Chikungunya virus (CHIKV) is a re-emerging mosquito-borne virus which causes epidemics of fever, severe joint pain and rash. Between 2005 and 2010, the East/Central/South African (ECSA) genotype was responsible for global explosive outbreaks across India, the Indian Ocean and Southeast Asia. From late 2013, Asian genotype CHIKV has caused outbreaks in the Americas. The characteristics of cross-antibody efficacy and epitopes are poorly understood.We characterized human immune sera collected during two independent outbreaks in Malaysia of the Asian genotype in 2006 and the ECSA genotype in 2008–2010. Neutralizing capacity was analyzed against representative clinical isolates as well as viruses rescued from infectious clones of ECSA and Asian CHIKV. Using whole virus antigen and recombinant E1 and E2 envelope glycoproteins, we further investigated antibody binding sites, epitopes, and antibody titers. Both ECSA and Asian sera demonstrated stronger neutralizing capacity against the ECSA genotype, which corresponded to strong epitope-antibody interaction. ECSA serum targeted conformational epitope sites in the E1-E2 glycoprotein, and E1-E211K, E2-I2T, E2-H5N, E2-G118S and E2-S194G are key amino acids that enhance cross-neutralizing efficacy. As for Asian serum, the antibodies targeting E2 glycoprotein correlated with neutralizing efficacy, and I2T, H5N, G118S and S194G altered and improved the neutralization profile. Rabbit polyclonal antibody against the N-terminal linear neutralizing epitope from the ECSA sequence has reduced binding capacity and neutralization efficacy against Asian CHIKV. These findings imply that the choice of vaccine strain may impact cross-protection against different genotypes.Immune serum from humans infected with CHIKV of either ECSA or Asian genotypes showed differences in binding and neutralization characteristics. These findings have implications for the continued outbreaks of co-circulating CHIKV genotypes and effective design of vaccines and diagnostic serological assays. Chikungunya virus (CHIKV) has caused large epidemics of fever, rash, and joint pain around the world in recent years. Three different CHIKV genotypes exist. Infection with one genotype is likely to lead to immune protection (or cross-protection) against future infections with a different genotype. However, little is known about the nature of this cross-protection. In this study, we used serum from Malaysian patients infected with CHIKV of either Asian or East/Central/South African (ECSA) genotypes. We compared the ability of the serum antibodies to bind to and neutralize two different viruses, from either Asian or ECSA genotypes. We found that both Asian and ECSA serum were more effective in binding and neutralizing ECSA virus. We identified the key amino acids/epitopes within the E1-E2 surface glycoprotein, and showed that variation of these impacts the efficacy of antiserum in cross-neutralizing different genotypes of CHIKV. We showed how sequence variation of a known linear neutralizing epitope could alter the cross-neutralization efficacy. This study aids understanding of the importance of different circulating genotypes within a country and has implications for the design of vaccines and diagnostic antibody tests. Chikungunya virus (CHIKV) is a re-emerging, mosquito-borne arbovirus which has caused unprecedented worldwide epidemics in recent years . There aTogaviridae. A CHIKV virion is 60-70nm in diameter, with a single-stranded positive RNA genome of approximately 11.8 kb in a capsid with a phospholipid envelope carrying glycoproteins E1 and E2. Its genome has 2 open reading frames encoding the non-structural (nsP1-nsP2-nsP3-nsP4) and structural polyproteins (C-E3-E2-6K-E1) [CHIKV is an alphavirus from the family 2-6K-E1) . The E1 2-6K-E1) . These p2-6K-E1) , 14. Aft2-6K-E1) , CHIKV-s2-6K-E1) . Among o2-6K-E1) , althoug2-6K-E1) .Currently, treatment for CHIKV is supportive and no licensed vaccine or antiviral are available. Phase I clinical trials have demonstrated the safety and efficacy of vaccination with virus-like particles using structural proteins derived from the West African genotype , and a rIn this study, we analyzed the neutralizing capacity of CHIKV ECSA and Asian immune sera against representative clinical isolates and rescued viruses of ECSA and Asian CHIKV. We demonstrated that both sets of serum panels have stronger neutralizing capacity against the ECSA isolate, which corresponded to strong epitope-antibody interaction. E1-E211K enhances the neutralization activity of ECSA serum, while E2-I2T, H5N, G118S and S194G within linear epitopes improve the neutralization activity of both sets of sera panels. Rabbit polyclonal antibody targeting a known linear neutralizing epitope (LP1) from ECSA virus could only neutralize homotypic virus, but not heterotypic Asian virus due to sequence variation. These findings indicate the antigenic variation of ECSA or Asian CHIKV genotypes in naturally-acquired infection alters the spectrum of cross-genotype protective antibody immunity.n = 15) with no past infection of CHIKV served as negative controls. Serum neutralization assay was performed on all the sera. To determine the neutralizing activity due to IgG, heat-inactivated sera were treated for 1 hour with dithiothreitol (DTT) (Life Technologies) at a final concentration of 5mM at 37°C.This study included 63 human samples from two independent outbreaks in Malaysia. The Asian serum panel comprised 40 samples collected from patients 11–14 months after an Asian CHIKV outbreak in Bagan Panchor in 2006 . The ECSThis study was approved by the Medical Ethics Committee of the University Malaya Medical Centre (reference no. 800.70). Our institution does not require informed consent for retrospective studies of archived and anonymized samples.Baby hamster kidney (BHK-21) cells (ATCC no. CCL-10) were maintained in Glasgow minimum essential medium (GMEM) (Life Technologies) supplemented with 5% heat-inactivated fetal bovine serum (Flowlab), 10% tryptose phosphate broth, 20mM HEPES, 5mM L-glutamine, 100 U/ml penicillin and 100μg/ml streptomycin. Infected cells were maintained in GMEM containing 2% FBS. The clinical isolates used, which have been previously characterized , were MYhttp://www.european-virus-archive.com/article147.html). Both molecular clones have ZsGreen gene incorporated as reporter and duplication of the subgenomic promoter. The ECSA molecular clone was named “ICRES1”, while the Asian molecular clone was designated as “CAR”.To study the neutralizing epitopes, viruses rescued from two different infectious clones, derived from ECSA and Asian genotypes of CHIKV, were included. The plasmid vectors capable of producing infectious viruses were constructed under the control of the human cytomegalovirus immediate-early promoter. The CHIKV infectious clone derived from the ECSA genotype was based on LR2006-OPY1, isolated in Reunion Island in 2006,and has been described previously . The fulFor construction of the chimeric viruses, the ectodomain regions of envelope glycoprotein genes E1 (amino acids 1–381) and E2 (amino acids 1–341) in the ICRES1 backbone were replaced with those of Semliki Forest virus (SFV) E1 (amino acids 1–381) and E2 (amino acids 1–340) from icDNA SFV6  using NE2 flasks were infected with P0 stocks at a multiplicity of infection (MOI) of 1 plaque forming unit/cell and maintained in 2% FBS GMEM. P1 stocks were harvested after 24 or 48 hours, titrated and used for the neutralization assay. Infectious center assay was performed on all the viruses rescued from icDNA. Details on virus rescue and related protocols are shown in The viruses were rescued from icDNA by electroporation. Stocks of rescued viruses (P0) were harvested and titrated by plaque assay on BHK-21 cells. To obtain P1 stocks, confluent BHK-21 cells grown in T75-cm4 cells in 96-well CellCarrier-96 optic black plates (Perkin Elmer), and further incubated for 1.5 hours at 37°C. The inocula were decanted and 2% FBS GMEM was added to the plates. The plates were fixed with 4% paraformaldehyde after 6 hours of incubation at 37°C, permeabilized with 0.25% Triton X-100 for 10 minutes, and immunostained using anti-CHIKV E2 monoclonal antibody B-D2(C4) [50) was expressed as the serum dilution that reduced infectivity by 50% using non-linear regression fitting in GraphPad Prism 5 (GraphPad Software).Seroneutralization was performed with a previously described immunofluorescence-based cell infection assay in BHK-21 cells , 27, witB-D2(C4)  at 1μg/mFor seroneutralization using rescued viruses, diluted sera were mixed with viruses pre-diluted with 2% FBS GMEM (with infection performed at an MOI of 50), followed by the steps described above. The plates were fixed after 7 hours of incubation at 37°C. The plates were only counter-stained with DAPI prior to acquisition of ZsGreen fluorescence.To investigate the cross-reactivity of CHIKV sera against another alphavirus, SFV was rescued from icDNA SFV6 as previously described . DilutedIKDHFNVYKATRPY) and anti-LP47 (NHKKWQYNSPLVPRN) were produced commercially (GenScript). LP1 is similar to E2EP3, an immunogenic peptide (from an ECSA virus) previously reported to elicit neutralizing antibodies [To investigate the effect of sequence variation of neutralizing epitopes in ECSA and Asian genotypes, polyclonal rabbit anti-LP1 (STKDNFNVYKATRPY), anti-LP1A (Stibodies . LP1A isFor indirect IgG ELISA (antibody end-point assay) and Western blot, the antigen was partially purified virus prepared by sucrose-cushion ultra-centrifugation, treated with 1% Triton X-100 in TE buffer, clarified by centrifugation, and stored in 50% glycerol at -20°C.BamH1 and Not1 restriction sites. Each plasmid construct together with a pIE1-neo vector were co-transfected into Sf9 cells (Novagen) using Cellfectin II reagent (Invitrogen) [2PO4, 300mM NaCl, pH 8.0). The proteins were stored at -20°C in 50% glycerol, except for the proteins used in the competitive protein blocking assay, which were filter-sterilized and kept at 4°C. Fusion sequences expressing for rE2 and rE1 was generated by overlapping PCR; recombinant proteins encoded by obtained sequence were linked via linker with sequence GGGS-His (8X)-GGGG  and E2 , viral RNA was extracted from clinical isolates (Asian MY/06/37348 and ECSA MY/08/065). cDNA was synthesized using reverse-transcription, and the genes were amplified using high fidelity Platinum Taq (Invitrogen) with designed primers . The traitrogen) . Stable 8X)-GGGG . The fusThe proteins were resolved with 12% SDS-PAGE under reducing and non-reducing conditions and electro-transferred onto a nitrocellulose membrane (GE). The membrane was blocked with 10% skimmed milk in 0.05% PBS-Tween 20 (PBST). The