Following phylogenetic analysis of 29 specimens in the complete S region, 28 belonged to subgenotype A1 and one to D3. Mutations affecting HBeAg expression at the transcriptional (1762T1764A), translational , or post translational levels (G1862T), were responsible for the high HBeAg-negativity observed. The G1862T mutation occurred only in subgenotype A1 isolates, which were found in one third (7/21) of HBsAg−ve participants, but in none of the 18 HBsAg+ve participants (p<0.05). Pre-S deletion mutants were detected in four HBsAg+ve and one HBsAg−ve participant/s. The following mutations occurred significantly more frequently in HBV isolated in this study than in strains of the same cluster of the phylogenetic tree: ps1F25L, ps1V88L/A; ps2Q10R, ps2 R48K/T, ps2A53V and sQ129R/H, sQ164A/V/G/D, sV168A and sS174N (p<0.05). ps1I48V/T occurred more frequently in females than males (p<0.05). Isolates with sV168A occurred more frequently in participants with viral loads >200 IU per ml (p<0.05) and only sS174N occurred more frequently in HBsAg−ve than in HBsAg+ve individuals (p<0.05). Prior to initiation of ART, ten percent, 3 of 29 isolates sequenced, had drug resistance mutations rtV173L, rtL180M+rtM204V and rtV214A, respectively. This study has provided important information on the molecular characteristics of HBV in HIV-infected southern Africans prior to ART initiation, which has important clinical relevance in the management of HBV/HIV co-infection in our unique setting.Hepatitis B virus (HBV) and human immunodeficiency virus (HIV) are hyperendemic in sub-Saharan Africa. The HBV genotypes prevailing in HIV-infected Africans are unknown. Our aim was to determine the HBV genotypes in HIV-infected participants and to identify clinically significant HBV mutations. From 71 HBV DNA Hepadnaviridae, is endemic in several parts of the world, including sub-Saharan Africa, which accounts for at least 65 of the 360 million people in the world chronically infected with the virus Hepatitis B virus (HBV), with a genome of ∼3,200 base pairs, is the smallest DNA virus infecting humans, yet it is one of the most important human pathogens, causing major health problems globally. HBV, the prototype member of the family The two viruses share a common mode of transmission and can co-exist in the same host −ve (having resolved the infection) and 23.8% being HBVDNA+ve+ve cohorts −ve participants met the “Taormina” definition of true occult HBV infection  HBsAg-covert (HBsAg-cryptic overt) infections, having higher viral loads (>200 IU/ml) We have recently shown that of approximately 300 HIV-infected individuals from a rural cohort in Mpumalanga Province, 77.5% had at least one HBV marker, with 53.7% being HBVDNA−5 nucleotide substitutions/site/year, which results in genetic heterogeneity HBV replicates by reverse transcription of the pregenomic RNA using a viral-encoded polymerase that lacks proof-reading activity. The genome may evolve at an estimated error rate of 1.4–5×10Very few studies have been conducted on the genotypes and molecular characterization of HBV in HIV-infected individuals in the Africa +ve individuals prior to the initiation of antiretroviral therapy (ART), 71 plasma samples were shown to be positive for HBV DNA Of the 298 samples obtained from HIVEcoRI site EcoRI site) DNA was extracted from 200 µl blood plasma with the QIAamp DNA Blood Mini Kit  and eluted into 75 µl of best-quality water (BQW). Known positive and negative sera and BQW were used as controls for the extraction. The basic core promoter/precore (BCP/PC) region and complete S open reading frame (ORF) were amplified in a MyCycler™ thermocycler  using Promega Taq DNA polymerase  as described previously in detail The amplicons were prepared for direct sequencing using the BigDye Terminator v3.0 Cycle Sequencing Ready Reaction Kit  and sequencing was performed by the Central Analytical Facility, Stellenbosch University, South Africa, using the ABI 3130XL Genetic analyser . BCP/PC sequences were analysed in both the forward and reverse directions of a single fragment, whilst the complete S sequences were analysed in 3 overlapping fragments. In addition to the primers used for amplification, HBV-specific primers EcoRI site) and complete surface DNA sequences  were assembled and aligned manually using GeneDoc Both BCP/PC  did not show any BCP/PC mutations, which can down-regulate or abolish the expression of the HBeAg. These had wild type sequences relative to the consensus for subgenotype A1 of HBV, that is, 1762A/1764G, 1809–1812 (TCAT Kozak), 1862G and 1888A. One isolate from a HBeAg+ve participant, SHH253, had Kozak sequence TCCT, which would down- regulate but not abolish, HBeAg expression. Another isolate, SHH274, from a HBeAg+ve participant, had a start codon mutation, together with 1762T/1764A. It is possible that HBeAg was expressed from a minor population.Of the 49 cases, 5 were HBeAg−ve individuals were analysed for mutations . Two of three isolates obtained from true occult infections were sequenced in this region: SHH060 and SHH107 and were wild-type for subgenotype A2 and A1, respectively , but in none of the isolates from 18 HBsAg+ve participants (p<0.05).Of the 39 samples belonging to subgenotype A1, for which the BCP/precore region was sequenced successfully, 18 were from HBsAgEcoRI site) was sequenced successfully for HBV isolates from 29 participants. Following phylogenetic analysis, 28 of 29 isolates clustered with subgenotype A1, whereas one isolate, SHH055, clustered with genotype D (data not shown). For the isolate from SHH167, a discordant result was obtained between the HBV genotyping deduced using BCP/PC sequences and the S region phylogenetic analysis of HBV i.e. “not genotype A”and “subgenotype A1”, respectively.The complete S region (position 2854–835 from the notype D  and belored) and the remaining 11 isolates clustered with the African cluster (green). There was no clustering with respect to whether the isolates were derived from HBsAg+ve or HBsAg−ve samples.Subgenotype A1 isolates from HBV/HIV co-infected individuals were compared to subgenotype A1 isolates from Asian and African countries using a circular unrooted phylogenetic tree . Seventeadw2, eight, including the genotype D isolate, to ayw2 and one to adr (red) had S5, S6, F25 in the pre-S1. The isolates in the African cluster (green) displayed greater variation with three geographically distinct clades: the largest consisting of southern African strains with S5, A6, F25 in the pre-S1, a second consisting of eastern African strains with L5, P6, F25 and a third one consisting of central African strains, which like the “Asian” strains, had S5, S6, F25 in the pre-S1 . sQ129R was the only mutation detected in the only isolate sequenced from a true occult HBV infection  The following mutations occurred significantly more frequently in HBV isolated from HIV-co-infected individuals in this study than in strains of the same cluster of the phylogenetic tree: in the pre-S1, ps1F25L, ps1V88L/A; in the pre-S2, ps2Q10R, ps2 R48K/T, ps2A53V and in the S region sQ129R/H, sQ164A/V/G/D, sV168A and sS174N (p<0.05). In the pre-S1, ps1I48V/T occurred more frequently in females than males (p<0.05). Isolates with sV168A occurred more frequently in participants with viral loads greater than 200 IU per ml (p<0.05). A mutation in ps2M1 of the preS2 abolished the start codon in four isolates. When comparing the frequency of mutations in HBsAg+ve and HBsAg−ve individuals. Only one of three isolates (SHH107) obtained from true occult infections was sequenced in this region and found to contain mutations only in the spacer region of the polymerase.In the polymerase region, the following mutations occurred significantly more frequently in HBV isolated from HIV-co-infected individuals than in isolates of the same cluster on the phylogenetic tree: spQ23K, spL28P, spS91I, spP132Q, spQ125E and rtE1D (p<0.05). The unusual start codon mutation, rtE1D, was seen in eight isolates, which grouped in the “Asian” cluster following phylogenetic analysis. This mutation occurred together with rtS105T+rtH122N in 7 isolates and with rtQ125E in three isolates. Analysis of 457 sequences from GenBank revealed that the rtE1D mutation was found in genotype B and G isolates from Asian countries. Glutamic acid (E) and aspartic acid (D) have similar chemical structures and properties; therefore this mutation is not expected to introduce a significant functional change to the reverse transcriptase polymerase and the isolates are probably replicative. Three isolates had drug resistant mutations: SHH011 had rtV214A, SHH074 had rtL180M+rtM204V and SHH130 had rtV173L. There was no significant difference in the frequency of polymerase mutations in HBV from HBsAg+ve, either alone  or together with anti-HBc  +ve participants with previously resolved HBV infection. Of the 231 participants exposed to HBV, 53.7% were HBV DNA−ve (resolved) and 23.8% HBV DNA+ve (current) [8.7% HBsAg+ve: 15.1% HBsAg−ve] Compared to areas of low endemicity, where HIV and HBV are most likely transmitted at the same time during sexual maturity, in southern Africa, where both viruses are endemic, HBV infection occurs before the age of 5 years and these children become chronic carriers of HBV in adulthood −ve blood donors +ve blood donors (90%∶10%). Discordant results between the genotypes deduced when the BCP/PC region and when the S region was sequenced were obtained for isolate SHH0167 . This mutation causes intracellular retention of the HBeAg precursor, aggresome formation and impaired secretion of HBeAg The HBeAg negativity found in 44/49 Shongwe participants  could be accounted for by the following HBV mutations: the basic core promoter mutations A1762T/G1764A, which can down-regulate transcription of precore mRNA −ve. All five had pre-S2 deletions, ranging in size from 11 to 22 amino acids, with one isolate having, in addition, a 10 amino acid pre-S1 deletion. Deletion mutants in the pre-S region have been previously found to occur more frequently in HIV-coinfected individuals Pre-S/S sequence data were analyzed in an attempt to explain the high HBsAg-negativity, which was a feature of this rural cohort of HBV/HIVco-infected individuals +ve and HBsAg−ve participants, there were a number of mutations that could account for the inability to detect HBsAg. These included ps1F25L, ps1I48V, ps1V88L/A in the pre-S1 region, ps2M1I, ps2Q10R, ps2R48K/T, ps2A53V in the pre-S2 and sY100C, sP120T/A, sQ129R/H, sE164D and sS174N in the S region , to which these mutants are sensitive. This study has provided important information on the molecular characteristics of HBV in HIV-infected South Africans prior to the initiation of ART. Our findings have important clinical relevance in the management of HBV and HIV co-infection in our unique setting, where subgenotype A1 HBV is hyperendemic and usually transmitted horizontally in childhood, before HIV infection occurs in adulthood.In conclusion, the study showed that subgenotype A1 predominates in HBV/HIV co-infected individuals from rural South Africa. Subgenotype A1 HBV isolates had mutations that can affect HBeAg-expression at the transcriptional, translational and posttranslational levels and these mutations can account for the HBeAg negativity seen in the majority of HBV/HIV infected individuals in this cohort. Although there were no significant differences between all S region mutations occurring in HBV from HBsAg


