Integrating phylogeographic patterns of microsatellite and mtDNA divergence to infer the evolutionary history of chamois (genus Rupicapra)

<p>Abstract</p> <p>Background</p> <p>The chamois, distributed over most of the medium to high altitude mountain ranges of southern Eurasia, provides an excellent model for exploring the effects of historical and evolutionary events on diversification. Populations have been grouped into two species, <it>Rupicapra pyrenaica </it>from southwestern Europe and <it>R. rupicapra </it>from eastern Europe. However, a previous study of cytochrome b revealed that the two proposed species were non-monophyletic. The reconstruction of phylogenetic relationships between animal species often depends on the markers studied. To further elucidate the evolutionary history of chamois, we extended earlier studies by analysing DNA sequences of four mitochondrial regions (ND1, 12S, tRNApro and Control Region) and microsatellites (20 loci) to include all subspecies and cover its entire distribution range.</p> <p>Results</p> <p>We found discordant microsatellite (μsat) and mitochondrial (mt) DNA phylogenies. Mitochondrial phylogenies form three clades, West, Central and East (mtW, mtC and mtE), at variance with taxonomic classification. Our divergence age estimates indicate an initial separation into branches mtW-mtC and mtE 1.7 million years ago (mya), in the late Pliocene-early Pleistocene, quickly followed by the split of clades mtW and mtC. Clade mtW contains haplotypes from the Iberian peninsula and the western Alps, Clade mtC includes haplotypes from the Apennines and the Massif of Chartreuse and Clade mtE comprises populations to the east of the Alps. Divergence among populations within these three major clades is recent (< 0.5 mya). New microsatellite multilocus genotypes added to previously published data revealed differences between every pair of subspecies, forming three well defined groups (μsatW, μsatC and μsatE) also with a strong geographic signature. Grouping does not correspond with the mitochondrial lineages but is closer to morphology and taxonomic classification. Recent drastic reductions in population size can be noted for the subspecies <it>ornata </it>as an extremely low diversity.</p> <p>Conclusions</p> <p>The phylogeographic patterns for mtDNA and microsatellites suggest an evolutionary history with limited range contractions and expansions during the Quaternary period and reflect a major effect of the Alpine barrier on west-east differentiation. The contrasting phylogenies for mtDNA and microsatellites indicate events of hybridization among highly divergent lineages in the central area of distribution. Our study points to the importance of reticulate evolution, with periods of isolation and reduction of population size followed by expansions and hybridizations, in the diversification at the level of close species or subspecies.</p

Why Do Membranes of Some Unhealthy Cells Adopt a Cubic Architecture?

Nonlamellar lipid arrangements, including cubosomes, appear in unhealthy cells, e.g., when they are subject to stress, starvation, or viral infection. The bioactivity of cubosomes-nanoscale particles exhibiting bicontinuous cubic structures-versus more common vesicles is an unexplored area due to lack of suitable model systems. Here, glycodendrimercubosomes (GDCs)-sugar-presenting cubosomes assembled from Janus glycodendrimers by simple injection into buffer-are proposed as mimics of biological cubic membranes. The bicontinuous cubic GDC architecture has been demonstrated by electron tomography. The stability of these GDCs in buffer enabled studies on lectin-dependent agglutination, revealing significant differences compared with the vesicular glycodendrimersome (GDS) counterpart. In particular, GDCs showed an increased activity toward concanavalin A, as well as an increased sensitivity and selectivity toward two variants of banana lectins, a wild-type and a genetically modified variant, which is not exhibited by GDSs. These results suggest that cells may adapt under unhealthy conditions by undergoing a transformation from lamellar to cubic membranes as a method of defense

The major histocompatibility complex homozygous inbred Babraham pig as a resource for veterinary and translational medicine

The Babraham pig is a highly inbred breed first developed in the United Kingdom approximately 50 years ago. Previous reports indicate a very high degree of homozygosity across the genome, including the major histocompatibility complex (MHC) region, but confirmation of homozygosity at the specific MHC loci was lacking. Using both direct sequencing and PCR‐based sequence‐specific typing, we confirm that Babraham pigs are essentially homozygous at their MHC loci and formalise their MHC haplotype as Hp‐55.6. This enhances the utility of the Babraham pig as a useful biomedical model for studies in which controlling for genetic variation is important