immunoreactivity of recombinant proteins was evaluated with pools of CHIKV immune sera applied at indicated dilutions in the blocking buffer. The bound antigen-antibody complex was detected by anti-human IgG-HRP (DakoCytomation) at 1:5000 dilution in 1% bovine serum albumin (BSA)-0.05% PBST. The membrane was visualized by chemiluminescence (Bio-Rad) and images were acquired with a BioSpectrum AC imaging system (UVP). Mouse anti-His tag antibody (Merck Millipore) was included as a loading control. Mouse anti-E2 monoclonal antibody (clone: B-D2(C4); EIEVHMPPDT)  was alsoAll incubation steps were performed at 37°C for 1 hour, using 1% BSA-0.05% PBST as diluent for serum and antibodies. The plates were washed 4 times with 0.05% PBST after each incubation step. To determine the relative level of anti-E2 antibodies, the plates were coated with 250 ng of virus antigen or 100 ng of rE2 in 0.05M carbonate-bicarbonate buffer (pH 9.6). The antigens were normalized with monoclonal antibody B-D2(C4) to determine the relative level of anti-E2 antibodies. The plate was blocked with 3% BSA in 0.05% PBST. The sera were tested at 2-fold serial dilutions from 1:512 to 1:1,048,000 or 1:640 to 1:655,000. The IgG end-point titer was determined as the reciprocal of the highest dilution that produced an optical density (OD) reading of three times greater than that of the negative control. Anti-human IgG-HRP at 1:5000 dilution was added to detect the bound antibodies. TMB substrate (KPL) was added to each well and the plates were incubated at room temperature for 5 min. The reaction was terminated by adding 1M phosphoric acid. The absorbance was measured at 450nm with 630nm as the reference wavelength using an automated ELISA reader (Biotek Instruments). The cut-off value was established as the OD obtained from healthy controls sera plus three standard deviations (SD). The relative level of anti-rE2 antibodies was calculated with the following formula: (end point titer for rE2/end point titer for whole virus antigen) × 100.Soluble recombinant CHIKV proteins (15μg) were mixed with heat-inactivated immune sera diluted at 1:200, and incubated for 1 hour at 37°C. CHIKV (MY/08/065) in amounts corresponding to an MOI of 10 was mixed with the samples, which were incubated for a further 2 hours at 37°C.Synthetic peptides were obtained from GenScript ; 60μg of each peptide was mixed with immune sera diluted with 1X DPBS at 1:100 and incubated for 1.5 hours at 37°C. All the synthetic peptides for the blocking assay have a purity grade greater than 95% and are soluble in high-grade water. ICRES1 (sucrose-cushion purified virus in TE buffer pre-diluted using 2% FBS GMEM) at an amount corresponding to an MOI of 1 was mixed with the samples, which were incubated for a further 2 hours at 37°C prior to infection of BHK-21 cells. The plate was replenished with plaque medium (2% FBS GMEM containing 0.8% of carboxymethylcellulose), fixed with 4% paraformaldehyde after 15 hours of incubation, and this was followed by ZsGreen fluorescence acquisition. Infectivity corresponded to the fluorescence intensity acquired with a Cellomics HCS reader. The effect on infectivity of antibodies in the presence and absence of blocking peptides was compared.Biotinylated synthetic peptides covering the E2 glycoprotein sequence from amino acids 1–362 from a previous study  were useP-value of <0.05 was considered significant. Statistical analyses were performed with GraphPad Prism 5.Data are presented as means ± SD or means ± standard error of the mean (SEM). Differences between groups and controls were analyzed using appropriate statistical tests. A 50 against MY/08/065 that was a median 2.67  times greater than the NT50 against MY/06/37348  times greater than the NT50 against MY/06/37348  and MY/06/37348 (Asian)  Figs. Th06/37348 . Unexpec06/37348 , with a 06/37348 . The gre06/37348 . Seroneu06/37348 . Immunob06/37348 . Asian s06/37348 . Under r06/37348 , and theTo determine if CHIKV immune serum targets E1, recombinant E1 glycoprotein (rE1) was probed in ELISA with serially diluted pooled sera, and signal was detected at low serum dilutions from 1: 100 to 1:800 . A compeTo further determine the importance of conformational epitopes resulting from interactions between E1 and E2, four sets of fusion E1-E2 glycoproteins were constructed. Each hybrid fusion protein contained E1 and E2 sequences from either MY/06/37348 (ECSA) or MY/08/065 (Asian), transiently expressed as secreted native recombinant proteins in insect cells . The antAsian-E1ECSA recombinant proteins (Between ECSA (MY/08/065 and ICRES1) and Asian (MY/06/37348 and CAR) genotypes of CHIKV in this study, there are 10 amino acids differences in E1 . Using tproteins . Recombiproteins . The criproteins .To study the linear epitopes in the immunodominant E2 glycoprotein , overlapping synthetic peptides covering amino acids 1–362 were mapped by peptide-ELISA using the ECSA and Asian sera . Both ECIKDHFNVYKATRPY), derived from the sequence of the Asian virus. Rabbit polyclonal antibodies were commercially prepared against LP1A and LP1. Peptide-ELISA was performed using human ECSA and Asian serum with LP1A and LP1 as antigens. Human ECSA serum bound to LP1 but not LP1A  is similar to E2EP3, a peptide derived from ECSA virus which has been found to be highly immunogenic in eliciting neutralizing antibodies in an animal model . We genenot LP1A . Rabbit not LP1A . Anti-LPnot LP1A , which hnot LP1A .Asian serum could recognize LP1A, although binding was marginally higher to LP1 , which sCHIKV has become a major public health concern worldwide and causes considerable socio-economic burden. Protective adaptive immunity is mainly provided by specific antibodies, particularly those directed against epitopes on the E2 and E1 glycoproteins , 38. UndAedes albopictus vector [In this study, we showed differences in cross-genotypic neutralization efficacy of immune sera against ECSA and Asian genotypes of CHIKV. Both ECSA and Asian serum had greater neutralizing capacity against ECSA genotype (MY/08/065 and ICRES1) than Asian genotype (MY/06/37348 and CAR), indicating that neutralizing antibodies regardless of initial infecting genotype preferentially recognized the epitopes presented by the ECSA genotype. The presence of cross-genotype neutralization was clearly shown lasting up to 14 months post-infection. The clinical significance of the differential cross-protective capacity of ECSA and Asian sera remains unclear, as all the immune sera had more than the minimum neutralizing titer (≥10) which appears to correlate with immune protection from symptomatic CHIKV infection in humans . This his vector , 41.Apart from the stronger antigenicity of epitopes of the ECSA genotype, we also showed that neutralizing capacity was also affected by the target and the amount of neutralizing antibodies. Both ECSA and Asian sera contain high levels of neutralizing antibodies to numerous linear epitopes on the E2 glycoprotein as well as conformational epitopes on the E1-E2 heterodimer complex. This supports recent findings that most of the reported CHIKV neutralizing monoclonal antibodies target conformational epitopes on the exposed, topmost outer surfaces of the E2/E1 spike, particularly in domain A and domain B –45. Our The loss of neutralization activity against chimeric CHIKV is in line with the finding that total IgG and anti-rE2 antibody titers correlate with the neutralizing titer of Asian serum , suggestAmino acid changes in key epitope regions, such as naturally occurring mutations or antigenic variation between different genotypes could affect surface charge distribution and electrostatic interactions between epitopes and antibodies, affect binding affinity and ultimately alter neutralizing capacity . The E21It was interesting that the Asian serum had greater neutralizing capacity against the heterologous ECSA isolates. The previously reported human CHIKV monoclonal antibodies 5F10 and 8B10 had a broad neutralization activity against isolates of the ECSA and West African genotypes, but were also less potent against an Asian isolate from Indonesia . MonkeysTwo amino acids in LP1  of the ECSA virus are critical for binding and neutralization activity, and this further highlights the fact that sequence variation could impact vaccine development. The rabbit polyclonal antibody targeting the linear neutralizing epitope LP1 from the ECSA virus showed reduced cross-neutralization against the Asian genotype, and unexpectedly, rabbit anti-LP1A poorly neutralized the homotypic CAR Asian virus, despite immunization of 4 rabbits. The linear neutralizing epitope LP1A from the Asian virus was not recognized by the ECSA sera. However, clearly there are preexisting antibodies against LP1 and LP1A in the Asian sera. LP47, another linear neutralizing epitope in humans , which hIn conclusion, immune serum from humans infected with CHIKV of either ECSA or Asian genotypes showed differences in neutralization and binding capacities. Our findings are relevant to current outbreaks with co-circulating genotypes and provide insights into antibody-mediated immunity resulting from infections with CHIKV of different genotypes.S1 FigRepresentative acquired immunofluorescence microscopic images of pooled serum at dilutions of 1:100, 1:400, and 1:1600, and virus control against clinical CHIKV isolates MY/08/065 (ECSA) and MY/06/37348 (Asian). Each image contains 9 combined fields within a well (96-well format). Objective magnification: 5X.(PDF)Click here for additional data file.S2 FigRepresentative acquired immunofluorescence microscopic images of pooled serum at dilutions of 1:100, 1:400, and 1:1600, and virus control against clinical CHIKV isolates MY/08/065 (ECSA) and MY/06/37348 (Asian). Each image contains 9 combined fields within a well (96-well format). Objective magnification: 5X.(PDF)Click here for additional data file.S3 Fign = 23 for ECSA sera, n = 40 for Asian sera.DTT-treated sera (containing IgG only) have similar neutralizing capacity to intact sera, which have a mixture of IgG and IgM. All sera were assayed up to 1:6400 dilution. The neutralization data was based on experiments performed against ECSA CHIKV (strain MY/08/065). Data are presented as means ± SEM; (PDF)Click here for additional data file.S4 FigP< 0.01, ***P<0.001, Mann-Whitney U test relative to virus control. (B) Representative acquired immunofluorescence microscopic images of pooled serum at dilutions of 1:25 or 1:100 and virus control against SFV rescued from icDNA SFV6. Objective magnification: 10X.