microRNA levels in the LV. The sGC stimulator riociguat exerted beneficial effects on cardiac structure and function during pressure overload, which was accompanied by a reversal of TAC-induced changes of the cardiac proteome and microRNA profile. Our data support the potential of riociguat as a novel therapeutic agent for heart failure.

Kank1 Is Essential for Myogenic Differentiation by Regulating Actin Remodeling and Cell Proliferation in C2C12 Progenitor Cells.

Actin cytoskeleton dynamics are essential regulatory processes in muscle development, growth, and regeneration due to their modulation of mechanotransduction, cell proliferation, differentiation, and morphological changes. Although the KN motif and ankyrin repeat domain-containing protein 1 (Kank1) plays a significant role in cell adhesion dynamics, actin polymerization, and cell proliferation in various cells, the functional significance of Kank1 during the myogenic differentiation of progenitor cells has not been explored. Here, we report that Kank1 acts as a critical regulator of the proliferation and differentiation of muscle progenitor cells. Kank1 was found to be expressed at a relatively high level in C2C12 myoblasts, and its expression was modulated during the differentiation. Depletion of Kank1 by siRNA (siKank1) increased the accumulation of filamentous actin (F-actin). Furthermore, it facilitated the nuclear localization of Yes-associated protein 1 (YAP1) by diminishing YAP1 phosphorylation in the cytoplasm, which activated the transcriptions of YAP1 target genes and promoted proliferation and cell cycle progression in myoblasts. Notably, depletion of Kank1 suppressed the protein expression of myogenic regulatory factors (i.e., MyoD and MyoG) and dramatically inhibited myoblast differentiation and myotube formation. Our results show that Kank1 is an essential regulator of actin dynamics, YAP1 activation, and cell proliferation and that its depletion impairs the myogenic differentiation of progenitor cells by promoting myoblast proliferation triggered by the F-actin-induced nuclear translocation of YAP1.

KANK4 Promotes Arteriogenesis by Potentiating VEGFR2 Signaling in a TALIN-1-Dependent Manner.

Arteriogenesis plays a critical role in maintaining adequate tissue blood supply and is related to a favorable prognosis in arterial occlusive diseases. Strategies aimed at promoting arteriogenesis have thus far not been successful because the factors involved in arteriogenesis remain incompletely understood. Previous studies suggest that evolutionarily conserved KANK4 (KN motif and ankyrin repeat domain-containing proteins 4) might involve in vertebrate vessel development. However, how the KANK4 regulates vessel function remains unknown. We aim to determine the role of endothelial cell-specifically expressed KANK4 in arteriogenesis. The role of KANK4 in regulating arteriogenesis was evaluated using Kank4-/- and KANK4iECOE mice. Molecular mechanisms underlying KANK4-potentiated arteriogenesis were investigated by employing RNA transcriptomic profiling and mass spectrometry analysis. By analyzing Kank4-EGFP reporter mice, we showed that KANK4 was specifically expressed in endothelial cells. In particular, KANK4 displayed a dynamic expression pattern from being ubiquitously expressed in all endothelial cells of the developing vasculature to being explicitly expressed in the endothelial cells of arterioles and arteries in matured vessels. In vitro microfluidic chip-based vascular morphology analysis and in vivo hindlimb ischemia assays using Kank4-/- and KANK4iECOE mice demonstrated that deletion of KANK4 impaired collateral artery growth and the recovery of blood perfusion, whereas KANK4 overexpression leads to increased vessel caliber and blood perfusion. Bulk RNA sequencing and Co-immunoprecipitation/mass spectrometry (Co-IP/MS) analysis identified that KANK4 promoted EC proliferation and collateral artery remodeling through coupling VEGFR2 (vascular endothelial growth factor receptor 2) to TALIN-1, which augmented the activation of the VEGFR2 signaling cascade. This study reveals a novel role for KANK4 in arteriogenesis in response to ischemia. KANK4 links VEGFR2 to TALIN-1, resulting in enhanced VEGFR2 activation and increased EC proliferation, highlighting that KANK4 is a potential therapeutic target for promoting arteriogenesis for arterial occlusive diseases.

Comparative Transcriptome Analysis of Onion in Response to Infection by Alternaria porri (Ellis) Cifferi.

