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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Bloom syndrome helicase contributes to germ line development and longevity in zebrafish.

RecQ helicases-also known as the "guardians of the genome"-play crucial roles in genome integrity maintenance through their involvement in various DNA metabolic pathways. Aside from being conserved from bacteria to vertebrates, their importance is also reflected in the fact that in humans impaired function of multiple RecQ helicase orthologs are known to cause severe sets of problems, including Bloom, Werner, or Rothmund-Thomson syndromes. Our aim was to create and characterize a zebrafish (Danio rerio) disease model for Bloom syndrome, a recessive autosomal disorder. In humans, this syndrome is characterized by short stature, skin rashes, reduced fertility, increased risk of carcinogenesis, and shortened life expectancy brought on by genomic instability. We show that zebrafish blm mutants recapitulate major hallmarks of the human disease, such as shortened lifespan and reduced fertility. Moreover, similarly to other factors involved in DNA repair, some functions of zebrafish Blm bear additional importance in germ line development, and consequently in sex differentiation. Unlike fanc genes and rad51, however, blm appears to affect its function independent of tp53. Therefore, our model will be a valuable tool for further understanding the developmental and molecular attributes of this rare disease, along with providing novel insights into the role of genome maintenance proteins in somatic DNA repair and fertility.

Development and In-Depth Characterization of Bacteria Repellent and Bacteria Adhesive Antibody-Coated Surfaces Using Optical Waveguide Biosensing.

Bacteria repellent surfaces and antibody-based coatings for bacterial assays have shown a growing demand in the field of biosensors, and have crucial importance in the design of biomedical devices. However, in-depth investigations and comparisons of possible solutions are still missing. The optical waveguide lightmode spectroscopy (OWLS) technique offers label-free, non-invasive, in situ characterization of protein and bacterial adsorption. Moreover, it has excellent flexibility for testing various surface coatings. Here, we describe an OWLS-based method supporting the development of bacteria repellent surfaces and characterize the layer structures and affinities of different antibody-based coatings for bacterial assays. In order to test nonspecific binding blocking agents against bacteria, OWLS chips were coated with bovine serum albumin (BSA), I-block, PAcrAM-g-(PMOXA, NH2, Si), (PAcrAM-P) and PLL-g-PEG (PP) (with different coating temperatures), and subsequent Escherichia coli adhesion was monitored. We found that the best performing blocking agents could inhibit bacterial adhesion from samples with bacteria concentrations of up to 107 cells/mL. Various immobilization methods were applied to graft a wide range of selected antibodies onto the biosensor's surface. Simple physisorption, Mix&Go (AnteoBind) (MG) films, covalently immobilized protein A and avidin-biotin based surface chemistries were all fabricated and tested. The surface adsorbed mass densities of deposited antibodies were determined, and the biosensor;s kinetic data were evaluated to divine the possible orientations of the bacteria-capturing antibodies and determine the rate constants and footprints of the binding events. The development of affinity layers was supported by enzyme-linked immunosorbent assay (ELISA) measurements in order to test the bacteria binding capabilities of the antibodies. The best performance in the biosensor measurements was achieved by employing a polyclonal antibody in combination with protein A-based immobilization and PAcrAM-P blocking of nonspecific binding. Using this setting, a surface sensitivity of 70 cells/mm2 was demonstrated.

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.

Mutations linked to loss of cell cycle control can render cells responsive to local differentiation cues.

Cell behaviors such as survival, proliferation, and death are governed by a multitude of cues, both intrinsic and extrinsic. To test whether a wild-type environment could encourage the survival and/or differentiation of neuronal progenitor cells with impaired cell cycle progression, we transplanted cells from cdk1, dtl, slbp, fbxo5, ahctf1, gins2, hdac1, mcm5, ssrp1a, and rbbp6 mutant zebrafish embryos into wild-type embryos, creating chimeric zebrafish with mutant cells in the developing eye. We found that when cells from cdk1, dtl, slbp, gins2, mcm5, or rbbp6 mutants were transplanted into wild-type hosts, survival and/or differentiation was almost always compromised in a manner consistent with cell-autonomous cell death. Interestingly, we observed that fbxo5, ahctf1, hdac1, or ssrp1a mutant cells survived and sometimes exhibited signs of differentiation when grafted into wild-type eyes.

