APOL1) high-risk genotypes (HRG), G1 and G2, increase the risk of various non-diabetic kidney diseases in the African population. To date, the precise mechanisms by which APOL1 risk variants induce injury on podocytes and other kidney cells remain unclear. Trying to unravel these mechanisms, most studies have used animal or cell models created by gene editing. We developed and characterised conditionally immortalised human podocyte cell lines derived from urine of a donor carrying APOL1 HRG G2/G2. Following induction of APOL1 expression by polyinosinic-polycytidylic acid (poly(I:C)), we assessed functional features of APOL1-induced podocyte dysfunction. As control, APOL1 wild type (G0/G0) podocyte cell line previously generated from a Caucasian donor was used. Upon exposure to poly(I:C), G2/G2 and G0/G0 podocytes upregulated APOL1 expression resulting in podocytes detachment, decreased cells viability and increased apoptosis rate in a genotype-independent manner. Nevertheless, G2/G2 podocyte cell lines exhibited altered features, including upregulation of CD2AP, alteration of cytoskeleton, reduction of autophagic flux and increased permeability in an in vitro model under continuous perfusion. The human APOL1 G2/G2 podocyte cell model is a useful tool for unravelling the mechanisms of APOL1-induced podocyte injury and the cellular functions of APOL1.Apolipoprotein L1 ( APOL1) gene [APOL1 risk variants (RVs), termed G1 and G2, have been reported to significantly increase the risk of various kidney diseases [Individuals with African ancestry have been reported to develop kidney failure at a higher rate than other races . A portiL1) gene . Inheritdiseases ,4,5,6,7.APOL1 was known to protect against certain trypanosome species in humans and some primates [Trypanosoma brucei (T.b.) [T.b. rhodesiense and T.b. gambiense have evolved a mechanism to evade the TLF lysis. T.b. rhodesiense evades lysis by expressing serum resistance associated (SRA) protein that binds to the C-terminus domain of the APOL1, in the process neutralising its lytic activity leading to infection of the host and the development of African sleeping sickness [T.b. gambiense resists TLF via multifactorial defence mechanisms [APOL1 risk variants G1 and G2 restore the lytic activity of human serum, providing selective advantage to carriers against African sleeping sickness caused by T.b. rhodesiense, but not T.b.gambiense [Prior to establishing its role in kidney diseases, primates . APOL1 ci (T.b.) ,9. T.b. sickness ,12,13,14chanisms . APOL1 rambiense .APOL1 RVs have been mostly limited to podocytes [APOL1 is expressed in various tissues such as lung, placenta, pancreas, liver and kidney. In the kidney, different cell types express APOL1, including extra glomerular vascular endothelial cells, podocytes and proximal tubular cells ,17. Howeodocytes ,19.APOL1 RVs do not develop kidney disease; this suggests that additional cellular stressors such as inflammation are required to upregulate APOL1 and induce podocyte injury [Most individuals carrying two e injury ,20. There injury . As suche injury ,22,23,24e injury , alterede injury , increase injury , increase injury . In mostAPOL1 high-risk genotype (HRG), G2/G2. Using this model, we demonstrate pathologic features of APOL1 G2/G2 podocytes in comparison with a reference podocyte cell line, carrying APOL1 allele G0/G0.In other in vitro cell models, APOL1 overexpression was generated using gene editing techniques such as viral vector transductions or CRISPR-Cas9 technology ,23,27. DThis study  was approved by the Ethical Committee of the University Hospital Leuven  and 1% Triton X-100, complete EDTA-free protease inhibitor tablets  were used to prepare protein lysate. Fluorescein isothiocyanate (FITC)-Annexin V/Propidium Iodide (PI) for flow cytometry  were used for apoptosis assay. WST-1 reagent  was used for cytotoxicity assay.All reagents were of analytical grade or used as specified. The antibodies used in this study include anti-synaptopodin , anti-podocin , anti-paxillin , anti-podocalyxin , anti-LC3-II , anti-β-Actin , polyclonal Goat Anti-mouse IgG-HRP  and polyclonal Goat Anti-rabbit IgG-HRP . Alexa Fluor-488-conjugated phalloidin was obtained from Invitrogen , as well as Alexa-546-conjugated secondary antibodies. Poly (I:C) (TLRL-PIC) was purchased from Invivogen  and used in a concentration of 50 µg/mL. Radioimmunoprecipitation assay (RIPA) buffer containing 10 mM sodium phosphate, 150 mM NaCl, 1.5 mM MgCln = 89). From the whole blood samples collected, DNA was extracted using QIAGEN kit following the manufacturer’s instructions  in the laboratory of Paediatric Nephrology, Katholieke Universiteit (KU) Leuven, Belgium. APOL1 genotyping was performed for two kidney risk alleles (G1 and G2) as previously described [APOL1 high-risk genotype (HRG) was defined by the presence of two risk variants  while low-risk genotype (LRG) was defined by the presence of zero or one risk variant.Since the podocyte isolation requires fresh urine, participants were recruited among African community living in Belgium (escribed . APOL1 hBlood and urine samples were collected from those who were identified as carrying HRG to assess kidney function via estimated glomerular filtration rate (eGFR) and urinary protein excretion. Serum creatinine and random spot urine protein/creatinine (P/C) ratio were measured according to standard laboratory protocols. eGFR was calculated using the Schwartz formula  for partAPOL1 HRG has been detected (6 out of 89), freshly voided urine was collected, and exfoliated cells were cultured as previously described [From the participants in whom escribed . In brieescribed . CD2AP) was analysed by qPCR in the clonal cell lines isolated from G2/G2 carrier and compared with APOL1 G0/G0 podocytes. qPCR was also used to analyse the expression of APOL1 in cell clones before and after 24 h of incubation with Poly I:C (50 µg/mL). Beta-actin was used as reference gene . qPCRs were performed in triplicate using StepOnePlus™ real-time PCR system  with Platinum SyberGreen qPCR Supermix , 10 µM of primers and 1.5 µL of cDNA. Results were analysed using StepOnePlus™ software. The expression of the specific podocyte markers synaptopodin, podocalyxin and Cluster of Differentiation 2-Associated Protein , podocalyxin  and podocin , as well as for analysing the podocyte cytoskeleton  and adhesion site . Briefly, cells grown on glass coverslips were fixed with 4% paraformaldehyde for 10 min at room temperature and washed once in PBS. They were permeabilised with 0.1% Triton X-100  for 5 min on ice and washed twice with PBS. Blocking solution  was added to cells for 1 h. Cells were incubated with primary antibodies overnight at 4 °C and further with AlexaFluor secondary antibodies for 1 h in dark at room temperature. 4′, 6-diamidino-2-phenylindole (DAPI) was diluted to 1:1000 in mounting medium  for nuclear staining. The cells were washed 5 times with PBS between steps. Samples were examined with a fluorescence microscope . Image processing and analysis were performed using Image J software. To analyse detachment of cells in culture upon upregulation of APOL1 induced by poly(I:C), 20,000 APOL1 HRG podocytes and control podocytes were seeded in a 6-well plate. After 7 days at 37 °C, cells were incubated with poly(I:C) (50 µg/mL) for 24 h. The supernatant was collected, and detached cells were counted using the TC20™ Automated Cell Counter , which estimates the number of live and dead cells in the cells’ suspension. Percentage of detachment was represented as (number of detached cells)/(number of detached + attached cells). Three independent experiments were performed with three technical replicates in each.APOL1-mediated cytotoxicity in G2/G2 podocyte cell lines and in control cells was measured using a water-soluble tetrazolium salt (WST-1) cytotoxicity assay  according to manufacturer instructions. WST-1 is reduced in metabolically active cells to produce a blue precipitate, formazan. The detection of formazan level in the cells allows quantifying the number of viable cells. Briefly, cells were grown in culture, trypsinised and seeded at a density of 20,000 cells per well in a 96-well plate. Cells were incubated overnight in 33 °C to allow attachment, and then transferred to 37 °C for 10 days. Poly(I:C) (50 µg/mL) was added for APOL1 induction. After at least 16 h of incubation, 10 µL of WST-1 was added in 100 µL of medium per well and cells were incubated further for 2 h at 37 °C. Then, absorbance was measured at 450 nm versus a 620 nm reference by using an ELISA reader. Three independent experiments were performed with three technical replicates in each. To analyse the APOL1-induced apoptotic cell death pathway, human HRG podocyte cell lines and controls treated or not with poly(I:C) were stained using the FITC-AnnexinV apoptosis detection kit and PI kit according to the manufacturer’s instructions. Samples were analysed by fluorescence-activated cell sorting (FACS) within 30 min. The analysis allowed obtaining the proportion of live, early apoptotic, late apoptotic and necrotic cells contained in each sample. Two independent experiments were performed.TM 4–12% Bis-Tris Protein Gel  and followed by blotting on polyvinylidene difluoride (PVDF) membrane. Primary and secondary antibodies were used according to the manufacturer’s instructions. Blots were developed using HRP-conjugated secondary antibodies and analysed using Image J software. Three independent experiments were performed. Full-length blots are presented in Western blot was used to investigate the expression and possible modifications of LC3-II involved in autophagy. Immunodetection of LC3-II  was performed in basal conditions and after 24 h of incubation with poly(I:C) in G0/G0 and G2/G2 podocytes. However, detection of LC3-II only shows a snapshot of protein levels in the cell without reproducing the dynamic information of the autophagic process, so-called autophagic flux. As autophagy is a continuously ongoing process, an increased level of LC3-II may result from either increased production or a decreased degradation of autophagosomes. To assess autophagic flux, cells were treated with 100 nM Bafilomycin A1 (Baf A1)  or dimethylsulfoxide (DMSO)  as vehicle for 3 h before collecting pellet, as previously described . Baf A1 TM Track Etched Membrane, it4ip S.A., Louvain-la-Neuve, Belgium) embedded in the central part of a small (2.5 mL of volume capacity) bioreactor. The membrane divides the cell-chamber into 2 compartments, each with an inlet and outlet channel, to allow, once connected the circuit, the continuous flow of fluid in one direction only. In case of administration of poly(I:C), the day after the setup of culture in the bioreactor, podocyte-medium were changed with medium plus 50 μg/mL poly I:C, otherwise a normal change in medium was carried out. After 16 h of poly(I:C) incubation, or 2 days after setting the culture, the perfusion assay was performed. Firstly, in an incubator, the bioreactor was connected to the main body of the system, capable of delivering liquid at a certain flow rate , directly into the bioreactor. During the filtration assay, in each circuit, a peristaltic pump allows the entry of medium with 1 mg/mL Albumin–fluorescein isothiocyanate conjugate  in the lower part of the bioreactor and in the upper part instead of normal medium. After 3 h of perfusion, the liquid coming out of the two compartments of the bioreactor were collected separately and fluorescence was measured in triplicate. A higher fluorescence indicates lower podocyte integrity.To perform the filtration assay, a dynamic millifluidic culture system  was used. A total of 80,000 differentiated podocytes were seeded on PET porous membrane  for detecting differences between more than two groups. Analysis was considered to be statistically significant when p < 0.05 .Statistical analysis was performed using Microsoft ExcelAPOL1 sequencing analysis was performed. Out of 89 recruited participants, six (6.7%) carried HRG, being G1/G1 (n = 1), G2/G2 (n = 1) and G1/G2 (n = 4)  . The lowAPOL1 for the variant G2 from gDNA isolated from the APOL1 G2/G2 clones and a podocyte control cell line (APOL1 G0/G0). Then, we evaluated the expression of specific podocyte genes synaptopodin, podocalyxin and CD2AP in twelve clones isolated from APOL1 G2/G2 carrier in comparison with APOL1 G0/G0 podocytes (We amplified and sequenced the exon 7 (883 bp) of odocytes A. All teodocytes B. Intereodocytes A. Based odocytes A. Overexpression of APOL1-G1 and G2 variants has been proposed as a mechanism of APOL1-associated kidney diseases mainly affecting podocytes ,24,26. WWe then tested whether poly(I:C) treatment influenced expression of podocyte-specific genes and observed a significant upregulation of CD2AP B. There Cell detachment: Normal podocyte density (number of cells per glomerular tuft volume) is necessary to maintain the integrity of glomerular filtration barrier. Reduction in podocyte density, caused by increased glomerular volume and/or reduced podocyte number is associated with proteinuria, glomerulosclerosis and kidney failure in various progressive glomerular diseases [diseases . It has diseases . We analCell viability: Upregulation of APOL1 in G0, G1 and G2 variants was shown to cause cell death in mammalian cell lines or model organisms [APOL1 genotypes. rganisms ,22,23,35Cell death: Enhanced cell death rate has been reported among various pathways leading to