(A) Seroneutralization was performed against SFV at 1:25 and 1:100 serum dilutions using pooled sera. Data are expressed as percentages of infectivity over infection control, and are presented as means ± SD from 3 independent experiments. **(PDF)Click here for additional data file.S5 Fig(A) Schematic representation of the E2 glycoprotein with the numbers indicating the amino acid positions of the glycoprotein and its domain proteins. The amino acid differences between E2 glycoproteins of ECSA  and Asian  strains were tabulated and mapped (from amino acids 1–362). Amino acid differences within a genotype are underlined. Amino acid changes which fall within the identified linear epitopes are color-coded. (B) Immunoblotting was performed against recombinant E2 glycoproteins under reducing conditions, with each named amino acid change from the Asian to the ECSA sequence introduced independently. Mouse anti-His was used as a control. Site-directed mutagenesis was not performed for amino acid positions 312, 317 and 318 as these are predicted not to be exposed on the protein surface.(PDF)Click here for additional data file.S6 Fig50 and antibody titers against (A) MY/08/065 and (B) recombinant E2 glycoprotein were assessed. Spearman’s rank correlation coefficients (ρ) and P-values are shown. ns, not significant.The relationships between NT(PDF)Click here for additional data file.S7 FigP<0.01, Mann-Whitney U test relative to virus control.Two antibodies were prepared commercially; anti-E1DII, which targets a linear epitope of E1 , and anti-LP1, which targets a linear epitope of E2 . Seroneutralization was performed at 25μg/ml against ICRES1. Data are presented as means ± SD from 2 independent experiments, run in triplicate. ** (PDF)Click here for additional data file.S8 FigP< 0.01, Mann-Whitney U test, relative to unblocked control. (C) The color-coded mapped neutralizing epitopes (LP19 and LP47) are localized on the structural E1-E2 heterodimer complex (based on PDB 3J2W).(A) Schematic diagram of the E2 protein showing the positions of the four mapped epitopes  which have higher OD relative to LP1. The numbers refer to the amino acid positions demarcating the E2 domains. N, N-link; C.arch, central arch. (B) Competitive peptide blocking assay was performed at 1:100 dilution with either pooled ECSA or Asian sera against ICRES1 at an MOI of 1. Sera blocked by LP19v and LP47 resulted in increases in infectivity. LP19v is a soluble peptide without cysteine residues at N- and C-terminuses of LP19. Data are expressed as percentages of infectivity of an infection control, and are presented as means ± SD from 2 independent experiments, run in triplicate. **(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Three random samples from a single loin chop and surrounding adipose were collected and analyzed. Serum concentrations of 25(OH)D3 did not differ (P≥0.376) between treatments prior to sun exposure in either group, but was increased  with SUN exposure. Total vitamin D content (D3 plus 25(OH)D3) of loin tissue was increased (P < 0.001) with sun exposure and averaged 0.997±0.094 μg/100g and 0.348±0.027 μg/100g for sun and control pigs, respectively. While exposure to sunlight increased (P = 0.003) tissue content of 25(OH) D in subcutaneous adipose tissue, vitamin D3 content was similar between treatments (P = 0.56). Sunlight exposure in pigs increased the vitamin D content of loin, and may provide an additional source of dietary vitamin D.There is a high prevalence of vitamin D insufficiency and deficiency worldwide likely because of both limited sun-exposure and inadequate dietary intake. Meat, including pork, is not typically considered a dietary source of vitamin D, possibly because of management practices that raise pigs in confinement. This experiment determined the vitamin D content of loin and subcutaneous adipose tissue in sun-exposed finisher pigs. Two separate groups of pigs were used. The first group (28 white Landrace-Duroc) was assigned at random to either sunlight exposure (SUN) in spring and summer or confinement per standard practice (Control). The second (24 Yorkshire-Duroc-Landrace) underwent the same exposure protocol but was exposed in summer and fall or assigned to control (Control). A subsample of five SUN and four Control pigs, matched for weight and body condition score, was selected for slaughter from each group. Pigs  had blood drawn for analysis of 25(OH)D Vitamin D is known for its critical role in bone health . Increas3 [Vitamin D is unique among vitamins in that it can be obtained from diet as well as synthesized in the body from exposure to UVB radiation from sunlight. Endogenous synthesis, however, is considered the most available source of vitamin D3 . Compare3  exposure3 . Concern3 .3 and D2 content of food sources [3 [3 and D2. Later work from Heaney has suggested that solar inputs of vitamin D are less than previously thought. Instead, there is a significant input of vitamin D from undocumented food sources such as preformed 25(OH)D3 in animal products[3 is estimated to be about five times as potent as vitamin D3 in raising serum 25(OH)D3 concentrations [3 is readily sequestered by adipose tissue [3 is distributed throughout the body and is taken up by skeletal muscle tissue [Meat, in general, is not considered a good source of vitamin D. The vitamin D content of pork may be particularly low because of husbandry practices, which confine pigs indoors with limited exposure to sunlight. Furthermore, the USDA nutrient database only evaluates vitamin D sources . Heaney urces [3 , the imm products. Moreovetrations . Recent e tissue , while 2e tissue ,20.Although swine are generally raised in confinement, they , like otAll experimental protocols were approved by the University of Wyoming (UW) institutional animal care and use committee (IACUC).White Landrace-Duroc cross pigs  were assigned at random to either sunlight exposure (SUN) in the spring and summer (n = 14) or confined to indoors as per standard practice (Control) with limited exposure to outdoor sunlight (n = 14). The study was replicated with a second group of white Yorkshire-Duroc-Landrace cross-bred pigs  aged 80±13 d, 31.6±2.1 kg with sunlight exposure (SUN) in the summer and fall (n = 12) or Control (n = 12). From each group of pigs, a subsample of sun-exposed (n = 5 per group) and control (n = 4 per group) pigs, matched for body condition score, were selected for blood and tissue analysis; these analyses were performed only in a subsample due to cost constraints. Pig breeds selected are commonly used for pork production. Waldo Farms  supplied the spring-summer group of pigs. The summer-fall group of pigs was born to the University of Wyoming Research and Extension Center farm.ad libitum water and feed. Diets were formulated as a growth ration with a corn and soybean base and vitamin and trace mineral premixes added  by using a Beckman TJ-6 . Serum was collected and stored at -20°C until analysis.Spring-summer pigs assigned to sun exposure were exposed to natural sunlight one hour each day during solar noon (12:30–1:30 PM standard time) for two weeks surrounding the spring equinox (March 20–April 3) and summer solstice (June 13–26). Summer-fall pigs were exposed to sunlight for an hour daily during solar noon for two weeks surrounding the summer solstice (June 17–30) and fall equinox (September 18-October 1). Sun pigs traveled down a short alley to an outdoor pen on the southern side of the facility. In both groups of SUN pigs, the first exposure period occurred when the pigs were young growers and the second exposure occurred when the pigs were near slaughter weight. Control pigs were raised conventionally, and kept in confinement, with limited sun exposure, throughout the duration of the experiment. Total UV radiation was measured during the exposure period by using a UV spectrometer  and a logging data acquisition system . UVB was calculated by using a UVA/UVB ratio of 20:1,i.e. 5% of total . Blood sth and last rib. Percentage of fat-free lean was calculated by using the equation of Boggs et al [The selected pigs from spring-summer and summer-fall were slaughtered at the University of Wyoming abattoir on the morning following the last day of sun exposure. At slaughter the pigs were killed by electrical stun followed by exsanguination as per standard slaughter procedures and as approved by the IACUC. Three random samples (≈ 3 g) from a single pork chop  were collected and frozen on dry ice, and stored in eppendorf tubes at -80°C until analysis. Back fat thickness was taken by the average of measurements at the 10gs et al .3 were determined by radioimmunoassay (RIA) with tissue content of vitamin D2, D3 and 25(OH)D determined via liquid chromatography-mass spectrometry (LC/MS/MS).Serum and tissue vitamin D analyses were conducted by Heartland Assays  who were blinded for treatment group. Serum concentrations of 25(OH)D2 and 25(OH)D3. Following incubation at room temperature, antigen-antibody complexes were separated with a second antibody precipitating complex followed by centrifugation. 25(OH)D equivalent values were calculated directly by the γ-radiation counting system via a smooth-spline method [The 25(OH)D assay is a two-step procedure  using rae method . The 25D2/D3 intra-assay %CV = 5.0% and inter-assay %CV = 8.6% with extraction efficiencies of >68% and assay range of 0.5–312.5ng/g. Vitamin D2/D3 intra-assay %CV = 9.9% and inter-assay %CV = 11.2% with extraction efficiencies of >64%. Limit of quantification was 0.5ng/g and assay range of 0.5-625ng/g with a limit of detection of 0.1ng/g for all analytes. All values were adjusted for extraction efficiency prior to statistical analysis.All samples were weighed and saponified with methanolic potassium hydroxide  and spik3 data for the second exposure period when the pigs were close to slaughter are included. Data are presented as mean ± standard error unless otherwise specified. Data were analyzed by using Minitab statistical software version 17 . Two sample t-test with equal or unequal variances were used to compare differences in body weights and body composition and pre-treatment serum 25(OH)D3 concentrations between SUN and Control pigs. Repeated Measures ANOVA was used to test for a time (pre versus post sun exposure) by treatment (SUN versus Control) effect on serum 25(OH)D3 concentrations. A two-sample t-test with equal or unequal variances was used to test for differences in vitamin D3 and 25(OH)D3 in loin and subcutaneous adipose from the SUN versus control pigs at slaughter and to test for differences between summer and fall exposure pigs for serum and tissue vitamin D concentrations. Pearson correlation coefficients were used to determine associations between tissues and serum concentrations of vitamin D.Only serum 25(OH)DStarting weights, slaughter weights, and average daily gain (ADG) were not different