Open Access

Highlights • Charge states of titratable residues analyzed based on cryo-EM and molecular simulations. • Conformational dynamics of sidechains is coupled to protonation states. • Mechanistic aspects of respiratory complex I are discussed. The charge states of titratable amino acid residues play a key role in the function of membrane-bound bioenergetic proteins. However, determination of these charge states both through experimental and computational approaches is extremely challenging. Cryo-EM density maps can provide insights on the charge states of titratable amino acid residues. By performing classical atomistic molecular dynamics simulations on the high resolution cryo-EM structures of respiratory complex I from Yarrowia lipolytica, we analyze the conformational and charge states of a key acidic residue in its ND1 subunit, aspartic acid D203, which is also a mitochondrial disease mutation locus. We suggest that in the native state of respiratory complex I, D203 is negatively charged and maintains a stable hydrogen bond to a conserved arginine residue. Alternatively, upon conformational change in the turnover state of the enzyme, its sidechain attains a charge-neutral status. We discuss the implications of this analysis on the molecular mechanism of respiratory complex I.

Eukaryotic gene expression is a well-regulated process, with the dynamics of chromatin state serving as a crucial control system. Chromatin condensation, known as heterochromatin formation, represses gene expression by limiting accessibility for the transcriptional machinery. Conversely, euchromatin represents a more accessible chromatin state allowing active gene expression. The transition between these chromatin states is facilitated by coregulatory proteins, which recruit specific enzymes. Coactivators recruit histone acetyltransferases (HATs) to acetylate lysine residues on histones, while corepressors recruit histone deacetylases (HDACs) to remove these acetyl groups. Decreased acetylation leads to increased positive charge on histones, thereby strengthening the interaction between DNA and histones. The nuclear receptor corepressor 1 (NCoR1) stands out as one of the best-studied members within the family of nuclear corepressors. Its strong evolutionary conservation underscores its significance in vertebrate development and diverse cellular fate determinations. Research indicates that tissue-specific deletion yields unique effects that cannot be compensated for by another corepressor. Notably, NCoR1 showed the highest expression levels among all corepressors in single-cell sequencing data from Tabula Sapiens and Tabula Muris. These data also highlighted NCoR1 as the most abundant corepressor in endothelial cells (ECs). Despite its remarkably high expression, its exact role in ECs remained so far unclear. This study aimed to investigate the role of NCoR1 in ECs, with a specific focus on its involvement in angiogenic processes. To this end, NCoR1 was depleted from ECs using a tamoxifen-inducible knockout mouse model, as well as knockdown approaches in HUVECs. The impact of NCoR1 loss on the endothelial phenotype was assessed by examining key angiogenic features such as sprouting, migration, and proliferation. Interestingly, although proliferation and undirected migration were decreased upon NCoR1 depletion, a significant increase in the angiogenic capacity of HUVECs was noted. This effect seemed to be driven by loss of growth factor sensing and a VEGF independent mechanism, which therefore appeared to cause random sprouting in a 3D environment. Notably, loss of NCoR1 promoted the number of tip cells. Similarly, increased angiogenic capacity was also observed in organ culture of mouse aortae of Ncor1 knockout mice. Following the establishment of the phenotype, the underlying mechanism was explored using RNA- and ATAC-seq techniques. Depletion of NCoR1 revealed genome wide chromatin opening with a clear enrichment in gene regulatory regions, especially promotor regions. This finding, together with an overlay of the RNA-seq data indicated a strong gene regulatory relevance of NCoR1, especially on genes associated with angiogenesis. Since NCoR1 itself is incapable of direct DNA binding, a subsequent transcription factor analysis was performed. With the aid of a bioinformatic tool (TEPIC2), multiple transcription factors, especially from the Fos gene family and Jun family, were predicted to bind in regions of differentially expressed genes. Both families are known to be part of the AP-1 transcription factor complex, which was previously shown to interact with NCoR1. Interestingly, in ECs, AP-1 was shown to regulate vascular development and migration. This could potentially explain why the loss of NCoR1 promotes angiogenesis. As the loss of NCoR1 particularly increased the number of tip cells during angiogenic sprouting, direct implications for this phenotype were investigated. Notch signalling is the main pathway, determining whether an EC differentiates into a highly migratory tip cell or highly proliferative stalk cell. Activation of Notch signalling involves the nuclear translocation of NICD, the intracellular domain of the transmembrane Notch receptor, with subsequent binding to RBPJκ. This interaction leads to a conformational change releasing an initially bound corepressor, followed by recruitment of a coactivator. In fact, NCoR1 was observed to bind RBPJκ, whereas depletion of NCoR1 – and hence reduced interaction between both proteins – directly increased RBPJκ promotor activity. Moreover, target gene expressions including DLL4 were upregulated. Hence, loss of NCoR1 leads to heightened Notch signaling by both derepressing the pathway and enhancing ligand expression, resulting in "superactive" Notch signaling. In ECs, the distinction between tip and stalk cells relies on competitive interaction and lateral inhibition. In NCoR1 depleted cells, uniform hyperactive Notch signaling potentially disrupts the reshuffling mechanism that tip and stalk cells usually undergo, thus locking in cell fate once a tip cell is established, promoting the overall number and thus angiogenesis. In functional ECs, activation of Notch signaling suppresses angiogenesis. However, this creates a paradox response as NCoR1 depletion clearly enhances Notch activity, but at the same time elicits a pro-angiogenic effect. Interestingly, Notch4, when expressed in cis with Notch1, was shown to act as an endogenous inhibitor of Notch1, thus resulting in a contradictory Notch response. This was confirmed by a spheroid outgrowth assay, where only knocking down NOTCH4, but not NOTCH1, in NCoR1 depleted cells blocked the hypersprouting phenotype. In summary, this study revealed that NCoR1 is crucial for maintaining precise endothelial gene expression programs. Loss of NCoR1 triggers an angiogenic response, but compromises the focused maintenance of gene expression programs, as highlighted by genome-wide chromatin opening. Consequently, prolonged depletion of NCoR1 may not be a favorable strategy for sustaining angiogenesis. However, in acute scenarios like diabetic wound healing, targeting NCoR1 could offer a potential therapeutic approach to restore angiogenesis.

