Brassica species, the candidate gene regulating synthesis of 3C GS remains ambiguous. Earlier reports of GSL-PRO, an ortholog of Arabidopsis thaliana gene At1g18500 as a probable candidate gene responsible for 3C GS biosynthesis in B. napus and B. oleracea could not be validated in B. juncea through genetic analysis. In this communication, we report the isolation and characterization of the gene CYP79F1, an ortholog of A. thaliana gene At1g16410 that is involved in the first step of core GS biosynthesis. The gene CYP79F1 in B. juncea showed presence-absence polymorphism between lines Varuna that synthesizes sinigrin and Heera virtually free from sinigrin. Using this presence-absence polymorphism, CYP79F1 was mapped to the previously mapped 3C GS QTL region (J16Gsl4) in the LG B4 of B. juncea. In Heera, the gene was observed to be truncated due to an insertion of a ~4.7 kb TE like element leading to the loss of function of the gene. Functional validation of the gene was carried out through both genetic and transgenic approaches. An F2 population segregating only for the gene CYP79F1 and the sinigrin phenotype showed perfect co-segregation. Finally, genetic transformation of a B. juncea line (QTL-NIL J16Gsl4) having high seed GS but lacking sinigrin with the wild type CYP79F1 showed the synthesis of sinigrin validating the role of CYP79F1 in regulating the synthesis of 3C GS in B. juncea.Among the different types of methionine-derived aliphatic glucosinolates (GS), sinigrin (2-propenyl), the final product in 3C GS biosynthetic pathway is considered very important as it has many pharmacological and therapeutic properties. In  Brassica crops primarily contain methionine derived aliphatic GS . Aliphatic GS can be broadly divided into propyls , butyls  and pentyls  on the basis of side chain length. Brassica species consist of various combinations of the above three types of aliphatic GS namely, 2-propenyl (commonly known as sinigrin) of 3C GS, 3-butenyl (known as gluconapin) and 2-hydroxy-3-butenyl (known as progoitrin) of 4C GS and 4-pentenyl (known as glucobrassicanapin) of 5C GS. The diploid B. nigra  contains 3C GS sinigrin, B. oleracea  contains either sinigrin or 4C GS, and B. rapa  contains both 4C and 5C GS. The GS profile of the three allotetraploid Brassica species, B. juncea , B. napus  and B. carinata  is a combination of the GS profiles of the progenitor diploid Brassica species  and Heera [a low GS east European line   cv. YSPB-24 and B. nigra (BB) cv. IC 257 with high seed GS content. For genetic analyses and mapping, two bi-parental populations–(i) A Varuna x Heera F1DH mapping population (VH) consisting of 123 lines (the population was previously used in the lab to construct a high-density comparative linkage map in B. juncea) [2 population of 95 segregants derived from a cross between Varuna x QTL-NIL J16Gsl4. The line QTL-NIL J16Gsl4 is a near isogenic line (NIL) which contains Heera allele of QTL J16Gsl4 in the genetic background of Varuna and is high in seed GS (>110 μmol g-1 seed) with trace amount of sinigrin. The NIL J16Gsl4 was developed by following a recurrent selection backcross (RSB) scheme [J16Gsl4 through genotyping for all GS QTL/genes from a population of 785 BC4DH lines segregating for aliphatic GS trait [J16Gsl4 (high seed GS) and EH-2  were used. We used EH-2 in place of Heera because the lab has standardized transformation protocol for EH-2.Several genetic stocks consisting of different oilseed  J16Gsl4  were use juncea)  and QTL GS trait . Finally2, 10 pmol each of forward and reverse primers and enzyme Taq polymerase . The PCR profile included initial denaturation at 94°C for 5 min followed by 36 cycles of 94°C for 30 sec, annealing at 62°C for 30 sec and 72°C for 1 min 30 sec and a final extension at 72°C for 12 min. For the purpose of sequencing, amplified fragments were cloned in the pGEM-T Easy TA cloning vector . Sequencing was done by capillary electrophoresis on Applied Biosystems 3730 Genetic Analyzer. Sequencing of each sample was done in a minimum of 3 independent replicates to remove chances of any PCR related error. Sequence files were assembled and analysed using EditSeq, SeqMan and MegAlign software packages of DNAStar (DNAStar Inc.). For mapping, marker genotyping data of the mapping population were added to the existing map of B. juncea [Genomic DNA was isolated from well-expanded leaves from the field grown plants following the protocol of Rogers and Bendich . All PCR. juncea  using th. juncea . Genome Development of BAC libraries used in the study has been described by Sharma et al. . BAC scrBrassica BAC annotation pipeline (http://brassica.nbi.ac.uk/annotate.html). Transposable elements (TE) in the BAC were predicted and located by using the program RepeatMasker (http://www.repeatmasker.org/). Sequence of the BAC was aligned to the corresponding A. thaliana sequences obtained from TAIR (https://www.arabidopsis.org/) and B. rapa sequences obtained from BRAD (http://brassicadb.org/brad/) using NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&PROG_DEF=blastn&BLAST_PROG_DEF=megaBlast&BLAST_SPEC=blast2seq) to analyze gene conservation.Sizing of the BAC insert and Sanger sequencing of BAC was outsourced to Amplicon Express, USA. BAC sequencing by 454/Roche GS-FLX Titanium pyrosequencing and sequence assembly using GS De Novo Assembler (v 2.9) was performed at Macrogen Inc., South Korea. Annotation of the assembled contigs was performed using the 2, 10 pmol each of forward and reverse primers and 1 Unit of Taq polymerase enzyme . The RT-PCR profile included initial denaturation at 94°C for 5 min followed by 30 cycles of 94°C for 30 sec, annealing at 60°C for 30 sec and 72°C for 30 sec and a final extension at 72°C for 5 min. The PCR products were analysed by agarose gel electrophoresis. Actin gene forward primer Actin-FP (5’–GGC TCC TCT TAA CCC AAA GG– 3’) and reverse primer Actin-RP (5’–TTC TCG ATG GAA GAG CTG GT– 3’) was used as endogenous control for equal template loading.Total RNA was extracted from frozen tissues using the RNeasy Plant Mini Kit (Qiagen), complying with manufacturer’s instructions. Total RNA isolated was treated with DNase using the RNase-Free DNase Set (Qiagen) and column purified. cDNA library was synthesized by reverse transcribing 2 μg of total RNA with High Capacity cDNA Reverse Transcription Kit  in a 20 μl reaction volume using mixture of random primers and oligo-dTs in 1:1 ratio according to manufacturer’s instructions. PCR amplification was carried out in a 20 μl reaction volume containing 1 μl of cDNA, 800 μM of dNTPs, 2 mM of MgClAgrobacterium tumefaciens mediated genetic transformation of B. juncea was performed using hypocotyl explants following the protocol described by Jagannath et al. [h et al. . Extracth et al. .BjuB.CYP79F1 from B. juncea cv. Varuna) and KT254223 (BniB.CYP79F1 from B. nigra cv. IC 257).The sequences reported in this paper are deposited in the National Center for Biotechnology Information GenBank database with accession numbers KT254222  encompassing the syntenous region of QTL J16Gsl4 (B. juncea [B. rapa ([http://brassicadb.org/brad/) was undertaken. The search identified a total of 15 genes of aliphatic GS biosynthetic pathway (GSL-PRO not included). A total of 14 degenerate primers (wherever necessary) to accommodate the multiple copies of the genes were used (B. juncea (AABB) lines Varuna and Heera having contrasting phenotype for sinigrin and also from the diploid parental species B. rapa cv. YSPB-24 (AA) and B. nigra cv. IC257 (BB). None of these primer pairs showed clear-cut polymorphism between Heera and Varuna (J16Gsl4 regulating the sinigrin synthesis was located in the LG B4 of B. juncea. The process was initiated with the genes CYP79F1 (At1g16410) and CYP79F2 (At1g16400) (tandemly duplicated in Arabidopsis) involved in the first step of synthesis of core GS structure. Priority was given to these two genes because of the fact that these gene IDs (At1g16400 and At1g16410) were assumed to be located nearer to the peak LOD value marker (At1g16740) of the QTL J16Gsl4 [B. juncea [An idopsis  and one reverse primer (GS1C-NS-RP) were designed for amplification (for detailed primer sequences see B. nigra-specific nested primers led to the amplification of only one band of different molecular weights by each primer. B. nigra (B genome) specificity of these three primers was confirmed by the fact that none of the primer pairs amplified any bands from B. rapa (A genome), whereas all the three primers amplified one band each from B. juncea line Varuna with the same molecular weight as that from B. nigra  showing dominant polymorphism between Varuna and Heera, the gene i et al.  Fig 1)..CYP79F1/CYP79F1/ CYP79F2 from B. juncea cv. Varuna and from B. nigra cv. IC257 were derived by using a common set of nested primers and through 5’ and 3’ genome walking , B. nigra  and other Brassica species. The gene was almost identical to BniB.CYP79F1 (99.8%) and also showed high sequence identity (>93%) with CYP79F1 of other Brassica species , a stretch of hydrophobic amino acids constituting the transmembrane helix, which anchors the protein in the ER; a positively charged domain; a proline-rich motif and a heme binding domain towards the C-terminal [https://www.predictprotein.org/; [The full length 1626 bp terminal ,31. Whenin.org/; ,33), subBjuB.CYP79F1 from the parent Heera. Hence, the gene organization of BjuB.CYP79F1 in Heera was resolved through BAC sequencing. Initially, a sequence of 1177 bp starting from -645 to +532 bp was established by 5’genome walking which showed that the 5’ end of the gene in Heera is intact. The sequence was identical to the corresponding sequence of the gene in Varuna. Subsequently, a primer pair that would amplify -428 to +532 bp was used for screening the two BAC libraries of the Heera genome [Our initial PCR experiment (previous section) could not succeed in amplifying the a genome . Of the BjuB.CYP79F1 was observed to be truncated in the first exon with next two exons missing. The truncated first exon was completely identical up to 855 bp from the 5’ end with B. rapa CYP79F1 (Bra026058) and up to 858 bp from the 5’ end with wild type BjuB.CYP79F1 gene of Varuna. Following the truncation point, presence of a ~4.7 Kb long En-Spm like DNA transposon downstream of BjuB.CYP79F1 was detected   . The gen2 segregants revealed a perfect co-segregation between the phenotype and the genotype wherein F3 seeds from 76 F2 segregants showed the synthesis of sinigrin ranging from 8.1–31.5 μmol g-1 (mean 13.7 μmol g-1) and the presence of ‘Varuna’ allele for BjuB.CYP79F1. The remaining 19 segregants showed no synthesis (or traces) of sinigrin and the presence of ‘Heera’ allele for BjuB.CYP79F1  PCR. Eight randomly selected F2 plants that showed the presence of gene BjuB.CYP79F1  and four F2 plants that did not  were chosen for the expression analysis. All the eight F2 segregants showing the presence of the gene BjuB.CYP79F1 also showed amplification from BjuB.CYP79F1 cDNA. On the other hand, the four lines that did not amplify the gene BjuB.CYP79F1 did not show amplification from cDNA as well  of the F as well . This laBjuB.CYP79F1 from Varuna in the binary vector pPZP200  and QTL-NIL J16Gsl4 (high seed GS content) both almost free from sinigrin and containing Heera allele of the gene, were used for transformation.A gene construct for plant transformation was developed under the control of CaMV35S promoter and terminator that contained wild-type  pPZP200 . The bar0) transgenic plants of EH-2 and QTL-NIL J16Gsl4, respectively, were transferred to a containment net house during the mustard growing season. Basta (200 mg l-1) was sprayed 30 days after transplantation. Basta resistant plants were grown to maturity and were allowed to set seeds through selfing as well as open pollination. The presence of