SignaLink3: a multi-layered resource to uncover tissue-specific signaling networks.

Signaling networks represent the molecular mechanisms controlling a cell's response to various internal or external stimuli. Most currently available signaling databases contain only a part of the complex network of intertwining pathways, leaving out key interactions or processes. Hence, we have developed SignaLink3 (http://signalink.org/), a value-added knowledge-base that provides manually curated data on signaling pathways and integrated data from several types of databases (interaction, regulation, localisation, disease, etc.) for humans, and three major animal model organisms. SignaLink3 contains over 400 000 newly added human protein-protein interactions resulting in a total of 700 000 interactions for Homo sapiens, making it one of the largest integrated signaling network resources. Next to H. sapiens, SignaLink3 is the only current signaling network resource to provide regulatory information for the model species Caenorhabditis elegans and Danio rerio, and the largest resource for Drosophila melanogaster. Compared to previous versions, we have integrated gene expression data as well as subcellular localization of the interactors, therefore uniquely allowing tissue-, or compartment-specific pathway interaction analysis to create more accurate models. Data is freely available for download in widely used formats, including CSV, PSI-MI TAB or SQL.

The paradise fish, an advanced animal model for behavioral genetics and evolutionary developmental biology.

Paradise fish (Macropodus opercularis) is an air-breathing freshwater fish species with a signature labyrinth organ capable of extracting oxygen from the air that helps these fish to survive in hypoxic environments. The appearance of this evolutionary innovation in anabantoids resulted in a rewired circulatory system, but also in the emergence of species-specific behaviors, such as territorial display, courtship and parental care in the case of the paradise fish. Early zoologists were intrigued by the structure and function of the labyrinth apparatus and a series of detailed descriptive histological studies at the beginning of the 20th century revealed the ontogenesis and function of this specialized system. A few decades later, these fish became the subject of numerous ethological studies, and detailed ethograms of their behavior were constructed. These latter studies also demonstrated a strong genetic component underlying their behavior, but due to lack of adequate molecular tools, the fine genetic dissection of the behavior was not possible at the time. The technological breakthroughs that transformed developmental biology and behavioral genetics in the past decades, however, give us now a unique opportunity to revisit these old questions. Building on the classic descriptive studies, the new methodologies will allow us to follow the development of the labyrinth apparatus at a cellular resolution, reveal the genes involved in this process and also the genetic architecture behind the complex behaviors that we can observe in this species.

Pseudouridylation defect due to DKC1 and NOP10 mutations causes nephrotic syndrome with cataracts, hearing impairment, and enterocolitis.

RNA modifications play a fundamental role in cellular function. Pseudouridylation, the most abundant RNA modification, is catalyzed by the H/ACA small ribonucleoprotein (snoRNP) complex that shares four core proteins, dyskerin (DKC1), NOP10, NHP2, and GAR1. Mutations in DKC1, NOP10, or NHP2 cause dyskeratosis congenita (DC), a disorder characterized by telomere attrition. Here, we report a phenotype comprising nephrotic syndrome, cataracts, sensorineural deafness, enterocolitis, and early lethality in two pedigrees: males with DKC1 p.Glu206Lys and two children with homozygous NOP10 p.Thr16Met. Females with heterozygous DKC1 p.Glu206Lys developed cataracts and sensorineural deafness, but nephrotic syndrome in only one case of skewed X-inactivation. We found telomere attrition in both pedigrees, but no mucocutaneous abnormalities suggestive of DC. Both mutations fall at the dyskerin-NOP10 binding interface in a region distinct from those implicated in DC, impair the dyskerin-NOP10 interaction, and disrupt the catalytic pseudouridylation site. Accordingly, we found reduced pseudouridine levels in the ribosomal RNA (rRNA) of the patients. Zebrafish dkc1 mutants recapitulate the human phenotype and show reduced 18S pseudouridylation, ribosomal dysregulation, and a cell-cycle defect in the absence of telomere attrition. We therefore propose that this human disorder is the consequence of defective snoRNP pseudouridylation and ribosomal dysfunction.

