Kenya introduced the monovalent G1P [8] Rotarix® vaccine into the infant immunization schedule in July 2014. We examined trends in rotavirus group A (RVA) genotype distribution pre- (January 2010–June 2014) and post- (July 2014–December 2018) RVA vaccine introduction.Stool samples were collected from children aged < 13 years from four surveillance sites across Kenya: Kilifi County Hospital, Tabitha Clinic Nairobi, Lwak Mission Hospital, and Siaya County Referral Hospital (children aged < 5 years only). Samples were screened for RVA using enzyme linked immunosorbent assay (ELISA) and VP7 and VP4 genes sequenced to infer genotypes.P < .001) and G3P [8]  genotypes in the post-vaccine introduction period. Additionally, we observed a decline in prevalence of genotypes G8P [4]  and G9P [8]  in the post-vaccine introduction period. Phylogenetic analysis of genotype G1P [8], revealed circulation of strains of lineages G1-I, G1-II and P [8]-1, P [8]-III and P [8]-IV. Considerable genetic diversity was observed between the pre and post-vaccine strains, evidenced by distinct clusters.We genotyped 614 samples in pre-vaccine and 261 in post-vaccine introduction periods. During the pre-vaccine introduction period, the most frequent RVA genotypes were G1P [8] (45.8%), G8P [4] (15.8%), G9P [8] (13.2%), G2P [4] (7.0%) and G3P [6] (3.1%). In the post-vaccine introduction period, the most frequent genotypes were G1P [8] (52.1%), G2P [4] (20.7%) and G3P [8] (16.1%). Predominant genotypes varied by year and site in both pre and post-vaccine periods. Temporal genotype patterns showed an increase in prevalence of vaccine heterotypic genotypes, such as the commonly DS-1-like G2P [4] (7.0 to 20.7%, Genotype prevalence varied from before to after vaccine introduction. Such observations emphasize the need for long-term surveillance to monitor vaccine impact. These changes may represent natural secular variation or possible immuno-epidemiological changes arising from the introduction of the vaccine. Full genome sequencing could provide insights into post-vaccine evolutionary pressures and antigenic diversity. Childhood diarrhea caused by rotavirus group A (RVA) infection remains a leading cause of morbidity and mortality in young children globally . In 2016Upon infection by the virus, immune response to RVA by the host is directed to the highly variable VP7 and VP4 genes found on two separate segments of the double-stranded RNA genome . MoleculIn 2009, the World Health Organization (WHO) recommended the inclusion of RotaTeq®  or Rotarix®  vaccines in the national immunization programs of countries that experience high diarrhea morbidity and mortality burden due to RVA disease . The intRVA surveillance was carried out in four health facilities in coastal, western and central regions of Kenya. These surveillance sites were: Kilifi County Hospital (KCH) in Kilifi County, Tabitha Clinic (TC), in Kibera, Nairobi County, Saint Elizabeth Lwak Mission Hospital (LMH) and Siaya County Referral Hospital (SCRH) in Siaya County  and < 5 years (in SCRH) of age presenting with acute gastroenteritis (AGE). AGE was defined as ≥3 watery stools passed within a 24-h period during the illness for KCH, TC and LMH, while for SCRH, AGE was defined as ≥3 loose stools and/or ≥ 1 episode of unexplained vomiting followed by loose stool within a 24-h period beginning no more than 7 days before the visit to SCRH.RVA was tested by use of commercially available enzyme immunoassays. The ProSpecT™ Rotavirus Kit  was used to test samples collected from KCH while the Rotaclone® kit  was used to test samples collected from LMH, SCRH, and TC. For samples collected from LMH, KCH and TC, partial fragments of the segments encoding the outer capsid proteins, VP4 (660 bp) and VP7 (881 bp), were amplified in a One-step Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) using previously described primer pairs , 24. Suchttps://www.ncbi.nlm.nih.gov/genbank/. Sequence accession numbers for these genes are provided in supplementary Table n = 113) of the samples were typed for only one of the genes due to failure in sequencing and/or contig assembly, supplementary Table n = 15/168) and P  genotypes and mixed infections  were observed and subsequently excluded from analysis.Reads from the sequencer were trimmed (removing regions including primer sequences) and assembled into contigs  using Sequencher version 5.4.6 . The cleaned sequences for the VP7 and VP4 genes have been deposited to GenBank database: http://tree.bio.ed.ac.uk/software/figtree/). Nucleotide distances matrixes were prepared using the p-distance algorithm inferred in MEGA v10 [Maximum likelihood (ML) method was used to determine the phylogenetic relationship of the partial sequences of genotypes G1 and P [8] observed during the pre- and post-vaccine periods in Kenya, and further compared to RVA strains circulating globally. Global contemporaneous sequences were retrieved from GenBank (as of May 2020) and compared to local strains. Sequences were aligned using MAFFT v7.2  and visuMEGA v10 . The treP-values < 0.05 were considered statistically significant.Data collating and analyses were performed in Microsoft Excel and R version 3.5. Genotype data collected from LMH and SCRH were merged to constitute data from Siaya County. The summary of proportions was conducted for each genotype by site and period of detection. Frequency distribution and temporal pattern graphs were generated. Two-sided proportional tests of the most common genotypes during the pre-vaccine (January 2010 thru June 2014) and post-vaccine (July 2014 thru December 2018) eras were performed in R, and n = 38), SCRH (n = 161), TC (n = 84), and KCH (n = 592)]. Of these, 614 (70.2%) were observed in the pre-vaccine (January 2010 – June 2014) and 261 (29.8%) in the post-vaccine (July-2014 – December 2018) introduction periods, respectively.A total of 6562 stool samples were collected from health facilities in LMH, SCRH, TC and KCH. Of the 1312 (20.0%) RVA positive samples, individual G and P genotypes were identified in 928 (70.7%) and 904 (68.9%) samples, respectively, while 875 (66.7%) samples were successfully genotyped for both G and P genes  (45.8%) was the predominant genotype observed in this population, followed by G8P [4] (15.8%), G9P [8]13.2%), G2P [4] (7.0%) and G3P [6] (3.1%). After vaccine introduction, G1P [8] remained the dominant genotype (52.1%), followed by G2P [4] (20.7%), G3P [8] (16.1%), G9P [8] (5.4%) and G3P [6] (2.7%). Multiple other genotypes were also observed in pre- and post-vaccine periods, although in low proportions (< 2%), Tables %, G2P  and G1P [8] Fig. . After vP < .001) and G3P [8]  and significant decline in prevalence of genotypes G8P [4]  and G9P [8] , Fig. P = .35) was measured before and after vaccine introduction. A decline in genotype diversity was observed after vaccine introduction (11 genotypes) as compared to pre-vaccine period (21 genotypes).In the post-vaccine introduction period, relative to the pre-vaccine period, we observed a significant increase in prevalence of genotypes G2P [4]  clustered within pre-vaccine strains, which showed high nucleotide sequence identities of up to 99% to pre-vaccine strains. Additionally, a single pre-vaccine strain, observed in 2014, grouped within the post-vaccine cluster, which showed high nucleotide identities of up to 100% to other post-vaccine strains. The lineage G1-I strains also clustered with other strains isolated from Kenya (2009), Malawi (2012), Togo (2011), Russia (2013), India (2013), Kuwait (2016), USA (2011) and Italy (2015), with high nucleotide identities ranging between 98 and 99%. Lineage II cluster contained strains observed in pre-vaccine period (n = 14) which clustered with other globally isolated strains from Pakistan (2013), India (2012), USA (2013) and Hungary (2012), with nucleotide identities of up to 99%. In contrast, a single post-vaccine strain, isolated in Kibera (KBR337), was observed within lineage G1-II cluster, which showed high nucleotide and amino acid identity of up to 100% to the Rotarix vaccine and other vaccine-derived strains from Japan, Brazil and Korea.As shown in Fig. As shown in Fig. Prior to vaccine introduction, G1P [8] was the predominant genotype. However, this genotype’s prevalence varied substantially from year to year , emphasizing the limits of short-term surveillance and the potential for misrepresentation of patterns. During 2010 and 2012, a large proportion of children were infected with genotype G8P [4], which was not detected in any years following vaccination (except during 2017). Genotypes G1P [8], G2P [4] and G9P [8] have also been identified as dominant genotypes in a study involving six countries from Eastern and Southern Africa  showed no overall change in prevalence compared to the pre-vaccine period, it predominated only in the first year after vaccine introduction (2015), and declined in prevalence thereafter, in particular to be replaced by G2P [4] in 2016, and by G3P [8] in 2018. Genotypes G8P [4] and G9P [8], which were the second and third common genotypes in pre-vaccine period, significantly declined in the post-vaccine period. Predominance of G2P [4] and G3P [8] has been reported worldwide following universal Rotarix vaccine introduction. For instance, G2P [4] was observed in Australia   , hence, Additional file 1: Supplementary Figure 1; Geographical boundaries and location of the health facilities participating in the rotavirus genotype surveillance program represented by the diamond shapes. Green – Siaya County Referral Hospital, blue – Lwak Mission Hospital, red – Tabitha Clinic and orange – Kilifi County Hospital.Additional file 2: Supplementary Table 1; Frequency of partially typed G and P genotypes. Gx and P [x] were unsuccessfully typed due to failure in sequencing and/or contig assembly.Additional file 3: Supplementary Table 2; GenBank accession numbers of all VP7 G gene sequences.Additional file 4: Supplementary Table 3; GenBank accession numbers of all VP4 P gene sequences.


