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Dendritic cells (DCs) are major players for the induction of immune responses. Apart from plasmacytoid DCs (pDCs), human DCs can be categorized into two types of conventional DCs: CD141 DCs (cDC1) and CD1c DCs (cDC2). Defining uniquely expressed surface markers on human immune cells is not only important for the identification of DC subpopulations but also a prerequisite for harnessing the DC subset-specific potential in immunomodulatory approaches, such as antibody-mediated antigen targeting. Although others identified CLEC9A as a specific endocytic receptor for CD141 DCs, such a receptor for CD1c DCs has not been discovered, yet. By performing transcriptomic and flow cytometric analyses on human DC subpopulations from different lymphohematopoietic tissues, we identified CLEC10A (CD301, macrophage galactose-type C-type lectin) as a specific marker for human CD1c DCs. We further demonstrate that CLEC10A rapidly internalizes into human CD1c DCs upon binding of a monoclonal antibody directed against CLEC10A. The binding of a CLEC10A-specific bivalent ligand (the MUC-1 peptide glycosylated with N-acetylgalactosamine) is limited to CD1c DCs and enhances the cytokine secretion (namely TNFα, IL-8, and IL-10) induced by TLR 7/8 stimulation. Thus, CLEC10A represents not only a candidate to better define CD1c DCs-due to its high endocytic potential-CLEC10A also exhibits an interesting candidate receptor for future antigen-targeting approaches.

Cross-presentation of antigen by dendritic cells (DCs) to CD8(+) T cells is a fundamentally important mechanism in the defense against pathogens and tumors. Due to the lack of an appropriate lineage marker, cross-presenting DCs in the mouse are provisionally classified as \"Batf3-IRF-8-Id2-dependent DCs\" or as \"CD8(+) DCs\" in the spleen, and as \"CD103(+)CD11b(-) DCs\" in the periphery. We have now generated a mAb to XCR1, a chemokine receptor which is specifically expressed on CD8(+) DCs and a subpopulation of double negative DCs in the spleen. Using this antibody, we have determined that only XCR1(+)CD8(+) (around 80% of CD8(+) DCs) and their probable precursors, XCR1(+)CD8(-) DCs, efficiently take up cellular material and excel in antigen cross-presentation. In lymph nodes (LNs) and peripheral tissues, XCR1(+) DCs largely, but not fully, correspond to CD103(+)CD11b(-) DCs. Most importantly, we demonstrate that XCR1(+) DCs in the spleen, LNs, and peripheral tissues are dependent on the growth factor Flt3 ligand and are selectively absent in Batf3-deficient animals. These results provide evidence that expression of XCR1 throughout the body defines the Batf3-dependent lineage of DCs with a special capacity to cross-present antigen. XCR1 thus emerges as the first surface marker characterizing a DC lineage in the mouse and potentially also in the human.

Embryonic losses constitute a major burden for reproductive efficiency of farm animals. Pregnancy losses in ungulate species, which include cattle, pigs, sheep and goats, majorly occur during the second week of gestation, when the embryo experiences a series of cell differentiation, proliferation, and migration processes encompassed under the term conceptus elongation. Conceptus elongation takes place following blastocyst hatching and involves a massive proliferation of the extraembryonic membranes trophoblast and hypoblast, and the formation of flat embryonic disc derived from the epiblast, which ultimately gastrulates generating the three germ layers. This process occurs prior to implantation and it is exclusive from ungulates, as embryos from other mammalian species such as rodents or humans implant right after hatching. The critical differences in embryo development between ungulates and mice, the most studied mammalian model, have precluded the identification of the genes governing lineage differentiation in livestock species. Furthermore, conceptus elongation has not been recapitulated in vitro , hindering the study of these cellular events. Luckily, recent advances on transcriptomics, genome modification and post-hatching in vitro culture are shedding light into this largely unknown developmental window, uncovering possible molecular markers to determine embryo quality. In this review, we summarize the events occurring during ungulate pre-implantation development, highlighting recent findings which reveal that several dogmas in Developmental Biology established by knock-out murine models do not hold true for other mammals, including humans and farm animals. The developmental failures associated to in vitro produced embryos in farm animals are also discussed together with Developmental Biology tools to assess embryo quality, including molecular markers to assess proper lineage commitment and a post-hatching in vitro culture system able to directly determine developmental potential circumventing the need of experimental animals.

Specification of embryonic lineages is an important question in the field of early development. Numerous studies analyzed the expression patterns of the candidate transcripts and proteins in humans and mice and clearly determined the markers of each lineage. To overcome the limitations of human and mouse embryos, the expression of the marker transcripts in each cell has been investigated using in vivo embryos in pigs. In vitro produced embryos are more accessible, can be rapidly processed with low cost. Therefore, we analyzed the characteristics of lineage markers and the effects of the DAB2 gene (trophectoderm marker) in in vitro fertilized porcine embryos. We investigated the expression levels of the marker genes during embryonic stages and distribution of the marker proteins was assayed in day 7 blastocysts. Then, the shRNA vectors were injected into the fertilized embryos and the differences in the marker transcripts were analyzed. Marker transcripts showed diverse patterns of expression, and each embryonic lineage could be identified with localization of marker proteins. In DAB2-shRNA vectors injected embryos, HNF4A and PDGFRA were upregulated. DAB2 protein level was lower in shRNA-injected embryos without significant differences. Our results will contribute to understanding of the mechanisms of embryonic lineage specification in pigs.

