Project description:The specificity of protein-protein interactions in cellular signaling cascades is dependent on the sequence and intramolecular location of distinct amino acid motifs. We used the two-hybrid interaction trap to identify proteins that can associate with the PDZ motif-rich segment in the protein tyrosine phosphatase PTP-BL. A specific interaction was found with the Lin-11, Isl-1, Mec-3 (LIM) domain containing protein RIL. More detailed analysis demonstrated that the binding specificity resides in the second and fourth PDZ motif of PTP-BL and the LIM domain in RIL. Immunohistochemistry on various mouse tissues revealed a submembranous colocalization of PTP-BL and RIL in epithelial cells. Remarkably, there is also an N-terminal PDZ motif in RIL itself that can bind to the RIL-LIM domain. We demonstrate here that the RIL-LIM domain can be phosphorylated on tyrosine in vitro and in vivo and can be dephosphorylated in vitro by the PTPase domain of PTP-BL. Our data point to the presence of a double PDZ-binding interface on the RIL-LIM domain and suggest tyrosine phosphorylation as a regulatory mechanism for LIM-PDZ associations in the assembly of multiprotein complexes. These findings are in line with an important role of PDZ-mediated interactions in the shaping and organization of submembranous microenvironments of polarized cells.
Project description:We have used the RIL-seq technique to map the global RNA-RNA interactome on the sRNA chaperone Hfq in Salmonella enterica. Overall design: RIL-seq (Melamed et al. 2016) with Hfq in Salmonella
Project description:Most E. coli sRNAs interact with their mRNA targets through simultaneous binding to the Hfq chaperon. In this experiment we cross-linked RNA to proteins in-vivo then did Hfq IP followed by ligation of bound RNAs and sequencing to identify sRNA-mRNA interactions. We termed the method RIL-seq for RNA Interactions by Ligation - sequencing.
Project description:IL-10 has been classically defined as a broad-spectrum immunosuppressant and is thought to facilitate the development of regulatory CD4(+) T cells. IL-10 is believed to represent one of the major suppressive factors secreted by IDO(+)FoxP3(+)CD4(+) Tregs. Contrary to this view, we have previously reported that PEGylated recombinant IL-10 (PEG-rIL-10) treatment of mice induces potent IFN? and CD8(+) T-cell-dependent antitumor immunity. This hypothesis is currently being tested in clinical trials and we have reported that treatment of cancer patients with PEG-rHuIL-10 results in inhibition and regression of tumor growth as well as increased serum IFN?. We have continued to assess PEG-rIL-10's pleiotropic effects and report that treatment of tumor-bearing mice and humans with PEG-rIL-10 increases intratumoral indoleamine 2, 3-dioxygenase (IDO) in an IFN?-dependent manner. This should result in an increase in Tregs, but paradoxically our data illustrate that PEG-rIL-10 treatment of mice reduces intratumoral FoxP3(+)CD4(+) T cells in an IDO-independent manner. Additional investigation indicates that PEG-rIL-10 inhibits TGF?/IL-2-dependent in vitro polarization of FoxP3(+)CD4(+) Tregs and potentiates IFN?(+)T-bet(+)CD4(+) T cells. These data suggest that rather than acting as an immunosuppressant, PEG-rIL-10 may counteract the FoxP3(+)CD4(+) Treg suppressive milieu in tumor-bearing mice and humans, thereby further facilitating PEG-rIL-10's potent antitumor immunity.