the transgene and its expression were confirmed by PCR amplification of the genomic DNA (0 plants of QTL-NIL J16Gsl4 and EH-2. Control (untransformed) parental lines namely, Varuna, EH-2 and QTL-NIL J16Gsl4 were also grown in the containment net house for comparative seed GS profile analysis.A total of 37 and 211 independent putative  grown under containment net house showed an upward trend in the seed GS content  of BjuB.CYP79F1 gene did not enhance synthesis of sinigrin in the low GS EH-2 transgenics. On the other hand, the range of sinigrin content in the 20 transgenic lines of QTL-NIL J16Gsl4 varied from 0.5 to 25.5 μmol g-1 dry weight wherein five transgenic lines showed sinigrin content varying from 17.8 to 25.5 μmol g-1 dry weight in the T1 seeds. Selfed T1 seeds of these five QTL-NIL J16Gsl4 transgenic lines showing high sinigrin content were grown in containment in the next growing season to obtain T2 seeds. After Basta (200 mg l-1) spraying, resistant segregants of each line were grown to maturity and selfed seeds from each line were harvested separately. Analysis of open-pollinated T2 seeds by HPLC revealed consistency in the sinigrin content and was comparable to the sinigrin content of the wild type B. juncea line Varuna  to the LG B4 [B. juncea genome [B. rapa helped in searching for the CYP79F1 gene in the QTL region. The genome search of B. rapa (AA) [B. oleracea (CC) [B. napus (AACC) [CYP79F1 in all these Brassica species. The gene in the A genome of B. napus was observed to be tandemly duplicated. In the present study, we detected only CYP79F1 (BjuB.CYP79F1) from the B genome of B. juncea and also one each from B. nigra and B. rapa. It indicated that at least two copies of CYP79F1, one in each homoeolog, should be present in B. juncea.Prior information on QTL mapping of the 3C aliphatic GS  , B. olercea (CC)  and B. ns (AACC)  revealedBjuB.CYP79F1 in B. juncea line Heera  of B. nigra and showed high sequence identity (>93%) with CYP79F1 of other Brassica species. The BjuB.CYP79F1 protein was predicted to be localized on the ER membrane inside the cell. It is known that methionine derivatives with carbon side chains are synthesized inside the chloroplast [A. thaliana suggests that these are orthologous genes and in all probability perform similar function.The observed truncation of the gene ne Heera  due to tne Heera  and 6B. oroplast  and are oroplast . High leBjuB.CYP79F1 (a B genome homoeolog of CYP79F1) as the gene responsible for regulating sinigrin biosynthesis in B. juncea was first undertaken by genotype-phenotype co-segregation analysis. All the Varuna x QTL-NIL J16Gsl4 F2 segregants containing the Varuna allele of the gene (wild type) showed synthesis of sinigrin while the segregants having Heera allele did not show synthesis of sinigrin  having mutated MYB28, that controls the basal level synthesis of aliphatic GS [BjuB.CYP79F1 transcripts. Such uncoupling of GS metabolite levels from the level of transcript accumulation for aliphatic GS biosynthetic genes has been shown in A. thaliana [MYB28 was knocked out [B. juncea by down regulating MYB28 through transgenic approach to develop low GS lines [The EH-2 transgenics did not show any significant increase in sinigrin content over that of the wild type EH-2, although there is accumulation of transcript of the transgene . EH-2 ishatic GS ,20. Tranthaliana . It has cked out ,38. ThisGS lines . This coArabidopsis [CYP79F1 (At1g16410) in modulating the level of short-chain methionine-derived aliphatic GS. Hansen et al. [CYP79F1 of Arabidopsis was heterologously expressed in E. coli, there was conversion of dihomo- and trihomomethionine to their corresponding aldoximes. In the same year, an Arabidopsis mutant bus1-1f that contained an En-1 insertion in the gene CYP79F1 revealed a complete lack of short-chain methionine-derived GS. Conversely, the overexpression of CYP79F1 in Arabidopsis led to an increased level of short-chain methionine-derived GS [Our results also lend credence to the earlier reports from bidopsis ,40 regarrived GS .B. juncea and the sequence analyses of other Brassica species, that CYP79F2 is absent in all these cases. It has been demonstrated in A. thaliana that CYP79F1 catalyses synthesis of both short and long chain aliphatic GS, while CYP79F2 catalyses that of only long chain aliphatic GS [CYP79F2 in Brassica species corresponds well with the fact that all profiles of aliphatic GS in brassicas are composed of short chain GS [It was inferred from the present study on hatic GS ,41. Therchain GS .CYP79F1 between a sinigrin rich B. juncea var. tumida and a B. rapa line free of sinigrin [CYP79F1 expression was found to be upregulated in leaves of B. juncea as compared to B. rapa, suggesting difference in aliphatic GS content between B. juncea and B. rapa. However, it was not clear from the study whether the comparison of CYP79F1 of B. rapa was with CYP79F1 of the A or B genome of B. juncea. RNA-seq analysis of transcriptome and GS metabolism in a broccoli  line rich in 4C GS could not detect orthologs for both CYP79F1 and CYP79F2 [Some recent genomic studies provide indirect evidence in support of our findings. Comparative genome analysis using genome survey sequences identified non-synonymous SNPs in sinigrin . The CYP CYP79F2 .BjuB.CYP79F1 located in the LG B4 of B genome as the candidate gene responsible for the 3C GS biosynthesis in B. juncea. B. nigra (BB) is the provider of B genome in allopolyploid B. juncea (AABB) and contributes a functional 3C GS pathway to B. juncea because sinigrin is the predominant aliphatic GS fraction in B. nigra. On the other hand, the A genome contributor B. rapa (AA) does not synthesize sinigrin and hence does not contribute to sinigrin biosynthesis in B. juncea. The fact was earlier confirmed by QTL analysis by Ramchiary et al. [J16Gsl4 in LG B4) and none in the A genome. Moreover, the observation of restoration of the phenotype in transgenic lines of NIL J16Gsl4 in the present study not only identified the CYP79F1 as the candidate gene for sinigrin biosynthesis in B. juncea but also revealed the fact that genes upstream and downstream to CYP79F1 in aliphatic GS biosynthesis are functional in NIL J16Gsl4. It could be one of the major reasons for not being able to map a B genome paralogue of GSL-PRO  to the NIL J16Gsl4 QTL region [GSL-PRO has been proposed as the candidate gene controlling sinigrin biosynthesis in B. oleracea [Brassica species, one that operates at the chain elongation level in the C genome and the other that works at core GS structure formation level in the B genome.The present study resolved the genetic control of 3C GS and identified y et al.  where 3CL region . On the oleracea –17. ThisBjuB.CYP79F1 in all probability is involved in conversion of methionine substrate (homomethionine) to aldoxime in the 3C GS biosynthesis pathway. If so, it puts forth the obvious question—are there specific CYP79F1 homologs that are involved in the 4C and 5C pathways in B. juncea? A detailed molecular analysis of A genome specific CYP79F1 which has been shown to be functional from the transcriptome data of B. rapa [B. juncea [The study also indicates that  B. rapa  and B. j. juncea  might thB. juncea can be classified into two chemo-types for aliphatic GS [BjuB.CYP79F1 elaborated in the present study in B. juncea can be used as functional foreground selection marker for the efficient transfer of the trait  through marker-assisted breeding.Natural germplasm of hatic GS . DependiS1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.S5 Fig(DOCX)Click here for additional data file.S6 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


EZH2) is involved in cancer development through gene silencing by trimethylation of lysine 27 of histone 3 (H3K27me3). The C/C genotype for the EZH2 rs3757441 single-nucleotide polymorphism (SNP) is linked with poor prognosis in metastatic colorectal cancer (CRC), but molecular and pathological characterization of this SNP is lacking.The enhancer of zeste-homolog 2  and H3K27me3  staining intensity compared with C/T and T/T. EZH2 3+ staining significantly correlated with stronger H3K27me3 expression (P = 0.039). KRAS and BRAF mutations were not associated with EZH2 or H3K27me3 expression.The C/C genotype was significantly associated with higher EZH2 (100 EZH2 rs3757441 C/C genotype is associated with stronger EZH2 and H3K27me3 immunoreactivity in primary CRC: this SNP may serve as a promising biomarker for EZH2-targeting agents and may add independent information to KRAS and BRAF testing.The online version of this article (doi:10.1186/s12885-015-1889-2) contains supplementary material, which is available to authorized users. RAS and BRAF mutations  was analyzed through real-time PCR, as previously described [KRAS exon 2 (codon 12–13) and BRAF exon 15 (codon 600) mutations were detected via real-time sequencing using PyroMark Gold Q96 reagents (QIAGEN) on PyroMarkTM Q96 ID instrument , as reported elsewhere [Researchers who were not aware of EZH2 and H3K27 immunohistochemistry results and were blinded to patients’ clinical data performed genetic analyses. escribed . KRAS exlsewhere . ResultsImmunohistochemistry was performed on formalin fixed, paraffin-embedded tumor tissues, using previously validated protocols for both antibodies . InvolveTwo pathologists were responsible for immunohistochemical evaluation and provided independent scoring results for both EZH2 and H3K27. In case of discrepancy, joint revision of the ambiguous samples was performed to assure the consistency of definitive results.i) percentage of positive cells : the percentage of positive cells for each sample was determined as the average value of positive cells in 10 fields (approximately 100 cells per field were counted) at 10 High Power Field (HPF); ii) staining intensity, evaluated by a validated system [iii) Staining Index (SI), a semiquantitative method, evaluating both the heterogeneous distribution of positive cells and the differing intensity of the staining simultaneously: as described by Fluge et al. [EZH2 expression was categorized as: d system : we clase et al. , SI is ci) percentage of positive cells (0 - 100 %); ii) staining intensity (as described for EZH2). In both cases, the pathologist performing immunohistochemistry analyses was blinded from genetic data.H3K27me3 expression was scored as follows: P < 0.05 for two-tailed tests).EZH2 and H3K27 expression intensities were correlated with clinical and molecular features by means of chi-square test, T-test or ANOVA  and H3K27me3  staining intensity. When the C/T and T/T cases were grouped, the samples harbouring the C/C genotype showed significantly stronger EZH2 positivity compared to other genotypes . The same significant association was reported for H3K27me3 . Moreover, we observed a trend toward higher percentage of EZH2 positive cells  and higher SI  in C/C cases compared to C/T or T/T cases.We found significant differences among the three genotypes in terms of both EZH2 (3+ samples: 100 % P = 0.021) .The C/C genotype was associated with higher tumor grade: all the 5 cases were scored as grade 3–4 tumors, while undifferentiated histology was reported in 37 % and 38 % of the C/T and T/T cases, respectively P = 0.02   and staining intensity  in poorly differentiated tumors.None of the investigated tumor features was associated with H3K27me3 expression, with the exception of tumor site . These results further corroborate the notion that higher EZH2 expression in CRC samples results in increased histone H3K27 methylation.Of note, we found a significant association between EZH2 and H3K27me3 staining intensity  and evaluated EZH2 expression in tumor cells by immunohistochemistry in a series of 119 primary CRC. In this context, we found that the C/C genotype was associated with significantly higher EZH2 expression .EZH2 rs3757441 SNP, showing that the C/C variant is associated with higher EZH2 expression and activity in the neoplastic tissue. This SNP genotype has been linked with worse prognosis among mCRC patients treated with chemotherapy with or without an anti-vascular endothelial growth factor antibody: our data confirm in primary tumors that the C allele is linked with increased EZH2 expression and activity, thus providing additional evidence for a functional relevance of this SNP in CRC biology. As anti-EZH2 drugs are under development and early clinical evaluation [To conclude, we characterized the aluation , this SN