kidney damage associated with APOL1 RVs [POL1 RVs . To evalAutophagy: Additionally, APOL1 overexpression has been associated with altered autophagic cell death mechanisms [chanisms . Therefochanisms . TherefoPodocytes exhibit a unique cytoskeletal architecture, which is linked to their function in maintaining the kidney filtration barrier . Normall−5 dyn/cm2 shear stress, as previously described [Impairment of the podocyte cytoskeletal apparatus by genetic mutations results in proteinuria, dysfunctional glomerular filtration and consequent glomerular disease . We usedescribed . PermeabAPOL1 HRG, representing a novel tool to support the investigation of APOL1-related kidney diseases and to test new therapies.Taken together, these data indicate that human APOL1 G2/G2 podocyte model can mirror in vitro the renal cytopathology observed in patients carrying APOL1 gene expression with several non-diabetic chronic kidney diseases and its mechanisms have been extensively investigated using animal or cell models created by gene editing [APOL1 HRG (G2/G2). The obtained podocyte cell lines were characterised by showing expression of podocyte genes and proteins and by demonstrating functional features of APOL1-induced injury when treated with poly(I:C). To our knowledge, this is the first G2/G2 human podocyte cell model derived from a patient carrying this genotype and it can be of great interest for researchers investigating cellular functions and mechanisms of APOL1.The strong association between  editing ,23,27. O editing ,41,42,43Using the new G2/G2 cell model, we confirmed several cellular phenotypes demonstrated in previous studies, indicating the auxiliary value of our model for unravelling molecular pathways of APOL1-related kidney cell damage. In fact, our G2/G2 podocyte cell model strongly supported the findings of Uzureau et al. , who recAPOL1 genotype. Subsequently, we examined the risk variant-dependent cytotoxicity induced by the upregulation of APOL1 in G2/G2 podocytes compared with control G0/G0 cells. We found that APOL1 overexpression induced podocytes detachment, decreased cells viability and increased apoptosis rate in both genotypes at similar levels. Using a tetracycline-regulated system in human embryonic kidney 293 (HEK293) cells, O’Toole et al. also suggested that APOL1 overexpression drives variant-independent cytotoxicity [The endogenous expression of APOL1 is induced under inflammatory conditions, such as viral infections, primarily activating the Toll-like receptor 3 (TLR3) via increased type 1 interferons, which is mimicked in vitro by incubation of cells with poly(I:C) ,44. Indetoxicity . In conttoxicity ,27 or trtoxicity  showed AAPOL1 gene region containing multiple coding variants organised into distinct haplotypes, including at least seven relatively common non-risk haplotypes with different coding sequences [In our study, as we were developing new podocyte cell lines, we chose to compare them with well-established human immortalised podocytes, which are the APOL1 G0/G0 control podocytes donated by Prof. Saleem . Howeverequences . The refequences . TherefoInterestingly, the patient from whom we derived the APOL1 G2/G2 cell line was born prematurely due to pre-eclampsia and suffered post-neonatal asphyxia, which might have contributed to the decreased eGFR and mild proteinuria . AlthougIn addition, treatment with poly(I:C) induces high overexpression of APOL1 in all podocyte genotypes, which might be too harsh for the cells, hiding the specific toxicity related to variant specific APOL1 overexpression. We suggest that the direct induction of APOL1 with interferon  might beInterestingly, our podocyte cell model showed an overall reduction in the autophagic flux in G2/G2 compared with G0/G0 podocytes in basal conditions, which was enhanced after treatment with poly(I:C). Several studies reported that overexpression of APOL1 led to altered autophagic cell death in different cell types ,47. UsinVarious mechanisms underpinning glomerular pathologies converge on abnormal podocyte cytoskeleton organisation . DiffereUsing a lentivirus transduced human podocyte cell model, Lan et al. reported that overexpression of APOL1-G1 and G2 dramatically increased lysosomal membrane permeability and the leakage of lysosomal components into the cytosol, which was accompanied by APOL1-induced F-actin degradation and necrotic cell death in podocytes . As a poIn conclusion, APOL1 HRG (G2/G2) podocyte cell lines generated from a human donor represent a robust and powerful tool to further study the mechanisms of APOL1-induced kidney injury.


COL1A1  and COL1A2 (c.1-1677_133-441del) as well as three cases with spondyloepiphyseal dysplasia congenita in COL2A1 . Co-occurrence of COL1A1 and WNT1 mutations was found in a patient with a mild OI phenotype but severe osteoporosis. These findings extended the pathogenic variant spectrum of COL1A1, COL1A2, and COL2A1 for type I and type II collagenopathies. Although WES provides a fast and accurate method to identify the genetic causes in most of the patients with type I and type II collagenopathies, its limitation of detecting CNVs because of variable capturing uniformity should be kept in mind when interpreting the results. Taken together, we demonstrate that multiple genetic characterizing technologies can provide an accurate and efficient molecular diagnostic of new genetic variants in disease-causing genes that are compatible with clinical phenotypes.Collagenopathy is a rare genetic condition characterized by abnormality in either collagen structure or metabolism. Variations in its clinical presentations highlight diversity in the genetic causes and potential existence of concurrent mutations. Through whole exome sequencing (WES) complemented with multiplex ligation-dependent probe amplification, we identified the genetic etiologies for six cases with osteogenesis imperfecta (OI) in  Collagenopathy is a group of disorders characterized by molecular abnormality either in the structure or metabolism of collagen proteins. Its clinical findings vary widely in affected tissue types and severity according to involved collagens and mutations . BecauseCOL1A1, COL1A2, BMP1); or defects in post-translational modification ; folding or crosslinking defects ; or ossification or mineralization defects ; or osteoblast differentiation defects with collagen insufficiency ; endoplasmic reticulum function ; Ca2 + sensitivity (PLS3) and other mechanisms   . ObtainiCOL2A1, encoding type II collagen, may results in spondyloepiphyseal dysplasia congenita (SEDC) that is rare chondrodysplastic condition characterized by skeletal deformities such as short-trunk dwarfism, odontoid hypoplasia, cervical spine subluxation, scoliosis, kyphosis, lumbar lordosis, coxa vara, genu valgum, clubfoot, pes planus, and metaphyseal changes 2. Variants were annotated with ANNOVAR3 and novel variants were filtered against 1000 Genomes, dbSNP, and Genome Aggregation Database4. Variants were sorted according to the Combined Annotation Dependent Depletion (CADD) score , which covered approximately 37 Mb of the exonic regions, to capture the fragmented genomic DNA. Pair-end sequencing 2 × 75 bp was performed on the Illumina NextSeq 500 sequencer. Sequencing reads were aligned to human genome reference Hg19 using BWA-memD) score , and thevarSEAK9 . Segrega10 and the aligned reads were counted with HTSeq11 and annotation GTF file from ENSEMBL (Homo_sapiens.GRCh38.99). Genes with fewer than 10 reads in all genotypes were not included in further analyses. Differential expression of genes in probands when compared with control was analyzed with DESeq212. For gene set enrichment analysis (GSEA), normalized expression level of genes was analyzed with gene set collections from the Molecular Signatures Database v7.013.Peripheral blood mononuclear cells (PBMCs) were isolated from 5 ml of blood using Ficoll-Paque PLUS. Blood samples from the same family were collected and processed to the library construction at the same time. Total RNA was extracted from the same amount of PBMCs using RNeasy Micro Kit (QIAGEN). Sequencing libraries from mRNA was prepared with SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara Bio USA) and Nextera XT DNA Library Prep Kit (Illumina), and then sequenced with Illumina NextSeq 500 sequencer with 2 × 75 pair-end sequencing reaction with Illumina NextSeq500 system. On average, 39.46 million reads were obtained for each sample. Reads were aligned with GRCh38 using HISAT2COL1A2 exon 1 and exon 4 were used to confirm the results of MLPA. Concentration of genomic DNA was measured by Qubit dsDNA BR Assay (Thermo Fisher Scientific). All reaction sets were conducted in triplicate, and a non-template control was run simultaneously. The amount of COL1A2 was normalized to the genomic GAPDH and quantified using the comparative CT method (ddCT).Probands with no variants detected with WES were subjected for multiplex ligation-dependent probe amplification (MLPA) analysis . SYBR GrTM Thermal Cycler (Applied Biosystems) and was run on the program 95°C for 2 m followed by 40 cycles of 20 s at 95°C, 20 s at 57°C, and 3 m at 72°C. PCR products were visualized on 1.5% agarose gel using SYBR Safe DNA gel stain (Invitrogen) and purified by QIAquick Gel Extraction Kit (QIAGEN).The concentration of genomic DNA was quantified by Qubit dsDNA BR Assay Kit before used. Each reaction mix consisted of 2.5 μl of genomic DNA (150 ng), 0.5 μl of PfuUltra II Fusion HS DNA Polymerase (Agilent), 0.5 μl of dNTP (25 mM), 5 μl of 10X PfuUltra II reaction buffer, 1 μl of forward primer (10 μM), 1 μl of reverse primer (10 μM), and 14.5 μl nuclease- free water in a total volume of 25 μl. PCR was performed using Veritipair 3 PCR amplicons was sequenced using the specific forward and reverse primers for break point identification  may be the underlying cause. In order to identify potential long-range duplication or deletion in the region of e family . Quantite family . Copy nue family .COL1A2 deletion, we designed multiple pairs of primer recognizing upstream of exon 1 and downstream of exon 4 to the assumptive breakpoints of the deleted segment. Polymerase chain reaction (PCR)-based electrophoresis revealed an aberrant product of 783 bp in the affected members   .COL1A2 deletion includes 1540 bp of the proximal promoter region, which is required for transcriptional regulation, and the transcription start site , making it difficult to compare the expression of COL1A2 mRNA in cells with the COL1A2 deletion to the normal cells. However, when comparing the expression profile between the family members with COL1A2 deletion  vs. those with normal COL1A2 (mother), we identified that 5 genes out of the 27 most differentially expressed (DE) genes  were related to bone development or remodeling   . The tremutation . In addimutation . Taken tLRP5 c.2318 + 2T > C  was found in these twin sisters but not in their parents  were found to have mutations in COL1A1 . P3 and COL1A1 . They alCOL1A1 . A previ parents .COL1A1 c.3839_3841delAAGinsCA; p.Q1280Pfs∗51  and a single allelic WNT1 c.677C > T (p.S226L)  . He presp.S226L) . The WNTs mother . Howevere allele .de novo heterozygous mutations, COL2A1 c.3121G > A (p.G1041S), COL2A1 c.1960G > A (p.G654S), and COL2A1 c.1322C > G (p.G441A), respectively (COL2A1 c.3121G > A (p.G1041S) has been reported to be a pathological mutation clinically associated with spondyloepimetaphyseal dysplasia (SEMD) that was also noted in P7  were found to have ectively . They alectively . Mutatioectively . COL2A1 ed in P7 .COL1A2, four in COL1A1, and three in COL2A1. These results extended the current knowledge of genotype-phenotype correlation in type I and type II collagenopathies.In this report, we confirmed the genetic diagnosis of 9 patients with type I and type II collagenopathies. Their clinical presentations fell into two main categories: OI (6 cases) and SEDC (3 cases). The pathogenic variants included one in COL1A2 may cause symptoms ranging from severe OI to a milder Ehler-Danlos syndrome , depending on the location of variant  that is involved in mineralization of the bone (ALPL may cause hypophosphatasia (HPP), a rare inherited skeletal dysplasia  has been reported in the database of collagen mutation in OI and EDS14. However, with the breakpoint of the deletion not determined, the expression of truncated COL1A2 protein is not clear. In our case, the deleted COL1A2 allele is likely to produce no COL1A2. While mutations in COL1A2 rendering premature stop codon usually have no phenotypes in heterozygote, modifying variant may contribute to the phenotypes of primary osteoporosis , one known frameshift mutation , and one novel frameshift mutation  in COL1A1  and WNT1 c.677C > T (p.S226L). This may explain that P12 had a mild OI phenotype, due to the presence of frameshift COL1A1 mutation, but severe osteoporosis. The c.1668delT (p.G557Vfs∗23) mutation also locates > 55 bp from the start of the last exon of COL1A1 and therefore is predicted to be targeted by NMD. Indeed, read with COL1A1 c.1668delT was not found in the RNA sequencing. The mutation, WNT1 c.677C > T (p.S226L), was expressed in the patient, has been recently indicated in severe recessively-inherited OI phenotypes in patients with homozygous mutations or compound heterozygous mutations  might exist in the local population as its compatibility with life in affected individuals. Further research may be required to understand the clinical