Background Jeju Island is the largest island of South Korea, located southwest far from the mainland of Korea, and has a unique history and its own cultures that are distinguished from those of the other regions of the Korean mainland. However, the Jeju population has not been deeply investigated to date to understand their genetic structure, which may reflect their historical and geographical background. Objective To identify the genetic characteristics and biogeographic origin of people of Jeju Island based on the statistical analysis of genetic data using lineage markers. Methods 17 Y-STRs data for 615 unrelated males and mitochondrial DNA haplogroup data for 799 unrelated individuals residing on Jeju Island were generated, and analyzed to investigate genetic diversity and genetic characteristics using statistical methods including pairwise Fst or Rst, Analysis of molecular variance (AMOVA) and Multidimensional scaling (MDS). Results For male individuals of Jeju Island, unique genetic characteristics were observed in the analysis of Y-STRs, including low haplotype diversity, strong association with surnames, genetic difference from other regions of Korea, and common genetic variation of the Y-STR loci known to be predominant in Northern populations, such as Mongolians. Statistical analysis of the mitochondrial DNA haplogroups also revealed similar results that showed low haplogroup diversity and high frequency of haplogroup Y prevalent mostly in ethnic populations around the Sea of Okhotsk in Northeastern Asia. All these results suggest that Jeju Island is genetically distinct from other regions of Korea, possibly being a subpopulation in Korea, and related closely to Northern Asian populations. Conclusion The findings in the genetic approach could support understanding of the historical background of Jeju Island that is consistent with evidence from other multidisciplinary studies.

The chronological period from the beginning of the Chalcolithic Age to the end of the Bronze Age on the Iberian northern sub-plateau of the Iberic Peninsula involves interesting social and cultural phenomena, such as the appearance of the Bell Beaker and, later, the Cogotas I cultures. This work constructs a genetic characterisation of the maternal lineages of the human population that lived on the northern sub-plateau between 5000 and 3000 years ago through an analysis of mitochondrial DNA (mtDNA), a kind of genetic marker that is inherited through maternal lineages, unaltered from generation to generation. Population and cultural questions are investigated through mtDNA analyses. This study intends to shed light on the following questions. Were individuals who were buried together in multiple or collective burials biologically related through their maternal lineages? Were there distinct maternal human lineages in the same or different geographical areas if different material cultures (Bell Beaker and Cogotas I) were associated with the arrival of new human populations who established close biological relationships with the endogenous populations? Or could this be the result of the transmission of knowledge without human populations mixing? Another important question is whether the material cultures were related to the female populations. We analysed 91 individuals from 28 different archaeological sites of the Iberian northern sub-plateau from four different chrono-cultural periods (Pre-Bell Beaker, Bell Beaker, Proto-Cogotas I, and Cogotas I), from the end of the Chalcolithic Age up to the Bronze Age. There were two historical moments of new populations arriving: the first during the Pre-Bell Beaker period, associated with the K mtDNA haplogroup, and the second during the Proto-Cogotas I culture, with new lineages of the H, HVO, and T haplogroups. Neither of these new population flows were directly associated with the maximum development of the two main material cultures Bell Beaker and Cogotas I, so they must have occurred immediately beforehand, during the Pre-Bell Beaker and Proto-Cogotas I periods, respectively. However, we cannot discard an association between the populations and material cultures. Curiously, it has also been observed that there was also a tendency towards multiple burials, in which the individuals who were buried together belonged to the same maternal lineage, during these two periods of population change. This study has shed some light on the populational changes that occurred through these different periods in this specific geographical area of the northern sub-plateau of the Iberian Peninsula.

Prostate cancer (PCa) is the second leading cause of cancer-related mortality and morbidity among males worldwide. Deciphering the biological mechanisms and molecular pathways involved in PCa pathogenesis and progression has been hindered by numerous technical limitations mainly attributed to the limited number of cell lines available, which do not recapitulate the diverse phenotypes of clinical disease. Indeed, PCa has proven problematic to establish as cell lines in culture due to its heterogeneity which remains a challenge, despite the various in vitro and in vivo model systems available. Growth factors have been shown to play a central role in the complex regulation of cell proliferation among hormone sensitive tumors, such as PCa. Here, we report the isolation and characterization of novel patient-derived prostate epithelial (which we named as AUB-PrC) cells from organoids culture system. We also assessed the role of epidermal growth factor (EGF) in culturing those cells. We profiled the AUB-PrC cells isolated from unaffected and tumor patient samples via depicting their molecular and epithelial lineage features through immunofluorescence staining and quantitative real-time PCR (qRT-PCR), as well as through functional assays and transcriptomic profiling through RNA sequencing. In addition, by optimizing a previously established prostate organoids culture system, we were able to grow human prostate epithelial cells using growth medium and EGF only. With these data collected, we were able to gain insight at the molecular architecture of novel human AUB-PrC cells, which might pave the way for deciphering the mechanisms that lead to PCa development and progression, and ultimately improving prognostic abilities and treatments.