Pandemic influenza A(H1N1)pdm09 emerged in Thailand in 2009. A prospective longitudinal adult cohort and household transmission study of influenza-like illness (ILI) was ongoing in rural Thailand at the time of emergence. Symptomatic and subclinical A(H1N1)pdm09 infection rates in the cohort and among household members were evaluated.A cohort of 800 Thai adults underwent active community-based surveillance for ILI from 2008–2010. Acute respiratory samples from ILI episodes were tested for A(H1N1)pdm09 by qRT-PCR; acute and 60-day convalescent blood samples were tested by A(H1N1)pdm09 hemagglutination inhibition assay (HI). Enrollment, 12-month and 24-month follow-up blood samples were tested for A(H1N1)pdm09 seroconversion by HI. Household members of influenza A-infected cohort subjects with ILI were enrolled in household transmission investigations in which day 0 and 60 blood samples and acute respiratory samples were tested by either qRT-PCR or HI for A(H1N1)pdm09. Seroconversion between annual blood samples without A(H1N1)pdm09-positive ILI was considered as subclinical infection.The 2-yr cumulative incidence of A(H1N1)pdm09 infection in the cohort in 2009/2010 was 10.8% (84/781) with an annual incidence of 1.2% in 2009 and 9.7% in 2010; 83.3% of infections were subclinical (50% in 2009 and 85.9% in 2010). The 2-yr cumulative incidence was lowest (5%) in adults born ≤1957. The A(H1N1)pdm09 secondary attack rate among household contacts was 47.2% (17/36); 47.1% of these infections were subclinical. The highest A(H1N1)pdm09 secondary attack rate among household contacts  occurred among children born between 1990 and 2003.Subclinical A(H1N1)pdm09 infections in Thai adults occurred frequently and accounted for a greater proportion of all A(H1N1)pdm09 infections than previously estimated. The role of subclinical infections in A(H1N1)pdm09 transmission has important implications in formulating strategies to predict and prevent the spread of A(H1N1)pdm09 and other influenza virus strains. Pandemic influenza A(H1N1)pdm09 emerged in North America in March 2009 and rapidly spread to other parts of the world, eventually replacing previous seasonal H1N1 virus strains. Several studies from different countries established a pattern of higher A(H1N1)pdm09 infection rates in school-age children relative to young adults, with older adults having the lowest infection rates In Thailand, like other tropical countries, influenza activity tends to occur in a biphasic seasonal pattern (June to August and January to March) with sporadic infection throughout the year At the time of pandemic onset in 2009, a prospective longitudinal cohort study was ongoing in Kamphaeng Phet, Thailand in which 800 adults (age range 20–85 years) were undergoing active surveillance for influenza-like illness (ILI) from 2008 to 2010. The study also included a household transmission component triggered by confirmed influenza virus (IFV) infections in the cohort The study was approved by the institutional review boards of the Thai Ministry of Public Health (MOPH), Walter Reed Army Institute of Research (WRAIR), Naval Medical Research Unit No. 2 (NAMRU-2), US Army Medical Research and Materiel Command, University of Iowa, and University of Florida. Written informed consent was obtained from all study subjects (or their parents if applicable) and assent was obtained from children older than seven years.Although the study was designed to focus chiefly upon studying subclinical avian influenza infections, reviewing IRBs approved the study to include all influenza A infections.Approximately 800 adult subjects living in Kamphaeng Phet province (KPP), Thailand, a rural province located about 350 km north of Bangkok, were enrolled in a prospective longitudinal cohort study. Details of study methods have been previously published Annual follow-up visits at 12 and 24 months after enrollment were performed in 2009 and 2010 during which clinical questionnaires were administered and blood samples were obtained. The questionnaires assessed changes in demographic or health status during the preceding year. Serological testing of the enrollment and annual follow-up blood samples was conducted by influenza HI to monitor for changes in influenza antibody titers over time. An IFV infection was considered to be subclinical if IFV HI seroconversion occurred between annual blood samples without an IFV-positive ILI detected during the intervening surveillance period.When a cohort subject developed an ILI in which an acute respiratory sample tested positive for influenza A by qRT-PCR, that cohort subject’s household members ≥6 months old were enrolled in a household transmission investigation. A blood sample was collected at enrollment (day 0) from each household member . If a contact subject developed ILI during the following 60 days, acute respiratory samples  were additionally collected from that contact subject for qRT-PCR for influenza A and B. A questionnaire was administered to each contact subject to assess the degree of contact with the cohort subject and identify any illnesses during the preceding 7 days. Weekly follow-up visits were conducted with the contact subjects for 60 days after initiation of the household investigation. At day 60, a convalescent blood sample was collected from each contact subject.Blood samples were transported at room temperature and respiratory swabs were transported in Micro Test M4RT Viral Transport Media  at 4°C within the same day to the field laboratory at Kamphaeng Phet-AFRIMS Virology Research Unit (KAVRU) in KPP, Thailand, where qRT-PCR for IFV was performed. Serological testing by HI was performed at the University of Florida Global Pathogens Laboratory in Gainesville, Florida, USA.Enrollment and annual follow-up serum samples were tested using the World Health Organization (WHO) or US Centers for Disease Control and Prevention (CDC) HI protocol as previously described The procedures have been previously described Infection with A(H1N1)pdm09 was considered to have occurred if (a) a respiratory sample was positive for A(H1N1)pdm09 by qRT-PCR, or (b) a 4-fold or greater increase in HI titer (seroconversion) against A(H1N1)pdm09 occurred between paired annual sera or paired ILI sera, with a second titer of at least 1∶40. If paired sera were not available, a single high titer of at least 1∶40 was considered to indicate recent infection.Risk factors for bivariate associations with HI results were initially examined using binary logistic regression and proportional odds modeling Between April and October 2008, field staff enrolled a total of 800 adults (100 from each of 8 sites). Details of participant demographics at enrollment have been previously described Seroprevalence of elevated HI antibodies against A(H1N1)pdm09 increased from 1.7% and 1.3% in 2008 and 2009, respectively, to 10.3% in 2010 . Based oFrom January 2009 to December 2010, 53 cohort subjects met the case definition for ILI; 10 of these subjects were confirmed to have A(H1N1)pdm09 infection by qRT-PCR. Most  of these subjects were born after 1957. Prior to ILI occurrence, none of the 10 subjects had pre-existing A(H1N1)pdm09 antibody. Only 3 of these subjects had A(H1N1)pdm09 seroconversion in the paired ILI sera. Among the remaining 7 subjects with A(H1N1)pdm09-positive qRT-PCR, 5 were seronegative in both of the paired ILI sera and 2 did not have sufficient paired sera for HI testing.When comparing A(H1N1)pdm09 seroconversions from the enrollment and annual follow-up blood samples and the qRT-PCR data from ILI acute respiratory samples, 5 subjects had A(H1N1)pdm09 infection by both qRT-PCR and HI, 5 by qRT-PCR only, and 74 by HI alone. Among these 84 cohort subjects with A(H1N1)pdm09 infection, only 14 subjects reported ILI . Most  of the A(H1N1)pdm09 infections among the cohort subjects were subclinical. The proportion of subclinical infections among cohort subjects born after 1957 was higher than those born ≤1957 .Using binary logistic regression analysis, several covariates were associated with an increased risk for seroconversion against A/Mexico/4108/2009(H1N1) . Cohort Thirty-six contact subjects (age range 1–70 years) living in the same household as the 10 cohort subjects with A(H1N1)pdm09-positive qRT-PCR were enrolled. Paired day 0 and 60 sera from these contact subjects were evaluated by HI against A(H1N1)pdm09 [A/Mexico/4108/2009(H1N1)]; three contact subjects missed their day 60 blood draw. Of the 36 contact subjects, 6 had ILI of which all were A(H1N1)pdm09-positive by qRT-PCR . Five ofOur report is one of the few combined prospective longitudinal cohort and household transmission studies conducted during the A(H1N1)pdm09 pandemic that describes the epidemiology of A(H1N1)pdm09 in a tropical region. The proportion of A(H1N1)pdm09 infections in our cohort that were subclinical was very high (83.3%). This proportion was higher than some prior estimates (approximately 50%) from a few comparable studies Cohort subjects born ≤1957 (age ≥52 years) were less likely to have A(H1N1)pdm09 seroconversion, comparable to the findings from other studies The cumulative incidence of A(H1N1)pdm09 infection by qRT-PCR and HI seroconversion in the cohort from January 2009 to December 2010 was 10.8% with an annual incidence of 1.2% and 9.7% in 2009 and 2010, respectively. Previous studies estimated the incidence of A(H1N1)pdm09 infection among adults to range from 6% to 13% during the early pandemic (May to September 2009) The A(H1N1)pdm09 secondary attack rate (SAR) among household members of cohort subjects was 47.2% . Previous A(H1N1)pdm09 household transmission studies conducted during the early pandemic (April to August 2009) reported a wide range (4% to 45%) of SARs Our study had some limitations. Only adults over 20 years of age were enrolled in the cohort. Since children are at high risk of A(H1N1)pdm09 infection Our study was unique in evaluating the 2009 influenza pandemic in several ways. First, it combined a prospective longitudinal cohort study undergoing active ILI surveillance with a household transmission component. Second, it was already fully operational at the outset of the pandemic. Third, it continued operating for the full duration of the pandemic. Keeping these aspects of the study in mind, our results demonstrate that subclinical A(H1N1)pdm09 infection occurred frequently among Thai adults in this rural population. The role of these subclinical infections in A(H1N1)pdm09 transmission has important implications in formulating strategies to predict and control the spread of A(H1N1)pdm09 and other IFV strains. For instance, if a large portion of viral infections are subclinical, hospital-based surveillance will not well-represent a population’s influenza ecology. Hence, public health officials should consider these large numbers of subclinical infections in developing future influenza surveillance and control programs. The effort to capture subclinical data need not be as extensive and expensive as this cohort study. For example, subclinical infection data might be collected through modest household transmission studies, where secondary attack rates  among persons in the same household as index cases may be used as a surrogate for general population data.


