PPM1D locus or gain-of-function truncating mutations of PPM1D occur in various cancers. Here we used a transgenic mouse model carrying a truncating mutation in exon 6 of PPM1D (Ppm1dT). As with human cell lines, we found that the truncated PPM1D was present at high levels in the mouse thymus. Truncated PPM1D did not affect differentiation of T-cells in the thymus but it impaired their response to ionizing radiation (IR). Thymocytes in T/+Ppm1d mice did not arrest in the checkpoint and continued to proliferate despite the presence of DNA damage. In addition, we observed a decreased level of apoptosis in the thymi of T/+Ppm1d mice. Moreover, the frequency of the IR-induced T-cell lymphomas increased in T/+Ppm1d+/−Trp53 mice resulting in decreased survival. We conclude that truncated PPM1D partially suppresses the p53 pathway in the mouse thymus and potentiates tumor formation under the condition of a partial loss of p53 function.Genome integrity is protected by the cell-cycle checkpoints that prevent cell proliferation in the presence of DNA damage and allow time for DNA repair. The transient checkpoint arrest together with cellular senescence represent an intrinsic barrier to tumorigenesis. Tumor suppressor p53 is an integral part of the checkpoints and its inactivating mutations promote cancer growth. Protein phosphatase magnesium-dependent 1 (PPM1D) is a negative regulator of p53. Although its loss impairs recovery from the G2 checkpoint and promotes induction of senescence, amplification of the  Indeckpoint . Replicaeckpoint . Mainteneckpoint . Recovereckpoint ,11,12,13eckpoint ,15. The eckpoint ,16,17. Iint [p21 ,19. PPM1int [p21 . Importaint [p21 ,23.PPM1D is common in various cancer types and results in overexpression of enzymatically active PPM1D [PPM1D has been reported in about 10% of breast cancers, mainly those that retain a wild-type p53 status [PPM1D knock-out mice demonstrate that PPM1D promotes tumor growth by inhibiting p53 and p38/MAPK pathways [PPM1D that result in production of the C-terminally truncated PPM1D protein [PPM1D show G1 checkpoint override upon exposure to the mild level of IR [PPM1D in human non-transformed retinal pigment epithelial (RPE1) cell lines using CRISPR/Cas9 technology, we observed decreased ability to induce the G1 checkpoint after exposure to IR [PPM1D using the Transcription activator-like effector nuclease (TALEN) technology [TPPM1D allele protected intestinal stem cells from apoptosis by suppressing the p53 pathway [T/+PPM1D mice showed a higher amount of intestinal polyps and increased frequency of colon adenocarcinoma induced by constitutively active Wnt signaling in minAPC background [TPPM1D allele alone did not induce the formation of colon tumors, it significantly potentiated the phenotype and reduced the survival of minAPC mice [Amplification of the chromosomal locus 17q23.2 carrying the ve PPM1D . Amplifi3 status ,25,26. Dpathways ,27,28. Ipathways ,30. Rece protein ,32,33. T protein ,34. Impo protein . Cancer el of IR . Similarre to IR . Howeverchnology . We have pathway . In addickground . Althougmin mice .−CD8− double-negative (DN) and CD4+CD8+ double-positive (DP) stages and leave the medulla as single positive CD4+ or CD8+ cells with a fully assembled T-cell receptor (TCR) [TCR gene present in DN cells trigger the p53 response and are eventually repaired by V(D)J recombination allowing the transition to the DP stage [T-cells differentiate in the thymus cortex from early progenitors by sequentially progressing through CD4or (TCR) ,37. SiteDP stage ,39,40. SDP stage . In addiDP stage ,43,44.TPpm1d mouse model to study the impact of truncated PPM1D on cell survival and tumorigenesis in murine thymus. We find that thymocytes carrying truncated PPM1D escape apoptotic cell death and continue proliferation despite the presence of DNA damage. Although the truncated PPM1D did not significantly drive tumorigenesis upon exposure of mice to IR, it promoted the formation of thymic lymphoma in +/−Trp53 heterozygotes. We propose that truncation of PPM1D prevents full activation of p53 upon genotoxic stress and promotes tumor formation in cells exhibiting partial loss of p53.Here we used the knock-in Ppm1dT allele with a premature stop codon in exon 6 of the Ppm1d was described previously [tm1Tyj/JTrp53 mouse strain was obtained from the Jackson Laboratory (stock #002101) and was described previously [All animal experiments were approved by the ethical committee of the Institute of Molecular Genetics (c.j. 1/2016) and were performed in C57Bl/6 mice. The mouse strain carrying a eviously . Trp53tmeviously . Where iProtein concentration was measured by the Bicinchoninic Acid (BCA) protein assay . Proteins (30 μg) were separated on 4–20% gradient gels using SDS-PAGE and blotted onto a nitrocellulose membrane. Membranes were blocked in 3% milk in phosphate-buffer saline (PBS) for 2 h and incubated overnight with primary antibodies at 4 °C. The following antibodies were used for immunoblotting: rabbit monoclonal to PPM1D/WIP1 , p53-pS15 , p21 , KAP1-pS824 , γH2AX-pS319 , p53 (Leica-CM5P-L), H2AX , Cleaved Caspase-3-Asp175 , KAP1  and Importin β . After the washing, membranes were incubated with HRP-conjugated secondary antibodies (BioRad) and signal was detected using Enhanced chemiluminiscence (ECL) Western Blotting Substrate .Analysis of the developing T-cells by flow cytometry was performed as described . BrieflyTissues embedded in paraffin were sectioned on a Leica RM2255 microtome at a thickness of 3 μm. To examine the morphological changes in the tissues, the standard hematoxylin and eosin (H&E) staining protocol was followed. For staining with the Ki-67 proliferation marker  and the Cleaved Caspase-3-Asp175 apoptotic marker , tissues were deparaffinized with xylene, isopropanol, and decreasing concentrations of ethanol. Heat-induced epitope retrieval was performed in 10 mM citrate buffer (pH 6.0). Samples were blocked with 0.3% hydrogen peroxide in methanol and in bovine-serum albumin (1%) with goat serum (5%) for 2h for Ki-67 staining or 5% BSA and 0.1% TX-100 in PBS for cleaved caspase staining. Incubation with the primary antibody was done overnight at 4 °C and with the biotin-conjugated secondary antibody for 1 h at room temperature. Signal was developed using VECTASTAIN ABC HRP kit (Vector Laboratories) and 3,3′-Diaminobenzidine (DAB). Apoptosis was detected using Click-iT™ Plus terminal deoxynucleotidyl transferase (TUNEL) Assay . Briefly, tissues were deparaffinized, fixed with 4% formaldehyde, and permeabilized with Proteinase K. The TdT reaction with Ethynyl-2′-Deoxyuridine, 5′-Triphosphate (EdUTP) incorporation into the dsDNA breaks was performed for 60 min at 37 °C. Click-iT™ Plus reaction for detecting EdUTP with Alexa Fluor 594 picolyl azide was performed for 30 min and nuclei were stained using 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI). Slides were mounted using Q Path Coverquick mounting medium and visualized on a Leica DM6000 microscope (Leica) equipped with HC PL FLUOTAR 5X, and HC PLAN APO 20X and 40X objectives. Images were analyzed using ImageJ software . After tCDKN1A/p21 TGAGGAGGAGCATGAATGGAGACA and AACAGGTCGGACATCACCAGGATT; PUMA CCT-GGAGGGTCATGTACAATCT and TGCTACATGGT-GCAGAAAAAGT; PPM1D AGCCAGGAG-ACCTGTGTGAT and GGCATTACTGCGAACAAGGG; GAPDH ACAGCCGCATCTTCTTGTGCA-GTG and GGCCTTGACTGTGCCGTTGAATTT. The LightCycler480 software was used to determine Ct values. The data is shown as a ratio between the tested mRNA and GAPDH mRNA.cDNA was generated from 0.5 μg RNA using random hexamers and RevertAid Reverse Transcriptase . RT-qPCR was performed using LightCycler 480 SYBR Green I Master mix (Roche) as described . The folt-test was used and p values < 0.05 are considered statistically significant. Kaplan-Meier survival plot was evaluated using a log-rank test. Error bars indicate standard deviation.Several animals used in each experimental group is indicated. Immunoblots were performed using tissues from at least 3 animals and representative images are shown. Statistical significance was evaluated using Graph Pad Prism 5.04 software. Unless stated otherwise, two-tailed unpaired Ppm1d blocked progression of thymocytes from a DN to DP stage resulting in a decreased population of differentiated T-cells and in reduction of the thymus size in −/−Ppm1d mice [TPPM1D knock-in mouse model [Ppm1d and truncated TPpm1d were expressed at comparable levels suggesting that introducing a stop codon in the last exon does not affect transcriptional control of Ppm1d mRNA  and collected the thymi and lymph nodes after 6 h  and induction of the p53 transcriptional target CDKN1Ap21 after exposure to IR  wild-type mice  of whole-body irradiation  developed lymphoma  and enlarged kidney also referred to as hydronephros) that has previously been linked with T-cell lymphomas [Ppm1dT/++/−Trp53 double heterozygotes compared to +/−Trp53 indicating that truncated Ppm1d may promote tumorigenesis in cells with a partial loss of p53 function.Under the same conditions, one out of 12 d thymus  and showd thymus C,D indiclymphoma . Similar+/− mice C,D. Immu+/− mice E. In addosarcoma . Anotherymphomas   are heterozygotes, and also colon carcinoma samples do not contain both mutant alleles [PPM1D observed in Acute Myeloid Leukemia (AML) patients spread throughout the whole exon 6 which results in many variants of the PPM1D protein with differently a sized C-terminal tail [Ppm1dT/T homozygote could suppress p53 function more efficiently leading to increased probability of tumor formation. We speculate that another Ppm1d truncation leading to higher protein stability could possibly reach the threshold required for efficient suppression of p53 already in a heterozygous state. It will be interesting to address this possibility in the future by generating other mouse models with nonsense mutations at various positions in exon 6 of the Ppm1d.In this study, we exploited our recently generated transgenic model of the truncated Ppm1d to study its impact on DNA damage response in T lymphocytes and to evaluate its role in tumorigenesis . As withan cells . Moreove alleles ,35. Multnal tail ,56. WhenPPM1D locus leading to high expression levels of the full-length PPM1D protein is relatively common in human breast cancer and was observed mainly in tumors retaining the wild-type p53 [Amplification of the type p53 ,57. Data