Nicotiana benthamiana plants results in the recovery of GFP, a proxy for suppression of TGS. This study provides new insights into the molecular mechanisms by which geminiviruses suppress TGS, and uncovers a new viral strategy based on the inactivation of the methyltransferase NbDRM2.In plants, cytosine DNA methylation is an efficient defense mechanism against geminiviruses, since methylation of the viral genome results in transcriptional gene silencing (TGS). As a counter-defense mechanism, geminiviruses encode viral proteins to suppress viral DNA methylation and TGS. However, the molecular mechanisms by which viral proteins contribute to TGS suppression remain incompletely understood. In this study, we found that the C4 protein encoded by tomato leaf curl Yunnan virus (TLCYnV) suppresses methylation of the viral genome through interacting with and impairing the DNA-binding ability of NbDRM2, a pivotal DNA methyltransferase in the methyl cycle. We show that NbDRM2 catalyzes the addition of methyl groups on specific cytosine sites of the viral genome, hence playing an important role in anti-viral defense. Underscoring the relevance of the C4-mediated suppression of NbDRM2 activity, plants infected by TLCYnV producing C4(S43A), a point mutant version of C4 unable to interact with NbDRM2, display milder symptoms and lower virus accumulation, concomitant with enhanced viral DNA methylation, than plants infected by wild-type TLCYnV. Expression of TLCYnV C4, but not of the NbDRM2-interaction compromised C4(S43A) mutant, in 16c-TGS  Geminiviridae family encode a C4 protein capable of inhibiting TGS, hence promoting virulence. However, the molecular mechanism underlying the C4-mediated TGS suppression is still incompletely understood. This study demonstrates that the C4 protein encoded by tomato leaf curl Yunnan virus interacts with NbDRM2, a pivotal DNA methyltransferase in the methyl cycle, and interferes with its ability to bind the viral DNA. As a result, methylation of the viral genome is impaired, which in turn favours viral replication and spread. Our results uncover a novel strategy deployed by geminiviruses to counter anti-viral DNA methylation, hence shedding new light onto the arms race between viruses and hosts.Interfering with transcriptional gene silencing (TGS) is essential for DNA viruses to infect and replicate in plant cells. Some members of the  Cytosine DNA methylation is a conserved epigenetic mark in plants, animals, and fungi that modulates a number of biological activities including genome imprinting, transposon control, and transcriptional gene silencing (TGS) –4. ImporBecurtovirus, Begomovirus, Capulavirus, Curtovirus, Eragrovirus, Grablovirus, Mastrevirus, Topocuvirus, and Turncurtovirus) based on genome structure, host range, and insect vector , or the NbDRM2-interaction compromised C4 mutant [C4(S43A)]. Consistent with our previous results, TLCYnV expressing C4(T35A) did not infect emically , while Tsymptoms . NtGRS1. tabacum , was selC4(S43A) . To furtm plants . Taken tN. benthamiana plants, in which the GFP transgene driven by the CaMV 35S promoter is transcriptionally silenced, by using a potato virus X (PVX)-based expression vector. 16c-TGS plants infected by PVX-TLCYnV C4 or PVX-TLCYnV C4(S43A) showed abnormal development, including foliar distortion and longer internodes and petioles -CFP, or C4(S43A)-CFP in 16c-TGS transgenic N. benthamiana plants and ChIP-qPCR assays were conducted. Our results show that the interaction between TLCYnV C4 and NbDRM2 indeed impairs the 35S promoter-binding ability of NbDRM2 (N. benthamiana plants with TLCYnV or the TLCYnV-C4(S43A) mutant, we found that infection by TLCYnV restores GFP expression at 12 dpi, but the TLCYnV mutant fails to do so  in NbDRM2-silenced 16c-TGS transgenic N. benthamiana plants when compared with those in 16c-TGS transgenic N. benthamiana plants inoculated with the control (TRV-GUS) (GUS) and NbDRM2-silenced (TRV-NbDRM2) 16c-TGS transgenic N. benthamiana plants. As shown in NbDRM2-silenced 16c-TGS plants. These results suggest that the TLCYnV C4/NbDRM2 interaction plays a critical role in TGS suppression.Given that TLCYnV C4 inhibits NbDRM2 function through protein-protein interaction, we assumed that it might suppress the NbDRM2-mediated TGS. We expressed TLCYnV C4 and C4 mutants in 16c-TGS transgenic petioles . These pa plants , while ta plants ; this via plants . To idenf NbDRM2 . When weto do so . In ordeTRV-GUS) . PVX-TLCGeminiviruses replicate their genome via dsDNA intermediates in the nucleus, which could be the target of methylation-mediated TGS . In thisHow does the interaction between TLCYnV C4 and NbDRM2 lead to suppressed TGS? Our results show that NbDRM2 localizes in the nucleus and forms speckles, which correlates with its DNA-binding capacity. However, TLCYnV C4 can interfere with the DNA-binding ability of NbDRM2, inhibiting the formation of DNA-NbDRM2 complexes and retaining NbDRM2 in the nucleoplasm. Inhibition of NbDRM2 DNA binding by TLCYnV C4 interferes with the NbDRM2-mediated DNA methylation. This model is supported by several pieces of evidence: (1) The nuclear speckles potentially containing DNA-bound NbDRM2 disappear in the presence of TLCYnV C4; (2) A higher proportion of soluble NbDRM2 can be detected in the presence of TLCYnV C4; (3) The TLCYnV infectious clone expressing the NbDRM2-interaction compromised C4 mutant has weaker pathogenicity and higher methylation levels of the viral genome; (4) TLCYnV C4, but not the mutant deficient in NbDRM2 interaction, decreases the methylation level of a DRM2 target locus. Taken together, these results strongly suggest that TLCYnV C4 impacts NbDRM2-mediated TGS through the interaction with this protein.NbDRM2-silenced 16c-TGS transgenic N. benthamiana plants ] produces developmental abnormalities similarly to WT C4 ] does not impact the DNA-binding ability of NbDRM2, as opposed to the wild-type C4 Figs  and 6C. NtGRS1.3) in N. tabacum  were kindly provided by Dr. Michael M. Goodin   for 3 h at room temperature. The suspensions were adjusted to OD600 = 0.5. For co-expression experiments, the individual cultures were adjusted to OD600 = 0.4 and equal volumes were mixed before leaf infiltration. The suspensions were infiltrated into leaves of 4- to 6-week old N. benthamiana leaves using 1-ml needleless syringes.Agroinfection assays were conducted essentially as described previously ,23. The g for 10 min. The supernatant containing soluble nucleoplasmic proteins was collected. The remaining pellet was resuspended in 3 volumes of 1% SDS and sonicated, followed by centrifugation at 14,000 g for 10 min. The supernatant containing chromatin-bound proteins was collected. Total nuclear protein was isolated by adding 3 volumes of 1% SDS directly to the nucleus pellet, followed by boiling at 95°C for 10 min. Equal volumes of each fraction were mixed with loading buffer, boiled, gel-separated, and subjected to SDS-PAGE analysis.Isolation of chromatin-bound protein assays were performed as described previously –53. LeavN. benthamiana plants infected by TLCYnV at 14 dpi were harvested and prepared for total genomic DNA isolation. Genomic DNA was isolated by using DNeasy Plant Minikit (Qiagen). Then, 100 ng of genomic DNA was digested for 1 h at 37 with 2U of HpaII, MspI, or McrBc in 20 μl reaction mixtures. The enzymes were heat inactivated, and 2 μl of the cleaved DNA was loading into PCR mixtures containing primers for full-length TLCYnV. PCR products of undigested genomic DNA served as control.Methylation-sensitive PCR assays were performed as described previously . SystemiBamHI, which acts outside of interest to decrease the size of DNA, followed by overnight treatment with proteinase K. Bisulfite modification was carried out using the EZ DNA Methylation Gold Kit (Zymo Research). Bisulfite-modified DNA was purified using a Zymo-Spin IC column and dissolved in 20 μl of Elution Buffer according to the manufacturer’s instructions. PCR polymerase and products were cloned into a pLB vector. Individual clones were sequenced. Primers were designed against templates are listed in Bisulfite sequencing assays were performed essentially as described previously –44. GenoChIP quantitative PCR was preformed essentially as described previously –56. ChroE. coli strain BL21(Codonplus). The concentration of purified proteins was measured with a NanoDrop instrument . To generate Alexa Fluor 680-labeled TLCYnV IR, Alexa Fluor 680-labeled oligonucleotides corresponding to the viral sequence were mixed with the PCR mixture to amplify TLCYnV IR. GST-NbDRM2 (0.5 μg) was mixed with Alexa Fluor 680-labeled TLCYnV IR, then steadily increasing amounts  of GST Tag, GST-C4, or GST-C4(S43A) were added into the GST-NbDRM2/TLCYnV IR mixture containing the binding buffer . The reaction mixtures were incubated at room temperature for 45 min, resolved on a 1% Tris-borate-EDTA (pH 8.5) agarose gel, and visualized by using an Odyssey infrared imaging system in the 700-nm channel.EMSA assays were performed essentially as described previously , 58. TheS1 Table(DOC)Click here for additional data file.S1 Fig(A) Phylogenetic analysis of NbDRM2 homologues from different species based on the amino acid sequences using Clustal W from MegAlign software. (B) Schematic representation of NbDRM2 deduced from the SMART online software. (C) Sequence alignment of the catalytic DNA methyltransferase domain of NbDRM2 and orthologues using Meglign software.