Gram-negative bacteria inhabit a broad range of ecological niches. For Escherichia coli, this includes river water as well as humans and animals where it can be both a commensal and a pathogen1–3. Intricate regulatory mechanisms ensure bacteria have the right complement of β-barrel outer membrane proteins (OMPs) to enable adaptation to a particular habitat4,5. Yet no mechanism is known for replacing OMPs in the outer membrane (OM), a biological enigma further confounded by the lack of an energy source and the high stability6 and abundance of OMPs5. Here, we uncover the process underpinning OMP turnover in E. coli and show it to be passive and binary in nature wherein old OMPs are displaced to the poles of growing cells as new OMPs take their place. Using fluorescent colicins as OMP-specific probes, in combination with ensemble and single-molecule fluorescence microscopy in vivo and in vitro, as well as molecular dynamics (MD) simulations, we established the mechanism for binary OMP partitioning. OMPs clustered to form islands of ~0.5 μm diameter where their diffusion was restricted by promiscuous interactions with other OMPs. OMP islands were distributed throughout the cell and contained the Bam complex, which catalyses the insertion of OMPs in the OM7,8. However, OMP biogenesis occurred as a gradient that was highest at mid-cell but largely absent at cell poles. The cumulative effect is to push old OMP islands towards the poles of growing cells, leading to a binary distribution when cells divide. Hence the OM of a Gram-negative bacterium is a spatially and temporally organised structure and this organisation lies at the heart of how OMPs are turned over in the membrane.

Abstract:The development of the northern Danube Basin (nDB) was closely related to the Late Miocene geodynamic evolution of the Pannonian Basin System. It started with a wide rifting which led to subsidence of several basin depocenters which were gradually filled during the Late Miocene and Early Pliocene. In the Late Pliocene the subsidence continued only in the basin's central part, while the northern marginal zone suffered inversion and the uplifted sedimentary fill began to be eroded. Individual stages of the basin development are well recorded in its sedimentary succession, where at least three great tectono-sedimentary cycles were documented. Firstly, a lacustrine cycle containing Lower, Middle and lowermost Upper Pannonian sediments (A-F Zones; sensu Papp 1951) deposited in the time span 11.6-8.9 Ma and is represented in the nDB in Slovakia by the Ivanka and Beladice Formations. In the Danube Basin of the southern part in Hungary, where the formations are defined by the appearance of sedimentary facies in time and space, the equivalents are: (1) the deep-water setting marls, clays and sandy turbidites of the Endrőd and Szolnok Formations leading to the overlying strata deposits of the basin paleoslope or delta-slope represented by the Algyő Formation, and (2) the final shallow-water setting deposits of marshes, lagoons and a coastal and delta plain composed of clays, sands and coal seams, represented by the Újfalu Formation. The second tectono-sedimentary cycle was deposited in an alluvial environment and it comprises the Upper Pannonian (G and H Zones; sensu Papp 1951) and Lower Pliocene sediments dated 8.9-4.1? Ma. The cycle is represented in the nDB, by the Volkovce Formation and in the southern part by the Zagyva Formation in Hungary. The sedimentary environment is characterized by a wide range of facies from fluvial, deltaic and ephemeral lake to marshes. The third tectono-sedimentary cycle comprises the Upper Pliocene sediments. In Slovakia these are represented by the Kolárovo Formation dated 4.1-2.6 Ma. The formation contains material of weathering crust preserved in fissures of Mesozoic carbonates, diluvial deposits and sediments of the alluvial environment.