Exploring functional pairing between surface glycoconjugates and human galectins using programmable glycodendrimersomes

Precise translation of glycan-encoded information into cellular activity depends critically on highly specific functional pairing between glycans and their human lectin counter receptors. Sulfoglycolipids, such as sulfatides, are important glycolipid components of the biological membranes found in the nervous and immune systems. The optimal molecular and spatial design aspects of sulfated and nonsulfated glycans with high specificity for lectin-mediated bridging are unknown. To elucidate how different molecular and spatial aspects combine to ensure the high specificity of lectin-mediated bridging, a bottom-up toolbox is devised. To this end, negatively surface-charged glycodendrimersomes (GDSs), of different nanoscale dimensions, containing sulfo-lactose groups are self-assembled in buffer from a synthetic sulfatide mimic: Janus glycodendrimer (JGD) containing a 3′-O-sulfo-lactose headgroup. Also prepared for comparative analysis are GDSs with nonsulfated lactose, a common epitope of human membranes. These self-assembled GDSs are employed in aggregation assays with 15 galectins, comprising disease-related human galectins, and other natural and engineered variants from four families, having homodimeric, heterodimeric, and chimera architectures. There are pronounced differences in aggregation capacity between human homodimeric and heterodimeric galectins, and also with respect to their responsiveness to the charge of carbohydrate-derived ligand. Assays reveal strong differential impact of ligand surface charge and density, as well as lectin concentration and structure, on the extent of surface cross-linking. These findings demonstrate how synthetic JGD-headgroup tailoring teamed with protein engineering and network assays can help explain how molecular matchmaking operates in the cellular context of glycan and lectin complexity

Flowcytometric data of intermediate-large cell gastrointestinal lymphoma presenting a gross mass in 32 cats – “let them glow in the flow”

Gastrointestinal lymphoma is the most common form of lymphoma in domestic cats. Aggressive phenotypes are much less common but do bear and unfavorable prognosis. Immunophenotyping by flow cytometry (FCM) is not systematically performed in these patients, because of difficulties in the acquisition of suitable sample material from the gastrointestinal tract. A multimodal diagnostic approach is recommended to improve identification of subtypes targeting patient tailored therapeutic strategies. The aim of this prospective study was to present results of multicolor FCM immunophenotyping in surgically removed gastrointestinal mass and relate them with histopathology using the World Health Organization (WHO) classification and clonality PCR testing. Thirty-two patients were included. Eight cats (25%) had gastric, 23 (72%) had intestinal lymphoma and 1 (3%) had gastric/jejunal lymphoma. Intestinal lymphoma sites were represented by 18 small intestinal, 4 ileocaecal, 1 large intestinal. All gastric lymphomas were diffuse large B-cell lymphoma (DLBCL). Small intestinal lymphomas were 10 enteropathy associated T-cell lymphoma type I (EATL I), 2 enteropathy associated T-cell lymphoma type II (EATL II), 2 peripheral T-cell lymphoma (PTCL), 3 DLBCL and one DLBCL+EATL II. The most common small intestinal FCM T-cell phenotype was CD3+CD21− CD4−CD8−CD18+ CD5−CD79− in 7/10 EATL I and one EATL II. The most frequent FCM B-cell phenotype was CD3−CD21+ CD4−CD8−CD18+ CD5−CD79+ in 13/17 DLBCL and the DLBCL+EATL II. Clonality PCR results were positive in 87.5% (28/32) of all cases. No cross-lineage rearrangement was observed. IHC and FCM results agreed in 87.5% (28/32) of all cases. When all 3 methods were combined, consistent results were seen in 75% (24/32). This is the first demonstration of a multicolor FCM approach set in context to the gold standard histopathology and clonality testing results

A local drug delivery system prolongs graft survival by dampening T cell infiltration and neutrophil extracellular trap formation in vascularized composite allografts.