impacts on heterozygous carriers and allele frequency in osteoporotic patients due to the founder effect.Another co-occurrence of mutations was found in P6, who had a heterozygous utations . Modelin variant . In the  variant . WNT1 isevere OI . As repoevere OI . AlthougCOL2A1 were more compatible with SEDC (p.G654S and p.G441A) and SEMD (p.G1041S) in that they were short in stature with a marked spinal kyphotic curvature. The alpha-1 chain of type II collagen encoded by COL2A1 has three domains: an N-propeptide which may be involved in the regulation of primary fibril diameters, a triple-helical domain which contains 330 Gly-X-Y repeats and is the predominant motif and a C-propeptide which is considered to be an important factor in the initiation of triple helix formation ’ legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.M-CT and Y-YC recruited the patients and collected their clinical information. C-YL, Y-CW, H-WY, and C-HC conducted molecular research. P-CC conducted bioinformatic analysis and supervised the entire research, critically reviewed and revised the manuscript. M-CT and C-HC drafted the manuscript. All authors have read and approved the final version of manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Long non-coding RNAs (lncRNAs) play key roles in various human cancers. We aimed to determine the key lncRNAs mediating colorectal cancer (CRC) progression. We identified some lncRNAs aberrantly expressed in CRC tissues by using lncRNA microarrays and demonstrated that SH3PXD2A-AS1 was one of the most highly overexpressed lncRNAs in CRC. We further aimed to explore the roles and possible molecular mechanisms of SH3PXD2A-AS1 in CRC. RNA ISH revealed that SH3PXD2A-AS1 was overexpressed in CRC compared with adjacent normal colon tissues and indicated poor prognosis in CRC. Functional analyses showed that SH3PXD2A-AS1 enhanced cell proliferation, angiogenesis, and metastasis. Mechanistically, SH3PXD2A-AS1 can directly interact with p53 protein and regulate p53-mediated gene transcription in CRC. We provided mechanistic insights into the regulation of SH3PXD2A-AS1 on p53-mediated gene transcription and suggested its potential as a new prognostic biomarker and target for the clinical management of CRC. Colorectal cancer (CRC) is a major lethal malignancy worldwide, the incidence rate of which has rapidly increased recently in China Long noncoding RNA (lncRNA) is a class of large transcripts (>200 nucleotides) with limited protein-coding potential. LncRNAs play a key role in both physiological and pathological processes, including cancer In the present study, we identified lncRNAs aberrantly expressed in CRC tissues by using lncRNA microarrays. SH3PXD2A antisense RNA 1 (SH3PXD2A-AS1) was identified as one of the most upregulated lncRNAs in the lncRNA microarrays. Although it had been reported as a biomarker in colon and ovarian cancers The CRC cell lines FHC, HCT116, DLD1, HT29, SW620, and SW480 were purchased from the cell bank of Chinese academy of sciences from 2014 to 2016. The cells were cultured following the instructions recommended by the cell bank. The cells were tested by short tandem repeat (STR) analysis and were validated to be mycoplasma-free.CRC and matched adjacent tissues used for tissue microarray (TMA) construction and qRT-PCR were enrolled at Affiliated Hospital of Xuzhou Medical University from 2010 to 2015 in China. The TMAs slides included examination of 568 pairs of tissue specimens including 568 CRC and the matched corresponding adjacent normal colorectal tissues from CRC patient cohorts . RNA quantity and quality were measured by NanoDrop ND-1000. RNA integrity was assessed by standard denaturing agarose gel electrophoresis. LncRNA microarray was performed by . Arraystar Human LncRNA Microarray V3.0 is used for detecting the global profiling of human LncRNAs. Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. LncRNAs were deemed differentially expressed if their fold change between the CRC and NCT  groups exceed 2.0 and their P-values were less than 0.05.http://kobas.cbi.pku.edu.cn). Gene Set Enrichment Analysis (GSEA) software RNA-seq was performed to detect the mRNA expression profiles of SH3PXD2A-AS1-silenced CRC cells at CapitalBio Technology  using Illumina HiSeq sequencer (Illumina). DESeq2 was used to analyze the DEGs between samples. The differential genes were selected with fold change > 1.5. A GO functional enrichment analysis and KEGG pathway enrichment analysis were performed for the DGEs using the Goseq R package and KOBAS 3.0 software  was synthesized by GENEWIZ Biotechnology (China) and cloned to the lentivirus vector pCDH1-CMV-MSC-EF1-GFP-Puro vector. The shRNA sequences of SH3PXD2A-AS1 was synthesized and cloned into the pLKO-Tet-On vector. The SH3PXD2A-AS1 overexpression/knockdown or the control vectors were co-transfected into HEK293T with the packing plasmids (psPAX and pMD2G) for producing viral particles using Lipofectamine 2000 (Invitrogen) as described before in vitro transcription were cloned from SH3PXD2A-AS1 expression vector by PCR using primers containing a T7 promoter. Then, 500 ng DNA templates were used synthesize RNA using T7 RNA polymerase Transcription system (Promega) and Biotin RNA Labeling Mix (Roche), according to the manufacturer's instructions. The RNA pull-down assays were performed as previously described TM mass spectrometer (Thermo Fisher Scientific). For Flag-MS2bp-MS2bs-based RNA pull-down assay, SH3PXD2A-AS1 was cloned to the pCDNA3-12xMS2bs. Specifically, pcDNA3-FlagMS2bp and pcDNA3-SH3PXD2A-AS1-MS2bs were co-transfected to 293T cells, and the cells were harvested and performed RNA immunoprecipitation with anti-Flag antibody (Sigma) as previous described The templates for in vitro transcription were cloned from SH3PXD2A-AS1 expression vector by PCR using primers containing a T7 promoter and purified using DNA Gel Extraction Kit. Full-length SH3PXD2A-AS1 cDNA temple was cloned using paired primers P1 and P8, SH3PXD2A-AS1 cDNA temple fragment 1-580 was cloned using paired primers P1 and P5, fragment 1-1077 was cloned using paired primers P1 and P6, fragment 1-1650 was cloned using paired primers P1 and P7, fragment 581-2023 was cloned using paired primers P2 and P8, fragment 1078-2023 was cloned using paired primers P3 and P8, fragment 1651-2023 was cloned using paired primers P4 and P8. Then, 500 ng DNA temples were used synthesize RNA using T7 RNA polymerase Transcription system (Promega) and Biotin RNA Labeling Mix (Roche), according to the manufacturer's instructions.P1: GATCACTAATACGACTCACTATATTCTAGAGCTAGCGAATTC;P2: GATCACTAATACGACTCACTATATTCACCAACTACCAACTATT;P3: GATCACTAATACGACTCACTATAAGACGTTCTCATGCCACCCT;P4: GATCACTAATACGACTCACTATAGGGGAGGCCAAGTGTCTGCAT;P5: GATGGCTTAAGTGAGACCTATGTGG;P6: AAAGCCTATGTCTGGAGTTA;P7: CAAGATCTGTAAGTCACCGT;P8: CATAGTTGTGTGACTCTGG.The temples for The RNAscope® probe targeting SH3PXD2A-AS1 was designed and synthesized by Advanced Cell Diagnostics, and the expression of SH3PXD2A-AS1 was detected in TMAs and cells using the RNAscope® 2.5 HD Reagent Kit (Red) according to the manufacturer's instructions. The ISH score of SH3PXD2A-AS1 staining was semi-quantitative according to the manufacturer's instructions of RNAscope® 2.5 HD Reagent Kit (Red).st Strand cDNA Synthesis Kit . Then, relative RNA expression was detected by q-RT-PCR as described above.Nuclear and cytoplasmic RNA were purified by PARIS™ Kit (Thermo Fisher) followed the according to the manufacturer's instructions. The purified RNA was used as temple to generate cDNA using the HiScript 1Western blots were performed as previously reported st Strand cDNA Synthesis Kit . Realtime PCR was carried out on ABI-7500 using UltraSYBR One Step RT-qPCR Kit . The relative lncRNA and mRNA expression levels were normalized to 18s rRNA and GAPDH. The primers are listed in Total RNA from cell and tissue samples was isolated using TRIzol Reagent  according to the manufacturer's protocols, and cDNA was synthesized using the HiScript 1CCK-8 assay was applied to measure the cell proliferation according to the Cell Counting Kit-8 manufacturer's protocol (Dojindo). Annexin V-FITC/PI apoptosis detection kit was used to detect the apoptosis rates according to the manufacturer's protocol .The Colony formation, Cell migration and invasion assays were performed as described previously 6 cell/well onto six well plates. cell layers were wounded using a sterile 10 μl pipette tip after the cells adhering to the plate; the suspended cells were washed away with PBS, and then the cells were cultured in medium with 1% FBS. Gap areas were photographed with a light microscope at different time periods.Wound-healing assay was performed as previously described 4 HUVECs were suspended in 100 μl conditioned medium and seeded into the matrigel-coated 96-well plate and cultured for 4 hours, photos were taken under a microscope, and the complete tubular structures were counted.Tube formation assay was performed following the protocol as described previously 2. Fresh media was added every 3 days. Spheres >50 μm in diameter were counted at day 7.Mammosphere formation assays were performed as previously reported The RIP experiments were carried out with the EZ-Magna RIP Kit (Millipore) according to the manufacturer's protocol. The primers using for quantitative RT-PCR analysis was listed in Supplementary ChIP assay was performed according to the manufacturer's instructions of ChIP assay kit . Briefly, the anti-p53 antibody immunoprecipitated DNA was obtained by eluting the Protein A/G beads with ChIP elution buffer, and was purified for analysis using the promoter-specific primers through qPCR. Meanwhile, anti-IgG antibody was used in CHIP assays as negative control. The primers sequences of the promoters were provided in Supplementary 6) and shSH3PXD2A-AS1 control (1×106) stable SW620 cells, and 2 mg/ml of Dox administered in 5% drinking water was used to induce shRNA expression. Seven days later, the subcutaneous tumors can be observed by vision. Tumors volumes were measured by multiplying long diameter and wide diameter every two days. The tumor volume was calculated using the formula V =a × (b × b)/2, where a is the largest and b is the smallest diameter. Lung metastasis model was established by tail vein injection. The total of 16 female BALB/c nude mice (6-8 weeks old) were randomly divided into three groups: control, SH3PXD2A-AS1 overexpression HCT116 cells. 1 × 106 HCT116 cells suspended with 200 μL PBS were injected through tail vein. 8 weeks later, mice were sacrificed by cervical dislocation. The lungs of the mice were excised, and each lung was fixed by 4% paraformaldehyde for further H.E. staining and ISH assays and metastatic nodules on the surface of each lung were counted. All animal experiments were approved by the Animal Care Committee of the Xuzhou Medical University.The female BALB/c nude mice (6-8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. . The BALB/c nude mice were fed under specific pathogen-free condition. The animals were maintained in a controlled environment with controlled temperature (~25 °C), humidity (50 70%) and . Specially, for the xenograft model, the total of 18 female mice were injected with control  and GraphPad Prism 7. The Student's t test, χin situ hybridization  in an expanded CRC cohort. The ISH arrays showed that SH3PXD2A-AS1 was overexpressed in CRC tissues relative to adjacent NCTs  to screen differentially expressed lncRNAs by using lncRNA microarray. We identified numerous differently expressed lncRNAs and SH3PXD2A-AS1 was one of the most expressed lncRNAs in the microarray Endogenous SH3PXD2A-AS1 expression was measured in colorectal cell lines. Results showed that SH3PXD2A-AS1 expression was higher in CRC cells than in normal colon cells A. SubseqTo identify whether SH3PXD2A-AS1 is involved in colorectal cancer stem cell (CSC), we cultured CRC cell lines in suspension cultured to acquire tumor spheres that may enrich CSCs. SH3PXD2A-AS1 levels were dramatically increased in tumor spheres compared with those in adherent colon cancer cells . Results showed that some known p53 target genes were upregulated . Our CHIP assay showed that SH3PXD2A-AS1 knockdown enhanced the binding of p53 to the promoters of CD82, CXCL1, DR5, SCD, and IER3 and decreased the binding of p53 to the promoters of MET, TGFA, SFN, PCNA, and GDF15 (Figure LncRNAs exert their functions via diverse mechanisms, including co-transcriptional regulation, modulation of gene expression, scaffolding of molecular complexes, pairing with other RNAs, and coding short peptides SH3PXD2A-AS1's interaction can regulate p53-mediated gene transcription, suggesting a possible broad function for RNAs in the regulation of p53 signaling. The lncRNAs DINO and MEG3 can also regulate p53 signaling In summary, this study provided novel evidence that SH3PXD2A-AS1 was upregulated in CRC and associated with poor prognosis. SH3PXD2A-AS1 enhanced CRC cell proliferation, angiogenesis, and metastasis. Mechanistic studies revealed that SH3PXD2A-AS1 directly interacted with p53 protein and regulated p53-mediated gene transcription in CRC. These results highlighted a critical role for SH3PXD2A-AS1 in regulating CRC growth and metastasis through the p53 signaling pathway and suggested that SH3PXD2A-AS1 may serve as a clinical diagnostic and prognostic biomarker and as a potential therapeutic target in CRC.Supplementary figures and tables.Click here for additional data file.