OsAAP1 positively regulates growth and grain yield in rice by influencing amino acid uptake and reallocation. OsAAP1 gene is highly expressed in rice axillary buds, leaves, and young panicles, and that the OsAAP1 protein is localized to both the plasma membrane and the nuclear membrane. Compared with the wild-type ZH11, OsAAP1 overexpression (OE) lines exhibited increased filled grain numbers as a result of enhanced tillering, while RNAi and CRISPR  knockout lines showed the opposite phenotype. In addition, OsAAP1-OE lines had higher concentrations of neutral and acidic amino acids, but lower concentrations of basic amino acids in the straw. An exogenous treatment with neutral amino acids promoted axillary bud outgrowth more strongly in the OE lines than in the WT, RNAi, or Osaap1 lines. Transcriptome analysis of Osaap1 further demonstrated that OsAAP1 may affect N transport and metabolism, and auxin, cytokinin, and strigolactone signaling in regulating rice tillering. Taken together, these results support that increasing neutral amino acid uptake and reallocation via OsAAP1 could improve growth and grain yield in rice.Nitrogen (N) is a major element necessary for crop yield. In most plants, organic N is primarily transported in the form of amino acids. Here, we show that amino acid permease 1 (AAP1) functions as a positive regulator of growth and grain yield in rice. We found that the  Nitrogen (N) is one of the major nutrients for plant growth and crop yield. Most non-legume plants absorb inorganic N, including nitrate and ammonium, which are then utilized in amino acid synthesis. These amino acids, which are the primary forms of transported organic N, are then transported and reallocated from the source organs (e.g. developing roots and leaves) to the sinks  through the xylem and phloem . Recent AAT genes have been identified in Arabidopsis  to absorb and translocate amino acids. A large number of bidopsis , rice  were identified  inhibit shoot branching in Arabidopsis  is localized on both the plasma membrane and nuclear membrane of rice protoplasts. Overexpression of OsAAP1 led to an increased number of filled grains per plant as a result of an increased tiller number in rice. Knockout of OsAAP1 affected N transport and metabolism, and many hormonal pathways, resulting in the inhibition of axillary bud outgrowth and reduced tiller number. Thus, our results provide a valuable candidate gene for the improvement of rice yield.In this study, we found that OsAAP1 overexpression (OE) vector, a 1464 bp OsAAP1 (LOC_Os07g04180) cDNA was amplified using the primers 5'-GGGGTACCATGGGGATGGAGAGGCCGCAA-3' and 5'-GCTCTAGATGAGGAGACGCTGAATGGCTT-3′, and inserted downstream of the 35S promoter in pCAMBIA1306  for 4 h in the dark under gentle shaking (80 rpm). After incubation, protoplasts were passed through a Centauri. A 10 ml aliquot of W5 solution  was added and the solution was centrifuged for 5 min at 80 g to pellet the protoplasts.Our protoplast isolation protocol was based on the protocol for Arabidopsis protoplasts provided online by J. Sheen’s laboratory 9http://genetics.mgh.harvard.edu/sheenweb/0 with several modifications. At 7 d to 14 d post-germination, rice plants were ~10–20 cm tall. The plant materials were put in balance liquid (0.6 M mannitol), and then shoot tissue was cut into 0.5 mm pieces using very sharp razors and placed into the balance liquid for 10 min. Tissue was immediately incubated in an enzyme solution , and 100 μl of rice protoplast suspension (~5×105 cells) was transfected with 60 μg of HBT and OsAAP1-GFP with the polyethylene glycol (PEG) transformation method ; 0.8 ml of each filtrate was analyzed using the amino acid analyzer. Total N content was determined using the semi-micro Kjeldahl method using an N analyzer   . Nitroge2, 5 mM KCl, and 2 mM MES, pH 5.8) containing each FITC-labeled amino acid to a final concentration of 1 mM in the dark at 28 °C for 4 h, the protoplasts were centrifuged at 100 g for 5 min and the supernatant was carefully removed, and then the protoplasts were suspended with 1 ml of W5 solution and centrifuged at 100 g for 5 min. This step was repeated three times. Finally, the fluorescence signals were observed using a confocal laser scanning microscope.Amino acids with fluorescein isothiocyanate (FITC) markers  were synthesized by Yuan Peptide Biotechnology Company. A protoplast amino acid uptake assay was performed as described previously 2SO4, and NH4NO3 for 4 h, respectively. To investigate the effect of neutral amino acids on the OsAAP1 transgenic plants, seedlings were grown in basic rice culture solution with 1 mM NH4NO3 as the N source for 1 week, then transferred to the basic rice culture solution supplemented with 0.5 mM neutral amino acids  as the N source for an additional 3 weeks. To further explore the effect of different amino acid groups on the OsAAP1 transgenic plants, seedlings were grown in basic rice culture solution for 1 week, then transferred to basic rice culture solution with 1 mM basic amino acids Lys and Arg, 1 mM acidic amino acids Asp and Glu, or 1 mM neutral amino acids Tyr and Pro for an additional 3 weeks, respectively. To analyze the phenotype of OsAAP1 transgenic plants in the presence of different concentrations of NH4NO3, 1-week-old seedlings were cultivated in basic rice culture solution supplemented with 0.25, 1.0, or 4.0 mM NH4NO3 for an additional 3 weeks. The nutrient solution was renewed every 3 d.To detect whether the OsAAP1 CRISPR  vector construct was prepared using CRISPR/CRISPR-associated protein 9 (Cas9)-based multiplex genome editing for monocot and dicot plants (OsAAP1 were designed and added to the U6IPAT/U6OPST or the U3IPAT/U3IPST primers. A 422 bp fragment from target sequence 1 was amplified by PCR using primers U6OPST and OsAAP1-U6IPAT (5'-GGTTATGACCGAGAACGCCACAACCTGAGCCTCAGCGCAGC-3′) and the U6 plasmid. In the same way, a 348 bp fragment was amplified by PCR from target sequence 2 using primers ZRO589 and OsAAP1-U3IPAT (5'-GGTTATGACCGAGAACGCCATGCCACGGATCATCTGCACAACTC-3′) with the U3 plasmid. A 476 bp fragment from target sequence 1 was amplified by PCR using primers OsAAP1-U6OPST (5'-GTGGCGTTCTCGGTCATAACCGTTTTAGAGCTAGAAATAGCAAGTTA-3′) and U6IPAT with the U6 plasmid. Similarly, a 463 bp fragment from target sequence 2 was amplified by PCR using primers OsAAP1-U3IPST (5'-ATGGCGTTCTCGGTCATAACCGTTTTAGAGCTAGAAATAGCAAGTTA-3′) and ZRO282 with the U3 plasmid. Complete U6 and U3 fragments were amplified by fusion PCR containing U6 promoter–short guide RNA (sgRNA) and U3 promoter–sgRNA. The U6 fragment was digested with KpnI, and the U3 fragment was digested with KpnI and SacI, and then the U6 and U3 fragments were connected. The complete U6 and U3 sequence of 1600 bp was amplified by PCR using U6OPST and ZRO282 primers with the U6–U3 plasmid. Finally, the entire U6–U3 fragment of 1600 bp was inserted into the Per8–Cas9 vector using KpnI and SacI, and cloning kits. The vector was transformed into the ZH11 background. The T0 CRISPR transgenic lines were selected by PCR and sequencing using the corresponding primers 5'-GTCCGGTGTCCATGGATGAGGTAG-3′ and 5'-CACCTCAGAAGGTGTACACAGTC-3′.The OsAAP1-CRISPR lines and the wild-type ZH11 plants were collected for RNA sequencing (RNA-seq), analysis and two biological replicates were performed for each sample by Novogene. The clean data were aligned to the rice genome reference sequence (Oryza_sativa.IRGSP-1.0) by HiSAT2 (v2.1.0) (The axillary buds (<0.5 mm) from t-test. Significance levels: ***P < 0.001; **P < 0.01; *P < 0.05.Differences were analyzed using Student’s OsAAP1, we first generated pOsAAP1::GUS transgenic plants in the ZH11 background; the GUS staining results showed that OsAAP1 was highly expressed in root tips (JXB online). Then, we performed qRT-PCR using RNAs isolated from diverse tissues collected from the wild-type ZH11, including roots, leaf sheaths, axillary buds, tiller bases, and leaf blades at the vegetative stage, and stems, panicles, and leaf blades at the reproductive stage. These results showed that OsAAP1 was widely expressed in many rice tissues, but was much more highly expressed in roots, mature leaf blades, and panicles ] to treat wild-type rice seedings to explore whether they could affect the expression of OsAAP1. Interestingly, we found that ABA and the SL analog rac-GR24 could significantly inhibit the OsAAP1 expression, while indole-3-acetic acid (IAA) and 6-benzyl adenine (6-BA) did not affect its expression (Supplementary OsAAP1 (2500 bp upstream of ATG) and found two ACGT-containing elements (ACEs) and one ABRE, which may be responsible for ABA’s regulatioin of OsAAP1 expression  to have nine transmembrane helixes with the N-terminus in the cytoplasmic side. To investigate the subcellular localization of OsAAP1, a 35S promoter-driven GFP fused to OsAAP1 was transiently expressed in rice protoplasts. We found that the GFP signal was widely observed in rice protoplasts (35S promoter-driven OsAAP1 with a GFP tag at the C-terminus (2302-OsAAP1-GFP) was transiently expressed in pavement cells of N. benthamiana. The GFP signal was detected not only on the plasma membrane but also in the endomembrane system in the cytosol. DAPI staining further confirmed that OsAAP1–GFP was also localized on the nuclear membrane  of Ser, Gly, Ala, Cys, Val, Met, Ile, Leu, Tyr, Phe, and Pro were mixed and exogenously applied to the OE, RNAi, and wild-type ZH11 plants, and their growth phenotypes were evaluated after 3 weeks. The results showed that the growth of the OE lines was notably improved as compared with the wild-type ZH11, but the RNAi lines had much poorer growth than ZH11  between the two samples, including 911 up-regulated genes and 2420 down-regulated genes , could promote axillary bud outgrowth and enhance grain yield and N use efficiency , OsCYCP4;3, CYCP4;4, and OsCDKA1 were all down-regulated in Osaap1 compared with the wild type .Table S1. Amino acid concentrations in the straw (mg g DW–1).Table S2. Amino acid concentrations in the grain (mg g DWeraa256_suppl_Supplementary_MaterialClick here for additional data file.