(TIF)Click here for additional data file.S2 FigNbDRM2 transcripts is normalized to the actin transcript. Error bars represent standard deviation of three biological replicates.Relative accumulation level of (TIF)Click here for additional data file.S3 FigNbDRM2-silenced (TRV-DRM2) and mock (TRV-GFP) N. benthamiana plants, respectively. X-axis indicates days post-inoculation (dpi); Y-axis represents the percentage of symptomatic N. benthamiana plants. Over 60 N. benthamiana plants were used in this experiment. Error bars represent standard deviation of three biological replicates.Red and blue curves represent the time course of TLCYnV symptom development in (TIF)Click here for additional data file.S4 Fig(A) Nuclear distribution of NbDRM2 in isolated nuclei; (B) TLCYnV C4 influences the NbDRM2 nuclear distribution pattern in isolated nuclei. Chromosomal DNA was stained by propidium iodide (PI). Scale bar = 20 μm. (C) Immunoblot analysis of TLCYnV C4 accumulation in 35S::TLCYnV C4 transgenic N. benthamiana plants. Two independent transgenic lines were used. Rubisco was used as loading control.(TIF)Click here for additional data file.S5 FigThe yeast strain Gold co-transformed with the indicated plasmids was subjected to 10-fold series dilution, and grown on a SD/-Leu/-Trp/-His medium.(TIF)Click here for additional data file.S6 FigLeaves co-expressing Flag-NbDRM2 with GFP, TLCYnV C4-, or TLCYnV C4(S43A)-GFP were harvested at 2 dpi for co-immunoprecipitation (Co-IP) assays. Immunoblot analysis was conducted with antibodies specific to detect the indicated proteins.(TIF)Click here for additional data file.S7 Fig(A) Phenotype of N. benthamiana plants expressing TLCYnV C4 or C4 mutants systemically by using a CMV-based vector. The TLCYnV C4 mutant [C4(T35 A)], which does not induce viral symptoms, is used as control. Photographs were taken at 8 days post-inoculation. (B) Accumulation of TLCYnV C4 or C4 mutants in leaves of N. benthamiana plants under different treatments determined by Western blot using antibodies specific to the indicated proteins.(TIF)Click here for additional data file.S8 FigThe yeast strain Gold co-transformed with the indicated plasmids was subjected to 10-fold series dilution, and grown on a SD/-Leu/-Trp/-His/-Ade medium.(TIF)Click here for additional data file.S9 FigC4-, C4 mutants-GFP and Flag-NbDRM2 proteins were detected using GFP and Flag specific monoclonal antibodies. Rubisco was used as the loading control.(TIF)Click here for additional data file.S10 Fig(A) Phenotype of N. tabacum plants expressing TLCYnV C4 or C4 mutants by using a CMV-based vector. Photographs were taken at 12 days post-inoculation. (B) Accumulation of TLCYnV C4 and C4 mutants in N. tabacum leaves under different treatments determined by Western blot using the antibodies specific to the indicated proteins. (C) Southern blot analysis of the methylation level of NtGRS1.3. Genomic DNAs from N. tabacum plants expressing TLCYnV C4 and C4 mutants were digested with MspI, then resolved in a 1.5% agarose gel. (D) Detection of the methylation level of NtGRS1.3 by using methylation-sensitive PCR. Genomic DNA was digested with MspI and then used as template for PCR. Undigested DNA was used as the loading control.(TIF)Click here for additional data file.S11 FigTLCYnV C4-, C4 mutants-GFP and Flag-NbDRM2 proteins were detected using monoclonal antibodies specific for GFP and Flag. Rubisco was used as the loading control.(TIF)Click here for additional data file.S12 FigNbDRM2 transcripts is normalized to the actin transcript. Error bars represent standard deviation of three biological replicates.Relative accumulation of (TIF)Click here for additional data file.


Bladder cancer (BC) is one of the most prevalent cancers worldwide and has high rates of relapse and progression. Cell division cycle associated 5 (CDCA5), a substrate of the anaphase-promoting complex, was reported to be upregulated in several types of cancer; however, the function of CDCA5 in BC remains unclear. In this study, we observed that BC tissues had higher levels of CDCA5 expression than adjacent normal tissues. We also found that high CDCA5 expression in patients was associated with poor survival rates. An in vitro study showed that knockdown of CDCA5 in T24 and 5637 cells reduced cell proliferation and induced apoptosis in T24 and 5637 cells, while overexpression of CDCA5 in UMUC3 cells caused the opposite effects. In an additional experiment, we found that CDCA5 promoted cell proliferation by upregulating two key cell cycle factors, cell division cycle protein 2 (CDC2) and cyclin B1, and by activating the PI3K/AKT/mTOR pathway. Furthermore, CDCA5 regulate cancer cell apoptosis through the mitochondrial apoptosis pathway. In conclusion, CDCA5 plays a pivotal role in the proliferation of BC cells. A better understanding of CDCA5 may provide new insights into its role as a therapeutic target for BC. Bladder cancer (BC) is the most common tumor arising from the urinary system, with approximately 400000 cases diagnosed annually The phosphoinositide 3-kinase (PI3K)/AKT signaling pathway is involved in numerous cellular processes, including cell proliferation, metabolism, invasion and migration cdh1)-dependent ubiquitination. Recent studies have reported that CDCA5 is overexpressed in some tumor tissues, suggesting that it may act as a promoter in tumor progression Cell division cycle associated 5 (CDCA5), also known as sororin, is a key player in sister chromatid cohesion and separation and was first identified as a substrate of the anaphase-promoting complex In the current study, we found that BC cells had significantly higher CDCA5 expression than normal bladder cells, and this expression was related to advanced clinical stage and poor prognosis. In addition, knocking down or overexpressing CDCA5 in BC cells affected their proliferation and apoptosis. Taken together, our results showed that CDCA5 may act as a potential therapeutic target in BC.Tissue samples and immunohistochemistry.Twenty BC patients sourced from the First Affiliated Hospital of Zhejiang University School of Medicine were enrolled in the study from July 2017 to December 2018. All of these patients had histologically confirmed muscle-invasive urothelial cancer and no metastatic disease present. Patients with metastatic disease who have received prior radiotherapy for BC may be excluded. All participants signed informed consent. Immunohistochemical staining was performed according to the kit instructions. In brief, after paraffin section dewaxing, the sections were rehydrated and subjected to high-pressure antigen retrieval. Next, 3% H2O2 was used to eliminate the influence caused by endogenous peroxidase activity, followed by blocking with normal goat serum. The sections were incubated with primary antibodies against CDCA5 , Ki67 , cleaved caspase-3 , cyclin B1  and p-Akt  overnight at 4°C. Following incubation with the secondary antibody (goat anti-rabbit IgG antibody-HRP), DAB was used to stain the slides, and hematoxylin counterstaining and alcohol dehydration were performed. Finally, the prepared sections were examined under a microscope.Online database.The Oncomine database (http://www.oncomine.org) was used to identify CDCA5 mRNA expression in BC and normal tissues with the following search terms: CDCA5 and BChttps://cancergenome.nih.gov/) using the following search terms:project, TCGA-bladder urothelial carcinoma; primary site, bladder; access date, January 2018.Cell culture.The human BC cell lines RT4, UM-UC-3, 5637, T24, TCCSUP and the normal uroepithelial cell line SV-HUC-1 were purchased from the Cell Bank of the Chinese Academy of Sciences . RT4, 5637, T24 and TCCSUP cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum ; UM-UC-3 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FBS, and SV-HUC-1 cells were cultured in Minimum Essential medium (MEM) containing 10% FBS. All of the above cell lines were preserved at 37°C in 5% CO2 in a stable incubator.Cell transfection.shRNA vectors for  and a control vector were purchased from Guangzhou GeneCopoeia Co., Ltd. . The sequences of shRNAs were as follows: sh-CDCA5-1 forward, 5'CCGGGGACGCCAGAGACTTGGAAATCTCGAGATTTCCAAGTCTCTGGCGTCCTTTTTG3' and reverse, 5'AATTCAAAAAGGACGCCAGAGACTTGGAAATCTCGAGATTTCCAAGTCTCTGGCGTCC3'; CDCA5-2 forward, 5'CCGGGACATGACTCTCCCTGGAATCCTCGAGGATTCCAGGGAGAGTCATGTCTTTTTG3' and reverse, 5'AATTCAAAAAGACATGACTCTCCCTGGAATCCTCGAGGATTCCAGGGAGAGTCATGTC3'. The