Autophagy is a double-edged sword in tumorigenesis and plays an important role in the resistance of cancer cells to chemotherapy. S100A8 is a member of the S100 calcium-binding protein family and plays an important role in the drug resistance of leukemia cells, with the mechanisms largely unknown. Here we report that S100A8 contributes to drug resistance in leukemia by promoting autophagy. S100A8 level was elevated in drug resistance leukemia cell lines relative to the nondrug resistant cell lines. Adriamycin and vincristine increased S100A8 in human leukemia cells, accompanied with upregulation of autophagy. RNA interference-mediated knockdown of S100A8 restored the chemosensitivity of leukemia cells, while overexpression of S100A8 enhanced drug resistance and increased autophagy. S100A8 physically interacted with the autophagy regulator BECN1 and was required for the formation of the BECN1-PI3KC3 complex. In addition, interaction between S100A8 and BECN1 relied upon the autophagic complex ULK1-mAtg13. Furthermore, we discovered that exogenous S100A8 induced autophagy, and RAGE was involved in exogenous S100A8-regulated autophagy. Our data demonstrated that S100A8 is involved in the development of chemoresistance in leukemia cells by regulating autophagy, and suggest that S100A8 may be a novel target for improving leukemia therapy. Autophagy is a catabolic process involving the degradation of intracellular aggregated or misfolded proteins, and damaged organelles through lysosomal machinery in response to stress or starvation via regulating the autophagy in leukemia.It has been well documented that autophagy plays an important role in the resistance of cancer cells to chemotherapy In this study, we found that S100A8 enhances drug resistance by upregulating autophagy through promoting the formation of BECN1-PI3KC3  complex, providing a novel potential target for the treatment of leukemia.The antibodies against S100A8 and p62 were obtained from Santa Cruz Biotechnology . The antibodies to Actin, BECN1, PI3KC3, C-PARP, ULK1, Bcl-2 and P-ULK1 were from Cell Signaling Technology . The antibodies to LC3 and TLR-4 were purchased from Abcam . Anti-Atg7 antibody was from Novus . Vincristine (VCR), adriamycin (ADM), rotenone (Rot), thenoyltrifluoroacetone (TTFA), antimycin A (AA), E64D, anti-RAGE antibody and pepstatin were from Sigma . Full-length human S100A8 cDNA (pLPCX-S100A8) was a gift from Dr. RW Stam . FITC-Annexin V Apoptosis Detection kit and the Nuclear and Cytoplasmic Protein Extraction kit were purchased form Beyotime Institute of Biotechnology . S100A8 protein was obtained from Novus Biologicals. Contaminating LPS was removed by Triton X-114 extraction. LPS content was always below 0.5 ng/mg protein, which did not cause an effect in our assays.The human leukemia cell lines, K562 (chronic myeloid leukemia cells), HL-60 (acute myeloid leukemia cells), MV-4-11 (biphenotypic B myelomonocytic leukemia cells), Jurkat (T-cell acute lymphoblastic leukemia cells), and K562/A02 (multidrug resistance K562) were from the American Type Culture Collection; HL-60/ADR (multidrug resistance HL-60) was from the Institute of Hematology & Blood Diseases Hospital of Chinese Academy of Medical Sciences & Peking Union Medical College. Cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS and 2 mM glutamine in a humidified incubator with 5% CO2 and 95% air.Cell viability was assessed by MTT assay. Briefly, leukemia cells were seeded in 96-well plates (4000 cells/well) the day before treatment. Following treatment with ADR for 72 h, 25 µL MTT  was added to each well and incubated for 3.5 h, followed by the addition of 100 µL of N,Ndimethylformamide . The plates were left at room temperature overnight to allow complete lysis of the cells, and read at 450 nm the following day. Half-maximal inhibitory concentration (IC50) was calculated using MS Excel, as previously described via semidry transfer. The membrane was blocked with 5% dried milk or 3% bovine serum albumin in Tris-buffered saline and Tween 20 , incubated with primary antibodies, and then with horseradish peroxidase–conjugated secondary antibodies. The signals were visualized by enhanced chemiluminescence.Cell lysates were prepared with cell lysis buffer , and cleared by centrifugation. Total protein concentration was determined with the bicinchoninic acid assay Kit (Bio-Rad). Proteins were resolved on a denaturing 10% SDS-PAGE gel and subsequently transferred to polyvinylidene fluoride membranes 5′- CCTAACCGCTATAAAAAGGAG -3′, reverse primer 5′- ATGATGCCCACGGACTTGCC -3, probe 5′ FAM-CCTCTCAGCCCTGCATGTCTCTT -TAMRA 3′; ACTB: forward primer 5′- GGCACCCAGCACAATGAAGA-3, reverse primer 5′-CGTCATACTCCTGCTTGCTG-3′, probe 5′FAM-CTGGAAGGTGGACAGCGAGGC-TAMRA 3′.) in an LightCycler 480 System (Roche). Quantification was determined by the standard curve and 2-ΔΔCt methods Total RNA was extracted using TRIzol  according to the manufacturer's instructions. Reverse transcription (RT) was performed with 2 µg of total RNA with HiFi-MMLV Enzyme Mix . Twenty ng cDNA was subjected to real-time quantitative PCR (TaqMan probes) for the evaluation of the relative S100A8 mRNA, with beta actin as an internal control with gene specific primers and fluorogenic probes . Cell lysates were cleared by a brief centrifugation . Protein concentration in the supernatant was determined by bicinchoninic acid assay. Before immunoprecipitation, equal amounts of proteins were pre-cleared with Protein A or protein G agarose/sepharose (Millipore) at 4°C for 3 h and subsequently incubated with various irrelevant immunoglobulin-G or specific antibodies (5 µg/ml) in the presence of protein A or G agarose/sepharose beads for 2 h or overnight at 4°C with gently shaking. Following incubation, agarose/sepharose beads were washed extensively with phosphate buffered saline. Proteins were eluted by boiling in 2×SDS sample buffer before SDS–polyacrylamide gel electrophoresis and immunoblot analysis, as previously described Cells were transfected with S100A8 pLPCX constructs by square-pulse electroporation at 600 V for 2 msec and cultured under selection of neomycin  and puromycin  in order to obtain a pure population of transfected cells +/PI).Apoptosis was assessed using the FITC Annexin V Apoptosis Detection kit, which involves staining cells with Annexin V-FITC (a phospholipid-binding protein that binds to disrupted cell membranes) in combination with PI . Flow cytometric analysis (FACS) was performed to determine the percentage of apoptotic cells  Cells were collected, fixed and permeabilized with 0.3% triton X-100 for 10 min, incubated with anti-LC3 for 1 h and then FITC-conjugated Anti-LC3A/B antibody  for 1 h at room temperature. Cell nuclei were stained using ProLong Gold Antifade Reagent with DAPI (Life Technologies). Samples were examined under an Olympus FV1000 confocal microscope. For evaluating tandem fluorescent LC3 puncta, cells were washed with PBS, fixed with 4% paraformaldehyde, mounted with DAPI and viewed under a confocal microscope To analyze autophagic flux, we monitored the formation of autophagic vesicles by the mRFP–GFP–LC3 method (Invitrogen). Due to the quenching of GFP in the acidic lysosomal environment t-test or ANOVA least significant difference test, and P<0.05 was considered significant.All experiments were performed in at least triplicates per group, and data are reported as mean±SEM, unless otherwise indicated. Data were analyzed by 2-tailed Student S100A8 was differentially expressed in different cell lines, with relatively low S100A8 in Jurkat cells. We found that S100A8 protein was significantly increased in the drug resistance leukemia cell line K562/A02, relative to the nondrug resistant cell line K562 . ElevateAt the same time, we found the basal authophagy level in the drug resistant leukemia cells was higher compared to the non-drug resistant cell lines. We evaluated basal authophagy in leukaemia cells and compared the levels of autophagy with S100A8 expression. Basal authophagy in leukaemia cells was low. Moreover, we found there were no significant difference in levels of LC3-II/LC-I in HL-60, K562 and MV4-11 cells in comparison to Jurkat cells. However, the authophagy level were increased in K562/A02 relative to K562, and HL-60/ADR compared to HL-60 .To explore the functions of S100A8 in drug resistance, we next analyzed the relationship between the IC50 of adriamycin and the expression level of S100A8. Increased expression of S100A8 was correlated with higher IC50 of adriamycin . OverexpIt was reported that chemotherapeutic drugs induce S100A8 in the supernatants of cell cultures To evaluate whether overexpression of S100A8 results in drug resistance, we knocked down S100A8 by shRNA in HL60/ADR and K562/A02. S100A8 shRNA transfection led to a significant decrease of both S100A8 protein and mRNA in these cells . KnockdoTo further determine the role of S100A8 in leukemia cells after chemotherapy, we transfected K562 leukemia cells with a plasmid containing full-length human S100A8 cDNA . EctopicWe further found that increase of S100A8 during anticancer therapy could induce autophagic flux in leukaemia cells. Accumulation of LC3-II was observed in the presence of bafilomycin A1 (an inhibitor of late phase autophagy) in k562 cells both before and after treatment with adriamycin for 24 h compared with the absence of bafilomycin A1 . At the Bafilomycin A1 increased the induction of LC3-II by S100A8 , whereasS100A8 is required for the initiation of As2O3-reduced autophagy in leukemia cells Rapamycin induces autophagy by inhibiting the mammalian target of rapamycin complex 1 (mTORC1) Mammalian autophagy is a multi-step process including initiation, nucleation, elongation, closure, maturation, and finally degradation or extrusion, and regulated by a core family of ATG proteins To investigate whether extracellular S100A8 induce autophagy in leukemia cells, we treated K562 cells with 1 µg/ml S100A8 protein for 24 h and detected LC3-I/LC3-II and P62 by immunoblot analysis. We found that S100A8 treatment increased the