Dean-Flow Affected Lateral Focusing and Separation of Particles and Cells in Periodically Inhomogeneous Microfluidic Channels.

The purpose of the recent work is to give a better explanation of how Dean vortices affect lateral focusing, and to understand how cell morphology can alter the focusing position compared to spherical particles. The position and extent of the focused region were investigated using polystyrene fluorescent beads with different bead diameters (Ø = 0.5, 1.1, 1.97, 2.9, 4.8, 5.4, 6.08, 10.2, 15.8, 16.5 µm) at different flow rates (0.5, 1, 2 µL/s). Size-dependent focusing generated a precise map of the equilibrium positions of the spherical beads at the end of the periodically altering channels, which gave a good benchmark for focusing multi-dimensional particles and cells. The biological samples used for experiments were rod-shaped Escherichia coli (E. coli), discoid biconcave-shaped red blood cells (RBC), round or ovoid-shaped yeast, Saccharomyces cerevisiae, and soft-irregular-shaped HeLa cancer-cell-line cells to understand how the shape of the cells affects the focusing position at the end of the channel.

Geometry-Dependent Efficiency of Dean-Flow Affected Lateral Particle Focusing and Separation in Periodically Inhomogeneous Microfluidic Channels.

In this study, inertial focusing phenomenon was investigated, which can be used as a passive method for sample preparation and target manipulation in case of particulate suspensions. Asymmetric channel geometry was designed to apply additional inertial forces besides lift forces to promote laterally ordered particles to achieve sheathless focusing or size-dependent sorting. The evolving hydrodynamic forces were tailored with altered channel parameters (width and height), and different flow rates, to get a better understanding of smaller beads' lateral migration. Fluorescent beads (with the diameter of 4.8 µm and 15.8 µm) were used to distinguish the focusing position in continuous flow, and experimental results were compared to in silico models for particle movement prediction, made in COMSOL Multiphysics. The focusing behaviour of the applied microfluidic system was mainly characterised for particle size in the range close to blood cells and bacteria.

Conserved Serotonergic Background of Experience-Dependent Behavioral Responsiveness in Zebrafish (Danio rerio).

Forming effective responses to threatening stimuli requires the adequate and coordinated emergence of stress-related internal states. Such ability depends on early-life experiences and, in connection, the adequate formation of neuromodulatory systems, particularly serotonergic signaling. Here, we assess the serotonergic background of experience-dependent behavioral responsiveness using male and female zebrafish (Danio rerio). For the first time, we have characterized a period during behavioral metamorphosis in which zebrafish are highly reactive to their environment. Absence of social stimuli during this phase established by isolated rearing fundamentally altered the behavioral phenotype of postmetamorphic zebrafish in a challenge-specific manner, partially due to reduced responsiveness and an inability to develop stress-associated arousal state. In line with this, isolation differentially affected whole-brain serotonergic signaling in resting and stress-induced conditions, an effect that was localized in the dorsal pallium and was negatively associated with responsiveness. Administration of the serotonin receptor 1A partial agonist buspirone prevented the isolation-induced serotonin response to novelty in the level of the whole brain and the forebrain as well, without affecting catecholamine levels, and rescued stress-induced arousal along with challenge-induced behaviors, which together indicates functional connection between these changes. In summary, there is a consistent negative association between behavioral responsiveness and serotonergic signaling in zebrafish, which is well recognizable through the modifying effects of developmental perturbation and pharmacological manipulations as well. Our results imply a conserved serotonergic mechanism that context-dependently modulates environmental reactivity and is highly sensitive to experiences acquired during a specific early-life time window, a phenomenon that was previously only suggested in mammals.SIGNIFICANCE STATEMENT The ability to respond to challenges is a fundamental factor in survival. We show that zebrafish that lack appropriate social stimuli in a sensitive developmental period show exacerbated alertness in nonstressful conditions while failing to react adequately to stressors. This shift is reflected inversely by central serotonergic signaling, a system that is implicated in numerous mental disorders in humans. Serotonergic changes in brain regions modulating responsivity and behavioral impairment were both prevented by the pharmacological blockade of serotonergic function. These results imply a serotonergic mechanism in zebrafish that transmits early-life experiences to the later phenotype by shaping stress-dependent behavioral reactivity, a phenomenon that was previously only suggested in mammals. Zebrafish provide new insights into early-life-dependent neuromodulation of behavioral stress-responses.

