We have developed a novel set of reagents based on Pseudorabies Virus (PRV) for efficient and long-term genetic tagging of neurons based on their projection targets. By deleting IE180, the master transcriptional regulator in the PRV genome, we have produced a mutant virus capable of infection and transgene expression in neurons but unable to replicate in or spread from those neurons. IE180-null mutants showed no cytotoxicity, and infected neurons exhibited normal physiological function more than 45 days after infection, indicating the utility of these engineered viruses for chronic experiments. To enable rapid and convenient construction of novel IE180-null recombinants, we engineered a bacterial artificial chromosome (BAC) shuttle-vector system for moving new constructs into the PRV IE180-null genome. Using this system we generated an IE180-null recombinant virus expressing the site-specific recombinase Cre. This Cre-expressing virus (PRV-hSyn-Cre) efficiently and robustly infects neurons in vivo and activates transgene expression from Cre-dependent vectors in local and retrograde projecting populations of neurons in the mouse. We also generated an assortment of recombinant viruses expressing fluorescent proteins . These viruses exhibit long-term labeling of neurons in vitro but transient labeling in vivo. Together these novel IE180-null PRV reagents expand the toolkit for targeted gene expression in the brain, facilitating functional dissection of neuronal circuits in vivo.Brain regions contain diverse populations of neurons that project to different long-range targets. The study of these subpopulations in circuit function and behavior requires a toolkit to characterize and manipulate their activity  Neuroinvasive viruses have become an essential part of the systems neuroscience toolkit as a vehicle for delivery of genetic material into neurons. Their applications range from anatomical tracing with fluorescent proteins to monitoring and manipulating the activity of defined populations of cells  vector (pBecker3) described previously  promoter driving expression of Green Fluorescent Protein, mCherry or Cyan fluorescence protein .To make an IE180- and Us9- null pBecker (PZ57) BAC, we used recombineering to replace Us9 in PZ42 with a neo-rpsL cassette (Gene Bridges) amplified with Us9 homology arms (primers GGAGAACCGGCCCGCCCGCATTCCGACATGCCCGCCGCCGCCCCCGCCGACATGGACACGGGCCTGGTGATGATGGCGGGATCG and GCGGCGGGGCGGGCGGCCACCACCCGCTCGCTACACGTGCCGGGCGATGATGCCCCCGATTCAGAAGAACTCGTCAAGAAGGCG). Positive clones were selected on kanamycin plates and verified by diagnostic digest. The shuttle vector was constructed by subcloning the em7 (promoter)-zeocin cassette into a minigene containing a multiple cloning site flanked by homology arms matching the rpsL sequence, which was synthesized by IDT [HA–MCS–HA (GGCCTGGTGATGATGGCGGGATCGTTGTATATTTC TTGACACCTTTTCGGCATCGCCCTAAAATTCGGCGTCCTCATATTGTGTGAGGACGTTTTATTAC—AACGTTACGCGTAAGCTTCTAGAATTCATTAATGCATGC–GGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGA)]. Transgenes were subcloned into the MCS of the shuttle vector and the recombination cassette was amplified using the shuttle forward (GGCCTGGTGATGATGGCGGGATCGTTG) and shuttle reverse (TCAGAAGAACTCGTCAAGAAGGCGATAGAAG) primers. Transgenes constructs cloned into this BAC include the synapsin promoter expressing Cre (hSyn-Cre), and the elongation factor 1a . Immediately after transfection 2 μg/ml doxycycline was added to the media. When ~80% of cells exhibited cytopathic effects  the virus stock was harvested, and used to infect a larger volume of cells. Subsequently, new batches of viral stocks were prepared by amplifying them in PK15-IE180 cells. Virions were concentrated and titered as previously described . The tissue was dissociated and plated at approximately 16,000 cells/cm9 pfu) in left auditory cortex  with a single injection at a depth of 500 μm from the pia. ~250 nl of PRV EF1a-EGFP, PRV- EF1a-Mcherry and PRV-EF1a-ECFP were injected at titers of ~7 × 108 pfu.All animal protocols were approved by the Cold Spring Harbor Laboratory Animal Care and Use Committee and carried out in accordance with National Institutes of Health standards. Mice were anesthetized with a mixture of ketamine/medetomidine . For each injection a small craniotomy was made above the injection site and a glass micropipette was used to deliver ~250 nl of the virus. Injections were performed in mice of either sex by delivering brief pulses of pressure at 0.2 Hz using a Picospritzer II (Parker), each pulse delivering ~2 nl. Ai14 mice  were injected with PRV-hSyn-CRE IE180-null PRV . The brains were removed and post-fixed overnight in 4% PFA before being sectioned into 100 μm slices. Slices were mounted in VectaShield with DAPI (Vector Laboratories) and imaged using the LSM710 confocal microscope (Zeiss). Z-stacks spanning 50 μm were acquired of the entire brain slices or regions of interest. For presentation of the distribution of labeled neurons, maximum intensity projection images were calculated using ImageJ (NIH) software.3, 25 mM D-glucose, 2.5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, and 1.25 mM NaH2PO4, aerated with 95% O2 5% CO2. The brains were transferred to a chilled slicing solution composed of 110 mM choline chloride, 25 mM NaHCO3, 25 mM D-glucose, 11.6 mM sodium ascorbate, 7 mM MgCl2, 3.1 mM sodium pyruvate, 2.5 mM KCl, 1.25 mM NaH2PO4, and 0.5 mM CaCl2. We made 300 μm thick horizontal slices that were incubated in ACSF at 34° for 20–30 min and then kept at room temperature during the experiments.Ai14 mice were injected with PRV-hSyn-Cre in the left auditory cortex at postnatal day 30. At 40–70 days post-injection, animals were anesthetized with isofluorane and perfused intracardially with cold artificial cerebrospinal fluid (ACSF) containing 127 mM NaCl, 25 mM NaHCO2, 10 mM HEPES, 1 mM EGTA, 4 mM Na2ATP, 0.4 mM Na2GTP, 10 mM Na-phosphocreatine, and 0.015 mM Alexa-594 , pH 7.25, 300 mOsm). The membrane resting potential and input resistances reported were all measured in whole-cell configuration immediately after break-in. Cells were included in analysis if their resting membrane potential did not become depolarized during the recording, and series resistance did not increase above 35 MΩ. Statistical comparisons were done using Wilcoxon sign-rank test comparing pairs of nearby PRV-infected and control neurons.To examine the physiological properties of retrogradely labeled neurons in the right auditory cortex, we made current-clamp recordings of excitatory neurons in layers 3 and 5 45–80 μm below the surface of the slice. Neurons were patched with electrodes (4–6 MΩ) containing intracellular solution -EYFP supplied from UNC vector core. 2–4 weeks after injection, the mice were anesthetized using a mixture of ketamine/medetomidine  and placed in an orbitonasal stereotaxic apparatus. A cranial window ~2 mm in diameter was made to expose the left primary auditory cortex and the dura was resected. For light stimulation, 1 ms pulses of blue light at 10 mW from a 473 nm laser were projected on the cortical surface. Spike responses were recorded extracellularly using acute NeuroNexus probes. Signals were amplified, filtered 600–9000 Hz and acquired using the Cheetah 32 data acquisition system . Single units were identified after spike sorting using MClust software.Two FVB mice were injected with AAV-Herpes viruses have three phases of gene expression: immediate early (IE), early (E) and late (L). IE gene expression occurs in the absence of new protein synthesis and is activated by viral transactivators that enter the cell in the viral tegument, a proteinaceous layer between the capsid and envelope. IE genes are required for E gene expression, whose gene products promote the replication of viral DNA. Newly replicated DNA is the substrate for L gene expression. Herpes Simplex Virus-1 (HSV-1) encodes five immediate early genes, and the recombinant HSV-1 used for retrograde tagging has deletions in two of the essential IE genes; ICP4 and ICP27, and also carries a mutation in the tegument protein VP16 that blocks transactivation of the other 3 IE genes  from pBecker3, a self-excising PRV BAC carrying the PRV genome   Figure . On the ) Figure . InfectiR/neoR selection cassette that confers resistance to kanamycin and sensitivity to streptomycin into the IE180-null PRV genome disrupting the Us9 locus. Us9 is required for anterograde viral spread in vivo  but its deletion has no effect on primary infection properties of PRV , mCherry (PRV HKO242) and ECFP (PRV HKO243).To test expression of transgenes in the PRV IE180-null background, we used the shuttle system to produce several recombinants carrying transgenes encoding different fluorescent proteins. We used the constitutive in vivo injection of PRV HKO128 (expressing EGFP) into auditory cortex labeled only neurons near the injection site but did not label input structures such as the contralateral auditory cortex or the medial geniculate body (MGB) (not shown). Injection into other structures, including auditory striatium and cerebellum, also failed to label input structures. Thus in their current form, these reagents are useful only for in vitro experiments (see Section Discussion).These viruses efficiently drove fluorescent protein expression in PK15 cells Figures  and dissin vivo expression of fluorescent proteins from the recombinants we had generated was either too weak or too transient to be useful in vivo. However, because even weak and transient expression of Cre recombinase can effectively activate gene expression, we expected that an IE180-null PRV expressing Cre recombinase would be a useful reagent.We hypothesized that the To produce a PRV IE180-null recombinant that could be used in combination with Cre-dependent viruses and Cre-reporter mice that are already a widely used component of the systems neuroscience toolkit, we inserted the Cre-recombinase gene under control of the neuron specific synapsin-1 promoter (hSyn). Moreover, tdTomato