expression of LC3-II, and decreased the expression of p62. To test the role of RAGE and TLR4 in S100A8-induced autophagy, we knocked down RAGE and TLR4 by shRNA in K562 cells. We found that silencing RAGE inhibited LC3-II formation and prevented autophagic p62 degradation. In contrast, knock down of TLR4 did not influence S100A8-induced autophagic flux. These findings indicate that RAGE is involved in exogenous S100A8-induced autophagy Chemotherapy is the major treatment strategy for nearly all types of childhood leukemia S100A8 is expressed in a wide variety of cell types and abundant in myeloid cells. Elevated expression of S100A8 has been found in disorders including rheumatoid arthritis, inflammatory bowel disease, and vasculitis A number of anticancer therapies, including DNA-damaging chemotherapeutic drugs, induce the accumulation of autophagosomes in tumor cell lines, while pharmacologic inhibition of autophagy or genetic knockdown of phylogenetically conserved autophagy-related genes, such as Atg5 and Atg7, enhanced drug-induced cytotoxicities Cancer cells respond to chemotherapy in a variety of ways, ranging from the activation of survival pathways to the initiation of cell death. Increased autophagy is observed in leukemia cells when exposed to chemotherapy drugs We previously found that S100A8 interacts with BECN1 and displaces Bcl-2 In conclusion, we showed that chemotherapy-induced S100A8 expression in leukemia cells promoted autophagy, which in turn inhibited apoptosis and increased drug resistance, indicating that S100A8 is an important regulator of autophagy. Moreover, suppression of S100A8 expression significantly increased drug sensitivity of leukemia cells, suggesting that S100A8 may be a novel target for leukemia therapy. .


HyPRP1 gene encodes a protein with proline-rich domain, and an eight-cysteine motif was identified from our previous microarray experiments on drought-tolerant tomato. In this study, the expression of the HyPRP1 gene in tomato was suppressed under various abiotic stresses, such as drought, high salinity, cold, heat, and oxidative stress. Transgenic functional analysis showed no obvious changes in phenotypes, but enhanced tolerance to various abiotic stresses  was observed in RNAi transgenic plants. Interestingly, several SO2 detoxification-related enzymes, including sulfite oxidase, ferredoxins (Fds), and methionine sulfoxide reductase A (Msr A), were revealed in HyPRP1-interacting proteins identified by Yeast Two-Hybrid screening. More sulfates and transcripts of Msr A and Fds were accumulated in HyPRP1 knockdown lines when wild-type plants were exposed to SO2 gas. Our findings illustrate that the tomato HyPRP1 is a negative regulator of salt and oxidative stresses and is probably involved in sulfite metabolism.Many hybrid proline-rich protein (HyPRP) genes respond to biotic and abiotic stresses in plants, but little is known about their roles other than as putative cell-wall structural proteins. A  Hybrid proline-rich proteins (HyPRPs) comprise a dynamically evolving protein family unique to seed plants  in tobacco increased its tolerance to freezing, chilling, osmotic stress, and methyl viologen (MV)-induced oxidative stress . Although, cultivated tomato (Solanum lycopersicum) is sensitive to drought and salt  shows strong adaptation to arid environments owing to its high water-use efficiency  were grown in a naturally illuminated glasshouse. Tissues from the roots, stems, leaves, flowers, and fruits at various developmental stages were collected from untreated control plants, immediately frozen in liquid nitrogen, and stored at −80°C. For gene expression profiling analysis, identical 2-month-old tomato plants were subjected to various stresses or plant growth regulator treatments. Salt, drought, cold, heat, wounding, ABA treatments, and oxidative stress were simulated as previously described . The three replicates in qRT-PCR assay were three technical replicas, each assayed sample represents three independently collected samples. The qRT-PCR assays are from one of two different experiments that yielded essentially identical results. The expression of tomato oxidative related gene SlCAT (Solyc12g094620.1.1), SlSOD (Solyc09g082690.2.1), and SlMSR B (EF144171) was analyzed by real-time PCR in wild-type (WT) and transgenic plants. The qRT-PCR primer sequences are listed in Table Total RNA was isolated using TRIzol reagent . Dnase I  treated RNA is reverse-transcribed using ReverTra Ace reverse transcriptase . Real-time quantitative RT-PCR (qRT-PCR) was performed on a LightCycler Roche 480  with a LightCycler 480 SYBR Green I Master kit (Roche) used in accordance with the supplier's instructions. The PCR amplification consisted of an initial incubation at 95°C for 5 min, followed by 40 cycles for 10 s at 95°C, 15 s at 58°C, and 20 s at 72°C. Data were gathered during the extension step. Melting-curve acquisition and analyses were also performed on the cycler. Each sample included three replicates, and the data were normalized against the reference βSpHyPRP1  cDNA from S. pennellii LA0716 was amplified with the forward primer 5′-CAATCTTTGTACCAA ATTATTTAACCA-3′ and reverse primer 5′- AACAATT CCACAAAGCCAAAA-3′. The PCR product was cloned into the pMD18-T vector  and then sequenced. pMD18-T-SpHyPRP1 was digested with restriction enzymes SalI and KpnI. The resulting product was inserted into the XhoI and KpnI sites of the binary vector pMV (pBI121 reformed) to yield the overexpressing construct with SpHyPRP1, which was driven by a cauliflower mosaic virus 35S promoter (CaMV35S). To construct the RNA interference vector, a 451 bp fragment was amplified from the SlHyPRP1 coding sequence (Solyc12g009650.1.1) by using gene-specific primers with a 5′-attB1 extension forward primer GGGGACAAGTTTGTACAAAAAAGCAGGCTTCTTTGTACCAAATTATTTAACCA CA and a 5′-attB2 extension reverse primer GGGGACCACTTTGTACAAGAAAGCTGGGTCAATTGGTGGAACTGT GACC (5′-attB1 and 5′-attB2 extensions are underlined). A recombination reaction between the PCR product and the pHellsgate 2 vector  was performed using BP clonase (Invitrogen) in accordance with the manufacturer's instructions. Both constructs were used to transform tomato S. lycopersicum M82 mediated by the Agrobacterium tumefaciens strain C58. A copy of the T0 transgenic tomato plant was detected through Southern blot hybridization by using neomycin phosphotransferase II gene as the probe. The expression of HyPRP1 in HyPRP1-RNAi and overexpressed transgenic  plants was examined by qRT-PCR as described above. The transgenic T2 or T3 lines with HyPRP1 transcripts that increased or decreased significantly were used for further analysis.A full length of Positive transgenic seedlings from three lines were germinated in 1/2 MS medium for 2 days and then subcultured in 1/2 MS containing 150 mM NaCl, 200 mM mannitol, or 3 μM ABA. The seedlings were grown for 12 days, each with three replicates.To evaluate the salt tolerance of transgenic lines, uniform-sized positive seedlings that were confirmed through PCR were transplanted into cylindrical pots  and grown up to the five-leaf stage. Afterward, the seedlings were treated with either 75 μM MV with 1 mg/L Tween-20 sprayed on the leaves until run off or 200 mM NaCl by watering the plants, each with three to four replicates. The relative seedling growth and root weight were measured by dividing the treated seedling height and root weight by those of the untreated plants and multiplying the result by 100 . Briefly, all the leaves were stained with DAB solution (1 mg/mL) and incubated for 4 h at 25°C in the dark. Samples were then cleaned with 70% alcohol and incubated at 70°C for 10 min.To determine the water loss rate, 15 leaves were collected from the same location in transgenic and WT plants. The weights of the leaves were measured progressively at specified time points. After 3 h of dehydration, the accumulation of H2 stress treatment. SO2 exposure was carried out in a transparent 1 m3 growth chamber. The corresponding weight (2 or 5 g) of sublimed sulfur was burned in the chamber to produce ~4 or 10 ppm SO2. The chambers were sealed with transparent adhesive tape and gently shaken to keep the SO2 evenly distributed inside. Both the control (plants in a chamber without SO2 treatment) and treated plants were kept under continuous light at 25°C with ~85 to 95% relative humidity. To determine the inner sulfate level, HyPRP1 knockdown, overexpression lines, and WT control plants were exposed to 10 ppm SO2 for 2 h. The leaves were then cut and extracted immediately in double-distilled water, followed by heating for 5 min at 95°C  and confirmed by sequencing. The correct plasmid was digested with SmaI and PstI, and the fragment was fused to the frame with the GAL4 DNA-binding domain into the SmaI and PstI-digested pGBKT7 vectors. The bait construct pGBKT7-SpHyPRP1 was transformed into the yeast strain Y187 through lithium acetate method. Interacting clones were screened through mating in accordance with the manufacturer's instructions , and 60 randomly selected positive clones were sequenced and analyzed.For the yeast two-hybrid (Y2H) screenings, the full coding sequence of SpHyPRP1 without the stop codon was amplified using PCR and cloned into the N-terminal 155 amino acid portion of yellow fluorescent protein (YFPN) in the pUC-SPYNEG vector  cells by using Biolistic PDS-1000 . All samples were observed under a Leica TCSST2 confocal laser microscope  after 24 h of bombardment.The full-length cDNA of rs Table . The fraSpHyPRP1 and SlHyPRP1 was amplified through PCR by using the forward primer 5′-GG ATCCATGGAGTTCTCTAAGATAACTTCA C-3′ and the reverse primer 5′-CTCGAGCTAGATGG AACAAGTGTAGCCAG-3′ from S. lycopersicum cv. M82 and S. pennellii LA0716, correspondingly. The amplicons were inserted into the pEASY-E1 vector  through TA cloning, in which the exogenous gene was under the control of a T7 RNA polymerase promoter. The resulting constructs pET-SlHyPRP1 and pET-SpHyPRP1, together with the empty vector pEASY-E1, were introduced into E. coli BL21 (DE3) cells. The