between control and SUN pigs for spring-summer and summer-fall groups . At slauAverage UVB radiation at solar noon (±30 minutes) for the 14 days of exposure around the summer solstice and 14 days around the fall equinox is shown in 3 between SUN (46.1±1.7 ng/ml) and Control pigs (39.9±5.6 ng/ml) prior to sun exposure. Serum 25(OH)D3, however, was increased (P<0.001) following sun exposure in SUN but not Control pigs  in serum concentrations of 25(OH)Deraction ) Fig 2)3 between3 content was higher . Total vitamin D content (D3 plus 25(OH)D3) of loin was also higher (P = 0.001) in SUN pigs (0.997±0.094 μg/100g) compared to control pigs (0.348±0.027 μg/100g)  in the μg/100g) .3 content was greater in SUN pigs (0.814±0.1 μg /100g) versus that in control pigs . Total vitamin D content in subcutaneous adipose, as well, did not differ (P = 0.35) between SUN (5.42 μg/100g) and control (4.513 μg/100g) pigs.Adipose content of 25(OH)D = 0.003 ). Vitami2 was not detectable in any tissue samples.Vitamin D3 concentrations were positively associated with loin vitamin D3  loin 25(OH)D3  and subcutaneous fat 25(OH)D3  content, but not with subcutaneous adipose vitamin D3 content .Serum 25(OH)DWe have demonstrated sun exposure prior to slaughter in pigs significantly increases serum concentrations of vitamin D and vitamin D content in the lean tissue of the loin. To our knowledge, this is the first study demonstrating the effects of natural sunlight in pigs on the vitamin D content of pork products.3 concentrations of 20 ng/ml  such circulating concentrations may not provide further health benefits including reduced risk of certain cancers [3 should be at least 30 ng/ml and further stresses that maintaining circulating concentrations of 25(OH)D3 in the range of 30–50 ng/ml may produce added health benefits [3 may be optimal for many physiological functions such as fully suppressed parathyroid hormone, efficient intestinal calcium absorption, and optimized vitamin D concentrations in breastmilk [3 are believed to approach those of our hominid ancestors based on research in African tribes [3 status, or at a minimum, meeting the RDA for vitamin D. Many people in the US [The main finding of this experiment is of significant importance when considering dietary intake of vitamin D. The current recommended dietary allowance (RDA) for vitamin D is 600 IU/d for children and adults <70 years old . Despite cancers ,32,33 an cancers . Heaney benefits . Furthereastmilk . These hn tribes . Considen the US  and worln the US  fail to 3 and 25(OH)D3 content in meat cuts from pigs[Strategies to fortify or naturally add vitamin D to the food supply are currently being explored. Green and colleagues  have sugfrom pigs. Alterin3 concentrations of egg yolks and meat. Another study in a small sample of Gottingen minipigs reported that UVB exposure was more effective than dietary administration of vitamin D3 at increasing vitamin D tissue content [A recent study found that exposing hens to artificial sunlight via 23-W UVB lamps for 3 hr daily for four weeks increased the vitamin D content of egg yolks by 337% and the content of fibularis longus muscle to detectable concentrations of 0.16 to 0.96 μg/100g [ content . Our dat3 and 25(OH)D, content of 0.4 and 0.5 ng/g in the loin tissue, and 5 and 3.1 ng/g in the adipose, respectively. In contrast, sun-exposed pigs in our experiment had an average D3 content of 7.16 ng/g in the loin and 46.05 ng/g in subcutaneous adipose. This increase is a near 18-fold of D3 in loin tissue and a 9-fold increase in subcutaneous adipose tissue. In the present experiment 25(OH)D content averaged 2.81 ng/g (11.2 IU/100g) in the loin and 8.14 ng/g (32.6 IU/100g) adipose of sun-exposed pigs. Interestingly, our control pigs also had higher vitamin D content than pigs in the Clausen et al. analysis, which is likely attributable to the dietary supplementation of our pigs. The USDA nutrient database reports the vitamin D3 content of a raw, lean center loin to be 14 IU/100g. Our sun-exposed pigs had an average total vitamin D content of 39.9 IU/100g. The USDA nutrient database currently does not include the 25(OH)D3 content of meat products. Considering that 25(OH)D3 is present in natural animal-based products, and has five times the biological activity of vitamin D3, 25(OH)D3 should be measured and accounted for in future vitamin D analysis.In a previous study of Clausen and colleagues , a thoro3 content of subcutaneous adipose was not further improved by sun exposure and was similar between the sun-exposed and control pigs, despite a 100% percent increase in 25(OH)D3. The reason for this is unclear. It is possible that a ceiling effect for D3 storage in adipocytes was reached by the content of D3 in feed which produced near optimal serum concentrations even before sun exposure , and could not be further increased with sun exposure. Another possibility is that 25(OH)D3 concentrations increased in subcutaneous adipose as a direct result of higher serum 25(OH)D3, which may enhance adipose hydroxylation of 25(OH)D3. Further research is needed to address these possibilities.Interestingly, in the current study, the vitamin DThis study has shown that daily sun exposure for two weeks in pigs prior to slaughter increases the vitamin D content of the loin. Lean pork, recently shown to be an effective replacement for chicken and fish in the DASH (Dietary Approaches to Stop Hypertension) diet, can be an additional source of dietary vitamin D especially for individuals with high blood pressure . This reS1 TableStarting weights were taken on same day as first blood sample collected before first sun exposure. Slaughter weights were measured immediately before slaughter. Average daily gain in kg/d was also calculated, covering the entire study period. Weights are presented as means ± the standard error. Fat-free lean percentage was calculated as noted in Boggs et al.(XLSX)Click here for additional data file.S1 Fig3 concentrations before (Pre) and after (Post) sun exposure for one hour daily at solar noon for 2-weeks (SUN) or standard confinement (Control) in the summer and fall. Summer exposure occurred in the spring-summer group and fall exposure occurred in the summer-fall group in finisher pigs 2-weeks prior to slaughter.Serum 25(OH)D(XLSX)Click here for additional data file.S2 Fig3 and 25(OH)D3. The black bar represents the value presented in the USDA national nutrient database for the vitamin D content of “raw pork, center loin.” Center loin samples were collected on the day of slaughter. Error bars represent the standard error. (1 ug vitamin D3 = 40 IU).Total vitamin D content includes both vitamin D(XLSX)Click here for additional data file.


Arabidopsis plastidic HSP90C is an HSP90 family molecular chaperone that is required for chloroplast development and function. To understand the mechanism of action of HSP90C within the chloroplast, we conducted a yeast two-hybrid screening and revealed it interacts directly with the photosystem II extrinsic protein PsbO1, which performs a canonical function in the thylakoid lumen. To understand the biological significance of HSP90C-PsbO1 interaction, we investigated the role of HSP90C in modulating the stromal and thylakoid distribution of PsbO1GFP fusion protein. Fusion to GFP significantly delays the PsbO1 thylakoid transport and induces a variegation phenotype. Overexpression of HSP90C promotes the thylakoid distribution of PsbO1GFP and alleviates the leaf variegation. By tracking the chloroplast maturation during photomorphogenesis, we observed PsbO1GFP tends to form distinct fluorescent clusters within the stroma with delayed thylakoid membrane biogenesis, while HSP90C overexpression corrects these adverse effects. We also demonstrated that active HSP90C function is specifically required for stable accumulation of mature PsbO1GFP in thylakoid by using specific inhibitor geldanamycin. This study therefore not only identified novel HSP90C interactors, but also reports for the first time that PsbO1 enroute from the cytoplasm to thylakoid lumen is tightly regulated by the HSP90C chaperone complex in plastid stroma; whereas the proper HSP90C homeostasis is also critical for chloroplast maturation and function. The plates were incubated at 30°C for 4 days.mPsbO1T200A. In vitro pulldown of HA-tagged mature PsbO1T200A using anti-HA affinity resin from EGY48 cell lysate. Immunoprecipitated samples were immunoblotted using anti-LexA antibody or anti-HSP90C to detect the presence of HSP90C-MC protein. Anti-HA antibody was used to test the efficiency of co-immunoprecipitation.(B) Co-purification of HSP90C-MC and (TIF)Click here for additional data file.S2 Figa and b in extract was calculated by formula (μg/mL) = 20.2 (A645) + 8.02 (A663) after spectrophotometric measurement of the absorbance at 645 and 663 nm.The concentration of chlorophyll (TIF)Click here for additional data file.S3 FigThe homozygous psbO1 T-DNA insertion knockout line was confirmed by immunoblotting with anti-PsbO1 antibody. The seedlings were grown for 4, 7, and 11-days.(TIF)Click here for additional data file.S4 Fig(A) Top, Expression of PsbO1GFP results in formation of GFP clusters that do not overlap with chlorophyll fluorescence in palisade mesophyll cells. Middle, PsbO1GFP expression was found to induce formation of PsbO1GFP-containing stromule-like extensions. Bottom, smaller plastids above mesophyll chloroplasts were found to contain many small GFP clusters. Scale bar = 2μm.(B) Stromule-like structures were observed to connect from one plastid to another using z-stack analysis. Each slice is imaged 0.79μm in depth apart. Scale bar = 2μm.(TIF)Click here for additional data file.S5 FigData are represented as the mean ± STD. n = 100. *p < 0.05; Student’s t-test. Error bars represent standard deviation.(TIF)Click here for additional data file.S6 FigFLAG overexpression line and propagated to F3 generation. Siblings expressing PsbO1GFP with or without HSP90CFLAG were identified and grown at 22°C 110μmol/m2s and 16h light 8h dark cycle for 5 and 12-DAG (A), 28 and 35-DAG (B) and 41-DAG.Two independent transgenic lines OEX1 and OEX9 were crossed with an HSP90C(TIF)Click here for additional data file.S7 FigSeedlings were first grown on MS medium for 3.5 days in the dark and then under constant light. The chlorophyll contents were measured for seedlings grown under light for different times.(TIF)Click here for additional data file.S8 Fig1-58GFP (top) and PsbO11-85GFP (bottom).Seedlings were first grown on MS medium for 3.5 days in the dark and then switched to constant light. Confocal fluorescence images were taken for cotyledon chloroplasts in seedlings expressing PsbO1(TIF)Click here for additional data file.