expressing neurons were observed at the earliest tested time point of 3 days after infection (n = 3 mice) and persisted as long as 6 months .The application of PRV in the study of neural circuits has been limited by its rapid cytotoxicity; wild-type PRV alters neuronal properties within hours of infection and cell death follows shortly thereafter  were indistinguishable from adjacent uninfected control cells  curves of neurons 40–70 days after infection . We found that the intrinsic properties of IE180-null PRV infected cells (n = 2 mice). When AAV FLEX-ChR2-YFP was injected into the left auditory cortex and PRV-hSyn-Cre in the right auditory cortex  technique  required for virion infection and cell-cell spread, or thymidine kinase (TK) required for viral DNA synthesis in terminally differentiated cells . However, a few weeks are needed for optimization of injection and validation of labeling in vivo.To facilitate the construction of recombinant IE180-null PRV mutants, we used a shuttle-vector based strategy that avoids many of the pitfalls of BAC recombineering and reliably yields recombinant IE180-null PRV mutants carrying transgenes of interest with a turnaround time of ~13 days , this strategy could be applied to enable the transneuronal spread of the virus from anatomically or genetically defined populations of cells to their inputs. Like RV lentivirus and AAV, herpesviruses are amendable to pseudotyping, which may with further development allow for targeting infection to particular cell types  classification can be an important determinant in the selection of viral tools to be used for a given experiment, as implementation of the appropriate safeguards can be expensive and time consuming. There are no specific official  references to how replicating PRV should be classified. However, it is reasonable that IE180-null PRV should be classified in the lowest risk group BSL-1. Unmodified PRV does not infect humans, and thus by definition is classified as Risk Group 1  can accommodate much larger expression cassettes than is possible with other commonly used viral vectors. Unlike its cousin HSV-1, wild-type PRV has a broad host range from some avian species to a number of mammals including Rhesus monkeys and marmosets. However, PRV is not pathogenic in humans, and fewer safety concerns are associated with its application in the laboratory (Kluge et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hypothalamic ribosomal S6K1 has been suggested as a point of convergence for hormonal and nutrient signals in the regulation of feeding behavior, bodyweight, and glucose metabolism. However, the long-term effects of manipulating hypothalamic S6K1 signaling on energy homeostasis and the cellular mechanisms underlying these roles are unclear. We therefore inactivated S6K1 in pro-opiomelanocortin (POMC) and agouti-related protein (AgRP) neurons, key regulators of energy homeostasis, but in contrast to the current view, we found no evidence that S6K1 regulates food intake and bodyweight. In contrast, S6K1 signaling in POMC neurons regulated hepatic glucose production and peripheral lipid metabolism and modulated neuronal excitability. S6K1 signaling in AgRP neurons regulated skeletal muscle insulin sensitivity and was required for glucose sensing by these neurons. Our findings suggest that S6K1 signaling is not a general integrator of energy homeostasis in the mediobasal hypothalamus but has distinct roles in the regulation of glucose homeostasis by POMC and AgRP neurons.  •Mice lacking S6K1 in POMC and AgRP neurons have impaired glucose homeostasis•S6K1-deficient POMC and AgRP neurons have altered excitability•S6K1 in POMC and AgRP neurons does not regulate food intake and bodyweight POMC and AgRP neurons regulate systemic energy homeostasis. Smith et al. demonstrate that S6K1 signaling in these cells regulates neuronal excitability and peripheral glucose homeostasis in mice. However, POMC and AgRP S6K1 signaling does not play a major role in feeding behavior and the control of bodyweight. Obesity and its associated diseases such as type 2 diabetes and cancer are major causes of morbidity and premature mortality . The CNSA significant body of work has focused on identification of common intracellular signaling pathways that respond to a range of metabolic signals and may couple hormone and nutrient sensing by neurons to the regulation of organismal metabolism. One such pathway is the mechanistic target of rapamycin (mTOR) signaling network . mTOR exHypothalamic mTORC1 signaling and S6K1 in particular have been linked to the regulation of energy homeostasis . S6K1 isRps6kb1) specifically in either POMC or AgRP neurons. In contrast to published studies, we found a minimal role for S6K1 in the long-term regulation of food intake and bodyweight. S6K1 in POMC and AgRP neurons, however, played a key role in neuronal excitability and the regulation of peripheral glucose homeostasis.Despite these findings, a number of issues remain unclear with respect to the role of S6K1 in the hypothalamus. For example, the use of acute non-targeted viral systems to manipulate S6K1 signaling has not permitted long-term studies on energy balance and glucose homeostasis or revealed any specific contribution of POMC and AgRP neurons. The effects of hypothalamic S6K1 on peripheral glucose metabolism also require clarification . FinallyRps6kb1 flanked by loxP sites (Rps6kb1 floxed (S6K1fl/fl) mice were phenotypically indistinguishable from WT animals and displayed normal Rps6kb1 expression in the absence of cre-recombinase (data not shown). In mice with either germ-line deletion or with global neuronal deletion (using either nestin-cre or synapsin-cre transgenic mice), western blotting confirmed complete loss of S6K1 in all tissues or specifically in the CNS, respectively , and the recombination event was restricted to the hypothalamus in both knockout (KO) lines (Rps6kb1-deleted neurons (Mice were generated on a C57Bl/6 background with exons 3 and 4 of xP sites A. When b/fl mice C. S6K1flO) lines D and 1E. neurons F and 1G.Rps6kb1 signaling in POMC and AgRP neurons does not affect food intake or long-term bodyweight regulation.Food intake, ad libitum and in response to a fast, was unaltered in POMCS6K1KO and AgRPS6K1KO mice on chow A–2D, andAs POMC and AgRP neurons regulate peripheral glucose homeostasis, we studied this in POMCS6K1KO or AgRPS6K1KO mice on chow and HFD. Fed or fasted serum glucose levels, fasted insulin levels , and gluHyperinsulinemic-euglycemic clamp studies were conducted to reveal any potential tissue-specific regulation of glucose homeostasis. Serum glucose concentrations, glucose infusion rate (GIR), and insulin concentrations before and at the end of the clamp were equivalent between male WT and POMCS6K1KO or AgRPS6K1KO mice (data not shown). Under basal conditions, whole-body glucose utilization, equivalent to endogenous hepatic glucose production (HGP), did not differ between WT and POMCS6K1KO or AgRPS6K1KO mice I and 2J.Rps6kb1 in POMC neurons impairs central regulation of HGP and also impacts upon peripheral lipid metabolism, while loss of Rps6kb1 in AgRP neurons impairs skeletal muscle insulin sensitivity.In POMCS6K1KO compared with control mice, fasted plasma free fatty acid (FFA) levels were lower but not suppressed by the hyperinsulinemic clamp M and 2N.Pck1), glucose-6-phosphatase, and interleukin-6 (a cytokine implicated in the control of HGP) were unaltered in either POMCS6K1KO or AgRPS6K1KO mice  POMC neurons  and  neurons , insulin neurons G and S3J studied H. Howevet change I and S3JRps6kb1-deleted POMC and AgRP neurons were less excitable in 5 mM external glucose  and lower spike frequency than control POMC neurons while input resistance was not different  channel subunit expression, and we found that Kcjn11 expression was significantly increased in POMCS6K1KO and reduced in AgRPS6K1KO when compared with WT mice; however, Abcc9 expression was unchanged  and inhibitory (mIPSC) post-synaptic currents in the presence of tetrodotoxin , and KO (Cre+/−/Rps6kb1fl/fl) mice for each line. To generate mice lacking floxed alleles but expressing GFP or YFP in cells harboring the deletion event, mice were bred with POMC-GFP or Rosa26YFP indicator mice and bred to homozygosity for the floxed allele. Mice were maintained on a 12-hr light/dark cycle with free access to water and standard mouse chow  or HFD  and housed in specific-pathogen free barrier facilities in individually ventilated cages of mixed genotypes. All KO and transgenic mice were studied with appropriate littermates of the three control genotypes. Mice were handled and all in vivo studies performed in accordance to the United Kingdom Animals (Scientific Procedures) Act (1986) and approved by Imperial College’s Animal Welfare and Ethical Review Body.The use and genotyping of POMCCre, AgRPCre (both on a C57Bl/6 background), and NestinCre (on a mixed C57Bl/6 × 129sv background) mice have been previously described . Mice wiData are expressed as mean ± SEM unless otherwise stated, and the single animal was the unit of analysis unless otherwise stated. Statistical significance was calculated at the 95% level of confidence using parametric  or non-parametric  tests with post hoc Bonferroni’s or Dunn’s multiple comparison analysis, where appropriate. Statistical significance was calculated from all electrophysiological recordings (responsive and non-responsive).M.A.S., L.K., E.E.I., M.K.H., S.M.A.P., and M.W.G. generated and phenotyped the mouse lines. M.A.S. and E.E.I. performed i.c.v. feeding studies. M.A.S. performed and analyzed the electrophysiological studies. L.K., M.K.H., S.M.A.P., and A.I.C. performed and analyzed PCR analysis. L.K., A.W., and D.C. undertook and conceived western blot analysis. P.J.V. and A.V.-P. designed and conducted hyperinsulinemic-euglycemic clamp studies. M.A.S. and D.J.W. conceived and designed the study and wrote the manuscript. All authors contributed to the editing of the manuscript.