Purple blotch (PB) is one of the most destructive foliar diseases of onion and other alliums, caused by a necrotrophic fungal pathogen Alternaria porri. There are no reports on the molecular response of onion to PB infection. To elucidate the response of onion to A. porri infection, we consequently carried out an RNAseq analysis of the resistant (Arka Kalyan; AK) and susceptible (Agrifound rose; AFR) genotype after an artificial infection. Through differential expression analyses between control and pathogen-treated plants, we identified 8,064 upregulated and 248 downregulated genes in AFR, while 832 upregulated and 564 downregulated genes were identified in AK. A further significant reprogramming in the gene expression profile was also demonstrated by a functional annotation analysis. Gene ontology (GO) terms, which are particularly involved in defense responses and signaling, are overrepresented in current analyses such as “oxidoreductase activity,” “chitin catabolic processes,” and “defense response.” Several key plant defense genes were differentially expressed on A. porri infection, which includes pathogenesis-related (PR) proteins, receptor-like kinases, phytohormone signaling, cell-wall integrity, cytochrome P450 monooxygenases, and transcription factors. Some of the genes were exclusively overexpressed in resistant genotype, namely, GABA transporter1, ankyrin repeat domain-containing protein, xyloglucan endotransglucosylase/hydrolase, and PR-5 (thaumatin-like). Antioxidant enzyme activities were observed to be increased after infection in both genotypes but higher activity was found in the resistant genotype, AK. This is the first report of transcriptome profiling in onion in response to PB infection and will serve as a resource for future studies to elucidate the molecular mechanism of onion-A. porri interaction and to improve PB resistance in onions.

The role of Hippo pathway signaling and A-kinase anchoring protein 13 in primordial follicle activation and inhibition

To determine whether the mechanotransduction and pharmacomanipulation of A-kinase anchoring protein 13 (AKAP13) altered Hippo signaling pathway transcription and growth factors in granulosa cells. Primary ovarian insufficiency is the depletion or dysfunction of primordial ovarian follicles. Invitro activation of ovarian tissue in patients with primary ovarian insufficiency alters the Hippo and phosphatase and tensin homolog/phosphatidylinositol 3-kinase/protein kinase B/forkhead box O3 pathways. A-kinase anchoring protein 13 is found in granulosa cells and may regulate the Hippo pathway via F-actin polymerization resulting in altered nuclear yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif coactivators and Tea domain family (TEAD) transcription factors. Laboratory studies. Translational science laboratory. None. COV434 cells, derived from a primary human granulosa tumor cell line, were studied under different cell density and well stiffness conditions. Cells were transfected with a TEAD-luciferase (TEAD-luc) reporter as well as expression constructs for AKAP13 or AKAP13 mutants and then treated with AKAP13 activators, inhibitors, and follicle-stimulating hormone. TEAD gene activation or inhibition was measured by TEAD-luciferase assays. The messenger ribonucleic acid levels of Hippo pathway signaling molecules, including connective tissue growth factor (CTGF), baculoviral inhibitors of apoptosis repeat-containing 5, Ankyrin repeat domain-containing protein 1, YAP1, and TEAD1, were measured by quantitative real-time polymerase chain reaction. Protein expressions for AKAP13, CTGF, YAP1, and TEAD1 were measured using Western blot. Increased TEAD-luciferase activity and expression of markers for cellular growth were associated with decreased cell density, increased well stiffness, and AKAP13 activator (A02) treatment. Additionally, decreased TEAD-luc activity and expression of markers for cellular growth were associated with AKAP13 inhibitor (A13) treatment, including a reduced expression of the BIRC5 and ANKRD1 (YAP-responsive genes) transcript levels and CTGF protein levels. There were no changes in TEAD-luc with follicle-stimulating hormone treatment, supporting Hippo pathway involvement in the gonadotropin-independent portion of folliculogenesis. These findings suggest that AKAP13 mediates Hippo-regulated changes in granulosa cell growth via mechanotransduction and pharmacomanipulation. The AKAP13 regulation of the Hippo pathway may represent a potential target for regulation of follicle activation.

Effects of N-Acetyl cysteine on gene expression of Protein kinase C-α and Ankyrin repeat domain-containing protein 1 in the cardiac tissue of rats exposed to lead