The lysis of red blood cells was shown to occur in human ruptured atherosclerotic lesions and intraventricular hemorrhage (IVH) of the brain. Liberated cell-free hemoglobin was found to undergo oxidation in both pathologies. We hypothesize that hemoglobin-derived peptides are generated during hemoglobin oxidation both in complicated atherosclerotic lesions and IVH of the brain, triggering endothelial cell dysfunction. Oxidized hemoglobin and its products were followed with spectrophotometry, LC-MS/MS analysis and detection of the cross-linking of globin chains in complicated atherosclerotic lesions of the human carotid artery and the hemorrhaged cerebrospinal liquid of preterm infants. The vascular pathophysiologic role of oxidized hemoglobin and the resultant peptides was assessed by measuring endothelial integrity, the activation of endothelial cells and the induction of proinflammatory genes. Peptide fragments of hemoglobin (VNVDEVGGEALGRLLVVYPWTQR, LLVVYPWTQR, MFLSFPTTK, VGAHAGEYGAELERMFLSFPTTK, and FLASVSTVLTSKYR) were identified in ruptured atherosclerotic lesions and in IVH of the human brain. Fragments resulting from the oxidation of hemoglobin were accompanied by the accumulation of ferryl hemoglobin. Similar to complicated atherosclerotic lesions of the human carotid artery, a high level of oxidized and cross-linked hemoglobin was observed in the cerebrospinal fluid after IVH. Haptoglobin inhibited hemoglobin fragmentation provoked by peroxide. The resultant peptides failed to bind haptoglobin or albumin. Peptides derived from hemoglobin oxidation and ferryl hemoglobin induced intercellular gap formation, decreased junctional resistance in the endothelium, and enhanced monocyte adhesion to endothelial cells. Enhanced expression of TNF and the activation of NLRP3 and CASP1 followed by the increased generation of IL-1β and nuclear translocation of the NF-κβ transcription factor occurred in response to hemoglobin-derived peptides, and ferryl hemoglobin in endothelium was upregulated in both pathologies. We conclude that the oxidation of hemoglobin in complicated atherosclerotic lesions and intraventricular hemorrhage of the brain generates peptide fragments and ferryl hemoglobin with the potential to trigger endothelial cell dysfunction.

Eurasian steppes have an essential role in conserving biodiversity, but due to the huge habitat loss in the past centuries they are often preserved only in small refuges. Among such refuges are the ancient steppic burial mounds (the so called ‘kurgans’) which have a high cultural and historical importance and are also essential sites of nature conservation. Despite their high number (approximately half million) and conservational importance there is a huge lack of knowledge on the locality and conservational state of the kurgans in most regions of Eurasia. To fill this knowledge gap, we built a public database which allows to record and query basic information on their cultural values and factors (such as land cover type, threatening factors, cover of woody species) that might serve as a basis for their effective conservation. The database provides a transparent, public and easy-to-use source for conservation managers and landscape planners focussed on grassland conservation. In addition, it also provides background information for other associate disciplines and public agencies dealing with the protection of cultural heritage.

The aim of our study was to explore the pathophysiologic role of oxidation of hemoglobin (Hb) to ferrylHb in human atherosclerosis. Results:We observed a severe oxidation of Hb to ferrylHb in complicated atherosclerotic lesions of carotid arteries with oxidative changes of the globin moieties, detected previously described oxidation hotspots in Hb (β1Cys93; β1Cys112; β2Cys112) and identified a novel oxidation hotspot (α1Cys104). After producing a monoclonal anti-ferrylHb antibody, ferrylHb was revealed to be localized extracellularly and also internalized by macrophages in the human hemorrhagic complicated lesions. We demonstrated that ferrylHb is taken up via phagocytosis as well as CD163 receptormediated endocytosis then transported to lysosomes involving actin polymerization.Internalization of ferrylHb was accompanied by up-regulation of heme oxygenase-1 and Hferritin and accumulation of iron within lysosomes as a result of heme/iron uptake.Importantly, macrophages exposed to ferrylHb in atherosclerotic plaques exhibited proinflammatory phenotype, as reflected by elevated levels of IL-1β and TNFα. To find further signatures of ferrylHb in complicated lesions we performed RNA-seq analysis on biopsies from patients who underwent endarterectomies. RNA-seq analysis demonstrated that human complicated lesions had a unique transcriptomic profile which was different from arteries and atheromatous plaques. Pathways affected in complicated lesions included gene changes associated with PI3k signaling, lipid transport, tissue remodeling and vascularization. Targeted analysis of gene expression associated with calcification, apoptosis and hemolytic-specific clusters indicated an increase in the severity of complicated lesions compared to atheroma. A 39% overlap in the differential gene expression profiles of human macrophages exposed to ferrylHb and the complicated lesion profiles was uncovered. Among these 547 genes, we found inflammatory, angiogenesis, and iron metabolism gene clusters regulated in macrophages. Innovation and Conclusion:We conclude that oxidation of Hb to ferrylHb contributes the progression of atherosclerosis via polarizing macrophages into a pro-atherogenic phenotype.