adenoviruses used to express CDCA5 or the negative control (NC) were purchased from GenePharma . Before the infection step, the cells were cultured in 6-well plates (5×104 cells/well) to 60% confluence. Then, T24 and 5637 cells were treated with lentivirus at a multiplicity of infection of 50 pfu/cell in 1640 medium with 10% FBS and polybrene ; UMUC3 cells were subjected to the same treatment in DMEM. After 48 hours, virally transduced cells were subjected to 1 mg/ml puromycin (Thermo Fisher Scientific) for screening successfully infected cells. Both GFP levels and Western blotting were conducted to validate the transfection efficiency.Cell proliferation assay and colony formation assay.The proliferation rates of cells were investigated using the Cell Counting Kit-8  according to the manufacturer′s protocol. Cells were cultured in 96-well plates at a density of 1000 cells per well at 37°C in 5% CO2. Then, the cells were treated with 10% CCK-8 reagent for another 1 h at 37°C before the absorbance was measured at 450 nm. The colony formation assay was performed as follows: cells were prepared in 6-well plates at a density of 500 cells per well. After discarding the supernatants after 8-10 days, the cells were washed 3 times with phosphate-buffered saline (PBS), followed by 15 min fixation at room temperature; finally, the cells were stained with 0.5% crystal for 15 min, and the 6-well plate was photographed and analyzed.Quantitative real-time PCR.Total RNA was isolated by TRIzol  from the BCa cell lines or frozen tissue samples. cDNA was synthesized from 2 µg total RNA using TaKaRa PrimeScriptTM RT and SYBR EX TaqTM kits , and qRT-PCR was conducted using a Bio-Rad CFX96 real-time system according to the manufacturer's instructions. The primers were designed as follows: for CDCA5, forward primer, 5'CGCCAGAGACTTGGAAATGT3' and reverse primer, 5'GTTTCTGTTTCTCGGGTGGT3'; for β-actin, forward primer, 5'GCAAGCAGGAGTATGACGAG3' and reverse primer, 5'CAAATAAAGCCATGCCAATC3'. Control groups were used to confirm the absence of the pollution of agents or primer dimers, and melt-curve analysis was used to identify the specificity of amplification. All genes were normalized to β-actin expression, and the gene mRNA relative expression levels were calculated using the ΔΔCq method (18).Western blotting.T24, 5637 and UMUC3 cell lines were harvested and washed twice with PBS and lysed using radio-immunoprecipitation assay lysis buffer  containing 1% of a protease inhibitor cocktail  for 1 hour at 4°C and collecting the supernatants after centrifugation . Protein concentrations were measured using a bicinchoninic acid protein assay . The samples were boiled at 95°C for 5 min after mixing with 4x sodium dodecyl sulfate (SDS) loading buffer (Invitrogen). The proteins (20 μg/10 μl) were loaded in 12% Tris-acetate gels  and then separated by electrophoresis on SDS-PAGE gels; the proteins were subsequently transferred onto a polyvinylidene fluoride membrane. The membranes were blocked with 5% nonfat milk for 1 hour at room temperature, and the membranes were blocked and incubated with the corresponding primary antibodies overnight at 4°C. Immunoblots were washed with Tris-buffered saline containing 1% Tween-20 (TBST) three times and incubated with conjugated anti-mouse or anti-rabbit antibodies (1:5000) for 1 hour at room temperature. The primary antibodies used in this experiment were: rabbit polyclonal anti-CDCA5 antibody ; rabbit monoclonal anti-phosphorylated-protein kinase B antibodies , anti-AKT , anti-P21 , anti-P27 , anti-cyclin B1 , anti-Chk2 , anti-Parp , anti-Caspase-9 , anti-Caspase-3 , anti-Bcl-xL , anti-Bcl2 , anti-Bax , anti-PI3k , anti-phospho-PI3 kinase , anti-mTOR , anti-phospho-mTOR , mouse monoclonal anti-β-actin antibody , and anti-CDC2 . Autoradiography with the ChemiDoc MP Imaging System (Bio-Rad) with an EZ-ECL Chemiluminescence Detection Kit , and β-actin was selected as an internal reference.Flow cytometry analysis of cell apoptosis.Stably transfected T24, 5637 and UMUC3 cells were seeded in a 6-well plate at a density of 2x105 cells/well. Cells were harvested by non-EDTA trypsin , washed twice with PBS and subsequently incubated using a BD Annexin V-APC/7-aminoactinomycin D Apoptosis Detection Kit . Finally, the apoptosis cells were measured by staining with Annexin V (10 μl/200 μl) along with 7-aminoactinomycin D (10 μl/200 μl) After incubating for 15 min at room temperature, the stained cells were detected using BD FACSCantoTM II (BD Biosciences), and apoptotic cells were analyzed using FlowJo 7.6 software .Flow cytometry analysis of the cell cycle.Stably transfected T24, 5637 and UMUC3 cells were seeded in 6 cm dishes at a density of 60%. A total of 1.0×106 transfected cells were collected in PBS, fixed in 70% ethanol overnight at -20°C, subsequently washed twice with PBS and harvested by centrifugation at 1000 rpm for 5 min. Cells were resuspended in a cell cycle staining kit  according to the manufacturer's protocol and incubated for 30 min in the dark at room temperature. All cells were measured using BD FACSCantoTM II (BD Biosciences), and the results were analyzed using FlowJo 7.6 software .Experimental animals and tumor growth in nude mice.Ten four-week-old female NOD-SCID mice were purchased from Vital Laboratory Animal Technology Co., Ltd. . They were raised in the animal facility of Zhejiang University School of Medicine. All experimental procedures and protocols were performed in compliance with the Animal Experimental Ethical Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University and were approved by the Zhejiang Medical Experimental Animal Care Commission. To establish the subcutaneous model, lentivirus-transfected T24 cells (5×106 cell/ml) with a low expression of CDCA5 and the NC cells were suspended in 0.2 mL of PBS, which was subcutaneously injected into the left flank of each mouse. We observed the survival of these mice and the growth of the tumors. The mice were monitored once every 2 days. All nude mice were sacrificed by cervical dislocation 7 weeks later; the tumors were removed; and the relative data, such as the length, width and weight, of the tumor were recorded.Statistical analysis.SPSS 22.0 (IBM Corp.) was used to analyze the data. The normality of the data was initially determined using a Kolmogorov-Smirnov test. Data are shown as the mean ± standard deviation. The correlations between CDCA5 expression and clinicopathological characteristics were analyzed using a Pearson's χ2 test or a continuity correction χ2 test. The survival rate was evaluated using the Kaplan-Meier method, and data were analyzed by a log-rank test. The significant differences between two groups were determined using a two-tailed Student's t-test. Student-Newman-Keuls test was used as a post hoc test. P<0.05 was recognized as statistically significant. Data were obtained from triplicate and three or more independent experiments.CDCA5 is upregulated in BCa tissues, and high CDCA5 expression indicates poor survival.Recently, a study revealed that overexpression of CDCA5 was an indicator of poor prognosis in BCa Expression of CDCA5 in BC cell lines. CDCA5 expression was high in the BC cell lines RT4, UMUC3, 5637, T24, and TCCSUP and low in the normal uroepithelial cell line SV-HUC-1  and NC (n=5) groups developed xenograft tumors in the right flank, and xenograft tumors were harvested 6 weeks after injection ; CDK-interacting protein/kinase inhibitory protein (cip/kip) family members, such as p21 and p27; and inhibitor of kinase 4/alternative reading frame (INK4a/ARF) family members. The cell cycle contains G1/S, S and G2/M checkpoints Further, we investigated the oncogenic function of CDCA5 in human BCa. Our results showed that CDCA5 knockdown inhibited cell proliferation, promoted cell apoptosis and induced G2/M arrest. With the knockdown of CDCA5 in BCa cells, the expression of cell cycle-related proteins  was downregulated, while p21, p27 and p-Chk2 were upregulated. CDC2 may accelerate the cell cycle G2/M transition when binding to cyclin B1 Apoptosis, also called programmed cell death, can be divided into extrinsic and intrinsic  pathways The PI3K/AKT/mTOR signaling pathway is important for multiple cellular processes, including proliferation, apoptosis, metabolism, migration, invasion and differentiation In conclusion, we have identified CDCA5 as a novel functional oncogene in BC, and high CDCA5 expression is associated with an advanced clinical stage and poor prognosis. Our results indicate that CDCA5 regulates apoptosis through the mitochondrial apoptosis pathway and that CDCA5 promotes cell proliferation by regulating cyclins and CDKs; CDCA5-knockdown arrests BC cells in G2/M. All these findings indicated that CDCA5 acts as a tumor promoter in BC. Moreover, CDCA5 may serve as a potential epigenetic biomarker and a novel molecular target for BC.Supplementary tables.Click here for additional data file.