The detailed metabolism of copper-induced pathology in WD is still unknown. Gene mutations as well as the possible pathways involved in the ATP7B deficiency were documented. The ATP7B gene was analyzed for mutations in 18 Chinese Han families with WD by direct sequencing. Cell viability and apoptosis analysis of ATP7B small interfering RNA (siRNA)-treated human liver carcinoma (HepG2) cells were measured by 3--2,5-diphenyltetrazolium bromide (MTT) assay and Hoechst 33342 staining. Finally, the expression of B-cell CLL/lymphoma 2 (BCL2), BCL2-associated X protein (BAX), sterol regulatory element binding protein 1 (SREBP1), and minichromosome maintenance protein 7 (MCM7) of ATP7B siRNA-treated cells were tested by real-time polymerase chain reaction  and Western blot analysis. Twenty different mutations including four novel mutations  in the ATP7B gene were identified in our families. Haplotype analysis revealed that founder effects for four mutations  existed in these families. Transfection of HepG2 cells with ATP7B siRNA resulted in decreased mRNA expression by 86.3%, 93.1% and 90.8%, and decreased protein levels by 58.5%, 85.5% and 82.1% at 24, 48 and 72 hours, respectively (All P<0.01). In vitro study revealed that the apoptotic, cell cycle and lipid metabolism pathway may be involved in the mechanism of WD. Our results revealed that the genetic cause of 18 Chinese families with WD and ATP7B deficiency-induce apoptosis may result from imbalance in cell cycle and lipid metabolism pathway.Wilson's disease (WD) is an autosomal recessive inherited disorder caused by mutations in the ATPase Cu The incidence of WD among different populations varies from 1/30,000 to 1/100,000 ATP7B disrupts copper homeostasis, particularly in the liver, by greatly decreasing the ability of exporting excess copper from hepatocytes to bile. Copper accumulation causes severe morphological and functional changes, including cirrhosis, hepatitis and liver failure. There is wide variability in clinical manifestation and age at the onset (from 3 to 70 years) of this disease, and typical biochemical features may not always be present. Therefore, genetic analysis provides the potential for more reliable early diagnosis, and prompt treatment http://www.wilsondisease.med.ualberta.ca/database.asp), but a detailed mechanistic understanding of copper-induced pathology in WD is still lacking. Knowledge of the distribution of particular mutations may help to design shortcuts for genetic screening strategies of WD. To evaluate the frequency of the ATP7B mutations in Chinese Han patients with WD, to explore genotype-phenotype correlations and to possibly unveil the pathways involved in the ATP7B deficiency, we screened 18 families with WD and inhibited the ATP7B gene expression in human liver carcinoma (HepG2) cells.Wilson's disease (WD) is an autosomal recessive disorder of copper metabolism. The disease is caused by mutations in the ATPase CuEighteen Chinese Han WD families , consistATP7B gene. The primers sequences are available on demand (RefSeq NG_008806). PCR products were directly sequenced on 3130 Genetic Analyzer. Given that WD is an autosomal recessive disorder with an estimated carrier frequency of 1/90 Polymerase chain reaction (PCR) amplified all coding regions and intron/exon boundaries of the Haplotype analysis was conducted in families harbored the same mutation with single nucleotide polymorphisms (SNPs) including rs1801243, rs1801244, rs1061472, rs732774, rs1801249, rs2282057 and rs9535795 by sequencing.In vitro transient transfection was done as described previously ATP7B siRNA were purchased from Qiagen. The target sequence was 5′-CCAATTGATATTGAGCGGTTA-3′. Briefly, Cells from passages 10 to 20 were used and they were seeded at 60% confluency into six-well or 96-well dish. The ATP7B siRNA and 5.0 μl HiPerFect Reagent  were diluted into a final volume of 100 μl in Opti-MEM , respectively, and gently mixed and incubated at room temperature for 10 min, then 800 μl Opti-MEM was added to the mixture. The above transfection solution was overlaid onto cells at a final concentration of 5 nM siRNA. Transfection of HepG2 cells with AllStars Negative Control siRNA  served as a negative control. After 12-hour incubation at 37°C in the presence of 5% CO2, 2 ml of complete medium with 10% FBS was added to each well of transfected cells to replace transfection solution.HepG2 cells  were cultured in Dulbecco's modified Eagle's medium  supplemented with 10% heat-inactivated fetal calf serum, 1% L-glutamine and 1% penicillin/streptomycin. All experiments were repeated at least three times. Total RNAs were prepared and cDNA was synthesized by using the SuperScript First-Strand kit  as per the manufacturer's instructions. The cDNA samples were amplified by the following primers . PCR wasATP7B polyclonal antibody , rabbit anti- minichromosome maintenance protein 7 (MCM7) monoclonal antibody , rabbit anti- sterol regulatory element binding protein 1 (SREBP1) polyclonal antibody , mouse anti- B-cell CLL/lymphoma 2 (BCL2) polyclonal antibody , mouse anti- BCL2-associated X protein (BAX) polyclonal antibody  and mouse anti-β-actin polyclonal antibody . The membrane was washed three times with PBS and then incubated for 1 h with 1000-fold diluted horseradish peroxidase-conjugated anti-rabbit IgG  to detect ATP7B, MCM7 and SREBP1, and horseradish peroxidase-conjugated anti-mouse IgG  to detect BCL2, BAX and β-actin, respectively. Immunoreactive proteins were visualized using enhanced chemiluminescence .Cells grown to 90% confluence were harvested and lysed in lysis buffer. Protein (30–50 μg) was heated at 95°C for 5 min, separated by SDS-PAGE and electrophoretically transferred onto PVDF membranes. The blots were blocked for 1 h in 5% milk in TBST and incubated at 4°C overnight with rabbit anti-2) for 12 h, then transfected with 5 nM ATP7B siRNA for 24 h and recovery for 3 h with cell culture medium. Copper sulfate  at various concentration was added for 24 hours. The supernatant was aspirated and 200 μl medium mixed with 3 μl 3--2,5-diphenyltetrazolium bromide (MTT) reagent (5 mg/ml) was added to each well. After a 3-h incubation at 37°C, 200 μl of DMSO was added to dissolve the formazan crystals, and the absorbance was then measured at 490 nm using a Digiscan Microplate Reader HepG2 cells were seeded per well in a 96-well plate and incubated  to evaluate membrane integrity. About 100 cells were examined in each field at 200× magnification, and at least five random fields were chosen P<0.05 and P<0.01, as indicated.MTT and cell apoptosis assays were performed in triplicate in three separate experiments. Statistical analysis was performed using SPSS17.0. Results are presented as means ± SD. Data analysis was performed using one-way ANOVA. The level of significant difference was set at Twenty patients with WD were included in our study. The mean age of disease onset was 17±10 years (range 2–41 years), seven (35%) were males. The 20 patients were the offsprings of 18 couples, and two of the 18 couples (11.1%) were consanguineous. Patients were classified phenotypically into hepatic  and neurological  forms of WD based on initial symptoms or features. The most frequent symptoms or signs in our WD patient population were of hepatic origin, including hepatomegaly , splenomegaly , jaundice , abdominal distension , abdominal pain , and bleeding . Neurological manifestations were observed in 12/20 patients (60%). K-F rings was observed in 19 (95%) patients. Low serum ceruloplasmin (64.7±29.7 mg/dl) and elevated 24 h urinary copper excretion (256.7±80 μg/24 h) were present in all patients. All patients were based on clinical manifestations, laboratory findings , and met the criteria of the Leipzig score ATP7B gene were observed by screening 18 probands of our 18 Chinese Han families with WD ATP7B gene mutations ATP7B gene were the cause of WD in our families. Single heterozygous mutation in one of the two copies of the ATP7B gene was insufficient to cause this disorder, consistent with a loss-of-function mechanism of the ATP7B mutations.Thirty-six sequence variants, including 16 polymorphisms (7 novel polymorphisms: c.411C>A (p.Ser137Ser), c.2289C>T (p.Phe763Phe), c.2502C>G , c.3973C>T (p.Leu1325Leu), c.3999G>T (p.Leu1333Leu), IVS8+26A>G, and IVS8+27G>A), in the Haplotype analysis of different families with the same mutation revealed that clear founder effects for p.Arg778Leu , p.Pro992Leu , p.Ile1148Thr (Families M1807 and M1856) and p.Ala1295Val (Families M41 and M1407) mutations existed in these families (data not shown).ATP7B siRNA resulted in decreased mRNA expression by 86.3%, 93.1% and 90.8% . When incubated with 5 nM ATP7B siRNA for 48 hours, the cell viability decreased by 17.2% and 16.6%  by MTT analysis , and by 44.0% as compared with ATP7B siRNA treatment alone (P<0.05). Cell viability decreased more obviously when ATP7B-deficient cells were exposed to higher concentration of copper sulfate (decreased by 79.8% for 200 μM) as compared with AllStars Negative Control siRNA treated cells alone (P<0.05), and by 77.6% as compared with ATP7B siRNA treatment alone  treatment also increased copper-induced apoptosis accompanying the higher dose of copper sulfate . Whereas BAX expression increased by 5.9-fold, 6.4-fold and 7.2-fold at 24, 48 and 72 hours, respectively comparing to those of the AllStars Negative Control of siRNA treated cells (All P<0.05). Consistently, Western blot showed that BCL2 decreased to 54.6%, 19.3% and 3.1%, and BAX increased to1.8-fold, 1.7-fold and 3.2-fold for 24 h, 48 h and 72 h  and lipid metabolism-related gene(s). The mRNA levels of SREBP1, a lipid metabolism-related gene, were significantly decreased by 49.7%, 59.9% and 80.0% in the ATP7B siRNA silenced cells at 24, 48 and 72 hours, respectively, compared with those of AllStars Negative Control siRNA treated cells (P<0.05). Consistently, the protein levels were decreased by 42.7%, 52.2% and 94.9%, respectively. The mRNA levels of the MCM7, a cell cycle-related gene, was significantly decreased by 89.2%, 95.1% and 95.5%, and the MCM7 protein levels were significant decreased by 32.8%, 92.4% and 93.6% at 24, 48 and 72 hours, respectively  and their unaffected mother was in heterozygous status (M1279). p.Thr498Ser was found in a 20-year female WD patient  with compound heterozygous mutations (p.Thr498Ser and p.Pro992Leu), and the age at the onset was 12 years with neurological presentation. Compound heterozygous ATP7B mutations (p.Gly837X/p.Pro992Leu) were found in an 18-year WD female  presented with liver disease at the onset age of 2 years old, the earliest reported onset age. All these four novel mutations were not detectable in 100 normal controls. Some reports suggested that the early onset of severe liver disease occurs with frameshift or nonsense mutations ATP7B and hepatic phenotype Wilson's disease, a genetic disorder of copper metabolism, is caused by mutations in the ATP7B mutation may cause WD ATP7B mutation was detected. It is possible that a second mutation escaped identification by the methods employed or that some mutations in heterozygous forms are sufficient to cause disease ATP7B mutations in WD. Therefore, caution should be exercised in the interpretation of heterozygous forms in WD individuals.Several studies suggest that a single ATP7B gene silencing is able to effectively inhibit endogenous expression of ATP7B mRNA and protein. ATP7B deficiency may reduce cell viability by inducing apoptosis in HepG2 cells. The ATP7B deficiency, coupled with exposure to copper, may further aggravate apoptosis in a dose-dependent manner for copper. The increased BAX expression and decreased BCL2 expression in HepG2 cells treated with ATP7B siRNA correlates with reduced cell viability and apoptosis.Our study indicates that ATP7B silencing in HepG2 cells provides evidence that the deficiency of the ATP7B gene is involved in the initiation of the lipid metabolism and cell cycle pathway, although the precise mechanism is unknown. SREBP1 is a fundamental regulator of fatty acid desaturation, elongation and phospholipid biosynthesis. Our results of decreased expression of SREBP1 mRNA and protein by the ATP7B inhibition is consistent with the findings in a recent publication of 45 WD patients with the lower levels of total cholesterol and LDL cholesterol compared with the control group Atp7b knockout mice Our discovery of the reduced expression of SREBP1 and MCM7 mRNA and protein by Atp7b knockout mouse in cDNA microarray studies ATP7B deficiency cells is consistent with previous observation of overexpression of MCM7 in cancers MCM7 is involved in replicative licensing and synthesis of DNA ATP7B silencing on hepatocytes may be attributed to its role in promoting reactive oxygen species generation via Cu2+ accumulation induced oxidation of biomolecules such as lipids, proteins, and nucleic acids The detrimental effects of ATP7B deficiency-induced imbalance in cell cycle and lipid metabolism pathway. The results add data to the spectrum of the mutations in the ATP7B gene in Chinese Han population.In summary, 4 novel mutations were identified in our study and apoptosis may result from Table S1List of real-time RT-PCR primers.(DOC)Click here for additional data file.Table S2Polymorphisms detected in Chinese Wilson disease chromosomes.(DOC)Click here for additional data file.Table S3Mutations detected in Chinese Wilson disease chromosomes.(DOC)Click here for additional data file.Table S4GenBank accession numbers.(DOC)Click here for additional data file.


