Cannabinoid agonists stimulate feeding in rodents and increased levels of endocannabinoids can disrupt lipid metabolism. Therefore, the hypothesis that sustained endocannabinoid signaling can lead to obesity and diabetes was examined in this study using S426A/S430A mutant mice expressing a desensitization-resistant CB1 receptor. These mice display exaggerated and prolonged responses to acute administration of phytocannabinoids, synthetic cannabinoids, and endocannabinoids. As a consequence these mice represent a novel model for determining the effect of enhanced endocannabinoid signaling on metabolic disease. S426A/S430A mutants consumed equivalent amounts of both high fat (45%) and low fat (10%) chow control diet compared to wild-type littermate controls. S426A/S430A mutants and wild-type mice fed either high or low fat control diet displayed similar fasting blood glucose levels and normal glucose clearance following a 2 g/kg glucose challenge. Furthermore, S426A/S430A mutants and wild-type mice consumed similar amounts of chow following an overnight fast. While both THC and JZL195 significantly increased food intake two hours after injection, this increase was similar between the S426A/S430A mutant and wildtype control mice Our results indicate that S426A/S430A mutant mice expressing the desensitization-resistant form of CB1 do not exhibit differences in body weight, food intake, glucose homeostasis, or re-feeding following a fast.Multiple lines of evidence implicate the endocannabinoid signaling system in the modulation of metabolic disease. Genetic or pharmacological inactivation of CB Additionally, the Emax for CP55,940-stimulated [35S]GTPγS binding in the spinal cord was also similar in S426A/S430A mutant compared to wild-type mice demonstrating that the mutant form of CB1 can be activated by agonist to an equivalent extent [1 in the cerebellum were detected in S426A/S430A mutant mice. This raises the possibility that S426A/S430A mutants might possess region-specific dysregulation of endocannabinoid signaling in brain regions responsible for metabolic regulation such as the hypothalamus. Alternatively, CB1 receptor desensitization might be less critical in some regions, a hypothesis that is supported by findings demonstrating that repeated Δ9-THC administration resulted in significant CB1 desensitization in the prefrontal cortex, amygdala and hippocampus but no CB1 desensitization was observed in the striatum [1 desensitization is behavior and region specific [1 desensitization in response to agonist is sex and age-specific [1 receptor hyperactivity on specific metabolic parameters.Early during the phenotypic characterization of S426A/S430A mutant mice we hypothesized that the relative lack of metabolic phenotype might be due to down-regulation of CBolerance ,40. Therant mice . The numt extent . Howeverstriatum . Additiospecific . A furthspecific , raising9-THC was administered or endocannabinoid levels were increased using an inhibitor of endocannabinoid breakdown [9-THC-induced feeding. As expected, lower doses of Δ9-THC significantly and dose-dependently increased food intake 1 and 2 hours after its administration Click here for additional data file.S2 File(PZF)Click here for additional data file.S3 File(PZF)Click here for additional data file.S4 File(PZF)Click here for additional data file.S5 File(PZFX)Click here for additional data file.S6 File(PZFX)Click here for additional data file.S7 File(PZF)Click here for additional data file.S8 File(PZF)Click here for additional data file.S9 File(PZFX)Click here for additional data file.S10 File(PZF)Click here for additional data file.S11 File(PZFX)Click here for additional data file.S12 File(PZFX)Click here for additional data file.


Background. Reliable genotyping that provides an accurate description of diversity in the context of pathogen emergence is required for the establishment of strategies to improve disease management. MultiLocus variable number tandem repeat analysis (MLVA) is a valuable genotyping method. It can be performed at small evolutionary scales where high discriminatory power is needed. Strains of the Ralstonia solanacearum species complex (RSSC) are highly genetically diverse. These destructive pathogens are the causative agent of bacterial wilt on an unusually broad range of host plants worldwide. In this study, we developed an MLVA scheme for genotyping the African RSSC phylotype III.Methods. We selected different publicly available tandem repeat (TR) loci and additional TR loci from the genome of strain CMR15 as markers. Based on these loci, a new phylotype III-MLVA scheme is presented. MLVA and multiLocus sequence typing (MLST) were compared at the global, regional, and local scales. Different populations of epidemiologically related and unrelated RSSC phylotype III strains were used.Results and Discussion. Sixteen polymorphic TR loci, which included seven microsatellites and nine minisatellites, were selected. These TR loci were distributed throughout the genome (chromosome and megaplasmid) and located in both coding and intergenic regions. The newly developed RS3-MLVA16 scheme was more discriminative than MLST. RS3-MLVA16 showed good ability in differentiating strains at global, regional, and local scales, and it especially highlighted epidemiological links between closely related strains at the local scale. RS3-MLVA16 also underlines genetic variability within the same MLST-type and clonal complex, and gives a first overview of population structure. Overall, RS3-MLVA16 is a promising genotyping method for outbreak investigation at a fine scale, and it could be used for outbreak investigation as a first-line, low-cost assay for the routine screening of RSSC phylotype III. Genotyping with appropriate genetic markers  is a valuable tool that has been effectively used to study infectious diseases, resolve the epidemiology of pathogens, and improve disease management strategies . DiseaseMultilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) typically determine sequence polymorphisms in 7–8 housekeeping genes. These genotyping methods are consistently used to infer genetic relationships and evolutionary history on a global scale. Therefore, these methods are recognized as the gold standard for the global epidemiology of bacterial pathogens . HoweverMultilocus variable number tandem repeat analysis (MLVA) is an alternative genotyping method that targets tandem repeats (TRs), which are some of the most polymorphic loci found in genomes . VariatiRalstonia solanacearum species complex (RSSC) is one of the most destructive plant diseases worldwide. RSSC affects a wide range of host species—primarily vegetable, woody, and ornamental species. This soil-borne and xylem-limited bacterium is an unusually heterogeneous and highly disparate organism  and MLSA (n = 14) studies (Solanum scabrum), tomato (Solanum lycopersicum), potato (Solanum tuberosum), and pepper (Capsicum annuum) (Three populations of RSSC phylotype III isolates were used . All 65  studies . Populat annuum) . Populat annuum) .Isolates were routinely grown at 28 °C on Nutrient Broth, Kelman’s triphenyl tetrazolium chloride agar medium , and modThe design of the optimized MLVA scheme involved the following three major steps: the TR loci to be characterized were selected, the primers for gene amplification were designed, and the multiplex PCR reactions were optimized.The RS3-MLVA11 scheme  consisteRS2AL03 was identified from the phylotype IIA strain CFBP2957. RS2BL23 was identified from the IIB strain MOLK2. RS1L05, RS1L09, RS1L10, and RS1L12 were identified from the phylotype I strain GMI1000. RS4L26 was identified from the phylotype IV strain PSI07. RS3L17, RS3L18, RS3L19, and RS3L20 were identified from the phylotype III strain CMR15. The average nucleotide identity (ANI) values are as follows: 91.2% between CMR15 and CFBP2957 (IIA-36), where RS2AL03 is identified; 90.9% between CMR15 and Molk2 (IIB-3), where RS2BL23 is identified; 96.3% between CMR15 and GMI1000 (I-18), where RS1L05, RS1L09, RS1L10, and RS1L12 are identified; and 92.4% between CMR15 and PSI07 (IV-10), where RS4L26 is identified . We examFP885895 for the chromosome and FP885896 for the megaplasmid). The CMR15 genome was screened for repetitive DNA sequences using the Tandem Repeats Finder software package (https://tandem.bu.edu/trf/trf.html) ((i) No indel occurred between adjacent copies. (ii) The sequence identity within the TR array was greater than 85%. (iii) The size of the consensus pattern was ≤20 bp. (iv) The copy number was greater than 3. In the event of multiple TR assignations reported for a same locus, the shorter repeat was retained. (v) Regions containing overlapping or successive TR arrays were not considered. (vi) The TR was found only once within the genome. (vii) The PCR products ranged in length from 100 to 500 bp for matching the ladder used during multiplex capillary electrophoresis.We used the complete genome of the phylotype III strain CMR15 to identify new TR loci (accession numbers rf.html) . Candidahttp://primer3.ut.ee/)  . Oligonucleotides were synthesized by Macrogen, Inc. . The primers were tested via simplex PCR using RSSC isolates from population P35. PCR amplification was performed in 15-µl reaction volumes containing 7.5 µl 2 × QIAGEN Type-it Multiplex PCR Master Mix, 3 µl 5 × Q-solution , 1.5 µl of a forward and reverse primer mix (2 µM each), 2 µl sterile HPLC-grade water, and 1 µl of a bacterial suspension as a template. The PCRs were performed in a GeneAmp® PCR System 9,700 thermal cycler  using the following conditions: an initial denaturation step at 95 °C for 5 min; 30 cycles of denaturation at 95 °C for 30 s, annealing at a temperature gradient  for 90 s, and extension at 72 °C for 30 s; and a final extension step at 60 °C for 30 min. Six microliters of the PCR product were mixed with 1 µl of loading dye solution and loaded into a 1.5% (w/v) SeaKem® LE Agarose  gel for electrophoresis. The gels were stained with ethidium bromide. The bands were visualized/photographed under ultraviolet (UV) light using the G:BOX gel imaging system . The molecular weights were estimated by comparison with a 100-bp DNA ladder . Loci with poor amplification and/or that lacked diversity were discarded. Sequences of the primers used to amplify previous TR loci were obtained from a previous report (Primer pairs flanking the TR candidates were designed using the Primer3 v4.0.0 software package (.ut.ee/) . The cons report ; one excs report . DescripAccording to the amplicon size, four sets of primer mixes, which consisted of four specific primer pairs each, were created for the simultaneous amplification of multiple TR loci (multiplex PCR) . The sui® Size Standard (Applied Biosystems). The samples were denatured at 95 °C for 5 min and cooled immediately on ice for 5 min before being loaded onto a 3130xl Genetic Analyzer. Capillary electrophoresis was performed using 36-cm capillaries filled with POP7-polymer (Applied Biosystems). The assays were run at 60 °C for 30 min, with a running voltage of 15 kV. Injection was performed at 15 kV for 23 s. Each TR locus was identified as a peak according to its colour and size (® v4.0 software package (Applied Biosystems) using the settings for microsatellite analysis.Prior to the size analyses, 1 µl of fluorescent PCR products was diluted in sterile HPLC-grade water; the dilutions were between 1:10 and 1:50, as determined from the test runs. The diluted aliquots (1 µl) were mixed with 10.7 µl Hi-Di formamide (Applied Biosystems) and 0.3 µl GeneScanTM-500 LIZand size . Peaks wThe amplicon sizes were converted into repeat numbers. The repeat numbers for two to four alleles (depending on the range of repeat numbers) from each locus were confirmed by sequencing. The sequence analysis results were also used to check for patterns in the flanking sequences and internal repeat variations . The calculated number of repeats, referred to as alleles, were combined into a string and ordered according to the position of the loci in the CMR15 genome. The allele strings were reported as MLVA profiles. Each unique MLVA profile was given a haplotype designation, called an MLVA type (MT), using the GenAlEx v6.5 software package . Each isgdhA, mutS, adk, leuS, rplB, gyrB, and egl) were chosen from a previous MLSA scheme (gyrB primers were redesigned to address amplification failures with some isolates. Descriptions of the loci and the corresponding oligonucleotide primer sets are presented in 2 solution (25 mM), 1 µl dNTP mix (10 mM each), 1.25 µl of a primer pair mix (10 µM each), 0.25 µl of GoTaq® G2 Flexi DNA Polymerase (5 µ.