Eif2s3y, a Y-linked gene that promotes proliferation of spermatogonia, had an inhibitory effect on oogenesis. This led us to the concept that male and female gametogenesis appear to be in mutual conflict at an early stage. This study provides a deeper understanding of oogenesis under a sex-reversal condition.A set of sex chromosomes is required for gametogenesis in both males and females, as represented by sex chromosome disorders causing agametic phenotypes. Although studies using model animals have investigated the functional requirement of sex chromosomes, involvement of these chromosomes in gametogenesis remains elusive. Here, we elicit a germ cell-intrinsic effect of sex chromosomes on oogenesis, using a novel culture system in which oocytes were induced from embryonic stem cells (ESCs) harboring XX, XO or XY. In the culture system, oogenesis using XO and XY ESCs was severely disturbed, with XY ESCs being more strongly affected. The culture system revealed multiple defects in the oogenesis of XO and XY ESCs, such as delayed meiotic entry and progression, and mispairing of the homologous chromosomes. Interestingly,  in vitro culture system, in which female (XX) ES cells differentiate into functional oocytes. Our results showed that oogenesis from XO and XY ES cells was severely disturbed due to defective processes such as delayed meiotic initiation/progression, aberrant gene expression and mispairing of homologous chromosomes. In addition, our results demonstrate that a Y-linked gene, Eif2s3y that promotes spermatogenesis, has conversely a negative impact on oogenesis. This study provides a culture system that serves as a useful model of sex reversal and contributes the concept of a substantial germ cell-intrinsic conflict between male and female gametogenesis. Further study using the culture system will reveal molecular insights into infertility with sex chromosome disorder(s).Oogenesis is a highly sex-dependent differentiation process that commences at the mid-gestation stage. Oogenesis is severely disturbed in XO and XY females, although the mechanisms underlying the disturbance are still elusive. In this study, we subjected XO and XY ES cells to an  Oogenesis is a unique sequence of differentiation processes that finally confers totipotency to eggs. The process is strictly regulated in a sex-dependent manner in mammals. In mice, primordial germ cells (PGCs) arise from the pluripotent epiblast and migrate into embryonic gonads, which eventually become either ovaries or testes , 2. The Zfy2, disturbed meiotic progression in oocytes [Several potential causes of the defective gametogenesis of “sex-mismatched” germ cells have been proposed. In the case of defective oogenesis in XY females, a half dosage of X chromosomes, a heterologous pairing of the sex chromosome and resulting asynapsis during meiosis, and a negative impact of Y-linked gene(s) are the main postulated mechanisms. It is known that one of the X chromosomes is inactivated in nascent PGCs, but reactivated prior to meiotic entry. Therefore, XY oocytes are essentially insufficient of transcripts from the X chromosome. During meiosis, all homologous chromosomes align by the pachytene stage. In spermatogenesis, a unique regulatory action takes place between X and Y chromosomes via a structure known as the XY body, in which the transcriptional activity of the sex chromosome is silenced in the spermatocytes . This si oocytes . Althoug oocytes . In addiin vitro differentiation (IVDi), largely reproduced that of in vivo differentiation in terms of gene expression, morphology and functionality: eggs produced in the culture system gave rise to live pups. This culture system seems advantageous from the following standpoints: (1) the functions of germ and somatic cells can be separately evaluated in the reconstitution system; (2) cells without mutation/deletion can be used; and (3) it is easy to evaluate gene function by manipulating the gene(s) in ESCs/iPSCs. Therefore, in this study, we used this novel culture system as a tool to gain insight into the germ cell-intrinsic role of sex chromosomes on oogenesis.Recently, we reported that oogenesis can be reconstituted in culture using pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). In the reported culture system, PGC-like cells (PGCLCs) derived from ESCs/iPSCs were aggregated with female gonadal somatic cells of embryonic day (E) 12.5 embryos , thereafBlimp1-mVenus (BV), a reporter gene of PGCs, and stella-ECFP (SC), a reporter gene of PGCs and oocytes, were clearly observed in aggregates at 2 days of culture (agg-2). As expected, in XX PGCLC, BV was downregulated from 7 days of culture onward (agg-7)  in rOvaries composed of E12.5 female gonadal somatic cells . XO ESCs (agg-7) , during  (agg-7) . At 21 d (agg-7) . It was  (agg-7) . The red (agg-7) . The num (agg-7) , suggest (agg-7) . HoweverNobox, Figla and Sohlh1, were enriched in XX oocytes , 5 (agg-5), 7 (agg-7), 9 (agg-9), 11 (agg-11) and 13 days (agg-13) of culture. Principal component analysis (PCA) of the expression profiles showed that the expression profiles of XX, XO and XY were similar before and at agg-3 , suggest oocytes . In cont oocytes . In cont oocytes . The DEGand Sox2 . This oband Sox2 . Moreoveand Sox2 . Collectin vivo  [Nanog expression was higher in E13.5 XYΔSry PGCs than in E13.5 XX PGCs, and Sycp3 expression was lower in in E13.5 XYΔSry PGCs than in E13.5 XX PGCs , which are markers of meiotic entry, pluripotency and PGCs/oocytes from ESCs, respectively, were examined at various stages of IVDi culture by immunofluorescence analysis. Among XX PGCLCs/oocytes, almost all (95.5%) were positive for SYCP3, demonstrating that they entered meiosis by agg-7 . In contin vivo . When E1in vivo . Althougan in XX . These r(XYΔSry) : Nanog e XX PGCs . Collectde novo entered meiosis at this stage. Based on these observations, XO and XY oocytes were likely arrested at the zygotene stage. Since meiotic arrest at the zygotene stage is caused by mispairing of homologous chromosomes, we analyzed the asynapsis rate in XX, XO and XY oocytes. We counted oocytes that have more than 1 pair of homologous chromosomes that are completely aligned, based on immunofluorescence analysis of SYCP1 and SYCP3. Under this criterion, 44.4% of XX oocytes completed the synapsis of all homologous chromosomes  , 24, gloΔSry female [Eif2s3y were most abundant in XY oocytes ΔSry femates Figs  and S8. tes Figs  and S8.  XO ESCs , followe XO ESCs . The decvel Figs  and S9B. XY ESCs . Althougt agg-21 . These fEif2s3y on the X chromosome, named Eif2s3x, which encodes an amino acid sequence that is 97.9% (461/471 amino acids) identical to EIF2S3Y. Based on the transcriptome analysis, Eif2s3x was expressed from ESCs to agg-13 at a different level  and Eif2s3y-middle (E19) clones, respectively  [Yy2 could be an interesting candidate for involvement in the timing of meiotic entry. Yy2 is a paralog of Yy1 and in some cases plays roles opposite to those of Yy1 by competing with the DNA-binding activity of Yy1 [Yy1 is involved in regulation of X chromosome inactivation [Yy2 to reactivation of the X chromosome, a hallmark of XX PGC-specific epigenetic reprogramming. It could be interesting to elucidate the function of Yy2 in the proper timing of meiotic initiation. In our experiments, it appeared that the expression profiles of XY oocytes were closer to that of XX oocytes than XO oocytes   S4 Fig)TIR femal(XYΔSry) –31. In P(XYΔSry) . Moreove(XYΔSry) . Therefoy of Yy1 –36. Intetivation , suggest oocytes . The reaAnother possible reason for the loss of XO and XY oocytes is the mispairing of homologous chromosomes during meiotic prophase I  11, 20,, 24. Indin vivo differed from that in vitro was that the culture system was less efficient for the pairing of homologous chromosomes in meiotic prophase I than the endogenous condition in vivo. Specifically, it was shown that there was tendency for mispairing of X chromosome(s) not only in XO and XY but also in XX oocytes in culture  responsible for the reduction in the oocyte pool of XY females has remained unclear. Here we showed that Eif2s3y is a novel inhibitory factor of early oogenesis  [sry was positively correlated with the expression level of exogenous Eif2s3x, indicating that a higher expression of Eif2s3x than the 3x72 clone (XX ESCs carrying a high copy number of exogenous Eif2s3x in this study) would show an inhibitory effect on oogenesis.These conflicting functions led us to focus on  XO mice . In additivation . These fnd agg-9 . The repnd agg-9 . More imogenesis . These r (XOsry) , 45. TheEif2s3y has a negative role in oogenesis. Further analyses are expected to reveal the detailed function of various genes. This culture system will also be a useful tool for the screening of small molecules that restore oogenesis in XO and XY cells/animals. Such a screening could provide a strategy to overcome infertility due to disorders involving the sex chromosomes.Here, we evaluated oocyte formation from ESCs harboring various sets of sex chromosomes in a novel culture system. The advantage of this culture system is that it recombines germ cells and somatic cells from independent sources, thus allowing us to evaluate germ cell-intrinsic effects on oogenesis. Experiments using animal models are less efficient than those using culture systems, especially for the evaluation of gene function through the overexpression or targeted deletion of a gene. Our culture system facilitates the functional analysis of genes, and here it was indeed revealed that All animal experiments were performed under the ethical guidelines of Kyushu University for animal use (#A28-109-4) and recombinant DNA experiments (#26–74).129X1/svj, C57Bl/6J, BDF1 and ICR mice were purchased from Japan SLC. E10.5 PGCs and E12.5 gonadal somatic cells were collected from ICR embryos. E11.5 BVSC PGCs were collected from embryos obtained by mating BDF1 female mice with C57Bl/6J male mice harboring BVSC.Eif2s3y XX cell lines. BVSC H18 #39RC4 h4, h36 and E7 are over expressed Eif2s3y XO cell lines. These ESCs were maintained under a 2i+LIF N2B27 medium condition without feeder cells.BVSC reporter ESCs were established as described previously . BVSC H1XO ESCs from XX ESCs were generated according to a previous report . Briefly15 μg genomic DNA of H18, #28, #30 and #39 was loaded onto 0.8% TBE agarose gel after EcoRV enzyme reaction. The DNA was then transferred to a membrane  and the membrane was hybridized with DIG-labeled probe DNA (15ng/ml) overnight at 42°C. After washing the membrane, we performed chemiluminescent detection with CDP-Star Substrate (Thermo Fisher Scientific T2305). Samples were analyzed by a chemiluminescence Imaging system (Fusion Solo).To identify the sex chromosomes, PCR amplification was performed using the following primers: for Sexing-F, TGG