Loxhd1/DFNB77 gene as being required for the mechanotransduction process.Sound detection happens in the inner ear via the mechanical deflection of the hair bundle of cochlear hair cells. The hair bundle is an apical specialization consisting of actin-filled membrane protrusions  connected by tip links (TLs) that transfer the deflection force to gate the mechanotransduction channels. Here, we identified the hearing loss-associated  Loxhd1/DFNB77 gene as being required for the mechanotransduction process. LOXHD1 consists of 15 polycystin lipoxygenase α-toxin (PLAT) repeats, which in other proteins can bind lipids and proteins. LOXHD1 was distributed along the length of the stereocilia. Two LOXHD1 mouse models with mutations in the 10th PLAT repeat exhibited mechanotransduction defects (in both sexes). While mechanotransduction currents in mutant inner hair cells (IHCs) were similar to wild-type levels in the first postnatal week, they were severely affected by postnatal day 11. The onset of the mechanotransduction phenotype was consistent with the temporal progression of postnatal LOXHD1 expression/localization in the hair bundle. The mechanotransduction defect observed in Loxhd1-mutant IHCs was not accompanied by a morphologic defect of the hair bundle or a reduction in TL number. Using immunolocalization, we found that two proteins of the upper and lower TL protein complexes (Harmonin and LHFPL5) were maintained in the mutants, suggesting that the mechanotransduction machinery was present but not activatable. This work identified a novel LOXHD1-dependent step in hair bundle development that is critical for mechanotransduction in mature hair cells as well as for normal hearing function in mice and humans.Sound detection happens in the inner ear via the mechanical deflection of the hair bundle of cochlear hair cells. The hair bundle is an apical specialization consisting of actin-filled membrane protrusions  connected by tip links (TLs) that transfer the deflection force to gate the mechanotransduction channels. Here, we identified the hearing loss-associated SIGNIFICANCE STATEMENT Hair cells detect sound-induced forces via the hair bundle, which consists of membrane protrusions connected by tip links. The mechanotransduction machinery forms protein complexes at the tip-link ends. The current study showed that LOXHD1, a multirepeat protein responsible for hearing loss in humans and mice when mutated, was required for hair-cell mechanotransduction, but only after the first postnatal week. Using immunochemistry, we demonstrated that this defect was not caused by the mislocalization of the tip-link complex proteins Harmonin or LHFPL5, suggesting that the mechanotransduction protein complexes were maintained. This work identified a new step in hair bundle development, which is critical for both hair-cell mechanotransduction and hearing. Hair cells are inner ear mechanosensors that detect the forces generated by sound and head motions. Hair-cell apical membrane protrusions, called stereocilia, are filled with F-actin and are organized into rows of increasing height . The talLOXHD1 cause DFNB77, a human nonsyndromic recessive form of hearing loss (Loxhd1 encodes a protein consisting of 15 polycystin lipoxygenase α-toxin (PLAT) repeats , exhibit hearing loss at 3 weeks of age  were able to mechanotransduce at P7 . The SbaLoxhd1 mouse strain was provided by the Scripps Research Institute. Genotyping was performed by PCR around the missense mutation using the primers indicated below, followed by fragment purification and sequencing . The SbaLoxhd1 strain was produced and backcrossed onto the C56BL/6J background >20 times.The Administrative Panel on Laboratory Animal Care (APLAC) at Stanford University (APLAC protocols #28278 and #30305) approved all animal procedures. Mice of both sexes were used in all experiments. No sex-specific phenotype was observed in any of the mutants. WT or heterozygous littermates served as controls. The eviously  and carrT1308XLoxhd1 vector was designed by inserting a stop codon after the two first coding codons of exon 30, and the remaining sequence of exons 30 and 31 was removed (p.T1308X_ P1420del of the reference cDNA GenBank accession no. FJ750876) and replaced by the FRT-pGK-Neo-pA-FRT cassette. The homology arms were amplified from BAC DNA bMQ-372L7  using the following primers: 5′ arm, ClaI-SnaBI-5′-arm-S: AGATCGATTACGTAGCATCAGAGCAAAGCCAAGACCAC; with 5′ arm-STOP-EcoRV-AS: TTGATATCATCAGTCAGCCTGGAGGGTCCAAATATG; 3′ arm, SacII-MfeI-3′arm-S: GTCCGCGGCAATTGTTAGAAAATGAGCAGGCACAGG; with 3′ arm-PacI-SacII-AS: TACCGCGGTTAATTAATTCCCCAATGAGCAAGACAAA; followed by cloning into a pBluescript vector containing the FRT-pGK-Neo-FRT cassette. The targeting vector was electroporated into 129P2/OlaHsd-derived E14TG2a ES cells. ES clones were screened by PCR for the homologous integration of the 3′ arm (ES-screen-Neo-S: CTTGGCGGCGAATGGGCTGACC with ES-screen-outside-3′ arm-AS: GGGCCAGCACCTCTATTTTCTTGATGA). Positive clones were analyzed by Southern blotting using an internal probe. Two confirmed clones were injected into OLA129 blastocysts at the Scripps Research Institute transgenic facility. The resulting chimeras were mated to C57BL/6J females to obtain germline transmission. Heterozygous F1 mice were mated with B6.Cg-Tg(ACT-FLPe) mice  to remove the pGK-Neo-cassette, and the resulting offspring were subsequently mated to C57BL/6J mice to remove the FLPe transgene, followed by backcrossing onto the C57BL/6J background >14 times. The T1308XLoxhd1 strain was provided by the Scripps Research Institute. T1308XLoxhd1 genotyping was performed by tail-DNA PCR using the following primers: Loxhd1-T1308X-S: GGTCCCCTGCCAAGCCCTCAT; with Loxhd1-T1308X-S: CAGGGTGAGGGCAGAGTTTAGGAC. The mutant allele produced a 457 bp band, while the WT allele produced a 787 bp band.Targeting was conducted by homologous recombination in embryonic stem (ES) cells. A m sense and antisense primers specific for different PLAT domains, as follows: PLAT1-S: TACGAGGAGGAGCTGCTGAACTACGA; with PLAT1-AS: CATCCCCTGTGGCTGTGACC, PLAT5-S: GGAATGGCCCGGTATCGTGTGA; with PLAT5-AS: GGTCGGTGTTGTTCCTGCAGTTGTAAA; PLAT10-S: GCAAGTCAGAGAACCGCACCAACAAG; with PLAT10-AS: AAGACAAAGGCACGGCTATCAGT; PLAT15-S: AGGCTGGCTGGTGGAGAAGGTG; with PLAT15-AS: TGCCGCAGGAGAAGATTGTGG; GAPDH-S: CCACCCAGCCGAGAGGAATGA; with GAPDH-AS: GCAAACGGGAAGGAAATGAATGAA. Triplicate quantitative real-time PCRs were performed on P26 Sba/SbaLoxhd1 (n = 6), Loxhd1Sba/+ (n = 4), Loxhd1+/+ (n = 4), T1308X/T1308XLoxhd1 (n = 4), Loxhd1T1308X/+ (n = 3), and Loxhd1+/+ (n = 3) littermates. The relative expressions of PLAT-coding exons were calculated as 2−ddCt by normalization against Gapdh and WT PLAT1 and plotted as whisker plots (±SEM). The relative values were compared using two-way ANOVA with the three genotypes as independent variables. When a genotype-specific difference was identified, the mean values were compared using Tukey's multiple-comparison test. RT-PCR was performed around exons 26/32 on P25 cochlear total RNA using a poly-dT primer for the single-stranded cDNA and the following primers: Ex26-S: GGCTTCCCGCTTCATTGTGG; with Ex32-AS: AGTGGCTGCCTCCTTGTGTTTC. The PCR products were gel purified, Sanger sequenced, and analyzed using LaserGene (DNASTAR) software.Total RNA was extracted from cochlea using the NucleoSpin RNA XS Kit  and converted to cDNA by reverse transcription using Superscript III (Thermo Fisher Scientific). To quantify PLAT domain expression levels, equal amounts of cDNA were mixed with Maxima SYBR Green qPCR Master Mix (Thermo Fisher Scientific) and 300 nin vitro on 293T cells by immunofluorescence and Western blotting . The expression vector pcDNA3-mPKD1-PLAT-HA, which contains the PLAT domain of mouse PKD1, was transfected as a control to evaluate possible cross-reactions. Twenty-four hours after transfection, the cells were fixed with 4% PFA for 10 min at room temperature (RT) and then blocked and permeabilized with PBS containing 0.5% saponin and 4% BSA for 1 h at RT. The rabbit anti-PLAT11/12 (1:500) antibody was used for costaining with the mouse anti-Myc  or rat anti-HA  antibody. Alexa Fluor 488-conjugated donkey anti-rabbit , Alexa Fluor 568-conjugated goat anti-mouse , and Alexa Fluor 568-conjugated goat anti-rat  antibodies were used as secondary antibodies (1:500 dilution). The cell nuclei were stained with DAPI. The images were captured on an LSM 880 Confocal Microscope (Zeiss).293T cells  were transfected with pcDNA3-mLOXHD1-Myc, which encodes the mouse full Loxhd1 coding sequence was cloned from P7 mouse organ of Corti using the following primers: NG-138, ATGATGGCCCAGAAGAAGAAGCGGAG; and NG-64, ACACCCTGCAGCAAGTCCCAACC; followed by reamplification using primers containing XhoI and EcoRI sites, for cloning into pcDNA3. A Myc tag was added using NEBuilder HiFi DNA assembly (New England Biolabs) with the following primers: PW-83, GAACAAAAACTCATCTCAGAAGAGGATCTTGAGAATTCCACCACACTG; and PW-84, TCTGAGATGAGTTTTTGTTCAACGGCCGCGACAGACGGGAAGAGCTC.To generate pcDNA3-LOXHD1-Myc, the full To generate pcDNA3-PKD1-PLAT-HA, a DNA fragment encoding the PLAT domain of PKD1 was cloned from P10 mouse cochlea cDNAs using the following primers: PW-284-CCCTCTAGACTCGAGACCATGATGTTCAAATATGAAATACTTGTTA; and PW-285, TCTGGAACATCGTATGGGTAAACGGCCGCAAGCACCTCCTTCTCCACTA. The pcDNA3 vector was amplified with the primers PW-85, TACCCATACGATGTTCCAGATTACGCTTGAGAATTCCACCACACTG; and PW-219, GGTCTCGAGTCTAGAGGG. An HA tag was included in the primers, and expression vector was constructed by assembling the two fragments using the NEBuilder HiFi DNA assembly (New England Biolabs).m HEPES buffer, pH 7.2, 10 mm CaCl2, 5 mm MgCl2, and 0.9% NaCl) at RT and were placed in a dish containing fixative (4% PFA in WB). A hole was poked in the cochlear apex using a needle, fixative was perfused slowly through the round window, and the sample was fixed for 30 min at RT. Samples were transferred to WB and the cochlear shell, stria vascularis, Reissner's membrane, and tectorial membrane were removed. The samples were washed three times, 5 min/wash, with WB containing no calcium or magnesium  and permeabilized at RT in WB containing 0.02% Triton X-100 for 10 min (for anti-PLAT11/12 antibodies), or 0.5% Triton X-100 for 20 min with 4% BSA Fraction V (for anti-Harmonin and anti-LHFPL5). They were then blocked with 4% BSA fraction V in WB for 1 h at RT. The samples were subsequently incubated with the primary antibodies diluted in 1% BSA fraction V in WB containing 0.02% Tween-20 overnight at 4°C with gentle rocking. After three washes with WB, 5 min/wash, the secondary antibody anti-rabbit Alexa Fluor-564 or Alexa Fluor-488 (Fisher) at 1:500 and Phalloidin-488 or Phalloidin-568 (Fisher) at 1:300 were added to the samples, which were then incubated at RT with gentle rocking for 1 h. After 3 × 5 min washes with WB, samples were mounted using a slide and coverslip with ProlongGold antifade medium (Thermo Fisher Scientific). Samples were imaged using a Zeiss LSM880 microscope in Airyscan mode and a 63× Plan-Apochromat 1.4 oil lens. Staining was repeated on more than three animals per condition.The inner ears of P7 and P11 mice were dissected from temporal bones in washing buffer  was determined. If the region of interest was not visible, the stereocilium was disregarded. The 2 envelope rise and fall times of 0.5 ms. The repetition time was 50 ms, and 260 trials were averaged. At each frequency, the peak-to-peak voltages of ABR signals at stimulus intensities ranging from 10 to 80 dB sound pressure levels (SPLs) were measured in 10 dB steps and fitted and interpolated to find thresholds that were 5 SDs above the noise floor. For statistical purposes, we defined the threshold as 80 dB SPL, if no ABR was detected.We measured auditory brainstem responses (ABRs), distortion products of otoacoustic emissions (DPOAEs), and cochlear microphonics (CM) in anesthetized mice by administering 100 mg/kg ketamine and 10 mg/kg xylazine intraperitoneally, and their body temperature was held constant at 37°C until they had recovered fully. Sound stimuli were generated digitally by a self-written software  in MATLAFor the stimulation of DPOAEs, two sine wave tones of equal intensity (l1 = l2) and a duration of 1 s were presented to the ear. The tones ranged from 20 to 80 dB SPL attenuated in 10 dB increments, and the two frequencies (f2 = 1.2f1) ranged from f2 with 4–32 kHz. The acoustic signal detected by the microphone in the ear bar was sampled at 200 kHz, and the magnitude of the cubic distortion product (2f1 – f2) was determined by fast Fourier transform. The noise floor was determined by averaging 20 adjacent frequency bins surrounding the distortion product frequency. DPOAE thresholds were reached when the signal was >3 SDs above the noise floor.For CM, the head of the anesthetized animal was secured in a holder. The left tympanic bulla was surgically uncovered and opened to expose the round window. The CM was measured from the ball-ended tip of a Teflon-coated silver wire  that was advanced onto the round window membrane with a micromanipulator. The signal was referenced to a silver wire inserted under the skin near the vertex of the skull. The ground electrode was placed in the hindleg. A bioamplifier was used  to amplify the signals 100 times, which were then bandpass filtered between 1 Hz and 10 kHz. The sound stimuli were 20 ms, 6 kHz tones with an intensity ranging from 10 to 100 dB in 5 dB steps. The stimuli were synthesized in a software and output by a speaker  inserted into the ear canal. We calibrated the intensity using a probe tip microphone in the ear bar, as described previously . The CM Recordings were performed on the apical cochlear turn, which corresponded to a 6−10 kHz frequency in the adult mouse.m NaCl, 2 mm KCl, 2 mm CaCl2, 1 mm MgCl2, 10 mm HEPES, 6 mm glucose, 2 mm pyruvate, 2 mm ascorbic acid, and 2 mm creatine monohydrate. The pH of the external solution was adjusted to 7.4 by the addition of NaOH, and the osmolality ranged from 304 to 308 mOsm. The internal patch solution contained the following: 116 mm CsCl, 3.5 mm MgCl2, 3.5 mm ATP, 5 mm creatine phosphate, 0.1 mm tetracesium BAPTA, 10 mm HEPES, and 20 mm ascorbic acid. The internal solutions were adjusted to pH 7.2 with CsOH, and the osmolality ranged from 282 to 286 mOsm. All recorded currents are included in the summary plots.Whole-cell patch-clamp recordings were performed at RT (19–22°C) with borosilicate patch pipettes with a resistance of 2.5–3.5 MΩ. MET currents were low-pass filtered at 100 kHz, measured with an Axopatch 200B patch-clamp amplifier, digitized with a daq3000 instrument (IOtech) at 500 kHz, and recorded using jClamp (SciSoft). MET currents were filtered offline at 10 kHz for further visualization and analysis. Voltages were adjusted offline for the liquid junction potential, and cells were held at −84 mV. Cells with >80 pA of leak current were discarded. The uncompensated series resistance was between 6 and 10 MΩ. The organ of Corti was perfused with external solution via an apical perfusion pipette placed 20–30 µm away from the cell that was being patched. Recordings were visualized and analyzed using jClamp (SciSoft) and OriginPro 2018 (OriginLab). Graphs were generated using OriginPro 2018 (OriginLab) and Adobe Illustrator . For fluid jet stimulation of hair bundles, we used a piezoelectric disk bender driving the fluid stimulation through a pipette with a diameter of 8–12 µm. The voltage stimulus that was used to drive the disk bender was filtered at 1 kHz with an 8-pole Bessel filter (Frequency Devices). Stiff-probe recordings were conducted as described previously . The tism HEPES buffer, pH 7.2, 10 mm CaCl2, 5 mm MgCl2, and 0.9% NaCl) and fixed in 4% PFA in 0.05 mm HEPES buffer, pH 7.2, 10 mm CaCl2, 5 mm MgCl2, and 0.9% NaCl for 30 min at RT. The inner ears were then dissected to remove the stria vascularis and Reissner's and tectorial membranes. The samples were refixed in 2.5% glutaraldehyde and 4% PFA in 0.05 mm HEPES buffer, pH 7.2, 10 mm CaCl2, 5 mm MgCl2, and 0.9% NaCl overnight at 4°C, then washed, dehydrated in ethanol , and processed to the critical drying point using Autosamdri-815A (Tousimis). Cochleae were mounted on studs using silver paint and coated with 5 nm of iridium . Samples were imaged at 5 kV on an FEI Magellan 400 XHR Field Emission Scanning Electron Microscope at the Stanford Nano Shared Facilities. The microscope is periodically calibrated for measurements using a SIRA-type calibration specimen for ultra-high-resolution modes with ≤2% error between 50 and 350k× magnification at our imaging settings.Inner ears were isolated in WB  pictures of apical turn IHCs were deidentified from genotype information before counting. TLs that connected the second row of stereocilia to the first  row were counted in P7 and P11 mice. Only second-row stereocilia that were designated as paired with their corresponding first-row stereocilia (based on their relative position from the center of the hair bundle and the distance between the two stereocilia) were used for quantification. If it was unclear whether a second-row stereocilium was paired, it was not included in the statistics. Second-row stereocilia that appeared to be paired, but with tips that were not in the field of view of the image , were also discounted. For all paired stereocilia, only visible links that connected stereocilia in the direction of stimulation were tallied as TLs. Paired stereocilia with no visible link were considered to have no TL. The To compare stereociliary heights between control (WT and heterozygous littermates) and mutant animals, we chose to maximize sampling by quantifying 12–21 apical IHCs at P7 and P11. From scanning EM pictures almost orthogonal to the face of the hair bundle, we measured the height of each staircase step, up to the fourth row, using Fiji software. If more rows were present, the entire height of row 4 was determined. As an approximation of the absolute height, we summed the corresponding steps, likely leading to a slight overestimation of height across all measurements. Five to 12 stereociliary columns were measured per cell and plotted as the average per cell. Unpaired nonparametric Mann−Whitney scores were obtained to compare stereocilia height from IHCs of different genotypes.n values were obtained between groups. Although gender was noted, no gender differences were observed between groups, and data are presented with genders combined. The statistical analyses are described in the Results section and were performed using the Origin (OriginLab) or Prism7 (GraphPad) software. In all cases, p < 0.05 was considered significant.Data collection was performed by experimentalists who were blinded to the genotypes of the animals. Genotype was revealed during the analytical phase to ensure that appropriate Qualitative PCR results are described in Materials and Methods.Loxhd1T1308X/+ littermate animals and Sba/SbaLoxhd1 animals for comparison, as follows: P21: T1308X/T1308XLoxhd1 (n = 13), Loxhd1T1308X/+ (n = 16), Loxhd1+/+ (n = 8), and Sba/SbaLoxhd1 (n = 9); and P60: T1308X/T1308XLoxhd1 (n = 9), Loxhd1T1308X/+ (n = 8), Loxhd1+/+ (n = 4), and Sba/SbaLoxhd1 (n = 9). In T1308X/T1308XLoxhd1 animals, we observed heterogeneity in the responses to 11 kHz sound stimulation at 80 dB, which was the highest sound intensity tested: some animals responded while others did not. The number of animals in each responder group are indicated. The mean threshold values are indicated in decibels SPL and SDs.ABR and DPOAE thresholds were determined at 4, 6, 8, 11, 24, and 32 kHz for P21 and P60 T1308X/T1308XLoxhd1 (n = 4), Loxhd1T1308X/+ (n = 4), Sba/SbaLoxhd1 (n = 3), and Loxhd1Sba/+ (n = 4) littermates. Heterozygous littermates gave similar results to those of age-matched WT animals.The magnitude of the CM signal (peak-to-peak amplitude in microvolts ± SD) was measured in P26 Loxhd1T1308X/+, two Loxhd1Sba/+, and one WT; P11 pooled animals: three Loxhd1T1308X/+, one Loxhd1Sba/+, and two WT animals), T1308X/T1308XLoxhd1 mice (n = 4), and Sba/SbaLoxhd1 mice (n = 4). Similar recordings were performed on P9 IHCs using a stiff probe on littermates . All recorded currents are included in the summary plots. Student's t tests were performed to compare current characteristics across genotypes.We used whole-cell voltage-clamp and hair bundle fluid jet mechanical stimulation in apical IHCs between P7 and P11 on littermate control animals . We then stained the cochlear apical turn at different ages to localize the endogenous LOXHD1. At P2, anti-PLAT11/12 staining was restricted to weak labeling at the junctions between hair cells and supporting cells (C). Some hair bundle staining was detected at P7, together with some staining at the supporting cell apical surfaces. The latter is likely to be nonspecific because LOXHD1 mRNA was detectable only in the hair cells (D). The smallest rows appeared to be entirely stained. In optical sections of the stereocilia, anti-PLAT11/12 staining formed a ring around the actin core . Overall, we conclude that LOXHD1 PLATs 11 and 12 are expressed gradually in cochlear hair-cell hair bundles, with an increase from an intermediate level to a high level occurring between P7 and P11.To gain new insights into the function of LOXHD1 in hair cells, we revisited its expression onset in the hair bundle . We usedA fusion B. We theng cells C. Some hir cells . At P11,ir cells D. The smtin core D, inset.SbaLoxhd1, carries a missense mutation affecting the PLAT10 repeat (I1342N), but otherwise produces a full-length protein . This approach was expected to truncate the protein at the center of the PLAT10 repeat and to provide an in vivo control for the anti-PLAT11/12 antibody. The T1308XLoxhd1 allele (p.T1308X_ P1420del) was produced by homologous recombination in ES cells and injected into blastocysts to generate chimera . After removing the selection cassette using an FLPe deleter mouse, the allele was backcrossed over 14 generations onto the C57BL/6J background and produced genotypes in Mendelian ratios.The vast majority of the pathologic mutations in LOXHD1/DFNB77 patients correspond to nonsense mutations . The onlFig. 