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Polymeric matrices mimicking multiple functions of the ECM are expected to enable a material induced regeneration of tissues. Here, we investigated the adipogenic differentiation of human adipose derived mesenchymal stem cells (hADSCs) in a 3D architectured gelatin based hydrogel (ArcGel) prepared from gelatin and L-lysine diisocyanate ethyl ester (LDI) in an one-step process, in which the formation of an open porous morphology and the chemical network formation were integrated. The ArcGel was designed to support adipose tissue regeneration with its 3D porous structure, high cell biocompatibility, and mechanical properties compatible with human subcutaneous adipose tissue. The ArcGel could support initial cell adhesion and survival of hADSCs. Under static culture condition, the cells could migrate into the inner part of the scaffold with a depth of 840 +/- 120 mu m after 4 days, and distributed in the whole scaffold (2mm in thickness) within 14 days. The cells proliferated in the scaffold and the fold increase of cell number after 7 days of culture was 2.55 +/- 0.08. The apoptotic rate of hADSCs in the scaffold was similar to that of cells maintained on tissue culture plates. When cultured in adipogenic induction medium, the hADSCs in the scaffold differentiated into adipocytes with a high efficiency (93 +/- 1%). Conclusively, this gelatin based 3D scaffold presented high cell compatibility for hADSC cultivation and differentiation, which could serve as a potential implant material in clinical applications for adipose tissue reparation and regeneration.
Ecosystem boundaries, such as the Arctic-Boreal treeline, are strongly coupled with climate and were spatially highly dynamic during past glacial-interglacial cycles. Only a few studies cover vegetation changes since the last interglacial, as most of the former landscapes are inundated and difficult to access. Using pollen analysis and sedimentary ancient DNA (sedaDNA) metabarcoding, we reveal vegetation changes on Bol’shoy Lyakhovsky Island since the last interglacial from permafrost sediments. Last interglacial samples depict high levels of floral diversity with the presence of trees (Larix, Picea, Populus) and shrubs (Alnus, Betula, Ribes, Cornus, Saliceae) on the currently treeless island. After the Last Glacial Maximum, Larix re-colonised the island but disappeared along with most shrub taxa. This was probably caused by Holocene sea-level rise, which led to increased oceanic conditions on the island. Additionally, we applied two newly developed larch-specific chloroplast markers to evaluate their potential for tracking past population dynamics from environmental samples. The novel markers were successfully re-sequenced and exhibited two variants of each marker in last interglacial samples. SedaDNA can track vegetation changes as well as genetic changes across geographic space through time and can improve our understanding of past processes that shape modern patterns.
Mosses are a major component of the arctic vegetation, particularly in wetlands. We present C / N atomic ratio, delta C-13 and delta N-15 data of 400 brown-moss samples belonging to 10 species that were collected along hydrological gradients within polygonal mires located on the southern Taymyr Peninsula and the Lena River delta in northern Siberia. Additionally, n-alkane patterns of six of these species (16 samples) were investigated. The aim of the study is to see whether the inter-and intraspecific differences in C / N, isotopic compositions and n-alkanes are indicative of habitat, particularly with respect to water level. Overall, we find high variability in all investigated parameters for two different moisture-related groups of moss species. The C / N ratios range between 11 and 53 (median: 32) and show large variations at the intraspecific level. However, species preferring a dry habitat (xero-mesophilic mosses) show higher C / N ratios than those preferring a wet habitat (meso-hygrophilic mosses). The delta C-13 values range between 37.0 and 22.5% (median D 27.8 %). The delta N-15 values range between 6.6 and C 1.7%(median D 2.2 %). We find differences in delta C-13 and delta N-15 compositions between both habitat types. For some species of the meso-hygrophilic group, we suggest that a relationship between the individ-ual habitat water level and isotopic composition can be inferred as a function of microbial symbiosis. The n-alkane distribution also shows differences primarily between xeromesophilic and meso-hygrophilic mosses, i. e. having a dominance of n-alkanes with long (n-C29, n-C31 /and intermediate (n-C25 /chain lengths, respectively. Overall, our results reveal that C / N ratios, isotopic signals and n-alkanes of studied brown-moss taxa from polygonal wetlands are characteristic of their habitat.