The detailed mechanism of HIV-1 latency remains unclear. We investigated histones and their post-translational modification associated with HIV-1 latency in novel HIV-1 latently infected cell lines established previously, NCHA cells.The persistence of latently Human immunodeficiency virus-1 (HIV-1) infected cellular reservoirs in resting CD49 (H3K9) and Lys27 (H3K27) in HIV-1 latently infected cells were analyzed by western blot and chromatin immunoprecipitation-sequencing (ChIP-seq).To examine histones and their modification linked with HIV-1 latency, the expression profiles for core histone proteins and histone deacetylases (HDACs) in NCHA cells were characterized by RT-PCR, ELISA, and western blot. The levels of histone acetylation and methylation at histone H3 LysThe expression levels for four core histone proteins  and HDACs (HDAC1-8) in NCHA cells were not significantly different from those in their parental cells. Histone H3K9 and H3K27 acetylations in NCHA cells showed no difference in parental and NCHA cells, whereas the levels of di- and tri-methylation were increased in NCHA cells. The expression of EED which is a component of polycomb repressive complex 2 (PRC2), and BMI1 and RING2 which are constituents of PRC1, were upregulated in NCHA cells. In addition, more ubiquitylation at histone H2A was detected in NCHA cells.Our results suggest that tri-methylation of histone H3K27 and H2A ubiquitylation via polycomb group protein may play a crucial role in epigenetic silencing accounting for HIV-1 latency in NCHA cells. HIV-1 can evade the immune responses of host cells and establish latent infection among HIV-infected patients, which leads to AIDS despite treatment referred to a highly active antiretroviral therapy (HAART). Latently infected memory T cells, which are generally established within days of the initial infection, are very rare (~1 million) in a patient and have a long half-life of over 44 months on average. The complete elimination of HIV reservoirs seems to take over 60 years under the HAART regimen [276 enhances the recruitment of coactivator p300/HAT, leading to increased transcriptional activation in NF-κB dependent genes by the acetylation of histones [In some HIV-1 latency studies, phosphorylation of NF-κB p65 activates the long terminal repeat (LTR) of HIV-1 provirus in latent cells directly, which in turn drives HIV-1 transcription. Phosphorylation of p65 at serhistones . Even if4 tri-methylation (H3K4me3) is mostly enriched at active gene promoters, whereas histone H3K9 and H3K27 di-/tri-methylation (H3K9me2/me3 and H3K27me2/me3) are highly enriched at inactive loci in mammalian cells [The eukaryotic genome is organized into the highly complex nucleoprotein structure of chromatin. Epigenetic studies show that this chromatin structure can be modified by DNA methylation, covalent histone modifications, and nucleosome remodeling. In particular, histone proteins consisting of the nucleosome core are subject to modifications such as methylation, acetylation, ubiquitylation, sumoylation, and phosphorylation on specific residues . Posttraan cells .Recent cancer studies show that broad alteration of gene activation or silencing by epigenetic alterations can lead to cancer development and carcinogenesis . ElevateReference cell lines, A3.01 cells and ACH2 cells, were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program. Latently infected A3.01-derived cell lines, NCHA1 and NCHA2, were established from our laboratory . Cells wTRIZol reagent  was used for isolation of RNA according to the manufacturer's protocol. Extracted RNA was stored at -20°C prior to use for RT-PCR. Total RNA (2ug) collected from each cell line was reverse transcribed to cDNA with Superscript II Reverse Transcriptase (Invitrogen) and an oligo(dT) primer.Nuclear extracts were prepared with Nuclear Extraction Kit  and protein concentration of these nuclear extracts was measured with Protein DC assay Kit  according to the manufacturer's protocol. Nuclear extracts were applied to ELISA assay immediately and the rest were stored at -80°C prior to use.Acetylation levels of four core histones were measured with Pathscan Acetyl Histone H2A, H2B, H3 and H4 Sandwich ELISA Kit  according to the manufacturer's instruction.To identify HDAC proteins, total cellular protein extracts were prepared in ice-cold RIPA buffer  supplemented with fresh Complete™ protease inhibitor cocktail . Protein extracts were separated by SDS-PAGE, transferred to an Immobilon™-P membrane , and probed with specific antibodies . GAPDH  and Lamin B  were used as protein loading control. After incubation with the HRP-conjugated secondary antibody, proteins were visualized using West Pico chemiluminescence substrate  according to the manufacture's protocol.Immunoprecipitation was performed using IP Kit  with mouse anti-BMI1 . The interaction of RING2 with BMI1 was examined with western blot using rabbit anti-RING2 .Chromatin from uninfected and latently infected CD4+ T cells was digested with micrococcal nuclease into nucleosome-size unit. ChIP was performed using antibodies to histone H3K4me3 , H3K9me3 , H3K27me3 , and H3K9ac . Following treatment of proteinase K, the DNA fragments were purified by phenol/chloroform extraction and ethanol precipitation.The DNA sequencing library preparation and sequencing on the Genome Analyzer II were performed according to the manufacturer's instruction . Briefly, the ChIPed DNA was polished by blunting and 'A'-base addition followed by adapter ligation. After sequencing, the GA pipeline 1.4 software provided by Illumina was used for image analysis, base calling, and sequence alignment. The uniquely aligned sequence tags to the human genome (hg18/NCBI build 36) were used in further analysis. The sequence tags were counted in promoters (transcription start site ± 1kb), genebodies (from transcription start site + 1kb to transcription end site), and intergenic regions. To analyze the enrichment patterns of histone modifications on HIV-1 provirus, we aligned the sequencing tags to the HIV-1 genome and counted tags in a non-0verlapping 200bp window.The expression and acetylation levels of four core histones  in NCHA cells did not exhibit significant differences as shown in Figrue 1A and 1B, respectively. The mRNA levels for HDACs in Class I  were measured by RT-PCR analysis shown in Figure As shown in Figure To understand HIV-1 latency related with gene silencing by polycomb group protein complex, we examined the expression levels of polycomb group proteins in NCHA cells. Firstly, the expression level of EED, which is part of PRC2 that recognizes histone H3K27 and/or H3K9 residues for histone methylation, was investigated by western blot using an antibody specific to EED. As shown in Figure Current antiretroviral therapies, including HAART, do not completely eradicate latent reservoirs of HIV-1 from patients, even though they suppress new HIV-1 replication effectively. The best way to eliminate this reservoir completely is to reactivate HIV-1 latently infected cells by inducing the expression of HIV-1 provirus. Many studies have reported potential antireservoir therapies to reactivate HIV-1 latently infected cells using HDAC inhibitors -17, inteInterestingly, H3K9me2/me3 and H3K27me2/me3 were highly enriched in HIV-latent cells. To determine whether the enrichment of H3K9me3 and H3K27me3 in NCHA cells was specific to certain genomic regions, the distribution of histone methylations was examined in a genome-wide level. H3K4me3 and H3K9ac showed high enrichment in promoter regions, whereas H3K9me3 and H3K27me3 were spread out over gene bodies and intergenic regions. However, these patterns were not significantly changed in ACH2 and NCHA cells compared with those of A3.01 cells. To identify the chromatin states of HIV-1 proviral genome, we analyzed the histone methylation patterns at H3K9 and H3K27. High enrichment of H3K9me3 was observed in HIV-1 infected latent cells, whereas there was no enrichment H3K27me3. Our results show the possibility that the HIV-1 genome silencing may be mainly caused by H3K9me3-mediated process and the host cell genomes, especially in NCHA cells, can be regulated by both H3K9me3 and H3K27me3. These gene repression through histone methylations were consistent with the previous report showing that histone H3K27 trimethylation correlated with gene silencing in human T cells .Drosophila melanogaster, are required for the maintenance of HOX gene cluster repression during development. They induce transcriptional gene silencing in higher eukaryote and act as a global chromatin regulator [Polycomb group (PcG)-proteins mediated gene silencing is mainly established by histone H3K27 methylation. PcG proteins, which were first identified in egulator -28. PcG egulator ,30. Receegulator . Accordiegulator . To elucNewly established NCHA cells harboring latent HIV-1 showed the enrichment of histone H3K27 methylation, histone H2A ubiquitylation, and PcG proteins expression. Therefore, our result demonstrates that tri-methylation of H3K27 and H2A ubiquitylation via polycomb repressive complexes should be involved in HIV-1 latency and contribute to epigenetic gene silencing.The authors declare that they have no competing interests.BSC and HGK conceived the project and designed experiments. HGK and KCK carried out research, wrote the manuscript and prepared the figures. KMJ, TYR and JP carried out CHIP and CHIP-seq. JSL, SYC and SSK helped to draft the manuscript. All authors approved the final manuscript.