Project description:F2 and recombinant inbred lines (RILs) populations are very commonly used in plant genetic mapping studies. Although genome-wide genetic markers like single nucleotide polymorphisms (SNPs) can be readily identified by a wide array of methods, accurate genotype calling remains challenging, especially for heterozygous loci and missing data due to low sequencing coverage per individual. Therefore, we developed Genotype-Corrector, a program that corrects genotype calls and imputes missing data to improve the accuracy of genetic mapping. Genotype-Corrector can be applied in a wide variety of genetic mapping studies that are based on low coverage whole genome sequencing (WGS) or Genotyping-by-Sequencing (GBS) related techniques. Our results show that Genotype-Corrector achieves high accuracy when applied to both synthetic and real genotype data. Compared with using raw or only imputed genotype calls, the linkage groups built by corrected genotype data show much less noise and significant distortions can be corrected. Additionally, Genotype-Corrector compares favorably to the popular imputation software LinkImpute and Beagle in both F2 and RIL populations. Genotype-Corrector is publicly available on GitHub at https://github.com/freemao/Genotype-Corrector .
Project description:Maize rough dwarf disease (MRDD) is a significant viral disease caused by rice black-streaked dwarf virus (RBSDV) in China, which results in 30% yield losses in affected summer maize-growing areas. In this study, two connected recombinant inbred line (RIL) populations were constructed to elucidate the genetic basis of resistance during two crop seasons. Ten quantitative trait loci (QTLs) for resistance to MRDD were detected in the two RILs. Individual QTLs accounted for 4.97-23.37% of the phenotypic variance explained (PVE). The resistance QTL (qZD-MRDD8-1) with the largest effect was located in chromosome bin 8.03, representing 16.27-23.37% of the PVE across two environments. Interestingly, one pair of common significant QTLs was located in the similar region on chromosome 4 in both populations, accounting for 7.11-9.01% of the PVE in Zheng58×D863F (RIL-ZD) and 9.43-13.06% in Zheng58×ZS301 (RIL-ZZ). A total of five QTLs for MRDD resistance trait showed significant QTL-by-Environment interactions (QEI). Two candidate genes associated with resistance (GDSL-lipase and RPP13-like gene) which were higher expressed in resistant inbred line D863F than in susceptible inbred line Zheng58, were located in the physical intervals of the major QTLs on chromosomes 4 and 8, respectively. The identified QTLs will be studied further for application in marker-assisted breeding in maize genetic improvement of MRDD resistance.
Project description:A retrospective analysis was conducted on data from four open-label, nonrandomised, phase II trials of recombinant interleukin-2 (rIL-2) in patients with metastatic renal cell carcinoma to compare the safety and efficacy of administration by subcutaneous (s.c.) and continuous intravenous (c.i.v.) infusion (n=103 s.c. and n=225 c.i.v.). No statistically significant differences were found between the cohorts in terms of overall response rate (s.c.: 13.6% vs c.i.v.: 12.4%, P=0.77), response duration (s.c.: 9.8 months vs c.i.v.: 10.1 months, P=0.99), and overall survival (P=0.08). Compared with c.i.v. administration, more patients in the s.c. cohort experienced stable disease (50.5 vs 29.8%) and fewer underwent disease progression (35.0 vs 43.6%). Subcutaneous administration was associated with a significantly lower incidence of grade 3 or 4 adverse events (46 vs 76%; P<0.001), and fewer s.c. patients required dose reductions because of toxicity (20 vs 82%). At the doses and within the schedules tested, this comparative analysis did not detect any difference in efficacy between s.c. and c.i.v. administration of rIL-2 in terms of overall survival, duration of response and response rate in patients with metastatic renal cell carcinoma. However, s.c. delivery of rIL-2 was associated with improved tolerability.