Objective: This study aimed to evaluate the effects of N-acetylcysteine (NAC) on the oxidative damage, inflammation and expression of PKC-α and ANKRD1 genes in the cardiac tissue of rats exposed to lead (Pb). Methods: Rats were randomly divided into five groups including G1 (control), G2 (single dose of Pb), G3 (continuous dose of Pb), G4 (single dose of Pb + NAC), and G5 (continuous dose of Pb + NAC). Levels of malondialdehyde (MDA), total antioxidant capacity (TAC), IL-10 and TNF-α were measured. Expression of PKC-α and ANKRD1 genes was considered using RT-PCR. Results: Continuous exposure to Pb caused a significant decrease in serum levels of TAC and IL-10, but increased MDA and TNF-α contents (p<0.001). Continuous dose of Pb also dramatically increased the expression of PKC-α and ANKRD1 genes in the cardiac tissue by 4.27-fold and 3.07-fold, respectively (p<0.001). NAC treatments not only improved morphological changes, oxidative stress and inflammatory biomarkers, but also compensated antioxidant capacity and expression of PKC-α and ANKRD1 genes in cardiac tissue. Conclusion: Pb exposure is strongly associated with cardiotoxicity through inducing oxidative stress, inflammation, and antioxidant depletion. NAC ameliorates Pb-induced cardiotoxicity by elevating antioxidants capacity, mitigating oxidative stress and down-regulating of PKC-α and ANKRD1 genes.

Mask, the Drosophila ankyrin repeat and KH domain-containing protein, affects microtubule stability.

ABSTRACTProper regulation of microtubule (MT) stability and dynamics is vital for essential cellular processes, including axonal transportation and synaptic growth and remodeling in neurons. In the present study, we demonstrate that the Drosophila ankyrin repeat and KH domain-containing protein Mask negatively affects MT stability in both larval muscles and motor neurons. In larval muscles, loss-of-function of mask increases MT polymer length, and in motor neurons, loss of mask function results in overexpansion of the presynaptic terminal at the larval neuromuscular junctions (NMJs). mask genetically interacts with stathmin (stai), a neuronal modulator of MT stability, in the regulation of axon transportation and synaptic terminal stability. Our structure–function analysis of Mask revealed that its ankyrin repeats domain-containing N-terminal portion is sufficient to mediate Mask's impact on MT stability. Furthermore, we discovered that Mask negatively regulates the abundance of the MT-associated protein Jupiter in motor neuron axons, and that neuronal knocking down of Jupiter partially suppresses mask loss-of-function phenotypes at the larval NMJs. Taken together, our studies demonstrate that Mask is a novel regulator for MT stability, and such a role of Mask requires normal function of Jupiter.

Label-free proteomic analysis reveals differentially expressed Wolbachia proteins in Tyrophagus putrescentiae: Mite allergens and markers reflecting population-related proteome differences

Tyrophagus putrescentiae is an astigmatid mite of great economic, medical and veterinary importance. The microbiome, especially intracellular bacteria, may affect allergy/allergen expression. We targeted Wolbachia proteins, allergen comparisons and markers in Wolbachia–mite interactions in three mite populations. A decoy database was constructed by proteogenomics using the T. putrescentiae draft genome, Wolbachia transcriptome assembly and current T. putrescentiae-related sequences in GenBank. Among thousands of mite-derived proteins, 18 Wolbachia proteins were reliably identified. We suggest that peroxiredoxin, bacterioferritin, ankyrin repeat domain-containing protein and DegQ family serine endoprotease indicate a higher-level bacterium–bacterium–host interaction. We produced evidence that the host–Wolbachia interaction is modulated through pattern recognition receptors (PRRs), mannose-binding lectins/mannose receptors, the cholinergic anti-inflammatory pathway with TNF-α, and others. We observed Tyr p 3 suppression in mites with Wolbachia, linking trypsin to PRR modulation. Nine out of the 12 current WHO/IUIS official allergens were reliably identified, but the remaining three allergens, Tyr p 1, 8 and 35, were detected as only trace hits. This study provides numerous markers for further Wolbachia–host interaction research. For accuracy, mite allergens should be considered according to abundance in species, but mite populations/strains, as well as their microbiome structure, may be key factors. SignificanceThe astigmatid mites occurring in homes are significant producers of allergens that are highly dangerous to humans and domesticated animals. Mites are tightly associated with microorganisms that affect their biology and consequently allergy signatures. Mite populations were found to be infected with certain intracellular bacteria, but some populations lacked an intracellular bacterium. Our previous research showed that some populations of Tyrophagus putrescentiae are infected with Wolbachia, but some populations host additional bacteria of interest. Thus, there are not only interactions between the mites and Wolbachia but also likely an additional level of interaction that can be found in the interaction between different bacteria in the mites. These “higher-level” signatures and consequences that bacteria affect, including allergen production, are not understood in mites. In this study, we identified Wolbachia-specific proteins in mites for the first time. This study provides Wolbachia- and mite-derived markers that can be clues for describing “higher-level” mite-bacterium-bacterium interactions. Indeed, the microbiome contribution to allergies can potentially be derived directly from bacterial proteins, especially if they are abundant.