Human embryonic stem cells (hESCs) are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV) single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication. It is becoming increasingly appreciated that human embryonic stem cells (hESCs) have an altered DNA damage response compared to multipotent and differentiated cells: i) hESCs display high rates of spontaneous apoptosis and induce rapid apoptosis in response to, generally, sub-lethal forms of DNA stress (1), ii) apoptotic induction in hESCs is often elicited via a p53-transcription independent mitochondrial pathway Parvoviridae members B19, minute virus of mice and adeno-associated virus (AAV) in manners both dependent and independent of viral gene expression Parvoviridae genus Dependovirus. The protein capsid is packaged with a 4.7 kb single-strand DNA genome flanked at both ends by 145 nucleotide (nt) inverted terminal repeats (ITRs) that are necessary for the initiation of replication and packaging, among other processes  of 100,000 . Of the analyzed serotypes, AAV3B demonstrated the highest transduction at 46% GFP+ cells after 24 h  CMV-ter 24 h . AAV2, Ater 24 h .egfp transgene among the most efficient serotypes directly correlated with viral gene copy number/cell, total DNA was extracted (including that from intact intracellular AAV particles) and quantitated by PCR (Q-PCR). The results were normalized to the copy number of the human lamin B2 gene and are presented as viral genomes/cell. In general, the copy number of the egfp transgene directly correlated with the percentage of GFP+ cells determined by flow cytometry  were observed as early as 8 h after rAAV infection. At 24 h post-infection, hESC viability was assessed by microscopy and quantitated by dye exclusion . At 24 h post-infection, the majority of the differentiated cells demonstrated a GFP+ phenotype with negligible toxicity consistent with a previous report  was analyzed by Western blot 8 h post-infection. The results demonstrate a dramatic increase in the phosphorylation of p53ser-15 for rAAV transduced cells while no phosphorylation at that site was observed for untreated cells , a phenomenon reported for both differentiated cells and hESCs ed cells . Another for 8 h . Collectegfp  and harvested for determination of cell viability 24 h post-infection. At this time point there was a 2-fold decrease in cell viability for rAAV treated hESC/p53- cells compared to a 10-fold decrease for wild type hESCs when compared to their respective untreated control , Btg2, Bcl-2, Bax, and Puma. The p21 and Btg2 transcripts both increased >10-fold 8 h post-infection . The cel+ cells with 4N DNA content were considered mitotic. Immortalized human diploid fibroblasts, which tolerate rAAV transduction, were initially investigated after vector treatment and displayed an increased fraction of cells in G2 phase, consistent with previous reports  and the AAV capsid were not involved in hESC apoptosis  induces Our investigation using AAV vectors for the genetic modification of hESCs encountered an unexpected phenomenon: rAAV transduction directly correlated with hESC apoptosis. This phenotype was demonstrated to be independent of the viral capsid , 7, but In contrast to reports of AAV-induced toxicity in p53 deficient cancer cells Within the generalized term of pluripotency there appears to be various stages which can be delineated based on their response to DNA damaging agents, the significance of which is under current investigation. This notion is supported by hESCs of different origins which display distinct properties, such as levels of the Oct4 transcript and replication indices, which directly correlate to the induction of rapid apoptosis in response to rAAV transduction or etoposide . A similFurther characterization of the AAV induced DNA damage response in human pluripotent, cancer and differentiated cell types is currently underway and has strong implications for understanding the basis of oncogenesis and differentiation, as well as for the optimization of AAV as a DNA delivery vector in stem cells.2 atmosphere in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum and penicillin–streptomycin (100 U/ml). These experiments employ the commonly used WiCell H9 cells (hESCs unless otherwise indicated) from the National Stem Cell Bank, initially obtained from excess human embryos following in vitro fertilization. Cells were cultured in conditioned hESC complete medium and grown on fibronectin coated plates. The experiments herein were performed 2 days after passage at a time when hESC cell colonies are isolated and are predominantly monolayers. At this time point, Oct4 is constitutively expressed and localized within the nucleus. The hESC line H9 , was cultured on feeder-free fibronectin coated plates and fed with mouse embryonic fibroblasts (mEF) conditioned human ES medium. mEFs were mitomycin-c inactivated and plated in fibroblast medium [Dulbeccos modified eagles medium (Invitrogen)], 10% fetal bovine serum (Invitrogen), 2 mM L-glutamate (Invitrogen), and 1% Penicillin/Streptomycin (Invitrogen). 24 h after attachment, the medium was changed to human ES complete medium (77% DMEM:F12 (Sigma), 20% Knockout SR (Invitrogen), 1% Non-Essential amino acids (Invitrogen), 1% Penicillin/Streptomycin (Invitrogen), 1 mM L-Glutamine (Invitrogen), 0.1 mM beta-mercaptoethanol (Sigma), 4 ng/ml basic Fibroblast Growth Factor (Invitrogen). After 24 hours, the medium was removed, filtered and used as conditioned medium for human ES cultures. Cells were cultured in 5% CO2 at 37°C and manually passaged every 5–6 days to maintain undifferentiated cultures. H9 hESCs were differentiated towards a neural lineage using procedure SOP-CH-207 Rev A from the national stem cell bank.Human embryonic kidney cells  for rAAV production, and normal human fibroblasts (NHF) were maintained at 37°C in a 5% COrep2 of AAV and individually the capsid genes of serotypes 1–9 (ex. pXR1 is prep2 cap1). The phpaTRsk+ plasmid egfp gene expressed from the CMV promoter. Following AAV production and cesium chloride gradient separation egfp primers: forward primer: 5′-AGC AGC ACG ACT TCT TCA AGT CC-3′ and reverse 5′-TGT AGT TGT ACT CCA GCT TGT GCC-3′.A previously described triple transfection method was used to generate the vectors used herein Production of empty and full capsids followed the rAAV production scheme described above, except that an ITR containing plasmid was not used. For these experiments AAV2 empty capsids were produced and tittered by Western dot blotting as described egfp (above) or the human lamin B gene: Forward 5′-GTT AAC AGT CAG GCG CAT GGG CC-3′ and reverse 5′-CCA TCA GGG TCA CCT CTG GTT CC-3′.At 24 h post-infection cells were harvested and total DNA was purified using a Qiagen DNEasy preparation kit. This DNA then served as the template for a Q-PCR reaction using either primers for amplification of The indicated amount of rAAV particles were added in an equal volume of PBS to hESCs grown in a 24 well plate. At the indicated time points, cells were analyzed by microscopy and flow cytometry.GFP+ cells were visualized by fluorescent microscopy and quantitated by flow cytometry. For flow cytometry, a minimum of 10,000 similar sized/shaped single cells that were 7-AAD negative were counted, at an event per second rate of 1,500–2,000 for each replicate at the indicated times. The GFP+ gate was designed such that untreated cells did not give a single GFP+ event beyond a million counted cells. It was also positioned away from false, or transitioning, GFP+ cells to obtain no false positives. Visualization of pooled GFP+ cells after FACs sorting confirmed the GFP+ phenotype (Flow Cytometry Core at UNC-Chapel Hill). These experiments had a N = 3 and were performed in 8 independent experiments. The standard deviation is depicted in the figure.Sample preparation for Western analysis followed standard techniques To quantitate the transcript level of selected genes hydrolysis probes-primer set was used. The probes are provided by Roche Universal Probe Library Set, Mouse (UPL # 04 683 641 001), and Roche assay design center program was used to find ideal pair of primer and probe for each transcript (available upon request). The RT-qPCR was done on LightCycler® 480 Instrument in 10 ul of reaction volume in a 96 well plate. In each reaction 2 ul (50 ng) of cDNA, 5 ul of the probe master mix (2×), 0.5 ul of 10 uM each forward and reverse primers, 0.5 ul of 10 uM of specific probe and 2.5 ul of RNAase/DNAase free water was used. No template control was included in each run to rule out the possibility of contamination for each primer-probe set. The reaction has a hot start step at 95°C for 10 min for 1 cycle and 45 cycles of denaturation at 95°C for 10 seconds and annealing and extension at 60°C for 30 seconds. The baseline and the cp (crossing points) values were already calculated by the LC software 1.5 (Roche applied biosystem). The results were imported to the MS excel to perform relative quantitation analysis on each transcript using the delta-delta CT method. The experiments contained a N = 3 and standard deviation is depicted in the corresponding Figures.2 dishes and were microinjected on day three of culture using a Narishige micromanipulator mounted on a Leica inverted fluorescent microscope using needles pulled on a Narishige PC-10 micropipette puller. Approximately 50 hESCs were injected for each experimental condition in 2 independent experiments (note that the A and A' injections were performed only on one occasion). The microinjection buffer contained 100 mM KCl and 10 mM KPi, pH 7.4. DNA microinjections contained 100 ng/ul of a DNA oligonucleotide containing AAV ITR sequence (5′-GGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCA-3′) or a control a DNA oligonucleotide with no ITR homology (5′-GTTAGTTCACTGGGTTTATCCATATGCCAAATTGAGGGACCCAAATGTTATTTCAACTATCAATGTTATGAGCTTAGCCG-3′) as well as A (5′- GGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGC-3′) and A' (GCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCC). To block caspase activation, cells were pre-treated for 30 min with 50 uM zVAD-FMK (Promega), a pan-caspase inhibitor. The cells were co-injected with 5 mg/ml rhodamine dextran for visualization. Following injections, viable cells were identified as rhodamine positive and intact using phase bright microscopy. For investigation of oligonucleotide intra- and inter-molecular interactions, the A and A' 39-mers were denatured and then allowed to anneal as previously described Human embryonic stem cells (H9) were plated onto 35 mmgfp gene fragment 3) containing 0.5% tween 20. Cells were then re-suspended in the primary antibody staining solution  with 2 ug/ml anti-H2AX phospho-Ser 139 (Millipore) FITC conjugated antibody. Following O/N incubation at 4°C, cells were washed again in IFA-0.5%-Tween 20 and resuspended in the flow analysis solution . Single cells with 2-4N DNA content were gated on for quantitation of γ-H2AX-FITC labeling using a N = 3 in 3 independent experiments. The standard deviation is presented in the To quantitate the levels of γ-H2AX (Ser 139), hESCs were treated with single-strand AAV2 particles harboring a partial egfp. 8 h post-infection cells were harvested and stained with an annexin V – Alexafluor 647 conjugate according to the manufacturer's instructions (Invitrogen). The degree of labeling was determined by flow cytometry using a N = 3 on 2 different occasions. The standard deviation is depicted in the figure.For annexin V staining, hESCs were pre-treated with a caspase inhibitor prior to infection with 10,000 particles of scAAV3B-CMV-Dividing hESCs were treated with 200 ug/mL nocodazole and 10 uM EdU was added to each sample 2 h before harvest. Samples were harvested 0, 2, 4, or 6 h after nocodazole addition with TrypLE  and cells were pelleted by centrifugation. All samples were fixed in 95% ethanol/5% acetic acid, and stained with a Cy5-labeled MPM-2 primary antibody  to identify mitotic cells Figure S1Chromosome condensation in rAAV treated hESCs. Chromosome condensation was evaluated by DAPI staining to determine whether the hESCs were undergoing apoptosis in response to AAV infection. Eight hours after scAAV3B infection, hESCs treated with 100,000 scAAV3B-egfp particles demonstrated a dramatic increase in punctate nuclear staining compared to cells that were not treated.