The all-female Amazon molly (Poecilia formosa) is the result of a hybridization of the Atlantic molly (P. mexicana) and the sailfin molly (P. latipinna) approximately 120,000 years ago. As a gynogenetic species, P. formosa needs to copulate with heterospecific males including males from one of its bisexual ancestral species. However, the sperm only triggers embryogenesis of the diploid eggs. The genetic information of the sperm donor typically will not contribute to the next generation of P. formosa. Hence, P. formosa possesses generally one allele from each of its ancestral species at any genetic locus. This raises the question whether both ancestral alleles are equally expressed in P. formosa. Allele-specific expression (ASE) has been previously assessed in various organisms, e.g., human and fish, and ASE was found to be important in the context of phenotypic variability and disease. In this study, we utilized Real-Time PCR techniques to estimate ASE of the androgen receptor alpha (arα) gene in several distinct tissues of Amazon mollies. We found an allelic bias favoring the maternal ancestor (P. mexicana) allele in ovarian tissue. This allelic bias was not observed in the gill or the brain tissue. Sequencing of the promoter regions of both alleles revealed an association between an Indel in a known CpG island and differential expression. Future studies may reveal whether our observed cis-regulatory divergence is caused by an ovary-specific trans-regulatory element, preferentially activating the allele of the maternal ancestor.
Transient Catalytic Voltammetry of Sulfite Oxidase Reveals Rate Limiting Conformational Changes
(2017)
Sulfite oxidases are metalloenzymes that oxidize sulfite to sulfate at a molybdenum active site. In vertebrate sulfite oxidases, the electrons generated at the Mo center are transferred to an external electron acceptor via a heme domain, which can adopt two conformations: a “closed” conformation, suitable for internal electron transfer, and an “open” conformation suitable for intermolecular electron transfer. This conformational change is an integral part of the catalytic cycle. Sulfite oxidases have been wired to electrode surfaces, but their immobilization leads to a significant decrease in their catalytic activity, raising the question of the occurrence of the conformational change when the enzyme is on an electrode. We recorded and quantitatively modeled for the first time the transient response of the catalytic cycle of human sulfite oxidase immobilized on an electrode. We show that conformational changes still occur on the electrode, but at a lower rate than in solution, which is the reason for the decrease in activity of sulfite oxidases upon immobilization.
For the first time a molecularly imprinted polymer (MIP)-based sensor for tyrosinase is described. This sensor is based on the electropolymerization of scopoletin or o-phenylenediamine in the presence of tyrosinase from mushrooms, which has a high homology to the human enzyme. The template was removed either by treatment with proteinase Kor by alkaline treatment. The measuring signal was generated either by measuring the formation of a product by the target enzyme or by evaluation of the permeability of the redox marker ferricyanide. The o-phenylenediamine-based MIP sensor has a linear measuring range up to 50 nM of tyrosinase with a limit of detection of 3.97 nM (R 2 = 0.994) and shows good discrimination towards other proteins, e.g., bovine serum albumin and cytochrome c.
Shifts among Eukaryota, Bacteria, and Archaea define the vertical organization of a lake sediment
(2017)
Background: Lake sediments harbor diverse microbial communities that cycle carbon and nutrients while being constantly colonized and potentially buried by organic matter sinking from the water column. The interaction of activity and burial remained largely unexplored in aquatic sediments. We aimed to relate taxonomic composition to sediment biogeochemical parameters, test whether community turnover with depth resulted from taxonomic replacement or from richness effects, and to provide a basic model for the vertical community structure in sediments. Methods: We analyzed four replicate sediment cores taken from 30-m depth in oligo-mesotrophic Lake Stechlin in northern Germany. Each 30-cm core spanned ca. 170 years of sediment accumulation according to Cs-137 dating and was sectioned into layers 1-4 cm thick. We examined a full suite of biogeochemical parameters and used DNA metabarcoding to examine community composition of microbial Archaea, Bacteria, and Eukaryota. Results: Community beta-diversity indicated nearly complete turnover within the uppermost 30 cm. We observed a pronounced shift from Eukaryota- and Bacteria-dominated upper layers (<5 cm) to Bacteria-dominated intermediate layers (5-14 cm) and to deep layers (>14 cm) dominated by enigmatic Archaea that typically occur in deep-sea sediments. Taxonomic replacement was the prevalent mechanism in structuring the community composition and was linked to parameters indicative of microbial activity (e.g., CO2 and CH4 concentration, bacterial protein production). Richness loss played a lesser role but was linked to conservative parameters (e.g., C, N, P) indicative of past conditions. Conclusions: By including all three domains, we were able to directly link the exponential decay of eukaryotes with the active sediment microbial community. The dominance of Archaea in deeper layers confirms earlier findings from marine systems and establishes freshwater sediments as a potential low-energy environment, similar to deep sea sediments. We propose a general model of sediment structure and function based on microbial characteristics and burial processes. An upper "replacement horizon" is dominated by rapid taxonomic turnover with depth, high microbial activity, and biotic interactions. A lower "depauperate horizon" is characterized by low taxonomic richness, more stable "low-energy" conditions, and a dominance of enigmatic Archaea.