PGCC formation is closely related to the abnormal expression of cell cycle-related proteins and cell fusion. In this study, we investigated the molecular mechanism of PGCCs formation by detecting the expression of cell cycle-related proteins in mutant and wild-type p53 cancer cell lines.Our previous studies have confirmed that cobalt chloride (CoCl2 and the cell cycle was detected by flow cytometry. The expression and subcellular localization of cell cycle-related proteins, kinases, and P53 were compared before and after CoCl2 treatment. Immunoprecipitation was used to analyze the interacting proteins of pCDC25C-Ser216 and pCDC25C-Ser198. The clinicopathologic significances of these cell cycle-related proteins and protein kinases expression were studied.HEY, BT-549, SKOv3 and MDA-MB-231 cells were treated with CoCl2 induced the formation of PGCCs and G2/M arrest. CDC25C, cyclin B1, and CDK1 expressions after CoCl2 treatment were lower than that in control cells. Cytoplasmic CDC25C was degraded by ubiquitin-dependent proteasome. The expression of P53 and phosphokinases including CHK1, CHK2, PLK1, and Aurora A increased after CoCl2 treatment. The expression of pCDC25C-Ser216 and pCDC25C-Ser198 depended upon the genotype of p53. The expressions of cell cycle-related proteins and kinases gradually increased with the development of ovarian cancer and breast cancer.CoClp53.CHK1, CHK2–pCDC25C-Ser216–cyclin B1–CDK1, and Aurora A–PLK1–pCDC25C-Ser198–cyclin B1–CDK1 signaling pathways may participate in the formation of PGCCs and different phosphorylation sites of CDC25C may be associated with the genotype of  When recovered from CoCl2 treatment, PGCCs can generate daughter cells via asymmetric cell division. Daughter cells from PGCCs have strong invasion and metastatic abilities [We previously reported that polyploid giant cancer cells (PGCCs) had the properties of cancer stem cells (CSCs)  and commbilities , 3. In mbilities . Our prebilities . Furtherbilities , 6.p53-deficient cells decreased the total CDC25C and pCDC25CSer216 expression level, suggesting that DNA damage response (DDR) regulated CDC25C expression in a p53-dependent manner [2 treatment were studied and the clinicopathological significance of CDC25C expression-related proteins were evaluated in human ovarian and breast cancer tissues.PGCC formation is associated with abnormal expression and change in subcellular localization of CDC25C . CDC25C t manner , 14. In p53) and SKOv3 with lack of p53, human breast cancer cell line BT-549 (mutant-type p53) and MDA-MB-231 (mutant-type p53), were obtained from the American Type Culture Collection  and the culture conditions were described previously [The human ovarian cancer cell line HEY  for 24 and 48 h, respectively, based on their hypoxia-resistance abilities. Only scattered PGCCs survived after CoCl2 treatment and most regular-sized cancer cells were dead. Fifteen to twenty days later, PGCCs began to produce daughter cells via asymmetric division. After treatment with CoCl2 3–4 times, the PGCCs occupied 20–30% total cells and 70–80% were the daughter cells derived from PGCCs. The cells treatment process was unified to ensure that the proportion of PGCCs and daughter cells were constant in this range. For the definition and counting of PGCCs, PGCCs were defined as tumor cells with a nucleus more than three times larger than that of diploid tumor cells, usually appearing as multinucleated and giant nucleated cells. Five microscopic fields were randomly counted at 400× magnification and the average PGCC percentage in slides was calculated after hematoxylin–eosin (H&E) staining. In this study, “control” represents the untreated control cells, “treatment” is used to indicate that HEY, BT-549, SKOv3 and MDA-MB-231cells with CoCl2 treatment.The induction, definition and counting of PGCCs were described previously . HEY, SK–339 were proved to have the strongest inhibitory efficiency and used in this study. Detailed information about transient siRNA transfection is provided in the CDC25C and P53 were knockdown by transient siRNA transfection. The siRNA oligonucleotides were synthesized by Gene-pharma , including three siRNA interference sequences, one positive control sequence (GAPDH), one negative control (NC) sequence, and one mock control (MC) with only transfection reagents. Three CDC25C transfection sequences including 1103, 1343, 1531and three P53 transfection sequences including 339, 886, 985 were used to inhibit the expression of CDC25C  transfection HEY and BT-549 cells before and after CoCl2 treatment were cultured on slides for H&E staining. The detailed information about H&E staining is provided in the Control and CDC25C-siRNA (CDC25Ci) transfection HEY and BT-549 cells before and after CoClIHC and ICC staining was carried out as described previously . DetaileThe protein expression was evaluated as previously described , 6. DetaThe abilities of cell migration and invasion assay were evaluated as previously described and detailed information about these assay is provided in the Plate clone formation experiment was evaluated as previously described . Detailen = 81) and breast cancer (n = 229) tissue samples were collected from the Department of Pathology in Tianjin Union Medical Center. All cases had complete clinicopathological data. The 81 human ovarian cancer tissues were divided into four groups: 20 primary ovarian cancer with lymph node metastasis (group I), 20 lymph node metastatic foci of group I (group II), 31 primary ovarian cancer without lymph node metastasis (group III), and 10 serious borderline serous cystadenoma (group IV). The 242 cases of human breast cancer were divided into two groups: 167 cases of invasive breast cancer without lymph node metastasis (group I) and 62 cases of breast cancer with lymph node metastasis (group II). The groups and clinical features of both ovarian tumor and breast cancer were listed in supplementary table Paraffin-embedded human ovarian tumor  was added in the wells for 6 h, followed by lysis buffer for western blot analysis.Cell cycle status was detected by flow cytometry and the detailed information is provided in the 2 treatment in vitro. Detailed information about kinase activity assay is provided in the The kinase activity assay was performed using the EnzyChrom Kinase Assay Kit  to test the differences in protein kinase activity before and after CoCl2 treatment. The detailed information about Co-IP is provided in the Co-IP was performed to determine the direct or indirect interaction with pCDC25CSer216 and pCDC25CSer198 in HEY and BT-549 cells after CoClt-test and Pearson chi-square (χ2) test. A two-tailed P value less than 0.05 was defined as statistically significant.SPSS 22  was used for all statistical analysis in this study. All histogram data are expressed as mean ± SD, and all tabular data are presented as mean ± SEM. Kruskal-Wallis test was performed to compare the differences in cell cycle-related protein expressions in ovarian cancer tissues. The Mann-Whitney test was used to analyze the differences in cell cycle-related protein expressions in breast cancer tissues. Other comparisons were performed using a two-tailed Student’s 2 was added to HEY of CoCl HEYFig. A a for 4 HEYFig. A d for 2ent Fig. A b, e. Tent Fig. A c, f. Fent Fig. B a, e anent Fig. B c, g, a0 μM of Coup Fig. C a, b. T2 treatment in HEY, BT-549, SKOv3 and MDA-MB-231 cells compared with those in control cells  expression levels and subcellular localization. The average number of PGCCs in 5 high-power-fields (400×) occupied 28% of the total cell and 72% was the daughter cells based on the H&E staining. Western blot results showed that the total protein level of CDC25C, cyclin B1 CDK1 and decreased after CoClCells after CDC25Ci (siRNA CDC25C-1531) treatment for 24–48 h were used to measure the migration ability of cells in control, PGCCs with their daughter cells, control-CDC25Ci, and PGCCs with daughter cells-CDC25Ci by wound-healing assay. The average number of PGCCs in 5 high-power-fields (400×) occupied 25% of the total cell and 75% was the daughter cells based on the H&E staining. Fig. S2 treatment, PLK1 and Aurora A expression decreased compared to that in the cells without CDC25C knockdown. However, the expression levels of CHK1 and CHK2 in the control group were lower than them in the siRNA control. Also, CDC25C expression inhibition increased CHK1 and CHK2 expression in PGCCs with their daughter cells  occupied 27% of the total cell and 73% was the daughter cells based on the H&E staining. In addition, pCDC25CSer216, pCDC25CSer198, CHK1, CHK2, PLK1, Aurora A, and P53 expressions increased in HEY control cells and PGCCs after MG132 treatment. In BT549 PGCCs with daughter cells after MG132 treatment, pCDC25CSer216, CHK1, and CHK2 expressions decreased and pCDC25CSer198, PLK1, Aurora A, and P53 expressions increased compared with those in the control cells  than in untreated control cells  . In breast cancer, P53 and CDC25CSer-216 were positively and negatively expressed in 97 and 35 cases of 167 samples, respectively. Both positive and negative expression between P53 and CDC25CSer-216 in breast cancer had statistical significance .Results of IHC staining analysis showed that P53 and pCDC25CSer216 expressions in the primary ovarian and breast cancer tumors were highly associated in multiple cases, as shown in Fig. P = 0.000), pCDC25CSer216 (P = 0.001), CDK1 (P = 0.000), CHK1 (P = 0.000), CHK2 (P = 0.000), PLK1 (P = 0.000) and Aurora A (P = 0.000) expression had statistical significances among these ovarian tumor groups. For breast cancer, the differences of CDC25C (P = 0.001), CDK1 (P = 0.000), CHK1 (P = 0.001), CHK2 (P = 0.000), PLK1 (P = 0.000) and Aurora A (P = 0.000) expression between primary cancer with and without metastasis also had statistical differences , pCDC25CSer216 (P = 0.001), CDK1 (P = 0.000), CHK1 (P = 0.014), CHK2 (P = 0.000), PLK1 (P = 0.000), and Aurora A (P = 0.003) in lymph node metastatic carcinoma was significantly higher than that in primary carcinoma with metastatic lymph nodes and the difference was statistically significant. CDC25C (P = 0.015), pCDC25CSer216 (P = 0.004), CDK1 (P = 0.005), CHK1 (P = 0.030) and CHK2 (P = 0.000) expressions