(EPS)Click here for additional data file.Figure S2Recombinant AAV Transduction of hESC WiCell H1s.A) WiCell H1s were transduced with 100,000 particles of scAAV3B-CMV-egfp. The cells were harvested for GFP analysis by flow cytometry 24 h after virus addition. B) Cell viability was measured by dye exclusion at the indicated timepoints following transduction of H1 cells by 100,000 scAAV3B-CMV-egfp particles. C) Total RNA was harvested from H9 or H1 hESCs and used as a template for reverse transcription. cDNA was investigated by Q-PCR to determine the amount of Oct4 transcript in each line normalized to the housekeeping transcript GAPDH. D) Rates of mitotic entry for H1 and H9 hESCs. Dual color flow cytometry using a mitosis-specific antibody to phospho-MPM2 epitope (Cy5) and DAPI to analyze DNA content was employed to measure mitotic entry rates. Samples were subjected to nococdazole treatment for 2, 4, or 6 hours. Mitotic entry rates were calculated as linear regression slopes generated from scatter plots of the percentage of MPM2+ cells over time. .(EPS)Click here for additional data file.Figure S3Recombinant AAV Transduction of H9 cells Differentiated Towards a Neural Lineage. A) hESCs were grown to embryoid bodies and differentiated towards a neuronal lineage for 15 days. Then, 100,000 scAAV3b-CMV-egfp particles were used for transduction and viability was determined 24 h later by dye exclusion. B) Immuno-fluorescence of cells following scAAV3B-CMV-egfp transduction using an anti-vimentin antibody and the native eGFP fluorescence.(TIF)Click here for additional data file.Figure S4Characterization and Transduction of p53 deficient hESCs. (A) Equal amounts of protein from wild type H9 hESCs and those knocked down for p53 transcript [described in (B)] were analyzed for total p53 abundance by Western blotting. Alpha-tubulin served as additional loading controls. (B) Wild type or p53 knockdown hESCs were analyzed for the Oct4 transcript by reverse transcriptase followed by quantitative PCR using cDNA template. The values were normalized to levels of the housekeeping transcript GAPDH. Results are presented as transcript induction which is the value determined for AAV infected cells divided by the value determined for the no treatment group . (C) H9 hESCs deficient for p53 were transduced with transduced by rAAV3B-gfp  and images of colony integrity and GFP fluorescence are provided at the time points following transfection.(EPS)Click here for additional data file.Figure S5Densitometry Analysis of Western Blots. The indicated Western blotting experiments were analyzed for a relative abundance using a storm scanner and Image Quant 5.2. Internal loading controls were used for normalization. .(EPS)Click here for additional data file.Figure S6Transcript Abundance of Apoptotic Effectors. H9 hESCs were treated with rAAV3B-CMV-egfp  or equal volume of PBS. RNA from treated cells was harvested, converted to cDNA and copy number of the indicated transcripts was determined by Q-PCR. Lamin B2 transcript abundance was used for normalization to cell number and the data is presented as the normalized transcript abundance of the rAAV treated cells divided by the vehicle control. In all cases the transcript abundance change in cells treated with AAV was not significantly different than the non-treated (NT) controls .(EPS)Click here for additional data file.Figure S7Quantitation of the DNA Microinjection Experiment. For each of the indicated injection regimens approximately 100 hESCs were injected with the indicated DNA oligonucleotide (oligo) as described in the results and methods. Two hours post-injection rhodamine positive cells were tallied and are presented as a percentage of the total injected. .(EPS)Click here for additional data file.


The familial form of PD, PARK2, is caused by mutations in the Here, we generated induced pluripotent stem cells (iPSCs) from two PARK2 patients. PARK2 iPSC-derived neurons showed increased oxidative stress and enhanced activity of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. iPSC-derived neurons, but not fibroblasts or iPSCs, exhibited abnormal mitochondrial morphology and impaired mitochondrial homeostasis. Although PARK2 patients rarely exhibit Lewy body (LB) formation with an accumulation of α-synuclein, α-synuclein accumulation was observed in the postmortem brain of one of the donor patients. This accumulation was also seen in the iPSC-derived neurons in the same patient.Thus, pathogenic changes in the brain of a PARK2 patient were recapitulated using iPSC technology. These novel findings reveal mechanistic insights into the onset of PARK2 and identify novel targets for drug screening and potential modified therapies for PD. Parkin is a causative gene of autosomal recessive juvenile Parkinson’s disease (PARK2). It encodes a component of an E3 ubiquitin ligase involved in mitochondrial homeostasis. Expression levels were normalized to that of KhES1 (set as 1). (D) Cont A (YA9), Cont B (WD39), PA , and PB  iPSCs gave rise to teratomas with all three germ layers, confirming pluripotency. Scale bar, 100 μm. (E) Silencing of transgenes in control and PARK2 iPSC clones. Expression levels were normalized to the positive control of fibroblasts in cultures assayed 6 days after retroviral infection ( = 100). Cont A, Control A; Cont B, Control B.Click here for fileConfirmation ofparkindeletions and genomic stability of PARK2 iPSCs using comparative genomic hybridization (CGH) microarray analysis. (A) Exons 2–4 were deleted in the PA9 and PA22 iPSC lines. Exons 6 and 7 were deleted in the PB2, 18, and 20 iPSC lines. (B) Copy number profiles of whole chromosomes in PARK2 iPSCs assessed by CGH microarray analysis revealed that no genomic aberrations were introduced during the process of establishing PARK2 iPSCs.Click here for fileExpression level of MAO-A and -B showed no difference among Control and PARK2 iPSC-derived neurons.  qRT-PCR measurement of MAO-A and -B transcripts in PARK2  and PB ) iPSC-derived neurons showed no difference compared to those in Cont A (B7 and YA9). ContA; Control A, ContB; Control B.Click here for fileHealthy mitochondria in PARK2 fibroblasts and iPSCs.  Electron micrographs of fibroblasts (upper panels) and iPSCs (lower panels) from Control (Cont A and Cont B) and PARK2 patients (PA and PB). Mitochondria in the fibroblasts and iPSCs from both groups showed long, cylindrical profiles with well-organized cristae, and the electron density of the matrix was relatively low (asterisks). Scale bar, 0.25 μm. Cont A, Control A; Cont B, Control B. (C) Fibroblasts were treated with 30μM CCCP or DMSO for 48 h, followed by staining for CIII coreI (magenta) to label the internal mitochondrial membrane (IMM) and counterstaining with Hoechst . Mitochondrial size decreased after CCCP treatment in both Control (Cont A and Cont B) and PARK2 (PA and PB) fibroblasts. Scale bar, 20 μm. (D) iPSCs were treated with 30 μM CCCP or DMSO for 48 h and then stained for CIII coreI (magenta) to label IMM, Oct4 (blue) to label iPSCs, and Hoechst . Mitochondrial size in Control (Cont A (B7), Cont B (WD39)), and PARK2 (PA9 and PB2) iPSCs decreased after CCCP treatment. Scale bar, 20 μm (E) CCCP/DMSO ratios in Control , Cont B (WD39)), and PARK2 (PA9 and 22 and PB2 and 20) iPSCs (Mann Whitney U-test). Data represent the mean and SEM (n > 3 for each group).Click here for fileMitochondrial membrane potential after CCCP treatment in control and PARK2 iPSC-derived neurons. (A) iPSC-derived neurons were treated with 30 μM CCCP or DMSO for 48 h, after which they were stained for the mitochondrial membrane potential marker, TMRE. The intensity of TMRE (yellow) was clearly reduced in control (Cont A (B7), Cont B (WD39)), and PARK2 (PA9 and PB2) iPSC-derived neurons. Scale bar, 50 μm.Click here for fileConfirmation ofparkindeletions carried by the father of patient PB. (A) Deletion of exons 6 and 7 was confirmed in blood samples from PB and the father of PB by PCR.Click here for fileα-Synuclein signals are not seen in PARK2 iPSCs. (A) Quantitative genomic PCR analysis for SNCA exons 1 and 4 demonstrated a normal copy number in PARK2  iPSCs. The copy number was the same as that observed for Cont A (B7 and YA9) and Cont B (WD39). The SNCA gene copy number was normalized to β-globin (HBB) and β2-microglobulin (B2MG). (B) iPSCs were stained for α-synuclein (red), Oct4  and Hoechst (blue). No α-synuclein signals were observed in Cont A (B7 and YA9), Cont B (WD39), or PARK2  iPSCs. Scale bar, 50 μm.Click here for file