Purpose The clinical relevance and mechanisms of local bone loss early post-anterior cruciate ligament (ACL) reconstruction remain unclear. The early spatial and temporal changes of peri-tunnel bone, its molecular mechanisms and its relationships with graft–bone tunnel healing were investigated in a 12-week-old rat model. Methods At various times, the reconstructed ACL complex was harvested for vivaCT imaging, biomechanical test, histology and immunohistochemical staining of CD68+ cells (a monocyte–macrophage lineage marker), MMP1 and MMP13. Results The peri-tunnel bone resorbed simultaneously with improvement of graft–bone tunnel healing. There were 30.1 ± 17.4, 46.8 ± 10.5 and 81.5 ± 12.3 % loss of peri-tunnel BMD as well as 43.2 ± 21.7, 78.7 ± 8.5 and 92.4 ± 17.7 % loss of peri-tunnel bone volume/total volume (BV/TV) at week 6 at the distal femur, epiphysis and metaphysis of tibia, respectively. MMP1, MMP13 and CD68+ cells were expressed at the graft–bone tunnel interface and peri-tunnel bone and increased with time post-reconstruction at the tibia. The ultimate load and stiffness of the healing complex positively correlated with tibial tunnel bone formation and negatively correlated with tibial peri-tunnel bone. Tunnel BV/TV at the tibial metaphysis and epiphysis showed the highest correlation with ultimate load ( ρ  = 0.591; p  = 0.001) and stiffness ( ρ  = 0.427; p  = 0.026) of the complex, respectively. Conclusion There was time-dependent loss of peri-tunnel bone early post-reconstruction, with the greatest loss occurring at the tibial metaphysis. This was consistent with high expression of MMP1, MMP13 and CD68+ cells at the graft–bone tunnel interface and the peri-tunnel region. The significant loss of peri-tunnel bone, though not critically affecting early tunnel healing, suggested the need to protect the knee joint early post-reconstruction.

Background and AimsTo date chloroplast genomes are available only for members of the non-protein amino acid-accumulating clade (NPAAA) Papilionoid lineages in the legume family (i.e. Millettioids, Robinoids and the ‘inverted repeat-lacking clade’, IRLC). It is thus very important to sequence plastomes from other lineages in order to better understand the unusual evolution observed in this model flowering plant family. To this end, the plastome of a lupine species, Lupinus luteus, was sequenced to represent the Genistoid lineage, a noteworthy but poorly studied legume group.MethodsThe plastome of L. luteus was reconstructed using Roche-454 and Illumina next-generation sequencing. Its structure, repetitive sequences, gene content and sequence divergence were compared with those of other Fabaceae plastomes. PCR screening and sequencing were performed in other allied legumes in order to determine the origin of a large inversion identified in L. luteus.Key ResultsThe first sequenced Genistoid plastome (L. luteus: 155 894 bp) resulted in the discovery of a 36-kb inversion, embedded within the already known 50-kb inversion in the large single-copy (LSC) region of the Papilionoideae. This inversion occurs at the base or soon after the Genistoid emergence, and most probably resulted from a flip–flop recombination between identical 29-bp inverted repeats within two trnS genes. Comparative analyses of the chloroplast gene content of L. luteus vs. Fabaceae and extra-Fabales plastomes revealed the loss of the plastid rpl22 gene, and its functional relocation to the nucleus was verified using lupine transcriptomic data. An investigation into the evolutionary rate of coding and non-coding sequences among legume plastomes resulted in the identification of remarkably variable regions.ConclusionsThis study resulted in the discovery of a novel, major 36-kb inversion, specific to the Genistoids. Chloroplast mutational hotspots were also identified, which contain novel and potentially informative regions for molecular evolutionary studies at various taxonomic levels in the legumes. Taken together, the results provide new insights into the evolutionary landscape of the legume plastome.

Recent reports of a new generation of ubiquitous transgenic chimaera markers prompted us to consider the criteria used to evaluate new chimaera markers and develop more objective assessment methods. To investigate this experimentally we used several series of fetal and adult chimaeras, carrying an older, multi-copy transgenic marker. We used two additional independent markers and objective, quantitative criteria for cell selection and cell mixing to investigate quantitative and spatial aspects of developmental neutrality. We also suggest how the quantitative analysis we used could be simplified for future use with other markers. As a result, we recommend a five-step procedure for investigators to evaluate new chimaera markers based partly on criteria proposed previously but with a greater emphasis on examining the developmental neutrality of prospective new markers. These five steps comprise (1) review of published information, (2) evaluation of marker detection, (3) genetic crosses to check for effects on viability and growth, (4) comparisons of chimaeras with and without the marker and (5) analysis of chimaeras with both cell populations labelled. Finally, we review a number of different chimaera markers and evaluate them using the extended set of criteria. These comparisons indicate that, although the new generation of ubiquitous fluorescent markers are the best of those currently available and fulfil most of the criteria required of a chimaera marker, further work is required to determine whether they are developmentally neutral.