Neurospora crassa translocations into N. tetrasperma, we constructed heterokaryons bearing haploid nuclei of opposite mating types, and either the translocation and normal sequence chromosomes  or a duplication and its complementary deficiency . The [T + N] heterokaryons result from alternate segregation of homologous centromeres, whereas adjacent-1 segregation generates [Dp + Df]. Self-cross of either heterokaryon produces [T + N] and [Dp + Df] progeny. Occasionally during N. tetrasperma ascus development, a pair of smaller homokaryotic ascospores replaces a heterokaryotic ascospore. Crosses with the Eight-spore mutant increase such replacement, and can generate asci with eight homokaryotic ascospores, either 4T + 4N from alternate segregation, or 4Dp + 4Df from adjacent-1 segregation. Crosses of some of the introgressed translocation strains with normal sequence N. tetrasperma produced more Dp than T or N homokaryotic progeny. We suggest this is due to an insufficiency for a presumptive ascospore maturation factor, which increases the chance that, in asci with > 4 viable ascospores, none properly mature. Since only four viable ascospores (Dp or [Dp + Df]) share the limiting factor following adjacent-1 segregation, whereas four to eight ascospores compete for it following alternate segregation, this would explain why Dp homokaryons outnumber T and N types, whereas the heterokaryons are not as affected. We believe that this novel form of transmission ratio distortion is caused by a Bateson–Dobzhansky–Muller Incompatibility (BDMI) triggered by an N. crassa gene in the N. tetrasperma background. Heterokaryons tend not to out-cross, and crosses of Dp strains are barren, thus the BDMI impedes interspecies gene flow.By introgressing  Neurospora crassa and N. tetrasperma are related fungal species but they differ strikingly in ascus development. N. crassa asci form eight initially uninucleate ascospores, whereas N. tetrasperma asci make four initially binucleate ascospores  mutant increases such replacement, and can generate asci with eight homokaryotic ascospores  by chance, and mycelia from homokaryotic conidia and ascospores can out-cross with like mycelia of the opposite mating type  in their constituent nuclei  and normal sequence (N) chromosomes. [Dp + Df] and [NtT + N] heterokaryons share identical genes and hence are expected to share identical phenotypes. Were they, however, to display different phenotypes, it might flag the absence of one or more ‘nucleus-limited’ genes from the Df nuclei (WT) in [WT + Δ] heterokaryons . We have determined the breakpoint junctions of several insertional, quasi-terminal, and reciprocal translocations , viable parental ascospores, while ADJ gives eight nonparental ascospores, with the four duplication spores being black and viable, and the four deficiency spores white (W) and nonviable. Since either segregation is equally likely, asci with 8B:0W and 4B:4W ascospores occur in equal numbers. Additionally, some asci can have 6B:2W ascospores as a result of recombination in the interstitial regions between the centromeres and breakpoints, but asci with 2B:6W and 0B:8W ascospores are not expected . Thus, equal numbers of asci with 8B:0W and 0B:8W ascospores characterize heterozygosity for a reciprocal translocation. Most asci from isosequential crosses  have 8B:0W ascospores , having either both normal sequence or both translocation chromosomes, or unbalanced if segregation is adjacent-1 (ADJ), where one has a duplication and the other the complementary deficiency . In N. cexpected . ALT segcospores .N. tetrasperma, crosses heterozygous for a nonreciprocal translocation ALT and ADJ generate 4B:0W asci that contain, respectively, [NtT + N] and [Dp + Df] heterokaryotic ascospores. The constituent nuclei in these ascospores are of opposite mating type  and, hence, the resulting mycelia can undergo a self-cross. The [NtT + N] and [Dp + Df] genotypes can interconvert through self-crosses  from N. crassa to N. tetrasperma, and the introgressed translocation strains were designated as NtT(EB4); NtT(IBj5); NtT(UK14-1) and NtT(B362i)  and NtT(UK14-1) with E yielded comparable numbers of asci with 8B:0W and 4B:4W ascospores, and also some with 6B:2W ascospores. Surprisingly, the crosses of NtT(IBj5) and NtT(B362i) with E did not yield any asci with 8B:0W and 6B:2W ascospores , and NtT(B362i) strains, and blocked ascospore maturation in eight-spored asci formed following ALT, but not in asci formed following ADJ .N. crassa/N. tetrasperma hybrid strain C4,T4 a was used as a bridging strain in the introgression crosses. It has four N. crassa great-grandparents and four N. tetrasperma great-grandparents. The N. crassa great-grandparents were of the OR background, whereas the N. tetrasperma great-grandparents were of the 343.6 A E background (The ckground .NtT strains with N. tetrasperma strain 85 derivatives of opposite mating type (NtT × 85) yield self-fertile heterokaryotic progeny of [NtT + N] or [Dp + Df] genotype. [NtT + N] mycelia yield homokaryotic (self-sterile) conidial derivatives of both mating types, whereas [Dp + Df] mycelia yield homokaryons of only the mating type of the Dp nucleus. The NtT strains are reisolatable as self-sterile conidial derivatives from the [NtT + N] mycelia.Crosses of N. tetrasperma strain FGSC 2508 A and FGSC 2509 a is publicly available (fungidb.org). The N. tetrasperma strains 85, EA, and Ea share considerable DNA sequence homology (data not shown), and their genome is more diverged from that of FGSC 2508A and FGSC 2509a. Table S2 lists the oligonucleotide primers used for PCR and restriction enzymes used to digest the resulting amplicons to obtain polymorphisms between the 85/EA/Ea and FGSC 2508 A/FGSC 2509 a alleles on each of the seven N. tetrasperma chromosomes. Figure S2 gives the positions of the marker on the chromosomes.The genome sequence of the ITs are defined by breakpoint junction A on the donor chromosome, and junctions B and C  on the recipient chromosome, whereas QTs have only two breakpoint junctions, A and B, on the two participating chromosomes. Additionally, primers to PCR amplify genome segments from the N-derived donor and recipient chromosome homologs (designated ND and NR), but not from the translocation chromosomes, were used as positive controls. The primers are listed in Table S3. Genomic DNA from T progeny can PCR amplify across the A, B, and C breakpoints, but not with the ND and NR primers; DNA from Dp progeny can PCR amplify across B and C but not A, and also give products with the ND (but not NR) primers, while DNA from N does not amplify with primers for A, B, or C, but can give products with the ND and NR primers.N. crassa insertional translocation T(VR > VIL)UK3-41 translocates 1,879,356 bp, bearing 490 genes, from chromosome 5R to 6L (T(UK3-41) strain was digested with HaeIII, self-ligated, and the ligation product used as template in inverse PCR reactions with the primers listed in Table S4.The 5R to 6L . Its traN. crassa strains of the Oak Ridge (OR) background and N. tetrasperma strains of the 85 background are almost completely sterile A strain was crossed with C4,T4 a, and T progeny from these crosses (designated 1xC4T4T) were distinguished from their Dp and N siblings by PCR with A and C breakpoint junction-specific primers. Nominally, 50% of the genome of 1xC4T4T progeny is derived from the C4,T4 a parent. The 1xC4T4AT progeny strains were crossed with C4,T4 a to obtain the 2xC4T4T progeny in a like manner. Crosses of 2xC4T4T with the opposite mating type derivative of strain 85 were productive, and their T progeny were designated as 1x85T. Likewise, 1x85T × 85 yielded 2x85T, etc. Ordinarily, after two to three iterations of the crosses with 85, one can recover progeny ascospores that produce mycelia of dual mating specificity characteristic of N. tetrasperma  progeny for additional crosses by the f4 generation.The Accession numbers of nucleotide sequences determined in this work: KU554720 and KU599833 (Genbank). Nt aT(IBj5) × E A and Nt AT(B362i) × E a crosses showed the max-4- phenotype , whereas the Nt aT(EB4) × E A and Nt AT(UK14-1) × E a crosses produced asci with 8B:0W and 6B:2W ascospores  and NtT(B362i) strains, but not T(EB4)Nt, T(UK14-1)Nt, 85 A and 85 a, contain a recessive mutation whose homozygosity induces the max-4- phenotype. To test this hypothesis, we screened 103 and 58 f1 progeny from E A × 85 a and E a × 85 A and found, respectively, 57 and 21 homokaryons , crossed them with Nt aT(IBj5) or Nt AT(B362i), and examined the crosses for the max-4- phenotype. We expected that the progeny inheriting the E-derived recessive allele would show the max-4- phenotype, and those inheriting the 85-derived wild-type allele would also produce some 8B:0W and 6B:2W asci. Unexpectedly, all 78 f1 progeny showed the max-4- phenotype (data not shown). This is possible if the chromosome bearing the E-derived mutant allele also exerts meiotic drive relative to its 85-derived homolog. However, we were unable to easily find polymorphic markers between the E and 85 strains to test if such was indeed the case.The EA/Ea strains on the one hand and the N. tetrasperma FGSC 2508A/FGSC 2509a strains on the other . We verified that these markers showed independent segregation in the homokaryotic progeny from the crosses Ea × 2508A (EA × 2509a (NtaT(IBj5) and NtAT(B362i) with 2508A and 2509a produced mostly four-spored asci, but the rare eight-spored asci included some 8B:0W, 4B:4W, and 6B:2W types  and EA × 2509a (N = 20) showed the max-4- phenotype in crosses with Nt aT(IBj5) or Nt AT(B362i) (data not shown). Given that all chromosomes segregated independently in the f1 progeny, these results are incompatible with the idea that the max-4- phenotype segregates with a specific chromosome from EA or Ea. Thus, the hypothesis that the max-4- phenotype is caused by homozygosity for a recessive mutation is not tenable.Instead, we found several polymorphic markers between the 85/ × 2508A  and EA × × 2509a . Further2W types . This shE mutation segregates with chromosome 6. Since this was easy to confirm, it seemed worthwhile to do so. We crossed the homokaryotic f1 progeny from Ea × 2508A and EA × 2509a with opposite mating type derivatives of strain 85 and examined the progeny asci for the eight- or four-spore phenotype. Our results (Table S5) confirmed that E segregates with chromosome 6.i.e., mating type homokaryons) were identified from crosses of the NtT strains with E and 85 strains of the opposite mating type, and their T, N, or Dp genotype was determined by PCR. As expected, and as can be seen in the results summarized in E strains. Additionally, we found three different phenotypes: the crosses of NtaT(EB4) with the N A strains produced comparable numbers of T, N, and Dp progeny (T = N = Dp), whereas those of NtaT(IBj5), and NtAT(B362i) × 85 a, produced far fewer T and N progeny than Dp type , and NtAT(B362i) × E a produced T << N, Dp (NtT(UK14-1), a QT, is not yet defined. Consequently, only the T progeny from Nt AT(UK14-1) × N a could be identified by their A breakpoint junction, whereas Dp and N were not distinguishable  in the homokaryotic progeny from crosses of E or 85 strains with NtT strains which gave the max-4- phenotype in NtT × E crosses. The distortion appeared to be specific to the homokaryotic progeny because [Dp + Df] and [NtT + N] heterokaryotic (self-fertile) progeny did not show comparably distorted ratios. The [Dp + Df]/[NtT + N] ratio was 3/3 and 7/3 in crosses with NtT(IBj5) and NtT(B362i) (which showed the max-4- phenotype in crosses with E), and 4/4 and 2/3 in crosses with NtT(EB4) and NtT(UK14-1) (which did not) (Our results show evidence for transmission ratio distortion (NtaT(IBj5) and NtAT(B362i) with 85A/85a or 2508A/2509a produced rare 8B:0W and 6B:2W asci, hardly any T progeny were obtained from the crosses of these NtT strains with 