However, this mechanism remains unclear largely due to the lack of an animal model that can support persistent HBV replication and allow for the investigation of the relevant immune responses. In this study, we used hydrodynamic injection to introduce HBV replicon DNA into the livers of three different mouse strains: BALB/c, C57BL/6, and FVB/N. Interestingly, we found that an HBV clone persistently replicated in the livers of FVB/N mice for up to 50 weeks but was rapidly cleared from the livers of BALB/c and C57BL/6 mice. Flow cytometric analysis and quantitative reverse transcription PCR analysis of the mouse livers indicated that after DNA injection, FVB/N mice had few intrahepatic activated cytotoxic T lymphocytes (CTLs) and produced low levels of alanine aminotransferase, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and the CXCL9 and CXCL10 chemokines. These findings were in sharp contrast with those observed in BALB/c and C57BL/6 mice, reflecting a strong correlation between the degree of liver inflammation and viral clearance. Mutational analysis further demonstrated that a change of Asn-214 to Ser-214 in the HBV surface antigen rendered the persistent HBV clone clearable in FVB/N mice, which was accompanied by increased levels of activated CTL and upregulated expression of IFN-γ, CXCL9, and CXCL10 in the livers. These results indicate that the heterogeneity of the host factors and viral sequences may influence the immune responses against HBV. An inadequate activation of immune or inflammatory responses can lead to persistent HBV replication  More than 95% of acutely infected adults recover from the infection, whereas most of the neonatally transmitted infections become persistent The genetic background of the host and viral factors are believed to contribute to the different outcomes of HBV infection. Genetic polymorphisms of several host factors have been implicated in the susceptibility to chronic HBV infection, including estrogen receptor In addition to host factors, several viral factors have been reported to affect the innate or adaptive immune responses against HBV infection. The hepatitis e antigen (HBeAg) is a viral immunomodulatory protein that, via deletion or anergy, inhibits the HBV core (HBcAg)/HBeAg cross-reactive T-cell response An easily generated immunocompetent animal model is instrumental to the systematic investigation of host and/or viral factors pertinent to HBV persistence. Although mice cannot be infected by wild-type HBV, mouse hepatocytes can support HBV replication and produce infectious virions when viral DNA is directly delivered into the cells. Therefore, a genetically well-characterized inbred mouse should be an ideal model to study the mechanism of HBV persistence if HBV DNA can be efficiently and appropriately delivered to the mouse liver. We have previously employed hydrodynamic injection to deliver HBV replicon DNA, cloned in a pGEM4Z plasmid vector, into BALB/c, C57BL/6, and FVB/N mice. Surprisingly and interestingly, persistent HBV replication was maintained in FVB/N mice for up to 50 weeks but was rapidly diminished in BALB/c and C57BL/6 mice. Thus, we used these mouse strains to further investigate the host and viral factors pertaining to HBV persistence. In this study, we provide data demonstrating that mouse strains that elicit strong cytotoxic T lymphocyte (CTL) responses and induce strong inflammatory responses, e.g., BALB/c and C57BL/6, can clear HBV rapidly, whereas mice that induce low levels of CTL and weak inflammatory responses, e.g., FVB/N mice, tend to develop a persistent infection. In addition, we show that a single amino acid difference in the HBV surface protein can affect the activation of CTL responses and result in different rates of viral persistence.in vivo. BALB/c (H-2d), C57BL/6 (H-2b), and FVB/N (H-2q) mice were hydrodynamically injected with a pHBV1.3-B6 replicon DNA that contains a 1.3-fold overlength of a genotype B HBV genome  staining were performed on the livers. HBV replicative intermediates and the episomal form of the injected DNA were detected in the livers of BALB/c mice injected with the pHBV1.3-B6 replicon DNA for up to two weeks  and in tTo pursue the mechanisms underlying the different clearance rates of HBeAg in BALB/c, C57BL/6, and FVB/N mice, we examined the severity of liver damage, the presence of activated CTLs, and the status of liver inflammation. Serum ALT levels in the DNA-injected animals were measured in order to determine the severity of liver damage. As shown in + CTLs are thought to play an important role in the clearance of HBV infection; they are also implicated in the pathogenesis of liver diseases +CD8+CD69+ activated CTLs were elevated as early as 2 wpi in the BALB/c mice (∼40%), and they peaked at 3 wpi in the C57BL/6 mice (∼25%). IFN-γ enzyme-linked immunospot (ELISPOT) assays demonstrated that the CTLs isolated from the peak levels displayed significant reactivities to the HBs, HBc, or polymerase peptides . In both the BALB/c and C57BL/6 mouse strains, IFN-γ  and TNF-et al. have shown that the IFN-γ expressed by activated CTLs not only clears HBV non-cytopathically but also induces the expression of chemokines such as CXCL9 and CXCL10, which in turn recruit antigen non-specific mononuclear cells, resulting in liver pathogenesis and viral clearance Previous studies by Guidotti Collectively, these data indicate that the injection of HBV DNA into the liver induces different degrees of T-cell activation and liver inflammation in different mouse strains, and the mice can be ordered by the degree of inflammation as follows: BALB/c mice >C57BL/6 mice >FVB/N mice. The degree of liver inflammation strongly correlated with the animal's ability to clear the HBV DNA.The low levels of IFN-γ and inflammatory cells in the FVB/N mice may suggest that this mouse model has little or no ability to induce CTL activity in response to HBV replication. However, although clone B6 and another HBV isolate (B22) persistently replicated in the FVB/N mouse livers, other HBV isolates from different patients  were cleared within 10–20 weeks . The lowWe next investigated the impact of viral factors on HBV persistence. The persistence rates were analyzed among the six sub-clones  contained in the B6 clone. These sub-clones had <0.5% nucleotide sequence diversity compared to the B6 consensus sequences (data not shown). Interestingly, when injected separately into the FVB/N mice, clone B6.9 demonstrated a lower persistent rate than did the others . Sequencvs. B6.2) can lead to significantly different persistence rates.Therefore, we investigated whether the variation of these two amino acid residues conferred the susceptibility of clone B6.9 to immune responses. The sequence of clone B6.2, one of the highly persistent sub-clones , is the Next, we examined whether the clearance of clone B6.2S was also associated with the induction of inflammation. Although serum ALT levels were not significantly elevated in the FVB/N mice injected with pHBV1.3-B6.2 or -B6.2S , the levq have not been reported. Thus, it is not clear whether the single N214S mutation may generate a potential CTL epitope leading to the clearance of clone B6.2S in the FVB/N mice. To test this possibility, we performed ELISPOT assays using the IHLs isolated from the animals injected with B6.2S. The IHLs were pulsed with two 15-mer peptides that had the wild-type or the N214S mutated residue located in the center of the 10-amino acid overlapped region . The animal study was carried out in strict accordance with the recommendations in the “Guide for the Care and Use of Laboratory Animals” of the Council of Agriculture, Taiwan, and that of the National Institutes of Health, USA. The protocol has been reviewed and approved by the Institutional Animal Care and Use Committee of National Yang-Ming University . All surgery was performed under ketamine and xylazine anesthesia, and all efforts were made to minimize suffering.SapI  per µg of DNA for at least 12 h and self-ligated with T4 DNA ligase. The self-ligated DNAs were used as the templates for the next PCR. Two sets of primer pairs, Primer-A1 and -A2 and Primer-B1 and -B2  via a tri-molecular ligation. The resulting pGEM4Z/HBV1.3 replicons contain a 1.3-fold overlength HBV genome spanning from nucleotide (nt) 971 to nt 3215/1 and nt 1 to nt 1984  following the manufacturer's instructions and was then amplified by polymerase chain reaction (PCR) using the primer pair P1–P2  and -B2 , were us nt 1984 . To reprSite-directed mutagenesis was performed to generate the pHBV1.3-B6.2 mutant clones using the previously described method BALB/c, C57BL/6, and FVB mice  obtained from the National Laboratory Animal Center, Taiwan were housed in a specific pathogen-free room. Ten micrograms of the HBV plasmid DNA in a volume of PBS equivalent to 8% of the mouse body weight was introduced by tail vein injection in 6–8 s. Serum samples were collected at the time points indicated after DNA injection and assayed for HBeAg and ALT.Serum levels of HBeAg in the injected mice were determined using the AXSYM system kit . Serum ALT activity was measured by an automated clinical chemistry analyzer TBA-200FR  using the ALT/GPT reagent .HindIII and separated on a 1% agarose gel. The gel was blotted onto a positively charged nylon membrane , which was then hybridized with a digoxigenin-labeled probe encompassing the HBx coding region (nt 1372–1833), and the signals were detected by exposure to X-ray films.Twenty micrograms of liver genomic DNA were digested with Liver tissues were collected from the mice at the indicated time points, snap-frozen, and sectioned into 5-µm cryosections. Liver sections were stained with rabbit anti-HBcAg  or rabbit anti-HBsAg (ad+ay)  antibody and then developed using the Envision System-HRP, DAB (DAKO) following the manufacturer's instructions. The liver sections were counterstained with hematoxylin.g for 5 min at 4°C. The supernatant was collected and centrifuged at 300×g for 10 min at 4°C. The cell pellet was resuspended with ice-cold serum-free RPMI-1640 to a final volume of 4 ml, and 4 ml of 80% (v/v) Percoll  in RPMI-1640 was added and mixed well. The mixture was layered on the top of 2 ml of 60% (v/v) Percoll in HBSS and centrifuged at 1,200×g for 20 min at room temperature (RT). The IHLs were harvested from the interface, washed, and used for flow cytometric analysis.IHLs were isolated following the method described previously IHLs were stained with the following monoclonal antibodies: peridinin chlorophyll protein (PerCP)-conjugated anti-mouse CD3e, fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD8a, and R-Phycoerythrin (R-PE)-conjugated anti-mouse CD69 . Flow cytometry data were acquired using a FACSCanto apparatus with FACSDiva software (BD Biosciences) and were analyzed using FlowJo software .Liver RNA was extracted using TRIzol® reagent , and 1 µg of RNA was subjected to reverse transcription. One twentieth of the cDNA product was then incubated with specific primers  using Fat-test was used . A P-value<0.05 was considered statistically significant.The HBeAg clearance rates in different groups were compared using the logrank test. For statistical analysis of two variables, two-tailed Student's Figure S1The pGEM4Z/HBV1.3 replicon plasmid. The pGEM4Z/HBV1.3 vector contains the HBV fragment spanning from nt 971 to nt 3,215 and from nt 1 to nt 1,984, which contains a 1.3-fold overlength HBV genome. The restriction enzyme sites used for construction of pGEM4Z/HBV1.3 are shown.