INTRODUCTION The standard treatment for preventing rejection in vascularized composite allotransplantation (VCA) currently relies on systemic immunosuppression, which exposes the host to well-known side effects. Locally administered immunosuppression strategies have shown promising results to bypass this hurdle. Nevertheless, their progress has been slow, partially attributed to a limited understanding of the essential mechanisms underlying graft rejection. Recent discoveries highlight the crucial involvement of innate immune components, such as neutrophil extracellular traps (NETs), in organ transplantation. Here we aimed to prolong graft survival through a tacrolimus-based drug delivery system and to understand the role of NETs in VCA graft rejection. METHODS To prevent off-target toxicity and promote graft survival, we tested a locally administered tacrolimus-loaded on-demand drug delivery system (TGMS-TAC) in a multiple MHC-mismatched porcine VCA model. Off-target toxicity was assessed in tissue and blood. Graft rejection was evaluated macroscopically while the complement system, T cells, neutrophils and NETs were analyzed in graft tissues by immunofluorescence and/or western blot. Plasmatic levels of inflammatory cytokines were measured using a Luminex magnetic-bead porcine panel, and NETs were measured in plasma and tissue using DNA-MPO ELISA. Lastly, to evaluate the effect of tacrolimus on NET formation, NETs were induced in-vitro in porcine and human peripheral neutrophils following incubation with tacrolimus. RESULTS Repeated intra-graft administrations of TGMS-TAC minimized systemic toxicity and prolonged graft survival. Nevertheless, signs of rejection were observed at endpoint. Systemically, there were no increases in cytokine levels, complement anaphylatoxins, T-cell subpopulations, or neutrophils during rejection. Yet, tissue analysis showed local infiltration of T cells and neutrophils, together with neutrophil extracellular traps (NETs) in rejected grafts. Interestingly, intra-graft administration of tacrolimus contributed to a reduction in both T-cellular infiltration and NETs. In fact, in-vitro NETosis assessment showed a 62-84% reduction in NETs after stimulated neutrophils were treated with tacrolimus. CONCLUSION Our data indicate that the proposed local delivery of immunosuppression avoids off-target toxicity while prolonging graft survival in a multiple MHC-mismatch VCA model. Furthermore, NETs are found to play a role in graft rejection and could therefore be a potential innovative therapeutic target

Emergence and intensification of dairying in the Caucasus and Eurasian steppes

Archaeological and archaeogenetic evidence points to the Pontic-Caspian steppe zone between the Caucasus and the Black Sea as the crucible from which the earliest steppe pastoralist societies arose and spread, ultimately influencing populations from Europe to Inner Asia. However, little is known about their economic foundations and the factors that may have contributed to their extensive mobility. Here, we investigate dietary proteins within the dental calculus proteomes of 45 individuals spanning the Neolithic to Greco-Roman periods in the Pontic-Caspian Steppe and neighbouring South Caucasus, Oka-Volga-Don and East Urals regions. We find that sheep dairying accompanies the earliest forms of Eneolithic pastoralism in the North Caucasus. During the fourth millennium Bc, Maykop and early Yamnaya populations also focused dairying exclusively on sheep while reserving cattle for traction and other purposes. We observe a breakdown in livestock specialization and an economic diversification of dairy herds coinciding with aridification during the subsequent late Yamnaya and North Caucasus Culture phases, followed by severe climate deterioration during the Catacomb and Lola periods. The need for additional pastures to support these herds may have driven the heightened mobility of the Middle and Late Bronze Age periods. Following a hiatus of more than 500 years, the North Caucasian steppe was repopulated by Early Iron Age societies with a broad mobile dairy economy, including a new focus on horse milking.Peer reviewe

Emergence and intensification of dairying in the Caucasus and Eurasian steppes

Archaeological and archaeogenetic evidence points to the Pontic-Caspian steppe zone between the Caucasus and the Black Sea as the crucible from which the earliest steppe pastoralist societies arose and spread, ultimately influencing populations from Europe to Inner Asia. However, little is known about their economic foundations and the factors that may have contributed to their extensive mobility. Here, we investigate dietary proteins within the dental calculus proteomes of 45 individuals spanning the Neolithic to Greco-Roman periods in the Pontic-Caspian Steppe and neighbouring South Caucasus, Oka-Volga-Don and East Urals regions. We find that sheep dairying accompanies the earliest forms of Eneolithic pastoralism in the North Caucasus. During the fourth millennium Bc, Maykop and early Yamnaya populations also focused dairying exclusively on sheep while reserving cattle for traction and other purposes. We observe a breakdown in livestock specialization and an economic diversification of dairy herds coinciding with aridification during the subsequent late Yamnaya and North Caucasus Culture phases, followed by severe climate deterioration during the Catacomb and Lola periods. The need for additional pastures to support these herds may have driven the heightened mobility of the Middle and Late Bronze Age periods. Following a hiatus of more than 500 years, the North Caucasian steppe was repopulated by Early Iron Age societies with a broad mobile dairy economy, including a new focus on horse milking.Peer reviewe