increased in the primary carcinoma with metastatic lymph nodes compared to those in primary carcinoma without metastasis, except for PLK1 (P = 0.199) and Aurora A (P = 0.194). In addition, the expression of CDC25C (P = 0.000), pCDC25CSer216 (P = 0.000), CDK1 (P = 0.000), CHK1 (P = 0.000), CHK2 (P = 0.000), PLK1 (P = 0.000) and Aurora A (P = 0.008) between the primary carcinoma without metastasis and serious borderline cystadenoma were also statistically different.We also compared the difference of cell cycle-related proteins expression in different ovarian tumor groups. The results showed that CDC25C  is a kind of variations which the normal mitotic cell cycle involves multiple rounds of DNA replication without an intervening mitosis step. In addition, cell fusion can contribute to the formation of PGCCs and play an important role in cancer progression [p53 genotypes in different cell lines may be associated with the formation of PGCCs by regulating the expression and subcellular location of CDC25C. CDC25C knockdown can induce the formation of PGCCs. Daughter cells derived from PGCCs had strong abilities of migration, invasion and proliferation which can be inhibited by CDC25C knockdown. IHC staining confirmed that CDC25C expression level was closely related to the grade and stage in human ovarian tumors and breast cancer.PGCCs are the key contributors to cancer heterogeneity, which can generate daughter cells with strong invasive ability via asymmetric division. PGCCs are the commonest histopathological characteristics of human malignant tumors. The number of PGCCs in high malignant tumor, tumor with lymph node metastasis, and recurrent tumor is more than that in low degree malignant tumor, tumor without lymph node metastasis, and primary tumor. Tumor tissue with more PGCCs has strong resistance to radiotherapy and chemotherapy . Our pregression . In thisgression . During gression , 17. Befgression . In this2 treatment. PGCCs with the daughter cells in HEY and BT-549, highly expressed CHK2 and the CHK1 expression decreased. CHK1 and CHK2 in the nucleus can prevent CDC25C from dephosphorylating CDK1. Once CDK1 is activated, CHK1 is phosphorylated by CDK1 at Ser286 and Ser301 sites in turn. This phosphorylation induces nucleus to cytoplasm CHK1 translocation and eventually the accumulated cytoplasmic CHK1 is degraded by the proteasome degradation pathway [When DNA damage occurs in cells, CDC25C acts as the main target of checkpoint kinase, is regulated by CHK1 and CHK2 by phosphorylation, and its activity is inhibited , 18. CDC pathway , 22. CHK pathway , 24.CDC25C phosphorylation can also be regulated by PLK1 and Aurora A. PLK1 expression level is different in different cell cycle stages and reaches the peak in the G2/M stage . Activat2-treated HEY PGCCs and increased in BT-549 PGCCs, which may be related to p53 genotype. Mutant p53 not only cannot exert its anti-cancer function, but also affects wild-type p53 normal function and promotes cancer progression [p53 expression, induce P53 protein accumulation and stabilization, reduce P53 degradation, and trigger p53-dependent apoptosis [2 could increase mutant-type p53 expression and high P53 expression can inhibit CDK1, cyclin B1, and cyclin D1 expressions, which results in cell cycle G2/M arrest and PGCCs formation. In addition, co-immunoprecipitation and siRNA interference assays showed that P53 directly regulated pCDC25CSer216 and pCDC25CSer198 phosphorylation. P53 knockdown could reduce pCDC25CSer216 and pCDC25CSer198 expression. Kim et al. have reported that wide-type p53 could inhibit phosphorylation at CDC25C-Ser216 and prevent the polyploid generation caused by mitosis failure. However, mutant-type p53 decreased mitotic checkpoint function and increased CDC25C-Ser216 phosphorylation, which resulted in aneuploidy due to chromosome segregation failure [p53 decreased pCDC25CSer216 and pCDC25CSer198 expression levels. In BT-549, mutant p53 can promote PGCCs formation by mediating pCDC25CSer216 and pCDC25CSer198 phosphorylation level, increase tumor heterogeneity, and accelerate tumor progression.The expression of pCDC25CSer198 decreased in CoClgression . CDC25C gression . Hypoxiapoptosis –38. Beyfpoptosis . In this failure . In HEY,p53 genotype.The abnormal expression and subcellular location of CDC25C associated with the formation of PGCCs. CHK1, CHK2–pCDC25CSer216–cyclin B1–CDK1 and Aurora A–PLK1–pCDC25CSer198–cyclin B1–CDK1 signaling pathways may participate in PGCCs formation and the various phosphorylation sites of CDC25C may depend on Additional file 1: Supplementary figures and figure legends.Additional file 2. Supplementary materials and methods.Additional file 3: Supplementary tables.


The block in apoptosis in the mosaic mutant tissue was achieved by using a 82 FRT42D Darkchromosome as a starting point for the EMS mutagenesis . The 82Dark allele was generated by an imprecise excision of the P{lacW}ArkCD4, this allele retains the +mCw  One of the mutants identified was L.3.1 which generated a small rough eye mosaic phenotype, with a smaller percentage of pigmented tissue than the 82 FRT42D, Darkcontrol . The ‘rough eye’ phenotype indicates a disruption in the ommatidial organization. In both images the pigmented (+mCw) tissue is homozygous mutant and the unpigmented tissue is homozygous wild type.An EMS screen was conducted utilizing the Flp/FRT system to identify mutations that caused an array of phenotypic alterations in the size of the eye including the ratio of mutant to wild type tissue (red over white) or the developmental patterning of the mosaic eye. This screen was done in the genetic background of blocked apoptosis in the homozygous mutant cells to identify conditional regulators of cell growth and eye development . Complementation mapping was conducted independently and the results confirmed between groups. Virgin females from the 82/CyOFRT42D L.3.1 Dark stock were mated in series to male flies from the 87 deficiency stocks that comprise the Bloomington Stock Center 2R Deficiency Kit  . Mutant L.3.1 failed to complement Deficiency stock Df(2R)BSC664 , while complementing the flanking overlapping deficiencies Df(2R)BSC821 and Df(2R)BSC597. This left a region of failure to complement of 2R:21,497,290..21,806,350, which is pictured above in L.3.1 to test for complementation. L.3.1 failed to complement an apparent loss of function allele k05115 , indicating that L.3.1 is likely a novel Egfr allele, L.3.1.EgfrThe genetic mapping of the location of mutant 82/CyOFRT42D Dark 82 EgfrL.3.1/CyOFRT42D Dark (this manuscript)Ey>Flp; FRT42D (BDSC 5616)1 w67c23y; P{+mCw=lacW}k05115Egfr/CyO (BDSC 10385)et al., 2012):Bloomington Drosophila Stock Center 2R Deficiency Kit BSC664/SM6a1118w; Df(2R)BSC821, P+PBac{ +mCw =XP3.RB5}BSC821/SM6a111];w Df(2R)BSC597/SM6a


Restriction of foreign DNA is a fundamental defense mechanism required for maintaining genomic stability and proper function of mammalian cells. APOBEC cytidine deaminases are crucial effector molecules involved in clearing pathogenic DNA of viruses and other microorganisms and improperly localized self-DNA (DNA leakages). Mastering the expression of APOBEC provides the crucial means both for developing novel therapeutic approaches for combating infectious and non-infectious diseases and for numerous research purposes. In this study, we report successful application of a CRISPRa approach to effectively and specifically overexpress APOBEC3A and APOBEC3B deaminases and describe their effects on episomal and integrated foreign DNA. This method increased target gene transcription by >6–50-fold in HEK293T cells. Furthermore, CRISPRa-mediated activation of APOBEC3A/APOBEC3B suppressed episomal but not integrated foreign DNA. Episomal GC-rich DNA was rapidly destabilized and destroyed by CRISPRa-induced APOBEC3A/APOBEC3B, while the remaining DNA templates harbored frequent deaminated nucleotides. To conclude, the CRISPRa approach could be readily utilized for manipulating innate immunity and investigating the effects of the key effector molecules on foreign nucleic acids. Intracellular sensing, recognition, and clearance of foreign DNA are fundamental defense mechanisms required for the proper functioning of human cells. Both pathogenic  and non-pathogenic DNAs  pose a threat to mammalian cells and must be cleared by innate immune responses ,2. ForeiPreviously, APOBEC proteins were shown to be foreign DNA restriction factors that can directly deaminate cytidine nucleotides in single-stranded or double-stranded DNA, resulting in C-to-T and/or G-to-A hypermutation, generation of deletions, and decay of foreign DNA . The humThe antiviral properties of APOBEC3s have been extensively studied over the past decade in regard to retroviruses  , hepadnaHowever, in the evolutionary arms race between viruses and their hosts, viruses have evolved numerous mechanisms to counteract innate immunity, impair recognition of cytosolic and nuclear sensors, prevent triggering of innate immune responses , and directly inhibit effector antiviral proteins . Activation of APOBEC3 proteins is largely complex and cell type-dependent, and requires an intricately regulated cascade of reactions that may contribute to successful elimination of foreign DNA as a threat to the host cell or, upon dysregulation, to genetic instability, mutations, and development of cancer. Overall, APOBEC3s are important effector molecules in the innate immune response that protect human cells from foreign DNA. Developing the strategies to precisely regulate APOBEC3 expression in a timely manner is important for novel antiviral drug development and fundamental studies. Here, we utilized a novel dead Cas9 (dCas9)-based approach (CRISPR activation or CRISPRa ) to direA3A and A3B genes, we utilized a previously established CRISPR-activation (CRISPRa) system based on a dCas9 protein, a nucleolytically dead Cas9 protein harboring point mutations in RuvC and HNH domains, which is fused to a p300 core, a catalytic histone acetyltransferase core domain of the E1A-associated protein p300 [In order to activate transcription of target ein p300 . Recruitein p300 . The dCaein p300 .A3A and A3B genes lie in a tandem gene cluster on chromosome 22 