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Biomembranes fulfill several essential functions. They delimitate cells and control the exchange of compounds between cells and the environment. They generate specialized cellular reaction spaces, house functional units such as the respiratory chain (RC), and are involved in content trafficking. Biomembranes are dynamic and able to adjust their properties to changing conditions and requirements. An example is the inner mitochondrial membrane (IMM), which houses the RC involved in the formation of adenosine triphosphate (ATP) and the superoxide anion as a reactive oxygen species (ROS). The IMM forms a characteristic ultrastructure that can adapt to changing physiological situations. In the fungal aging model Podospora anserina, characteristic age-related changes of the mitochondrial ultrastructure occur. More recently, the impact of membranes on aging was extended to membranes involved in autophagy, an important pathway involved in cellular quality control (QC). Moreover, the effect of oleic acid on the lifespan was linked to basic biochemical processes and the function of membranes, providing perspectives for the elucidation of the mechanistic effects of this nutritional component, which positively affects human health and aging.
Determination of tumor volume in subcutaneously inoculated xenograft models is a standard procedure for clinical and preclinical evaluation of tumor response to treatment. Practitioners frequently use a hands-on caliper method in conjunction with a simplified formula to assess tumor volume. Non-invasive and more precise techniques as investigation by MR or (μ)CT exist but come with various adverse effects in terms of radiation, complex setup or elevated cost of investigations. Therefore, we propose an advanced three-dimensional sonographic imaging technique to determine small tumor volumes in xenografts with high precision and minimized observer variability. We present a study on xenograft carcinoma tumors from which volumes and shapes were calculated with the standard caliper method as well as with a clinically available three-dimensional ultrasound scanner and subsequent processing software. Statistical analysis reveals the suitability of this non-invasive approach for the purpose of a quick and precise calculation of tumor volume in small rodents.
Acinetobacter baumannii infections pose a grave risk to hospitalized patients due to their resistance to antimicrobial substances. This necessitates the urgent development of novel therapeutic strategies. By comparing the genetic repertoire between pathogenic and non-pathogenic Acinetobacter we discovered genetic traits that offer surprising insights into how the pathogen interacts with humans. However, much remains to be learned about the pathogen’s phenotypic diversity.
Polyunsaturated fatty acids (PUFAs) are organic acids, including more than one double bond in their carbon long chains. They can be further classified in ω-3 and ω-6 fatty acids according to the localization of the double bound within the molecule and comprise a wide group of bioactive compounds involved in the regulation of the cardiovascular homeostasis.
PUFAs can be metabolized Cytochrome P450 which can use several metabolic precursors, to synthetize ω-3 and ω-6 epoxides. Epoxides can be further converted to the corresponding diolss via the soluble epoxide-hydrolase (sEH). ω-3/ω-6 epoxides and diols exert multiple functions in the modulation of endothelial physiology and pathophysiology including regulation of vascular tone, angiogenesis and tumor-associated vasculogeneis. However, little is known about how these metabolites can impact on vascular specification and lymph-angiogenesis.
In the present study, we aimed to investigate the role of ω-3/ω-6 epoxides and diols on endothelial cells specification, and subsequently assessed the effects of their metabolic imbalance on angiogenesis and lymph-angiogenesis.
To address these questions, we chose the ω-6 12,13-EpOME and the derived 12,13-DiHOME, while 19,20-EDP and corresponding diol, 19,20 DHDP, were selected from the ω-6 pool. To investigate effect of ω-3/ω-6 epoxides and diols on the establishment of blood (BEC) and lymphatic (LEC) cell identity, endothelial specification was recapitulated in vitro via differentiation of both human induced and mouse pluripotent embryonic cells (hIPSCs and mESCs). Differentiation was performed with or without administration of ω-3/ω-6 epoxides or diols respectively. Subsequently, BEC/LEC differentiation was confirmed via immunohistochemistry and quantitative flow cytometry, while PUFAs-dependent transcriptional changes were assessed in the differentiated cells via single-cells RNA sequencing.
We were able to demonstrate that the ω-6 epoxide and the ω-3 diol were able to mainly promote blood endothelial differentiation of pluripotent cells by favoring expression of vasculogenic mediators. Differently, the ω-6 diol and the ω-3 epoxide enhanced lymphatic specification via up-regulation of lymph-angiogenic mediators such Prox-1, Lyve-1 and Inregrin-α9. Moreover, the same compounds were able to favor an increase of the lymphatic cell portion in murine lung endothelial cells ex vivo.
Using mice harboring a constitutive deletion of the sEH enzyme, we have shown that the subsequent imbalance of the physiologic epoxides/diols ratio, resulted in altered angiogenic and lymph-angiogenic responses both in vivo and vitro. Indeed, whole mount fluorescent-immunohistochemistry and three-dimensional imaging analysis confirmed that sEH-/- mice exhibited an increased blood endothelial vascular angiogenesis in the ear skin as well as enhanced sprouting of aortic explants in culture. Thus, linked to the accumulation of endogenously produced EpOMEs.