(TIF)Click here for additional data file.Figure S2Flow cytometric analysis of the levels of activated CTLs in the livers of different mouse strains after DNA injection. Mice were injected with pHBV1.3-B6 DNA (10 µg/mouse), and three animals were sacrificed at the indicated time points after DNA injection. Intrahepatic lymphocytes were isolated and stained for CD3 (PerCP), CD8 (FITC), and CD69 (PE). Representative histograms show the CD69 expression by CD3+CD8+ cells within a forward and side scatter gate appropriate for lymphocytes (solid lines). Lymphocytes were stained for CD3 (PerCP) and CD8 (FITC) as fluorescence-minus-one controls (shaded plots).(TIF)Click here for additional data file.Figure S3HBV specific CTL responses detected by IFN-γ ELISPOT assay. The results of IFN-γ ELISPOT assays for BALB/c (A) or C57BL/6 (B) mice are shown. Mice were injected with pGEM4Z vector or pHBV1.3-B6 DNA (10 µg/mouse), and three animals were sacrificed at 2 wpi (BALB/c mice) or 3 wpi (C57BL/6 mice), respectively. The isolated IHLs and splenocytes of BALB/c mice were stimulated with a peptide pool containing 5 µg/ml of each of the following: P140 (Pol140–148), C131 (HBcAg131–139), and S28 (HBsAg28–39), whereas those of C57BL/6 mice were stimulated with 5 µg/ml of S190 . After 18–20 h of peptide stimulation, the frequencies of IFN-γ-secreting cells were determined and measured as the number of spot-forming cells (SFC) per 5×105 cells. Asterisks mean significant difference between the HBV DNA- and the vector-injected animals. Results are shown as the mean ± SD. **P<0.01; *** P<0.001.(TIF)Click here for additional data file.Figure S4The replication and gene expression efficiencies of different HBV isolates. One microgram of pHBV1.3 DNA was transfected into 7×105 HuH-7 cells. Sixteen hours later, the transfected cells were subcultured for RNA, DNA, protein, and luciferase activity analyses. (A) Northern blot analysis shows HBV gene expression efficiency. Two days after HBV DNA transfection, total RNA was extracted and analyzed by Northern blot with an HBV-specific probe and a GAPDH probe. The sizes corresponding to the HBV transcripts are indicated to the right. (B) Southern blot analysis shows viral DNA replication efficiency. Total genomic DNA was harvested three days after HBV DNA transfection, digested with HindIII, and then analyzed by Southern blot with an HBV-specific probe. The HBV replicative intermediates are indicated to the right. (C) Western blot analysis shows viral antigens expression. Total proteins were extracted four days after HBV DNA transfecton and subjected to western blot analysis. The HBV core protein, S proteins, and α-tubulin are indicated to the right.  ELISA determines the HBsAg and HBeAg levels, respectively. The supernatants were collected on the 3rd day after HBV DNA transfection, and subjected to ELISA analysis. (F) Renilla luciferase activity assay shows the transfection efficiency. Protein lysates were collected on the 4th day after HBV DNA transfection and Renilla luciferase activities were analyzed.(TIF)Click here for additional data file.Figure S5No peptide-specific T cell response in FVB mice. (A) The sequences of the synthetic 15-mer peptides. The sequences covering the Asn-214 region are shown at the top of the figure. The four peptides covering the mutated site are depicted by the lines, namely, LS205-N (L-HBsAg205–219-N), LS210-N (L-HBsAg210–224-N), LS205-S (L-HBsAg205–219-S), and LS210-S (L-HBsAg210–224-N), where N and S represent Asn or Ser at the 214th residue, respectively. (B) No T cell response specific to the mutated region was observed in the FVB mice injected with HBV DNA. FVB mice were hydrodynamically injected with pGEM4Z vector or pHBV1.3-B6 DNA (10 µg/mouse), and three animals were sacrificed at 4 wpi. The IHLs were isolated and stimulated with 10 µg/ml of LS205-N, LS210-N, LS205-S, or LS210-S. After 18–20 h of peptide stimulation, the frequencies of IFN-γ-secreting cells were determined and measured as the number of spot-forming cells (SFC) per 5×105 cells. (C) No T cell response specific to the mutated region was observed in the FVB mice intramuscularly immunized with HBsAg-expressing DNA. FVB mice were immunized with control vector or the plasmid DNA encoding HBsAg-B6.2 or HBsAg-B6.2S (100 µg/mouse). The animals were boosted with the same dose of DNA every two weeks for three times. One week after the final boost, HBsAg-specific T-cell response was examined by IFN-γ ELISPOT assays using the aforementioned peptides. The splenocytes isolated from the immunized mice (n = 3) were stimulated with 10 µg/ml of the 15-mer synthetic peptides as described above. After 18–20 h of incubation, the frequencies of IFN-γ-secreting cells were counted. (D) A significant HBsAg-specific T cell response was observed in the C57BL/6 mice. The C57BL/6 mice immunized with a plasmid DNA encoding the HBsAg-B6.2S served as the positive control for DNA immunization. One week after the second immunization, an HBsAg-specific IFN-γ ELISPOT assay was performed on the splenocytes isolated from the immunized mice (n = 3), which were stimulated with 10 µg/ml of HBsAg190–197. After 18–20 h of peptide stimulation, the spots of IFN-γ-secreting cells were counted. The results are shown as the mean ± SD. Asterisks mean significant difference between the peptide stimulated- and medium control- groups. *** P<0.001.(TIF)Click here for additional data file.Figure S6Long-term follow up of serum HBeAg in DBA/1 mice injected with pHBV1.3-B6 DNA. Ten micrograms of pHBV1.3-B6 DNA were injected into DBA/1 mice. (A) Serum HBeAg was monitored regularly for up to 16 weeks. Each line represents one animal. (B) The HBeAg clearance curves in FVB mice and DBA/1 mice are shown. DBA/1 vs. FVB, *** P<0.001, logrank test.(TIF)Click here for additional data file.Table S1The sequences of primers used for construction of HBV replicons, site-directed mutagenesis, and real-time PCR analysis.(DOC)Click here for additional data file.Methods S1IFN-γ ELISPOT assay. DNA immunization.(DOC)Click here for additional data file.


A/J and 129P3/J mouse strains have different susceptibilities to dental fluorosis due to their genetic backgrounds. They also differ with respect to several features of fluoride (F) metabolism and metabolic handling of water. This study was done to determine whether differences in F metabolism could be explained by diversities in the profile of protein expression in kidneys. Weanling, male A/J mice  and 129P3/J mice  were housed in pairs and assigned to three groups given low-F food and drinking water containing 0, 10 or 50 ppm [F] for 7 weeks. Renal proteome profiles were examined using 2D-PAGE and LC-MS/MS. Quantitative intensity analysis detected between A/J and 129P3/J strains 122, 126 and 134 spots differentially expressed in the groups receiving 0, 10 and 50 ppmF, respectively. From these, 25, 30 and 32, respectively, were successfully identified. Most of the proteins were related to metabolic and cellular processes, followed by response to stimuli, development and regulation of cellular processes. In F-treated groups, PDZK-1, a protein involved in the regulation of renal tubular reabsorption capacity was down-modulated in the kidney of 129P3/J mice. A/J and 129P3/J mice exhibited 11 and 3 exclusive proteins, respectively, regardless of F exposure. In conclusion, proteomic analysis was able to identify proteins potentially involved in metabolic handling of F and water that are differentially expressed or even not expressed in the strains evaluated. This can contribute to understanding the molecular mechanisms underlying genetic susceptibility to dental fluorosis, by indicating key-proteins that should be better addressed in future studies. The widespread use of F has contributed to the caries decline, but excessive intake may affect both bone metabolism and enamel development, causing skeletal and dental fluorosis, respectively. There are many sources of F intake, such as drinking water, dental products, dietary supplements and infant formulas To determine whether such differences were due to differences in F metabolism, we conducted a metabolic study in which total F intake and excretion were measured. Our results showed that, compared to A/J mice, 129P3/J mice ingested less water, excreted less urine, had lower urinary F excretion and consequently had higher F retention and plasma and femur F levels Kidneys represent the major route of removal of F from the body ad libitum access to low-F food  and water, to allow analysis of water and food consumption Male mice from the A/J and 129P3/J inbred strains (3-week-old) were randomly distributed into three groups (n = 6/strain) based on the F concentrations in the drinking water. All animals were housed in pairs in metabolic cages with Kidneys were homogenized in deionized water for 2 min using a homogenizer . Kidney F concentrations were determined in duplicate (100 mg of kidney tissue) after overnight hexamethyldisiloxane (HMDS)-facilitated diffusion g for 30 min at 4°C for supernatants collection. The proteins were precipitated by using the kit PlusOne 2D Cleanup , as recommended by the manufacturer. The pellets were resuspended in rehydration buffer . Protein concentration was measured in each sample by Bradford protein assay. After quantification, 1000 µg of kidney proteins from each animal of the same group and strain were combined to constitute a pool Kidney samples were homogenized using mortar and pestle in liquid nitrogen. Denaturation buffer  was added. After 1 h vortexing at 4°C, samples were centrifuged at 25000×Ettan DALTsix   with homemade12.5% acrylamide gels was used for the second dimension separation. Electrophoresis was performed at 15 mA/gel (80V) for 1 h and at 60 mA/gel (500V) until bromophenol blue line had reached the bottom of the gels. The resolved protein spots were stained with Colloidal Coomassie Brilliant Blue G-250 Renal proteins (1000 µg) were taken from each pooled sample and mixed in rehydration buffer to a volume of 400 µL which was then loaded onto 24-cm IPG strips (linear pH 3–10). Rehydration and first-dimensional IEF were performed on IPGphor IEF system at 20°C with the following parameters: 50V for 12 h, 500V for 1 h, 1000V gradient for 1 h, then 10000 V for a total 40,000 V. http://www.matrixscience.com) (version 2007.12.04) based on IPI  protein database restricted to taxonomies Mus musculus (Mouse). The search was limited with a mass tolerance of 100 ppm and only one missed cleavage per peptide was allowed. For modification of peptides, cysteine carbamido-methylation (fixed) and methionine oxidation (variable) were considered. Significant matching protein required score of >60. Accuracy between the theoretical and experimental obtained mass and pI were also considered. When 2 or more proteins with high scores were identified in the same spot, they are excluded from analysis. Identified proteins were classified into 6 different categories