Most studies of aquatic plankton focus on either macroscopic or microbial communities, and on either eukaryotes or prokaryotes. This separation is primarily for methodological reasons, but can overlook potential interactions among groups. Here we tested whether DNA metabarcoding of unfractionated water samples with universal primers could be used to qualitatively and quantitatively study the temporal dynamics of the total plankton community in a shallow temperate lake. Significant changes in the relative proportions of normalized sequence reads of eukaryotic and prokaryotic plankton communities over a 3-month period in spring were found. Patterns followed the same trend as plankton estimates measured using traditional microscopic methods. The bloom of a conditionally rare bacterial taxon belonging to Arcicella was characterized, which rapidly came to dominate the whole lake ecosystem and would have remained unnoticed without metabarcoding. The data demonstrate the potential of universal DNA metabarcoding applied to unfractionated samples for providing a more holistic view of plankton communities.
Arsenic-containing hydrocarbons (AsHCs), natural products found in seafood, have recently been shown to exert toxic effects in human neurons. In this study we assessed the toxicity of three AsHCs in cultured human astrocytes. Due to the high cellular accessibility and substantial toxicity observed astrocytes were identified as further potential brain target cells for arsenolipids. Thereby, the AsHCs exerted a 5-19-fold higher cytotoxicity in astrocytes as compared to arsenite. Next we compared the toxicity of the arsenicals in a co-culture model of the respective human astrocytes and neurons. Notably the AsHCs did not show any substantial toxic effects in the co-culture, while arsenite did. The arsenic accessibility studies indicated that in the co-culture astrocytes protect neurons against cellular arsenic accumulation especially after incubation with arsenolipids. In summary, these data underline the importance of the glial-neuron interaction when assessing the in vitro neurotoxicity of new unclassified metal species.
Lying is an everyday moral phenomenon about which philosophers have written a lot. Not only the moral status of lying has been intensively discussed but also what it means to lie in the first place. Perhaps the most important criterion for an adequate definition of lying is that it fits with people’s understanding and use of this concept. In this light, it comes as a surprise that researchers only recently started to empirically investigate the folk concept of lying. In this paper, we describe three experimental studies which address the following questions: Does a statement need to be objectively false in order to constitute lying? Does lying necessarily include the intention to deceive? Can one lie by omitting relevant facts?
Arctic and alpine treelines worldwide differ in their reactions to climate change. A northward advance of or densification within the treeline ecotone will likely influence climate-vegetation feedback mechanisms. In our study, which was conducted in the Taimyr Depression in the North Siberian Lowlands, w present a combined field-and model-based approach helping us to better understand the population processes involved in the responses of the whole treeline ecotone, spanning from closed forest to single-tree tundra, to climate warming. Using information on stand structure, tree age, and seed quality and quantity from seven sites, we investigate effects of intra-specific competition and seed availability on the specific impact of recent climate warming on larch stands. Field data show that tree density is highest in the forest-tundra, and average tree size decreases from closed forest to single-tree tundra. Age-structure analyses indicate that the trees in the closed forest and forest-tundra have been present for at least similar to 240 yr. At all sites except the most southerly ones, past establishment is positively correlated with regional temperature increase. In the single-tree tundra, however, a change in growth form from krummholz to erect trees, beginning similar to 130 yr ago, rather than establishment date has been recorded. Seed mass decreases from south to north, while seed quantity increases. Simulations with LAVESI (Larix Vegetation Simulator) further suggest that relative density changes strongly in response to a warming signal in the forest-tundra while intra-specific competition limits densification in the closed forest and seed limitation hinders densification in the single-tree tundra. We find striking differences in strength and timing of responses to recent climate warming. While forest-tundra stands recently densified, recruitment is almost non-existent at the southern and northern end of the ecotone due to autecological processes. Palaeo-treelines may therefore be inappropriate to infer past temperature changes at a fine scale. Moreover, a lagged treeline response to past warming will, via feedback mechanisms, influence climate change in the future.