correct clones confirmed by sequencing were used for further analysis. To measure the growth rate under oxidative stress, E. coil cells with either of the above plasmids were grown in LB liquid media containing 100 μg/mL ampicillin with continuous shaking at 37°C. When the cells were grown to an absorbance value of A600nm = 0.6–0.8, 1 mM isopropyl-1-thio-β-galactopyranoside was used to induce gene expression for 4 h. Subsequently, the cells were diluted 10 times using new LB media with antibiotics and grown further to the mid-log phase (A600nm = 0.3–0.6). The cells were then challenged with 1.5 mM H2O2. The attenuance at 600 nm was measured at designated time points. Afterward, 2 μL of cells were also dotted into the LB agar plates supplemented with 1.5 mM H2O2. Colony formation was observed after 4 h of incubation at 37°C.The full-length ORF of SlHyPRP1 gene was observed between the drought-tolerant ILs and M82 . These two putative amino acids shared 96% similarity, differing only in eight residues. Threonine at site 43 (T) and isoleucine 85 (I), 120 (I), and 150 (I) in S. lycopersicum cv. M82 were substituted by serine 43 (S) and valine 85 (V), 120 (V), and 150 (V) in S. pennellii LA0716, correspondingly. These findings suggest that a major amino acid difference between the two species is the demethylation in S. pennellii LA0716 at a corresponding site. One exception was observed in site 115, where the V in S. lycopersicum cv. M82 was methylated to I. At sites 81 and 95, the positively charged residue lysine (K) in M82 was replaced by asparagine (N) with neutral residue in LA0716. The most prominent feature was found at site 95, where the hydrophobic isoleucine (I) in M82 was substituted by hydrophilic asparagine (N) in LA0716 , a differential expression profile of the 6 Figure .http://bp.nuap.nagoya-u.ac.jp/sosui/), SlHyPRP1 and SpHyPRP1 exhibited one putative transmembrane signal peptide and an average hydrophobicity of 0.52 and 0.48, respectively. This finding implies that HyPRP1 is a transmembrane protein, which may differ in hydrophobicity between S. lycopersicum and S. pennellii.A similar search of the GenBank database revealed that HyPRP1 in tomato shares a significant degree of sequence identity at 8CM residues with other HyPRPs from various species of plants. In addition, the proline-rich repetitive domain (PRD) at the N-terminus showed a varied repeated order and high proline content Figure . AccordiE. coli, transformants enhanced the host abiotic stress  were obtained, and three T2 homozygous lines with HyPRP1 transcripts 69.3-fold (OE3), 6.4-fold (OE8), and 9.1-fold (OE14) greater than those of WT were screened out for further analysis  were obtained from kanamycin-resistant calli. The expression levels were significantly knocked down in SlHyPRP1-RNAi transgenic T2 lines (Ri3: 0.02-fold and Ri9: 0.07-fold downregulated compared with WT) were obtained and used for the following abiotic stress analysis. At high salinity (200 mM NaCl), the growth rate and root weight of the seedlings of SlHyPRP1-RNAi transgenic lines were significantly higher than those of the WT control seedlings; the plants that exhibited overexpression showed slightly lower growth rate and root weight than those of the WT control seedlings, but no significant differences were observed  were increased twofold to fourfold in SlHyPRP1-RNAi transgenic plants compared with WT plants, whereas all genes were downregulated in the HyPRP1-overexpressed transgenic lines , UBQ10 (polyubiquitin: SGN-U580864), Fds (ferredoxins: Q43517), ZPR1 (ZPR1-type zinc finger protein: SGN-U576075), and SO (sulfite oxidase: ABI53846.1), were screened out. BiFC experiments were then performed to confirm the interactions. Pairwise expression of HyPRP1::YFPN with UBQ10::SPYCEC, Msr A::SPYCEC, Fds::SPYCEC, SO::SPYCEC, or ZPR1::SPYCEC all resulted in the accumulation of YFP fluorescence in the transformed BY2 cells, whereas no YFP fluorescence was observed in the control cells . At 12 h after stress treatment, the transcripts increased more than 10-fold , and MST1  are late-responsive (24 h) SO-dependent upregulated genes that catalyze the diversion of sulfite to other assimilatory pathways , whereas SiR uses NADPH in bacteria (Yonekura-Sakakibara et al., JZ and ZY conceived the study; JL and WS performed the experiments; TW and ZL analyzed the data; BO, CY, and HL provided the reagents and tools; JL and JZ wrote the paper. All the authors have discussed the results and contributed to improving the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Blind docking studies predict that RMNC6 could bind two different pockets in the RT: one in the DNA polymerase domain , and a second one close to the RNase H active site. Enzymatic studies showed that RMNC6 interferes with efavirenz (an approved NNRTI) in its binding to the RT polymerase domain, although NNRTI resistance-associated mutations such as K103N, Y181C and Y188L had a minor impact on RT susceptibility to RMNC6. In addition, despite being naturally resistant to NNRTIs, the polymerase activity of HIV-1 group O RT was efficiently inhibited by RMNC6. The compound was also an inhibitor of the RNase H activity of wild-type HIV-1 group O RT, although we observed a 6.5-fold increase in the IC50 in comparison with the prototypic HIV-1 group M subtype B enzyme. Mutagenesis studies showed that RT RNase H domain residues Asn474 and Tyr501, and in a lesser extent Ala502 and Ala508, are critical for RMNC6 inhibition of the endonuclease activity of the RT, without affecting its DNA polymerization activity. Our results show that RMNC6 acts as a dual inhibitor with allosteric sites in the DNA polymerase and the RNase H domains of HIV-1 RT.The DNA polymerase and ribonuclease H (RNase H) activities of human immunodeficiency virus type 1 (HIV-1) are needed for the replication of the viral genome and are validated drug targets. However, there are no approved drugs inhibiting RNase H and the efficiency of DNA polymerase inhibitors can be diminished by the presence of drug resistance mutations. In this context, drugs inhibiting both activities could represent a significant advance towards better anti-HIV therapies. We report on the mechanisms of allosteric inhibition of a newly synthesized isatin-based compound designated as RMNC6 that showed IC Since the identification of the human immunodeficiency virus (HIV) as a retrovirus causing AIDS , 2, it win vitro [Because of their pivotal role in viral replication, RDDP and RNase H activities are both validated targets for the identification of new RT inhibitors, needed to combat the emergence of multi-drug resistant strains, whose spreading in newly infected patients is an issue of increasing concern, causing a number of associated antiviral therapy failures . In thisin vitro . In partin vitro . More rein vitro –22.With the aim of identifying new HIV-1 RNase H inhibitors, we previously performed a successful ligand-based virtual screening identifying hydrazoindolin-2-one derivatives, active against RNase H and DNA polymerase functions of the RT, in the low micromolar range . This scCompound RMNC6 was synthesized and characterized as reported in E. coli strain M15 containing the p6HRT-prot vector was grown to an optical density at 600 nm of 0.7 and induced with 1.7 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 4 h. Protein purification was carried out with a BioLogic LP system (Biorad), using a combination of immobilized metal affinity and ion exchange chromatography. Cell pellets were resuspended in lysis buffer , incubated on ice for 20 min, and after adding NaCl to a final concentration of 0.3 M, were sonicated and centrifuged at 30,000×g for 1 h. The supernatant was loaded onto a Ni2+-NTA-Sepharose column pre-equilibrated with loading buffer  and washed thoroughly with wash buffer . RT was eluted with an imidazole gradient in wash buffer (0–0.5 M). Fractions were collected, protein purity was checked by SDS-PAGE and found to be higher than 90%. The 1:1 ration between the p66/p51 subunits was also verified. Enzyme-containing fractions were pooled and diluted 1:1 with 50 mM sodium phosphate buffer pH 7.0, containing 10% glycerol; and then loaded into a Hi-trap heparin HP GE  pre-equilibrated with 10 column volumes of loading buffer . The column was then washed with loading buffer and the RT was eluted with Elute Buffer 2 . Fractions were collected, protein was dialyzed and stored in buffer containing 50 mM Tris HCl pH 7.0, 25 mM NaCl, 1 mM EDTA, and 50% glycerol. Catalytic activities and protein concentrations were determined. Enzyme-containing fractions were pooled and aliquots were stored at −80°C.Heterodimeric RT was expressed essentially as described . BrieflyRecombinant HIV-1 group O RT was expressed and purified as previously described , 27. It Amino acid substitutions were introduced into the p66 HIV-1 RT subunit coded in a p6HRT-prot plasmid using the QuikChange mutagenesis kit by following the manufacturer’s instructions .2, 1 mM dithiothreitol (DTT), 80 mM KCl, 0.25 μM hybrid RNA/DNA 5'-GAUCUGAGCCUGGGAGCU-Fluorescin-3'  (available from Metabion) 5'-Dabcyl-AGCTCCCAGGCTCAGATC-3', increasing concentrations of inhibitors, whose dilution were made in water, and different amount of enzymes according to a linear range of dose-response curve: 20 ng of WT RT; 60 ng K103N RT; 37.5 ng V106A RT; 75 ng V108A RT; 5 ng Y181C RT; 50 ng Y188A RT; 30 ng Y188L RT; 100 ng E224A RT; 37.5 ng P225A RT; 20 ng P226A RT; 18 ng F227A RT; 30 ng L228A RT; 30 ng W229A RT; 10 ng M230A RT; 30 ng G231A RT; 62.5 ng N474A RT; 1 μg Y501A RT; 100 ng A502F RT; 37.5 ng A508V RT; 38 ng HIV-1 group O RT. The reaction mixture was incubated for 1 h at 37°C, stopped by addition of EDTA and products were measured with a multilabel counter plate reader Victor 3 (Perkin Elmer model 1420–051) equipped with filters for 490/528 nm (excitation/emission wavelength)HIV RT-associated RNase H activity was measured as described  in 100 μ2, 60 mM KCl, 13 mM DTT, 2.5 μM poly(A)-oligo(dT), 100 μM dTTP, increasing concentrations of inhibitors, whose