Following recent discussions around suspended life, this paper focuses on an endeavor that sought to arrest biological material in time and space and render it available on demand. It depicts the attempt to establish a collection of cryopreserved donated cells. The study offers rare insights into how this initiative was at odds with familiar politics significant in its field, those of innovation and preparedness, and therefore was suspended itself. In identifying parallels with accounts of unsuccessful biobanks, the paper makes a case for the analytical value of considering ill-fated projects of suspension along with those that prosper and attract public attention. The case of a novel cryo-collection, in particular, demonstrates how the idea and practice of suspension only gathers political momentum when it serves other well-established rationales. As such, it prompts two important conclusions. First, the power to arrest life as it comes with cryotechnologies is much more likely to unravel in entrenched constellations than to carry transformative or disruptive potential. Second, however, the paper also exemplifies that projects of suspension are not necessarily doomed to serve hegemonic ways of governing life. It advocates for preventing such mismatches from falling into oblivion.

1. Differences in ecosystem service (ES) priorities often lead to conflicts between stakeholders. While differences in priorities have often been described, the sociocultural factors, including differences in cultural worldview, which drive them have not. We propose that the cultural theory of risk and its ‘grid-group’ typology, which classifies people as individualists, hierarchists, egalitarians and fatalists, can provide a conceptual framework for doing this. 2. We examined the relationship between ES prioritisation by stakeholders and underlying cultural (cultural worldviews, and related environmental nature and risk perceptions), sociocultural (region, stakeholder group, political party preference) and socio-demographic factors. This was achieved by applying multivariate statistics to data from a survey with 321 respondents, conducted across 14 stakeholder groups in three German regions. 3. Results show that most stakeholders prioritised many services but gave the highest priority to services linked to their stakeholder group. We identified four ‘ES priority bundles’: cultural services, open-land provisioning services, environmental protection services and forest provisioning services. 4. Each ES priority bundle was consistently associated with particular cultural worldviews, perceptions of nature and sociocultural factors, meaning that we could identify ‘cultural types’. Two of these associations were particularly strong: Prioritisation of open-land provisioning ES was high for the agriculture stakeholder group, associated with individualism, a perception of nature as durable but unpredictable, and support for economic liberal, conservative political parties. In contrast, those who prioritised environmental protection tended to hold egalitarian cultural worldviews and perceive nature as tolerant and sensitive. They also often belonged to the research and nature conservation stakeholder groups, with a mostly left-leaning political party preference. 5. The identification of cultural types of stakeholder with consistent ES priorities and cultural worldviews may provide a useful construct in the future ES research. Furthermore, it may allow communications regarding ES to be tailored to improve their effectiveness, potentially aiding the promotion of sustainable management strategies.