together with the rest of the APOBEC3 genes. First, we designed a set of sgRNAs targeting A3A and A3B promoters. sgRNAs were designed using CCTop CRISPR/Cas9 target online predictor and UCSC genome browser [A3A- and A3B sgRNA-targeting sites are shown in A3A- and A3B-targeting sgRNAs is provided in A3A promoter with high predicted scores   in further experiments. We co-transfected dCas9-p300 with each of the individual sgRNAs and analyzed A3A and A3B levels on day 2 post transfection A. As concontrols B,C. In cnd A3A4) D. sgA3A3A3A and A3B transcription is maintained, we analyzed A3A and A3B levels dynamically .Further analysis of GFP-expressing cell populations  revealeds groups . The ratA3A and A3B activation, correspondingly  and CRISPRa platforms were generated based on a nucleolytically null Cas9 protein. Fusing this dCas9 with a transcriptional regulator enables precise regulation of gene expression . Recent A levels . SimilarA levels . Besidesomal DNA . HoweverA3A or A3B). Although in studies with systems other than dCas9p300 it was demonstrated that several sgRNAs demonstrate synergy and substantially increase activation of gene transcription [A3B baseline mRNA was expressed <50-fold lower compared to A3A   .A3A or A3B mRNA expression  cells were cultured in complete DMEM high-glucose medium with 10% FBS (Thermo Fisher), 100 U/mL penicillin, 100 µg/mL streptomycin , and 2 mM L-glutamine (Sigma Aldrich). The cells were seeded into 12-well plates at ~20% confluency and transfected the next day using 7.5 mM polyethylenimine. Briefly, a DNA mix containing 0.625 µg of AAV-GFP , 0.625 µg of pcDNA-dCas9-p300 Core or control pcDNA-dCas9-p300 Core (D1399Y) containing a mutation in p300 acetyltransferase domain , and 10 ng PCR product (with U6 promoter transcribed into a specific gRNA targeting A3A or A3B promoter) were gently added to 35 µL of NaCl solution (solution A). Solution B containing polyethylenimine (5.7 µL/well) in NaCl (29.8 µL/well) was prepared in parallel, incubated for 10 min, and gently mixed with solution A. Two solutions were incubated at ambient temperature for 10 min and then added to the cell culture medium. The day after transfection, cell culture medium was discarded, and the cells were gently washed twice in PBS and cultured in complete medium for the next 24–50 h before harvest for PCR analysis of analysis A. A3A and A3B promoter targets were assessed and sgRNA-designed using the open-access web tools CHOPCHOP and CCTop CRISPR/Cas9 target online calculator. Synthesis of sgRNA-encoding PCR products was performed as described in [ribed in . BrieflyA3A and A3B mRNA levels were assessed in relation to GAPDH mRNA. Specific sets of primers and probes are presented in Cell culture medium was discarded; cells were washed twice with PBS and lysed in AmpliSens Riboprep lysis buffer . RNA was isolated as described previously . BrieflyDNA samples were used for real-time PCR with Taq DNA polymerase, 300 nM 3D/DNA/RNA-GFP_fw/3D/DNA/RNA-GFP_rev primers , and SYBg and 4 °C. The supernatant was discarded, and the concentrated lentiviruses were dried at room temperature and resuspended in complete DMEM. Aliquots were stored at −70 °C until use. Lentiviruses were generated as described previously . In brieHEK-293T cells were plated onto 12-well plates in complete medium and allowed to adhere overnight. Lentiviruses were added to cells in complete DMEM in the presence of 8 µg/mL polybrene . After 48 h, media were discarded, and cells were washed twice in PBS and cultured in complete DMEM. At harvest, cells were counted on a BD FACSCanto II flow cytometer . Briefly, cell culture medium was discarded, and cells were washed twice in PBS, detached from the plates in trypsin-EDTA, resuspended in complete medium, and washed twice in PBS. EGFP-positive cells were detected in the FITC channel. Data were acquired with BD FACSDiva software and analyzed with NovoExpress software . Fluorescent and bright field images were taken on an Olympus IX71 . HEK-293T cells were seeded on 96-well plates to reach ~30–40% confluence on the day of transfection. Cells were co-transfected with AAV-GFP, pcDNA-dCas9-p300 Core/control, pcDNA-dCas9-p300 Core (D1399Y), and sgRNA-encoding PCR products using polyethylenimine. Cell cytotoxicity was analyzed using Abcam Cell Cytotoxicity Assay Kit  on day 5 post transfection according to manufacturer’s protocol. Optical density was measured using an iMark plate Spectrophotometer . Values were expressed as the mean ± standard deviation (SD) of triplicate experiments in GraphPad Prism 7.0 software. Student’s T-test with Tukey’s HSD post hoc test were used to compare variables and calculate p values to determine statistically significant differences in means.


Notospermus geniculatus, was recovered through Illumina sequencing data. This complete mitochondrial genome of N. geniculatus is 15,180 bp in length and has a base composition of A (14.4%), T (41.3%), C (14.6%), G (29.6%), demonstrating a bias of higher AT content (55.7%) than GC content (44.2%). The mitochondrial genome contains a typically conserved structure among Lineidae mitogenomes, encoding 13 protein-coding genes (PCGs), 23 transfer RNA genes (tRNA), 2 ribosomal RNA genes (12S rRNA and 16S rRNA), and a control region (D-loop region). All PCGs were located on the H-strand. ND4L gene and ND4 gene were overlapped by 6 bp. The whole mt genome of N. geniculatus and other Protostomia mitogenomes  were used for phylogenetic analysis. The result indicated N. geniculatus has the closest relationship with Lineus viridis (FJ839919.1) and clustered within Heteronemertea Clade, which representing a distinct order.In this study, the complete mitochondrial genome of  Notospermus geniculatus is one of the largest nemerteans present in Mediterranean waters, reaching 1 m in length and 15 mm in width. This species was originally described from Naples and is also widely distributed  and fixed in absolute ethanol. Genomic DNA was extracted from muscle tissues using DNEasy Extraction Kit (supplied by QIAGEN) following the manufactures instructions. The isolated DNA was stored in the sequencing company . Purified DNA was fragmented and used to construct the sequencing libraries following the instructions of NEBNext® Ultra™ II DNA Library Prep Kit . Whole genomic sequencing was performed by the Illumina HiSeq 2500 Sequencing Platform . Adapters and low-quality reads were removed using the NGS QC Toolkit , and manual correction for start and stop codons were conducted. This complete mitochondrial genome sequence together with gene annotations were submitted to GenBank under the accession numbers of MH714705.In this study, a specimen of Notospermus geniculatus was 15,180 bp in length and has a base composition of (14.4%), T (41.3%), C (14.6%), G (29.6%), demonstrating a bias of higher AT content (55.7%) than GC content (44.2%). The mitochondrial genome contains a typically conserved structure among Lineidae mitogenomes, encoding 13 protein-coding genes (PCGs), 23 transfer RNA genes (tRNA), 2 ribosomal RNA genes (12S rRNA and 16S rRNA), and a control region (D-loop region).All PCGs were located on the H-strand. ND4L gene and ND4 gene were overlapped by 6 bp.The complete mitochondrial genome of N. geniculatus has the closest relationship with Lineus viridis (FJ839919.1) and clustered within Heteronemertea Clade, which represents a distinct order.For phylogenetic analysis assessing the relationship of this mitogenome, we selected other 17 Protostomia mitogenomes from Lineidae (4 taxa), Monostilifera (4 taxa), Palaeonemertea (1 taxon), Molluscae (4 taxa), and Ecdysozoa (2 taxa) to construct genome-wide alignment. The genome-wide alignment of all mt genomes was done by HomBlocks (Bi et al.


Efficient radiolabeling of the aromatic ring of [18F]FPEB has been an ongoing challenge. Herein, five metal-free precursors for the radiofluorination of [18F]FPEB were compared, namely, a chloro-, nitro-, sulfonium salt, and two spirocyclic iodonium ylide (SCIDY) precursors bearing a cyclopentyl (SPI5) and a new adamantyl (SPIAd) auxiliary. The chloro- and nitro-precursors resulted in a low radiochemical yield (<10% RCY), whereas both SCIDY precursors and the sulfonium salt precursor produced [18F]FPEB in the highest RCYs of 25% and 36%, respectively. Preliminary PET/CT imaging studies with [18F]FPEB were conducted in a transgenic model of Alzheimer’s Disease (AD) using B6C3-Tg85Dbo/J (APP/PS1) mice, and data were compared with age-matched wild-type (WT) B6C3F1/J control mice. In APP/PS1 mice, whole brain distribution at 5 min post-injection showed a slightly higher uptake (SUV = 4.8 ± 0.4) than in age-matched controls (SUV = 4.0 ± 0.2). Further studies to explore mGluR5 as an early biomarker for AD are underway.