Expression of T-Bet, Eomesodermin, and GATA-3 Correlates With Distinct Phenotypes and Functional Properties in Porcine γδ T Cells

Unlike mice and humans, porcine γδ T cells represent a prominent subset of T cells in blood and secondary lymphatic organs. GATA-3, T-bet and Eomesodermin (Eomes) are transcription factors with crucial functions in T-cell development and functional differentiation, but their expression has not been investigated in porcine γδ T cells so far. We analyzed the expression of these transcription factors in γδ thymocytes, mature γδ T cells from blood, spleen, lymph nodes, and lung tissue as well as in vitro stimulated γδ T cells on the protein level by flow cytometry. GATA-3 was present in more than 80% of all γδ-thymocytes. Extra-thymic CD2− γδ T cells expressed high levels of GATA-3 in all investigated organs and had a CD8α−/dimCD27+perforin− phenotype. T-bet expression was mainly found in a subset of CD2+ γδ T cells with an opposing CD8αhighCD27dim/−perforin+ phenotype. Eomes+ γδ T cells were also found within CD2+ γδ T cells but were heterogeneous in regard to expression of CD8α, CD27, and perforin. Eomes+ γδ T cells frequently co-expressed T-bet and dominated in the spleen. During aging, CD2−GATA-3+ γδ T cells strongly prevailed in young pigs up to an age of about 2 years but declined in older animals where CD2+T-bet+ γδ T cells became more prominent. Despite high GATA-3 expression levels, IL-4 production could not be found in γδ T cells by intracellular cytokine staining. Experiments with sorted and ConA + IL-2 + IL-12 + IL-18-stimulated CD2− γδ T cells showed that proliferating cells start expressing CD2 and T-bet, produce IFN-γ, but retain GATA-3 expression. In summary, our data suggest a role for GATA-3 in the development of γδ-thymocytes and in the function of peripheral CD2−CD8α−/dimCD27+perforin− γδ T cells. In contrast, T-bet expression appears to be restricted to terminal differentiation stages of CD2+ γδ T cells, frequently coinciding with perforin expression. The functional relevance of high GATA-3 expression levels in extra-thymic CD2− γδ T cells awaits further clarification. However, their unique phenotype suggests that they represent a thymus-derived separate lineage of γδ T cells in the pig for which currently no direct counterpart in rodents or humans has been described

Depletion of Dendritic Cells Enhances Innate Anti-Bacterial Host Defense through Modulation of Phagocyte Homeostasis

Dendritic cells (DCs) as professional antigen-presenting cells play an important role in the initiation and modulation of the adaptive immune response. However, their role in the innate immune response against bacterial infections is not completely defined. Here we have analyzed the role of DCs and their impact on the innate anti-bacterial host defense in an experimental infection model of Yersinia enterocolitica (Ye). We used CD11c-diphtheria toxin (DT) mice to deplete DCs prior to severe infection with Ye. DC depletion significantly increased animal survival after Ye infection. The bacterial load in the spleen of DC-depleted mice was significantly lower than that of control mice throughout the infection. DC depletion was accompanied by an increase in the serum levels of CXCL1, G-CSF, IL-1α, and CCL2 and an increase in the numbers of splenic phagocytes. Functionally, splenocytes from DC-depleted mice exhibited an increased bacterial killing capacity compared to splenocytes from control mice. Cellular studies further showed that this was due to an increased production of reactive oxygen species (ROS) by neutrophils. Adoptive transfer of neutrophils from DC-depleted mice into control mice prior to Ye infection reduced the bacterial load to the level of Ye-infected DC-depleted mice, suggesting that the increased number of phagocytes with additional ROS production account for the decreased bacterial load. Furthermore, after incubation with serum from DC-depleted mice splenocytes from control mice increased their bacterial killing capacity, most likely due to enhanced ROS production by neutrophils, indicating that serum factors from DC-depleted mice account for this effect. In summary, we could show that DC depletion triggers phagocyte accumulation in the spleen and enhances their anti-bacterial killing capacity upon bacterial infection