Angiogenesis, the formation of blood vessels from pre-existing ones, is a vital process in growth, development, and wound healing. However, in the context of solid tumors, chronic inflammation, and wAMD, angiogenesis is often dysregulated, contributing to the progression of the disease. As a result, several anti-angiogenic therapies have been developed and used with some success in the treatment of wAMD and cancer. However, while these treatments have significantly improved the outcome of these diseases, their efficacy is still limited. This is because resistance is common in cancer, while in wAMD, the underlying inflammatory processes can still drive the disease. In addition, angiogenesis is still not routinely targeted in chronic inflammatory diseases. Thus, there is still a great need for anti-angiogenic agents, and combined inhibition of angiogenesis and inflammation is desirable. 2-Desaza-annomontine, also called C81, is a synthetically available derivative of the alkaloid annomontine, which is found in the plant family Annonaceae. Derivatives of annomontine have previously been characterized as bioactive compounds. As one of them, C81 has been shown to affect inflammatory processes of the endothelium and restore endothelial barrier function against immune cells by Krishnathas et al. (2021), who also discovered that C81 inhibits DYRK2, PIM3, and the splicing kinases CLK1 and CLK4. Therefore, we hypothesized that C81 could also inhibit endothelial angiogenic functionality, making it a promising compound for the treatment of diseases that depend on angiogenesis and inflammation. As an initial test of this hypothesis, a murine laser-induced choroidal neovascularization (CNV) model was used. In this model, 3 and 10 µM C81 strongly inhibited vascular permeability and endothelial infiltration into the choroid, indicating that C81 inhibits angiogenesis. Since VEGF is one of the most important pro-angiogenic signaling molecules in CNV, we next tested whether C81 specifically affects VEGF-induced angiogenesis. A murine aortic ring model was used for this purpose. In this model, 3 µM C81 significantly inhibited aortic sprouting, demonstrating that C81 is an inhibitor of VEGF-induced angiogenesis. To elucidate the processes by which C81 affects angiogenesis, assays testing individual steps of the angiogenic cascade were performed in human umbilical vein endothelial cells (HUVECs). In these assays, C81 inhibited the ability of HUVECs to form VEGF-induced sprouts from spheroids, reduced the formation of capillary-like structures on Matrigel, abrogated proliferation, and inhibited migration. Because angiogenesis in vivo is mediated by the microvascular endothelium, key experiments were repeated in the microvascular cell line HMEC-1. Since C81 also reduced proliferation, sprouting, and migration of HMEC-1, we concluded that C81 impedes angiogenesis by inhibiting angiogenesis-related cellular functions of the endothelium. We then investigated the pathways by which C81 affects angiogenic cellular functions. We found that C81 reduced the activity of the VEGF/VEGFR2 signaling pathway by abrogating VEGFR2 protein expression. This reduction in VEGFR2 protein expression was, in turn, caused by impaired VEGFR2 mRNA expression in C81-treated cells. Taken together, these results indicate that C81 impairs angiogenesis by downregulating VEGFR2 expression. To find the targets responsible for the C81-induced effects, we tested established chemical probes against the targeted kinases. This revealed that only inhibition of CLKs resulted in a significant downregulation of VEGFR2. Subsequently, we discovered that C81 is a pan-CLK inhibitor. Moreover, the CLK inhibitors MU1210 and T3-CLK were also able to phenocopy C81-derived effects on the sprouting of VEGF-stimulated HUVEC spheroids. These results suggest that the CLKs are the targets responsible for the anti-angiogenic effects of C81. This was confirmed by the knockdown of individual CLKs. Knockdowns of all CLK isoforms inhibited VEGFR2 mRNA expression and VEGF-induced sprouting of HUVEC spheroids. We could not identify an alternative splicing event associated with VEGFR2 in C81- or MU1210-treated HUVECs. Therefore, alternative splicing of VEGFR2 is unlikely to be the responsible mechanism. However, a GO term analysis followed by a reporter gene assay confirmed the previously published finding that CLK inhibitors abolish the WNT/β-catenin signaling pathway activity. This pathway has been shown to affect VEGFR2 expression in HUVECs and CNS endothelial cells. Therefore, we induced the activity of this pathway by using a GSK3β inhibitor, which increased the expression of VEGFR2. Conversely, knockdown of β-catenin inhibited VEGFR2 expression, confirming the involvement of this pathway. In summary, this work shows that CLK inhibitors are promising compounds in the treatment of angiogenesis related diseases. Especially in the therapy of inflammation related diseases and tumors they could be relevant, as CLK inhibitors have previously shown promising disease modifying properties in this context.