2A; . To deteT1308XLoxhd1 allele triggered nonsense-mediated mRNA decay. The relative Loxhd1 expression values were calculated as 2−ddCt (±SEM) using Gapdh for normalization and the WT PLAT1 expression level as a reference for the other PLAT domains. The values were compared using two-way ANOVA and Tukey's post hoc multiple-comparison test (95% CI) from triplicates  on a minimum of three animals stemming from different litters for each genotype. The premature termination codon of the T1308XLoxhd1 allele did not trigger nonsense-mediated mRNA decay, as quantitative PCR at different Loxhd1 mRNA positions revealed no significant differences between genotypes, other than the expected PLAT10 deleted area (D). Rather, we observed alternative splicing around the mutated area that rescued the Loxhd1 open reading frame in T1308X/T1308Loxhd1, which was not observed in WT or missense Sba/SbaLoxhd1 mice (E). The mRNA surveillance mechanism called “NAS” . By P21, T1308X/T1308XLoxhd1 animals exhibited thresholds that were elevated to at least 70 dB SPL for ABR and 60 dB SPL for DPOAE across frequencies, indicating severe hearing loss, and both IHCs and OHCs were affected, similar to the results obtained for our Sba/SbaLoxhd1 mice. However, we noted that the T1308X/T1308XLoxhd1 animals were less affected than the Sba/SbaLoxhd1 animals, with residual responses being stable between P21 and P60 (B–E), together with reduced penetrance of hearing loss in the T1308X/T1308XLoxhd1 animals. To determine whether the hair-cell dysfunction originated from a MET defect, we measured CM (F–H), which is a voltage measurement that reflects the summed currents across hair cells near the base of the cochlea on sound stimulation and is often used as an indirect in vivo readout of MET function , and no response was detected in Sba/SbaLoxhd1 animals . In conclusion, both nonsense and missense mutations affecting Loxhd1-PLAT10 lead to hearing loss, potentially originating from a hair-cell MET deficit. Strikingly, we also demonstrated that the nonsense mutation followed by a short deletion yielded an attenuated hearing loss phenotype compared with the missense mutation, likely reflecting the selective occurrence of splicing rescue by NAS.We then confirmed that the d DPOAEs A–E. By P and P60 B–E, togesured CM F–H, whicfunction . At P26,in vitro. We used whole-cell voltage clamping and hair bundle fluid jet mechanical stimulation in apical IHCs between P7 and P11 , we detected maximum MET current amplitudes (METmax) of 558 ± 72 pA (±SD) at P7 and 395 ± 85 pA at P11 . This 29% METmax reduction observed between P7 and P11 was in accordance with the findings of a previous report . However, at P11, the METmax was drastically reduced (95%) to 24 ± 16 pA  in T1308X/T1308XLoxhd1 and to 25 ± 18 pA in Sba/SbaLoxhd1 animals . Moreover, in both mutants, ∼25% of the hair cells did not exhibit any measurable current under these recording conditions.To determine whether LOXHD1 is critical for the functional maturation of the hair bundle, we measured the MET currents s report . At P7, max current amplitude compatible with kinetic studies. IHCs were mechanically stimulated with a glass probe according to a step stimulus protocol  was induced by deflection, which adapted first with a fast phase and then a slow phase. In homozygous mutant animals, the METmax at P9 was reduced to 162 ± 104 pA (n = 12) in Sba/SbaLoxhd1 animals and to 114 ± 40 pA (n = 5) in T1308X/T1308XLoxhd1 animals (D). The normalized current/displacement relationship , the fast and slow adaptation time constants , and the extent of adaptation were not altered between genotypes . In summary, these data provide evidence that mutations affecting LOXHD1-PLAT10 induce a hair-cell MET defect between P7 and P11 that is characterized by a drastic loss of METmax without modification of the kinetic properties.To determine whether the kinetic properties of the residual current were modified, we focused on the transition time point P9. At this age, most hair cells were expected to be affected while still maintaining a MET animals D. The noLoxhd1 mutants could potentially be explained by a progressive hair bundle structural defect that might prevent MET channel stimulation. To explore this possibility, we performed scanning EM on apical cochlear turns at P7 and P11. At both time points, hair bundles from T1308X/T1308XLoxhd1 and Sba/SbaLoxhd1 were indistinguishable from those of control animals at both low magnification  and high magnification (C). The staircase organization was also preserved, and the height of stereocilia rows was comparable across genotypes, with a minor reduction observed in rows 3 and 4 at P7 and P11 (D) in T1308X/T1308XLoxhd1 animals  and SbaLoxhd1/Sba animals ; P7, row 4: −16.0%; P11 row 3: −9.5% (p = 0.0118)]. In addition, the number of TLs bridging rows 1 and 2 was not reduced in T1308X/T1308XLoxhd1 or Sba/SbaLoxhd1 animals compared with WT animals at P7 and P11 (A–C), with even more TLs detected at P11 in Sba/SbaLoxhd1 versus control specimens . Finally, we did not observe any loss of the prolate shape of the second-row stereocilia tips, supporting the conclusion that TLs are present in these structures , as reported previously .We then studied the hair bundle at the molecular level to determine whether the protein complexes at each TL insertion point were maintained in mutant IHCs at P11 or later. First, we investigated the LTL insertion point, where the MET channel complex localizes. Using a validated antibody, we investigated the localization of LHFPL5, an essential component of the MET channel complex located at the tip of transducing stereocilia . LHFPL5 Fig. 8eviously . We deteUsh1c gene product, Harmonin, which is the most abundant protein of this complex and for which a validated antibody exists (Loxhd1 mutants (C). Within a given hair bundle, the percentage of stereocilia with Harmonin puncta at the UTL insertion point was similar between genotypes . Inactivation of the lhfpl5 and Ush1c genes led to early mechanotransduction defects in cochlear hair cells, which were detectable as early as P1–P3  or lower (LHFPL5) TL complex proteins. Together, these results suggest that the mechanotransduction machinery is present in the hair bundle of Loxhd1 mutants but does not function properly. Thus, LOXHD1 highlights the importance of the stereocilia microenvironment for mechanotransduction function. Furthermore, our findings reveal a time window during which late developmental changes in mechanotransduction occur at the molecular level.Mutations in LOXHD1 lead to recessive hearing loss in humans (DFNB77) and mice; however, its function remains unknown. We showed that LOXHD1 is required for mechanotransduction in mouse IHCs. Interestingly, this dependence began after the first postnatal week. In fact, mutations affecting LOXHD1 at the PLAT10 repeat led to a 95% reduction in the MET current between P7 and P11 . The latLoxhd1 function in hair cells. This approach also allowed us to compare the outcomes of the missense mutation in SbaLoxhd1 (I1342N) with those of the nonsense mutation followed by a short deletion in T1308XLoxhd1  (p.T1308X_ P1420del). Unexpectedly, hearing loss was attenuated in T1308X/T1308XLoxhd1 compared with Sba/SbaLoxhd1 mice, with residual CM at P24, and residual ABR and DPOAE responses remaining stable between P21 and P60 in some animals. The reason for this low penetrance is unclear and could have different origins. First, the genetic background of the strain could influence the Loxhd1 phenotype. The T1308XLoxhd1 allele was produced on a mixed background  and was subsequently backcrossed for 14 generations onto C57BL/6J, which should result in a 99.99% C57BL/6J backcrossed strain  is inactivated will be required to prevent compensation by splicing or the usage of alternative translational start sites and to prevent putative cross-reactivity between the PLAT repeats. Nevertheless, an indication that the anti-PLAT11/12 antibody exhibits a specific LOXHD1 signal arises from the gradual increase in the hair bundle staining observed between P7 and P11, which correlates with the occurrence of the MET phenotype. This signal is distributed along the height of the stereocilia, with enrichment at the lower stereocilia portions of rows 1 and 2, forming a ring around the actin core. Since LOXHD1 is expressed in all stereocilia rows, it could affect mechanotransduction from either side of the TL insertion points.To study LOXHD1 expression in the hair bundle, we used an Sba/SbaLoxhd1 and T1308X/T1308XLoxhd1 mice. Using ex vivo whole-cell voltage clamping of apical IHCs (whose location corresponded to a 6–10 kHz frequency range in the adult mouse), we timed the MET phenotype onset between P7 and P11 in the two mutants. At P11, a maximum MET current of 25 pA could be elicited by mechanical stimulation. This receptor current would correspond to a maximum depolarization of ∼2.5 mV (considering a hair-cell membrane resistance of 100 MΩ), which is unlikely to have a major effect on the calcium current that drives synaptic release. The loss of MET current will reduce the resting current, leading the IHC to hyperpolarize by ∼10 mV, and further lower the effect of the 2.5 mV depolarization .In this study, we primarily focused on the IHCs, but LOXHD1 is expected to be similarly required for MET in OHCs, which express LOXHD1 (mRNA and protein) and are functionally affected by the In conclusion, we have added LOXHD1 to the short list of proteins required for hair-cell MET. Mutations in PLAT10 led to an IHC MET defect that occurred only after the first postnatal week. Furthermore, this defect did not affect the hair bundle structure or the maintenance of Harmonin and LHFPL5 in the bundle, suggesting that MET channels are present but not activatable. Understanding this mode of MET regulation is critical, as it underlies both congenital and age-related forms of hearing loss in humans.