Patients with high B7-H4 expression in their primary pancreatic cancer had higher risk of developing liver metastases (p < 0.05). In univariate analysis, B7-H4 expression was significantly associated with the risk of death (p < 0.05). And the multivariate analysis identified that B7-H4 was an independent prognostic indicator (p < 0.05). Our results revealed B7-H4 to be associated with poor prognosis in patients with pancreatic cancer liver metastasis. B7-H4 may promote pancreatic cancer metastasis and was promising to be a potential prognostic indicator of pancreatic cancer.Liver metastasis development in pancreatic cancer patients is common and confers a poor prognosis. Clinical relevance of biomarker analysis in metastatic tissue is necessary. B7-H4 has an inhibitory effect on T cell mediated response and may be involved in tumor development. Although B7-H4 expression has been detected in pancreatic cancer, its expression in liver metastases from pancreatic cancer is still unknown. In this study, overall 43 pancreatic cancer liver metastases (with matched primaries in 15/43 cases) and 57 pancreatic cancer cases without liver metastases or other distant metastases were analyzed for their expression of B7-H4 by immunohistochemistry. Survival curves and log-rank tests were used to test the association of B7-H4 expression with survival. B7-H4 was highly expressed in 28 (65.1%) of the 43 liver metastases and 9 (60.0%) of the 15 matched primary tumors. The expression of B7-H4 in liver metastases was significantly higher than in the matched primary tumors ( Pancreatic cancer is one of the most fatal malignancies, while the five-year survival rate of about only 5% . PancreaThe prognosis for pancreatic carcinoma is poor. Liver metastasis is often occurred in 70% of patients with pancreatic cancer. Occult liver metastases may already have been present at the time of surgery are one of the most common sites of treatment failure and are associated with poor prognosis –7. In reB7-H4 (B7S1 or B7X) is a member of B7 family –15 and iAlthough our primary research has found that B7-H4 was expressed in pancreatic carcer, and high expression level of B7-H4 was positive correlation with lymph node metastasis , 22, theA total 43 patients (26 men and 17 women) with liver metastases from pancreatic cancer were enrolled in the research. All patients were followed until May 2016 when 43 patients were deceased. All tumor types of the primary pancreatic cancer were infiltrating ductal adenocarcinoma. The median age was 66 years (range: 36–82 years) at the time of transliver biopsy. Table B7-H4 expression was high in 28 (65.1%) of 43 liver metastases and nine (60.0%) of 15 matched primary pancreatic cancers. The clinical significance of B7-H4 expression in liver metastases was analysed through comparing the expression level of B7-H4 with patients' clinical characteristics. There was no significant relationship between B7-H4 expression and age, sex, differentiation, or treatment modality (Table p < 0.05)  Figure .p < 0.05; Figure The differential expression of B7-H4 was also detected by immunohistochemical stain score criterion between the primary pancreatic cancers and control. The matched primary pancreatic cancer scores were significantly higher than that of the pancreatic cancer without liver metastases or other distant metastases diagnosed during the same period   and were independent prognostic factors in the multivariate analysis.We determined the role B7-H4 played in survival using Kaplan–Meier analysis and log rank test. The median overall survival (OS) was 6.0 ± 0.5 months . The median OS for patients who had high expression of B7-H4 in liver metastases was 4.0 ± 0.6 months (95% CI: 2.9–5.1 months) as compared with a median OS of 7.0 ± 0.8 months (95% CI: 5.4–8.6 months) for patients who had low expression of B7-H4  Figure . Tables Recently, advances in biotechnology and immunologic techniques had led to progress in research for anti-tumor immunological therapy. B7-H4 is a significant costimulatory molecule responsible for T cell inactivation , which iIn this research, we found that the expression of B7-H4 was higher in liver metastases compared with matched primary pancreatic cancers. In addition, we measured the expression of B7-H4 in a patient series with pancreatic cancer with or without (control) liver metastases, demonstrating that the matched primary pancreatic cancer with liver metastases had higher B7-H4 expression than control. The relative effect of B7-H4 on survival in patients with liver metastases was also determined. The aberrant expression of B7-H4 in liver metastases indicated poorer survival. Thus, B7-H4 may be a prognostic factor for pancreatic cancer patients with liver metastases.in vitro, suggesting that B7-H4 expression may shield tumors from immune surveillance by suppressing the tumor-infiltrating CD8+ T lymphocytes in the tumor microenvironment [B7-H4 inhibits both innate immunity and T cell responses in the tumor microenvironment . B7-H4 oironment , 28. B7-in vivo, and the tumor burden reduced in a murine tumor model using anti-mouse B7-H4 antibodies, indicating proof of therapy for targeting B7-H4 in vitro [In addition, B7-H4 might be a target for immunotherapy in tumor . Therapein vitro , 15, 30.in vitro . There rThe differential expression of B7-H4 in primary tumors and metastases indicated that B7-H4 was positively associated with tumor development and poor survival , 33. B7-In conclusion, our results found that the expression level of B7-H4 was higher in liver metastases than in matched primary pancreatic cancer. The high B7-H4 expression in liver metastases was positively correlated to poorer prognosis. Additionally, B7-H4 might promote progression and metastasis of pancreatic cancer and was promising to be a potential prognostic indicator of pancreatic cancer.We analyzed 43 patients pathologically diagnosed with liver metastases from pancreatic cancer between January 2000 and December 2015 at The Second Affiliated Hospital, Zhejiang University School of Medicine. All patients underwent transliver biopsy for liver metastases resection. In addition, 15 matched corresponding primary pancreatic cancers were obtained. The clinical parameters included patient demography, tumor differentiation, and treatment modality. Fifty-seven patients with pancreatic cancer who were treated during the same period without liver metastases or other distant metastases were enrolled as the control group. All tissues were fixed in 10% buffered formalin and embedded in paraffin. Two pathologists reviewed all archival hematoxylin and eosin–stained sections. The Ethics Committee of The Second Affiliated Hospital, Zhejiang University School of Medicine approved the present study.Immunohistochemical staining was performed using the two-step EnVision method . Paraffin-embedded tissues were cut into 5-μm serial sections, transferred onto adhesive slides, and dried at 65°C for 2 hours. The sections were deparaffinized with xylene and rehydrated through graded alcohols. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide solution for 30 minutes at room temperature and antigen retrieval was performed at 100°C for 30 minutes in citrate buffer . After washing three times with PBS at 5 minutes each time, the sections were incubated with 10% normal goat serum to block nonspecific binding. Then, the sections were incubated with rabbit anti-human B7-H4 monoclonal antibody  at 4°C overnight, followed by immunodetection using the Dako EnVision detection system (K5007). The slides were counterstained with Mayer's hematoxylin, dehydrated in graded alcohol, and mounted with a neutral resin. The negative control was performed by replacing the primary antibody with PBS. Human tonsil tissue was used as the positive control.Two pathologists without knowledge of the patients' clinical records examined and scored the sections. Five tumor fields at ×400 magnification were randomly selected. Cytoplasmic and/or tumor cell membrane staining were considered to indicate positive expression. A previously reported semiquantitative system  was usedt-test was used to analyze the significance of B7-H4 expression scores between the matched corresponding primary pancreatic cancer group and the control group. The OS was calculated from the diagnosis of liver metastases until death or the date of the last follow-up. Survival data were analyzed by the Kaplan–Meier method and log rank test. Cox proportional hazard model analysis was used to identify the independent prognostic factors of survival. P < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS . The relationship between B7-H4 expression and clinicopathological characteristics was compared using the chi-square test or two-tailed Fisher's exact test. McNemar's test for paired data was used to compare the frequency of B7-H4 expression between liver metastases and the matched primary tumors. The two independent samples


Both NK cell populations showed down-modulation of NKG2C and NKG2D expression, which was associated with high serum levels of the soluble form of NKG2D ligands. In contrast, an expansion of “memory” CD57+ NKG2C+ NK cells and high cytomegalovirus antibody titers were detected in SS patients. Profiling of the TLR signaling genes in NK cells from SS patients showed an abundance of differentially expressed genes (DEGs) in NK cells in the unstimulated condition, with mostly up-regulation of NFκB/JNK p38 pathway genes, but there was down-regulation of type I (IFN-α/β) and II (IFN-γ) interferon and IL-12A. After activation of NK cells with TLR7/8 agonist, the down-regulated genes correlated with the IFN response, and IL-12 became up-regulated, together with other antitumor factors. NK cell activation with a dual agonist for TLR7 and TLR8 is able to induce the expression of IFN-γ and type I IFN, which can improve immunity in SS patients.Sézary syndrome (SS), an aggressive and leukemic form of cutaneous T-cell lymphoma, usually results in shortened survival. Improving innate immunity in SS by targeting natural killer (NK) cells with Toll-like receptor (TLR) agonists could be an interesting modulatory strategy. We evaluated the NK cell populations in SS patients assessing activating and inhibitory receptors expression and profiled the differential expression of TLR signaling pathway genes in unstimulated NK cells and after TLR7/8 stimulation. We observed preserved CD56 Sézary syndrome (SS) is an aggressive and advanced form of CTCL with circulating malignant T cells. Impaired immunity in SS , 2 probadim CD16brightNK cells are predominant in the peripheral blood, whereas NK cells from secondary lymphoid tissue and from other tissues are primarily CD56bright NK cells [in vivo, human CD56bright NK cells may undergo progressive differentiation toward CD56dim NK cells [Natural killer (NK) cells are cytotoxic type 1 innate lymphoid cells with potential for cancer immunotherapy and for treating viral infections. There are two NK cell populations: CD56NK cells . MoreoveDecreased NK cell activity has been described in CTCL –8, whereMajor NK cell-activating receptors involved in cancer cell recognition include NKG2D. This activating immune receptor has been identified on NK cells, CD8 T cells, NKT cells, and subsets of γδ T cells . NKG2D eExpansion of an NK cell subset carrying an activating receptor heterodimer, comprising CD94 and NKG2C (CD94/NKG2C) could be elicited by human cytomegalovirus (HCMV) infection , 14. CD5The scientific and clinical interest in TLR7 and TLR8 in cancer biology has originated from the antitumor activities of these molecules in preclinical models . ImiquimAn attempt to enhance NK cell function using synthetic agonists of TLRs has been explored in SS. The combined effects of synthetic oligodeoxynucleotides (ODN) with the CpG motif (TLR 9 agonist) and IL-15 on the activation of NK cells in patients with CTCL led to induced activation of NK cells and enhanced activation of CD8 T cells . The CD1Here, we propose to investigate innate immunity in SS by evaluating NK cell subsets, activation/inhibitory receptor expression, soluble NKG2D ligand levels, “memory” phenotype NK cells, and the effector response induced by a TLR7/8 agonist and differentially expressed genes (DEGs) related to the TLR signaling pathway.Disturbance of innate immunity has been described in SS, including a reduction of NK cells . Despitedim NK cells . We assessed purified NK cells to verify the direct effect of TLRs activation on NK cells. By PCR array, 84 genes of the TLR signaling pathway  were assFigure Figure The ratio between up/down-regulated genes changed from 2.52 in the unstimulated condition to 0.96 after TLR7/8 activation, revealing that in the unstimulated condition