µl−1), 31.25 µl sterile HPLC-grade water, and 2 µl of fresh bacterial suspensions used as a template. The optimized amplification protocol was as follows: an initial denaturation at 96 °C for 9 min; 30 cycles of denaturation at 95 °C for 1 min, an appropriate annealing temperature . The allelic richness (A) estimates the genetic diversity in a population; it was calculated per locus and population using the allelic.richness function in the “hierfstat” package (n = 17) drawn from the population.All analyses were performed with R v 3.0.2 software package , except  package . This cak loci (k = 1–16). A haplotype accumulation curve (HAC) for increasing numbers of loci was generated for each population tested. Reaching a plateau indicated that the locus set tested was sufficient to identify all unique haplotypes   using thy_power/ . This inr method . The strr method ; similarfunction  providedFinally, the ability of MLST and MLVA genotyping to link or differentiate strains was assessed. MLVA and MLST minimum spanning trees (MSTs) for collection C65 were analysed. MSTs were built using the goeBURST full MST algorithm, which is based on the Euclidean and goeBURST distances between two profiles; this was implemented in the PHYLOViZ v1.0 software package . Clonal Among the 11 TR loci previously described for phylotype III strains , seven TIn this study, we retained the following five TR loci from RS3-MLVA11: RS3L17, RS3L19, RS1L05, RS1L10, and RS1L12. These five TR loci fulfilled our selection criteria and were successfully amplified from collection C65 (100% typeability) .Additional TR loci were required to improve the resolution of MLVA typing. Because size homoplasy  is inherent to the TR loci, the use of a large number of TR loci could decrease the effect on genetic structure and population genetics indices . EighteeIn total, 16 loci (five from RS3-MLVA11 and 11 newly identified) were selected as candidate markers for the new MLVA scheme . A totalThe newly developed MLVA scheme for phylotype III strains is hereafter referred to as RS3-MLVA16. This scheme consists of 16 TR loci regularly scattered throughout the chromosome (RS3L27-RS3L28-RS3L29-RS3L17-RS3L19-RS3L30-RS3L31-RS3L32) and on the megaplasmid (RS3L33-RS3L34-RS1L05-RS3L35-RS3L36-RS1L10-RS3L37-RS1L12) of the phylotype III strain CMR15 . Seven lThe same MLVA scheme can be used for addressing different questions. The required precision level of a genotyping technique is typically objective dependent. The analysis of a few isolates during an outbreak investigation would require a much lower refinement in the markers’ characteristics than a more complex population genetics analysis for which the evolution mode of the markers would need to be accurately determined. The original design of an MLVA scheme should allow the accomplishment of various objectives and consequently a thorough selection of markers should be implemented. Ideally, a single typing technique answering every investigator’s needs is desirable because it could bring together communities that have different, non-overlapping objectives. In this study, we provide an MLVA scheme optimized for practicality and containing a sufficiently high number of markers for maintaining homoplasy at a reasonable level, while eliminating markers for which future users could not appropriately evaluate their evolution mode and mutation rate.HE ranging from 0.11 to 0.91; mean HE: 0.63 and mean A: 5.57). RS3-MLVA16 showed high discrimination between isolates from different countries (HGDI: 0.99). The set of 16 loci discriminated 32 haplotypes with 29 singletons by Minimum Spanning Tree , all loci were polymorphic with high indexes of diversity . Every isolate had a unique haplotype; of these, 18 were singletons (HGDI: 1) , as was found at the global scale. Two loci (RS1L10 and RS1L05) were monomorphic , geographically related isolates were discriminated well using the 16 loci (HGDI: 1) . Within omorphic . These lomorphic .n = 14) among isolates . However, these loci clearly resolved 4 haplotypes  without any singleton (HGDI: 0.42), suggesting the existence of outbreak-evolved strains that formed a complex clonal (MLVA-CC3) within P17 , most RS3-MLVA16 loci were monomorphic  were resolved in 48 haplotypes by RS3-MLVA16, and these haplotypes contained 39 singletons and 3 clonal complexes  that each consisted of epidemiologically related strains . The disThe resulting HACs of the locus combinations reached a plateau in all of the populations tested . This reAll MLST gene fragments were successfully amplified for the 65 isolates used in this study.HE ranging from 0.89 to 0.95; mean HE: 0.93; and mean A: 10.80; At a global scale (P35), high polymorphism was revealed for each of the 7 loci . The 20 isolates were resolved into 11 haplotypes with 3 singletons and 2 CCs containing ST18 and ST7 from AEZ4 and AEZ5; ST2, ST3, ST4, ST13, ST14 and ST17 from AEZ3 and AEZ4; (HGDI: 0.91) , all loci were polymorphic (I: 0.91) , respectAt a local scale (P17), all seven loci were monomorphic.egl is an exception). This may explain the phylogenetic relationships among related Cameroon strains and among Reunion strains. Each MLST-CC is most likely derived from a common ancestor. This complex most likely spread with clonal expansion, and a new variant arose from the source ST and propagated new clonal STs. Typically, haplotypes from Guinea and Zimbabwe were distantly related and differed by four to six loci within their respective countries (ST11 and S15 from Guinea were exceptions that differed at two loci (DLV)  were resolved in 32 haplotypes with MLST, including 22 singletons. The discriminatory index HGDI was 0.91. Based on the MLST data , the genci (DLV) ; howeverci (DLV) . Higher gl genes , AFLP angl genes , and MLSgl genes .The MLVA profiles for the 48 haplotypes generated by RS3-MLVA16 and the MLST profiles of the 32 haplotypes resolved by MLST are listed in p-value = 0.05). Coherence occurs primarily at the level of clonal clusters found in Reunion, Cameroon (AEZ4), and Ivory Coast. However, RS3-MLVA16 showed higher differentiation of the worldwide strains than MLST; RS3-MLVA16 predicted the STs in 74.4% , whereas MLST had a lower prediction of partition by RS3-MLVA16 . At the regional scale (P20), the genetic distances between the two methods were highly correlated , but they showed different partitions (aR was 0.00). RS3-MLVA16 was more discriminative and had high prediction of STs . At the local scale (P17), RS3-MLVA16 and MLST were not similar, resulting in higher resolution of closely related strains by RS3-MLVA16 . Combining the 65 isolates (C65), the congruence between RS3-MLVA16 and MLST was high . The MT to predict ST was 0.84 , and the ST to predict MTs was 0.50 . Although the correlation between RS3-MLVA16 and MLST was high, differences in the distributions of various haplotypes were observed. MLST structure was not fully maintained in the MST of RS3-MLVA16. Haplotypes from Zimbabwe and Guinea were scattered somewhat by the presence of haplotypes from Cameroon. RS3-MLVA16 confirmed that these haplotypes were epidemiologically unrelated. The Cameroon haplotypes were 5–6 LVs from the Zimbabwean haplotypes. The Guinean haplotypes were 8–10 LVs from strains from other countries , RS3-MLVA16 and MLST were slightly congruent. The aR coefficient was estimated at 0.35 , and the genetic distance matrices between the two methods were 0.15  of an inoculum; analysis can also provide insight into the manner in which strains are established in weed reservoirs, the pattern of spread for successful clonal lines, the scale of distribution, and other epidemiological traits associated with the fitness of strains and the extent of disease.Highly portable, RS3-MLVA16 can be a first-line assay for routine screening at low cost. We are currently using RS3-MLVA16 to investigate these molecular epidemiology traits in bacterial wilt disease caused by RSSC phylotype III strains in Madagascar.Genotyping data are useful for surveillance networks and outbreak investigations of infectious diseases. Currently, there is not a database for RSSC. The MLST and MLVA databases for RSSC phylotype III recovered from the global, regional, and local populations should be used as a starting point for generating such a database.10.7717/peerj.1949/supp-1Figure S1(A) The consensus repeat pattern in the RS2AL03 locus  is CGGACCTCGCACTT. TRs are not present in the CMR15 genome (a reference strain belonging to phylotype III). (B) The consensus pattern in the RS2LB23 locus  is ATCGGTGAC. TRs are absent in the strain CMR15. (C) RS1L09  matches with RS3L18 (found in the strain CMR15). In strain CMR15, the RS3L18 locus exhibits the following two consecutive TR sequences with different repeat patterns: GTGTCGGCT (9 bp) and GTCAGCGGGATCGGCAGG (18 bp).Click here for additional data file.10.7717/peerj.1949/supp-2Figure S2Mean identity of 51% was observed between repeat units.Click here for additional data file.10.7717/peerj.1949/supp-3Figure S3RS3L27 is a TR locus with TR units that are 8 bp in length. The lengths of the flanking regions are conserved. The number of TRs differs between different isolates, suggesting that polymorphisms based on size differences are due to variability in the number of repeats. Note the imperfect character of some of the repeats in loci from the CMR15 isolate where an “A” is substituted by a “G”; the single nucleotide differences are observed in the flanking regions and in sequences of the repeated units.Click here for additional data file.10.7717/peerj.1949/supp-4Figure S4g) detected and the number of loci sampled (n). The radius of each circle is equal to the ratio between different combinations of n loci detecting g haplotypes among the total number of combinations of loci. The bottom and top circles of each bubble-plot indicate the minimum and maximum number of haplotypes found in each population  and in the collection C65. The dotted line represents the upper limit of the maximum number of haplotypes observed.Each circle represents the relationship between the numbers of haplotypes . nd: not determined. AEZ: Agro-ecological zones of Cameroon from where RSSC strains originated ((A) Isolates included in collection P35. (B) Isolates included in collection P20. (C) Isolates included in collection P17. Collection C65 includes all isolates used in this study _start physical position expressed in kilobases in the CMR15 genome_TR unit size (bp)_ amplicon size in the genome of origin (bp)_number of repeats (units)  (FP885895) and megaplasmid ( FP885896). (C) Fluorescent dye labelled at 5′-end forward primer.(A) Nomenclature of VNTR locus was as follows: marker alias_6bp_12u) . Marker Click here for additional data file.10.7717/peerj.1949/supp-7Table S3FP885895) and megaplasmid ( FP885896) (B) defined based on the CMR15 genome F: F primer; R: R primer.(A) Rank according to location within the physical map of the CMR15 genome. Accession numbers: chromosome  The number of possible combinations for each set of loci are given in parentheses. (B) There are 120 possible combinations if 2 of the 16 loci are used, 560 possible combinations if 3 of the 18 loci are used, and so forth. The underlined combinations of loci are the optimal combinations proposed after simulations produced the same maximum number of haplotypes as all 16 loci combined. (C) For example, 4 combinations of 4 loci were optimal for the detection of the 32 haplotypes. Locus RS3L33 discriminated 14 haplotypes among the 32 detected. When combined with locus RS3L17, this 2-locus combination generated 29 haplotypes. Adding locus RS3L27 to the previous combination resolved 31 haplotypes. One of the five proposed loci  can be added to distinguish all 32 haplotypes. Thus, MLVA based on 4-locus combinations can be used to discriminate all haplotypes in P35. The combination could be RS3L33 + RS3L17 + RS3L27 + RS3L34 (or RS3L37 or RS3L12).Click here for additional data file.