85 (NtaT(IBj5) × 85A and NtAT(B362i) × 85a onto water agar. The majority of asci from these crosses were four- or five-spored, but we could also pick the rare seven- and eight-spored asci  × 85 A, including two 8B:0W, one 7B:1W, two 5B:3W, one 4B:4W, one 2B:6W, and three 0B:8W types. Two progeny were obtained from the 4B:4W ascus, and both amplified for junction B and normal A indicating that they had Dp genotype; one was mat A and the other was mat a. This was consistent with the expectation that 4B:4W asci yield only Dp progeny. Three progeny were obtained from one 8B:0W and one 7B:1W ascus. One appeared to be a [NtT + N] or [Dp + Df] heterokaryon bearing both mat A and mat a nuclei, one was Dp a, and one was T A. Normally, one does not expect heterokaryotic and Dp genotypes in progeny ascospores from 8B:0W and 7B:1W asci. Additionally, we examined six progeny from two seven-spored asci (one 7B:0W + one 5B:2W). Three were Dp a, one was N a, and two were [Dp + N] heterokaryons possessing both mat A and mat a nuclei. This result was also unexpected because one expects to find three Df A type progeny complementary to the three Dp a types, but the two asci together contained only two white ascospores. These results show that. while the progeny from the 4B:4W asci from Nt aT(IBj5) × 85 A have the expected Dp genotype, those from asci containing > 4 viable ascospores often display unexpected genotypes.Ten eight-spored asci were obtained from the cross NtAT(B362i) × 85a cross, including 10 8B:0W, six 7B:1W, five 6B:2W, four 5B:3W, eight 4B:4W, two 3B:5W, and one 2B:6W types. All three progeny examined from the 4B:4W asci had the expected Dp genotype. In contrast, none of the 17 progeny examined from the 8B:0W asci were T or N type. Six appeared to be [T + N] or [Dp + Df] heterokaryons. Four of them were mating type homokaryons, whereas two had mat A and mat a nuclei. Two progeny appeared to be [Dp + N] heterokaryons, and nine appeared to have the Dp genotype. None of these genotypes are expected in ascospores from 8B:0W asci. From the 6B:2W asci, one progeny appeared to be a [T + N] or [Dp + Df] heterokaryon, two appeared to be [Dp + N] heterokaryons, ten were Dp type, and one was N type. Heterokaryotic genotypes are not expected in ascospores from 6B:2W asci. Again, these results show that although progeny from the 4B:4W asci from the NtAT(B362i) × 85a cross have the expected Dp genotype, those from the 8B:0W and 6B:2W asci often display unexpected genotypes.We obtained 36 eight-spored asci from the HaeIII-digested genomic DNA of the T(VR > VIL)UK3-41A strain  was self-ligated, and used as a template for inverse PCR with primers complementary to sequences within the translocated segment. In this way, we retrieved the adjoining sequence on the recipient chromosome and thus defined the B and C breakpoint junctions (respective Genbank accession numbers KU599833 and KU554720). The flanking sequence of the B junction had a HaeIII site located merely 2 bp proximal to the breakpoint junction. Obtaining only 2 bp of proximal flanking sequence from the recipient chromosome was inadequate to design a primer to PCR amplify across the breakpoint junction. On normal sequence chromosome 6L, a 15,882 bp AT-rich (70.5%) sequence containing only a few 4 bp-cutting restriction sites separates the C breakpoint locus from its closest proximal ORF (ncu 07116). These restriction enzymes did not have any convenient sites within the UK3-41 translocated segment, making it difficult to design alternative inverse PCR strategies to retrieve additional sequences proximal to the translocated segment. Therefore, the B breakpoint remains undetermined. Figure S3 updates the breakpoints of 12 Dp-generating translocations on the N. crassa genome sequence.T(UK3-41) allowed us to distinguish the T, Dp, and N progeny from T(UK3-41) × N crosses. Therefore, we attempted to introgress this translocation into N. tetrasperma (T(UK3-41) strains with C4T4 a and 85 A showed the T = N = Dp phenotype, but subsequent to the first productive T a × 85 A crosses the crosses of the T progeny with 85 A or 85 a gave the T << N, Dp phenotype (see above). One introgression line , similar to that associated with the max-4- phenotype, precluded our obtaining sufficient numbers of T progeny. Defining the TRD-inducing gene will help us to identify early generation translocation progeny from which it is absent, and facilitate future introgression efforts.Knowledge of the A and C breakpoint junctions of rasperma . The ini left in , produceNtT × E crosses, wherein no asci contain more than four black ascospores  is that the N. crassa-derived T recipient chromosome and the N. tetrasperma-derived homolog of the T donor chromosome carry meiotic drive elements that induce inviability in progeny that do not inherit them. Meiotic drive elements are selfish genes that skew the 1:1 Mendelian segregation ratio to their own advantage and their presence, in turn, imposes selection for unlinked suppressors that restore the Mendelian ratio. Reproductively isolated taxa such as N. crassa and N. tetrasperma may have accumulated distinct drivers and suppressors that might get separated in the hybrid NtT strains, and allow drive to surface in their crosses  and NtT(B362i). It also does not readily account for the T << N, Dp phenotype. Chromosome 4 is the donor chromosome in NtT(B362i), and if a meiotic drive element was present on chromosome 4 of the E strain, then its segregation in NtAT(B362i) × E a could make the T progeny that do not inherit it unviable, whereas the N and Dp progeny that do would survive. However, chromosome 4 is the recipient chromosome in NtT(IBj5); therefore, the model predicts inviability of the Dp progeny from NtaT(IBj5) × E A, which is clearly not the case. germlings, thus masking the T, N << Dp phenotype in the NtAT(B362i) × E a cross. The T genotype has more N. crassa-derived sequences than N, especially on the translocation donor and recipient chromosomes. Conceivably, a second BDMI could select against the T nuclei during vegetative growth. However, we do not rule out the possibility that the second BDMI affects the viability of T ascospores in [NtT + N] asci to produce an effectively four-spored [lethalT + N] ascus. Thus, both the T, N << Dp and T << N, Dp phenotypes can potentially be explained by BDMIs that generate the max-4- phenotype. NtT strains ), from which the N. crassa-derived meiotic drive element or BDMI gene(s) is absent, are not expected to show transmission ratio distortion in crosses with N strains.How might one explain the NtT + N], [Dp + Df], and Dp progeny would act to impede gene flow between N. crassa and N. tetrasperma because heterokaryotic strains tend not to out-cross as females  used by a silencing complex to identify and degrade complementary mRNA as it exits the nucleus (The production of only  or [Dp + N] asci the nuclei might undergo additional mitoses beyond the first postmeiotic mitosis before being partitioned into the eight ascospores. This is a novel and unprecedented finding.Finally, our results revealed that the rare 8B:0W and 6B:2W asci from the


CHEK2 is a protein kinase that is involved in cell-cycle checkpoint control after DNA damage. Germline mutations in CHEK2 gene have been associated with increase in breast cancer risk. The aim of this study is to identify the CHEK2 gene germline mutations among high-risk breast cancer patients and its contribution to the multiethnic population in Malaysia. We screened the entire coding region of CHEK2 gene on 59 high-risk breast cancer patients who tested negative for BRCA1/2 germline mutations from UKM Medical Centre (UKMMC), Hospital Kuala Lumpur (HKL) and Hospital Putrajaya (HPJ). Sequence variants identified were screened further in case-control cohorts consisting of 878 unselected invasive breast cancer patients  and 270 healthy individuals . By screening the entire coding region of the CHEK2 gene, two missense mutations, c.480A>G (p.I160M) and c.538C>T (p.R180C) were identified in two unrelated patients (3.4%). Further screening of these missense mutations on the case-control cohorts unveiled the variant p.I160M in 2/172 (1.1%) Indian cases and 1/90 (1.1%) Indian control, variant p.R180C in 2/526 (0.38%) Chinese cases and 0/90 Chinese control, and in 2/180 (1.1%) of Malay cases and 1/90 (1.1%) of Malay control. The results of this study suggest that CHEK2 mutations are rare among high-risk breast cancer patients and may play a minor contributing role in breast carcinogenesis among Malaysian population. CHEK2) (OMIM +604373) encodes a checkpoint serine/threonine kinase and is the human homolog of Saccharomyces cerevisiae RAD53 and S. pombe CDS1 [CHEK2 gene is critical in DNA damage induced cell-cycle checkpoint control by activating P53 and BRCA1, which in turn play an important role in controlling the cell cycle checkpoints, apoptosis and DNA damage repair [Cell cycle checkpoint kinase 2 gene,  in the French-Canadian population [The ity gene . Howeverpulation , 2.9%  High-risk breast cancer patients for sequencingBRCA1 and BRCA2 mutations were included in the study. The recruitment of breast cancer patients for this study was done in three main breast clinics in the Klang Valley; UKM Medical Centre (UKMMC), Hospital Kuala Lumpur (HKL) and Hospital Putrajaya (HPJ). Each patient who presented with either one or a combination of the criteria set below was approached and given a consent form for the use of clinical records and blood specimens were collected. The inclusion criteria for CHEK2 testing are: 1) early-onset of breast cancer (≤ 40 years); 2) breast cancer patients with at least one or more family member (s) with breast and/ or ovarian cancer in first-, second- or third-degree relatives; 3) bilateral breast cancer and 4) male patients with breast cancer. Information regarding nature and objectives of the study had been explained to the patients and informed consent was taken. Each patient was interviewed to obtain data on family history and family pedigree. Ten milliliters of peripheral blood specimen from each of the selected patient was collected in EDTA tubes.A total of 59 high-risk breast cancer patients who tested negative for b) Breast cancer cases and controls for genotypingCHEK2 variants and of these, 878 samples were successfully genotyped. There were 526 Chinese (59.9%), 180 Malays (20.5%), and 172 Indians (19.6%). In addition, 270 healthy individuals  attending the diagnostic centre for non-cancer related health concerns in University Malaya Medical Centre were included in the case-control study.The recruitment of breast cancer patients to the Malaysian Breast Cancer Genetic Study started in January 2003 at the University Malaya Medical Centre and included both prevalent and incident breast cancer cases. In the present study, blood samples from 880 patients diagnosed with invasive breast cancer were available for genotyping to determine the prevalence of All study subjects provided written informed consent. Blood, demographic and family history data were collected from breast cancer patients who consented to participate in this study. The study was approved by the Medical Ethics Committee of Universiti Kebangsaan Malaysia Medical Centre (UKMMC).CHEK2 were applied to amplify 14 coding exons and intron-exon boundaries of the CHEK2 gene using AmpliTaq Gold DNA Polymerase . Because multiple copies of CHEK2 pseudogenes exist for exons 10–14, a nested PCR was performed to specifically amplify the chromosome 22 copy of the CHEK2 gene [Genomic DNA from blood was extracted using QIAamp DNA Midi Kit  according to the manufacturer’s protocol. Fourteen primer pairs of EK2 gene . Long raCHEK2 missense mutations identified in exon 3 were performed using high throughput Sequenom MassARRAY iPLEX platform . All mutations identified were confirmed by direct DNA sequencing in an independent DNA sample. The detail on the