dilution were made in water, and different amounts of enzymes according to a linear range of dose-response curve: 6 ng wt RT; 30 ng K103N RT; 12 ng V106ART; 19 ng V108A RT; 1.51.5 ng Y181C RT; 45 ng Y188A RT; 15 ng Y188L RT; 30 ng E224A RT; 15 ng P225A RT; 18 ng P226A RT; 23 ng F227A RT; 15 ng L228A RT; 30 ng W229A RT; 30 ng M230A RT; 15 ng G231A RT; 15 ng N474A RT; 15 ng Y501A RT; 15 ng A502F RT; 19 ng A508V RT; 38 ng HIV-1 group O RT. After enzyme addition, the reaction mixture was incubated for 30 min at 37°C and the stopped by addition of EDTA. Reaction products were detected by picogreen addition and measured with a multilabel counter plate reader Victor 3 (Perkin Elmer model 1420–051) equipped with filters for 502/523 nm (excitation/emission wavelength). The Yonetani-Theorell analysis was performed as described previously [RNA-dependent DNA polymerase (RDDP) activity was measured as described  in 25 μL2, 1 mM DTT, 80 mM KCl, fix amount of enzymes according to a linear range of dose-response curve: 20 ng of WT RT; 37.5 ng V106A RT; 75 ng V108A RT; 100 ng E224A RT; 100 RT ng A502F RT; 37.5 ng A508V RT, and increasing concentrations (from 0 to 500 nM) of hybrid RNA/DNA 5'-GAUCUGAGCCUGGGAGCU-Fluorescin-3'/ 5'-Dabcyl-AGCTCCCAGGCTCAGATC-3'. The reaction mixture was incubated for 1 h at 37°C, stopped by addition of EDTA and products were measured with a multilabel counter plate reader Victor 3 (Perkin Elmer model 1420–051) equipped with filters for 490/528 nm (excitation/emission wavelength). Results were represented according to Lineaweaver—Burke plot with the Sigmaplot10 software. Velocity (ν) was expressed as fmoles/min.Kinetic analysis of the DNA-polymerase independent RNase H activity was performed as already reported . Briefly2, 100 mM NaCl, 300 nM of HIV-1 RT and a 1:1000 dilution of Sypro Orange dye (Invitrogen). The mixture was heated from 30 to 90°C in increments of 0.2°C. Fluorescence intensity was measured using excitation and emission wavelengths of 483 and 568 nm, respectively. Changes in protein thermal stability (ΔTm) upon inhibitor binding were analyzed by using the LightCycler 480 software. All assays were performed in triplicate.Thermal stability assays were performed according to Nettleship et al. . In a Li-1.Theoretical 3D model of RMNC6 was built with the Maestro software . The inhibitor structure was optimized by energy minimization carried out using the MMFFs force field , the GB/The coordinates for RTs were taken from the RCSB Protein Data Bank   docking algorithm was applied for scoring theoretical poses. The other settings were left as default. The same protocol was applied for all the simulations.Molecular docking studies were performed using QMPL workflow protocol  (Schroed1. The binding free energies (ΔG(Bind)) were computed applying molecular mechanics and continuum solvation models with the molecular mechanics generalized Born/surface area (MM-GBSA) method [Ten thousand steps of the Polak-Ribier conjugate gradient (PRCG) minimization method were conducted on the top ranked theoretical complexes using OPLS_2005 force field. The optimization process was performed up to the derivative convergence criterion equal to 0.01 kJ/(mol*Å)) method .50 values of 1.4 and 9.8 μM, respectively.We synthetized a new derivative (Z)-4-(2-(2-(2-oxoindolin-3-ylidene)hydrazinyl)thiazol-4-yl)benzonitrile (RMNC6)  that, wh2, excluding the involvement of chelation in the mechanism of action (data not shown). RNase H inhibitors such as vynologous ureas (VUs) are known to destabilize the RT heterodimer by binding to an allosteric pocket in the RNase H domain at the interface between p66 and p51 subunits [2+, while the interface inhibitor VU, that has been shown to destabilize the HIV-1 RT [Since the mode of action of previously identified dual RT inhibitors was not determined, we decided to investigate the mechanism of RT inhibition by RMNC6 in comparison with available RNase H inhibitors and NNRTIs. Since many RNase H inhibitors act as chelators of cations needed in the active site (e.g. diketo acid derivatives ), we firsubunits . Therefosubunits  in the psubunits . In agresubunits , 50, theHIV-1 RT  decrease50 values in the range 5.4–20.1 μM). In contrast, HIV-1 group O RT and mutants K103N and Y188L showed decreased susceptibility to EFV   . These o50 values were 5 to 6.5 times higher than those obtained with the prototypic wild-type HIV-1 group M subtype B RT. These effects could be attributed to differences that affect the overall structure of the RNase H domain in both enzymes. Previous studies showed that HIV-2 RT was about 3.7 times less susceptible to BTP than the HIV-1 enzyme [Interestingly, the HIV-1 group O RT was also inhibited by BTP and RMNC6 in RNase H activity assays, although IC1 enzyme , while t1 enzyme . HoweverTo achieve further insights into the RMNC6 binding mode, we performed blind docking studies on the wt HIV-1 group M subtype B RT heterodimer and RMNC6, by using the QM-polarized ligand docking protocol  and usinDue to the flexibility of the target and different shapes of known inhibitors , we deciThe first and most energetically favored binding site (pocket 1)  is locatThe second putative binding pocket (pocket 2) is located in the RNase H domain, between the RNase H active site and the primer grip region, close to the p66/p51 interface. Docking modeling suggests that, in this site, RMNC6 could be partially sandwiched between different secondary structural units, such as namely β21 strand and the αH helix in p51, and the αB helix in p66 (residues 500–508) .in silico analysis supports alternative modes of action for RMNC6, since the drug could bind (i) preferentially only to one of the two pockets and have both short- and long-range effects; or (ii) to both sites and act by short-range effects on the two functions. According to the first mode of action, RMNC6 would inhibit RDDP and RNase H activities through binding into the sole pocket 1, by having a short-range inhibitory effect on RDDP activity and a long-range inhibitory effect on RNase H activity. Hence, it would act differently from classical NNRTIs such as nevirapine and EFV that were shown to destabilize the 3’-end of the DNA primer in the DNA polymerase active site and promote RT-mediated polymerase-independent RNase H cleavages [Overall, such leavages . Alterna50 values 4 and 10 times higher, respectively, in comparison with the wt RT. In contrast, we did not observed major differences in the IC50 values obtained with other mutant RTs . Hence, the results of the assays with the mutant RTs did not provide incontrovertible evidence of RMNC6 binding. However, the results could not exclude the RMNC6 binding to pocket 1. In fact, given the amplitude of the pocket and the high RT flexibility, the very limited IC50 reduction towards RDDP function observed could be determined by a different orientation of RMNC6 while accommodating in pocket 1, as a response to the introduced amino acid changes. It is worth noting that the kinetic analysis of the interaction between RMNC6 and EFV, showed a negative interference between the two compounds, even if they are not kinetically mutually exclusive, supporting the possibility that RMNC6 could accommodate in the large pocket 1 even in the presence of EFV.When the RDDP function of mutant RTs was assayed, a similar pattern emerged, even though the extent of the reduction in the IC50 value 4.5 times higher than the one obtained with the wt RT.Pocket 2, located in the RNase H domain, was already predicted  and inveNoteworthy, these amino acidic substitutions did not affect RMNC6 susceptibility in RDDP assays. These results strongly support the hypothesis that RMNC6 interacts with pocket 2 and that this binding is responsible for short-range inhibition of the RNase H function. The data are also consistent with the possibility of RMNC6 binding to both pockets 1 and 2 and acting by two different short-range effects. In fact, in these mutant RTs, RMNC6 binding to pocket 1 could be responsible for the inhibition of the RDDP activity, while RMNC6 binding to pocket 2 could be impeded. The amino acid sequence alignment of HIV-1 RTs of group O and group M subtype B , 56 revekcat values with respect to wt RT for all mutant RTs, and a slight reduction in template-primer Km values for A508V, V106A, V108A and E224A RTs, resulting in a reduced efficiency (kcat/Km) for V108A, E224A and A502F RTs   . OverallOur study demonstrates that RMNC6 is a novel dual function RT inhibitor that has an allosteric binding mode, different from that shown by known NNRTIs. Our results support the hypothesis that RMNC6 binds two different sites in HIV-1 RT. This double-site binding mode seems to confirm that it could be possible to target both RT-associated functions by a single molecule, retaining full potency of inhibition on drug-resistant mutant RTs such as K103N, Y181C and Y188L. RNase H inhibition by RMNC6 seems to be mainly due to its interaction with pocket 2, close to the RNase H active site. The long-range effects on RNase H inhibition produced by RMNC6, and observed when amino acids in the polymerase domain were mutated, are probably due to an alteration of the substrate binding pocket in the RNase H domain that, in turn, alters RMNC6 binding or efficacy. On the contrary, HIV-1 RT-associated RDDP inhibition by RMNC6 seems to be related to its binding in the polymerase domain. However, the size of the pocket and the plasticity of the enzyme may be responsible for the compound retained RDDP inhibition on mutant RTs. Overall, dual RT-associated functions inhibition by compounds binding to two sites is an appealing possibility to strongly reduce drug resistance occurrence. Furthermore, since pocket 2 is particular for HIV RT, compounds that target this site will not interfere with human RNase H1. These results prompt us to undergo further studies to better define the drug interactions within these two pockets in order to perform a rational drug design of dual inhibitors acting on both binding sites.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.


