BackgroundHuman primary myeloma (MM) cells do not survive in culture; current in vitro and in vivo systems for growing these cells are limited to coculture with a specific bone marrow (BM) cell type or growth in an immunodeficient animal model. The purpose of the study is to establish an interactive healthy donor whole BM based culture system capable of maintaining prolonged survival of primary MM cells. This normal BM (NBM) coculture system is different from using autologous BM that is already affected by the disease.MethodsWhole BM from healthy donors was cultured in medium supplemented with BM serum from MM patients for 7 days, followed by 7 days of coculture with CD138-selected primary MM cells or MM cell lines. MM cells in the coculture were quantified using flow cytometry or bioluminescence of luciferase-expressing MM cells. T-cell cytokine array and proteomics were performed to identify secreted factors.ResultsNBM is composed of adherent and nonadherent compartments containing typical hematopoietic and mesenchymal cells. MM cells, or a subset of MM cells, from all examined cases survived and grew in this system, regardless of the MM cells’ molecular risk or subtype, and growth was comparable to coculture with individual stromal cell types. Adherent and nonadherent compartments supported MM growth, and this support required patient serum for optimal growth. Increased levels of MM growth factors IL-6 and IL-10 along with MM clinical markers B2M and LDHA were detected in supernatants from the NBM coculture than from the BM cultured alone. Levels of extracellular matrix factors (e.g., MMP1, HMCN1, COL3A1, ACAN) and immunomodulatory factors (e.g., IFI16, LILRB4, PTPN6, AZGP1) were changed in the coculture system. The NBM system protected MM cells from dexamethasone but not bortezomib, and effects of lenalidomide varied.ConclusionsThe NBM system demonstrates the ability of primary MM plasma cells to interact with and to survive in coculture with healthy adult BM. This model is suitable for studying MM-microenvironment interactions, particularly at the early stage of engagement in new BM niches, and for characterizing MM cell subpopulations capable of long-term survival through secretion of extracellular matrix and immune-related factors.

BackgroundHuman primary myeloma (MM) cells do not survive in culture; current in vitro and in vivo systems for growing these cells are limited to coculture with a specific bone marrow (BM) cell type or growth in an immunodeficient animal model. The purpose of the study is to establish an interactive healthy donor whole BM based culture system capable of maintaining prolonged survival of primary MM cells. This normal BM (NBM) coculture system is different from using autologous BM that is already affected by the disease.MethodsWhole BM from healthy donors was cultured in medium supplemented with BM serum from MM patients for 7 days, followed by 7 days of coculture with CD138-selected primary MM cells or MM cell lines. MM cells in the coculture were quantified using flow cytometry or bioluminescence of luciferase-expressing MM cells. T-cell cytokine array and proteomics were performed to identify secreted factors.ResultsNBM is composed of adherent and nonadherent compartments containing typical hematopoietic and mesenchymal cells. MM cells, or a subset of MM cells, from all examined cases survived and grew in this system, regardless of the MM cells’ molecular risk or subtype, and growth was comparable to coculture with individual stromal cell types. Adherent and nonadherent compartments supported MM growth, and this support required patient serum for optimal growth. Increased levels of MM growth factors IL-6 and IL-10 along with MM clinical markers B2M and LDHA were detected in supernatants from the NBM coculture than from the BM cultured alone. Levels of extracellular matrix factors (e.g., MMP1, HMCN1, COL3A1, ACAN) and immunomodulatory factors (e.g., IFI16, LILRB4, PTPN6, AZGP1) were changed in the coculture system. The NBM system protected MM cells from dexamethasone but not bortezomib, and effects of lenalidomide varied.ConclusionsThe NBM system demonstrates the ability of primary MM plasma cells to interact with and to survive in coculture with healthy adult BM. This model is suitable for studying MM-microenvironment interactions, particularly at the early stage of engagement in new BM niches, and for characterizing MM cell subpopulations capable of long-term survival through secretion of extracellular matrix and immune-related factors.

BackgroundExploring genetic differentiation and genomic variation is important for both the utilization of heterosis and the dissection of the genetic bases of complex traits.MethodsWe integrated 1857 diverse maize accessions from America, Africa, Europe and Asia to investigatetheir genetic differentiation, genomic variation using 43,252 high-quality single-nucleotide polymorphisms(SNPs),combing GWAS and linkage analysis strategy to exploring the function of relevant genetic segments.ResultsWe uncovered many more subpopulations that recently or historically formed during the breeding process. These patterns are represented by the following lines: Mo17, GB, E28, Ye8112, HZS, Shen137, PHG39, B73, 207, A634, Oh43, Reid Yellow Dent, and the Tropical/subtropical (TS) germplasm. A total of 85 highly differentiated regions with a DEST of more than 0.2 were identified between the TS and temperate subpopulations. These regions comprised 79 % of the genetic variation, and most were significantly associated with adaptive traits. For example, the region containing the SNP tag PZE.108075114 was highly differentiated, and this region was significantly associated with flowering time (FT)-related traits, as supported by a genome-wide association study (GWAS) within the interval of FT-related quantitative trait loci (QTL). This region was also closely linked to zcn8 and vgt1, which were shown to be involved in maize adaptation. Most importantly, 197 highly differentiated regions between different subpopulation pairs were located within an FT- or plant architecture-related QTL.ConclusionsHere we reported that 700–1000 SNPs were necessary needed to robustly estimate the genetic differentiation of a naturally diverse panel. In addition, 13 subpopulations were observed in maize germplasm, 85 genetic regions with higher differentiation between TS and temperate maize germplasm, 197 highly differentiated regions between different subpopulation pairs, which contained some FT- related QTNs/QTLs/genes supported by GWAS and linkage analysis, and these regions were expected to play important roles in maize adaptation.

BackgroundStripe rust, a highly destructive foliar disease of wheat (Triticum aestivum), causes severe losses, which may be accompanied by reduced photosynthetic activity and accelerated leaf senescence.MethodsWe used suppression subtractive hybridization (SSH) to examine the mechanisms of resistance in the resistant wheat line L693 (Reg. No. GP-972, PI 672538), which was derived from a lineage that includes a wide cross between common and Thinopyrum intermedium. Sequencing of an SSH cDNA library identified 112 expressed sequence tags.ResultsIn silico mapping placed one of these tags [GenBank: JK972238] on chromosome 1A. Primers based on [GenBank: JK972238] amplified a polymorphic band, which co-segregated with YrL693. We cloned a candidate gene encoding wheat copper-binding protein (WCBP1) by amplifying the polymorphic region, and we mapped WCBP1 to a 0.64 cM genetic interval. Brachypodium, rice, and sorghum have genes and genomic regions syntenic to this region.DiscussionSequence analysis suggested that the resistant WCBP1 allele might have resulted from a deletion of 36-bp sequence of the wheat genomic sequence, rather than direct transfer from Th. intermedium. qRT-PCR confirmed that WCBP1 expression changes in response to pathogen infection.ConclusionsThe unique chromosomal location and expression mode of WCBP1 suggested that WCBP1 is the putative candidate gene of YrL693, which was involved in leaf senescence and photosynthesis related to plant responses to stripe rust infection during the grain-filling stage.