On the other hand, the same animals displayed significantly impaired dermal lymph-angiogenesis and decreased lymphatic sprouting responses in vitro. Subsequently, we were able to show that such phenotype could be mainly linked to the lack of DiHOMEs, as exogenous administration was able to rescue the sprouting responses of sEH-/- thoracic duct explants as well as to improve those from the corresponding wild-type controls.
In synthesis, we have shown that, ω-6/ω-3 epoxides diols can differentially contribute to endothelial cell specification as well as angiogenesis and lymph-angiogenesis. Specifically, while
ω-6 epoxides and ω-3 diols mainly favor blood endothelial cell (BECs) specification of pluripotent cells, ω-6 diols and ω-3 epoxides promoted lymphatic differentiation of angioblast cell progenitors.
Moreover, we were able to confirm that EpOME, and on a lower scale DHDP, were able to enhance angiogenesis in vitro and in vivo. On other hand, we have shown that DiHOME, and partially EDP (in vitro), can regulate lymph-angiogenesis and enhance lymph-angiogenic potential of WT and sEH-/- mice in vitro.
Highlights
• Cryptic species are a so far overlooked aspect of environmental risk assessment.
• Multiple populations of four genetic lineages exposed in acute toxicity assays.
• Thiacloprid exposure shows up to 4-fold differences in tolerance among populations.
• Recent substance exposure probably triggers population-specific adaptive mechanisms.
• Population-level vulnerability calls for a shift in ecotoxicological methodology.
Abstract
Cryptic species are rarely considered in ecotoxicology, resulting in misleading outcomes when using a single morphospecies that encompasses multiple cryptic species. This oversight contributes to the lack of reproducibility in ecotoxicological experiments and promotes unreliable extrapolations. The important question of ecological differentiation and the sensitivity of cryptic species is rarely tackled, leaving a substantial knowledge gap regarding the vulnerability of individual cryptic species within species complexes. In times of agricultural intensification and the frequent use of pesticides, there is an urgent need for a better understanding of the vulnerability of species complexes and possible differences in adaptive processes. We used the cryptic species complex of the aquatic amphipod Gammarus roeselii, which comprises at least 13 genetic mtDNA lineages and spans from small-scale endemic lineages in Greece to a large-scale widely distributed lineage in central Europe. We exposed eleven populations belonging to four lineages to the neonicotinoid thiacloprid in an acute toxicity assay. We recorded various environmental variables in each habitat to assess the potential pre-exposure of the populations to contaminants. Our results showed that the populations differed up to 4-fold in their tolerances. The lineage identity had a rather minor influence, suggesting that the cryptic species complex G. roeselii does not differ significantly in tolerance to the neonicotinoid thiacloprid. However, the observed population differentiation implies that recent pre-exposure to thiacloprid (or similar substances) or general habitat contamination has triggered adaptive processes. Though, the extent to which these mechanisms are equally triggered in all lineages needs to be addressed in the future. Our study provides two key findings: Firstly, it shows that observed phylogenetic differences within the G. roeselii species complex did not reveal differences in thiacloprid tolerance. Second, it confirms that differentiation occurs at the population level, highlighting that susceptibility to toxicants is population-dependent. The population-specific differences were within the range of accepted intraspecific variability from a regulatory standpoint. From an evolutionary-ecological perspective, it remains intriguing to observe how persistent stresses will continue to influence tolerance and whether different populations are on distinct pathways of adaptation. Given that the potential selection process has only lasted a relatively short number of generations, it is crucial to monitor these populations in the future, as even brief exposure periods significantly impact evolutionary responses.
Highlights
• AIS is a useful tool for assessing the effects of repeated pulse exposures.
• Deltamethrin impacts aquatic communities at reported environmental exposure levels.
• Gammarus pulex as most sensitive species to repeated pulse exposures.
• MEC exceed the RAC derived from our results indicating a risk for aquatic organisms.
Abstract
Pesticides are considered to be one of the main causes of the decline in macroinvertebrate biodiversity in small streams. In particular, pyrethroids are detected in agricultural surface waters worldwide and pose a high risk to aquatic invertebrates. Due to their knock-down effect, even short pyrethroid exposure pulses can have significant short- and long-term effects on macroinvertebrate communities. Therefore, it is necessary to consider more realistic exposure scenarios for the environmental risk assessment of pyrethroids and, consequently, to obtain more realistic effect data by using multi-stressor test systems.
In an experimental setup with artificial indoor streams (AIS), four pyrethroid pulses simulated the exposure scenario of heavy rainfall events. Effects of these 12 h-exposures at different concentrations of deltamethrin (0.64 ng/L, 4 ng/L, 16 ng/L, 64 ng/L) with intervening recovery periods of six days were assessed on an aquatic community consisting of Gammarus pulex, Ephemera danica, Lumbriculus variegatus and Potamopyrgus antipodarum with various lethal and sub-lethal endpoints.
