Functional specialization of duplicated AP3‐like genes in Medicago truncatula

The B–class of MADS box genes has been studied in a wide range of plant species, but has remained largely uncharacterized in legumes. Here we investigate the evolutionary fate of the duplicated AP3‐like genes of a legume species. To obtain insight into the extent to which B‐class MADS box gene functions are conserved or have diversified in legumes, we isolated and characterized the two members of the AP3 lineage in Medicago truncatula: MtNMH7 and MtTM6 (euAP3 and paleoAP3 genes, respectively). A non‐overlapping and complementary expression pattern of both genes was observed in petals and stamens. MtTM6 was expressed predominantly in the outer cell layers of both floral organs, and MtNMH7 in the inner cell layers of petals and stamens. Functional analyses by reverse genetics approaches (RNAi and Tnt1 mutagenesis) showed that the contribution of MtNMH7 to petal identity is more important than that of MtTM6, whereas MtTM6 plays a more important role in stamen identity than its paralog MtNMH7. Our results suggest that the M. truncatula AP3‐like genes have undergone a functional specialization process associated with complete partitioning of gene expression patterns of the ancestral gene lineage. We provide information regarding the similarities and differences in petal and stamen development among core eudicots.     

Weighted gene co‐expression network analysis can sort cancer‐associated fibroblast‐specific markers promoting bladder cancer progression

The role of cancer‐associated fibroblasts (CAFs) has been thoroughly investigated in tumour microenvironments but not in bladder urothelial carcinoma (BLCA). The cell fraction of CAFs gradually increased with BLCA progression. Weighted gene co‐expression network analysis (WGCNA) revealed a specific gene expression module of CAFs that are relevant to cancer progression and survival status. Fifteen key genes of the module were consistent with a fibroblast signature in single‐cell RNA sequencing, functionally related to the extracellular matrix, and significant in survival analysis and tumour staging. A comparison of the luminal‐infiltrated versus luminal‐papillary subtypes and fibroblast versus urothelial carcinoma cell lines and immunohistochemical data analysis demonstrated that the key genes were specifically expressed in CAFs. Moreover, these genes are highly correlated with previously reported CAF markers. In summary, CAFs play a major role in the progression of BLCA, and the 15 key genes act as BLCA‐specific CAF markers and can predict CAF changes. WGCNA can, therefore, be used to sort CAF‐specific gene set in cancer tissues.     

Tree dynamics and co‐existence in the montane–sub‐alpine ecotone: the role of different light‐induced strategies

Questions: Is light availability the main factor driving forest dynamics in Pyrenean sub‐alpine forests? Do pines and firs differ in growth, mortality and morphological response to low light availability? Can differences in shade tolerance affect predictions of future biome changes in Pyrenean sub‐alpine forests in the absence of thermal limitation? Location: Montane–sub‐alpine ecotones of the Eastern Pyrenees (NE Spain). Methods: We evaluated morphological plasticity, survival and growth response of saplings of Scots pine, mountain pine and silver fir to light availability in a mixed forest ecotone. For each species, we selected 100 living and 50 dead saplings and measured size, crown morphology and light availability. A wood disk at root collar was then removed for every sapling, and models relating growth and mortality to light were obtained. Results: Fir had the lowest mortality rate (<0.1) for any given light condition. Pines had comparable responses to light availability, although in deep shade Scots pine risked higher mortality (0.35) than mountain pine (0.19). Pines and fir developed opposing strategies to light deprivation: fir employed a conservative strategy based on sacrificing height growth, whereas pines enhanced height growth to escape from shade, but at the expense of higher mortality risk. Scots pine showed higher plasticity than mountain pine for all architectural and morphological traits analysed, having higher adaptive capacity to a changing environment. Conclusions: Our results support the prediction of future biome changes in Pyrenean sub‐alpine forests as silver fir and Scots pine may find appropriate conditions for colonizing mountain pine‐dominated stands due to land‐use change‐related forest densification and climate warming‐related temperature increases, respectively.     