Arabidopsis ortholog PUB13 negatively regulate programmed cell death (PCD) and defense response. However, the components involved in the SPL11/PUB13-mediated PCD and immune signaling pathway remain unknown. In this study, we report that SPL11-interacting Protein 6 (SPIN6) is a Rho GTPase-activating protein (RhoGAP) that interacts with SPL11 in vitro and in vivo. SPL11 ubiquitinates SPIN6 in vitro and degrades SPIN6 in vivo via the 26S proteasome-dependent pathway. Both RNAi silencing in transgenic rice and knockout of Spin6 in a T-DNA insertion mutant lead to PCD and increased resistance to the rice blast pathogen Magnaporthe oryzae and the bacterial blight pathogen Xanthomonas oryzae pv. oryzae. The levels of reactive oxygen species and defense-related gene expression are significantly elevated in both the Spin6 RNAi and mutant plants. Strikingly, SPIN6 interacts with the small GTPase OsRac1, catalyze the GTP-bound OsRac1 into the GDP-bound state in vitro and has GAP activity towards OsRac1 in rice cells. Together, our results demonstrate that the RhoGAP SPIN6 acts as a linkage between a U-box E3 ligase-mediated ubiquitination pathway and a small GTPase-associated defensome system for plant immunity.The ubiquitin proteasome system in plants plays important roles in plant-microbe interactions and in immune responses to pathogens. We previously demonstrated that the rice U-box E3 ligase SPL11 and its  Spin6 RNAi and mutant plants show enhanced resistance to rice pathogens and activate defense gene expression and ROS generation. Importantly, we found that SPIN6 is the RhoGAP of the small GTPase OsRac1, which is a key component in rice immunity. Our study provides further insights into the relationship between SPIN6 and its interacting proteins SPL11 and OsRac1, and its function in the control of cell death and immunity in rice.Rice diseases are the major threat for stable rice production and food security worldwide. Deep understanding of the disease resistance pathway in rice is essential for effective control of the diseases. Although rice contains many E3 ubiquitin ligases, the function of their substrates in immune responses is still not fully understood. We previously characterized U-box E3 ligase SPL11 in rice that is involved in the regulation of cell death, immune responses, and flowering. However, how SPL11 interacts with its substrates to control cell death and immunity is not clear. In this study, we found that the SPL11 interacts with SPIN6, ubiquitinates, and degrades the protein via the 26S proteasome pathway. Both the  To resist pathogen invasion, plants have evolved two layers of innate immunity: pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) . The firspl11 mutant confers broad-spectrum resistance to rice pathogens and has elevated defense gene expression and ROS levels [plant U-box protein 13), one of the SPL11 orthologs in Arabidopsis, was also found to negatively regulate PCD and resistance to biotrophic pathogens [The ubiquitin proteasome system (UPS) is involved in selective degradation of proteins in the cells of eukaryotic organisms. It consists of three main kinds of enzymes: ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). In plants, many ubiquitin E3 ligases have been implicated in growth, development, and responses to abiotic and biotic stresses ,6. The US levels ,9. Recenathogens ,11, indiathogens . These fNicotiana benthamiana, for example, by directly associating with the nucleotide-binding-leucine-rich repeat (NB-LRR) R protein Rx, regulates the partitioning of Rx in both the cytoplasm and nucleus, and thereby enhances resistance against Potato virus X (PVX) [Blumeria graminis penetration by influencing the polar organization of cortical microtubules [By enhancing active, GTP-bound small GTPases into the inactive GDP state, the small GTPase-activating proteins (GAPs) function as an important regulator in small GTPase-mediated cellular signaling . Like ot X (PVX) ,16. In botubules . OsGAP1 otubules ,19.In rice, the small GTPase OsRac1 is a critical defense component because it integrates multiple ETI and PTI signaling pathways . OsRac1 in vitro and degraded in vivo by SPL11 via the 26S proteasome-mediated pathway. Silencing of Spin6 in transgenic rice causes PCD and enhanced resistance to the blast pathogen Magnaporthe oryzae and to the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo), suggesting that Spin6 plays a negative role in the regulation of PCD and immunity in rice. Strikingly, we found that SPIN6 interacts with OsRac1 and catalyze the active form of OsRac1 into an inactive form in rice cells. Together, these results show that SPIN6 is a key molecule that transduces defense signals from the E3 ligase SPL11 to the small GTPase OsRac1 for the regulation of PCD and innate immunity in rice.In this study, we report that the RhoGAP SPIN6 protein interacts with SPL11 and is ubiquitinated Spin6 (Os07g46450) encodes a RhoGAP protein with a Pleckstrin Homology (PH) domain, a RhoGAP domain at the N-terminal and a C-terminal coiled coil (CC) motif   in a yeast two-hybrid (Y2H) screen . Among tC) motif . In a BLSpin6 putative splicing isoforms, i.e., Os07g46450.1/Spin6, Os07g46450.2/Spin6.2 and Os07g46450.3/Spin6.3. In this study, we used the longest isoform Spin6 because Spin6.2 lacks the PH and RhoGAP domains, and Spin6.3 only contains a truncated RhoGAP domain and a truncated C-terminal  [The interaction between SPIN6 and SPL11 in yeast was confirmed with a Y2H experiment with full-length SPIN6, full-length SPL11, the SPL11 ARM domain (ARM), and a truncated SPL11 with a three-amino-acid deletion at C(SPL11m) . The Y2Hin vitro GST pull-down assay. The results showed that both GST fusion SPL11 and ARM proteins bind to SPIN6  SPL11 was not stable in N. benthamiana cells due to self-ubiquitination  after tin vivo, we co-infiltrated different combinations of agrobacteria carrying the Spl11:Myc and GFP:Spin6 plasmids into N. benthamiana leaves with or without the 26S proteasome inhibitor MG132. Total protein was extracted 3 days after the infiltration, and MG132 was infiltrated into the same leaves 18 h before tissue collection. The immunoblot analysis revealed that the GFP:SPIN6 was degraded by SPL11:Myc  via Agrobacterium-mediated transformation. Over 20 independently transformed lines were obtained in the T0 generation. The presence of the RNAi transgene and the hygromycin gene was determined in both the T0 and T1 generation by PCR. We identified six homozygous lines that had a single transgene insertion in the genome and that showed cell death phenotypes in the greenhouse. To confirm the cell death phenotypes, we grew two homozygous lines of the T3 generation (16–2 and 22–2) in a growth chamber under normal growth conditions . About 4 weeks after planting, obvious cell death-like lesions were evident in both RNAi lines but not in the NPB plants  than in NPB  in the Spin6 RNAi line 22–2 and in NPB after treatment with chitin and flg22. At 1 h after chitin treatment, the expression of the genes PBZ1 and PAL was > 10-times greater in the Spin6 RNAi plants than in NPB, but the expression returned to the normal level in the RNAi plants at 3 h after chitin treatment  and long day (LD) conditions  was used for rice transformation. Rice seeds were germinated as previously described . After gAgrobacterium-mediated transformation was used to generate the transgenic rice plants as described previously [SI data  was applied to the injured area, which was sealed with cellophane tape. The inoculated plants were kept in darkness at 80% RH for 24 h before they were transferred to a growth chamber at 28°C, 80% RH, and a 12-h/12-h light/dark photoperiod. After the plants had been in the growth chamber for 10 days, lesion size, spore number per lesion, and fungal biomass per lesion were determined. A leaf-clipping method was used to inoculate rice plants at the booting stage with Xoo isolate race 6 as previously described [Xoo-inoculated plants were maintained as described for M. oryzae-inoculated plants. Lesion lengths were measured 14 days after inoculation.A punch inoculation method was used to inoculate rice plants with ribed by . Brieflyescribed . The XooRNA was extracted from rice leaves with Trizol reagent (Invitrogen) and was treated with Dnase I. A 1-μg quantity of RNA in a 20-ul reaction volume was used for cDNA synthesis using the Promega reverse transcription system. After the cDNA was diluted 5–10 times, 1 μl of the diluted cDNA in a 25-μl reaction volume with BioRad SYBR supermix buffer was used for real-time quantitative PCR with a BioRAD iQ2 machine and the following program: 40 cycles at 95°C for 15 s, 60°C for 30 s, and 72°C for 20 s. Three replications were performed. The data were normalized with ubiquitin, and the means of three replications are presented.2) and submerged in distilled water for 3 h. Three leaf pieces were then placed in a 1.5-ml microcentrifuge tube with 100 μl of luminol (Bio-Rad Immun-Star horseradish peroxidase substrate 170–5040), 1 μl of horseradish peroxidase (Jackson ImmunoResearch), and 100 nM flg22 or 8 nM hexa-N-acetyl-chitohexaose, or distilled water as a control. The samples were immediately placed in a Glomax 20/20 luminometer (Promega), and luminescence was measured at 10-s intervals for 20 min.ROS was detected as previously described . BrieflySpl11, Spin6, and OsRac1 were separately cloned into the BD vector pDBLeu or the AD vector pPC86. Corresponding BD and AD constructs were then co-transformed into the yeast strain Mav203 and selected on synthetic dextrose medium without Trp or Leu (SD-Leu-Trp). The single transformant was picked up and diluted 10, 100, or 1000 times in sterilized-distilled water. For the interaction assay, 1 μl of each dilution was plated on SD-Leu-Trp-His medium with 0 mM or 40 mM 3-amino-1,2,4,-triazole (3AT).The ProQuest yeast two-hybrid system (Invitrogen) was used according to the manufacturer’s protocol. SPL11-interacting genes were screened as previously described . For intSpin6 cDNA was cloned into the vector pET28a