ATG GTG TGG CCA ATG CYC T; for Sexing-R, CCA CCT GCA CGT TGC CCT TKG TGC CCA G). The expected sizes of the PCR fragments were 253 bp for the X chromosome and 355 bp/399 bp for the Y chromosome. To identify the origin of the X chromosome, the sequence of the locus  was amplEpiLCs were cultured with Demecolcine solution 20 ng/ml (Sigma D1925) for 3 h. After the Demecolcine treatment, EpiLCs were harvested, resuspended in 0.075M KCl and then incubated at 37°C for 15 min. Ice-cold Carnoy's solution was added to the cell suspension and then the cells were collected by centrifugation at 1,500 rpm for 7 min. After removal of the supernatant, the cell pellet was resuspended in ice-cold Carnoy's solution. After repeating the suspension of cells with Carnoy's solution three times, the cell suspension was dropped on the slides. The slides were dried and then incubated in PBS containing DAPI for 15 min at room temperature. The numbers of chromosomes were measured under a fluorescence microscope (OLYMPUS IX73).Il2) transcript presumably because of contamination, we therefore excluded Il2 for further analysis. For the differential gene expression analyses, edgeR [Eif2s3x and Eif2s3y expression levels were determined with multi-mapped reads. Hierarchical clustering and principal component analyses were performed with R, based on RefSeq gene expression levels.For RNA-seq analysis, cDNA libraries were constructed as described previously . Brieflys, edgeR  was usedst strand cDNA Synthesis Kit . Genes of interest were amplified with Power SYBR Green PCR Master Mix  on a real-time qPCR system .For quantitative PCR (Q-PCR) analysis, total RNAs were purified using an RNeasy Micro Kit  and reverse-transcribed by PrimeScript 1For H3K27me3, 2,000 EpiLCs were aggregated on a low binding U-bottom 96-well plate (NUNC) for 2 days. The aggregates were dissociated and spread on the slide glass by Cytospin (Thermo). Slides were fixed with 4% paraformaldehyde (PFA) for 30 min, washed three times in PBSTB (PBS containing 0.1% Triton X-100 and 0.1% BSA), and then incubated in anti-H3K27me3 rabbit polyclonal antibody (pAb) (Upstate 07–449) overnight at 4°C. The slides were then washed three times and incubated with anti-rabbit IgG goat pAb Alexa 488 (Invitrogen A11034) and DAPI overnight at 4°C. The slides were washed three times and covered with Fluoro-KEEPER antifade reagent  for analysis by a confocal microscope (Zeiss LSM 700).For whole-mount immunofluorescence analysis, rOvaries were fixed with 4% PFA, washed twice in PBS containing 3% BSA, permeabilized in PBS containing 0.5% Triton X-100, incubated in blocking reagent (PBS containing 0.1% BSA and 0.3% Triton X-100) and then incubated with anti-GFP chicken pAb (Abcam ab13970) or anti-TRA98 rat IgG monoclonal antibody (mAb) (Bioacademia 73–003), anti-NANOG rabbit mAb (CST #8822) and anti-SYCP3 mouse mAb (Abcam ab97672) overnight at 4°C. rOvaries were then washed twice for 15 min each and three times for 1 h each and incubated with anti-chicken IgY goat pAb Alexa 488 (Invitrogen A11039) or anti-rat IgG donkey pAb Alexa 488 (Invitrogen A21208), anti-rabbit IgG donkey pAb Alexa 568 (Invitrogen A10042), anti-mouse IgG donkey pAb Alexa 647 (Invitrogen A31571) and DAPI overnight at 4°C. Finally, rOvaries were washed twice in PBS containing 0.3% Triton X-100 and soaked in Fluoro-KEEPER antifade reagent  for analysis by a confocal microscope (Zeiss LSM 700).For frozen sections, freshly isolated P1 ovaries and rOvaries were fixed with 4% PFA for 1 hour at 4°C. After washing with PBS, fixed samples were sequentially soaked in 10%, 15%, and 20% sucrose and then treated with fresh 20% sucrose overnight. Then, the samples were embedded in optimal cutting temperature (O.C.T.) compound (Tissue-Tek) and sectioned at 7 μm thickness. Sections were incubated in blocking reagent (PBS containing 3% FBS and 0.1% TritonX-100) for 1 hour at room temperature. Then, primary antibodies for GFP or TRA98 and pCHK2 or active Caspase3 or cleaved PARP1 were reacted at 4°C overnight. The sections were washed three times in PBS containing 0.1% TritonX-100 and once in PBS, and then anti-chicken IgY goat pAb Alexa 488 (Invitrogen A11039) or anti-rat IgG donkey pAb Alexa 488 (Invitrogen A21208), anti-rabbit IgG goat pAb Alexa plus 555 (Invitrogen A32732) or anti-rabbit IgG donkey pAb Alexa 568 (Invitrogen A10042) and DAPI (1 μg/ml) were reacted for 1 hour at room temperature. The slides were washed three times in PBS containing 0.1% TritonX-100 and once in PBS and then mounted in Fluoro-KEEPER antifade reagent  for analysis by a confocal microscope (Zeiss LSM 900). The antibodies used in this study are listed in A Click-iT Plus EdU Alexa Flour 594 imaging kit (Thermo Fisher Scientific C10639) was used for EdU analysis. rOvaries were cultured in 5 μM EdU labeling solution for 4 h at 37°C. The following processes were the same as for the whole-mount immunofluorescence analysis of rOvaries except for the secondary antibody reaction. rOvaries were incubated with anti-chicken IgY goat pAb Alexa 488 (Invitrogen A11039), anti-rabbit IgG donkey pAb Alexa 647 (Invitrogen A31573) and DAPI. rOvaries were soaked in Fluoro-KEEPER antifade reagent  for analysis by a confocal microscope (Zeiss LSM 700).ICR female mice mated with ICR male mice. Genomic DNAs were extracted from the heads of the embryos. XX and XY were discriminated by PCR. After genotyping, female genital ridges were isolated from the embryos, dissociated by trypsin treatment, and then incubated with FITC-SSEA1  for 15 min on ice. SSEA1-positive PGCs were sorted using a FACS Aria II (BD Bioscience). Each rOvary was composed of 2,000 PGCs and 30,000 gonadal somatic cells. After 6 days of IVDi culture, the rOvaries were analyzed by immunofluorescence analysis.E10.5 embryos were collected from pregnant 2 in PBS) for 30 min at 37°C, followed by Accutase  for 5 min at 37°C. Dissociated single cells were suspended in hypotonic buffer and placed on glass slides. Slides were washed three times in PBS, incubated in blocking buffer (PBS containing 10% FBS) and incubated with anti-SYCP1 rabbit pAb  and anti-SYCP3 mouse mAb (Abcam ab97672) overnight at 4°C. The slides were then incubated with anti-rabbit IgG goat pAb Alexa 568 (Invitrogen A11036) and anti-mouse IgG donkey pAb Alexa 647 (Invitrogen A31571). Then the slides were washed three times in PBS containing 0.05% Tween 20 and incubated with anti-γH2AXSer139 mouse mAb directly conjugated with FITC (Millipore 16-202A) and DAPI for 1 h at room temperature. Slides were washed three times in PBS containing 0.05% Tween 20 and mounted in Fluoro- KEEPER antifade reagent  for analysis by a confocal microscope (Zeiss LSM 700).Chromosome spreads were processed for immunohistochemistry as previously described . BrieflyChromosome spreads and immunostaining were performed as described above. The primary antibody used the following anti-SYCP1 rabbit pAb  and anti-SYCP3 mouse mAb (Abcam ab97672). The secondary antibody used the following anti-rabbit IgG goat pAb Alexa 405 (Invitrogen A31556) and anti-mouse IgG donkey pAb Alexa 647 (Invitrogen A31571). After chromosome spreads and immunostaining, slides were washed two times in PBS containing 0.05% Tween 20, rinsed in PBS and then fixed in 4% PFA for 10 min at room temperature. The slides were then washed three times in PBS and dehydrated by a serial incubation in ethanol: 70% for 5 min, 70%, 80%, 90% and 100% for 1 min each, and 100% for 5 min. After dehydration, the slides were incubated with probes of sex chromosomes (MetaSystems D-1420-050-FI and D-1421-050-OR) and mouse Cot-1 DNA (Invitrogen 18440016) for 5 min at 75°C for denaturing, followed by 16 to 20 h at 37°C for hybridization. Slides were washed once in 2×SSC for 5 min at room temperature and then two times in 50% formamide-containing 2×SSC for 5 min at 42°C. After cooling down, slides were soaked two times in cold 70% ethanol for 5 min and two times in cold 100% ethanol for 5 min. After dehydration, the samples on the slides were mounted with Fluoro- KEEPER antifade reagent  for analysis by a confocal microscope (Zeiss LSM 700).Huwe1 at 37°C overnight. The coverslips were washed three times in 50% formamide-containing 2×SSC for 7 min at 42.5°C, washed twice in 2×SSC for 5 min at 42.5°C, and then stained with DAPI (1 μg/ml) for 20 min at room temperature. The coverslips and slides were mounted in Fluoro-KEEPER antifade reagent  for analysis by a confocal microscope (Zeiss LSM 900).200,000 ESCs and 200,000 d6PGCLCs were transferred onto a poly-L-lysine  coated glass coverslip in a drop of PBS. Coverslips were fixed for 10 min in 4% PFA, then permeabilized on ice for 3 min 30 sec in 0.5% TritonX-100/PBS. After dehydrating through an ethanol series, they were denatured in 70% formamide-containing 2×SSC for 30 min at 80°C and dehydrated again through an ice-cold ethanol series. They were then hybridized with fluorescent BAC probes (RP24-157H12) for Stages of meiotic prophase l were determined by immunostaining of SYCP3 and SYCP1 based on the method in a previous report. LeptoteBased on immunofluorescence of SYCP1 and SYCP3, oocytes with more than 10 completely paired homologous chromosomes were subjected to pairing analysis. Autosomes and sex chromosomes were distinguished by FISH. The numbers of completely paired autosome(s) and sex chromosomes were divided by 19 and 1, respectively, to determine the pairing rate. In the case of X and Y chromosomes, chromosomes showing partial pairing were counted as paired chromosomes. The average of the pairing ratio in cells analyzed in each experiment was calculated. The experiments were performed independently three or four times and statistically processed by Tukey’s HSD test. The total numbers of oocytes counted were 36, 22, and 42 in XX, XO and XY, respectively.S1 FigA) Targeted integration of an elimination cassette to the X chromosome. The elimination cassette containing the DsRed gene floxed with inverted loxP sequences was introduced into the X chromosome locus. The probes and restriction enzymes for Southern blot analysis are shown. (B) Southern blot analyses using the probe described in (A). Note that the vector was properly integrated in the locus in clone #28 and #39. M, size marker. (C) Schematic diagram of elimination of the X chromosome via Cre-loxP recombination and FACS analysis. The elimination process can be monitored by expression of DsRed. FACS analysis shows DsRed expression at the elimination process. (D) Detection of the inactive X chromosome. Images show the immunofluorescence analysis of H3K27me3 (red) in EpiLCs after 1 day of aggregation culture. Arrowheads indicate punctuate staining of H3K27me3 representing the inactive X chromosome. (E) PCR analysis to distinguish the parental X chromosome. There is a polymorphism that is sensitive to SfaNI in the genome of C57BL/6J. The region can be amplified by the primers (arrows). The image below the diagram is a gel-electrophoresis of the PCR product after digestion of the enzyme. Scale bars, 10 μm. (F) Karyotype of the ESC clones. The X-axis indicates the number of chromosomes. The number of nuclei counted is shown in each graph. (G) Loss of the X chromosome in PGCLC induction. Representative images of DNA-FISH analysis of PGCLCs at day 6 of induction from BVSC H18 ESCs (left) and the quantification of the analysis (right) are shown. Scale bars, 1 μm.