To explore the effects of postmastectomy radiotherapy (PMRT) on the locoregional failure-free survival (LRFFS) and overall survival (OS) of breast cancer patients under different tumor stages and with one to three positive axillary lymph nodes (ALNs).1-T2 N1 and T3-T4 N1 groups. The effects of PMRT on the LRFFS and OS of these two patient groups were analyzed using SPSS 19.0, Pearson’s χ2-test, Kaplan-Meier method, and Cox proportional hazard model.We conducted a retrospective review of 527 patients with one to three positive lymph nodes who underwent modified radical or partial mastectomy and axillary dissection from January 2000 to December 2002. The patients were divided into the T1-T2 N1 patients, no statistical significance was observed in the effects of PMRT on LRFFS  and OS  of the general patients. Extracapsular extension (ECE) and high histological grade were the risk factors for LRFFS and OS with statistical significance in multivariate analysis. Stratification analysis showed that PMRT statistically improved the clinical outcomes in high-risk patients  but not in low-risk patients . For T3-T4 N1 patients, PMRT effectively improved the local control  of the general patients, whereas no statistical effect was observed on OS . Absence of estrogen receptors and progesterone receptors (ER/PR) (–) was an independent risk factor. Further stratification analysis indicated a statistical difference in LRFFS and OS between the high-risk patients with ER/PR (–) receiving PMRT and not receiving PMRT . However, PMRT had a beneficial effect on the reduction of locoregional recurrence (LRR) but not in total mortality  in T3-T4 N1 patients with ER/PR (+) who received endocrine therapy.For T1-T2 N1 patients. T3-T4 N1 patients with ER/PR (–) could benefit from PMRT by improving LRFFS and OS. However, PMRT could only reduce LRR but failed to improve OS for T3-T4 N1 patients with ER/PR (+) who received endocrine therapy.PMRT could reduce ECE (+), histological grade III-related LRR, and total mortality of T Given that several clinical and pathological factors may affect prognosis of patients with intermediate-risk breast cancer, using T/N classification only is an imprecise method in determining whether a patient should be considered for PMRT--,-According to the National Comprehensive Cancer Network (NCCN) guidelines1-T2 N1 and T3-T4 N1 breast cancer patients. In addition, we compared the locoregional failure-free survival (LRFFS) and overall survival (OS) of the high-risk patients with and without PMRT to define a subgroup of patients who might benefit from PMRT.The function of PMRT is not clearly defined in breast cancer patients with one to three positive lymph nodes. In this retrospective study, we identified prognostic factors for LRR and mortality of T1-T2 disease and 95 patients with T3-T4 disease were included in the study, 75.7% (327/432) and 70.5% (67/95) of whom received PMRT, respectively. The study was approved by the institutional ethics committee.From January 2000 to December 2002, breast cancer patients with pathologically proven one to three positive axillary lymph nodes (ALNs) were treated with modified radical mastectomy plus axillary dissection at the Tianjin Cancer Hospital. Of the 527 patients with one to three positive lymph nodes, the median age was 48.73 years . The median number of involved ALNs was 1.93 . A total of 432 patients with TAll patients received TEC-based  or docetaxel-containing regimens as adjuvant chemotherapy. Adjuvant endocrine therapy was performed for 5 years in all patients who had positive hormone receptors. Among 527 patients, 74.8% (394/527) underwent PMRT, which was delivered to the breast, chest wall, internal mammary, supraclavicular, and axillary fossa drawing region by medial and lateral-tangential fields with external-beam irradiation (4 or 6 MV photons/60 Co). The standard dose to the entire chest wall was 50 Gy , 1.8 to 2 Gy/d, and five times weekly. The supraclavicular region and the full axilla were treated with a dose of 50 Gy using an anterior field. An additional external boost with electrons (2 Gy/10 Gy to 14 Gy) was performed in patients who had locally advanced disease.The median time of follow-up was 127.82 months . All intervals were calculated from the date of completion of surgery, and the endpoint was defined as the last follow-up or death. Evaluation of tumor control was performed for patients in 4-month intervals for the first 2 years and in 6-month intervals for the next 3 years. Subsequently, these patients were observed on a yearly basis. Clinical examinations, which included blood sampling, routine chest radiograph, mammograph, and ultrasound, were performed as evaluation during the follow-up. Further evaluations were conducted only if the clinical findings indicated a disease progression. Survival period was calculated from the date of surgical resection to the date of last follow-up. The endpoints of interest included LRFFS and OS.LRR was identified as local recurrence  or regional recurrence . Any recurrence outside these areas was defined as distant metastasis (DM).2-test was used to compare the proportions of categorical covariates among the groups of patients with different T stages. OS and LRFFS were analyzed with Kaplan-Meier method. Univariate and multivariate hazard ratios (HR) and their 95% confidence intervals (CIs) were calculated using Cox’s proportional hazard model. A probability level of ≤0.05 was considered statistically significant.All analyses were performed using SPSS 19.0. Pearson’s χ1-T2 N1 and T3-T4 N1 patient groups, respectively. OS was 93.5% (404/432) and 45.3% (43/95) in the T1-T2 N1 and T3-T4 N1 groups, respectively. The Kaplan-Meier curves of LRFFS and OS in different T stages confirmed the statistically significant difference in LRFFS and OS between the T1-T2 N1 and T3-T4 N1 patients (P<0.05).With a median follow up of 127.82 months , 3.7% (16/432) and 14.7% (14/95) of patients developed LRR in T1-T2 and T3-T4 patients were different. ECE  and histological grade III  were the risk factors in T1-T2 patients. For the T3-T4 patients, the risk factor was estrogen receptor and progesterone receptor (ER/PR) (–) tumors, whereas the protective factors were ER/PR (+) tumors  and PMRT   and histological grade III  were the risk factors in T1-T2 patients. However, the risk factor in T3-T4 patients was ER/PR (–) tumors. ER/PR (+) tumors  had a significant effect in improving OS ( and The factors affecting OS varied between the T1-T2 N1 patient subgroups: ECE (–) /PMRT (–), ECE (–) /PMRT (+), ECE (+) /PMRT (–), and ECE (+) /PMRT (+). The log-rank test results showed that PMRT had statistically positive effects on improving LRFFS (P=0.026) and OS (P=0.007) of T1-T2 N1 patients with ECE (+) but not ECE (–). We also performed a subgroup analysis according to the histological grade, and the results showed that PMRT could improve the LRFFS (P<0.001) and OS (P=0.007) of T1-T2 N1 patients with histological grade III  was a statistically significant factor on multivariate analysis. PMRT was beneficial on LRFFS of all patients regardless of the hormone receptor status. The effects of PMRT on LRFFS and OS of the patients with different ER/PR statuses were examined. All T3-T4 N1 patients were first stratified into subgroups of ER/PR (+) and ER/PR (–). We observed that PMRT was useful for the reduction of LRR (P<0.001) of T3-T4 N1 patients with ER/PR (+) but failed to improve OS (P=0.695). However, patients with ER/PR (–) could benefit from PMRT on improving LRFFS (P=0.046) and OS (P=0.039)  and a greater number of risk factors, such as pathological grade III and ECE. However, Geng et al.et al.1-T2 N1 breast cancer patients. However, PMRT might be beneficial in a subgroup of patients with histological grade III disease, ECE, or triple-negative subtype. PMRT is important in identifying the risk factors associated with increased risk of LRR and total mortality in patients with one to three positive axillary lymph nodes to establish its indications.Huang 1-T2 breast cancer and one to three positive lymph nodes. However, PMRT has been considered for T1-T2 N1 patients based on the NCCN guidelinesAccording to the American Society of Clinical Oncology1-T2 N1 and T3-T4 N1 patients. Previous studies have reported,1-T2 N1 breast cancer patients treated with radical mastectomy are dependent on several prognostic factors other than T and N stage. Our analysis revealed that ECE (+) and histological grade III were the high-risk factors for LRR and mortality of T1-T2 N1 patients. The stratification analysis results revealed that PMRT had a positive effect in reducing ECE (+) or histological grade III-related LRR and mortality. However, the remaining patients with ECE (–) or histological grade I-II experienced extremely low LRR and mortality rates after mastectomy treatment, and the benefit from PMRT was minimal. Although PMRT had no protective function in improving LRFFS and OS of the general T1-T2 N1 patients, high-risk patients with ECE (+) and histological grade III could benefit from PMRT.Based on our study, different effects of PMRT on improving LRFFS or OS were found between the T1-T2 N1 patients, the general T3-T4 N1 patients could benefit from PMRT in terms of LRFFS but not in OS. Stage T3-T4 is a high-risk factor in breast cancer patients, who are more likely to develop DM than patients with early T stage disease. Breast cancer tends to be a systemic disease with potential sub-clinical DM in Fisher’s theory3-T4 patients, but no statistically significant effect on OS was observed among these patients. In addition, patients with ER/PR (+) benefited from endocrine therapy. All patients with ER/PR (+) who were included in our study received endocrine therapy. Endocrine therapy was a protective factor to improve LRFFS and OS of T3-T4 N1 patients according to the multivariate analysis results. Thus, the risks of LRR and mortality were positively associated with ER/PR (–). NCCN guidelines3-T4 patients should receive PMRT. Rangan et al.3-T4 N1 patients with ER/PR (–) and who received endocrine therapy, respectively. The results of stratification analysis indicated that PMRT caused a statistically significant improvement in LRFFS and OS of T3-T4 N1 patients with ER/PR (–). For T3-T4 N1 patients who received endocrine therapy, PMRT could improve local control but no statistical change in OS was observed compared with non-PMRT.Contrary to Tet al.3-T4 N1 patients receiving endocrine therapy should be reconsidered because no statistical effect on OS was observed in this study despite the improvement in local control.PMRT alleviates local symptoms but often results in significant pathological damage to the heart, lungs, and skin. A meta-analysis by Taghian 1-T2 N1 patients with ECE (+) or pathological grade III as well as for T3-T4 N1 patients with ER/PR (–). However, PMRT has to be reconsidered for T3-T4 N1 patients with ER/PR (+) who benefited from endocrine therapy on improving LRFFS and OS. Other prognostic factors should be considered, and the decision has to be made individually on the basis of endocrine therapy and request of the patient because PMRT could control LRR but not total mortality.According to our results, PMRT is highly recommended to improve LRFFS and OS for T