genotyping analysis protocol was described previously [Multiplex genotyping of two eviously .http://agvgd.iarc.fr/alignments.php), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph/) and SIFT (http://blocks.fhcrc.org/sift/SIFT.html).In this study, the pathogenecity of the missense mutations (p.I160M and p.R180C) was predicted using three computational analyses based on sequence homology and physical properties of amino acids which are Align-GVGD  were Malays, 14 (23.7%) were Chinese and 8 (14.3%) were Indians. The median age of onset of breast cancer was 38 years old (range 17–70). Thirty-nine (66.1%) patients were diagnosed with breast cancer <40 years old, 4 (6.8%) had bilateral breast cancers, 15 (25.4%) had a positive family history and one (1.7%) was a male breast cancer patient . The CHEK2 p.I160M missense mutation was detected in a Malay patient who was diagnosed with breast cancer at the age of 43 years old. She had grade 1 infiltrating ductal carcinoma with estrogen receptor (ER) and progesteron receptor (PR) status positive while negative for HER-2 receptor  infiltrating ductal carcinoma with estrogen and progesterone receptor negative and HER-2 status positive  was detected in one patient (1.7%). This mutation is unlikely to have association with the disease due to the silent amino acid changes.Besides that, one synonymous mutation in exon 1, c.252A>G (p.E84E) was detected in 10 patients (16.9%). This variant has been previously reported in several other CHEK2 p.I160M variant in 2/172 (1.16%) Indian patients and 1/90 (1.11%) Indian control while p.R180C variant was found in 2/180 (1.11%) Malay patients and 1/90 (1.1%) Malay controls, and in 2/526 (0.38%) Chinese patients and 0/90 Chinese controls (p = 0.98). Notably, both Indian cases carrying the p.I160M variant were diagnosed with breast cancer at the age >45 while all four CHEK2 p.R180C carriers were diagnosed with breast cancer at the age of ≤45  German familial breast cancer patients [CHEK2 protein, which is important for dynamic interactions between upstream regulators or downstream targets of the CHEK2 [CHEK2 or structural abnormalities which may hinder protein-protein interactions. Notably, in silico analysis (SIFT and Polyphen-2) and an in vitro yeast-based assay suggested that this variant may have intermediate response to DNA damage [The he CHEK2 , this muA damage .CHEK2 p.R180C was previously reported in 0.58% (3/516) German familial breast cancer patients and 0.2% (1/500) controls [BRCA1/2 carriers [CHEK2 p.R180C variant was also previously reported in 2.1% (2/94) prostate tumor samples of Caucasian American patients who were diagnosed before the age of 59 and in germline DNA of 1/423 (0.24%) unaffected men [in silico analysis (SIFT and Polyphen-2) and has been demonstrated to have intermediate response to DNA damage in a yeast-based assay, the p.R180H variant is predicted to be likely benign , has little effect on protein expression, phosphorylation and stability, but has lower but not null kinase activity [The .58% 3/51 German fcontrols , 0.84% , 1.76% (16/909) and 0.73% (9/1228) in familial, unselected breast cancer cases and controls respectively (p = 0.041) [CHEK2 variants in the Asian population, further studies and family-based co-segregation analyses are required to determine the cancer risks associated with these variants.Taken together, three studies have determined the prevalence of  in Asia –34. In P= 0.041) . Our stuBRCA carriers with the frequency of 3.4%. Further screening in case-control cohorts showed that these variants were found in both cases and controls. The results of this study suggest that CHEK2 mutations are rare among high-risk breast cancer patients and may play a minor role in genetic predisposition to breast cancer in the Malaysian population.In summary, two potentially pathogenic missense mutations were found among 59 high-risk breast cancer patients who were non-


Loss of histone H4 lysine 20 trimethylation (H4K20me3) is associated with multiple cancers, but its role in breast tumors is unclear. In addition, the pathological effects of global reduction in H4K20me3 remain mostly unknown. Therefore, a major goal of this study was to elucidate the global H4K20me3 level in breast cancer tissue and investigate its pathological functions.in vitro.Levels of H4K20me3 and an associated histone modification, H3 lysine 9 trimethylation (H3K9me3), were evaluated by immunohistochemistry in a series of breast cancer tissues. Univariate and multivariate clinicopathological and survival analyses were performed. We also examined the effect of overexpression or knockdown of the histone H4K20 methyltransferases, SUV420H1 and SUV420H2, on cancer-cell invasion activity in vitro.H4K20me3, but not H3K9me3, was clearly reduced in breast cancer tissue. A reduced level of H4K20me3 was correlated with several aspects of clinicopathological status, including luminal subtypes, but not with HER2 expression. Multivariate analysis showed that reduced levels of H4K20me3 independently associated with lower disease-free survival. Moreover, ectopic expression of SUV420H1 and SUV420H2 in breast cancer cells suppressed cell invasiveness, whereas knockdown of SUV420H2 activated normal mammary epithelial-cell invasion H4K20me3 was reduced in cancerous regions of breast-tumor tissue, as in other types of tumor. Reduced H4K20me3 level can be used as an independent marker of poor prognosis in breast cancer patients. Most importantly, this study suggests that a reduced level of H4K20me3 increases the invasiveness of breast cancer cells in a HER2-independent manner. The heterogeneous nature of breast cancer has been well established. VariousEpigenetic alterations such as DNA methylation and histone modifications occur in many cancers (reviewed in-9). AberThe loss of H4K20me3 has been observed in animal models of breast carcinogenesis and H4K22O2 in methanol. The sections were blocked with 5% bovine serum albumin (BSA) and incubated with primary monoclonal antibodies (mAbs) against H3K9me3 (CMA318) and H4K20me3 (CMA423); the performance of these antibodies has been validated in Western blotting, immunofluorescence microscopy, and enzyme-linked immunosorbent assay (ELISA) . Next, sections were incubated with a biotinylated anti-mouse immunoglobulin G (IgG) antibody (DAKO) and further incubated with peroxidase-conjugated streptavidin (DAKO). Samples were visualized with 3,3-diaminobenzidine (DAB) solution  and counterstained with hematoxylin. The immunohistochemical staining of tissues were assessed by at least two pathologists.Samples were obtained from benign and tumor tissue from 112 patients with breast cancer who underwent operations between 2000 and 2004 at Osaka Police Hospital . The samples were immunostained with the informed consent of patients and the approval of the Osaka Police Hospital Ethics Committee according to the institutional ethics and legal rules. The detailed clinical subtypes of these patients are enumerated in Table S1 in Additional fileELISA)  = less than 5% of cancer cells positively stained; weak positive score 1) = 5 to 20% of cancer cells positively stained; strong positive (score 2) = more than 20% of cancer cells positively stained. Noncancerous mammary epithelial cells were scored as 2 . Multicollinearity within these predictor variables was examined by nonparametric test (Spearman’s rank correlation coefficient) using all combination (JMP software). PgR status and intrinsic subtype, were excluded because they correlated significantly with ER . Lymph node metastasis was also excluded because the number of samples exhibiting such metastasis was too small to analyze. Therefore, H4K20me3, nuclear grade, ER, and HER2 status were used for the multivariate analysis described in Table P value from a two-tailed test was <0.05.Results were tabulated as a Microsoft Excel worksheet, and then exported into and analyzed in Ekuseru-Toukei 2012 . For clinicopathological analysis  and negative-control siRNAs  were obtained from Thermo Fisher  and transfected into cells using RNAiMax (Life Technologies).The human breast cancer cell lines HBL-100, MDA-MB-231, and BT-474 (American Type Culture Collection) were grown in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% (w/v) fetal bovine serum (FBS) , 100 U/ml penicillin, and 100 μg/ml streptomycin . MCF-7 cells were grown in RPMI1640 (Nissui Seiyaku) supplemented with 10% (w/v) FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. MCF10A mammary epithelial cells were grown in DMEM/F12 (Life Technologies) supplemented with 5% (w/v) horse serum (Life Technologies), 20 ng/ml epidermal growth factor (EGF) , 0.5 μg/ml hydrocortisone (Sigma-Aldrich), 100 ng/ml cholera toxin , and 10 μg/ml insulin . Cells were purchased from ATCC via Sumitomo Pharmaceuticals International ; stocks were made at passage 2 or 3, and cells were used for fewer than 15 passages. Cells were transfected using Lipofectamine LTX (Life Technologies), Lipofectamine 2000 (Life Technologies), or Polyethylenimine ‘Max’ . Small interfering RNAs (siRNAs) against the Cells grown on glass coverslips were fixed and permeabilized for 10 minutes with 4% paraformaldehyde containing 0.5% Triton X-100, and then blocked with 5% BSA. Next, cells were incubated with a primary antibody against H4K20me3 and appropriate secondary antibodies . Finally, coverslips were mounted in ProLong Gold Antifade Reagent with 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies). Fluorescence intensity was analyzed using a fluorescence microscope . The staining intensities were measured using MetaMorph version 7.1 .SUV420H1 and SUV420H2 cDNAs were amplified from HBL-100 cDNA and inserted into the XhoI/BamHI sites and EcoRI/BamHI sites of vector pEGFP-C1, respectively. PCR reactions were performed using KOD Plus high-fidelity DNA polymerase . All cDNA constructs were verified by DNA sequencing.Human SUV420H1 and SUV420H2 were quantitated by real-time PCR using a LightCycler 480 System  and normalized to the mRNA level of GAPDH . Experiments were performed in triplicate. Sequences of the primers used are as follows: for SUV420H1, 5′-GCACGGCACTATTTTCTCAA-3′ and 5′-TCCACTGTCAGTTGCAAACA-3′; for SUV420H2, 5′-GGCCCGCTACTTCCAGAG-3′ and 5′-GCAGGATGGTAAAGCCACTT-3′; for GAPDH, 5′-CAATGACCCCTTCATTGACC-3′ and 5′-TTGATTTTGGAGGGATCTCG-3′.Total RNA was extracted from normal epithelial and breast cancer cell lines as previously described. The mRN5 or 1 × 105 cells were added to the upper chamber and incubated for 24 or 48 hours . Cells were fixed with 3.7% formaldehyde and stained with hematoxylin. Non-invading cells on the upper surface were removed by scrubbing with a cotton swab, and the invading cells were counted under a microscope (200×). Each assay was carried out in triplicate.Matrigel invasion assay was performed using Chemotaxicells  as described previously. BrieflySUV420H2-targeted siRNA or nontargeted negative control siRNA. Twenty-four hours after transfection, cells were seeded in the upper chamber of transwells. After an additional 48 hours , cells were fixed, stained, and counted in five randomly chosen microscopic fields per sample.For invasion assays using MCF10A, cells were transfected with Loss of H4K20me3 has been reported in multiple types of human tumors,13-15. Tin situ (DCIS), invasive ductal carcinoma (IDC), or more detailed classification of IDC[P <0.05) and MIB-1 index (P <0.01). H4K20me3 status also positively correlated with ER expression (P <0.05) and PgR expression (P <0.05), but not with HER2 expression. H4K20me3 staining score associated with each subtype . These results implied that the diversity of H4K20me3 staining patterns might result from the heterogeneous nature of breast cancer, as reflected by these aspects of clinicopathological status.Although loss of H4K20me3 was observed in many cancer cases (63.4%), in 36.6% of cases more than 20% of cancer cells