((TIF)Click here for additional data file.S2 FigA) Oocyte differentiation from XX and XY PGCs of E11.5 embryos. The PGCs were reaggregated with gonadal somatic cells of E12.5 female embryos. Note that the SC transgene was present in E11.5 PGCs, but not in E12.5 embryos. Scale bars, 200 μm. (B) The number of oocytes formed in culture. Each dot indicates the number of oocytes formed in one rOvary. The numbers in the graph indicate the average number of oocytes formed in each genotype. P values were calculated by t-test. ***P<0.001.((TIF)Click here for additional data file.S3 FigRplp0 and Ppia. The primer sequences used in this study are listed in The graph shows the expression of genes related to BMP and RA signaling in the somatic cells. Somatic cells were purified from rOvaries harboring XX, XO or XY oocytes. The Y-axis shows the ΔCt value of each gene relative to the average for (TIF)Click here for additional data file.S4 FigNanog, Stra8 and Sycp3 in XX and XYΔSry PGCs at E13.5. The expression profile was obtained from Sakashita et al. [The graph shows the expression of a et al. (TIF)Click here for additional data file.S5 FigA) Accumulation of γH2AX in the mispaired region. Three representative immunofluorescent images of SYCP3 (green), SYCP1(red), and γH2AX (white) and their merged images in XX, XO and XY oocytes are shown. The box in the merged image is shown on the right image. Note that the asynapsis regions, which are stained by SYCP3 but not SYCP1, are covered by γH2AX. (B) Pattern of autosomal asynapsis. The graph shows the percentage of each asynapsis pattern. Drawings at the right side of the graph illustrate a typical form of the chromosome in each asynapsis pattern.((TIF)Click here for additional data file.S6 FigA) Oocyte differentiation with CHK2-inhibitors. rOvaries harboring XX, XO or XY oocytes were cultured with the CHK2-inhibitors indicated at the left. Representative images at the day of culture indicated at the top are shown. Scale bars, 200 μm. (B) Immunostaining of phosphorylated CHK2 (pCHK2). Representative images of immunofluorescence analysis of pCHK2 in the P1 ovary and rOvaries harboring XX, XO or XY oocytes are shown. Scale bars, 10 μm. The graph shows the results of the immunostaining analysis.((TIF)Click here for additional data file.S7 FigA) The amounts of transcripts from autosomes and X chromosomes. Graphs show TPMs and SD of the amounts of transcripts from autosomes (left) and X chromosomes (right) in the cell type indicated. (B) X/A ratio during oogenesis in culture. The graph shows the X/A ratio in the cell type with a different set of sex chromosomes. (C) Relative values of X/A ratio between XX and XO. (D) DEGs between XX and XO oocytes. The list shows genes whose expression was 2-times higher or lower in XX oocytes compared to XO oocytes. The numbers in the heatmap are Log2(XX/XO). (E) DEGs between XX and XY oocytes. The list shows genes whose expression was 2-times higher in XX oocytes compared to XY oocytes. (F) Venn diagram of the DEGs.((TIF)Click here for additional data file.S8 FigThe expression data was extracted from the transcriptome analysis (in this study) and Sakashita et al. . The val(TIF)Click here for additional data file.S9 FigA) Structure of the Eif2s3y or Eif2s3x-transgene. Eif2s3y or Eif2s3x is driven by a c-kit promoter. (B) Expression level of exogenous Eif2s3y in the transgenic ESC lines. The transcript of Eif2s3y was detected by Q-PCR. The graph shows the ΔCt value and SD of Eif2s3y referenced by Rplp0. Eif2s3y-F, Eif2s3y-R, Rplp0-F and Rplp0-R primers were used for this study. (C) Expression level of exogenous Eif2s3x or Eif2s3y in the transgenic ESC lines. The transcript of Eif2s3x or Eif2s3y was detected by Q-PCR. The graph shows the ΔCt value and SD of Eif2s3x or Eif2s3y referenced by Rplp0. Eif2s3x3End Fw4, PB cKit 3'UTR Rv1, Rplp0-F and Rplp0-R primers were used for this study ((is study .(TIF)Click here for additional data file.S10 FigA) Deletion of the Eif2s3y gene by Cas9. gRNAs for deletion of exons of the Eif2s3y gene and primers for detection of the deletion are shown. The gRNA sequences used in this study are listed in B) Oocyte differentiation from Eif2s3y-KO XY ESCs in culture. Images are rOvaries at the days indicated. Note that SC-positive cells can be observed at agg-11. Scale bars, 200 μm. (C) The number of SC-positive cells in the rOvaries at agg-11. Note that the number of SC-positive cells at agg-11 were significantly higher  in the rOvaries with germ cells derived from Eif2s3y-KO XY ESCs (K39), compared with XY ESCs (N2).((TIF)Click here for additional data file.S11 FigA) Genomic structure of Eif2s3x and Eif2s3y. Note that the length of 3’UTR of Eif2s3x is longer than that of Eif2s3y. (B) Schematic diagrams of the reporter constructs. The mCherry gene with the 3’UTR of either Eif2s3x or Eif2s3y is driven by a c-kit promoter. (C) FACS analysis and images of ESC lines harboring the mCherry-reporter construct. FACS analysis provided a comparison of mCHERRY expression between transgenic ESC lines (red) and a non-transgenic ESC line (blue). Images below the FACS analysis show bright field (BF) and fluorescence images of mCHERRY (mCherry) in ESC lines. The copy numbers integrated in each ESC line are shown in brackets. Scale bars, 100 μm. (D) Correlation of mCHERRY intensity and the copy number integrated. Red and blue dots show ES clones harboring Eif2s3x or Eif2s3y. (E) Correlation of mCherry mRNA expression and the copy number integrated. The relative mRNA expression is the ΔCt value of mCherry referenced by Rplp0. (F) Correlation of mCHERRY intensity and mCherry mRNA expression. The primer sequences used in this study are listed in ((TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Leishmania requires proteasomal, autophagic and lysosomal proteolytic pathways to enact the extensive cellular remodelling that occurs during its life cycle. The proteasome is essential for parasite proliferation, yet little is known about the requirement for ubiquitination/deubiquitination processes in growth and differentiation. Activity-based protein profiling of L. mexicana C12, C19 and C65 deubiquitinating cysteine peptidases (DUBs) revealed DUB activity remains relatively constant during differentiation of procyclic promastigote to amastigote. However, when life cycle phenotyping (bar-seq) was performed on a pool including 15 barcoded DUB null mutants created in promastigotes using CRISPR-Cas9, significant loss of fitness was observed during differentiation and intracellular infection. DUBs 4, 7, and 13 are required for successful transformation from metacyclic promastigote to amastigote and DUBs 3, 5, 6, 8, 10, 11 and 14 are required for normal amastigote proliferation in mice. DUBs 1, 2, 12 and 16 are essential for promastigote viability and the essential role of DUB2 in establishing infection was demonstrated using DiCre inducible gene deletion in vitro and in vivo. DUB2 is found in the nucleus and interacts with nuclear proteins associated with transcription/chromatin dynamics, mRNA splicing and mRNA capping. DUB2 has broad linkage specificity, cleaving all the di-ubiquitin chains except for Lys27 and Met1. Our study demonstrates the crucial role that DUBs play in differentiation and intracellular survival of Leishmania and that amastigotes are exquisitely sensitive to disruption of ubiquitination homeostasis.The parasitic protozoan  Leishmania parasites require a variety of protein degradation pathways to enable the parasite to transition through the various life cycle stages that occur in its insect and mammalian hosts. Several enzymes involved in protein degradation in Leishmania are known to be essential, including a multi-protein complex, the proteasome, but little is known about how proteins are targeted to the proteasome for degradation. Here, we analyse components of the deubiquitination pathway, including twenty cysteine peptidases (DUBs) that remove the posttranslational modifier ubiquitin from substrates tagged for proteasomal degradation. We used chemical probes to measure active enzymes in parasite lysates and genome engineering to create DUB gene deletion mutants. We identified some DUBs that are essential for parasite viability and some that are required for life cycle progression. We carried out a detailed analysis of the essential DUB2, which has broad deubiquitinase activity and is found in the nucleus. This enzyme interacts with nuclear proteins associated with transcription/chromatin dynamics, mRNA splicing and mRNA capping. This work demonstrates the important role that DUBs play in Leishmania in vivo infection and further validates DUBs as potential drug targets in this parasite. Leishmania. The developmental process of Leishmania is closely linked with the ability of this parasite to survive and replicate in distinct environmental niches. The medical importance of one of these environmental niches is evident in the phagolysosomes of the infected host macrophages, where the Leishmania parasites differentiate from the motile promastigote to the non-motile amastigote and subsequently proliferate and establish an infection. The morphological and transcriptional changes that occur after differentiation of Leishmania promastigotes to amastigotes are well established , 1 x Penicillin/Streptomycin [Gibco] and 1 x BME Vitamins [Sigma-Aldrich] and adjusted to pH 5.25) up to a total concentration of 2 x 106 cells mL-1. Cultures were prepared in sextuplicate to provide technical replication. Next, the pooled cells were grown at 25°C for 7 d to allow for the enrichment of metacyclic promastigotes. To prepare axenic amastigotes, promastigotes from the 168 h time point were centrifuged and resuspended in amastigote medium at 2 x 106 cells per well. Cells were then cultured at 35°C with 5% CO2.Null mutant lines were grown to mid-log phase, spun down at 1,000 x g for 10 min and pooled in equal proportions in Grace’s medium , spun at 200 x g for 10 min and resuspended in macrophage medium (DMEM [Invitrogen] plus 10% FCS and 2 mM L-glutamine) in order to have 2.5 x 10per well . Enriche6 purified metacyclic promastigotes were injected into the left footpad of BALB/c mice. Mice were culled at 3 and 6 weeks post-inoculation. At the relevant time points, DNA was extracted from experimental samples and processed using the QIAGEN DNeasy Blood and Tissue Kit. DNA samples were prepared for next-generation sequencing by first amplifying unique barcodes by PCR using the following primers: 5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAgatgatgattacTAATACGACTCACTATAAAACTGGAAG-3’ and 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGAGAGACAGGCATGCCTT-3’ containing the Illumina adapter sequence. Reactions were set up with Q5 polymerase (NEB) as per the manufacturer’s instructions except for the following changes: just 0.5 x of Q5 Reaction Buffer and Q5 High GC Enhancer were used. The cycling conditions were 98°C for 5 min followed by 20–25 cycles of 98°C for 30 sec, 60°C for 30 sec and 72°C for 10 sec. Following purification of the PCR reactions using the QIAGEN MinElute PCR Purification Kit, a second PCR was performed using Illumina's Nextera XT indexing primers in order to add unique barcode sequences to each sample. 