for in vitro transcription/translation of SPIN6 protein using the TNT rabbit reticulocyte lysate translation system (Promega). The full-length Spll11 cDNA and the Spl11 ARM domain fragment were cloned into the vector pGEX-6p-1, and the GST:SPL11 fusion proteins were expressed and purified according to the manufacturer’s instructions (Amersham Biosciences). GST pull-down was performed as previously described [The full-length escribed . Bound SAgrobacterium strain GV3101 with corresponding constructs was incubated at 28°C with shaking (220 rpm) for 18 h. Bacteria were collected by centrifugation at 4000 rpm for 10 min and were resuspended to a final OD600 of 1.5 in MES buffer  with 150 nM acetosyringone. After 3 h, the suspensions were infiltrated into N. benthamiana leaves. After 3 days, leaf samples were collected for protein extraction or microscopic observation.Agroinfiltration was performed as described . BrieflySpin6, Spl11, and OsRac1 were separately cloned into vectors pSPYNE(R)173 and pSPYCE(M) for BiFC assay [N. benthamiana by the agroinfiltration method. A Leica confocal microscope was used for YFP fluorescence observation with excitation at 515 nm and emission at 525 nM; images were captured with a Leica Microsystems camera .Full-length cDNAs of FC assay . The conN. benthamiana leaves using a native buffer as previously described [For Co-IP, GFP:SPIN6 and Myc:OsRac1 protein was isolated from escribed  Co-IP waescribed . BrieflySPL11:Myc, or SPL11m:Myc(m) was co-expressed with GFP:SPIN6 plasmid by agroinfiltration in N. benthamiana. The RFP plasmid was co-infiltrated as a control. Tissues were harvested 3 days after infiltration for protein extraction. For protein level analysis, total proteins were detected using anti-GFP, anti-Myc, and anti-RFP antibody. For MG132 treatment, 50 μM MG132 was infiltrated with DMSO at 18 h before tissue was sampled.The plasmid Spin6 GAP domain fragment was cloned into the GST fusion vector pGEX-6p-1 . OsRac1 cDNA was cloned into the MBP vector pMAL-c2 (New England BioLabs). The proteins were expressed and purified according to the manufacturer’s protocols for GAP activity assay. OsRac1 protein at 1 μM was loaded with 10 μM mant-GTP  in the following buffer: 20 mM Tris/HCl pH 8.0, 50 mM NaCl, and 1 mM EDTA. After 10 min at 25°C, the Mant-GTP loaded OsRac1 was used for the GAP activity assay by adding 5 μM SPIN6GAP or GST (control) proteins in reaction buffer . Samples were immediately placed into a Glo-MAX luminometer (Promega) for fluorescence detection  at 25°C. Each reaction was repeated three times, and means and standard errors are presented.The in vivo GAP activity of SPIN6 toward OsRac1 [The Raichu intramolecular FRET sensor was used to analyze d OsRac1 . Ten to d OsRac1 .http://rice.plantbiology.msu.edu/) and the Arabidopsis TAIR database (http://www.arabidopsis.org/). For protein domain analysis, the SMART program was used (http://smart.embl-heidelberg.de/). The ClustalW 2.0 program [http://www.nrbsc.org/gfx/genedoc/) was used for conserved residue shading. For phylogenetic analysis, MEGA 3.1 [Sequences were collected using the BLAST program from the rice TIGR database -Rho GTPase-activating proteins (RhoGAPs) in rice and Arabidopsis. The marked regions with underlines are PH, GAP and Coiled-Coil domains, respectively. Figure S3. Protein sequence alignment of the three SPIN6 splicing isoforms. Figure S4. Phylogenic analysis of RhoGAP proteins in rice, Arabidopsis and other organisms. (A) Phylogenic relationship of RhoGAP proteins in rice and Arabidopsis. CRIB: Cdc42/Rac-interactive binding domain, PRLP: Prokar lipoprotein domain, RhoGAP: Rho GTPase-activating protein domain, PH: Pleckstrin Homology PH domain. (B) Phylogenic relationship of RhoGAP proteins in rice, Arabidopsis, Human and Drosophila. Figure S5. Interacting domain analysis of SPIN6 and SPL11 by yeast two hybrid. (A) The domains of SPIN6 and SPL11 were used for yeast two-hybrid. SPIN6FL: SPIN6 full length, PH: SPIN6 PH domain, GAP: SPIN6 GAP domain, NT: SPIN6 N-terminal, CT: SPIN6 C-terminal, SPL11FL: SPL11 full length, ARM: SPL11 ARM domain. (B) Interacting domain analysis of SPIN6 and SPL11 in yeast. Figure S6. Yeast two-hybrid assay of the interaction between SPL11 and SPIN6 homolog Os03g24180, and SPIN6 and SPL11 homology OsPUB12 (Os06g01304). A. The interaction between SPL11 and Os03g24180. The full-length cDNA of Os03g24180 was cloned in the AD vector. The Y2H assay was carried out as described in Materials and Methods. B. The interaction between SPIN6 and OsPUB12 (Os06g01304). The E3 ligase domain was mutated in Os06g01304 and cloned into the AD vector to avoid self-activation in yeast. The Y2H assay was carried out as described in Materials and Methods. Figure S7. The controls of BiFC assay of SPL11, SPIN6 and OsRac1. NeYFP and CeYFP are blank controls, NeYFP was fused with SPL11, SPL11m, ARM, OsRac1, respectively, and CeYFP was fused with SPIN6. (A). BiFC assay between NeYFP and SPIN6:CeYFP; (B) BiFC assay between NeYFP:ARM and CeYFP; (C) BiFC assay between NeYFP:SPL11m and CeYFP; (D) BiFC assay between NeYFP:SPL11 and CeYFP; (E) BiFC assay between NeYFP:OsRac1 and CeYFP. Figure S8. Confirmation of SPIN6 ubiquitination by SPL11 with GST pulldown and MG132 inhibition of SPL11 degradation in vivo. (A). Ubiquitination of SPIN6 by SPL11 was further confirmed by GST pulldown. After the E3 ligase assay reaction, the GST:SPIN6 beads were washed with the PBST solution and subjected to immunoblot either with anti-ubiquitin or anti-GST antibody. Asterisk denotes GST:SPIN6 detected by the anti-GST antibody. (B). The same samples in Figure S9.Spin6 RNAi construct map. A. An unique 302-bp 3’-UTR sequence of Spin6 cDNA with no similarity with other sequences in the rice genome was cloned into the pANDA vector by Gateway cloning method. The two reverse UTR sequence fragments are linked by a Gus linker intron (1.0 kb). The Spin6 RNAi fragment is under the control of the maize ubiquitin promoter (1.9 kb). NOSt is the transcription terminator. The NPTII gene is for bacterial selection. HPT gene is for rice transgenic selection. B. Location and sequence of the RNAi fragment. The bold, masked sequence is the fragment for making the RNAi construct. The sequence with red font is Spin6’s coding region. The region with black font is either 3’ or 5’ UTR. Figure S10. Whole genome similarity searches of the Spin6 RNAi fragment at the NCBI rice genome database. A. Mega BLAST search with the Spin6 RNAi fragment. B. BLASTN search with the Spin6 RNAi fragment. Figure S11. Phenotypes of the Spin6 RNAi silencing and T-DNA insertion mutants at seedling (A) and mature (B) stages. NPB: Nipponbare, wild type; 16–2, 22–2 are two Spin6 RNAi lines; Hwayoung: wild type of the T-DNA insertion mutant; spin6: T-DNA insertion mutant. Figure S12. Identification and genotyping of Spin6 T-DNA insertion mutant. A. The T-DNA insertion localization in the spin6 mutant. B. The PCR genotyping of the spin6 mutant. Hwayoung: Wild type; spin6: a homozygous line of the spin6 mutant. C. The expression pattern of Spin6 and Spl11 in the spin6 mutant. The data represent average data of three replicates, the bar was shown by standard deviation (SD). Figure S13. Cell death and disease resistance phenotypes, and ROS generation after flg22 and chitin treatments of spin6. A. Cell death phenotypes of spin6. Hwayoung is the wild type. B. Phenotypes of inoculation with blast isolate RO1–1. C,D. Spores number and fungal biomass of lesions infected by rice blast isolate RO1–1. Rice ubiquitin (UBQ) was used as the internal control. E. Lesion length of spin6 mutant and wild type  inoculated with Xoo race RB6. F, G. The ROS accumulation dynamcs in spin6 mutant and wild type(HY) treated with chitin (F) and flg22 (G).Data means the average of three or more than three replicates, Error bar is SD. * and ** represent significant level at P<0.05 and P<0.01,respectively. Figure S14. Endogenous H2O2 content detection in Spin6 RNAi lines(A) and T-DNA insertion mutant (B). NPB and Hwayoung are the wild type for the RNAi line and mutants, respectively. The data are the means of three replications with standard error as error bar. The significant level was at **P<0.01, n = 3 with t- test. Figure S15. Expression pattern of the defense-related genes PAL(A), PR1a(B), OsRac1 , OsSGT1 , and OsRAR1  in wild type Nipponbare (NPB) and Spin6 RNAi plants (22–2) after chitin and flg22 treatments. The ubiquitin (UBQ) gene was used as the internal control. Data represents the means of three replications with standard error as error bar. The significant level was at **P<0.01, n = 3 with t test. Figure S16. The expression pattern of Spin6- and defense-related genes in wild type Nipponbare (NPB) and Spin6 RNAi plants after inoculation with blast isolate R01–1. The leaf tissue was harvested at 0 (treated with water), 24 and 48 h after inoculated with M. oryzae isolate R01–1. Relative expression level of Spin6, Spl11, RbohB, OsRac1, PR1a, OsNAC4, PR5 and PBZ1 is shown in A, B, C, D, E, F, G and H, respectively. The relative transcriptional level of each gene was determined by real-time quantitative PCR using ubiquitin (UBQ) as the internal control. Error bars represent SD (n = 3). Significance was determined at *P<0.05 and **P<0.01 with a t-test. Figure S17. The flowering time of wild type Nipponbare (NPB) and Spin6 RNAi (22–2) plants under both short day (SD) (A) and long day (LD) (B) conditions, and expression pattern of flowering marker genes OsGI (C), Hd1 (D) and Hd3a (E) in both NPB and Spin6 RNAi plants. The ubiquitin (UBQ) gene was used as the internal control. The data of each lines is the average of 5 plants, error bar represents SD. * and ** represent the significant level at P<0.05 and P<0.001, respectively(PDF)Click here for additional data file.S2 TextTable S1. The cDNA sequence identity of Spin6 and its two rice homologous genes. Table S2. The protein sequence identity of SPIN6 and its two rice homologous genes. Table S3. Primers used in this study.(PDF)Click here for additional data file.