most of the DEGs were up-regulated in NK cells from SS patients.dim NK cells, while the CD56bright NK population was preserved. The equilibrium percentage of CD56bright NK cells in SS must be crucial in attempting to replace the number of terminally differentiated CD56dim cells, which seems to be due to an impaired output of NK cells from bone marrow. It is known that the percentages and absolute numbers of NK cells increase during healthy aging, but the decrease in the CD56 bright subset is interpreted as a lower output of such NK cells from the bone marrow [dim NK cell population is preferential for causing apoptosis in cancer [The innate lymphocytes NK cells are essential for innate immune responses against cancer and viral infections. Indeed, a genetic NK deficiency causes severe herpes virus infections and affects tumor clearance . In SS, e marrow . Moreoven cancer .NK cell receptors provide signals, which determine whether an NK cell becomes activated or inhibited. We detected a down-modulation of NK-activating receptors, such as NKG2D and NKG2C, regardless of NK population, while the inhibitory receptor NKG2A remained unchanged in SS subjects. In addition, decreased NKG2D mRNA expression was detected in the PBMCs from the SS group with wide NKG2D down-modulation, including by CD3+ T cells. Down-regulation of NKG2D could be due to factors such as TGF-β or metalloproteinases  and/or tbrightNKG2D+ NK cells and sMICB levels was detected. This finding suggests that NKG2D ligands may interact with receptors to induce internalization and degradation, contributing to its down modulation in SS patients.A broad variability of NKG2D expression has been described in some SS patients (2/6), some with severe down-regulation . In our An increased percentage of CD56+CD57+ NKG2C+NK cells was found in SS patients, together with high seropositivity to CMV. The subset of CD57+NKG2C+ NK cells has been described as exhibiting memory-like features and has potent effector functions ; HCMV inbright NK cells. Therefore, to better investigate the NK response following TLR7/8 activation, we assessed DEGs related to the TLR signaling pathway in NK cells. We used purified NK cells to verify the direct role of TLR activation, and we did not split the NK cells into subtypes due to the scarcity of NK cells in patients with SS. Of note, unstimulated NK cells from SS patients showed abundant DEGs of the TLR signaling pathway (48/84 genes). These up-regulated genes included NFκB, JNK/p38 and MyD88-dependent genes and revealed the activated status of NK cells. Among the TLRs, the most intensively expressed was TLR2, which was possibly a consequence of bacterial infection; this is not surprising considering that sepsis is a common cause of death among SS patients. This aligns with a previous finding that some patients with SS showed up-regulation of TLR2 mRNA expression in unstimulated PBMCs [Employment of adjuvants such as TLR agonists has been widely studied in APCs and other cells, including NK cells. Human NK cells express TLR3, TLR7, and TLR8 . Previoued PBMCs .Other up-regulated genes expression in unstimulated NK cells from SS patients were prostaglandin synthase 2 (PTGS2/COX2), which exerts a negative effect on NK cell function via PGE2 through PGE2 receptor EP4 . MoreoveAlthough DEGs were mostly up-regulated in NK cells from SS patients under normal conditions, the down-regulated DEGs included genes in the IFN pathway, such as IRF3, TLR7, IFNA1, TLR3 and IL-12A, and the IFN-γ-inducible chemokine, CXCL10. Importantly, NK cells seem to contribute to the deficit in IFN-γ, type I IFN and IL-12 genes, which are well characterized in SS patients , 57, forNotably, the profile of DEGs after TLR7/8 stimulation showed that several inhibited genes became up-regulated, including those related to the type I and II IFN responses  and IL-12 (IL-12A). We verified that CL097 was an interesting adjuvant to enhance the activity of NK cells in SS. Included in the NK genes up-regulated after CL097 activation was HMGB1, which can play an important role against tumor cells. NK cells invoke this strategy by employing HMGB1 protein, which specifically targets glycolysis in cancer cells and opens up new perspectives for cancer immunotherapy . MoreoveHowever, some genes are down-regulated after stimulation, including IRF-1, a pivotal factor in the regulation of NK cells during infection, inflammation and metastasis , while PMoreover, our data with TLR7-8 as adjuvant to potentiate the innate immunity, as NK cells, aligns with the data that topical resiquimod, an agonist of TLR7/8, is safe and effective in patients with stage IA-IIA CTCL, that can clear both treated and untreated skin lesions .dimNK population number, down-modulation of NKG2D, and impairment of IFN-γ response, and the other revealing a preserved CD56bright NK population and the presence of mature, memory CD57+NKG2C+ NK cells. In addition, NK cells exhibited a constitutively activated status with abundant DEGs; after CL097 activation, up-regulation of crucial genes of the IFN response and anti-tumor response occurred. These findings emphasize the use of TLR agonists as adjuvants to improve innate immunity with NK cells in CTCL.Our findings showed that NK cells in SS display different features than those in healthy subjects, with one aspect being decreased CD56We enrolled patients with Sézary syndrome , with a median age of 61 years (range 48-84 years), from the Clinic of Cutaneous Lymphomas of the Department of Dermatology, Hospital das Clínicas, University of São Paulo Medical School, Brazil (HC/FMUSP). Diagnosis of SS was performed according to the revised clinical, histological, and biological criteria proposed by the International Society for Cutaneous Lymphomas (ISCL) and the cutaneous lymphoma task force of the European Organization of Research and Treatment of Cancer (EORTC) . All SS To analyze activation/inhibition markers in the NK cells, venous blood was collected in EDTA anticoagulant tubes, and staining was performed using the following antibodies: CD3 (BV605/clone SK7), CD19 (Horizon V500/clone HIB19), CD16 (APC-Cy7/clone 3G8), CD56 (Alexa 700/clone B159), NKG2D (PECy7/clone 1D11) NKp46 (APC/clone 9-E2) and CD57 (APC/clone NK-1) from BD Biosciences  and NKG2A (PE/clone 131491) and NKG2C (Alexa Fluor 488/clone 134591) from R&D Minneapolis, MN, USA. Approximately 70 μL of whole blood was stained for 20 min and then incubated for 15 min with FACS lysing solution  to lyse the erythrocytes. After two washes in an isotonic solution , 300,000 events were acquired using a flow cytometer  and were analyzed using FlowJo Software .Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation with Ficoll-Hypaque density gradient . PBMCs (1.0×106 cells/mL) were incubated with 5.0 μg/mL TLR7/8 agonist , phorbol myristate acetate (50 ng/mL) and ionomycin (1 μg/mL) and CD107a PE-Cy5 (Pe.Cy5/clone H4A3) to detect degranulating NK cells and incubated at 37°C with 5% CO2 for 6 h. After 2 h of incubation, Brefeldin  was added to the cultures for another 4 h of incubation. After incubation, the cells were incubated with Live-Dead reagent  for 20 min at room temperature. Cells were then subjected to fixation with Cytofix/Cytoperm solution  for 20 min and permeabilization with Perm/Wash solution for 20 min at 4°C. The cells were then stained with CD3 (BV605/clone SK7), CD19 (Horizon V500/clone HIB19), CD16 (APC-Cy7/clone 3G8), CD56 (Alexa 700/ clone B159), IFN-γ (Horizon V450/clone B27) and TNF (PE-Cy7/clone MAB11) antibodies. Next, the samples were washed with Perm/Wash buffer  and diluted in isotonic solution. Fluorescence Minus One (FMO) controls were performed for all antibody panels to confirm proper compensation and define positive signals. A total of 300,000 events were collected and analyzed by flow cytometry .Total RNA was extracted from PBMCs using an RNeasyPlus Mini Kit , and reverse transcription was performed with a Sensiscript Reverse Transcriptase Kit (Qiagen). The primers used in the real-time PCR assay for NKG2C, NKG2D, MICA, HLA-E, and UBLPB-3 are shown in GAPDH mRNA levels were used to normalize the mRNA content from PBMCs. PCR was performed on an Applied Biosystems 7500 system using specific primers and SYBR Green  fluorescence detection reagents. The cycling protocol consisted of 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 60 s at 60°C. The amplification results were visualized and analyzed using Sequence Detection System (SDS) software (Applied Biosystems). Normalized expression was calculated as previously described by Livak .Serum levels of ligands for the receptor NKG2D, such as MICA and MICB, were assessed by ELISA according the manufacturer recommendations . Serology for HMCV was performed using a kit from Virion\Serion .Purified NK cells from the PBMCs of three SS patients  and three HC subjects by cell sorting were left in RPMI medium supplemented with 10% human AB serum  for 18 h at 37°C. Cells were stimulated with a TLR7/8 agonist (5.0 μg/mL) or with medium only (unstimulated) for 4 hours at 37°C. The cells were stored in RNA later solution  at -80°C. Total cellular RNA was extracted from NK cells using an RNeasy Micro Kit (Qiagen). The first strand of cDNA was synthesized based on the instructions for the RT2 First Strand Kit and PreAMP kit , and the final cDNA product was used for the RT2 Profiler PCR Array using SYBR Green-based real-time PCR according to the manufacturer's protocol . The array consisted ofa panel of 84 genes relevant to the TLR pathway  plus fivThe Mann-Whitney U-test was used to compare variables between the groups, the Wilcoxon matched pairs test was used to compare the baseline level with stimulated samples. p≤0.05 was considered significant.


Many genes are differentially expressed in the cortex of people with schizophrenia, implicating factors that control transcription more generally. Hormone nuclear receptors dimerize to coordinate context-dependent changes in gene expression. We hypothesized that members of two families of nuclear receptors (NR4As), and retinoid receptors (RARs and RXRs), are altered in the dorsal lateral prefrontal cortex (DLPFC) of people with schizophrenia. We used next generation sequencing and then qPCR analysis to test for changes in mRNA levels for transcripts encoding nuclear receptors: orphan nuclear receptors  in total RNA extracted from the DLPFC from people with schizophrenia compared to controls (n = 74). We also correlated mRNA levels with demographic factors and with estimates of antipsychotic drug exposure (schizophrenia group only). We tested for correlations between levels of transcription factor family members and levels of genes putatively regulated by these transcription factors. We found significantly down regulated expression of NR4A1 (Nurr 77) and KLF4 mRNAs in people with schizophrenia compared to controls, by both NGS and qPCR (p = or <0.01). We also detected decreases in NR4A2 (Nurr1) and RXRB mRNAs by using qPCR in the larger cohort . We detected decreased expression of RARG and NR4A2 mRNAs in females with schizophrenia (p<0.05). The mRNA levels of NR4A1, NR4A2 and NR4A3 were all negative correlated with lifetime estimates of antipsychotic exposure. These novel findings, which may be influenced by antipsychotic drug exposure, implicate the orphan and retinoid nuclear receptors in the cortical pathology found in schizophrenia. Genes down stream of these receptors can be dysregulated as well, but the direction of change is not immediately predictable based on the putative transcription factor changes. Schizophrenia is a serious psychiatric disorder adversely affecting the quality of life of a significant number of people . SchizopA group of 48 transcription factors play a key role in transducing extracellular  signals into intercellular signals, resulting in changes in expression of target genes. The nuclear receptors (NRs) are grouped into 6 functionally related sub-families (NR1—NR6) and include the estrogen and androgen receptors (NR3A1/ESR1 and NR3C4/AR), the glucocorticoid and mineralocorticoid receptors (NR3C1/GR and NR3C2/MR), the retinoid receptors (NR1B/RARs and NR2B/RXRs), the vitamin D receptor (NR1I1/VDR), the peroxisome proliferator-activated (fatty acid) receptors (NR1C/PPARs) and the orphan nuclear receptors (NR4A sub-family)–8, the rTo gain a more complete picture of alterations in nuclear receptor alterations, we have focused this study on the NR4A sub-family of nuclear receptors (NR4A1 (Nurr 77 or NGF1B), NR4A2 (Nurr1), NR4A3 (Nor1)), and their dimerization partners, the retinoid X receptors  and the retinoic acid receptors .The RAR proteins are activated by all-trans retinoic acid while the RXR proteins are activated by by 9-cis retinoic