Autophagy in zebrafish.

From a hitherto underappreciated phenomenon, autophagy has become one of the most intensively studied cellular processes in recent years. Its role in cellular homeostasis, development and disease is supported by a fast growing body of evidence. Surprisingly, only a small fraction of new observations regarding the physiological functions of cellular "self-digestion" comes from zebrafish, one of the most popular vertebrate model organisms. Here we review the existing information about autophagy reporter lines, genetic knock-down assays and small molecular reagents that have been tested in this system. As we argue, some of these tools have to be used carefully due to possible pleiotropic effects. However, when applied rigorously, in combination with novel mutant strains and genome editing techniques, they could also transform zebrafish into an important animal model of autophagy research.

Evidence for spontaneous level-2 perspective taking in adults.

Social interactions are fostered by humans' propensity to compute their partner's perspective online. However, due to the mindreading system's limited capacity perspective taking (PT) was argued to occur spontaneously only for level-1, but not level-2 perspectives. We propose that level-2 perspectives (containing aspectual information) can also be computed spontaneously if participants have reason to assume that the partner is indeed aware of the objects' aspectual properties. Pairs of adult participants took part in the modified version of Surtees, Butterfill, and Apperly's (2012) number verification paradigm. Participants had prior information on their partner's task, which either called for processing aspectual properties or did not. The partner's inconsistent perspective was found to interfere with RT-s providing evidence for spontaneous level-2 PT. However, such interference only occurred when the partner's task involved processing the perspective dependent object feature, suggesting that PT was sensitive to the other's awareness of the to be represented information.

Complex regulation of autophagy in cancer - integrated approaches to discover the networks that hold a double-edged sword.

Autophagy, a highly regulated self-degradation process of eukaryotic cells, is a context-dependent tumor-suppressing mechanism that can also promote tumor cell survival upon stress and treatment resistance. Because of this ambiguity, autophagy is considered as a double-edged sword in oncology, making anti-cancer therapeutic approaches highly challenging. In this review, we present how systems-level knowledge on autophagy regulation can help to develop new strategies and efficiently select novel anti-cancer drug targets. We focus on the protein interactors and transcriptional/post-transcriptional regulators of autophagy as the protein and regulatory networks significantly influence the activity of core autophagy proteins during tumor progression. We list several network resources to identify interactors and regulators of autophagy proteins. As in silico analysis of such networks often necessitates experimental validation, we briefly summarize tractable model organisms to examine the role of autophagy in cancer. We also discuss fluorescence techniques for high-throughput monitoring of autophagy in humans. Finally, the challenges of pharmacological modulation of autophagy are reviewed. We suggest network-based concepts to overcome these difficulties. We point out that a context-dependent modulation of autophagy would be favored in anti-cancer therapy, where autophagy is stimulated in normal cells, while inhibited only in stressed cancer cells. To achieve this goal, we introduce the concept of regulo-network drugs targeting specific transcription factors or miRNA families identified with network analysis. The effect of regulo-network drugs propagates indirectly through transcriptional or post-transcriptional regulation of autophagy proteins, and, as a multi-directional intervention tool, they can both activate and inhibit specific proteins in the same time. The future identification and validation of such regulo-network drug targets may serve as novel intervention points, where autophagy can be effectively modulated in cancer therapy.

para-Nitroblebbistatin, the non-cytotoxic and photostable myosin†II inhibitor.

Blebbistatin, the best characterized myosin II-inhibitor, is commonly used to study the biological roles of various myosin II isoforms. Despite its popularity, the use of blebbistatin is greatly hindered by its blue-light sensitivity, resulting in phototoxicity and photoconversion of the molecule. Additionally, blebbistatin has serious cytotoxic side effects even in the absence of irradiation, which may easily lead to the misinterpretation of experimental results since the cytotoxicity-derived phenotype could be attributed to the inhibition of the myosin II function. Here we report the synthesis as well as the in vitro and in vivo characterization of a photostable, C15 nitro derivative of blebbistatin with unaffected myosin II inhibitory properties. Importantly, para-nitroblebbistatin is neither phototoxic nor cytotoxic, as shown by cellular and animal tests; therefore it can serve as an unrestricted and complete replacement of blebbistatin both in vitro and in vivo.