The JAK2V617F mutation is found in the majority of patients with myeloproliferative neoplasms (MPNs). Transgenic expression of the mutant gene causes MPN-like phenotypes in mice. We have produced JAK2V617F mice with p53 null background. Some of these mice developed acute erythroleukemia. From one of these mice, we derived a cell line designated J53Z1. J53Z1 cells were stained positive for surface markers CD71 and CD117 but negative for Sca-1, TER-119, CD11b, Gr-1, F4/80, CD11c, CD317, CD4, CD8a, CD3e, B220, CD19, CD41, CD42d, NK-1.1, and FceR1. Real time PCR analyses demonstrated expressions of erythropoietin receptor EpoR, GATA1, and GATA2 in these cells. J53Z1 cells grew rapidly in suspension culture containing fetal bovine serum with a doubling time of ∼18 hours. When transplanted into C57Bl/6 mice, J53Z1 cells induced acute erythroleukemia with massive infiltration of tumor cells in the spleen and liver. J53Z1 cells were responsive to stimulation with erythropoietin and stem cell factor and were selectively inhibited by JAK2 inhibitors which induced apoptosis of the cells. Together, J53Z1 cells belong to the erythroid lineage, and they may be useful for studying the role of JAK2V617F in proliferation and differentiation of erythroid cells and for identifying potential therapeutic drugs targeting JAK2. The transgenic mice display MPN-like phenotypes with much increased numbers of red blood cell and platelets Ph- myeloproliferative neoplasms (MPNs) are clonal hematopoietic malignancies in which one or more myeloid lineages are abnormally amplified. These diseases represent a group of chronic conditions including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) Antibodies for flow cytometric analysis of cell surface markers were from BD Biosciences and eBioscience. Antibodies against signaling proteins, including phospho-ERK1/2, phospho-Akt, and phospho-STAT5, were from Cell Signaling Technology. JAK2 inhibitors AZD1480 and ruxolitinib were purchased from Chemietek. All other protein kinase inhibitors were from the Approved Oncology Drugs Set IV of NCI Chemotherapeutic Agents Repository.tm1TyjTrp53/J) were purchased from The Jackson Laboratory Line A JAK2V617F transgenic mice which carry 13 copies of the JAK2V617F transgene were used in this study as previously described −6) were collected from a JAK2V617F/p53-/- mouse and cultured in Iscove's modified Dulbecco's medium (IMDM) supplemented with 20% fetal bovine serum, and 20 µM 2-mercaptoethanol at 37°C with 5% CO2. The culture was maintained for three weeks with equal volumes of fresh medium added every 3–4 days. Cells were then subjected to colony culture in semisolid medium containing 1% methylcellulose. Colonies were picked after 7 days of culturing and further expanded in the liquid medium. A total of 12 clones were analyzed, and each showed essentially similar morphology and cell surface markers (see below) typical for erythroleukemia cells. Clone no. 1 was selected for detailed characterization as described below. HCD-57 erythroleukemia cells were obtained from Dr. Maurice Bondurant, Vanderbilt University, and cultured in the above liquid medium plus 1 unit/ml of erythropoietin (EPO) Bone marrow cells , and single strand cDNAs were synthesized with equal amounts of total RNAs by using the QuantiTect reverse transcription kit from Qiagen. Real time PCR was performed with iQ SYBR Green Supermix (Bio-Rad) and primers specific for transgenic human JAK2V617F, mouse Jak2, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), GATA1, GATA2, and erythropoietin receptor EpoR. Melting curves were analyzed to confirm specific amplification of desired PCR, and the identities of final PCR products were verified by separation on agarose gels. For quantification, standard curves were obtained by performing PCR with serial dilutions (covering 5 orders of magnitudes) of purified PCR products in salmon sperm DNA For Wright-Giemsa staining, cells were spun onto glass slides by cytocentrifugation. For histological analysis, tissues were fixed in formaldehyde and embedded in paraffin. Tissue sections (5 µm) were deparaffinized and then stained with Hematoxylin and eosin (H&E). Images were captured by using a DP71 digital camera attached to an Olympus BX51 microscope.Cells were stained with fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, or allophycocyanin (APC)-conjugated monoclonal antibodies specific for mouse CD71 (clone C2), CD117 (clone 2B8), Sca-1 (clone D7), TER-119 (clone Ter-119), CD11b (clone M1/70), Gr-1 (clone RB6-8C5), F4/80 (clone BM8), CD11c (clone N418), CD317 (clone eBio927), CD4 (clone RM4-5), CD8a (clone 53-6.7), CD3e (clone 145-2C11), B220 (clone RA3-6B2), CD19 (clone 1D3), CD41 (clone MWReg30), CD42d (clone 1C2), NK-1.1 (clone PK136), and FceR1 (clone MAR-1) (from BD Biosciences and eBioscience). Stained cells were washed and analyzed by 4-color flow cytometry on a FACSCalibur flow cytometer (BD Biosciences) at the Flow and Image Cytometry Laboratory of University of Oklahoma Health Sciences Center. Data were collected by using the Cell Quest software (BD Biosciences) and analyzed by using the Summit software . At least 15,000 total events were analyzed. Dead cells were excluded according to staining with 7-amino-actinomycin D. For apoptosis analysis, the cells were stained with FITC-Annexin V and propidium iodide.J53Z1 cells were incubated in plain IMDM for 4 hours and then stimulated with EPO (10 U/ml) or mouse stem cell factor SCF (50 ng/ml) for 10 min. Cells were collected in and washed with ice-cold phosphate-buffered saline (PBS). Protein samples were prepared by adding SDS gel sample buffer directly into the cell pellets. Western blotting analyses were performed with antibodies against phospho-ERK1/2, phospho-Akt, and phospho-STAT5 followed by horseradish peroxidase-conjugated secondary antibodies. Captures of enhanced chemiluminescence signals were done by using FluorChem SP imaging system from Alpha Innotech.J53Z1 cells were cultured in the presence of various tyrosine kinase inhibitors. After 72 hours of culture, cell viability was assessed by performing XTT assays with XTT and phenazine methosulfate following standard protocols t tests. P values less than 0.05 (2-tailed) are considered significant.Statistical analyses were performed using the Excel program. Differences between 2 groups of samples were assessed using -/- mice  at three to six months of age -/- mice died of apparent acute leukemia in 4 months with huge spleen and liver. Detailed characterization of these mice will be reported elsewhere. Bone marrow cells from one of these mice were collected and cultured in IMDM medium supplemented with 20% fetal bovine serum. Cell proliferation took off in 3 weeks as a single cell suspension. We then performed colony culture in semisolid medium containing 1% methylcellulose. A total of 12 clones were analyzed, and each showed essentially similar morphology and cell surface markers typical for erythroleukemia cells (see below). Clone no. 1 was selected for detailed characterization as described below. We designated the new cell line J53Z1. JAK2V617F and p53 genotyping verified the presence of JAK2V617F and the absence of wild type p53 in the cells as seen in the parental JAK2V617F/p53-/- mouse (+/- mouse and a previously established erythroleukemia cell line, namely, HCD-57 By crossing JAK2V617F transgenic and p53 knockout mice, we generated JAK2V617F/53-/- mice . It has /- mouse . For com-/- cell line. J53Z1 cells proliferated rapidly in suspension culture with a doubling time of ∼18 hr . At presFlow cytometric analyses of cell surface markers further verified the erythroid lineage of J53Z1 cells. Like HCD-57 cells, J53Z1 cells are strongly positive for immature erythroid cell marker CD71 but negative for TER-119, and the level of CD71 is comparable to that seen in HCD-57 cells and higher than the level observed in normal mouse bone marrow erythroid cells . J53Z1 cPrimary erythroid progenitor cells rely on two key growth factors, namely, EPO and SCF, for survival and expansion 6 cultured J53Z1 cells into wild type C57Bl/6 mice through retro-orbital injections. The recipient mice developed anemia and died within 4 weeks with enlarged spleen and liver (in vivo.J53Z1 cells were derived from a mouse with acute erythroleukemia. To find out if they still possess the ability to cause leukemia, we implanted 1×10As a constitutively active tyrosine kinase, JAK2V617F likely plays a crucial role in supporting the proliferation of J53Z1 cells. Naturally, inhibition of its kinase activity should block cell growth. We analyzed a total of 14 protein kinase inhibitors for their inhibitory effects on J53Z1 cells as shown In the present study, we generated a JAK2V617F-containing erythroleukemia cell line designated J53Z1. J53Z1 cells belong to the erythroid lineage and are arrested at a stage before proerythroblast during erythroid development. They are positive for surface marker CD71 and negative for TER-119. They express EpoR and c-Kit and are responsive to stimulation by EPO and SCF. They express both GATA1 and GATA2 transcription factors expression thereby representing a unique stage of erythroid development. These cells should have wide applications in basic research to define the regulation of erythroid development and in translational research to identify JAK2 inhibitors for therapeutic drug development.in vivo.Identification of JAK2V617F in MPNs has provided an excellent target for drug development. Many potent JAK2 inhibitors have been developed, and some have shown clinical benefits in treatment of myelofibrosis -/- erythroid cell line, our current study also demonstrated the absence of p53 in HCD-57 cells which were derived from a mouse infected at birth with Friend murine leukemia virus in vivo progression of Friend erythroleukemia By establishing a JAK2V617F-positive, p53-null, immortal cell line, our study provides further evidence that JAK2V617F collaborates with loss-of-function of p53 to cause leukemic transformation. Malignant transformation usually involves a gain-of-function mutation of oncogenes and a loss-of-function mutation of tumor suppressor genes. Among various tumor suppressors, p53 is the most frequently mutated


Rnd3/RhoE is a small Rho GTPase involved in the regulation of different cell behaviors. Dysregulation of Rnd3 has been linked to tumorigenesis and metastasis. Lung cancers are the leading cause of cancer-related death in the West and around the world. The expression of Rnd3 and its ectopic role in non-small cell lung cancer (NSCLC) remain to be explored. Here, we reported that Rnd3 was down-regulated in three NSCLC cell lines: H358, H520 and A549. The down-regulation of Rnd3 led to hyper-activation of Rho Kinase and Notch signaling. The reintroduction of Rnd3 or selective inhibition of Notch signaling, but not Rho Kinase signaling, blocked the proliferation of H358 and H520 cells. Mechanistically, Notch intracellular domain (NICD) protein abundance in H358 cells was regulated by Rnd3-mediated NICD proteasome degradation. Rnd3 regulated H358 and H520 cell proliferation through a Notch1/NICD/Hes1 signaling axis independent of Rho Kinase. Lung cancer is the leading cause of cancer-related deaths in developed countries. The main types of lung cancer are small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). Despite advances in current medicine, the overall 5-year survival of patients diagnosed with NSCLC is approximately 15%, which indicates that (1) we lack an efficient way to prevent NSCLC; and that (2) we do not completely understand NSCLC. The recurrence of malignancy and chemotherapy resistance are two major limitations in the treatment of lung cancers. Of interest, several malignancies that are resistant to therapy have been closely associated with up-regulated Notch signaling Rnd3, also known as RhoE, is a small GTPase involved in the regulation of a wide variety of cell behaviors, including the cytoskeleton, proliferation, migration and apoptosis The role of Rnd3 in regulating the cell cycle was reported a decade ago. Forced overexpression of Rnd3 inhibits cell proliferation and serum-induced S-phase entry, independent of RhoA-ROCK signaling, indicating that complicated signaling is involved in cell cycle regulation H358 , H520  and A549  cells were cultured in RPMI-1640  plus 10% fetal bovine serum  and 1% penicillin-streptomycin . HBEC cells were cultured in Keratinocytes serum-free medium  plus bovine pituitary extract  and recombinant human EGF .Rnd3 cDNA was subcloned into the lentiviral vector pWPI-polio-eGFP. 293 T  cells were transfected with the lentiviral vector expressing Rnd3 and two helper vectors, pMD2 and psPAX2, to produce lentivirus. H358 and H520 cells were then infected with the virus at an MOI of 10. The infection efficiency was verified by GFP expression using fluorescent microscopy. The infected cells were selected by puromycin (2 µg/ml). A single clone was chosen to produce stable cell lines, H358-Rnd3 and H520-Rnd3.5 cells from each group were cultured in growth medium. The cell number was counted every 24 h for a total of 5 days.For the cell number measurement experiments, cells were synchronized for 12 h and 103VO4, 4 mM sodium pyrophosphate). Commercially available antibodies were from the following sources: MYPT1 (2634 S), phospho-T853 MYPT1 (4563 S) and PDK1 (3062) from Cell Signaling Technology ; MLC2 (PA5-17624) and phospho-Ser20 MLC (PA5-17727) from Thermo Scientific Pierce ; ROCK1 (sc-5560) and Rnd3 (sc-53874) from Santa Cruz Biotechnology . Hes1 (ab71559) and