acid, and other ligands such as the omega 3 unsaturated fatty acids and various synthetic compounds , 12. NR4While NR4A2 has an important role in the cell body of dopaminergic neurons, the action of NR4A1 is more pronounced at target areas of the dopaminergic neurons, such as the prefrontal cortex. The NR4A1- RXR complex is suggested to function as an adaptive homeostatic regulator of dopamine neurotransmission . BlockerIn this study, we have quantified and compared the mRNA expression of genes encoding nuclear receptors with a focus on those in the NR4A and RXR/RAR families. This study aims to determine if altered levels of the mRNA expressions in orphan nuclear receptors and retinoid receptors exist in the brains of people with schizophrenia using next generation sequencing and by real time quantitative polymerase chain reaction (RT-qPCR) in the DLPFC.Dorsal lateral prefrontal cortex (DLPFC) from thirty-seven schizophrenia/schizoaffective cases and thirty-seven controls was obtained from the New South Wales Tissue Resource Centre. Of the thirty-seven schizophrenia/schizoaffective cases, eight were on first generation antipsychotics only, twenty-two had predominantly received first generation antipsychotics, one was one second generation antipsychotics only, five had predominantly received second generation antipsychotics, and one received equal first and second generation antipsychotics. Cases were matched for sample pH, age, post-mortem interval (PMI), and RNA integrity number (RIN) . DetailscDNA derived from total RNA from the DLPFC tissue of a cohort of 20 schizophrenia/schizoaffective cases (referred to as schizophrenia) and 20 control samples was sequenced using the ABI SOLiD platform as previously described . In thisIn carrying out this analysis, we have used read data (fastq files) obtained from RNA-Seq previously performed on the DLPFC of post-mortem brain. Tools for the analysis of RNA-Seq data have improved rapidly over recent years. Since the time when this data was first analyzed  commonlyIn the edgeR analysis, low count transcripts were excluded and only those genes with at least 1 count per million (cpm), in at least 10 samples, were used for analysis. This filtering retained 17,483 of the original 42,358 transcripts. Normalization was performed using the trimmed mean of M values (TMM) . The disIn the DESeq2 confirmatory analysis, normalization was performed using the median-of-ratios method . DispersThe differential expression between schizophrenia and control are distinct from those obtained in our earlier analysis  due to thttp://www.R-project.org) [www.cytoscape.org) [Clustering functions available in the gplots package in the R environment (v 3.0.2) (ect.org)  were useape.org)  was usedSuperScript® II/III First-Strand Synthesis System  was used for cDNA synthesis from 3 μg RNA. The protocol for SuperScript® II was followed by adding random hexamers, nucleoside triphosphate (dNTP), and RNase OUT to each sample. After incubating at 65°C, tris-hydrochloride (tris-HCl), potassium chloride (KCl), magnesium chloride (Mg2Cl), dithiothreitol (DTT) and SuperScript® II were added. All samples were incubated at room temperature for 10 min before heating it to 42°C for 50 min, followed by 70°C for 15 min. RNase H was added to all samples before incubating at 37°C. We followed the protocol for SuperScript® III First-Strand Synthesis System according to manufacturer’s instructions.cDNA was plated out with a seven-point serial diluted standard curve, followed by quantitative real time PCR, probing with various primers to amplify members of the nuclear receptor superfamily . SamplesNormalized data was analyzed using IBM SPSS Statistics 23.0. KLF4 and RXRB were log10 transformed for normal distribution within each diagnostic group. All data were tested for correlation with age, pH, PMI and RIN. The correlations between the gene expressions with each of these factors are listed in We sought to characterize nuclear receptor mRNA change in the context of the expression landscape of all nuclear receptor genes in the DLPFC. A cluster-based analysis revealed that the nuclear receptor genes expressed in the adult human prefrontal cortex fall into three main groups, highly expressed genes, moderately expressed genes and lowly expressed genes . Our anaUsing RNA-Seq, we analyzed gene expression in the DLPFC of 19 schizophrenia patients and compared that to 19 controls. The biological coefficient of variation (BCV) calculated using the methods available in edgeR produced a value of 0.3863 for the 19 control samples and 0.479 for the 19 schizophrenia samples. This does indicate a slightly greater degree of variability in samples from people with schizophrenia compared to samples from the controls; however it also reflects that there is also quite a bit of variability in samples from controls as well. Considerable gene expression variability was seen between individuals, which was unsurprising for human brain and patient-derived samples. This could be due to the uncontrolled factors in case-control studies  and also due to the heterogeneous nature of schizophrenia. Consequently, we did not see a strong distinction between the two diagnostic categories when examining global gene expression via a multidimensional scaling (MDS) plot . HoweverIn this analysis, the most significant differentially expressed gene was the nuclear receptor NR4A1 (Nur77), for which expression is reduced in schizophrenia . The next most significant gene expression change was KLF4 (kruppel-like factor 4), which is reduced by a similar amount in schizophrenia . We confirmed the edgeR results using a different analysis program, DESeq2, which also found NR4A1 and KLF4 as the most significantly differentially expressed genes with decreases in schizophrenia similar to that found with edgeR  .To further validate these changes, quantitative PCR was used to measure the expression of these genes in the complete cohort of 74 individuals (37 schizophrenia and 37 controls). This established that both genes were significantly decreased in schizophrenia,  = 8.101, p<0.01); NR4A1: F = 6.912, p = 0.01; Using qPCR in the expanded cohort (37 schizophrenia and 37 controls), we confirmed decreased expression of NR4A1 and also found a significant decrease in NR4A2 expression in schizophrenia  = 4.655, p<0.05) and RXRB: F = 10.256, p<0.01; We performed Pearson’s correlations of the mRNA expression found by qPCR for NR4 sub-family and the retinoid receptors in our extended cohort. Correlations were performed across the combined group of controls and schizophrenia patients. We found that NR4A1 mRNA was significantly correlated with the two other closely related mRNAs NR4A2 and NR4A3. RXRG mRNA was significantly correlated with the other two RXRs (A and B). RXRB mRNA was also correlated with RARG mRNA and NR4A3 mRNA was correlated with RARA mRNA .We found a negative correlation between expression of the NR4A family genes and age (NR4A1: r(71) = -0.419, p = 0.0003; NR4A2: r(71) = -0.515, p = 0.000004; NR4A3: r(71) = -0.330, p = 0.005; We found a significant negative correlation between daily chlorpromazine dosage with NR4A1 and NR4A3 (NR4A1: rho(35) = -0.594, p = 0.0002; NR4A3: rho(36) = -0.438, p = 0.008) and no significant correlations between the last recorded chlorpromazine dosage to any of the mRNAs measured. There was a significant negative correlation between estimated lifetime chlorpromazine dosage and expression of NR4A1, NR4A2 and NR4A3 mRNAs (NR4A1: rho(35) = -0.601, p = 0.0001; NR4A2: rho(35) = -0.383, p = 0.023; NR4A3: rho(36) = -0.403, p = 0.015; We also found significant negative correlations between NR4A1 and NR4A2 mRNAs with duration of illness, and a trend between NR4A3 mRNA levels and duration of illness (NR4A1: rho(35) = -0.461, p = 0.005; NR4A2: rho(35) = -0.372, p = 0.028; NR4A3: rho(36) = -0.310, p = 0.067). We did not find a significant correlation between duration of illness and any retinoid receptors mRNAs measured .Because there were significant correlations between the NR4A genes with age, we re-analyzed correlations between the NR4A mRNA expressions with illness duration and lifetime dosage in a partial correlation, factoring for age. We found NR4A1 mRNA expression remained significantly correlated with the estimated lifetime antipsychotic dosage (r(32) = -0.401, p = 0.019) and a trend in NR4A3 (r(33) = -0.311, p = 0.069).We found there was a decrease of RXRG mRNA in females with schizophrenia  = 4.97, p = 0.029) and of RARG mRNA in females with schizophrenia  = 3.942, p = 0.05; To investigate the biological effect of NR4A1, NR4A2 and RXRB down-regulation, we generated a network representation of the genes annotated as being transcriptionally regulated by the NR4 sub-family or RXRB . The dowGenerally, the annotated interactions involve transcriptional activation, although in some cases there is an inhibitory effect of the transcription factor on the target gene. We see some indication of decreased expression in genes activated by NR4A1, for instance PYGM  , consistent with a decrease in NR4A1 activity while genes thought to be inhibited by NR4A1, such as PPARG (peroxisome proliferator-activated receptor gamma) , have higher levels in schizophrenia. However, the overall picture is complicated with examples of the converse also being demonstrated. This leads us to suggest that complex mechanisms are involved in the transcriptional regulation of these genes and that down-regulation of the NR4A genes and RXRB alone may not be sufficient to induce all the expected changes in downstream gene expression.This study presents evidence for down-regulation of the nuclear receptors NR4A1, NR4A2, RXRB, and KLF4 mRNAs in the DLPFC in schizophrenia and evidence of reduced RARG and RXRG expression in females with schizophrenia. This study also found a negative correlation between the expression of NR4A genes and estimated levels of antipsychotic exposure. To our knowledge, this is the first study to report a correlation between clinical lifetime chlorpromazine dose and decreased expression of NR4A genes in the DLPFC. Our finding of decreased expression of NR4A1 and NR4A2 mRNA in a large cohort confirms and expands upon a previous finding in post-mortem schizophrenia in the same area of the brain , whereasThe therapeutic efficacy of various antipsychotic drugs depends upon antagonism of the D2 dopamine receptors . SchizopIt has previously been reported that NR4A1 and NR4A3 expression increases in the murine prefrontal cortex upon a single administration of chlorpromazine . Maheux It has been proposed that NR4A1 and RXR work together as adaptive homeostatic regulators of dopamine function by reducing the effect of alterations in dopamine neurotransmission . Given tFunctions ascribed to NR4A1 in neuronal differentiation and neurite outgrowth , 37, leaIn our analysis, we found all three NR4A receptor mRNA levels to be decreased with age. The role of NR4A receptors in brain aging is currently unknown. With human brain aging, increased DNA damage is found and since NR4A receptors may protect against DNA damage , 43 our The decrease in RXRB in schizophrenia is interesting as the genetic locus of RXRB (6p21.3) has been linked to schizophrenia . AblatioOur finding of decreased RARG and RXRG mRNA levels in females with schizophrenia may be related to changes found in estrogen and/or estrogen receptors (ER) signaling. Indeed, direct protein interaction between the retinoid receptors and the ERs via their ligand binding domain has been documented , 56. FurWe have also found KLF4 to be differentially expressed in schizophrenia compared to controls. KLF4 is a transcription factor in the kruppel-like factor family which regulates multiple biological functions and is involved in neurogenesis, neuronal differentiation and neurite outgrowth . KLF4 isTranscriptional regulation by the nuclear receptors is complicated by heterodimerization and their activation by multiple ligands. Furthermore, recent genome-wide studies reveal that NR binding regions are enriched for sequence motifs of other transcription factors, such as Sp1, AP-1, and C/EBP motifs, suggesting that NRs interact with other transcription factors to regulate target gene expression , 78. TheThis study reports significant changes in the nuclear receptors NR4A1, NR4A2, and RXRB and KLF4 in schizophrenia and provides further evidence of a role for the nuclear receptors in the disease process. Evidence is growing in support of an important role for NR4A1 and NR4A2 in neurogenesis, learning and memory, which may be associated with the role of NR4A family genes in dopaminergic pathways. Cognitive defects and changes to dopamine signaling are well known effects of schizophrenia and of current treatment protocols. These genes also play a role in immune function which is emerging as an important focus in schizophrenia research , 80, 81.S1 FigMultidimensional scaling (MDS) plot of all samples, SCZ samples in batch 1 (orange), Control samples in batch 1 (cyan), SCZ samples in batch 2 (red), Control samples in batch 2 (blue). (B) DEGs were identified using a glm model in both the edger and DESeq2 tools taking account of batch and SCZ, using the default settings for edgeR and DESeq2.(TIF)Click here for additional data file.S2 FigThe differentially expressed genes calculated using edgeR but varying the parameter prior.df., using the current default setting (prior.df = 10) and increasing this to prior.df = 70 , and prior.df = 175  and prior.df = 350 .(TIF)Click here for additional data file.S3 FigOverview of the normalized expressions of NR4A1, NR4A2, NR4A3, KLF4, RARA, RARB, RARG, RXRA, RXRB, and RXRG. Blue bars indicate control group and red bars indicate schizophrenia group, all showing standard error of mean (SEM). * represents significance.(TIF)Click here for additional data file.S4 FigNormalized expressions of a) NR4A1 b) NR4A2 and c) NR4A3 correlated against age.(TIF)Click here for additional data file.S5 FigTwo-way ANCOVA analysis of the normalized expression of diagnosis and gender of a) RARG and b) RXRG.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Tablea. EdgeR DE analysis using only common dispersion. b. EdgeR DE analysis using df = 350 equating to prion.n = 10. c. EdgeR DE analysis using df = 175 equating to prion.n = 5. d. EdgeR DE analysis using df = 70 equating to prion.n = 2. e. EdgeR DE analysis using current defaults (df = 10).(ZIP)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