stained positively  more strongly than either ER (P = 0.027) or PgR (P = 0.019) alone, and independently associates with disease-free survival, but not with luminal A and luminal B distribution  could positively regulate H4K20me3.in vivo. The results of previous studies support this idea: HBL-100 and MCF-7 cells, which endogenously express higher levels of SUV420H2 , is associated with prostate- and breast-cancer aggressiveness-45; and There are several possible explanations for the decreased H4K20me3 level in breast cancers. A recent chemogenetic analysis revealed that the human genome encodes 96 protein methyltransferases, which can be classified into two families. AccordiEpigenetic alterations, including DNA methylation and histone modifications, are thought to play important roles in carcinogenesis and cancer progression. Therefore, many drugs targeted at epigenetic pathways, such as DNA methyltransferase and histone deacetylase inhibitors, have been developed, and some have been approved as cancer therapies. Our resin vitro; therefore, this protein may be involved in regulation of breast cancer progression in vivo. Our results also indicate that the H4K20me3-associated invasiveness is independent of the HER2-signaling pathway.In this study, we demonstrated that the loss of H4K20me3 is a novel candidate for an independent prognostic marker for use in breast cancer patients. Moreover, the level of H4K20me3 influences cancer cell invasiveness in situ; DMEM: Dulbecco’s modified Eagle’s medium; EGFR: epidermal growth factor receptor; ELISA: enzyme-linked immunosorbent assay; ER: estrogen receptor; FBS: fetal bovine serum; HER2: human epidermal growth factor receptor; IDC: invasive ductal carcinoma; IgG: immunoglobulin G; mAbs: monoclonal antibodies; PgR: progesterone receptor; siRNA: small interfering RNA; SUV: suppressor of variegation EZH2 (enhancer of zeste homolog 2).BSA: bovine serum albumin; CK: cytokeratin; DCIS: ductal carcinoma The authors declare that they have no competing interests.in vitro experiments. MH wrote the manuscript. All authors read and approved the manuscript.YY, AM, YN, MHi, and NM designed the experiments. KY and MT performed immunohistochemical experiments and analysis of patient data. YY, AM, YS, EO, MHa, MHi, and HK performed immunochemical staining and all Clinical subtype of patients. The detailed clinical subtypes of the patients used in this study.Click here for fileH4K20me3 staining in benign tumor tissue. Specimens of benign tumor tissue were stained using anti-H4K20me3 (left) and HE staining (right).Click here for fileH4K20me3 staining score associates with subtype. H4K20me3 staining score was classified by the hormone receptor expression.Click here for fileH4K20me3 staining and Luminal A/Luminal B distribution. H4K20me3 staining score was classified by the Luminal A and Luminal B. H4K20me3 staining score did not associate with Luminal A/Luminal B distribution.Click here for fileKaplan-Meier analysis of estrogen receptor expression in breast cancer patients. The patient overall survival time and disease-free survival rate were compared between the estrogen receptor expression low- and high-staining groups by Kaplan-Meier analysis.Click here for file


The replication of picornaviruses has been described to cause fragmentation of the Golgi apparatus that blocks the secretory pathway. The inhibition of major histocompatibility complex class I upregulation and cytokine, chemokine and interferon secretion may have important implications for host defense. Previous studies have shown that disruption of the secretory pathway can be replicated by expression of individual nonstructural proteins; however the situation with different serotypes of human rhinovirus (HRV) is unclear. The expression of 3A protein from HRV14 or HRV2 did not cause Golgi apparatus disruption or a block in secretion, whereas other studies showed that infection of cells with HRV1A did cause Golgi apparatus disruption which was replicated by the expression of 3A. HRV16 is the serotype most widely used in clinical HRV challenge studies; consequently, to address the issue of Golgi apparatus disruption for HRV16, we have systematically and quantitatively examined the effect of HRV16 on both Golgi apparatus fragmentation and protein secretion in HeLa cells. First, we expressed each individual nonstructural protein and examined their cellular localization and their disruption of endoplasmic reticulum and Golgi apparatus architecture. We quantified their effects on the secretory pathway by measuring secretion of the reporter protein Gaussia luciferase. Finally, we examined the same outcomes following infection of cells with live virus. We demonstrate that expression of HRV16 3A and 3AB and, to a lesser extent, 2B caused dispersal of the Golgi structure, and these three nonstructural proteins also inhibited protein secretion. The infection of cells with HRV16 also caused significant Golgi apparatus dispersal; however, this did not result in the inhibition of protein secretion.IMPORTANCE The ability of replicating picornaviruses to influence the function of the secretory pathway has important implications for host defense. However, there appear to be differences between different members of the family and inconsistent results when comparing infection with live virus to expression of individual nonstructural proteins. We demonstrate that individual nonstructural HRV16 proteins, when expressed in HeLa cells, can both fragment the Golgi apparatus and block secretion, whereas viral infection fragments the Golgi apparatus without blocking secretion. This has major implications for how we interpret mechanistic evidence derived from the expression of single viral proteins. Viral respiratory tract infections represent a major health care burden, with an estimated 500 million cases and a cost of $40 billion per year in the United States alone . It has 4–9–Picornaviridae family, for which the best studied prototype is poliovirus (PV) and which includes other important pathogens, such as foot-and-mouth disease virus (FMDV), hepatitis A virus, and coxsackie virus (CV). Upon entering a host cell, the RNA genome of these viruses is translated into a polyprotein that is posttranslationally cleaved by the encoded viral proteases to generate the structural and nonstructural proteins required for viral replication. The developing virus forms replication complexes on the surface of intracellular membranes, believed to be derived either from the endoplasmic reticulum (ER)/Golgi secretory apparatus or from autophagosomes, and greatly remodels their morphology and lipid composition  alone can replicate this effect. In some cases, viral replication or expression of 3A has been reported to cause an inhibition of protein traffic through the secretory pathway, but in some cases it does not. Whether a particular virus inhibits the secretory pathway is of great importance in host antiviral defense, as it would inhibit the secretion of inflammatory cytokines, antiviral interferons, and the cell surface expression of major histocompatibility complex (MHC) class I. Indeed, inhibition of the secretory pathway often has been cited as a mechanism by which picornaviruses evade host defense mechanisms.Two independent studies showed that the 3A protein of CV and PV blocked secretion by binding and inhibiting the function of GBF1, the GTP exchange protein that controls the function of Arf1, whereas the 3A proteins of HRV2, HRV14, encephalomyocarditis virus, FMDV, and hepatitis A virus did not , 16. How18–The cervical epithelial HeLa Ohio (ECACC 93021013) and HeLa H1 (ATCC CRL-1958) cell lines were maintained in exponential growth in high-glucose Dulbecco's modified Eagle medium (DMEM) supplemented with glutamine, 1% sodium bicarbonate, 25 mM HEPES, and 10% fetal bovine serum. The transfection of HeLa cells with mammalian expression plasmids was performed using FuGENE HD transfection reagent (Promega), with a 3.5:1 FuGENE HD-to-DNA ratio and doubling the volume of transfection mix added per well compared to the manufacturer's instructions. The HeLa Ohio cell line stably expressing Gluc was created by transfection with the pCMV-GLuc 2 plasmid (New England BioLabs) and selection with 1 mg/ml Geneticin-G418 (Invitrogen).L24917) and an N-terminal Myc tag. The sequence identity and correct orientation of all inserts were verified by DNA sequencing.cDNA constructs for each HRV16 nonstructural protein (NSP) were made by PCR amplification using the following primer pairs (each NSP cDNA was cloned into pRK5-Myc [Clontech] using the restriction sites indicated below by standard molecular biology techniques): Myc-HRV162A, 5′-TATGATGGATCCGGGCCTAGTGACATGTATGTGC-3′ (BamHI) and 5′-GCGCGCGAATTCTCATTGTTCTTCAGCACAGTGAAAG-3′ (EcoRI); Myc-HRV16 2B, 5′-GACGACGGATCCGGAATCACTGATTACATACACATGC-3′ (BamHI) and 5′-CGCGCGAATTCTCATTCTTTGTGTATATAAGTTAATTGAG-3′ (EcoRI); Myc-HRV16 2C, 5′-GCGCGCGGATCCTCAGATTCATGGCTCAAAAAATTCAC-3′ (BamHI) and 5′-GCGCGCGAATTCTCATTGGAAAATTGCAGACATGACATC-3′ (EcoRI); Myc-HRV16 2BC, 5′-GACGACGGATCCGGAATCACTGATTACATACACATGC-3′ (BamHI) and 5′-GCGCGCGAATTCTCATTGGAAAATTGCAGACATGACATC-3′ (EcoRI); Myc-HRV16 3A, 5′-GACGACGGATCCGGGCCTATATCCATGGATAAACCC-3′ (BamHI) and 5′-GCGCGCGAATTCTCACTGTAGAGAGCAAAAGAGC-3′ (EcoRI); Myc-HRV16 3AB, 5′-GACGACGGATCCGGGCCTATATCCATGGATAAACCC-3′ (BamHI) and 5′-GCGCGCGAATTCTCATTGAGCTACCACTCTTCTCTCG-3′ (EcoRI); Myc-HRV16 3C, 5′-GAGTGCGGATCCGGTCCAGAAGAAGAATTTGGAATGTC-3′ (BamHI) and 5′-GCGCGCGAATTCTCATTGTTGTTCAGTGAAGTATGATCTC-3′ (EcoRI); Myc-HRV16 3D, 5′-GACGACGGATCCGGCCAAATTCAAATCTCTAAACATG-3′ (BamHI) and 5′-GCGCTCGCGATCAGAATTTTTCATACCATTCATGTCTTAG-3′ (NruI). All of the pRK5-Myc-HRV16 constructs contained the published HRV16 sequence  (GenBank50) in HeLa Ohio cells.HRV16 viral stocks (ATCC VR-283) were produced by infecting HeLa H1 cells, and their titers were determined by measuring the 50% tissue culture infectious dose (TCIDHeLa Ohio cells were infected with HRV16 at a multiplicity of infection (MOI) of 1 or 20. Infections were synchronized by virus adsorption on the cells for 1 h at room temperature, followed by one wash with phosphate-buffered saline (PBS) and the addition of new media before incubating the cells at 37°C for 2 to 24 h, as indicated.Cells grown on glass coverslips were washed with PBS, fixed for 15 min with 4% formaldehyde, and washed with PBS. After quenching residual formaldehyde with 0.1 M glycine, cells were washed with PBS, permeabilized for 10 min at room temperature with 0.1% Triton X-100, and then washed with PBS. After blocking in 5% fetal bovine serum, cells sequentially were incubated with primary and secondary antibodies diluted in 1% bovine serum albumin (BSA). Apart from the calnexin antibody, which was incubated overnight at 4°C, primary antibodies were incubated for 1 h at room temperature and secondary antibodies were incubated for 45 min at room temperature. Cells were washed in PBS after each antibody incubation. Coverslips were mounted in ProLong Gold antifade reagent (Invitrogen) and analyzed using an LSM 5 PASCAL laser scanning microscope (Carl Zeiss).The following primary antibodies were used at the indicated dilution: mouse anti-Myc tag , rabbit anti-calnexin , rabbit anti-Giantin , mouse anti-GM130 , sheep anti-TGN46 , and rabbit anti-HRV16 2C . Secondary antibodies were obtained from Jackson ImmunoResearch (1/200).g) for 5 min, resuspended in agar, and spun to form a pellet. The pelleted cells were rinsed in cacodylate buffer and postfixed in buffered 1% osmium tetroxide. After a water rinse, the samples were dehydrated by a graded (70 to 100%) methanol series. Sample transition to 100% araldite was through 50:50 and then 25:75 propylene oxide-araldite mixtures. Ultrathin (70-nm) sections of araldite-embedded samples were stained with uranyl acetate and lead citrate and then examined with a transmission electron microscope (Hitachi H7000).One