Following escape into the cytoplasm of host cells, Burkholderia pseudomallei and the related species Burkholderia thailandensis employ the type VI secretion system 5 ( T6SS-5) to induce plasma membrane fusion with an adjacent host cell. This process leads to the formation of multinucleated giant cells and facilitates bacterial access to an uninfected host cell in a direct manner. Despite its importance in virulence, the mechanism of the T6SS-5 and the role of host cell factors in cell-cell fusion remain elusive. To date, the T6SS-5 is the only system of bacterial origin known to induce host-cell fusion. To gain insight into the nature of T6SS-5-stimulated membrane fusion, we investigated the contribution of cholesterol and proteins exposed on the host cell surface, which were shown to be critically involved in virus-mediated giant cell formation. In particular, we analyzed the effect of host cell surface protein and cholesterol depletion on the formation of multinucleated giant cells induced by B. thailandensis. Acute protease treatment of RAW264.7 macrophages during infection with B. thailandensis followed by agarose overlay assays revealed a strong reduction in the number of cell-cell fusions compared with EDTA treated cells. Similarly, proteolytic treatment of specifically infected donor cells or uninfected recipient cells significantly decreased multinucleated giant cell formation. Furthermore, modulating host cell cholesterol content by acute cholesterol depletion from cellular membranes by methyl-beta-cyclodextrin treatment or exogenous addition of cholesterol impaired the ability of B. thailandensis to induce cell-cell fusions. The requirement of physiological cholesterol levels suggests that the membrane organization or mechanical properties of the lipid bilayer influence the fusion process. Altogether, our data suggest that membrane fusion induced by B. pseudomallei and B. thailandensis involves a complex interplay between the T6SS-5 and the host cell.
Citizen science projects have a long history in ecological studies. The research usefulness of such projects is dependent on applying simple and standardized methods. Here, we conducted a citizen science project that involved more than 3500 Swedish high school students to examine the temperature difference between surface water and the overlying air (T-w-T-a) as a proxy for sensible heat flux (Q(H)). If Q(H) is directed upward, corresponding to positive T-w-T-a, it can enhance CO2 and CH4 emissions from inland waters, thereby contributing to increased greenhouse gas concentrations in the atmosphere. The students found mostly negative T-w-T-a across small ponds, lakes, streams/rivers and the sea shore (i.e. downward Q(H)), with T-w-T-a becoming increasingly negative with increasing T-a. Further examination of T-w-T-a using high-frequency temperature data from inland waters across the globe confirmed that T-w-T-a is linearly related to T-a. Using the longest available high-frequency temperature time series from Lake Erken, Sweden, we found a rapid increase in the occasions of negative T-w-T-a with increasing annual mean T-a since 1989. From these results, we can expect that ongoing and projected global warming will result in increasingly negative T-w-T-a, thereby reducing CO2 and CH4 transfer velocities from inland waters into the atmosphere.
The bat-eared fox, Otocyon megalotis, is the only member of its genus and is thought to occupy a basal position within the dog family. These factors can lead to challenges in complete mitochondrial reconstructions and accurate phylogenetic positioning. Here, we present the first complete mitochondrial genome of the bat-eared fox recovered using shotgun sequencing and iterative mapping to three distantly related species. Phylogenetic analyses placed the bat-eared fox basal in the Canidae family within the clade including true foxes (Vulpes) and the raccoon dog (Nyctereutes) with high support values. This position is in good agreement with previously published results based on short fragments of mitochondrial and nuclear genes, therefore adding more support to the basal positioning of the bat-eared fox within Canidae.
The unusual mix of morphological traits displayed by extinct South American native ungulates (SANUs) confounded both Charles Darwin, who first discovered them, and Richard Owen, who tried to resolve their relationships. Here we report an almost complete mitochondrial genome for the litoptern Macrauchenia. Our dated phylogenetic tree places Macrauchenia as sister to Perissodactyla, but close to the radiation of major lineages within Laurasiatheria. This position is consistent with a divergence estimate of B66Ma (95% credibility interval, 56.64-77.83 Ma) obtained for the split between Macrauchenia and other Panperissodactyla. Combined with their morphological distinctiveness, this evidence supports the positioning of Litopterna (possibly in company with other SANU groups) as a separate order within Laurasiatheria. We also show that, when using strict criteria, extinct taxa marked by deep divergence times and a lack of close living relatives may still be amenable to palaeogenomic analysis through iterative mapping against more distant relatives.