MAML1: a coregulator that alters endometrial epithelial cell adhesive capacity

BackgroundAbnormalities in endometrial receptivity has been identified as a major barrier to successful embryo implantation. Endometrial receptivity refers to the conformational and biochemical changes occurring in the endometrial epithelial layer which make it adhesive and receptive to blastocyst attachment. This takes place during the mid-secretory phase of woman’s menstrual cycle and is a result of a delicate interplay between numerous hormones, cytokines and other factors. Outside of this window, the endometrium is refractory to an implanting blastocyst. It has been shown that Notch ligands and receptors are dysregulated in the endometrium of infertile women. Mastermind Like Transcriptional Coactivator 1 (MAML1) is a known coactivator of the Notch signaling pathway. This study aimed to determine the role of MAML1 in regulating endometrial receptivity.MethodsThe expression and localization of MAML1 in the fertile human endometrium (non-receptive proliferative phase versus receptive mid-secretory phase) were determined by immunohistochemistry. Ishikawa cells were used as an endometrial epithelial model to investigate the functional consequences of MAML1 knockdown on endometrial adhesive capacity to HTR8/SVneo (trophoblast cell line) spheroids. After MAML1 knockdown in Ishikawa cells, the expression of endometrial receptivity markers and Notch dependent and independent pathway members were assessed by qPCR. Two-tailed unpaired or paired student’s t-test were used for statistical analysis with a significance threshold of P < 0.05.ResultsMAML1 was localized in the luminal epithelium, glandular epithelium and stroma of human endometrium and the increased expression identified in the mid-secretory phase was restricted only to the luminal epithelium (P < 0.05). Functional analysis using Ishikawa cells demonstrated that knockdown of MAML1 significantly reduced epithelial adhesive capacity (P < 0.01) to HTR8/SVneo (trophoblast cell line) spheroids compared to control. MAML1 knockdown significantly affected the expression of classical receptivity markers (SPP1, DPP4) and this response was not directly via hormone receptors. The expression level of Hippo pathway target Ankyrin repeat domain-containing protein 1 (ANKRD1) was also affected after MAML1 knockdown in Ishikawa cells.ConclusionOur data strongly suggest that MAML1 is involved in regulating the endometrial adhesive capacity and may facilitate embryo attachment, either directly or indirectly through the Notch signaling pathway.

A de novo frameshift variant of ANKRD11 (c.1366_1367dup) in a Chinese patient with KBG syndrome

BackgroundKBG syndrome is a rare autosomal dominant genetic disease mainly caused by pathogenic variants of ankyrin repeat domain-containing protein 11 (ANKRD11) or deletions involving ANKRD11. Herein, we report a novel de novo heterozygous frameshift ANKRD11 variant via whole exome sequencing in a Chinese girl with KBG syndrome.Case presentationA 2-year-2-month-old girl presented with a short stature and developmental delay. Comprehensive physical examinations, endocrine laboratory tests and imaging examination were performed. Whole‐exome sequencing and Sanger sequencing were used to detect and confirm the variant associated with KBG in this patient, respectively. The pathogenicity of the variant was further predicted by several in silico prediction tools. The patient was diagnosed as KBG syndrome with a short stature and developmental delay, as well as characteristic craniofacial abnormalities, including a triangular face, long philtrum, wide eyebrows, a broad nasal bridge, prominent and protruding ears, macrodontia of the upper central incisors, dental crowding, and binocular refractive error. Her skeletal anomalies included brachydactyly, fifth finger clinodactyly, and left-skewed caudal vertebrae. Electroencephalographic results generally showed normal background activity with sporadic spikes and slow wave complexes, as well as multiple spikes and slow wave complexes in the bilateral parietal, occipital, and posterior temporal regions during non-rapid-eye-movement sleep. Brain MRI showed a distended change in the bilateral ventricles and third ventricle, as well as malformation of the sixth ventricle. Whole exome sequencing revealed a novel heterozygous frameshift variant in the patient, ANKRD11 c.1366_1367dup, which was predicted to be pathogenic through in silico analysis. The patient had received physical therapy since 4 months of age, and improvement of gross motor dysfunction was evident.ConclusionsThe results of this study expand the spectrum of ANKRD11 variants in KBG patients and provide clinical phenotypic data for KBG syndrome at an early age. Our study also demonstrates that whole exome sequencing is an effective method for the diagnosis of rare genetic disorders.