culture well of each HRV16-infected  and uninfected control HeLa cells was scraped and fixed with 2.5% glutaraldehyde in 0.05 M sodium cacodylate buffer (pH 7.2). Cells were spun at 2,500 rpm , washed a further time, and stained with a fixable near-IR live/dead discriminator (Life Technologies). Cells were fixed with 1% formaldehyde, washed, and incubated with 1% human serum (HS) overnight. Following permeabilization with 0.5% saponin-1% HS in PBS, cells were either stained with rabbit anti-HRV16 2C antibody  or purified rabbit IgG control . Cells were visualized with donkey anti-rabbit Alexa 488 secondary antibody . Photomultiplier tube (PMT) voltages were adjusted after standardized cytometer setup and tracking (CST) checks minimizing the spectral overlap to increase data precision. Cells were measured on a Becton Dickinson Fortessa LSR-SORP equipped with various lasers  and an ND1.0 filter in front of the forward scatter (FSC) photodiode and analyzed with FlowJo software (TreeStar).6 cells per well in a 24-well plate, and each time point was performed in triplicate. Cells were infected with HRV16 at an MOI of 20 for 1 h at room temperature and then washed in PBS before adding fresh medium and starting a 7-h time course. During the infection time course, culture medium was removed and replaced every 30 min and cells collected every 1 h. As controls, HeLa-Gluc cells either were not infected with virus or were not infected but treated with brefeldin A (BFA) at a final concentration of 5 μg/ml. To determine the Gluc activity in the cells, the wells were washed with PBS and the cells lysed in 250 μl of luciferase cell lysis buffer . Gluc secreted in the media and remaining in the cells was assayed using the BioLux Gaussia luciferase flex assay kit  according to the manufacturer's instructions. Luminescence was analyzed on a FLUOstar OMEGA plate reader (BMG Labtech) as recommended. Each time point was determined in triplicate, and the experiment was performed independently three times.For transfection experiments, HeLa Ohio cells were cotransfected in 24-well plates with pCMV-GLuc 2- and pRK5-Myc-derived plasmids at a ratio of 1:3 for 24 h. For infection experiments, HeLa Ohio cells stably expressing Gaussia luciferase (HeLa-Gluc) were plated at a density of 0.2 × 10Cells were lysed in Laemmli sample buffer, and their DNA content was measured with a NanoDrop. An equivalent amount of DNA was loaded for each sample, and proteins were separated by SDS-PAGE. After transfer to PVDF membranes, proteins were revealed with a rabbit anti-HRV16 3A antiserum (produced for this study), a rabbit anti-β-actin antibody , or a mouse anti-Myc tag antibody . Secondary antibodies were obtained from Jackson ImmunoResearch.Escherichia coli BL21(DE3) and purified from inclusion bodies. The identity of the purified protein was confirmed by peptide mass fingerprinting. Purified HRV16 2C protein was used to immunize two rabbits using standard protocols . All antiserum titers were quantified by enzyme-linked immunosorbent assay (ELISA), and their specificity was tested by Western blotting.A 29-amino-acid peptide corresponding to the N terminus of HRV16 3A (GPISMDKPPPPAITDLLRSVRTPEVIKYC) was synthesized and coupled to the carrier protein PPD, which had previously been derivatized to the heterobifunctional cross-linker MBS . The peptide-PPD conjugate was used to immunize two rabbits using standard protocols . A full-length cDNA sequence for HRV16 2C with an N-terminal 6-His tag was codon optimized and cloned into the bacterial expression plasmid pET-26b (Novagen) using the NdeI/XhoI sites. The full-length 2C protein was expressed in In order to assess the subcellular localization of the HRV16 nonstructural proteins, each individual protein was expressed in HeLa cells with an N-terminal Myc tag by transfection. The expression of the proteins was verified by Western blotting with an anti-Myc antibody, revealing the expression of all HRV16 nonstructural proteins apart from 2A . TransfeThe analysis of the transfected cells by immunofluorescence microscopy with an anti-Myc antibody revealed that the viral nonstructural proteins 2A, 3C, and 3D had a diffuse localization in the cytoplasm and the nucleus, with 2A and 3C accumulating particularly in the nucleus of transfected cells  and 3. HThe nonstructural proteins 2B, 2C, 2BC, 3A, and 3AB had cytoplasmic localization and colocalized to various extents with the ER marker calnexin . In partcis- and medial-Golgi marker Giantin, together with the Myc tag , whereas the effect of 2B expression did not reach significance  and the trans-Golgi marker (TGN46). This effect increased as the infection time and 2C expression levels progressed, as evidenced by the quantification of the Golgi structure in 2C-positive infected cells from 3 independent experiments at MOIs of 20 and 1  by 7 hpi  and used it for infection with HRV16 at an MOI of 20. We analyzed 2C expression following an infection time course as a marker for viral replication. HeLa cells were infected with HRV16, and 2C expression over a 7-h time course was detected by immunofluorescence confocal microscopy . The perby 7 hpi . The infby 7 hpi . As an aby 7 hpi , suggestby 7 hpi . At 6 hpby 7 hpi  and 80.6by 7 hpi ; thus, aIn this study, we have sought to characterize the subcellular localization of individually transfected HRV16 proteins and their influence on the structure and function of the secretory pathway and to compare these effects to those of HRV16-infected cells.We have shown that individual expression of HRV16 2B induced ER aggregates and slightly disrupted the Golgi structure, while expression of 3A and, to a lesser extent, 3AB, which also colocalized with the ER, induced significant Golgi structure dispersion. Expression of 2B, 3A, and, to a lesser extent, 3AB significantly inhibited cellular protein secretion, as evidenced by the reduction of the secretion of the Gluc reporter. The other nonstructural proteins did not significantly affect the ER or Golgi structure or Gluc secretion. These results show that inhibition of secretion is not directly related to the level of NSP expression (3A and 3AB being very high and 2B being very low) and not directly related to the degree of Golgi dispersal (3A and 3AB being significant and 2B less pronounced). Our results on the effect of HRV16 3A are consistent with the effect reported for HRV1A 3A , which i25–38–Given our concerns about studying the effects of individual NSPs on the secretory pathway, we decided to analyze the effect of HRV16 infection. We optimized an MOI and time course to give us as synchronous and uniform an infection as possible, and we found that over this time course viral replication led to a substantial dispersion of the Golgi structure but did not significantly block or reduce protein secretion. After 6 hpi, there was a trend for a reduction in the rate of Gluc secretion, although not a complete inhibition, and this corresponded with the onset of a CPE. The observation that infected and uninfected cells produced equivalent amounts of Gluc protein over the 7-h time course (medium plus cell lysate) suggests that there was not a noticeable translation shutoff of Gluc mRNA following infection. The Golgi structure fragmentation we observed is consistent with the many previously cited reports. It is well established that the infection of cells with picornaviruses induces dramatic rearrangements of membranes of the secretory pathway into double membrane-bound vesicles upon which viral replication complexes are thought to form, although the exact origin of these replication complexes still is unclear. Our electron microscopy (EM) studies with HRV16 do not reveal quite such dramatic membrane remodelling as that described for PV. We observed an increase in swollen single-membrane vesicles and multivesicular bodies and, by 7 hpi, frequent electron-dense structures of unknown origin but reminiscent of condensed mitochondria. There is evidence to suggest that PV and CV form replication complexes by modifying COPI vesicles through interference with GBF1/Arf1 –46, and –41–The absence of a significant cellular protein secretion defect in infected cells is consistent with previously published reports on HRV-infected human bronchial epithelial cells and studies in patients showing that infected cells can secrete a wide range of cytokines, chemokines, and interferons and can upregulate MHC class I –70. Comp–Our experiments with transfected HRV16 3A indicate that this nonstructural protein disrupts the Golgi structure and inhibits cellular protein secretion, whereas our infection experiments indicate that infection with the whole virus disrupts only the Golgi structure and does not inhibit cellular protein secretion. This suggests that studies based on the isolated expression of individual nonstructural picornaviral proteins are unlikely to reflect the complexity of the changes occurring during an infection, as proposed by others .The observation that HRV16 infection leads to the dispersion of the Golgi structure but does not block cellular protein secretion raises several unresolved questions. First, picornaviruses disrupt the Golgi structure by a mechanism that is thought to act through the 3A protein interacting with GBF1, although it is not clear if this results in activation or inhibition of Arf1. BFA also fragments the Golgi structure by inhibiting Arf1, yet it is a potent inhibitor of viral replication , 71. Wha


Systemic onset juvenile idiopathic arthritis (SoJIA) shows properties of autoinflammatory disease, and requires the presence of arthritis and fever for diagnosis. The pro-inflammatory proteins MRP8/14 (S100A8/9) and S100A12 are biomarkers which have been shown to detect ongoing subclinical disease activity in patients with clinical remission. We compared the MRP8/14 (S100A8/9) and S100A12 biomarker profile of patients in the AID-NET (German Auto-inflammatory Disease Network) register to further provide evidence for the usefulness of measurement of these biomarkers.To characterize levels of pro-inflammatory markers of innate immune activation, S100A12 and MRP8/14, during active and inactive disease status of patients with SoJIA.The AID-Net register, which includes patients with SoJIA diagnosed according to ILAR criteria, was searched for patients with clinically defined remission and flare episodes. Patients in remission were those documented as being either: 'non-acute', in remission, or with a clinician score of <1 . Flares were any cases scored as 'flare' or 'acute'. Statistical significance was measured using the Mann-Whitney-U test, using SPSS.55 patients with a median age of 13 years (range 5-20 years) at time of blood sampling were included. A total of 158 episodes occurred where disease activity status and MRP8/14 and S100A12 results were available. This included 38 episodes of flares (19 patients) and 120 episodes of remission (44 patients). Patients presenting with episodes of flare had significantly higher mean S100A12 values compared with patients in remission  ng/ml vs 575  ng/ml, respectively, p- < 0.01). MRP 8/14 values were also higher in patients who were clinically flaring than in those in remission  ng/ml vs 2,965  ng/ml, p < 0.01).The measurement of MRP8/14 and S100A12 biomarkers in patients with SoJIA in the AID-net register corresponded well with the recorded clinical disease activity in these patients. This provides further evidence for the measurement of these biomarkers.References: 1) Foell D et al. Methotrexate withdrawal at 6 vs 12 months in juvenile idiopathic arthritis in remission: a randomized clinical trial. JAMA 2010;303:1266-73. 2) Gerss J et al. Phagocyte-specific S100 proteins and high-sensitivity C reactive protein as biomarkers for a risk-adapted treatment to maintain remission in juvenile idiopathic arthritis: a comparative study. Ann Rheum Dis 2012;71:1991-7.None declared.