Mall database, comprising rare, diverse and unique compounds, was explored for molecular docking-based virtual screening against the DYRK1A protein, in order to find out potential inhibitors. Ligands exhibiting hydrogen bond interactions with key amino acid residues such as Ile165, Lys188 , Glu239 (gk+1), Leu241 (gk+3), Ser242, Asn244, and Asp307, of the target protein, were considered potential ligands. Hydrogen bond interactions with Leu241 (gk+3) were considered key determinants for the selection. High scoring structures were also docked by Glide XP docking in the active sites of twelve DYRK1A related protein kinases, viz. DYRK1B, DYRK2, CDK5/p25, CK1, CLK1, CLK3, GSK3β, MAPK2, MAPK10, PIM1, PKA, and PKCα, in order to find selective DYRK1A inhibitors. MM/GBSA binding free energies of selected ligand–protein complexes were also calculated in order to remove false positive hits. Physicochemical and pharmacokinetic properties of the selected six hit ligands were also computed and related with the proposed limits for orally active CNS drugs. The computational toxicity webserver ProTox-II was used to predict the toxicity profile of selected six hits . The selected six docked ligand–protein systems were exposed to 100 ns molecular dynamics (MD) simulations to validate their mechanism of interactions and stability in the ATP pocket of human DYRK1A kinase. All six ligands were found to be stable in the ATP binding pocket of DYRK1A kinase.The dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a novel, promising and emerging biological target for therapeutic intervention in neurodegenerative diseases, especially in Alzheimer’s disease (AD). The mol Alzheimer’s disease (AD) is distinguished by irremediable neurodegeneration as well as diminishing cognitive functions. About 60–70% cases of dementia is attributed to AD . There aDYRK1A is referred to as dual specificity kinase, because of its ability of self-activation by autophosphorylation of Tyr321 in its activation loop, as well as phosphorylation of a wide variety of exogenous protein substrates . It is o50 330 nM)  10 μM), was obtained by the structural modification of TG003   20 μM) [b]indole derivative), exhibited potent DYRK1A inhibition with an IC50 67 nM [c]quinoline-6-carboxylic acid analogue (4E2 in PDB ID: 4YLK) revealed the interactions in the ATP binding space of DYRK1A (IC50 6 nM), via (i) a salt bridge amid the carboxylate group of 4E2 and the amine of Lys188, (ii) water interceded hydrogen bonds with Ser242 & Asp307, and (iii) flipped P-loop residue Phe170 forming pi-pi hydrophobic interactions with the inhibitor. In addition, the iodine at position 10 was found located nearby gatekeeper (gk) Leu241 at a distance of 3.3 Å pyridin-5-yl]pyridine-2,6-diamine derivatives, among them the most promising and selective DYRK1A inhibitor has an IC50 value of 5 nM (DYRK2 IC50 = 195 nM)  20 μM) . A lamel50 67 nM . Leucett50 67 nM . The co-50 67 nM . An indo 195 nM) .The gene for DYRK1A is found to be located within DSCR on human chromosome 21, and studies revealed that upregulation of DYRK1A is the key factor responsible for cognitive decline in patients of AD and DS ,15. SincMall database containing 15,381 chemically diverse structures was checked for any duplicates by using OpenBabel (ver. 3.0.0) and found to be unique without any duplicates. It was prepared for docking-based virtual screening by the application LigPrep (ver. 3.4). The resulting docking library consisting of 54,594 structures was utilized for comprehensive molecular docking-based virtual screening against a set of DYRK1A protein kinases.The downloaded molIn order to increase the accuracy of the results, five DRYK1A protein crystal structures , available in the protein data bank (PDB) from diverse reputed laboratories and reported in different timelines as having different resolutions, were used for docking-based virtual screening. The difference in the crystal structure of these five DYRK1A kinases were visualized by superimposing with the help of the PyMOL Molecular Graphics System (v1.8.4.0). A slight variation near αC helix and β1, β2 and β3 sheets were observed among the crystal structures and is depicted in Each of the co-crystallized ligands, viz. EHB (of 3ANQ), 3RA (of 4AZE), D15 (of 2WO6), 4E2 (of 4YLK), and QIV (of 5A3X) of the DYRK1A protein kinase, were docked into the ATP pocket of their corresponding pdb structures by Glide extra precision (XP) docking. The docked ligand’s top poses based on their scores and interactions with the active site residues were aligned with the co-crystallized poses in order to validate the docking protocol. The heavy atom RMSD for EHB, 3RA, D15, 4E2, and QIV were found to be 0.742 Å, 0.932 Å, 0.978 Å, 0.573 Å and 0.466 Å, respectively . The docThe docking protocol was further validated by area under receiver operating characteristic (ROC) curves. Five active DYRK1A inhibitors  were used to generate 213 decoys (inactive inhibitors) by using DUDE webserver (A Database of Useful Decoys: Enhanced) . All theMall ID: 19394) was already reported previously as a DYRK1A inhibitor, so this molecule was not included in further study indole-1,2(3H)-dione-1-(phenylhydrazone)), 20271 -1,2,4-triazolidine-3,5-dione), and 18976 (2-hydroxy-4-(4-hydroxyphenyl)-1H-phenalen-1-one). Structures having molMall IDs: 15938 and 19037, were found to have slight variation from the qualifying limits in terms of FOSA and PISA only, and thus were retained. A total of six structures were retained for further studies. The QP properties of the final six selected identified hit molecules are presented in Ligands showing great variations from the qualifying limits for orally active CNS drugs were discarded . Among t50), hepatotoxicity, mutagenicity, carcinogenicity, cellular toxicity, immunotoxicity, and toxicological pathways  of a chemical compound is of great importance in the discipline of drug discovery. In silico toxicity models can accurately predict the toxicity effects of chemicals and thus minimize time, costs and the need for animal testing. The computational toxicity webserver ProTox-II  X-ray co-crystallized structures were downloaded from the RCSB Protein Data Bank (PDB) website , 3RA (4AZE), D15 (2WO6), 4E2 (4YLK), QIV (5A3X), DYK (4D2S), IRB (3KVW), 3O0 (3O0G), 9WG (5W4W), DBQ (1Z57), 3RA (3RAW), B4K (5OY4), B18 (2PZY), 34I (3RTP), 8GX (5VUA), M77 (5LCP), and PSF (1DSY), were drawn on ChemDraw Ultra (ver. 12.0) and saved in SDF format. These 2D structures in SDF format were imported into Maestro workspace and processed by the application LigPrep by the method discussed above. The prepared structures were docked into the ATP pocket of their corresponding receptor by Glide extra precision (XP) docking ,68. The Furthermore, the docking protocol was also validated by area under receiver operating characteristic (ROC) curves. Five known and potent DYRK1A inhibitors, namely INDY, a lamellarin D analogue, a quinazoline amine derivative, Leucettine L41, and harmine , were seVirtual screening workflow available in Schrödinger computational software  was usedThe ligand–protein complexes of top scoring ligands were analysed by Biovia Discovery Studio Visualizer (v20.1.0.19295) . Moleculbind) of selected ligand–protein complexes, using default parameters (OPLS_2005 force field and VSGB solvent model), to remove false positive hits  in order to find the selectivity of the hit ligands ,54,55,56The QikProp 4.4 module of Maestro–Schrödinger suite 2015.2 was utilized for the estimation of thirty-five QikProp (QP) attributions of the selected ligands . Chemica50), hepatotoxicity, mutagenicity, carcinogenicity, cellular toxicity, immunotoxicity, and toxicological pathways  were computed, along with the accuracy of the prediction.The computational toxicity webserver ProTox-II was used to predict and compute the toxicity profile of selected hits . Acute t−) ions. Furthermore, the ionic strength of the solution of the system was maintained by adding 0.15 M NaCl to realistically mimic the physiological conditions. During the MD simulation, periodic boundary conditions (pbc) were used. For energy minimization, a steepest descent method with a maximum step size of 0.01 nm and a tolerance of 1000 kJ/mol/nm was utilized. The Linear Constraint Solver (LINCS) method was utilized to limit bond lengths. The particle mesh Ewald (PME) technique was used to conduct electrostatic calculations. The system was equilibrated utilizing canonical ensembles NVT, followed by isothermal-isobaric ensemble (NPT) for 100 ps after energy reduction. All simulations were run at the same temperature and pressure . Trajectories were created every 2 fs and stored every 2 ps during the manufacturing run, which lasted 100 ns. GROMACS analysis programs were used to perform all preliminary analyses, comprising root mean square deviation (RMSD), radius of gyration (Rg), root mean square fluctuations (RMSF), potential binding energy, solvent-accessible surface area (SASA), hydrogen bond (H-bond), and principal component analysis (PCA).MD simulations were used to confirm the interactions, constancy, and activity of recognised ligand structures in the ATP pocket of human DYRK1A protein (PDB ID: 3ANQ), with reference to DYRK1A structure, co-crystallized with a benzothiazole derivative, EHB . GROMACShttp://www.swisstargetprediction.ch/; accessed on 23 January 2022) [https://www.molinspiration.com/cgi-bin/properties; accessed on 23 January 2022) [The identified six hits and reported three inhibitors were submitted to the Swiss Target Prediction webserver (ry 2022)  and moliry 2022)  in orderhttps://chemminetools.ucr.edu/; accessed on 23 January 2022) [The structure similarity between the six identified hits and co-crystallized ligand structures of DYRK1A and related kinases were studied by ChemMine Web Tools (ry 2022) . The strhttp://ambit.sourceforge.net/reactor.html; accessed on 5 February 2022) [The synthetic accessibility (SA) prediction of the identified molecules was performed by SyntheticAccessibiliyCli (Ambit-SA) software tool (ry 2022) .Mall database, consisting of 15,381 rare, diverse and unique compounds, was screened by docking-based virtual screening against a set of five DYR1A protein kinases. Hydrogen bond interaction with Leu241 (gk+3) were considered key determinants for the selection. Binding free energies (ΔGbind) of selected ligand–protein complexes were also calculated in order to remove false positive hits. The high scoring selected ligands were further screened for their binding affinity with the other twelve DYRK1A-related protein kinases in order to find selective inhibitors. Among the identified ligands, a bis-indole indigoid scaffold  was already reported previously as a DYRK1A inhibitor. Various physicochemical and pharmacokinetic attributions, as well as the toxicity profile of the nominated six hit ligands, were also computed and were found to be satisfactory for orally active CNS drugs. The selected docked ligand–protein complexes were exposed to MD simulations to validate their mechanism of interactions and stability in the ATP pocket of human DYRK1A kinase. The 100 ns simulation time appeared to be suitable, and it is concluded that all the selected ligand structures having molMall IDs: 9539, 11352, 15938, 19037, 21830 and 21878 are potential inhibitors of the DYRK1A protein.The discovery of selective and potent inhibitors of DYRK1A is an emerging field of investigation, as decades of research on neurodegenerative diseases, especially Alzheimer’s Disease, have recognized DYRK1A as a novel drug target for therapeutic intervention. In the present study, the mol