Notch1 (ab27526) were from Abcam . Equivalent protein loading was verified by the intensity of a GAPDH  blot.Protein samples for western blot analysis were extracted by RIPA buffer  plus a protease inhibitor cocktail  and phosphatase inhibitors . qRT-PCR was performed using a SYBR green method with a MasterMix buffer system. The primers sequences were as follows: Rnd3: CTATGACCAGGGGGCAAATA/TCTTCGCTTTGTCCTTTCGT; Jag-1: CTATGATGAGGGGGATGCT/CGTCCATTCAGGCACTGG; Jag-2: TGGGATGCCTGGCACA/CCGGCAGATGCAGGA; Dll-1: GAGGGAGGCCTCGTGGA/AGACCCGAAGTGCCTTTGTA; Dll-4: GCATTGTTTACATTGCATCCTG/GCAAACCCCAGCAAGAGAC; Notch1: CACTGTGGGCGGGTCC/GTTGTATTGGTTCGGCACCAT; Hes1: AGGCGGACATTCTGGAAATG/CGGTACTTCCCCAGCACACTT; GAPDH: GAGTCAACGGATTTGGTCGT/TTGATTTTGGAGGGATCTCG.Cells were synchronized and then cultured in growth media for 12 h followed by BrdU (bromodeoxyuridine) treatment for 30 min before harvesting. The anti-BrdU antibody  was incubated at 4°C for 16 h, and the secondary goat anti-mouse IgG antibody conjugated with Alexa Fluor 594 (1∶200) was purchased from Invitrogen (A11032). The incubation time was 1 h at room temperature, protected from light. Nuclei were visualized by DAPI  , and images were acquired by fluorescence microscopy .t test was used. All of the analyses were conducted by SigmaPlot 11.0 . A value of P<0.05 was considered statistically significant.The data are expressed as means ± S.D. In multiple group comparisons, one-way ANOVA followed by the Student-Newman-Keuls method was used. In 2-group comparisons, an unpaired, two-tailed student’s Two human NSCLC cell lines, H358 and H520, were used in this study. Human bronchial epithelial cells (HBEC) were used as a normal control. We detected the Rnd3 expression at both the mRNA and protein levels. Compared to HBEC cells, Rnd3 expression was significantly down-regulated in H358 and H520 cells . Rnd3 wa5 cells from each group were synchronized and then cultured. The cell number was counted at different time points . We observed a significant increase in cell number in H358 and H520 cells. However, this increase was remarkably attenuated in both H358-Rnd3 and H520-Rnd3 cells started at Day 2  and anti-GFP (detects exogenous Rnd3 only) antibodies . H358-Rnat Day 2 . Taken tWe showed higher Rho Kinase activities and NICD expression together with a reduced Rnd3 expression level in H520 and H358 cells compared to the normal lung epithelial cells, HBEC . We nextWe demonstrated that forced overexpression of Rnd3 by lentivirus in both H520 and H358 cells 1) inhibited proliferation  and 2) bNotch signaling is an important cell cycle regulator. Blocking Notch signaling inhibits the growth of different cells To define the relationship between Rnd3 and NICD, we performed a dosage dependent expression of Rnd3 in H358 cells. myc-Rnd3 was transiently transfected into H358 cells at different dosages . Then, we examined the expression of NICD and Hes1 by western blot. With increased Rnd3 expression, the NICD and Hes1 expression levels decreased . Not surH358 cell line was derived from lung bronchioalveolar carcinoma and H520 cell line was derived from lung squamouse cell carcinoma. However, the major subtype of NSCLC was lung adenocarcinoma. So we confirmed our study in one lung adenocarcinoma cells, A549, as shown in Rnd3 belongs to the Ras superfamily and is a small Rho GTPase that regulates a variety of cell functions and diseases. It was originally identified as a ROCK1 endogenous inhibitor. Rnd3 binds to ROCK1 to inhibit downstream signaling One of the most important strategies for curing cancer is inhibition of the uncontrolled tumor cell proliferation. However, a lack of specific inhibition is the major problem faced in the clinic. Understanding the unique regulatory mechanism of the cell cycle in cancer cells would favor the discovery of new drugs and new treatments for cancer. Rnd3 overexpression in fibroblasts inhibits S phase entry by blocking cyclin D1 expression at the post-transcriptional level Rnd3 expression is correlated with cell migration in development and diseases. Two recent studies found that Rnd3 regulated neuron migration through Rho Kinase dependent signaling Notch is an evolutionarily conserved transmembrane receptor involved in development and diseases. Upon ligand stimulation, Notch can be cleaved by γ-secretase. The intracellular part (NICD) can translocate into the nucleus to initiate transcription. Forced expression of Notch 1 inhibited the growth of A549 cells in culture A recently published study highlighted that Rnd3 was a downstream mediator of Notch signaling in squamous cell carcinomas (SCC) in skin epithelia Expression of Rnd3 express was precisely regulated in human tumor tissues. Rnd3 was up-regulated in colorectal cancer compared to normal human colorectal tissue Taken together, we are the first group to report that Rnd3 is down-regulated in H358, H520 and A549 cells, resulting in a hyper-activation of Notch and Rho Kinase signaling. Mechanistically, Rnd3 regulates the proliferation of H358, H520 and A549 cells through the NICD/Hes1 signaling axis.Figure S1Expression of PDK1, a Rho Kinase activator, remains unchanged in H358 and H520 cells compared to HBEC cells. PKD1 could compete with Rnd3 to bind to ROCK1, activating Rho Kinase signaling. (A) The protein expression level of PDK1 does not change in two cancer cell lines compared to HBEC cells. (B) Quantification of PDK1 expression suggested no statistical significance in PDK1 expression among the three cells. Data represent means ± S.D.(TIF)Click here for additional data file.Figure S2Quantification of the ratio of BrdU+ cells to GFP+ cells. (A) the ratio of BrdU+ cells to GFP+ cells are significant higher in H358 and H520 cells compared to H358-Rnd3 and H520-Rnd3 cells, respectively as shown (A) & (B). Data represent means ± S.D.(TIF)Click here for additional data file.Figure S3Expression of Notch ligands in H358, H520, H358-Rnd3 and H520-Rnd3 cells. (A) Expression of selected Notch ligands, Jag-1, Jag-2, Dll-1, Dll-4, did not change in H358 cells compare to H358-Rnd3 cells. (B) The expression of Notch ligands did not change in H520 cells compared to H520-Rnd3 cells. The data are representive for 3 experiment repeats. Data represent means ± S.D.(TIF)Click here for additional data file.Figure S4Effect of Rho Kinase inhibitor, Fasudil, on the proliferation rate of H358 and H520 cells. Treatment of Fasudil did not change the proliferation of H358 and H520 compared to DMSO treatment group. (A) H358 cells were treated with Fasudil followed by BrdU incorporation. (B) Quantification of BrdU intensity normalized by DAPI. (C) H520 cells were treated with Fasudil followed by BrdU incorporation. (D) Quantification of BrdU intensity normalized by DAPI. The experiments were repeated 3 times, and images were required under 20x objective. BrdU-positive cells were quantified from 8 images taken from four slides. Data represent means ± S.D.(TIF)Click here for additional data file.Figure S5Relative mRNA expression level of NICD and Hes1 in response to Rnd3 overexpression. NICD mRNA remains no change when Rnd3 was over expressed represented by empty bar. The Hes1 mRNA was down-regulated along with Rnd3 overexpression represented by the black filled bar. The mRNA was normalized to GAPDH expression. *p<0.05 compared to control (group 0); #p<0.05 compared to group 1; $p<0.05 compared to group 3. Data represent means ± S.D.(TIF)Click here for additional data file.Figure S6Cell viability was evaluated by Trypan Blue staining in response to Compound E. No cell death was detected in baseline condition and 5 nM compound E treatment in all three cell lines . Minimal (1%–4%) cell death was observed in response to 50 nM Compound E treatment in three cell lines . Massive cell death (>50%) was observed in all three cell lines treated by 500 nM compound E . White arrow indicates the dead cells.(TIF)Click here for additional data file.


Based on high throughput docking protocol, 250,000 compounds were computationally screened for putative PKR1 agonistic activity, using a homology model, and 10 virtual hits were pharmacologically evaluated. One hit internalizes PKR1, increases calcium release and activates ERK and Akt kinases. Among the 30 derivatives of the hit compound, the most potent derivative, IS20, was confirmed for its selectivity and specificity through genetic gain- and loss-of-function of PKR1. Importantly, IS20 prevented cardiac lesion formation and improved cardiac function after MI in mice, promoting proliferation of cardiac progenitor cells and neovasculogenesis. The preclinical investigation of the first PKR1 agonists provides a novel approach to promote cardiac neovasculogenesis after MI.Prokineticins are angiogenic hormones that activate two G protein-coupled receptors: PKR1 and PKR2. PKR1 has emerged as a critical mediator of cardiovascular homeostasis and cardioprotection. Identification of non-peptide PKR1 agonists that contribute to myocardial repair and collateral vessel growth hold promises for treatment of heart diseases. Through a combination of  Cardiovascular disease is the leading cause of morbidity and mortality worldwide . Heart fαq, Gαi and Gαs, mediating intracellular calcium mobilization, the activation of MAPK and Akt kinases, and cAMP accumulation, respectively -MIT (PerkinElmer) in binding buffer alone or in the presence of various concentrations of prokineticin-2 (Peprotech) or IS1. The reaction was stopped and the mixture was passed through Whatman GF/C filters. The filters were then washed and their radioactivity was counted. Saturation-binding curves were obtained by incubating membrane proteins with various concentrations of [125I]-MIT. The competitive binding profiles were analyzed by nonlinear regression analysis, with PRISM 3.02 (GraphPad Software).Membrane preparations from CHO cells stably expressing the wild-type human PKR1 receptor were prepared as previously described . The cel2+]I measurements in cell monolayers, we incubated the cells for 40 minutes at room temperature with 5 M fluo-4/AM (Molecular Probes). CHO cells expressing PKR1 were imaged with a Zeiss LSM-410 inverted confocal microscope  . Fo. Fo44]. in vitro and in vivo: i) neither IS1 or IS20 activated ERK in the absence of PKR1 in CHO cells, ii) IS compounds did not activate ERK in the cells expressing only PKR2, whereas prokineticin-2 a ligand for both receptors activated ERK in CHO-PKR2 cells, iii) IS20 was not able to activate Akt in PKR1-KO hearts where the PKR2 receptor is intact unlike total deletion of PKR1. It seems that IS20 acts as a positive allosteric modulator, since it elevates functional response of an orthosteric agonist (prokineticin-2), iv) the effect of IS20 was blocked by PC-7, a PKR1 antagonist.The allosteric modulation of GPCRs by a ligand has several key advantages over the traditional targeting of orthosteric sites . The mosα11 in endothelial cells and induces MAPK and PI3/Akt phosphorylation, promoting angiogenesis. By contrast, PKR2 is coupled to Gα12 in endothelial cells, in which it internalizes Gα12 and downregulates ZO-1 expression, leading to vacuole formation and the fenestration of these cells [We have previously shown that different PKR subtypes are coupled to different G proteins in endothelial cells. PKR1 is coupled to Gse cells . Thus, tse cells .in vivo activity of non-peptide PKR1 agonists. The administration of IS20 in mice induced no signs of toxicity, such as diarrhea or weight loss , indicating the same binding site of PKR1 as MIT. IS1 does not replace 125I-MIT, verifying that it has an allosteric binding site with the same binding site of PKR1 as MIT. B. IS1 at 1 nM concentration enhances the functional response (ERK kinase activity) of endogenous ligand PK2 (10 nM) when the CHO-PKR1 cells were treated with these two ligands together, clearly indicating that IS1 acts as positive allosteric modulator.(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 FigThe properties were calculated by means of QikProp .(PDF)Click here for additional data file.S4 FigA. IS20 (100 nM) cannot increase ERK activity in the presence of PKR2 in CHO cells. However, prokineticin-2 (10 nM) was able to activate ERK kinase via PKR2. B. IS20 acts as positive allosteric modulator by further increasing PK2 function on ERK activity. C. IS20 promotes Akt activity in a dose-dependent manner in CHO cells expressing PKR1 EC50 10 nM). * p<0.05.(PDF)Click here for additional data file.S5 FigERK activity induced by Prokineticin-2  and IS20 (10 nM) were completely abolished by PC7, a PKR1 specific antagonist (100 nM). *p<0.05 compare to control, **p<0.05 compare to PC7 alone.(PDF)Click here for additional data file.S6 FigA. Detection of cardiomyocytes death by active caspase-3 staining on heart sections. Illustration shows troponin positive (red) cardiomyocytes in caspase-3 positive (green) apoptotic cell population. Histogram shows the quantification of troponin+ cardiomyocytes in caspase-3 positive apoptotic cell population. *p<0.05). B. Representative illustration of Tunel (upper) and BrdU (lower) positive cells in the hearts of sham operated mice treated with vehicle or IS20, showing no differences between the groups Click here for additional data file.S7 Fign = 10 each). The body weight was measured every day for 40 days. No sign of toxicity was detected.IS20 and its vehicle were injected i.p. to male and female mice Click here for additional data file.S1 FileComputational DetailsHomology ModelingHigh Throughput Docking (HTD)ADME+T properties prediction(PDF)Click here for additional data file.