Silicon-Rhodamine Functionalized Evocalcet Probes Potently and Selectively Label Calcium Sensing Receptors In Vitro, In Vivo, and Ex Vivo.

The calcium sensing receptor (CaSR) is a ubiquitously expressed G-protein coupled receptor (GPCR) that regulates extracellular calcium signals via the parathyroid glands. CaSR has recently also been implicated in noncalcitropic pathophysiologies like asthma, gut inflammation, and cancer. To date, molecular tools that enable the bioimaging of CaSR in tissues are lacking. Based on in silico analyses of available structure-activity relationship data on CaSR ligands, we designed and prepared silicon-rhodamine (SiR) conjugates of the clinically approved drug evocalcet. The new probes EvoSiR4 and EvoSiR6, with differing linker lengths at the evocalcet carboxyl end, both showed a 6-fold and 3-fold increase in potency toward CaSR (EC50 < 45 nM) compared to evocalcet and the evocalcet-linker conjugate, respectively, in an FLIPR-based cellular functional assay. The specificity of the EvoSiR probes toward CaSR binding and the impact of albumin was evaluated in live cell experiments. Both probes showed strong albumin binding, which facilitated the clearance of nonspecific binding interactions. Accordingly, in zebrafish embryos, EvoSiR4 specifically labeled the high CaSR expressing neuromasts of the lateral line in vivo. EvoSiR4 was also assessed in human parathyroid tissues ex vivo, showing a specific absolute CaSR-associated fluorescence compared to that of parathyroid autofluorescence. In summary, functionalization of evocalcet by SiR led to the preparation of potent and specific fluorescent CaSR probes. EvoSiR4 is a versatile small-molecular probe that can be employed in CaSR-related biomedical analyses where antibodies are not applicable.

Age-dependent heat shock hormesis to HSF-1 deficiency suggests a compensatory mechanism mediated by the unfolded protein response and innate immunity in young Caenorhabditis elegans.

The transcription factor HSF-1 (heat shock factor 1) acts as a master regulator of heat shock response in eukaryotic cells to maintain cellular proteostasis. The protein has a protective role in preventing cells from undergoing ageing, and neurodegeneration, and also mediates tumorigenesis. Thus, modulating HSF-1 activity in humans has a promising therapeutic potential for treating these pathologies. Loss of HSF-1 function is usually associated with impaired stress tolerance. Contrary to this conventional knowledge, we show here that inactivation of HSF-1 in the nematode Caenorhabditis elegans results in increased thermotolerance at young adult stages, whereas HSF-1 deficiency in animals passing early adult stages indeed leads to decreased thermotolerance, as compared to wild-type. Furthermore, a gene expression analysis supports that in young adults, distinct cellular stress response and immunity-related signaling pathways become induced upon HSF-1 deficiency. We also demonstrate that increased tolerance to proteotoxic stress in HSF-1-depleted young worms requires the activity of the unfolded protein response of the endoplasmic reticulum and the SKN-1/Nrf2-mediated oxidative stress response pathway, as well as an innate immunity-related pathway, suggesting a mutual compensatory interaction between HSF-1 and these conserved stress response systems. A similar compensatory molecular network is likely to also operate in higher animal taxa, raising the possibility of an unexpected outcome when HSF-1 activity is manipulated in humans.

The reference genome of Macropodus opercularis (the paradise fish).

Amongst fishes, zebrafish (Danio rerio) has gained popularity as a model system over most other species and while their value as a model is well documented, their usefulness is limited in certain fields of research such as behavior. By embracing other, less conventional experimental organisms, opportunities arise to gain broader insights into evolution and development, as well as studying behavioral aspects not available in current popular model systems. The anabantoid paradise fish (Macropodus opercularis), an "air-breather" species has a highly complex behavioral repertoire and has been the subject of many ethological investigations but lacks genomic resources. Here we report the reference genome assembly of M. opercularis using long-read sequences at 150-fold coverage. The final assembly consisted of 483,077,705 base pairs (~483 Mb) on 152 contigs. Within the assembled genome we identified and annotated 20,157 protein coding genes and assigned ~90% of them to orthogroups.