8 cycles of PCR amplification were performed using NEBNext Q5 Polymerase 2X Master Mix (New England Biolabs), according to the manufacturer’s guidelines. Indexed amplicons were purified using 0.9 X AMPure XP beads (Beckmann Coulter) and eluted into low TE buffer before quantitation and pooling at approximately equimolar ratios. Amplicon pools were then sent for 150 base paired end sequencing on an Illumina HiSeq 3000 sequencer at the University of Leeds Next Generation Sequencing Facility.To perform the mouse infections, 10Illumina read sequences were analysed using a custom Python script to search for the 12 bp sequence preceding the barcode. If an exact match was found, the following 12 bp sequence was counted as a barcode sequence. A total count for each unique barcode sequence was generated for each sample. Next, the relative fitness of null mutants at each life cycle stage was calculated by taking the number of unique barcode reads for each null mutant line and dividing it by the total number of expected barcode reads. Significant increases or decreases in null mutant line fitness between adjacent samples in the experimental workflow were analysed using unpaired t-tests and the Holm- Šídák method in GraphPad Prism 7.DUB2 inducible gene deletion was performed as previously described .Peak lists in.raw format were imported into Progenesis QI  and LC-MS chromatograms aligned. Precursor ion intensities were normalised against total intensity for each acquisition. A combined peak list was exported in.mgf format for database searching against the g SAINTq , using a-1, to a 5 mL His- trap crude FF column (GE Life Sciences). This was washed with Buffer A  and the protein eluted with increasing concentration of imidazole (0.03–0.5 M). Protein was concentrated using Amicon Ultra-4 30k MWCO (Millipore). 3C protease was added to purified protein in a 1:50  ratio with 2 mM DTT. The aliquot was added to a 16 kDa dialysis tube and incubated at 4°C in buffer A overnight. Protein was loaded onto a 5 mL His-trap crude FF column and ran as described above. The flow-through from the His-tag cleavage containing the tag free protein was concentrated to 2 mL using Amicon Ultra-4 30k MWCO (Millipore). The concentrated protein was applied to a HiLoad 16/600 S75pg column (GE Life Sciences), equilibrated and eluted in 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM β-mercaptoethanol. Fractions containing the protein were identified by absorbance at 280 nm and analysed by 10% SDS-PAGE. These fractions were pooled and concentrated up to 2 mg mL-1. 1 mM of DTT was added to the sample and the protein stored at -80°C.A baculovirus expression system was used for the expression of DUB2 recombinant protein in sf9 insect cells as described in the Bac-to-Bac Baculovirus Expression protocol (Invitrogen). The gene was first cloned into the PFastBacNKI-his-3C-LIC vector using Ligation Independent Cloning (LIC). After three days, the insect cells were lysed in lysis buffer  by sonication. The samples were then centrifuged  and the supernatant applied to an AKTA Start (GE Life Sciences) at 5 mL min2O and 2 μL 0.25 μg mL-1 ubiquitin probe Cy5UbPRG (UbiQ) for 30 min at room temperature. Binding of probe was analysed using a NUPAGE 4–12% Bis-Tris Gel. The gel was then scanned using the Amersham Typhoon  and stained with InstantBlue Protein Stain.10 μL of the appropriate concentration of the recombinant DUB was incubated with 2 μL 50 mM NaOH, 5 μL dHvitro enzymatic assay with the fluorogenic substrate ubiquitin-7-amido-4- methylcoumarin  was assembled in a 384-well microplate (Thermo Scientific). Assays were performed in 20 μL reaction volumes and in triplicate. The reactions were initiated by the addition of 10 μL of substrate or enzyme. Substrate and enzymes were diluted in buffer containing 50 nM Tris, pH 8.0, 0.15 M NaCl, 5 nM DTT and 0.05% CHAPS. The fluorescence intensity was monitored with a Polarstar plate reader (Omega) equilibrated at 25°C at intervals of 20 sec for 20 min using excitation/emission filter pairs of 340/460 nm. The fluorescence value was then converted into concentration using a standard curve. The standard curve was determined by allowing the reactions to plateau and the maximum arbitrary fluorescence units were measured and plotted against a known substrate concentration. An AMC concentration against time graph was generated in Excel software and the initial slope of each reaction was determined by generating a linear regression. For the determination of the Km and kcat values of DUB2, the enzyme concentrations were kept constant, and the concentration of Ub-AMC varied from 0.25 μM to 32 μM. The initial rates in μM sec -1 were then plotted against substrate concentration (μM) in Prism 7 software. The data were fitted to the Michaelis-Menton equation so the kinetic parameters, Km and kcat were determined.The in S1 FigLeishmania DUBs and selected reference human DUB sequences showing their active residues. Human DUB sequences were obtained from UniProtKB: PAN2 (Q504Q3), USP7 (Q93009), USP8 (P40818), BAP1 (Q92560), UCHL1 (P09936), UCHL3 (P15374), UCHL5 (Q9Y5K5), OTUB1 (Q96FW1), OTUB2 (Q96DC9) and OTULIN (Q96BN8). C19, C12 and C65 DUB families were aligned using the T-Coffee multiple sequence alignment program. A yellow star represents the active cysteine and the blue star represents the active histidine.Multiple protein sequence alignment of (TIF)Click here for additional data file.S2 FigDiagnostic PCRs were performed to check the successful generation of null and facilitated null mutants. PCR analysis of genomic DNA of at least two individual clones  or two separate populations (facilitated null mutants), using the indicated set of primers: ORF-specific primers (A), UTR-specific primers (B) or ORF-specific, 5’-UTR-blasticidin or UTR-specific primers (C). As a control, the parental Cas9 T7 cell line was used. The resulting amplicons were resolved on a 1% agarose gel and stained with SYBR safe. The expected size of the resulting amplicons is presented in (TIF)Click here for additional data file.S3 FigL. mexicana promastigotes treated with or without Cy5UbPRG for 30 min. Proteins were separated by SDS-PAGE and in-gel fluorescence (Cy5) was captured using a Typhoon imager followed by Coomassie staining as a loading control. (B) Lysates extracted from null mutant lines of log-phase L. mexicana promastigotes treated with Cy5UbPRG for 30 min. In-gel fluorescence images were obtained as for (A). The red arrowhead shows the position where an active DUB is missing compared to the parental Cas9 T7 cell line. (C) Western blot analysis of 2 x 107 axenic amastigotes. Samples were separated in a 4–15% protein gel. The stain-free gel used contains trihalo compounds which, in the presence of the UV-light, react with tryptophan residues, producing fluorescence. The gel was activated by 45 sec UV exposure, proteins were transferred to a PVDF membrane and probed with 1:1,500 dilution of anti-HASPB. Finally, as a loading control the total protein was determined using the stain-free property of the gel. (D) Lysate extracted from differentiated promastigotes to axenic amastigotes (48 h and 144 h after initiation of axenic differentiation) treated with or without Cy5UbPRG for 30 min. Protein was separated in an SDS-PAGE gel, and the image was captured using a Typhoon imager and the gel stained with Coomassie.(A) Lysate extracted from log-phase (TIF)Click here for additional data file.S4 FigL. mexicana procyclic promastigotes expressing mNeonGreen (mNG) tagged DUB2. DNA is stained with Hoechst 33342 and a representative selection of images is shown. DIC, differential interference contrast.Live cell imaging of (TIF)Click here for additional data file.S5 FigHYG cassette. The replacement of DUB2 wild type allele with the HYG cassette was detected by PCR amplification using the primers shown in the schematic (right). The forward primer was designed to bind to the ORF of DUB2 whereas the reverse primer binds on the 3’ UTR of the target gene. Black arrows represent the primers. (B) PCR amplification of DUB2-/+FLOX Clone 4, 6, DUB2-/+FLOX [DUB2] and DUB2-/+FLOX [DUB2C312A] surviving parasites after treatment with RAP in the clonal assay  between different time points, comparing treated with RAP to untreated samples. Error bars indicate the standard deviation of a mean. ***p < 0.001, ****p < 0.0001.(A) Left: PCR analysis of extracted gDNA demonstrates successful integration of al assay . Schemat(TIF)Click here for additional data file.S6 Fig−1 in PBS).(A) DUB profiling using the ABP Cy5UbPRG. Cell extract from a WT and mNeon:DUB2 was incubated with Cy5UbPRG for 30 min. Proteins were then separated by SDS-PAGE and imaged. The yellow arrow represents the DUB2 WT band whereas the red arrow represents the mNeon-DUB2. (B) Western blotting analysis with anti-myc antibody  of protein cell extract from samples collected after immunoprecipitation of control cell line (WT) and mNeon:DUB2. E1, E2 were samples collected from the elution (myc peptide 0.5 mg mL(TIF)Click here for additional data file.S7 FigPurification of DUB2 active (left) and DUB2C256inactive protein (right). (A) Protein gel of the soluble fraction (L), flow though (FT) and eluted fractions across the peak that were collected after Ni2+ affinity chromatography. 10 μL of the protein samples were separated by SDS-PAGE and visualised by InstantBlue Protein Stain. (B) Protein gel of the sample before overnight dialysis with protease 3C for the removal of the his-tag (T), after overnight dialysis (U) and flow through and eluted fractions across the peaks collected after the second Ni2+ affinity chromatography. (C) Protein gel of the diluted (Di) and concentrated (Co) samples before application to a HiLoad 16/600 S75pg column and eluted fractions collected after size-exclusion chromatography.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.