Small nuclear ribonucleoproteins (snRNPs), which are required for pre-mRNA splicing, contain extensively modified snRNA. Small Cajal body-specific ribonucleoproteins (scaRNPs) mediate these modifications. It is unknown how the box C/D class of scaRNPs localizes to Cajal Bodies (CBs). The processing of box C/D scaRNA is also unclear. Here, we explore the processing of box C/D scaRNA 2 and 9 by coilin. We also broaden our investigation to include WRAP53 and SMN, which accumulate in CBs, play a role in RNP biogenesis and associate with coilin. These studies demonstrate that the processing of an ectopically expressed scaRNA2 is altered upon the reduction of coilin, WRAP53 or SMN, but the extent and direction of this change varies depending on the protein reduced. We also show that box C/D scaRNP activity is reduced in a cell line derived from coilin knockout mice. Collectively, the findings presented here further implicate coilin as being a direct participant in the formation of box C/D scaRNPs, and demonstrate that WRAP53 and SMN may also play a role, but the activity of these proteins is divergent to coilin. O-methylation. Two examples are rRNA and small nuclear RNA (snRNA), and these modifications are necessary for optimal activity of ribosomes and spliceosomes , an, anin vin signal , which ln signal , which dO-methylation of some U2 snRNA residues is reduced in coilin knockout MEF cells compared to wild-type.Since coilin’s identification as the CB marker protein in 1991 , numerouCollectively, the data shown here support the hypothesis that coilin is a box C/D scaRNP assembly factor. More studies will be needed to clarify the exact function of coilin in this process, but based on our previous results and the data presented here, we propose a possible model . CentralHeLa and WI-38 cells were from the American Type Culture Collection . MEF26 and MEF42 lines  were froATCGTCGCAGGATCCACGCACATGTGTTTATAAAGATAACAGC-3’ (forward) and 5’-GGATCCTGCGACGATTATGCCCTTATTGTTTTACATTGTTTTATAGTTTTGC-3’ (reverse). All DNA constructs were sequence verified.Clones of scaRNA2 and scaRNA9 in pBluescript KS+ vector were described previously . ScaRNA2in vitro transcribed as previously described [in vitro transcribed scaRNA2 and scaRNA9 or scaRNA9 del GU. Transcripts were incubated at 37°C for 30 min with no protein, pure nucleic acid free coilin or purified GST, as indicated. Coilin was purified as previously described [ScaRNA2 and scaRNA9 clones in pBluescript KS were escribed . RNA proescribed  with modescribed . Reactio2. The blot was then placed in a hybridization bottle and pre-hybridized using 15 ml of Ultrahyb Ultrasensitive Hybridization buffer  per blot for 30 min at 37°C with rotation in a hybridization oven. The following DNA oligo probes were used to detect scaRNA 2, scaRNA 9 and 5S ribosomal RNA [nd Generation  according to the manufacturer’s protocol. After pre-hybridization, 22 μl of tailed probe was added to 20 ml of Ultrahyb Ultrasensitive Hybridization buffer to a final concentration of 4.5 nM. The blot was then hybridized overnight at 37°C with slow rotation in a hybridization oven. The blots were then washed according to a published protocol [Hela cells transfected with control, SMN, WRAP53 or coilin siRNA for 24 hr were additionally transfected with scaRNA2 cloned in pcDNA 3.1+. The cells were harvested 24 hr after the DNA transfection and RNA was isolated using TRI Reagent . WI-38 cells were co-transfected with different combinations of GFP, myc-SMN, myc-coilin and pcDNA 3.1+ scaRNA2. WI-38 cells were harvested 24hrs after transfection followed by RNA isolation, again using TRI Reagent. 20 μg of each RNA isolated from HeLa cells and 10 μg of each RNA isolated from WI-38 cells was run on a 6% or 8% denaturing polyacrylamide gel in 1X TBE at 200V for 30–40 minutes. The gel was then washed in 200 ml 1x TBE for 10 minutes with gentle shaking and the RNA was transferred onto a positively charged nylon membrane using the DNA Stacks  and the iBlot Gel Transfer device . Program P8 for 7 minutes was used for optimal transfer of the smaller, processed RNA or 13 minutes for optimal transfer of full-length scaRNA 2. After transfer, the blot was rinsed quickly in distilled water and allowed to dry. The RNA was then cross-linked to the membrane using a UV cross-linker  at a setting of 120,000μJ/cmomal RNA : 5’-CTCGprotocol , using tin vitro transcribed scaRNA2, scaRNA9 and increasing amounts of pure nucleic acid free coilin (0 ng-150 ng) as stated above. The reactions were then run on a 6% denaturing polyacrylamide gel in 1X TBE at 200V for 30–40 minutes. Northern blotting was then conducted as mentioned previously. The following DNA oligo probes were used to detect scaRNA2 and scaRNA9: 5’-CTCGTCTATCTGATCAATTCATCACTTCT-3’ (scaRNA2), and 5’-GGAAAGACTTCTGATGCTCAGATTTGGCTA-3’ (scaRNA 9).Reactions were set up with HeLa cells were transfected with either control or coilin siRNA as described above. 24 hrs after siRNA transfection, cells were transfected with scaRNA 2-pcDNA 3.1+. Cells were then harvested 24hrs after DNA transfection. Cytoplasmic, nucleoplasmic and nucleolar fractions were obtained using a published protocol  and RNA WI-38 cells were grown on glass slides  and transfected using Lipofectamine 2000  with combinations of myc-SMN, myc-coilin, GFP-WRAP53 or empty GFP vector. Cells were fixed with 4% paraformaldehyde in PBS for 10 mins, permeabilized in PBS containing 0.5% Triton for 5 min, followed by washing in PBS. Cells were then stained for SMN and coilin using anti-SMN mouse monoclonal antibodies (1:100)  and rabbit polyclonal anti-coilin H300 (1:200) . DAPI staining was used to define the nucleus. Images were captured and processed as previously described .32P labeled primer complementary to U2 snRNA (5' CCATTTAATATATTGTCCTCGG 3'), as previously described [RNA was isolated from MEF26 (wild-type) and MEF42 (coilin knockout) cells. Additional phenol:chloroform:isoamyl alcohol (25:24:1) extraction and ethanol precipitation were performed to ensure high purity of RNA. 2 μg of purified RNA was subject to primer extension with high (1 mM), medium (0.01 mM) or low (0.005 mM) levels of dNTPs using a escribed . Primer-S1 FigHeLa cells were transfected with siRNA. 24 hrs after transfection, cells were harvested and lysate was generated. Equal amounts of protein were resolved on SDS-PAGE, followed by Western transfer and detection of SMN using anti-SMN antibodies. Tubulin was then detected using anti-tubulin to verify equal protein loading.(TIF)Click here for additional data file.


The removal of histone H3 trimethylation at lysine residue 27 (H3K27me3) plays a critical role in the transcriptional initiation of developmental genes. The H3K27me3-specific KDM6 demethylases JMJD3 and UTX are responsible for the transcriptional initiation of various developmental genes, but some genes are expressed in a KDM6 demethylase-independent manner. To address the role of H3K27me3 in the retinoic acid (RA)-induced differentiation of the human carcinoma NCCIT cell line, we inhibited JMJD3 and UTX using the H3K27me3 demethylase inhibitor GSK-J4. The commitment of JMJD3/UTX-inhibited cells to a specific fate was delayed, and transcriptome profiling also revealed the differential expression of genes related to cell fate specification in demethylase-inactivated cells; the expression levels of RA metabolism and HOX family genes significantly decreased. We observed a weak correlation between H3K27me3 enrichment and transcriptional repression in the control and JMJD/UTX-inhibited cells, except for a few sets of developmental genes that are indispensable for cell fate specification. Taken together, these results provide the H3K27me3 landscape of a differentiating cell line and suggest that both demethylase-dependent and demethylase-independent transcriptional regulation play a role in early differentiation and developmental gene expression activated by H3K27me3 demethylation. The differentiation of pluripotent stem cells requires dramatic changes in the initiation and suppression of gene transcription to accomplish cell specification. Numerous modifications modulate the regulation of gene expression programs, including post-translational histone modifications. These covalent modifications epigenetically regulate and maintain lineage-specific gene expression during development. Two specific histone modifications play a critical role in the regulation of developmental genes: a repressive marker, the trimethylation of histone H3 on lysine residue 27 (H3K27me3), and an activating marker, the trimethylation of histone H3 on lysine residue 4 (H3K4me3). These two modifications co-exist at promoters that regulate the expression of essential developmental genes in embryonic stem (ES) cells and other progenitor cell lines, forming epigenetic signatures called bivalent promoters –3. Due tThe Jumonji-C domain (JmjC)-containing histone demethylase family KMD6 is responsible for H3K27me3 demethylation. Three KDM6 demethylases, JMJD3 (KDM6B), UTX (KDM6A) and UTY, can remove one methyl residue from H3K27me3 and H3K27me2; however, the activity of UTY is significantly lower than that of other demethylases –13. JMJDPrevious studies have assessed the significance of JMJD3/UTX functions in biological processes using several techniques to disable the catalytic activities of these enzymes, including RNA-interference techniques and site-specific mutagenesis , 20, 24.2 at 37°C for 48 h. EBs treated with RA and RA plus GSK-J4 are hereafter referred to as EBRA and EBRA+GSK, respectively.Human embryonal carcinoma (EC) NCCIT  cells were cultured in RPMI 1640 and OPTIMEM media  to maintain and differentiate the cells, respectively. All media were supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin (Invitrogen) and 10% heat-inactivated fetal bovine serum . Embryonic bodies (EB) were formed as previously described . All-traet al.  supplemented with The following antibodies were used for western blotting and were all purchased from Abcam : anti-histone H3 (ab1791), anti-H3K27me3 (ab6002) and anti-histone H3 dimethylated lysine 27 .RA and EBRA+GSK was examined with an Epigenase JMJD3/UTX Demethylase Activity/Inhibition Colorimetric Assay Kit (Epigentek) using the nuclear extracts (20 μg) according to the manufacturer’s instructions. The inhibition ratio was measured in triplicate, and the percentage of inhibition was calculated based on three different assays.Nuclear extracts were prepared using an EpiQuik Nuclear Extraction Kit  according to the manufacturer’s instructions. The inhibition of JMJD3/UTX in NCCIT, EB2, 0.2% Triton X-100, 5 mM butyrate, 1X protease inhibitor cocktail, 0.5 mM PMSF), followed by incubation with 0.3 U Micrococcal nuclease  at 37°C for 5 min. The reaction was stopped with 50 mM EDTA and treated with RIPA buffer for 16 h, followed by incubation with an appropriate antibody (see below) and Dynabeads Protein A beads (Invitrogen) for 16 h at 4°C. The chromatin-antibody-Dynabeads Protein A complexes were consecutively washed with RIPA buffer supplemented with 0.3 M NaCl, lithium chloride (LiCl) buffer and TE buffer, and then incubated with proteinase K at 65°C for 16 h. The DNA from the complexes was purified via phenol/chloroform extraction and concentrated by ethanol precipitation. The resulting DNA pellets were dissolved in TE buffer for further