according to their primary function Protein spots of interest were excised from the gel and destained three times with 25 mM Ambic/Acetonitrile (50∶50 v/v) for 30 min. The destained gel pieces were dehydrated twice with acetonitrile (ACN) for 10 min and dried in a vacuum concentrator . The dried gel pieces were rehydrated with 20 mM DTT in 50 mM Ambic for 40 min at 56°C. Excess of reagent was removed and 55 mM iodoacetamide (IAA) in Ambic 50 mM was added for 30 min RT at dark. The remaining liquid was removed and washed out with 25 mM Ambic, followed by dehydration with ACN. After its removal, the gels were dried again. For digestion, dried gels were incubated with 10 ng/µL trypsin in 25 mM Ambic for 15 min . Peptides were sequentially extracted from the gels initially in 50% ACN (v/v) with 5% formic acid for 14 h at 37°C, then in 50% ACN (v/v) with 1% formic acid for 15 min, followed by 60% methanol (v/v) with 1% formic acid for 15 min and twice with 100% ACN at 45°C under sonication . Extracts were dried using a vacuum concentrator  and kept at -20°C. Prior to MS identification, dried peptides were dissolved in 12 µL 0.1% formic acid. The peptides were identified and quantified by LC-ESI-Q-TOF MS (Liquid Chromatography Electrospray Ionization Quadrupole Time of Flight Mass Spectrometry) . MassLynx 4.1 SCN662 software  was used to submit the combined MS and MS/MS data to MASCOT database search engine  was used. Data were analysed by 2-way ANOVA and Bonferroni test for individual comparisons (p<0.05).For proteomic data, statistical analysis was performed using ANOVA available at ImageMaster 2D Platinum 7.0 software . Only proteins with significantly altered levels were excised for identification by LC-MS/MS (p<0.05).Mean kidney F (±se) concentrations for A/J mice for control, 10 ppmF and 50 ppmF groups were: 0.126±0.008, 0.174±0.007 and 0.296±0.026 µg/g. The corresponding values for 129P3/J mice were 0.139±0.015, 0.163±0.010 and 0.198±0.046 µg/g, respectively. Two-way ANOVA revealed a significant difference among the treatments , but not between the strains  without significant interaction between these criteria . For both strains, significantly higher kidney F concentrations were found for the 50 ppmF group, when compared with control and 10 ppmF groups that did not significantly differ from each other.vs 129P3/J mice), 10 ppmF groups (A/J vs 129P3/J mice), and 50 ppmF groups (A/J vs 129P3/J mice). For the statistical analysis, comparisons were performed between the strains as follows: Control groups , followed by information pathways (23.6%), cell processes (17.6%), transport (11.8%) and structure (5.9%) . From thRegarding the comparison between the groups treated with 50 ppmF, 18 proteins were significantly up-regulated and 13 down-modulated in kidney of 129P3/J mice when compared with A/J mice. Fourteen of eighteen enhanced proteins are associated with metabolism (77.8%), followed by processes (11.1%), information pathways (5.6%) and processes pathways (5.6%). Among the down-modulated proteins, most are also related to metabolism (46.2%), followed by cell processes (23.0%), transport (15.4%), information pathways (7.7%) and structure (7.7%) . Among tAmong the 8 proteins differentially expressed between the mice strains, regardless of the treatment with F, catalase, medium-chain specific acyl-CoA dehydrogenase and alpha-aminoadipic semialdehyde dehydrogenase were up-regulated, while isovaleryl-CoA dehydrogenase, ornithine aminotransferase, lactoylglutathione lyase, meprin A subunit alpha and albumin were down-regulated in the kidney of 129P3/J mice.A/J and 129P3/J mice exhibited 11 and 3 exclusive proteins, respectively. From these, 9 (64.3%) are related to metabolism, followed by cell processes (4 or 28.6%) and information pathways (1 or 7.1%). This profile was not altered upon exposure to F .per se accounts for such differences between these two strains of mice. We have focused on identified proteins that may be associated with metabolic handling of F and water and renal functions. Unique metabolic proteins in kidney from A/J mice are involved in carbohydrate (probable D-lactate dehydrogenase), carbon , aminoacid , cystathionine gamma-lyase (CSE), thiomorpholine-carboxylate dehydrogenase), and fatty acid [short-chain specific acyl-CoA dehydrogenase (SCAD)] metabolism. CSE is an enzyme that breaks down cystathione into cysteine and α-ketobutyrate and catalyses elimination of L-homoserine, L-cystine and L-cysteine producing ammonia and hydrogen sulfide (H2S) − that remains in the tubule to be excreted in urine In the present study, we identified proteins potentially involved in renal F metabolism that are either exclusively or differentially expressed in A/J and 129P3/J mice. This highlights the molecular mechanisms underlying the differential metabolic handling of F by these two strains of mice. Exclusive proteins expressed in A/J or 129P3/J mice exhibited the same profile, regardless exposure to F. This suggests that the genetic background Besides presenting unique proteins involved in metabolism, A/J mice also expresses exclusive proteins involved in cell processes (phenazine biosynthesis-like domain containing protein 2 (PBLD), biliverdin reductase A (BVR) and sorting nexin-5) and information pathways [serum amyloid P-component (SAP)]. Among these, SAP constitutes amyloid deposits characterized by the ordered aggregation of normal globular proteins and peptides into insoluble fibrils, which disrupt tissue architecture and are associated with cell death From those proteins found exclusively in kidneys of 129P3/J mice, the peroxisomal acyl-coenzymeA oxidase 1 (AOX), a fatty acid metabolic protein, is shown to be expressed in proximal tubules and enhancement of its activity is associated with the preservation of kidney function during ischemia As mentioned above, F exposure did not alter the profile of unique proteins in either strain of mice. However, among the proteins differentially expressed in the comparisons between the two strains, only 8 were present in the control, 10 and 50 ppmF groups , isovaleryl-CoA dehydrogenase, ornithine aminotransferase, lactoylglutathione lyase, meprin A subunit alpha and albumin). Some of these significantly altered proteins with potential roles to contribute for the intrinsic differences in F and water handling by A/J and 129P3/J mice are highlighted below. Meprin A, an information pathways related protein, is an enzyme that hydrolyzes protein and peptide substrates including components of the extracellular matrix + channel, Na+-K+-Cl− cotransporter (NKCC1), CFTR, epithelial Na+ channel (ENaC), aquaporin-2 (AQP2) and Na+/H+ exchanger 3 (NHE3) Two and 6 proteins with differential expression between the two strains in the control group were also identified upon exposure to 10 and 50 ppmF, respectively. Low F level increased the expression of serine/threonine-protein phosphatase PP1 and ATP synthase subunit delta. High F level kidney up-expressed aconitate hydratase, ATP synthase subunit beta, hydroxyacid oxidase 2, homogentisate 1,2-dioxygenase and beta-lactamase-like protein 2 and down-expressed phosphotriesterase-related protein. Besides, 6 proteins presented altered expression only in F-treated groups. Aminoacylase-1 and aspartoacylase-2 were increased, whereas L-lactate dehydrogenase B chain, nucleoside diphosphate-linked moiety X motif 19, Na(+)/H(+) exchange regulatory cofactor NHE-RF3 (PDZK1) and actin-related protein 3 were diminished in 129P3/J kidney. These proteins may act as molecular targets for the differential F metabolism between these strains induced by the treatment. Protein phosphatase 1 (PP1) is a serine/threonine protein phosphatase involved in diverse cellular processes, such as transcription, replication, pre-mRNA splicing, protein synthesis, carbohydrate metabolism, neuronal signaling, cell survival, and cell cycle progression + and Cl− in the proximal tubule of the mammalian kidney. Besides regulating reabsorption of filtered solutes, PDZK1 also plays a direct and essential role in maintaining normal brush border expression and function of CFEX in the proximal tubule in vivoPDZK1 is a scaffold protein that connects plasma membrane proteins and regulatory components, regulating their surface expression in epithelial cells apical domains. Within the kidney, PDZK1 is localized exclusively in the brush border of the proximal tubule and interacts with several renal proteins including NHE3, a Na-H exchanger, and CFEX, a Cl-anion exchanger We conclude that the renal proteome indicates several specific target proteins, both strain and F-induced, which possibly regulate the water and F metabolism in kidney of mice with distinct susceptibilities to F. In addition, although we did not focus in the correlation between target kidney proteins and DF, we found that some of those changed proteins are also codified by chromosomes 2  and 11 , previously characterized to determine susceptibility and resistance to DF in A/J and 129P3/J mice, respectively Figure S12D gel analysis of renal proteome. Representative 2D maps of control kidneys. Selected spots in green represent those with differential expression in the comparison between control A/J (A) vs control 129P3/J mice (B). In Figure B, spot identification numbers in boundaries or not represents increases or decreases in protein expression when compared to A/J, respectively . Dashed lines represent unique spots in the AJ group (A) and 129P3/J group (B), regardless exposure to F.(TIF)Click here for additional data file.Figure S22D gel analysis of renal proteome. Representative 2D maps of 10 ppmF treated-groups. Selected spots in green represent those with differential expression in the comparison between 10 ppmF treated- A/J (A) vs 10 ppmF treated- 129P3/J mice (B). In Figure B, spot identification numbers in boundaries or not represents increases or decreases in protein expression when compared to A/J, respectively .(TIF)Click here for additional data file.Figure S32D gel analysis of renal proteome. Representative 2D maps of 50 ppmF treated-groups. Selected spots in green represent those with differential expression in the comparison between 50 ppmF treated- A/J (A) vs 50 ppmF treated- 129P3/J mice (B). In Figure B, spot identification numbers in boundaries or not represents increases or decreases in protein expression when compared to A/J, respectively .(TIF)Click here for additional data file.Figure S42D gel variability analysis. Scatter plot of binary comparisons among the ratios of relative spots volumes detected in the representative gel (replicate 1) and the respective replicates (replicates 2 and 3). (A) Control A/J mice. (B) 10 ppmF treated-A/J mice. (C) 50 ppmF treated-A/J. (D) Control 129P3/J mice. (E) 10 ppmF treated-129P3/J mice. (F) 50 ppmF treated-129P3/J.(TIF)Click here for additional data file.