CONSTANS (CO) is an important regulator of photoperiodic flowering and functions at a key position in the flowering regulatory network. Here, two CO homologs, MiCOL16A and MiCOL16B, were isolated from “SiJiMi” mango to elucidate the mechanisms controlling mango flowering. The MiCOL16A and MiCOL16B genes were highly expressed in the leaves and expressed at low levels in the buds and flowers. The expression levels of MiCOL16A and MiCOL16B increased during the flowering induction period but decreased during the flower organ development and flowering periods. The MiCOL16A gene was expressed in accordance with the circadian rhythm, and MiCOL16B expression was affected by diurnal variation, albeit not regularly. Both the MiCOL16A and MiCOL16B proteins were localized in the nucleus of cells and exerted transcriptional activity through their MR domains in yeast. Overexpression of both the MiCOL16A and MiCOL16B genes significantly repressed flowering in Arabidopsis under short-day (SD) and long-day (LD) conditions because they repressed the expression of AtFT and AtSOC1. This research also revealed that overexpression of MiCOL16A and MiCOL16B improved the salt and drought tolerance of Arabidopsis, conferring longer roots and higher survival rates to overexpression lines under drought and salt stress. Together, our results demonstrated that MiCOL16A and MiCOL16B not only regulate flowering but also play a role in the abiotic stress response in mango. Arabidopsis is thoroughly understood. According to recent research, the onset of flowering is regulated in a timely manner by an intricate network involving a series of regulatory pathways, such as gibberellin, photoperiod, autonomous, aging and vernalization pathways [In higher plants, floral transition is the process that describes the switch from the vegetative stage to the reproductive stage. The time for this process is referred to as flowering time. The flowering mechanism of the annual plant species pathways ,2,3. Of pathways .Many photoperiod pathway-related genes have been discovered, such as TIME OF CAB EXPRESSION 1 (TOC1), LATE ELONGATED HYPOCOTYL (LHY), EARLY FLOWERING 4 (ELF4), GIGANTEA (GI), CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), FLOWERING LOCUS T (FT) and CONSTANS (CO) . Among tCO orthologs vary across different species. In Arabidopsis, overexpression of the AtCO, AtCOL5 and AtCOL16 genes promotes flowering under long-day (LD) or short-day (SD) conditions [AtCOL7, AtCOL8 and AtCOL9 inhibit flowering under LD conditions [OsHd1 gene delays flowering under LD conditions and promotes flowering under SD conditions [OsCOL16 inhibits flowering under both SD and LD conditions [StCO, a CO homolog in potato, regulates flowering [CO orthologs in Fuji apple, MdCOL1 and MdCOL2, play a significant role in the growth and development of reproductive organs [CO genes have a wide range of effects on plant flowering and development.However, the functions of nditions ,20, but nditions ,22. In rnditions , and OsCnditions . StCO, alowering . Moreovee organs . Thus, hMangifera indica L.) is a member of the Anacardiaceae family and is an economically important fruit tree species. Flowering time has a considerable influence on the yield and quality of mango. Therefore, the discovery and identification of flowering-related genes are necessary for mango production. Mango flowering is the result of a complex process influenced by many factors, but it is not affected by daylength [MiFT, MiSOC1 and MiAP1 genes promote flowering, but the MiCO gene inhibits flowering in Arabidopsis [COL16 gene has not been studied in mango. Here, we isolated two COL genes, MiCOL16A and MiCOL16B, and verified their function. Our results indicated that these two genes strongly influence the flowering and abiotic stress responses of mango.Mango  and MiCOL16B (GenBank No: MW326762), were identified from M. indica. L. cv. SiJiMi. The full cDNA sequences of MiCOL16A and MiCOL16B were 1269 bp and 1251 bp, encoding 423 and 417 amino acids, respectively; the two genes were 73.06% identical. The DNA sequences of MiCOL16A and MiCOL16B were 2030 bp and 1751 bp, respectively, and each contained one intron . We obtained 15 and 10 independent transgenic lines of MiCOL16A and MiCOL16B, respectively, and we selected three homozygous lines for each construct from within the T3 generation and planted them under LD or SD conditions. The PCR analysis results showed that MiCOL16A and MiCOL16B were expressed in the transgenic plants but not in the WT or empty vector-transformed Arabidopsis plants under LD  were selected. Three-day-old seedlings of the overexpression and WT plants were transplanted onto half-strength MS media supplemented with mannitol and NaCl. The length of their roots was measured after 7 days of stress treatment. The untreated WT and overexpression plants did not significantly differ, but compared with the WT plants, both MiCOL16A and MiCOL16B overexpression plants grew better and had longer roots under all stress conditions  6000 treatment [MiCOL16A and MiCOL16B transgenic plants were all greater than those of the WT plants under salt or drought conditions. This result shows that MiCOL16A and MiCOL16B enhance the tolerance of the transgenic plants to abiotic stress. However, BnCOL2 increases sensitivity to drought conditions in Arabidopsis plants, and BnCOL2 overexpression results in a survival rate that is lower than that of the WT plants [The molecular responses of plants to abiotic stresses are complex and dynamic ,56, and T plants . The expT plants . These rBnCOL2 transgenic lines [MiCOL16A and MiCOL16B transgenic plants were tolerant to drought and salt stress; the expression levels of AtNHX1, AtRD20 and AtSOS1 increased under salt treatment; and the expression levels of AtCOR15A, AtRD20 and AtNHX1 increased under drought treatment. AtNHX1 and AtRD20 play vital roles in the response to salt and drought stress [AtSOS1 transcript levels substantially increase upon NaCl treatment [AtCOR15A gene is known as a marker of drought stress [MiCOL16A and MiCOL16B genes positively regulate the salt and drought stress tolerance of Arabidopsis.To some extent, the transcript levels of stress-related genes also indicate the tolerance of plants to abiotic stresses . For exaic lines . In our t stress ,64,65, Areatment , and thet stress . In summM. indica L. cv. SiJiMi) were collected from an orchard at Guangxi University, Nanning, Guangxi, China. The leaves, buds and stems of nonflowering and flowering branches were collected on 4 January 2019. Leaves were collected on 5 November 2018, 5 December 2018, 4 January 2019, 29 January 2019 and 6 March 2019. Leaves were collected every three hours on three separate days from 2-year-old mango trees grown under different conditions for diurnal rhythmic expression analysis (LD (16 h light/8 h dark), MD (12 h light/12 h dark) and SD (8 h light/16 h dark)). The plant samples were frozen in liquid nitrogen and kept at −80 °C. Wild-type (WT) Arabidopsis ecotype Columbia (Col-0) plants were used to study gene function and were grown in the laboratory.The samples (MiCOL16 gene was isolated from the transcriptomic data of SiJiMi mango (unpublished data), after which the sequence was verified. The primers designed for cloning included SCOL16Au (5′-GCCTTTGCAA AATGATCACCGG-3′), SCOL16Ad (5′-GCGCCATTTATTTCTTGAGG-3′), SCOL16Bu (5′-GTCTTTGCGGTATGATCACTG-3′) and SCOL16Bd (5′-CGGCCATCACCATTTAT TTC-3′). The PCR conditions were as follows: 95 °C for 3 min; 38 cycles of 95 °C for 30 s, 53 °C/52 °C for 30 s and 72 °C for 1 min 30 s; and 72 °C for 10 min. The final products were inserted into a pMD18-T vector and sequenced.Total RNA of SiJiMi mango plants was extracted by using an RNAprep Pure Kit  . cDNA was reverse transcribed with 1 µg of total RNA with PrimeScript™ Reverse Transcriptase M-MLV  and Oligo(dT)18 primers (TTTTTTTTTT TTTTTTTTTTTT). The cetyl-trimethylammonium bromide (CTAB) method was used to isolate genomic DNA from mango leaves . The MiCMiCOL16A and MiCOL16B were analyzed with the BLAST search tool . CO homologous sequences were downloaded from the NCBI database . Sequence alignment was subsequently performed using DNAMAN 7 software . A neighbor-joining tree based on the CO gene family in Arabidopsis and several other tree species was constructed via MEGA 6.06, with 1000 bootstrap replicates.The amino acid sequences of MiActin1  was used as an internal control gene for data processing [MiCOL16A and MiCOL16B were qCOL16Au (5′-TGAATCACCACTG GCAGCTGA-3′) and qCOL16Ad (5′-GGGGTTCCTGTTGTCCATGGA-3′) as well as qCOL16Bu (5′-ACTTCGGAGACAGCACAGTGA-3′) and qCOL16Bd (5′-TTGCATTCGG TGTTCGCCATT-3′). qRT–PCR was performed with an ABI 7500 Real-Time PCR System  in conjunction with SYBR Premix Ex Taq II . The expression data were normalized according to the 2−ΔΔCt method [First-strand cDNA of each sample was synthesized, and ocessing . The gent method .MiCOL16A and MiCOL16B coding DNA sequence (CDS) regions were cloned into a green fluorescent protein (GFP)-encoding gene via the cauliflower mosaic virus (CaMV) 35S promoter. GFP fusions were transferred into Agrobacterium tumefaciens EH105, which were then transiently injected into onion inner epidermal cells. Fluorescent cells were observed via laser confocal microscopy. An empty vector with only GFP was used as the control, and 4′,6-diamidino-2-phenylindole (DAPI) was used to visualize the location of the nucleus.The MiCOL16A and MiCOL16B were amplified via PCR and inserted into the pGBKT7 expression vector . An empty pGBKT7 vector was used as a control (BD-vector). These four plasmids and empty pGBKT7 vectors were subsequently transferred into Y2H Gold yeast cells. The seedlings were subsequently grown on sucrose–dextran (SD) media lacking tryptophan for 3 days. The yeast strains that grew successfully were then transferred onto SD/-Trp, SD/-Trp/X-α-gal and SD/-Trp/X-α-gal/AbA media and grown for another 3 days.To determine the transcriptional activation domains, BD-MiCOL16A, BD-MiCOL16B, BD-MiCOL16A-ΔMR and BD-MiCOL16B-ΔMR constructs were generated, and the full-length CDS and middle region (MR) deletion of MiCOL16A and MiCOL16B were amplified and inserted into a pBI121 vector. The gene-specific primers used were as follows: ZCOL16Au , ZCOL16Ad , ZCOL16Bu  and ZCOL16Bd . The pBI121 vector was subsequently digested by Xba I and Xma I restriction enzymes. Positive recombinant pBI121-MiCOL16A and pBI121-MiCOL16B plasmids were then transformed into Agrobacterium tumefaciens EH105. The primers JCOL16Au (5′-GCCTTTGCAAAATGATCACCGG-3′), JCOL16Bu (5′-GTCTTTGCGGTATGATCAC TG-3′) and 121GUSd (5′-TTGGGACAACTCCAGTGAAAAG-3′) were used to assess the target bacterial colonies. Arabidopsis plants were subsequently transformed with the floral-dip method [Arabidopsis plants growing on half-strength Murashige and Skoog (MS) media supplemented with 50 mg/L kanamycin were identified and then transplanted into square pots filled with a mixture of organic substrate, peat moss and vermiculite (2:2:1), after which they were allowed to grow unabated for 7 days. Seeds of T3-generation homozygous plants were sown at 22 °C under SD conditions (8 h light/16 h darkness) or LD conditions (16 h light/8 h darkness) to measure flowering time, plant height and rosette leaves. Two-week-old WT and transgenic plants were sampled for qRT–PCR analysis. The flowering-related genes AtSOC1  and AtFT  were used for qRT–PCR analysis.The complete CDSs of p method . TransgeSeeds of the transgenic lines and WT were sown evenly on the surface of half-strength MS media without antibiotics and stored at 4 °C for 3 days for vernalization.MiCOL16A and MiCOL16B transgenic lines were subjected to stress, and 3 days later, all the plants were transferred to vertically oriented square-shaped containers filled with half-strength MS agar media supplemented with NaCl  or mannitol  [For root growth assays, WT and  500 mM) ,73. The Arabidopsis, 7-day-old seedlings were transplanted into square pots. For salt stress, the WT and transgenic lines were treated with 300 mM NaCl solution every 2 days until obvious phenotypic differences occurred, and then the survival rate was determined. For drought treatment, water was withheld from the transgenic lines and WT plants until obvious phenotypic differences occurred. After rewatering for 3 days, the survival rates of the overexpression lines and WT plants were measured.To assess the stress tolerance of transgenic Arabidopsis plants in response to stress, seeds of the transgenic lines and WT were sown on half-strength MS media supplemented with NaCl or mannitol. Fifteen days later, total RNA was extracted. The specific primers used for the stress-related genes AtNHX1  [AtRD20  [AtSOS1  [AtCOR15A  [AtActin2  was used as a reference control.To investigate the expression patterns of related genes in transgenic  ATG-3′) , AtRD20 ACAG-3′) , AtSOS1 TTTC-3′) , and AtCCTTC-3′)  were usep < 0.05 (* in the figures) and p < 0.01 (** or Lowercase letters in the figures) levels. Bar charts were generated with GraphPad Prism 7 software.All experiments were repeated at least three times. The standard deviations (SDs) are represented by error bars in the figures. All statistical analyses were performed by SPSS 17.0, and significance was tested at the MiCOL16A and MiCOL16B genes from SiJiMi mango. Both the MiCOL16A and MiCOL16B proteins localize to the nucleus and have transcriptional activity through their MR domain. Overexpression of MiCOL16A and MiCOL16B repressed flowering in Arabidopsis under SD and LD conditions and improved tolerance to drought and salt stress conditions. Overall, our results clearly demonstrated that the MiCOL16A and MiCOL16B genes play key roles in mango flowering and abiotic stress responses.In this study, we isolated the