Beet curly top virus (BCTV). The C4 protein of BCTV is sufficient to induce hyperplasia and alter Arabidopsis development. It was previously shown that C4 interacts with two Arabidopsis Shaggy-like protein kinases, AtSK21 and 23, which are negative regulators of brassinosteroid (BR) hormone signaling. Here we show that the C4 protein interacts with five additional AtSK family members. Bikinin, a competitive inhibitor of the seven AtSK family members that interact with C4, induced hyperplasia similar to that induced by the C4 protein. The Ser49 residue of C4 was found to be critical for C4 function, since: 1) mutagenesis of Ser49 to Ala abolished the C4-induced phenotype, abolished C4/AtSK interactions, and resulted in a mutant protein that failed to induce changes in the BR signaling pathway; 2) Ser49 is phosphorylated in planta; and 3) plant-encoded AtSKs must be catalytically active to interact with C4. A C4 N-myristoylation site mutant that does not localize to the plasma membrane and does not induce a phenotype, retained the ability to bind AtSKs. Taken together, these results suggest that plasma membrane associated C4 interacts with and co-opts multiple AtSKs to promote its own phosphorylation and activation to subsequently compromise cell cycle control.Even though plant cells are highly plastic, plants only develop hyperplasia under very specific abiotic and biotic stresses, such as when exposed to pathogens like  Geminiviridae) cause extensive agricultural losses in crops worldwide ) C4 protein and the Tomato golden mosaic virus (TGMV-[BR:Com:84]) AC4 protein interacted with two members of the Arabidopsis SHAGGY-like protein kinase family (AtSK21 and AtSK23), which are involved in the brassinosteroid (BR)-signaling pathway ) was shown to interact with a novel SHAGGY-like protein kinase (SlSK) of tomato that is closely related to AtSK21 and AtSK23 ) infection ), Beet severe curly top virus BSCTV-[US:Cfh], Spinach curly top virus (SpCTV-[US:Sp3:36]), and two monopartite begomoviruses, Tomato yellow leaf curl virus (TYLCV-IL[ES:Alm:Pep:99]) and ToLCV-To (N. benthamiana plants following inoculation with genomic transcripts from a potato virus X based vector (pPVX) [C4 or the C4-mutants. A non-translatable C4 (C4nt) served as a negative control [To identify amino acids critical for C4 function, alanine-scanning mutagenesis  was perfToLCV-To . The C4 r (pPVX)  carryingin vitro at undefined serine and threonine residues [N. benthamiana plants, whereas the Thr18 (C4A18T) phenotype remained the same  or C4A49T (IPC4A49T-2) at levels similar to wild-type C4 in transgenic line IPC4-28 (see below) were chosen for further study. IPC4S49A-2 and IPC4A49T-2 phenotypes, with and without ß-estradiol induction, were compared to transgenic line IPC4-28. Seedlings of the three lines developed normally and were indistinguishable from wild-type Arabidopsis when germinated in the absence of the inducer  mutants of the sg2AtSKs. The catalytically inactive AtSK21K69R mutant (referred to hereafter as AtSK21-KD) was unable to phosphorylate BZR1 ,48. We gn planta . The expn planta . Taken tTo determine the biological significance of the loss of C4 function, we assayed the effect of C4 expression on BR signaling. Expression of C4 resulted in a shift between phosphorylated and hypophosphorylated BES1 levels, and a positive correlation between the steady state levels of C4 protein and hypophosphorylated BES1 . We hypoCPD and DWF4, are repressed in the presence of the C4 protein [CPD and DFW4 transcript steady state levels. In contrast, the reversion mutant, C4A49T, should mimic C4. CPD and DWF4 transcript levels were compared in induced IPC4-28, IPC4S49A-2, IPC4A49T-2 and IPC4nt-12 seedlings relative to non-induced seedlings by qRT-PCR at 24 h post-induction  in the presence of wild-type C4, while no significant changes in the expression levels of CPD and DWF4 (<1.5-fold) were detected in the presence of C4S49A  in the presence of the revertant, C4A49T. These results corroborate the conclusion from To further confirm the role of C4 in altering BR-signaling, we used qRT-PCR analysis to determine the expression levels of BR-signaling pathway target genes in seedlings expressing C4S49A. We previously showed that BZR1 target genes, nduction . Steady f C4S49A . As expeN. benthamiana . While the importance of a phosphoacceptor residue at non-conserved residue 21 was not further pursued in this report, phosphorylation of a phosphoacceptor residue at position 49 was found to be required for C4-induced symptoms. Studies on recombinant BCTV C4 and TGMV AC4 proteins previously identified Ser49 as one of four amino acids in the central region of the protein that play a role in the C4 phenotype, although the significance of each individual amino acid was not determined . In C4, function . Future in planta correlates with the loss of phenotype following induction of C4S49A in transgenic plants, the loss of the C4-induced phenotype when C4S49A was expressed from a chimeric PVX-vector following inoculation onto N. benthamiana plants, the inability of C4S49A to affect the phosphorylation status of phosphorylated BES1 and the inability to modify BZR1 target gene (CPD and DWF4) transcript steady state levels. Second, C4 and C4A49T interactions with sg2AtSKs localize to the plasma membrane and the nucleus, while the mutant C4G2A interacts strongly with AtSK23 in the cytosol and nucleus in planta, but does not localize to the plasma membrane and fails to induce a phenotype. Therefore, only C4/AtSK interactions at the plasma membrane are functionally important.Mutants of C4 that disrupt C4/AtSK interactions (C4S49A) or localization of C4 to the plasma membrane (C4G2A) fail to induce hyperplasia or alter seedling development. The biological importance of the C4/AtSK interactions at the plasma membrane was supported by the following findings. First, the lack of a detectable interaction between plasma membrane localized C4S49A and AtSK23 Arabidopsis. In contrast, BL, which negatively regulates the BR response by inhibiting sg2AtSK and AtSK32 activities [Our results show that bikinin, which specifically inhibits sg1AtSKs, sg2AtSKs and AtSK32 , inducestivities ,24,25, htivities .50 of 23.3 μM for bikinin in a hypocotyl elongation assay (56).The observed C4/AtSK interactions suggest that the C4 protein, similar to bikinin, binds directly to the same seven AtSKs, which could explain the phenocopying induced by C4 and bikinin. Bikinin induces hyperplasia in a concentration dependent manner with significant hyperplasia being observed at ≥30 μM bikinin. At bikinin concentrations of ≥50 μM, bikinin-induced hyperplasia was indistinguishable from C4-induced hyperplasia. Previously, bikinin (30 μM) was shown to induce significant developmental changes by 3 days post treatment  and to iThere is considerable complexity in the role of AtSKs in a variety of biological processes . HoweverThe C4 protein provides an invaluable tool for expanding our understanding of the host factors and regulatory pathways involved in cell cycle regulation and to provide insights into the relationship between the plant cell cycle and development. In this study, we provide evidence that C4 function is activated by phosphorylation of Ser49 by members of the AtSK family and suggest that the protein regulates multiple AtSK activities that may subsequently disrupt mitotic control. Our results are consistent with a model in which C4 functions by sequestering multiple members of the AtSK family to the plasma membrane or by competing with AtSK target proteins resulting in inhibition of an array of AtSK functions. The possible disruption of AtSK function(s) by C4, which might equate to a similar response by bikinin, results in hyperplasia. Alternatively, an intriguing possibility is that C4 has an as yet undefined function after being activated by phosphorylation, which may modulate one or more signal transduction pathways at the plasma membrane, that result in hyperplasia. Future studies aimed at defining the specific C4/AtSKs interactions that result in hyperplasia and the role of phosphorylation in C4 function will reveal additional insights into the mechanism by which the C4 protein regulates cell division.S1 FigN. benthamiana. For comparison, phenotypes of transgenic plants expressing the C4nt negative control and the C4 positive control. Third systemic leaf collected from plants expressing C4nt, C4, C4-cEYFP, or C4-nEYFP shown in the bottom panel. B. Confocal micrographs of C4 fused to EYFP agroinfiltrated into N. benthamiana CFP-H2B marker plants. C. Confocal micrographs of AtSK22, AtSK23, and AtSK41 fused to mCherry agro-infiltrated into N. benthamiana CFP-H2B plants. AtSK22-, AtSK23- and AtSK41-mCherry fusion proteins localize to the cytoplasm, evidenced by cytoplasmic strands (arrow) and cytoplasmic granules (arrowhead), and plasma membrane. The mCherry signal also co-localized to the nucleus with the CFP reference nuclear signal (CFP and overlay). Arrowhead, cytoplasmic granule; arrow, cytoplasmic strands. Scale bar = 50 μM.A. Phenotypes induced following inoculation of transcripts from pPVX-C4-nEYFP  or pPVX-C4-cEYFP  onto (TIF)Click here for additional data file.S2 FigNon-specific protein used as a loading control (LC) is shown below each immunoblot. Mw: AtSK21 = 43.01 kDa, AtSK23 = 46.53 kDa, AtSK41 = 47.68 kDa.(TIF)Click here for additional data file.S3 FigN. benthamiana plants. Transcripts from pPVX-C4 were used as a positive control. Transcripts from pPVX-C4nt and pPVX were used as negative controls. Plants shown at 14 days post inoculation. pPVX, potato virus X expression vector.Phenotypes induced following inoculation of transcripts from pPVX-C4 mutants onto (TIF)Click here for additional data file.S4 FigA. IPC4SII transgenic seedlings grown on solid media (Non-Induced) or solid induction media (Induced) at 12 days post-induction. Scale bars = 4 mm (Non-induced) and 2 mm (Induced). B. Immunoblot of extracts from induced IPC4-28, IPC4SII-6 and IPC4A49TSII-1 seedlings prior to affinity chromatography (Pre-affinity column) or following affinity chromatography (Post-affinity column). Non-specific protein used as a loading control (LC) is shown in the lower panel of Pre-affinity Column samples. Non-specific proteins were not present in Post-affinity Column samples. Mw: C4 = 9.69 kDa, C4SII = 10.89 kDa, C4A49TSII = 10.90 kDa.(TIF)Click here for additional data file.S5 Fig21DYSTWYPQPGQHISIR, ELNPAPTSpS49PTSTR, and ELNPAPTSpT49PTSTR, respectively. Their precursor ions are shown at the top of each spectrum. The key fragment ions are emphasized at the sequence diagrams for ambiguous phosphorylation site assignments.A-C are the Collisional Induced Dissociation MS/MS spectra of SQIpS(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.