[ L-Glutamate is the primary excitatory neurotransmitter at the majority of excitatory synapses in the mammalian central nervous system (CNS). Signaling processes involving metabotropic glutamate occur via membrane-bound G-protein coupled receptors (GPCRs) known as metabotropic glutamate receptors (mGluRs). There are eight mGluR subtypes, of which the mGluR5 subtype is involved in the excitatory signaling cascade of intracellular calcium release, playing a vital role in brain development, learning, memory, and in maintaining synaptic plasticity [18F]3-Fluoro-5-[(pyridin-3-yl)ethynyl] benzonitrile ([18F]FPEB) was developed as a mGluR5 PET ligand and is widely used in clinical research 3-research ,8.F3-resear18F]FPEB has traditionally been low yielding because nucleophilic aromatic substitution by [18F]fluoride is not favored as the nitrile group is positioned meta to the leaving group on the aromatic ring of the precursor. Furthermore, high temperature reactions are generally required, leading to decomposition products and radiochemical impurities. [18F]FPEB is commonly synthesized from a chloro- or nitro-precursor in low radiochemical yields  as shown in 18F]FPEB was prepared via a SNAr reaction of an aryl-chloro precursor (1) with labeled potassium cryptand fluoride, [K222][18F], and K2CO3 as the base [18F]FPEB for human use was initiated with the commercially available nitro-precursor and a low RCY (4%) was achieved [3 is a boronic acid precursor and 4 is an arylstannane precursor that were used in copper-mediated radiofluorinations to synthesize [18F]FPEB in decay-corrected automated radiochemical conversions  of 5% and 11% respectively [18F]FPEB will be challenging as lower RCY can be expected upon purification and formulation, while additional testing for residual metals will be required.The routine radiosynthesis of FPEB [m) of [18F]FPEB, compared with our traditional SNAr reaction with the nitro-precursor [In our efforts to optimize the efficiency of ecursors . Our devecursors  was succ18F]FPEB ,15. The recursor , and wasrecursor ,13.18F]FPEB, with our first-generation SCIDY precursor (cyclopentyl auxiliary (5)) and new second-generation SCIDY precursor (adamantyl auxiliary (7)) and a newly reported sulfonium salt precursor (6) for the routine production of [18F]FPEB [18F]FPEB binding in a patient with early mild cognitive impairment [18F]FPEB to detect early changes in mGluR5 expression in a transgenic murine model of AD.The aim of this study was to compare the chloro- and nitro-precursors for [18F]FPEB ,17. Onlypairment , we also1) was synthesized following a literature procedure, with the modification that conventional heating was used to drive the radiofluorination instead of the previously reported microwave conditions [18F]FPEB via the commercially available nitro-precursor (2) showed an RCC based on radio-HPLC analysis of 33% (n = 3) at 150 °C for a reaction time of 5 min, upon automation and isolation, the highest RCY was only 4%  as the precursor for [18F]FPEB production led to a five-fold increase in RCY and a three-fold increase in molar activity (Am) compared to the nitro-precursor. The SCIDY precursor enabled the displacement reaction with [18F]fluoride to be conducted at milder conditions (lower temperatures and shorter times), thereby minimizing the formation of radiochemical impurities. [18F]FPEB synthesis via the SCIDY SPI5 auxiliary precursor was conducted on a GE TRACERlab™ FX2 N automated synthesis module. Varying temperatures and reaction times resulted in RCYs of [18F]FPEB ranging from 19% to 23% after HPLC purification and formulation  which resulted in a RCY of 25% ± 2%, with a molar activity (Am) of = 37 ± 13 GBq/µmol (n = 3), consistent with our previous reports [We recently showed that using the SCIDY chemistry with a cyclopentyl auxiliary (SPI5) . Experimental and mechanistic studies have shown that radiofluorination with the bulkier SPIAd auxiliary in SCIDY precursors can improve the RCY compared to the SPI5 precursors in conventional nucleophilic aromatic substitution with [18F]fluoride [7) also resulted in a 24% RCY for [18F]FPEB, and is similar to that of the SPI5 precursor  based on a sulfonium salt was also applied to the manual synthesis of [18F]FPEB and gave a reported 55% RCY [18F] and the milder base (KHCO3) at 80 °C for 5 min (18F]FPEB with a RCY of 36% ± 6% and Am = 77 ± 35 GBq/µmol (n = 3).A recently reported precursor  controls. Axial, coronal, and sagittal images of the murine brains acquired at 20 min post-injection of [18F]FPEB are presented in 18F]FPEB retention was observed in the brain of transgenic mice (SUV = 4.8) versus WT controls (SUV = 4.0), and as the tracer cleared from normal tissues, a significant difference (p < 0.05) was apparent at <5 min post-injection. A comparison of the area under the curve (AUC) analysis (mGluR5 has emerged as an imaging target in AD pathogenesis. It has been demonstrated that soluble oligomeric amyloid-β (Aβo) induces an accumulation and over-stabilization of mGluR5, and that Aβo up-regulates mGluR5, leading to an abnormal increase in the release of intracellular Ca2+ ,21,22,23analysis C over ti18F]FPEB, and that radiotracer uptake in the brain was reflective of increased mGluR5 density [18F]FPEB uptake in a transgenic mouse model of AD also showed an increased radiotracer uptake and retention in the brain of the APP/PS1 mice, compared with wild-type controls. This preliminary work provides support that mGluR5 levels measured by [18F]FPEB are potentially useful as an early biomarker of AD. Further [18F]FPEB imaging studies and biological evaluations are underway including regional analysis of imaging data as well as ex vivo biodistribution and autoradiography studies to evaluate this functional link between mGluR5 and AD.In a pilot study, we found that patients with early mild cognitive impairment had an increased brain uptake of [ density . This ob density . Consist18F]fluoride (ca. 100 mCi). [18F]Fluoride was produced using a Scanditronix MC17 cyclotron from enriched [18O]H2O through the 18O18F reaction. Reverse-phase high performance liquid chromatography (HPLC) was used to isolate and purify [18F]FPEB. Dynamic PET/CT imaging experiments were conducted on a dedicated small-animal PET/CT scanner  equipped with VISTA-CT version 4.11 software.Unless otherwise stated, all reagents were obtained from commercially available sources and used without further purification. The identification of all radiochemical products was determined by HPLC co-elution with an authentic non-radioactive standard. All RCC and RCY values are reported as decay corrected, relative to starting [18F]fluoride was dissolved in DMSO (3.0 mL), while 400 μL aliquots  were used per reaction to dissolve the precursor in a 1 dram vial. The reaction was heated at 150 °C for 5 min, then quenched with water and cooled for 3 min. Product identity and radiochemical conversion were determined as the ratio of free [18F]fluoride to [18F]FPEB as integrated by radio-HPLC. The manual radiolabeling of the chloro- (1) and nitro- (2) precursors was performed as previously reported, with slight modification to the concentration and reaction time. One milligram of 2 was dissolved in 0.4 mL [18F]fluoride in DMSO for 5 min instead of 1.5 mL for 15 min + calcd. for C22H15IN2O4Na, 520.9974; found 520.9967.18F]fluoride in enriched [18O]H2O was delivered to a GE TRACERlab™ FX2 N automated synthesis module. The [18F]fluoride was trapped on a HCO3− anion exchange cartridge (Chromafix® [18F] SPE) without additional conditioning and eluted with one of the following base mixtures: Et4NHCO3 in CH3CN/H2O, 4,7,13,16,21,24-hexaoxa-1,-10,diazabicyclo [8.8.8]hexacosane (Kryptofix 222) and KHCO3 in CH3CN/H2O or Kryptofix 222 and K2CO3 in MeOH/H2O. The [18F]fluoride was released from the cartridge and dried under nitrogen gas at 90 °C, followed by azeotropic drying with acetonitrile under nitrogen gas at 110 °C, producing either dry [18F]Et4NF or [18F]KF/K222 for radiofluorination. Automated radiosyntheses were carried out with precursors 2, 5, 6, and 7. Each precursor was dissolved in either DMSO, DMF, or CH3CN and eluted into the reaction vial with the dry [18F]fluoride, where they were heated for the specified reaction time at varying temperatures. The reactions were then cooled to room temperature and quenched with HPLC buffer. The mixture was diluted with water and the crude reaction mixture was injected onto a semi-preparative HPLC column. The eluent was monitored by UV, at a wavelength of 254 nm, and radiochemical detectors in series. The desired radiochemical product was collected and formulated by dilution with sterile water and loaded onto a C18 Sep-Pak cartridge . The cartridge was washed with water to remove impurities and then eluted with dehydrated EtOH, and finally, diluted with 0.9% sodium chloride. The final radiochemical yield of this formulated product was determined . Molar activity measurements were carried out as described in [Cyclotron produced [ribed in .18F]FPEB  via intravenous (i.v.) tail-vein injection using a catheter. Approximately 5 min prior to recording PET images, the mice were anesthetized by inhalation of 3–4% isoflurane /oxygen gas mixture, and a catheter was inserted into the tail-vein, with the mice then transferred to the scanner bed and placed in the prone position. Anesthesia was maintained with 1–2% isoflurane/oxygen gas mixture (flow rate ~5 L/min). Co-registered dynamic PET/CT images were recorded for a total of 20 min post-injection radiotracer injection (n = 3 per group). List-mode data were acquired for 20 min per scan using a γ-ray energy window of 250–700 keV. To ensure that the activity bolus was measured, PET/CT data acquisition was initiated 20 s prior to injecting the radioactivity. Data were processed by 3-dimensional Fourier re-binning (3D-FORE), and images were reconstructed using the 2-dimensional ordered-subset expectation maximum (2D-OSEM) algorithm. Image data were normalized to correct for the non-uniformity of response of the PET, dead-time count losses, positron branching ratio, and physical decay to the time of injection, but no attenuation, scatter, or partial-volume averaging correction were applied. An empirically determined system calibration factor (in units of Bq/cps) combined with the decay corrected administered activity and the animals’ weights were used to parameterize image activity in terms of the standardized uptake value (SUV). Manually drawn 2-dimensional regions-of-interest (ROIs) or 3-dimensional volumes-of-interest (VOIs) were used to determine the maximum and mean SUV radiotracer uptake in various tissues. Time–activity curves (TACs) were generated from the ROI analysis on dynamic PET/CT data using 20 s frames. CT images were recorded using an X-ray current of 300 μA, 360 projections, and an image size of 63.8 mm × 63.8 mm × 46.0 mm. Data were acquired using the Vista CT 4.11 Build 701 software, and reconstructed images were analyzed by using ASIPro VMTM software  and VivoQuant® 1.23 .All animal experiments were conducted in compliance with Institutional Animal Care and Use Committee (IACUC) guidelines and the Guide for the Care and Use of Laboratory Animals. Female wild-type B6C3F1/J mice and female transgenic B6C3-Tg85Dbo/J  mice were obtained from Jackson Laboratory . Mice were provided with food and water ad libitum. Mice were subjected to PET/CT imaging studies after being aged to 10 months. Mice were administered formulations of [p < 0.05) were considered to be statistically significant.Data and statistical analyses were performed using GraphPad Prism 5.01  and Microsoft Excel spreadsheets. Differences at the 95% confidence level (