CDKN2B-AS1 & CDKN2B) are widely associated with Primary open-angle glaucoma (POAG). However, the functional importance of this locus in POAG pathogenesis is still unexplored. This study investigated the role of CDKN2BAS1-CDKN2B axis in POAG. We observed significant association of CDKN2B-AS1 SNP rs4977756 with POAG and its endophenotypic traits  and central corneal thickness (p = 0.008)) by screening African American POAG cases (n = 1567) and controls (n = 1600). A luciferase reporter assay in Human embryonic kidney 293T (HEK293T) cells revealed that the region surrounding rs4977756 likely serves as a transcriptional repressor. siRNA-mediated knockdown of CDKN2B-AS1 in HEK293T cells and trabecular meshwork (TM) cells resulted in significantly increased expression of CDKN2B, which was also observed in human POAG ocular tissues. Pathway focused qRT-PCR gene expression analysis showed increased cellular senescence, TGFβ signaling and ECM deposition in TM cells after CDKN2B-AS1 suppression. In conclusion, we report that CDKN2B-AS1 may act as a regulator, and it could function by modulating the expression of CDKN2B. In addition, increase in CDKN2B levels due to CDKN2B-AS1 suppression may result in the senescence of trabecular meshwork cells leading to POAG pathogenesis.The genes in the 9p21 locus ( Primary open-angle glaucoma (POAG) is the most common form of glaucoma, characterized by optic nerve (ON) degeneration and progressive loss of visual field leading to irreversible vision loss. POAG affects over 56 million people worldwide  and the MYOC), optineurin (OPTN), WD repeat domain 36 (WDR36), neurotrophic factor 4 (NTF4) and ankyrin repeat and SOCS box containing 10 (ASB10) with POAG and its endophenotypic traits [POAG is a complex genetic disorder influenced by the interaction of multiple genes, risk factors and endophenotypic traits such as intraocular pressure (IOP), central corneal thickness (CCT), retinal fiber layer thickness (RNFL), and cup-to-disc ratio (CDR). Genome-wide association studies (GWAS) and candidate gene screening studies have identified the association of many genes including myocilin  [CDKN2B-AS1 gene lies in the centromeric region of the 9p21 locus, and encodes a long noncoding RNA also known as ANRIL. This noncoding RNA plays a role in epigenetic regulation of gene expression through cis and trans-mechanisms [CDKN2A (cyclin dependent kinase inhibitor 2A) gene lies in the 5′ end of CDKN2B-AS1 and encodes two proteins, p16INK4A, and p14ARF that differ only in exon 1 due to alternative splicing. CDKN2B (cyclin dependent kinase inhibitor 2B) gene lies within exon 1 of CDKN2B-AS1 in the antisense direction and encodes a p15INK4B protein  ,15,16,17and IOP) ,19, typeand IOP) , intracrand IOP)  and infland IOP) . The CDKchanisms . The CDK protein . These ple cells , and epile cells , whereasle cells . This vaCDKN2B-AS1 with POAG and with endophenotypic traits in a large, deeply phenotyped AA cohort with POAG cases and controls. We investigate if the genomic region surrounding this SNP has a regulatory function. We characterize the expression of CDKN2B-AS1 and its ability to regulate CDKN2B and CDKN2A transcripts using relevant cell lines including HEK293T cells, human primary TM and human retinal tissues. Gene knockdown studies determine the function of CDKN2B-AS1 and its role in pathways relevant to POAG pathogenesis.Published studies have reported the association of the 9p21 locus with POAG mainly in Caucasian, Asian ,28 and on = 1567) as cases and age and ethnicity matched healthy subjects without POAG (n = 1600) as controls. The race, age, family history, comorbidities and relevant medical history of all participants was recorded on a pre-designed questionnaire for clinical correlation. This study was approved by the UPenn institutional review board (IRB) and adhered to the Declaration of Helsinki. Subjects with co-morbid eye disease were excluded from the study. The diagnosis of POAG was based on patients having an open iridocorneal angle along with characteristic glaucomatous optic nerve findings corresponding to visual field loss. Blood and saliva samples from the subjects were collected in heparinized vacutainers  by venipuncture with prior written informed consent and were immediately transferred to the laboratories in ice containers for genetic analysis [Subjects for this study were part of the Primary Open-Angle African American Glaucoma Genetics (POAAGG) cohort, which is a study investigating the genetic architecture of POAG in the over affected AA population. The POAAGG cohort includes self-identified African American individuals , aged 35 years or older, from the Philadelphia region. Eligible patients were enrolled from ophthalmology clinics at the University of Pennsylvania (UPenn) and two external sites in West and North Philadelphia. Our study samples include AAs subjects with POAG  with gene specific primers followed by bidirectional sequencing . The association of genetic variants or SNP rs4977756 with POAG was assessed after determining the SNP status in all the study subjects using appropriate statistical tests. ANOVA analysis was performed on pooled case and control data to determine if the following continuous phenotypic variables related to POAG were associated with variation at this locus: IOP, central corneal thickness (CCT), vertical cup to disc ratio (VCDR), and retinal nerve fiber layer thickness (RNFL). A type 1 error rate of α = 0.05 was chosen to delineate significance for logistic regression and ANOVA analyses.Genomic DNA was isolated from peripheral blood and saliva samples of the study participants using the phenol-chloroform method . The DNACDKN2B-AS1 region were amplified from genomic DNA isolated from study participants. To generate the A/G genotype from an insert containing the G/G genotype, we used QuikChange XL mutagenesis kit  as described previously [Acc65I restriction site was added to the sense primer and an EcoRV site to the antisense primer. Sequences containing the A/A, G/G, and A/G genotypes were each ligated into a pGL4.11 promoter vector and a pGL4.23 enhancer vector (Promega). The resulting plasmids were transformed into 10-beta Competent E. coli (High Efficiency) cells (NEB) and purified using GeneJET Plasmid Midiprep Kit (ThermoFisher). Plasmid sequences were confirmed using Sanger sequencing with Big Dye X Terminator (ThermoFisher).Inserts containing the following genotypes at SNP rs4977756  within the eviously . AmplifiUsing a dual luciferase reporter assay, the enhancer and promoter activity of insert sequences containing A/A, A/G, and G/G were determined by transfecting HEK293T cells in triplicate along with positive and negative control expression vectors (empty pGL4.11 and pGL4.23 vectors). The pGL4.74 renilla luciferase vector (Promega) was co-transfected with experimental plasmids to control for transfection efficiency.2 for 48 h. Cells were subsequently lysed, and luciferase activity was measured by performing a Dual-Luciferase assay (Pierce) on transferred lysates in a 96-well white opaque bottomed luminometric plates . Measurements obtained for firefly luciferase activity were divided by renilla luciferase activity to control for transfection efficiency, and normalized by the expression from empty vectors [t-test for differential luciferase activity.Briefly, HEK293T cells were transfected at approximately 60% confluency with the reporter plasmids using Lipofectamine 3000 (ThermoFisher), according to the manufacturer’s instructions. Following transfection, the cells were incubated at 37 °C with 5%CO vectors . pRLU anMYOC gene expression after inducing dexamethasone (n = 3) while control  retina tissues were obtained from the donor eyes collected at Ramayamma International eye bank, L. V. Prasad Eye Institute, Hyderabad. The diagnosis of controls samples was confirmed from the history of the patients obtained from the family.The HEK293T (human embryonic kidney-CRL3216) cells were obtained commercially from ATCC and primary human trabecular meshwork (hTM) cells (Lot # 3194) from Cell Applications. Characterization of TM cells was done by analyzing the ethasone  as descrw/v) BSA in 1X PBS for 60 min at room temperature. Thereafter, the slides were incubated for 60 min with rabbit Anti-p15INK4b antibody (ab53034) diluted to 1:100 in 0.1% BSA, followed by three washings with 1X PBS. Further incubation was carried out with Alexa 594 anti-mouse secondary antibody for one hour. Cells were washed in 1X PBS and mounted with prolong gold mounting medium with DAPI for nuclei staining. Images were captured by Olympus FV1000 Confocal microscope with the use of appropriate filters and lasers [For immunolocalization of CDKN2B, HEK293T and TM cells grown to 60–70% confluence in chamber slides were fixed with absolute methanol for 15 min in −20 °C and then permeabilized by incubating with 0.5% Triton X in PBS for 5 min at room temperature. Blocking was carried out with 5% normal goat serum in 1%  and 10 nM of non-targeting control pool siRNA  using Lipofectamine RNAiMAX . After 72 h, RNA was extracted from the transfected TMs and converted to cDNA using Superscript III reverse transcription kit  following manufacturer’s instructions. Quantitative real-time PCR with gene specific primers was done following the methodology described below. These experiments were done in triplicate.TM cells were grown to approximately 80% confluence in 6-well plates. The cells were transfected with 10 nM of pooled Senescence in TM cells was determined by using β-galactosidase staining kit . Briefly, TM cells at 80–90% confluence were washed twice with 1X PBS to remove growth media and fixed in a fixative solution for 10–15 min at room temperature. Cells were washed twice with 1X PBS and incubated in dark overnight at 37 °C with fresh β-galactosidase staining solution. Stained cells were imaged under a light microscope and examined for the development of blue color.Primary TM cells (Passage 6) at 70–80% confluence were treated with 2.0 ng/mL activated rhTGF-β1 and rhTGF-β2  for 72 h. The medium was replaced every 24 h with fresh medium containing recombinant human TGF-β1 and TGF-β2  and incubation was continued for a total of 72 h. Cells treated with vehicle, 4 mM HCl containing 1% BSA was used as a control.CDKN2B-AS1 siRNA and TGFβ induction) and POAG and normal eye tissues was extracted by the Trizol method (25). The cDNA was prepared using Superscript III One-Step RT-PCR kit . The RNA isolated from retinal tissues  was converted to cDNA using verso cDNA synthesis kit . A 10 μL reaction mixture was made using iTaq™ Universal SYBR® Green Supermix . Quantitative PCR was carried out using gene specific primers  following manufacturer’s instruction.The RNA from untreated and treated TMs, HEK293 cells , with an OR of 1.19 for homozygotes of the A allele . Specifically, the AA genotype was associated with a thinner CCT than the GG and AG genotypes. When all three genotypes were considered in the ANOVA analysis, no other quantitative trait reached the threshold of statistical significance . Additionally, the association between rs4977756 and CCT became stronger (p = 0.0081). This analysis suggests that individuals with the AA genotype have a significantly lower CCT measurements and greater VCDR measurements compared to individuals with the GG or AG genotype at rs4977756 (p = 0.011), however the relationship between VCDR and rs4977756 did not reach significance (p = 0.261)  .CDKN2B-AS1 transcripts. Hence, we performed a luciferase reporter assay to understand the regulatory nature of this region. Analysis of the luciferase expression revealed that the region cloned upstream to the luciferase gene in the pGL4.11 promoter vector resulted in extremely low mean luciferase expression when compared to renilla luciferase expression and empty vector. This suggests the 2.4 kb DNA insert containing rs4977756 is unlikely to be a promoter  and CDKN2B (p = 0.002) transcripts in POAG retina when compared to normal/control retina, further supporting their involvement in POAG pathogenesis , insulin growth factor  and p53/pRB signaling genes   after CDKN2B-AS1 siRNA treatment  b. Signifreatment c supportnescence d.ECM1, COL7A1, COL12A1, COL15A1, COL16A1, COL1A1, and COL6A2 along with MMPs suggests an accumulation of ECM products in TM cells after CDKN2B-AS1 suppression  composition by increased expression and degradation of extracellular matrix metalloproteinase, leading to the accumulation of ECM products that may have deleterious effects in tissue physiology. Moreover, stiffening of TM cells is the hallmark of glaucoma and is believed to be induced by ECM deposition. Significant increased expression of ECM molecules pression e. IncreaCDKN2B-AS1 increased the expression of p15 (INK4b) and transforming growth factor