Bisphosphonates induce the osteogenic gene expression in co‐cultured human endothelial and mesenchymal stem cells

Bisphosphonates (BPs) are known to affect bone homeostasis and also to have anti-angiogenic properties. Because of the intimate relationship between angiogenesis and osteogenesis, this study analysed the effects of Alendronate (AL) and Zoledronate (ZL) in the expression of endothelial and osteogenic genes on interacting endothelial and mesenchymal stem cells, an issue that was not previously addressed. Alendronate and ZL, 10⁻¹²–10⁻⁶ M, were evaluated in a direct co-culture system of human dermal microvascular endothelial cells (HDMEC) and human bone marrow mesenchymal stem cells (HMSC), over a period of 14 days. Experiments with the respective monocultures were run in parallel. Alendronate and ZL caused an initial dose-dependent stimulation in the cell proliferation in the monocultures and co-cultures, and did not interfere with their cellular organization. In HDMEC monocultures, the expression of the endothelial genes CD31, VE-cadherin and VEGFR2 was down-regulated by AL and ZL. In HMSC monocultures, the BPs inhibited VEGF expression, but up-regulated the expression of the osteogenic genes alkaline phosphatase (ALP), bone morphogenic protein-2 (BMP-2) and osteocalcin (OC) and, to a greater extent, osteoprotegerin (OPG), a negative regulator of the osteoclastic differentiation, and increased ALP activity. In co-cultured HDMEC/HMSC, AL and ZL decreased the expression of endothelial genes but elicited an earlier and sustained overexpression of ALP, BMP-2, OC and OPG, compared with the monocultured cells; they also induced ALP activity. This study showed for the first time that AL and ZL greatly induced the osteogenic gene expression on interacting endothelial and mesenchymal stem cells.     

Recombinant production of omega‐3 fatty acids in Escherichia coli using a gene cluster isolated from Shewanella baltica MAC1

Aim: To isolate eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) genes from Shewanella baltica MAC1 and to examine recombinant production of EPA and DHA in E. coli to investigate cost‐effective, sustainable and convenient alternative sources for fish oils. Methods and Results: A fosmid library was prepared from the genomic DNA of S. baltica MAC1 and was screened for EPA and DHA genes by colony hybridization using a partial fragment of the S. baltica MAC1 pfaA and pfaD genes as probes. Analysis of total fatty acids isolated from transgenic E. coli positive for pfaA and pfaD genes by gas chromatography and gas chromatography‐mass spectrometry indicated recombinant production of both EPA and DHA. Analysis of the complete nucleotide sequence for the isolated gene cluster showed 16 putative open reading frames (ORFs). Among those, four ORFs showed homology with pfaA, pfaB, pfaC and pfaD genes of the EPA and/or DHA biosynthesis gene clusters; however, the protein domains of these genes were different from other EPA/DHA biosynthesis genes. Conclusions: The EPA and DHA gene cluster was cloned successfully. The transgenic E. coli strain carrying the omega‐3 gene cluster was able to produce both EPA and DHA. The isolated gene cluster contained all the genes required for the recombinant production of both EPA and DHA in E. coli. Significance and Impact of the Study: These findings have implications for any future use of the EPA and DHA gene cluster in other micro‐organisms, notably those being used for fermentation. Recombinant production of both EPA and DHA by E. coli or any other micro‐organism has great potential to add economic value to a variety of industrial and agricultural products.     

The medaka sex-determining gene DMY acquired a novel temporal expression pattern after duplication of DMRT1

The male sex-determining gene, DMY, of the medaka is considered to have arisen via gene duplication of DMRT1. In the medaka, both genes are expressed in Sertoli cell lineage cells, but their temporal expression patterns are quite different. DMY expression starts just before the sex-determining period, whereas DMRT1 expression occurs during the testicular differentiation period. To evaluate the alterations to the expression patterns of the DMRT1 genes after duplication, we analyzed the morphological gonadal sex differentiation processes and expression patterns of DMRT1 in Oryzias luzonensis and Oryzias mekongensis, which are closely related to the medaka but do not have DMY. Male-specific upregulation of DMRT1 in these two species occurred during the testicular differentiation period, similar to the case for DMRT1 in the medaka. These findings suggest that DMY acquired a novel temporal expression pattern after duplication and that this event played a critical role in the evolutionary process of this gene.     