The invasion success of the cyanobacterium Cylindrospermopsis raciborskii in experimental mesocosms
(2017)
The potentially toxic, invasive cyanobacterium Cylindrospermopsis raciborskii, originating from sub-tropical regions, has spread into temperate climate zones in almost all continents. Potential factors in its success are temperature, light and nutrient levels. Grazing losses through zooplankton have been measured in the laboratory but are typically not regarded as a factor in (failed) invasion success. In some potentially suitable lakes, C. raciborskii has never been found, although it is present in water bodies close by. Therefore, we tested the invasive potential of three different isolates introduced into natural plankton communities using laboratory mesocosm experiments under three grazing levels: ambient zooplankton densities, removal of large species using 100 mu m mesh and a ca. doubling of large species. Three C. raciborskii isolates originating from the same geographic region (North-East Germany) were added separately to the four replicates of each treatment and kept in semi-continuous cultures for 21 days. Two isolates disappeared from the mesocosms and were also not viable in filtered lake water indicating that the lake water itself or the switch from culture medium to lake water led to the decay of the inoculated C. raciborskii. Only one out of the three isolates persisted in the plankton communities at a rather low level and only in the treatment without larger zooplankton. This result demonstrates that under potentially suitable environmental conditions, top-down control from zooplankton might hamper the establishment of C. raciborskii. Non-metric multidimensional scaling showed distinct variation in resident phytoplankton communities between the different grazing levels, thus differential grazing impact shaped the resident community in different ways allowing C. raciborskii only to invade under competitive (= low grazing pressure) conditions. Furthermore, even after invasion failure, the temporary presence of C. raciborskii influenced the phytoplankton community.
Trait-based approaches have become increasingly successful in community ecology. They assume that the distribution of functional traits within communities responds in a predictable way to alterations in environmental forcing and that strong forcing may accelerate such trait changes. We used high frequency measurements of phytoplankton to test these assumptions. We analyzed the seasonal and long-term dynamics of the community trait mean within a multi-dimensional trait space under alternating multifactorial environmental conditions. The community trait mean exhibited a distinct recurrent annual pattern that reflected minor changes in climate, herbivory and nutrients. Independent of early spring conditions, the community trait mean was repeatedly driven into a narrow confined area in the trait space under pronounced herbivory during the clear water phase. The speed of movement was highest at the onset and the relaxation of such strong unidirectional forcing. Thus, our data support the conceptual framework of trait-based ecology that alterations in environmental conditions are systematically tracked by adjustments in the dominant functional trait values and that the speed of trait changes depends on the kind and intensity of the selection pressure. Our approach provides a sensitive tool to detect small functional differences in the community related to subtle differences in forcing.
Reduced expression of the Indy ("I am Not Dead, Yet") gene in lower organisms promotes longevity in a manner akin to caloric restriction. Deletion of the mammalian homolog of Indy (mIndy, Slc13a5) encoding for a plasma membrane-associated citrate transporter expressed highly in the liver, protects mice from high-fat diet-induced and aging-induced obesity and hepatic fat accumulation through a mechanism resembling caloric restriction. We studied a possible role of mIndy in human hepatic fat metabolism. In obese, insulin-resistant patients with nonalcoholic fatty liver disease, hepatic mIndy expression was increased and mIndy expression was also independently associated with hepatic steatosis. In nonhuman primates, a 2-year high-fat, high-sucrose diet increased hepatic mIndy expression. Liver microarray analysis showed that high mIndy expression was associated with pathways involved in hepatic lipid metabolism and immunological processes. Interleukin-6 (IL-6) was identified as a regulator of mIndy by binding to its cognate receptor. Studies in human primary hepatocytes confirmed that IL-6 markedly induced mIndy transcription through the IL-6 receptor and activation of the transcription factor signal transducer and activator of transcription 3, and a putative start site of the human mIndy promoter was determined. Activation of the IL-6-signal transducer and activator of transcription 3 pathway stimulated mIndy expression, enhanced cytoplasmic citrate influx, and augmented hepatic lipogenesis in vivo. In contrast, deletion of mIndy completely prevented the stimulating effect of IL-6 on citrate uptake and reduced hepatic lipogenesis. These data show that mIndy is increased in liver of obese humans and nonhuman primates with NALFD. Moreover, our data identify mIndy as a target gene of IL-6 and determine novel functions of IL-6 through mINDY. Conclusion: Targeting human mINDY may have therapeutic potential in obese patients with nonalcoholic fatty liver disease. German Clinical Trials Register: DRKS00005450.