The role of post-transcriptional modifications during development.

While the existence of post-transcriptional modifications of RNA nucleotides has been known for decades, in most RNA species the exact positions of these modifications and their physiological function have been elusive until recently. Technological advances, such as high-throughput next-generation sequencing (NGS) methods and nanopore-based mapping technologies, have made it possible to map the position of these modifications with single nucleotide accuracy, and genetic screens have uncovered the "writer", "reader" and "eraser" proteins that help to install, interpret and remove such modifications, respectively. These discoveries led to intensive research programmes with the aim of uncovering the roles of these modifications during diverse biological processes. In this review, we assess novel discoveries related to the role of post-transcriptional modifications during animal development, highlighting how these discoveries can affect multiple aspects of development from fertilization to differentiation in many species.

Trehalose-Bearing Carriers to Target Impaired Autophagy and Protein Aggregation Diseases.

In recent years, trehalose, a natural disaccharide, has attracted growing attention because of the discovery of its potential to induce autophagy. Trehalose has also been demonstrated to preserve the protein's structural integrity and to limit the aggregation of pathologically misfolded proteins. Both of these properties have made trehalose a promising therapeutic candidate to target autophagy-related disorders and protein aggregation diseases. Unfortunately, trehalose has poor bioavailability due to its hydrophilic nature and susceptibility to enzymatic degradation. Recently, trehalose-bearing carriers, in which trehalose is incorporated either by chemical conjugation or physical entrapment, have emerged as an alternative option to free trehalose to improve its efficacy, particularly for the treatment of neurodegenerative diseases, atherosclerosis, nonalcoholic fatty liver disease (NAFLD), and cancers. In the current Perspective, we discuss all existing literature in this emerging field and try to identify key challenges for researchers intending to develop trehalose-bearing carriers to stimulate autophagy or inhibit protein aggregation.

3D cell segregation geometry and dynamics are governed by tissue surface tension regulation.

Tissue morphogenesis and patterning during development involve the segregation of cell types. Segregation is driven by differential tissue surface tensions generated by cell types through controlling cell-cell contact formation by regulating adhesion and actomyosin contractility-based cellular cortical tensions. We use vertebrate tissue cell types and zebrafish germ layer progenitors as in vitro models of 3-dimensional heterotypic segregation and developed a quantitative analysis of their dynamics based on 3D time-lapse microscopy. We show that general inhibition of actomyosin contractility by the Rho kinase inhibitor Y27632 delays segregation. Cell type-specific inhibition of non-muscle myosin2 activity by overexpression of myosin assembly inhibitor S100A4 reduces tissue surface tension, manifested in decreased compaction during aggregation and inverted geometry observed during segregation. The same is observed when we express a constitutively active Rho kinase isoform to ubiquitously keep actomyosin contractility high at cell-cell and cell-medium interfaces and thus overriding the interface-specific regulation of cortical tensions. Tissue surface tension regulation can become an effective tool in tissue engineering.

Using Selective Enzymes to Measure Noncanonical DNA Building Blocks: dUTP, 5-Methyl-dCTP, and 5-Hydroxymethyl-dCTP.

Cells maintain a fine-tuned balance of deoxyribonucleoside 5'-triphosphates (dNTPs), a crucial factor in preserving genomic integrity. Any alterations in the nucleotide pool's composition or chemical modifications to nucleotides before their incorporation into DNA can lead to increased mutation frequency and DNA damage. In addition to the chemical modification of canonical dNTPs, the cellular de novo dNTP metabolism pathways also produce noncanonical dNTPs. To keep their levels low and prevent them from incorporating into the DNA, these noncanonical dNTPs are removed from the dNTP pool by sanitizing enzymes. In this study, we introduce innovative protocols for the high-throughput fluorescence-based quantification of dUTP, 5-methyl-dCTP, and 5-hydroxymethyl-dCTP. To distinguish between noncanonical dNTPs and their canonical counterparts, specific enzymes capable of hydrolyzing either the canonical or noncanonical dNTP analogs are employed. This approach provides a more precise understanding of the composition and noncanonical constituents of dNTP pools, facilitating a deeper comprehension of DNA metabolism and repair. It is also crucial for accurately interpreting mutational patterns generated through the next-generation sequencing of biological samples.