The mortality rate of G. pulex significantly increased with increasing deltamethrin concentrations, whereas the mean number of offspring significantly decreased (NOECoffspring: 16 ng/L, LOECoffspring: 64 ng/L). The biomass of L. variegatus decreased with increasing deltamethrin concentrations (NOECdry weight: 16 ng/L, LOECdry weight: 64 ng/L).
The findings of this study clearly demonstrate that 12 h-deltamethrin pulses at environmentally relevant concentrations adversely affect an aquatic community. Based on the results of this study a RAC value of 5.33 ng/L is assumed, which is below the concentrations measured in rivers of up to 58.8 ng/L. Unacceptable effects on the entire freshwater environment can therefore not be ruled out. The experimental AIS approach is a useful tool for assessing the effects of repeated pulse exposures that occur during surface runoff events.
Highlights
• Fomes fomentarius is a key fungal decomposer in European beech forests.
• Its occupancy is promoted on logs and snags under open and closed canopies.
• We observe low F. fomentarius occupancy on cut stumps.
• F. fomentarius abundance is most promoted by snags under closed canopy conditions.
• Snag enrichment under closed canopies promotes microhabitats and nutrient cycles.
Abstract
Fomes fomentarius (L.) Fr., (commonly known as Tinder fungus) is an abundant fungus in European beech old-growth forests and is important for nutrient cycles, food web dynamics, and biodiversity. The species was heavily reduced during the last centuries by forestry. Modern silviculture strategies in Central Europe aim to balance both the extraction of wood and promoting dead wood habitats. Such an approach is key to sustaining Fomes fomentarius as well as up to 600 saproxylic arthropods associated with the fungus. The aim of this study was to assess how dead wood type in combination with microclimate, resulting from different forest management strategies, affect the presence (occupancy) and abundance (percent cover occupied) of Fomes fomentarius fruit bodies at the stand- and dead wood object-scale. We experimentally extracted a standardized proportion of trees within 50 m by 50 m patches creating stumps, logs, snags, and logs with snags under two microclimate treatments (open canopy gap versus closed canopy) in a random block design. As a control, we defined cut stumps under closed canopies, as this is the common thinning approach in mature beech production forests. We tested the effects of alternative management strategies against the control using Generalized Linear Mixed-effects Models. At the stand-scale, our model revealed a significantly lower occupancy of Fomes fomentarius in control stands compared to treatments in which dead wood was not removed. The average cover of Fomes fomentarius on snags under a closed canopy, at both the stand- and dead wood object-scale, was higher than in control plots. However, effect size was weak at the object-scale. To increase this principal decomposer and boost important microhabitats for many arthropods, silviculture should aim to increase snags while maintaining dense forest canopies. Yet, at landscape scale, diversifying dead wood types and light conditions may boost overall saproxylic diversity.
Biological movement patterns can sometimes be quasi linear with abrupt changes in direction and speed, as in plastids in root cells investigated here. For the analysis of such changes we propose a new stochastic model for movement along linear structures. Maximum likelihood estimators are provided, and due to serial dependencies of increments, the classical MOSUM statistic is replaced by a moving kernel estimator. Convergence of the resulting difference process and strong consistency of the variance estimator are shown. We estimate the change points and propose a graphical technique to distinguish between change points in movement direction and speed.
Highlights
• The XUT approach allows engineering of NRPS/PKS hybrids
• A syrbactin derivative was designed, inhibiting the immunoproteasome
• Rational NRPS/PKS engineering might be complementary to synthetic chemistry
The bigger picture
Natural products (NPs) are structurally complex chemical compounds with diverse biological activities, representing an important source of new therapeutic agents for human health. Many NPs are synthesized by megasynthetases, such as non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), both of which are modularly organized multifunctional enzymes, where each module incorporates one specific building block in an assembly line fashion. Our work describes the engineering of a designed NRPS/PKS hybrid via the assembly of different megasynthetase fragments to produce a rationally modified syrbactin derivative with a complex peptide-polyketide structure, acting as proteasome inhibitor. To produce and identify further improved proteasome inhibitors as well as other therapeutics used against cancer, such as immune-modulating or anti-infective drugs, the engineering of megasynthases holds huge potential as an environmentally friendly and cost-effective synthetic biology platform.
Summary
The natural product (NP) class of syrbactins are potent proteasome inhibitors produced by hybrids of non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). Here, we describe the stepwise reassembly of an entire NRPS/PKS hybrid to produce a new syrbactin derivative by utilizing the recently described “eXchange Unit between Thiolation domains” (XUTs) approach. Remarkably, XUT-based engineering allowed the direct assembly of PKS and NRPS modules to introduce an α,β-unsaturated Michael system in a macrolactam moiety, which represents the inhibitory warhead of syrbactins. The novel derivative was produced in E. coli, isolated, and examined for its ability to inhibit yeast (yCP), human constitutive (cCP), and immunoproteasome (iCP). The engineered NP maintained the inhibitory activities of the syrbactin class but, due to rational modifications, inhibited iCP most strongly. Moreover, analysis of the crystal structure of yCP in complex with the derivative revealed further design strategies for even more specific iCP inhibition.