Project description:Aluminum (Al3+) toxicity is a typical abiotic stress that severely limits crop production in acidic soils. In this study, an RIL (recombinant inbred line, F12) population derived from the cross of Zhonghuang 24 (ZH 24) and Huaxia 3 (HX 3) (160 lines) was tested using hydroponic cultivation. Relative root elongation (RRE) and apical Al3+ content (AAC) were evaluated for each line, and a significant negative correlation was detected between the two indicators. Based on a high-density genetic linkage map, the phenotypic data were used to identify quantitative trait loci (QTLs) associated with these traits. With composite interval mapping (CIM) of the linkage map, five QTLs that explained 39.65% of RRE and AAC variation were detected on chromosomes (Chrs) Gm04, Gm16, Gm17 and Gm19. Two new QTLs, qRRE_04 and qAAC_04, were located on the same region of bin93-bin94 on Chr Gm04, which explained 7.09% and 8.98% phenotypic variation, respectively. Furthermore, the results of the expression analysis of candidate genes in the five genetic regions of the QTLs showed that six genes (Glyma.04g218700, Glyma.04g212800, Glyma.04g213300, Glyma.04g217400, Glyma.04g216100 and Glyma.04g220600) exhibited significant differential expression between the Al3+ treatment and the control of two parents. The results of qRT-PCR analysis indicated that Glyma.04g218700 was upregulated by Al3+ treatment with the hundreds-fold increased expression level and may be a candidate gene with potential roles in the response to aluminum stress. Therefore, our efforts will enable future functional analysis of candidate genes and will contribute to the strategies for improvement of aluminum tolerance in soybean.
Project description:<h4>Background</h4>Different soybean (Glycine max L.) leaf chlorophyll-content traits are considered to be significantly linked to soybean yield. To map the quantitative trait loci (QTLs) of soybean leaf chlorophyll-content traits, an advanced recombinant inbred line (RIL, ZH, Zhonghuang 24?×?Huaxia 3) population was adopted to phenotypic data acquisitions for the target traits across six distinct environments (seasons and soybean growth stages). Moreover, the restriction site-associated DNA sequencing (RAD-seq) based high-density genetic linkage map of the RIL population was utilized for QTL mapping by carrying out the composite interval mapping (CIM) approach.<h4>Results</h4>Correlation analyses showed that most traits were correlated with each other under specific chlorophyll assessing method and were regulated both by hereditary and environmental factors. In this study, 78 QTLs for soybean leaf chlorophyll-content traits were identified. Furthermore, 13 major QTLs and five important QTL hotspots were classified and highlighted from the detected QTLs. Finally, Glyma01g15506, Glyma02g08910, Glyma02g11110, Glyma07g15960, Glyma15g19670 and Glyma15g19810 were predicted from the genetic intervals of the major QTLs and important QTL hotspots.<h4>Conclusions</h4>The detected QTLs and candidate genes may facilitate to gain a better understanding of the hereditary basis of soybean leaf chlorophyll-content traits and may be valuable to pave the way for the marker-assisted selection (MAS) breeding of the target traits.
Project description:Plant growth and development are tightly linked to primary metabolism and are subject to natural variation. In order to obtain an insight into the genetic factors controlling biomass and primary metabolism and to determine their relationships, two Arabidopsis thaliana populations [429 recombinant inbred lines (RIL) and 97 introgression lines (IL), derived from accessions Col-0 and C24] were analyzed with respect to biomass and metabolic composition using a mass spectrometry-based metabolic profiling approach. Six and 157 quantitative trait loci (QTL) were identified for biomass and metabolic content, respectively. Two biomass QTL coincide with significantly more metabolic QTL (mQTL) than statistically expected, supporting the notion that the metabolic profile and biomass accumulation of a plant are linked. On the same basis, three out the six biomass QTL can be simulated purely on the basis of metabolic composition. QTL based on analysis of the introgression lines were in substantial agreement with the RIL-based results: five of six biomass QTL and 55% of the mQTL found in the RIL population were also found in the IL population at a significance level of P < or = 0.05, with >80% agreement on the allele effects. Some of the differences could be attributed to epistatic interactions. Depending on the search conditions, metabolic pathway-derived candidate genes were found for 24-67% of all tested mQTL in the database AraCyc 3.5. This dataset thus provides a comprehensive basis for the detection of functionally relevant variation in known genes with metabolic function and for identification of genes with hitherto unknown roles in the control of metabolism.