Enhanced green emissions of Er3+/Yb3+ co‐doped Gd2(MoO4)3 by co‐excited up‐conversion processes

Improving the emission from rare earth ions doped materials is of great importance to broaden their application in bio‐imaging, photovoltaics and temperature sensing. The green emissions of Gd₂(MoO₄)₃:Er³⁺/Yb³⁺ powder upon co‐excitation with 980 and 808 nm lasers were investigated in this paper. Distinct enhancement of green emissions was observed compared with single laser excitation. Based on the energy level structure of Er³⁺, the enhancement mechanism was discussed. Moreover, the result of temperature‐dependent enhancement revealed that the enhancement factor reached its maximum (2.5) as the sample heated to 120°C, which is due to the competition of two major thermal effects acting in the co‐excited up‐conversion processes. In addition, the same enhancement of green emissions was also observed in Gd₂(MoO₄)₃:Er³⁺ powder and NaYF₄:Er³⁺/Yb³⁺ powder.     

Phylogenomic analysis of gene co‐expression networks reveals the evolution of functional modules

Molecular evolutionary studies correlate genomic and phylogenetic information with the emergence of new traits of organisms. These traits are, however, the consequence of dynamic gene networks composed of functional modules, which might not be captured by genomic analyses. Here, we established a method that combines large‐scale genomic and phylogenetic data with gene co‐expression networks to extensively study the evolutionary make‐up of modules in the moss Physcomitrella patens, and in the angiosperms Arabidopsis thaliana and Oryza sativa (rice). We first show that younger genes are less annotated than older genes. By mapping genomic data onto the co‐expression networks, we found that genes from the same evolutionary period tend to be connected, whereas old and young genes tend to be disconnected. Consequently, the analysis revealed modules that emerged at a specific time in plant evolution. To uncover the evolutionary relationships of the modules that are conserved across the plant kingdom, we added phylogenetic information that revealed duplication and speciation events on the module level. This combined analysis revealed an independent duplication of cell wall modules in bryophytes and angiosperms, suggesting a parallel evolution of cell wall pathways in land plants. We provide an online tool allowing plant researchers to perform these analyses at http://www.gene2function.de .     

sPAR-3, a splicing variant of PAR-3, shows cellular localization and an expression pattern different from that of PAR-3 during enterocyte polarization

PAR-3 (partitioning-defective) is a scaffold-like PDZ (postsynaptic density-95/discs large/zonula occludens-1) domain-containing protein that forms a complex with PAR-6 and atypical PKC, localizes to tight junctions, and contributes to the formation of functional tight junctions. There are several alternatively spliced isoforms of PAR-3, although their physiological significance remains unknown. In this study, we show that one of the major isoforms of PAR-3, sPAR-3, is predominantly expressed in the Caco-2 cells derived from colon carcinoma and is used as a model to investigate the events involved in the epithelial cell differentiation and cell polarity development. During the polarization of Caco-2 cells, the expression of PAR-3 increases as do those of other cell-cell junction proteins, whereas the expression of sPAR-3 decreases. Biochemical characterization revealed that sPAR-3 associates with atypical PKC, as does PAR-3. On the other hand, immunofluorescence microscopy revealed that sPAR-3 does not concentrate at the cell-cell contact region in fully polarized cells, whereas it concentrates at premature cell-cell junctions. This makes a contrast to PAR-3, which concentrates at tight junctions in fully polarized cells. These results provide evidence suggesting the difference in the role between sPAR-3 and PAR-3 in epithelial cells.