β1 (TGFβ1) resulting in the inhibition of cellular proliferation in human esophageal squamous cell carcinoma [CDKN2B-AS1 mediated regulation in cell proliferation. Therefore, to understand the role of TGFβ1-CDKN2B-CDKN2B-AS1 axis in glaucoma pathogenesis, we investigated the effect of CDKN2B-AS1 knockdown and TGFβ1/TGF β2induction on this axis. Knockdown of CDKN2B-AS1 transcript resulted in an increase of TGFβ1 and CDKN2B transcript expression in TM cells , and aqueous humor of glaucomatous eyes ,40,41. Karcinoma . This stTM cells a. Quantipression a. Downrend THBS1 a, furtheCDKN2B along with a slight increase in CDKN2B-AS1 . The 9p21 locus is known to be associated with normal tension glaucoma and various optic disc parameters, including CDR, but is not a genetic predictor of CCT ,43. CCT CDKN2B-AS1 and its sense transcripts [DHRS9)  [DHRS9 participates in the metabolism of all-trans retinoic acid in neurons and astrocytes, and can also serve as a nuclear transcriptional repressor [It is hypothesized that the intronic region containing rs4977756 may possess response elements that affect the expression of nscripts . Interesulation) . DHRS9 pepressor . Our lucepressor . Accordiepressor . These tepressor .CDKN2A, CDKN2B and CDKN2B-AS1 [CDKN2B-AS1 isoform expression in human endothelial cell lines, macrophage cell cultures and coronary smooth muscle cells [CDKN2B-AS1 and CDKN2A expression increased CDKN2B expression in peripheral blood cells [CDKN2B-AS1, CDKN2B and CDKN2A/ARF in TM cells, which are one of the primary cells affected in POAG, and to correlate their functions with downstream pathway regulation. So far three novel CDKN2B-AS1 splice variants have been reported in the human retina [CDKN2B-AS1 splice variants are reported, with exons 1 and 6 coinciding in most of them. Therefore, a pool of four siRNAs targeting exon 1, 2 and 6 were used for CDKN2B-AS1 knockdown studies in HEK 293T cells and TMs, which resulted in the efficient suppression of CDKN2B-AS1 expression.Disease associated variants have been reported to result in differential transcript expression of KN2B-AS1 ,47. Cardle cells , while aod cells . The pren retina  and  in TM cells may play an important role in inflammation, senescence and tissue remodeling  in 9p21 locus may have regulatory roles that could potentially translate to altered expression of genes in this locus in a cell specific and tissue specific manner. Significant increased expression of modeling . Further


Both the two G3 lineages have been recently detected in several countries. Our study is the first to document African children infected with G3 Lineage IX. These data highlight the global nature of RVA transmission and the importance of increasing global rotavirus vaccine coverage.Globally, rotavirus group A (RVA) remains a major cause of severe childhood diarrhea, despite the use of vaccines in more than 100 countries. RVA sequencing for local outbreaks facilitates investigation into strain composition, origins, spread, and vaccine failure. In 2018, we collected 248 stool samples from children aged less than 13 years admitted with diarrheal illness to Kilifi County Hospital, coastal Kenya. Antigen screening detected RVA in 55 samples (22.2%). Of these, VP7 (G) and VP4 (P) segments were successfully sequenced in 48 (87.3%) and phylogenetic analysis based on the VP7 sequences identified seven genetic clusters with six different GP combinations: G3P[8], G1P[8], G2P[4], G2P[8], G9P[8] and G12P[8]. The G3P[8] strains predominated the season ( All four vaccines were shown to be largely cross-protective against heterotypic strains in both clinical trials and following vaccine implementation in several settings , G3P[8] and G12P[8] genotypes in rotavirus vaccinating countries ,16,17,18n = 13), discharge or transfer before sample collection (n = 22), consent refusal (n = 52), or other (n = 16). Among study eligible children (n = 384), the distribution of the sampled and not sampled children differed significantly across age strata (p = 0.002) and discharge outcome (p < 0.001), ® vaccine while the remainder (19.4%) received one dose, Between January and December 2018, 384 children aged less than 13 years were admitted to KCH with diarrhea as one of their illness symptoms. Of these, 208 (54.2%) were Kilifi Health and Demographic surveillance system (KHDSS) area residents . A stoolp = 0.008), n = 37, 77.1%), G1P[8] , G2P[4] , G2P[8] , G9P[8]  and G12P[8] . The G3P[8] and G1P[8] strains were the only genotypes detected for > 2 months while the other four genotypes were detected sporadically (1–2 months), RVA prevalence was higher in female compared to male children admitted with diarrhea  compared to P types (n = 2) types, the range of pairwise nt differences was much greater in the VP7 (up to 203 nt differences) compared to VP4 segment (up to 87 nt differences), For the VP4 segment, a 579 nt long region (~25%) was recovered for 47 viruses (88.5%) while for the VP7 segment, a 644 nt long region (~65%) was recovered for 48 viruses (87.3%). One virus (KEN/KLF0879/2018), genotyped G9P[8], yielded a significantly shorter VP4 fragment relative to the other viruses (<500 nt) due to low quality sequencing data and was excluded from subsequent analyses. Consistent with the greater number of assigned G types  to define a molecular genetic cluster, seven G clusters were assigned (named Clu_1-7). Members of a cluster were universally of same G type. All G type sequences identified to be of the same type formed a single cluster except G3P[8] that occurred in three clusters, named Clu_3/G3P[8], Clu_4/G3P[8] and Clu_5/G3P[8]. The temporal pattern of the assigned clusters is shown in A few viruses in different VP7-based genetic clusters had identical VP4 sequences and we explored if these were spatially clustered. Twenty-eight of the 48 genotyped samples were from KHDSS area residents. The geographical distribution of all diarrhea admissions and the RVA positives by genetic cluster is shown in A total of 338 G3 sequences from 26 countries fully met the criteria for inclusion as comparison data, including 39 previously collected in Kenya. The phylogeny derived from the combined Kilifi and global G3 viruses is shown in n = 35, 94.6%) and equine-like G3 Lineage . Viruses of the genetic cluster Clu_4/G3P[8] clustered with the equine-like G3 Lineage while the Kilifi G3 Lineage I viruses separated into two groups that corresponded to the Clu_3/G3P[8] cluster (n = 30) and the Clu_5/G3P[8] cluster (n = 5).The distribution of the pairwise nt differences in the compiled global G3 sequences dataset, like for the Kilifi G3 viruses, showed a multi-modal distribution (figure not shown). The first major trough was observed at 27 nt differences. A majority of the global viruses fell within two of nine previously identified G3 lineages ; LineageOn applying the threshold used to identify the local molecular genetic clusters (>97% genetic similarity) on the global G3 dataset, 18 clusters were identified . Of thes® vaccine into its NIP, multiple RVA GP genotypes circulated during the 2018 season in Kilifi, Kenya, with the G3P[8] genotype predominating at 67.2%. At this study site, the preceding two years (2016 and 2017) were dominated by the G2P[4] and G1P[8] genotypes, respectively, with only six cases of G3P[8] detected from September 2009 to December 2017  strain. During 2018, this local G3P[8] predominance is consistent with the previously documented season-to-season spatial-temporal fluctuations in the prevalence of RVA genotypes  strains, e.g., in Australia , Japan , ThailanBased on sequence data deposited in GenBank, the predominant Kilifi G3 cluster (Clu_3/G3P[8]) was the second most common genetic cluster globally. The closest sequences were from Singapore and India, both countries that did not yet have RVA vaccine in their NIP in 2018. The second most prevalent Kilifi G3 genetic cluster was Clu_5/G3P[8]. Notably, this cluster has not been detected frequently around the globe and the closest genetic links were Kenyan strains collected in Kiambu County  in July and August 2014 , Kilifi This study had some limitations. First, the sequence data from the cohort represents a single site and one season. Second, we only sequenced portions of the VP4 and VP7 segments. Whereas these data were adequate to assign genotypes, lineages and estimate the number of genetic clusters, whole genome sequences provide a better resolution in examining reassortment events, evolution in internal genes and studying genetic clusters ,25,41. T® into Kenya’s NIP in 2014. The cross-continent detection of the emerging equine-like G3 viruses and other typical human G3 strains demonstrates the global nature of RVA transmission. Strikingly, strains found circulating in the Kilifi population were most closely related to strains circulating in countries that were yet to introduce RVA vaccines into their NIP. This observation reminds of the global connectedness regarding pathogen movement and emphasizes the importance of vaccinating all eligible populations across the world, as failure to do so builds a reservoir for strains that continue to seed transmission in vaccinated populations. Identifying factors responsible for RVA vaccine underperformance in low-income settings is a priority research area that may support efforts to further reduce RVA burden. Our study did not ascertain that viral genetic diversity is a contributor to the vaccine underperformance in this setting. Studies investigating the relationship between RVA vaccine immunogenicity and infant characteristics, such as malnutrition, age at first RVA dose, concomitant receipt of oral polio vaccine (OPV), enteric co-infections and enteric dysbiosis may provide better insight into RVA vaccine performance characteristics. In conclusion, the finding that >20% of diarrheal stools from children admitted to KCH with diarrhea in 2018 were RVA positive highlights that RVA is still a significant contributor to severe childhood diarrhea in coastal Kenya, despite the introduction of Rotarix2 with a population of ~300,000 people) is monitored by the KWTRP and is known as the KHDSS area [KCH is the main referral hospital in Kilifi County (population size ~1.5 million people). The major economic activities in the county are subsistence farming, fishing and tourism . An areaDSS area , Figure DSS area . VaccinaDSS area ,43,44.In the current analysis, stool samples were collected from eligible and consented pediatric patients admitted to KCH between January and December 2018 (the surveillance period), as part of the ongoing rotavirus surveillance program ,43. All RVA in the stool samples was detected using ProSpecT™ enzyme immunoassay (EIA) kit  following the manufacturer’s instructions. RVA positive samples were amplified in the VP4 and VP7 segments using One-step Reverse Transcriptase PCR Kit  using previously published primers ,46. SuccThe sequence reads were assembled using Sequencher v5.4.6 . Nucleotide (nt) sequence alignments were prepared using MAFFT v7.222 and visualized using Aliview v1.8. G and P genotypes were determined using Virus Pathogen Resource (ViPR) online classification tool . The beshttps://github.com/gtonkinhill/pairsnp/). Viruses within the same molecular genetic clusters were those which pairwise nt differences occurred within the first modal distribution. Using this threshold, clusters were identified using the USEARCH algorithm [Molecular genetic clusters were defined from the distribution of pairwise nt differences of VP7 segment sequences. Pairwise nt differences were determined using pairsnp  detection in a human stool/rectal swab specimen, (ii) sequence fully overlapping with the VP7 region sequenced for the Kilifi viruses, (iii) information on country and date of sampling available and (iv) sample collected in 2012–2018. G3 sequences collected previously from around Kenya including Kilifi were included in the analysis.χ2 or Fisher’s exact test (depending on group sample size). The 95% CI were presented for proportions and standard deviation for means. A p-value of <0.05 was considered significant. Numerical data were analyzed in STATA v15.1. Continuous variables were summarized using various measures of dispersion. Differences between groups were assessed using a t-test or Wilcoxon rank-sum test. Binary data were summarized using proportions and comparison between groups made using either Partial sequences for the VP7 and VP4 segments reported in this work have been deposited to GenBank database under the sequence accession numbers MN194408-MN194485 for VP7 and MN194325-MN194364 for VP4.Before sample collection informed written consent was obtained from the child’s parent or guardian. The Scientific Ethics Review Unit (SERU) board that sits at KEMRI, Nairobi, approved the study protocols (SERU#3049).