analyses.NCCIT cells were collected and resuspended in digestion buffer  were removed from total RNA using the RiboMinus Transcriptome Isolation Kit (Invitrogen). rRNA-depleted total RNAs were used to construct paired-end transcriptome libraries using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs). Briefly, first-strand cDNAs were synthesized from rRNA-depleted RNA samples, followed by second-strand synthesis with DNA polymerase I and RNase H. The double-stranded cDNAs were then end-repaired and ligated to adaptors. The ligated libraries were then separated on a 2% agarose gel , and fragments with sizes between 300–400 bp were purified using the MinElute Gel Extraction Kit . The fragments were amplified for further enrichment and purified by ethanol precipitation. Two biological replicates were prepared from each condition.Immunoprecipitated DNA was used to construct paired-end ChIP-sequencing libraries using the NEBNext Ultra DNA Library Prep Kit for Illumina. ChIP reactions using rabbit IgG and from untreated and RA-treated NCCIT were used as normalization controls.http://ngsc.med.upenn.edu/).The paired-end deep sequencing of the constructed libraries was performed using HiSeq 2000 and 2500  by the National Instrumentation Center for Environmental Management  and the Next-Generation Sequencing Core from the University of Pennsylvania (Homo sapiens hg19 using Tophat (version 2.0.10) [2 fold-change were selected for functional annotation using the Database for Annotation, Visualization and Integrated Discovery (DAVID) version 6.7 [http://www.ncbi.nlm.nih.gov/geo) under accession number SRP045949.For transcriptome expression profiling, the raw reads were trimmed using the FASTX-Toolkit  and alig 2.0.10) . The ali 2.0.10) . Differe 2.0.10) . All dat 2.0.10) . DEGs dision 6.7 , 38 and https://www.genome.ucsc.edu).For the ChIP-seq analysis, the raw reads were aligned using Bowtie2 version 2.2.1 . UCSC hgThe JMJD3/UTX inhibition assay and qPCR data were statistically analyzed using SPSS 21.0 . All experiments were conducted in triplicate, and data are presented as the means ± SE. The data were tested with a one-way ANOVA followed by Tukey’s HSD post hoc test.RA cells1 and 2 days after RA treatment. However, treating both RA and GSK-J4 partially inhibited cell attachment and delayed the differentiation of EBRA+GSK cells  were considered to be differentially expressed genes (DEGs). A total of 673 genes were differentially expressed between NCCIT and EBRA, with 316 up-regulated and 357 down-regulated genes  were down-regulated, whereas a relatively small number of genes (363 genes) were activated. GSK-J4 treatment affected genes involved in stress response, gene transcription and cell specification . Transfecting four target gRNA plasmids together into NCCIT cleaved a 1650-bp fragment ranging from exon 17 to 22, which significantly reduced JMJD3 transcription in all cases of NCCIT differentiation and inhibition by GSK-J4  mRNA expression of JMJD3 and UTX in WT NCCIT and JMJD3 knockout cell. (C) mRNA expression patterns of JMJD3 during RA-induced differentiation and GSK-J4 inhibition. qRT-PCR were normalized for GAPDH. The values are presented as the means ± SE (n = 3). The asterisk represents the significant difference analyzed by one-way ANOVA followed by Tukey’s HSD post hoc test .(A) Schematic illustration of constructing (TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 FigJMJD3 knockout cells. qRT-PCR were normalized for GAPDH. The values are presented as the means ± SE (n = 3).(A-D) mRNA expression levels of pluriopotency markers (A), neural markers (B) and RA-responsive genes (C-D) in RA-induced differentiation of (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.


Infection of poultry with diverse lineages of H5N2 avian influenza viruses has been documented for over three decades in different parts of the world, with limited outbreaks caused by this highly pathogenic avian influenza virus. In the present study, three avian H5N2 influenza viruses, A/chicken/Shijiazhuang/1209/2013, A/chicken/Chiping/0321/2014, and A/chicken/Laiwu/0313/2014, were isolated from chickens with clinical symptoms of avian influenza. Complete genomic and phylogenetic analyses demonstrated that all three isolates are novel recombinant viruses with hemagglutinin (HA) and matrix (M) genes derived from H5N1, and remaining genes derived from H9N2-like viruses. The HA cleavage motif in all three strains (PQIEGRRRKR/GL) is characteristic of a highly pathogenic avian influenza virus strain. These results indicate the occurrence of H5N2 recombination and highlight the importance of continued surveillance of the H5N2 subtype virus and reformulation of vaccine strains. Infection of poultry with diverse lineages of H5N2 avian influenza viruses has been documented for over three decades . One of HPAI H5N2 strains are generated from mutations in the hemagglutinin (HA) gene of the LPAI H5N2 virus strains. Examples of these H5N2 strains include the HPAI H5N2 virus strains in Pennsylvania 1983) and in Mexico 1994) 983 and i. In addi 983 and Reassortment frequently occurs within a confined geographic area, such as countries or continents. However, HA and neuraminidase (NA) genes of a recombinant strain (A/Ck/Taiwan/1209/03 (H5N2)) isolated from chickens in Taiwan were found to be closely related to an American H5N2 strain and the remaining genes correlated with the Eurasian lineage . FurtherTherefore, to control avian influenza in poultry, it is critically important to track the evolution and epidemiology of H5N2 strains in the field. In the present study, we conducted genomic and phylogenetic analysis of three H5N2 influenza virus strains isolated from chickens that showed symptoms of avian influenza virus (AIV) and succumbed to infection-related death in the Hebei and Shandong province from 2013 to 2014.Chickens from three farms (one from Hebei and two from Shandong) were immunized with an inactivated, bivalent influenza vaccine . Five rounds of immunization were applied to these birds at 3, 8, 16, 20, and 35 week-old ages, respectively. The birds of the three farms were 240–550 days old when ailing or dead chickens were observed.Ailing or dead chickens  with clinical symptoms of AIV infection were collected from one Hebei province farm and two Shandong province farms during 2013 and 2014. Liver, oviduct and lung tissue samples were collected separately from each chicken and were transported to the laboratory at 4 °C in media containing antibiotics. The tissue samples were then made into slurries, and tested by Matrix (M) gene specific RT (reverse transcription)-PCR . The pooled samples from five positive birds of each farm were used for inoculation into the allantoic cavities of 10-day-old specific-pathogen-free chicken embryonic eggs and incubated at 37 °C for 72 h. The allantoic fluid was collected and used to determine the presence of virus with hemagglutination assay. All virus isolates were further purified with a plaque assay, sequenced and used in subsequent research.Subtyping of the HA and NA genes, and genotyping of the other six viral genes, was carried out by RT-PCR followed by sequencing. Briefly, RNA was extracted from the allantoic fluid using a Viral RNA Mini Kit  and PCR amplification of the specific viral genes was carried out using a PrimeScript™ One-Step RT-PCR Kit . All viral genes were amplified with primers as described previously . PCR proTo explore the origin of the H5N2 virus isolates, all eight viral genes were subjected to genomic and phylogenetic analyses. Gene sequences of the AIVs reported in GenBank were selected to construct the phylogenetic trees by using the neighbor-joining method and bootstrapping with 1000 replicates in MEGA 5.05 software. H5N1, H5N2 and H9N2 classical strains of Eurasian and North American lineages, and H9N2 and H5N2 strains currently circulating in the field were selected into the phylogenetic analyses. A total of 39, 28 and 22 strains of AIVs from GenBank were used for polygenetic analyses of HA, NA, and M genes, respectively and GenBank accession numbers are listed before strain names in This study was carried out with pre-approval obtained by the Shandong Administrative Committee for Laboratory Animals. All procedures involving the handling of animals were reviewed and approved by the Shandong IACUC (Permission number: SDXK-LD-2014-0004).Out of 270 birds, 252 tested positive by M gene-specific RT-PCR. Once determined positive for H5N2, all birds from these three farms were depopulated. Three H5N2 avian influenza virus strains were then isolated from chickens with clinical symptoms of AIV from each farm. The three isolates were named as A/chicken/Shijiazhuang/1209/2013(H5N2) (Ck/SJZ/1209/13), A/chicken/Chiping/0321/2014(H5N2) (Ck/CP/0321/14), and A/chicken/Laiwu/0313/2014(H5N2) (Ck/LW/0313/14). All three isolates were sequenced and their sequences have been deposited into GenBank under accession numbers KP000004 to KP000027.All three H5N2 AIV isolates clustered with H5N1 subtype HA genes from clade 7.2, A and shaFor all three of the H5N2 isolates, the HA gene was found to comprise a 1704 nucleotides open reading frame (ORF), coding for 568 amino acids, including a 16 amino acid signal peptide. Molecular analysis revealed these isolates harbor amino acid sequences characteristic of HPAI strains at the HA cleavage site (PQIEGRRRKR/GL), a determinant of high pathogenicity in birds.It has been known that the consensus receptor binding site (RBS) for all identified HA proteins is composed of three major structural elements: a 190-helix (residues 188–194), a 220-loop (residues 221–228), and a 130-loop (residues 134–138). The conservative residues of the three H5N2 isolates within the RBS, including E190, R220, G225, Q226 and G228 (numbering according to H3), were all present, implying they retain typical avian virus-like receptor specificity .In this study, three H5N2 influenza virus strains isolated from chickens were identified as novel reassortants with a highly pathogenic viral genotype. Surprisingly, the affected birds had been vaccinated with killed influenza vaccines but still showed characteristic clinical symptoms of avian influenza and eventually died. These results are in agreement with previous work indicating that AIVs can continue genetic evolution under vaccination pressure . MoreoveSeveral recombinant H5N2 AIV isolates have been reported in China and other countries. In 2012, three recombinant H5N2 viruses were identified in China: A/ chicken/Hebei/1102/2010 with its HA gene derived from clade 7.2 (H5N1) and the other seven genes from endemic H9N2 , and A/dThe amino acid sequence (RRRKR) of the influenza HA gene product at the cleavage site for all three viruses meets the molecular criterion put forth by the World Organization for Animal Health for an HPAI, or highly virulent, influenza strain. Furthermore, the intravenous pathogenicity index for a similar recombinant (A/chicken/Qingdao/1/2014(H5N2)) is 2.84 , South KOverall, we have identified three H5N2 influenza strains isolated from chickens in northern China that represent reassortant products between an H5N1 strain and several H9N2 strains. Results from this study highlight and reinforce the importance of vaccine strain reformulation and continued monitoring of AIVs both in domestic and wild birds.