The reference genome of the paradise fish (Macropodus opercularis).

Over the decades, a small number of model species, each representative of a larger taxa, have dominated the field of biological research. Amongst fishes, zebrafish (Danio rerio) has gained popularity over most other species and while their value as a model is well documented, their usefulness is limited in certain fields of research such as behavior. By embracing other, less conventional experimental organisms, opportunities arise to gain broader insights into evolution and development, as well as studying behavioral aspects not available in current popular model systems. The anabantoid paradise fish (Macropodus opercularis), an "air-breather" species from Southeast Asia, has a highly complex behavioral repertoire and has been the subject of many ethological investigations, but lacks genomic resources. Here we report the reference genome assembly of Macropodus opercularis using long-read sequences at 150-fold coverage. The final assembly consisted of ≈483 Mb on 152 contigs. Within the assembled genome we identified and annotated 20,157 protein coding genes and assigned ≈90% of them to orthogroups. Completeness analysis showed that 98.5% of the Actinopterygii core gene set (ODB10) was present as a complete ortholog in our reference genome with a further 1.2 % being present in a fragmented form. Additionally, we cloned multiple genes important during early development and using newly developed in situ hybridization protocols, we showed that they have conserved expression patterns.

Gene expression changes during the evolution of the tetrapod limb.

Major changes in the vertebrate anatomy have preceded the conquest of land by the members of this taxon, and continuous changes in limb shape and use have occurred during the later radiation of tetrapods. While the main, conserved mechanisms of limb development have been discerned over the past century using a combination of classical embryological and molecular methods, only recent advances made it possible to identify and study the regulatory changes that have contributed to the evolution of the tetrapod appendage. These advances include the expansion of the model repertoire from traditional genetic model species to non-conventional ones, a proliferation of predictive mathematical models that describe gene interactions, an explosion in genomic data and the development of high-throughput methodologies. These revolutionary innovations make it possible to identify specific mutations that are behind specific transitions in limb evolution. Also, as we continue to apply them to more and more extant species, we can expect to gain a fine-grained view of this evolutionary transition that has been so consequential for our species as well.

Trehalose-releasing nanogels: A step toward a trehalose delivery vehicle for autophagy stimulation.

Trehalose has been widely studied as a treatment for a variety of human disorders due to its ability to stimulate autophagy. Trehalose, however, is poorly adsorbed and is hydrolyzed in the intestinal mucosa, and oral delivery requires relatively high doses to induce autophagy. The parenteral injection of trehalose-releasing nanogels proposed in this study offers an alternative mode of delivery. This study aimed to develop stable colloidal dispersions of trehalose-rich nanogels that could sustainably release trehalose under physiologically relevant conditions. The nanogel design was based on the covalent incorporation of 6-O-acryloyl-trehalose within a polymer network. A series of nine trehalose-rich nanogels with highly conjugated trehalose (up to 59 % w/w) were synthesized and shown to sustainably release trehalose at a rate that is not dose dependent. The nanogels were optimized to keep colloidal stability in serum-enriched cell culture media. The stable nanogels were not cytotoxic to primary HUVECs. Two selected nanogels with opposite surface charges were subjected to extended in vitro characterization that included a cellular uptake study and a hemocompatibility assay. Both nanogels were efficiently taken up by HUVECs during a short incubation. They also proved not to be hemolytic to human RBCs in concentrations up to 2.0 mg/mL. Finally, an in vivo autophagy stimulation study employing transgenic zebrafish and Drosophila larvae demonstrated that prolonged exposure to a cationic trehalose-releasing nanogel can induce autophagic activity in in vivo systems without any detectable toxicity.