Intraplaque hemorrhage frequently occurs in atherosclerotic plaques resulting in cell-free hemoglobin, which is oxidized to ferryl hemoglobin (FHb) in the highly oxidative environment. Osteoclast-like cells (OLCs) derived from macrophages signify a counterbalance mechanism for calcium deposition in atherosclerosis. Our aim was to investigate whether oxidized hemoglobin alters osteoclast formation, thereby affecting calcium removal from mineralized atherosclerotic lesions. RANKL-(receptor activator of nuclear factor kappa-Β ligand-) induced osteoclastogenic differentiation and osteoclast activity of RAW264.7 cells were studied in response to oxidized hemoglobin via assessing bone resorption activity, expression of osteoclast-specific genes, and the activation of signalization pathways. OLCs in diseased human carotid arteries were assessed by immunohistochemistry. FHb, but not ferrohemoglobin, decreased bone resorption activity and inhibited osteoclast-specific gene expression (tartrateresistant acid phosphatase, calcitonin receptor, and dendritic cell-specific transmembrane protein) induced by RANKL. In addition, FHb inhibited osteoclastogenic signaling pathways downstream of RANK (receptor activator of nuclear factor kappa-Β). It prevented the induction of TRAF6 (tumor necrosis factor (TNF) receptor-associated factor 6) and c-Fos, phosphorylation of p-38 and JNK (c-Jun N-terminal kinase), and nuclear translocation of NFκB (nuclear factor kappa-Β) and NFATc1 (nuclear factor of activated T-cells, cytoplasmic 1). These effects were independent of heme oxygenase-1 demonstrated by knocking down HO-1 gene in RAW264.7 cells and in mice. Importantly, FHb competed with RANK for RANKL binding suggesting possible mechanisms by which FHb impairs osteoclastic differentiation. In diseased human carotid arteries, OLCs were abundantly present in calcified plaques and colocalized with regions of calcium deposition, while the number of these cells were lower in hemorrhagic lesions exhibiting accumulation of FHb despite calcium deposition. We conclude that FHb inhibits RANKL-induced osteoclastic differentiation of macrophages and suggest that accumulation of FHb in a calcified area of atherosclerotic lesion with hemorrhage retards the formation of OLCs potentially impairing calcium resorption.

Prehistoric mounds of the Great Hungarian Plain often function as refuges for relic loess steppe vegetation and their associated fauna. The Zsolca mounds are a typical example of kurgans acting as refuges, and even though they are surrounded by agricultural land, they harbour a species rich loess grassland with an area of 0.8 ha. With a detailed field survey of their geomorphology, soil, flora and fauna, we describe the most relevant attributes of the mounds regarding their maintenance as valuable grassland habitats. We recorded 104 vascular plant species, including seven species that are protected in Hungary and two species (Echium russicum and Pulsatilla grandis) listed in the IUCN Red List and the Habitats Directive. The negative effect of the surrounding cropland was detectable in a three-metre wide zone next to the mound edge, where the naturalness of the vegetation was lower, and the frequency of weeds, ruderal species and crop plants was higher than in the central zone. The ancient man-made mounds harboured dry and warm habitats on the southern slope, while the northern slopes had higher biodiversity, due to the balanced water supplies. Both microhabitats had different assemblages of ground-dwelling invertebrates.

Due to large-scale agricultural intensification, grasslands are often restricted to habitat islands in human-transformed landscapes. There are approximately half a million ancient burial mounds built by nomadic steppic tribes in the Eurasian steppe and forest steppe zones, which act as habitat islands for dry grassland vegetation. Land use intensification, such as arable farming and afforestation by non-native woody species are amongst the major threats for Eurasian dry grasslands, including grasslands on mounds. After the launch of the Good Agricultural and Environmental Condition framework of the European Union, in Hungary there is a tendency for ceasing crop production and cutting non-native woody plantations, in order to conserve these unique landmarks and restore the historical grassland vegetation on the mounds. In this study, restoration prospects of dry grassland habitats were studied on kurgans formerly covered by croplands and Robinia pseudoacacia plantations. Soil and vegetation characteristics were studied in thespontaneously recovering grasslands. The following questions were addressed: 1; How does site history affect the spontaneous grassland recovery? 2; Do residual soil nutrients play a role in grassland recovery? In former croplands, excess phosphorus, while in former Robinia plantations, excess nitrogen was present in the soil even four years after the land use change and grassland vegetation was in an early or mid-successional stage both on the mounds. The results showed that, without proper management measures, recovery of grassland vegetation is slow on mounds formerly used as cropland or black locust plantation. However, restoration efforts, focused on the restoration of mounds formerly covered by croplands, can be more effective compared to the restoration of mounds formerly covered by forest plantations.