Schizosaccharomyces pombe as readout, to explore whether replication perturbation affects nucleosome-mediated epigenetic inheritance. We show that replication stresses, due to either hydroxyurea treatment or various forms of genetic lesions of the replication machinery, reduce the inheritance accuracy of CENP-A/Cnp1 nucleosome positioning within centromere. Mechanistically, we demonstrate that excessive formation of single-stranded DNA, a common molecular abnormality under these conditions, might have correlation with the reduction in fidelity of centromeric chromatin duplication. Furthermore, we show that replication stress broadly changes chromatin structure at various loci in the genome, such as telomere heterochromatin expanding and mating type locus heterochromatin spreading out of the boundaries. Interestingly, the levels of inheritable expanding at sub-telomeric heterochromatin regions are highly variable among independent cell populations. Finally, we show that HU treatment of the multi-cellular organisms C. elegans and D. melanogaster affects epigenetically programmed development and PEV, illustrating the evolutionary conservation of the phenomenon. Replication stress, in addition to its demonstrated role in genetic instability, promotes variable epigenetic instability throughout the epigenome.The fidelity of epigenetic inheritance or, the precision by which epigenetic information is passed along, is an essential parameter for measuring the effectiveness of the process. How the precision of the process is achieved or modulated, however, remains largely elusive. We have performed quantitative measurement of epigenetic fidelity, using position effect variegation (PEV) in  In this study, we found replication stresses reduce the fidelity of nucleosome-mediated epigenetic inheritance. Using Position Effect Variegation (PEV) in centromere as an indicator of chromatin epigenetic stability, we quantified the precision of nucleosomal inheritance and found replication stresses reduce the fidelity of nucleosome-mediated epigenetic inheritance. Further analysis of genome-wide heterochromatin distribution showed that replication stresses affect chromatin structure by expanding of heterochromatin with locus specificity. Mechanistically, we provide evidence suggesting that excessive formation of single-stranded DNA might have correlation with the reduction in fidelity of centromeric chromatin duplication. Finally, we demonstrated replication stress perturb the development process by reducing the fidelity of chromatin organization duplication in fruit fly and worm, illustrating the broadness and the evolutionary conservation of the phenomenon. Together, our results shed light on the importance of replication stresses cause epigenetic instability in addition to genetic stability. In eukaryotic cells, genomic DNA are packaged into arrays of nucleosomes , each co2 tetramers in specific–are recycled to assemble nucleosomes on one of the daughter strands behind the replication fork. Finally, newly synthesized histones are incorporated on the other daughter strand to form nucleosomes de novo. Although the two daughter strands can be distinguished based on whether they originate from the leading or lagging strand in replication, in general, there appears to be no strand-specificity for histone recycling and de novo nucleosome assembly.During cell division, chromatin is duplicated in conjunction with DNA synthesis at the replication fork, through a process called replication coupled (RC) nucleosome assembly ,5. The pde novo on the other strand need to be positioned at the corresponding sites. The newly incorporated histone molecules should also be of the proper variant type and obtain the biochemical modifications matching that of the parental histones. Currently, little is known on how the precision is achieved for any of the above steps. An important aspect of chromatin duplication is that the parental H3-H4 histones are transferred from the template chromatin to the replicated strands as intact (H3-H4)2 tetramers ). ). mcm4-M as 25°C . HoweverThe above mechanistic model highlights the contribution by impaired parental histone recycling to epigenetic instability. It is conceivable that such effect is broadly applicable throughout the epigenome. Although, this does not exclude the possible contribution of defects in other steps of RC nucleosome assembly in association with replication stalling, such as the maturation of newly assembled nucleosomes in which new histones obtain proper post-translational modification marks.cdc22 cause heterochromatin spreading beyond the silent mat locus.S1 Figcds1-D (LW21) and mcm4-84c (CY344) colonies exhibit enhanced sectoring at the presence of HU. Cells of the mutant strains and wild type (XL762) are planted at the density of one cell per cm2 on YE+4S plate containing HU as labeled, and incubated at 25°C for eight days. (B) High degrees of sectoring on the colonies of the replication mutants. Wild type, ctf8-D and ssb3-D cells are planted on YE+4S plates, and psf1-HBD cells on YE+4S+100nM β-estradiol and YE+4S+0.1nM β-estradiol, respectively, incubated at 25°C for eight days. Wild type and mcm4-M68 cells incubated at 29 oC for six days. One representative colony for each was shown. Scale bar is 2mm.(A) (TIF)Click here for additional data file.S2 FigThe protein levels of Ssb2-GFP are examined by western blot analysis, using ribosome protein S6 as a negative control. The gray value of each band is measured, and the ratios of GFP/S6 of the indicated strains were normalized to the value of GFP/S6 in wild type without HU treatment.(TIF)Click here for additional data file.S3 Figmcm4-M68 (LW22) mutant cells show elongated cell shape compare with wild type cells at 29°C. Both mutant and wild type cells are grown at 25°C over night, then shift to 29°C for 6h. Cells are harvested and fixed with methanol. After washing with PBS, cells are stained with Hoechst dye for microscopic examining of the nuclear morphology. Scale bar is 10μm. (B) mcm4-M68 mutant cells exhibit mild reduced survival rate at 29°C. Cells are grown at 25°C over night. Cell suspensions of both strains are plated onto YE+5S solid media with 400 colonies per plate, and incubated at 25°C and 29°C separately. The number of survival colonies was counted. (C) Increased levels of Ssb2-GFP signal in the mutant G2 phase cells. Wild type, mcm4-M68 cells are grown in the liquid YE+5S media at 25°C over night, then shift to 29°C for 6h. Ssb2-GFP signal is measured as in (A) (TIF)Click here for additional data file.S4 FigL(BglII)::ade6+and ura4::cenH-ade6+ strains were picked and the ade6+ reporter was PCR amplified and subjected to sequencing. No mutation was found in ade6+ in these colonies. DNA sequence alignment for one of the four sequencing results is shown. Query: DNA sequence of ade6+ PCR product from red colonies. Subject: DNA sequence of ade6+ gene downloaded from website (http://www.pombase.org).Four red colonies from (TIF)Click here for additional data file.S5 Figmat2-P at the mating type locus in clr1-D strain. mat1-msmt0 clr1-D haploid cells (LW63) and h-/h+ wild type diploid cells (LW52) are plated on sporulation medium (EMM-N) containing HU as labeled, incubated at 30°C for five days, stained with iodine vapor. Two colonies each are shown. Cell suspensions of the colonies are then stained with Hoechst dye for microscopic examining of the nuclear morphology. The percentage of clr1-D cells showing a haploid meiosis phenotype (H.M.) is quantified in five independent colonies with >100 cells scored in each. (C) HU treatment promotes heterochromatin in clr1-D strain. mat1-msmt0 clr1-D haploid cells (LW63) were cultured in EMM5S liquid media until OD600 reached 0.1, then shifted to EMM-N liquid media with or without 2mM HU for 17 hours. Cells were harvested and immuno-precipitated with anti-H3K9me2 antibody. Heterochromatin enrichment was examined by ChIP using quantitative real-time PCR. Recovery ratios of immuno-precipitated DNA to total DNA at the indicated loci were normalized to the value of tubulin gene. Data are mean ±s.d. (n = 3).(A) The diagram illustrates the genetic construct of the mating type locus. Light green box indicates a compromised silencing domain. (B) HU treatment restores the silencing of (TIF)Click here for additional data file.S6 Figcen), telomere (tel), and mating type (mat) locus, H3K9me2 peaks distribute across the genome. (B) H3K9me2 distribution is shown at individual loci. Chromosome positions in (A) and (B) correspond to Sanger Center pombe database 2015 assembly. (C) H3K9me2 enrichment DNA reads in three biological wild type cells were plotted over sub-telomeric region of all three chromosomes. Chromosome coordinates are indicated above the peaks. Scale bars on the right denote H3K9me2 enrichment DNA reads numbers normalized per one million reads.(A) Relative fold enrichment of dimethylated H3K9 (H3K9me2), as determined by ChIP-Seq, is plotted. Besides centromere ((TIF)Click here for additional data file.S7 Figade6+ and ura4+ transgenes flanking the silent domain (green box). (B) HU induces transgenes silencing. Cells are pre-treated with 0mM or 2mM HU for 24 hours then spread on YE+4S+5-FOA or YE+4S+5-FOA+2mMHU, respectively, and incubated at 25°C for 7 days. The 5-FOA resistance colonies in white or red color are counted. (C) Stability of the silenced states of the transgenes. Cell suspension of five different 5-FOA-resistent (IRR::ura4+-silent) colonies are plated on the YE+4S and YE+4S+5-FOA plates, and incubated at 25°C for 7 days. The total viable colonies and 5-FOA-resistent colonies are counted, and the percentages of IRR::ura4+-silent progeny are calculated. The error bars are 1 SD of percentages for four independent plates.(A) Diagram illustrates the genetic construct at the mating type locus of strain PSG552, carrying (TIF)Click here for additional data file.S8 Figcdc22-3 mutant cells. Atf1 mRNA is detected using RT-PCR. ß-actin mRNA is used as an internal standard. And the relative ration of Atf1 mRNA in wild type is normalized to 1. (B) The mRNA expression level of Atf1 is decreased in clr1D cells and is slightly increased in clr1D cells with HU treatment. (C) The diagram illustrates the genetic construct of the mating type locus. Green box indicates a heterochromatin region. (D-E) The bindings of Swi6 (D) and Epe1 (E) at mating type boundaries and sub-telomeric region are analyzed by ChIP as well as qPCR. Levels of Swi6 or Epe1 are assayed by ChIP as well as qPCR from cdc22-3 and wild type strains. The PCR primer in sub-telomeric region is in the chromosome 1 left arm end. Primers of tel-1 is in the chromosome 1 20000-25000bp, and tel-2 is in the chromosome 1 15000-20000bp. The primers chosen here is according to the previous study [actin locus. The error bars are 1 SD of percentages for three replicates.(A) The mRNA expression level of Atf1 is increased in us study . Recover(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.