Housing, Husbandry and Welfare of a "Classic" Fish Model, the Paradise Fish (Macropodus opercularis).

Thanks to its small size, external fertilization and fecundity, over the past four decades, zebrafish (Danio rerio) has become the dominant fish model species in biological and biomedical research. Multiple lines of evidence, however, suggest that the reliance on only a handful of genetic model organisms is problematic, as their unique evolutionary histories makes them less than ideal to study biological questions unrelated to their historically contingent adaptations. Therefore, a need has emerged to develop novel model species, better suited for studying particular problems. The paradise fish (Macropodus opercularis) has a much more complex behavioral repertoire than zebrafish and has been a favored model animal in ethological research during the last decades of the previous century. We believe that with currently available, easily adaptable genetic toolkits, this species could be easily developed into a popular model of behavioral genetics. Despite its earlier popularity, however, the description of a detailed housing and husbandry protocol for this species is still missing from scientific literature. We present here a detailed description of how to raise and breed paradise fish successfully under laboratory conditions, and also discuss some of the challenges we faced while creating a stable breeding population for this species in our facility.

Subcellular Dissection of a Simple Neural Circuit: Functional Domains of the Mauthner-Cell During Habituation.

Sensorimotor integration is a pivotal feature of the nervous system for ensuring a coordinated motor response to external stimuli. In essence, such neural circuits can optimize behavioral performance based on the saliency of environmental cues. In zebrafish, habituation of the acoustic startle response (ASR) is a simple behavior integrated into the startle command neurons, called the Mauthner cells. Whereas the essential neuronal components that regulate the startle response have been identified, the principles of how this regulation is integrated at the subcellular regions of the Mauthner cell, which in turn modulate the performance of the behavior, is still not well understood. Here, we reveal mechanistically distinct dynamics of excitatory inputs converging onto the lateral dendrite (LD) and axon initial segment (AIS) of the Mauthner cell by in vivo imaging glutamate release using iGluSnFR, an ultrafast glutamate sensing fluorescent reporter. We find that modulation of glutamate release is dependent on NMDA receptor activity exclusively at the AIS, which is responsible for setting the sensitivity of the startle reflex and inducing a depression of synaptic activity during habituation. In contrast, glutamate-release at the LD is not regulated by NMDA receptors and serves as a baseline component of Mauthner cell activation. Finally, using in vivo calcium imaging at the feed-forward interneuron population component of the startle circuit, we reveal that these cells indeed play pivotal roles in both setting the startle threshold and habituation by modulating the AIS of the Mauthner cell. These results indicate that a command neuron may have several functionally distinct regions to regulate complex aspects of behavior.

A New Zebrafish Model for Pseudoxanthoma Elasticum.

Calcification of various tissues is a significant health issue associated with aging, cancer and autoimmune diseases. There are both environmental and genetic factors behind this phenomenon and understanding them is essential for the development of efficient therapeutic approaches. Pseudoxanthoma elasticum (PXE) is a rare genetic disease, a prototype for calcification disorders, resulting from the dysfunction of ABCC6, a transport protein found in the membranes of cells. It is identified by excess calcification in a variety of tissues (e.g., eyes, skin, arteries) and currently it has no cure, known treatments target the symptoms only. Preclinical studies of PXE have been successful in mice, proving the usefulness of animal models for the study of the disease. Here, we present a new zebrafish (Danio rerio) model for PXE. By resolving some ambiguous assemblies in the zebrafish genome, we show that there are two functional and one non-functional paralogs for ABCC6 in zebrafish (abcc6a, abcc6b.1, and abcc6b.2, respectively). We created single and double mutants for the functional paralogs and characterized their calcification defects with a combination of techniques. Zebrafish deficient in abcc6a show defects in their vertebral calcification and also display ectopic calcification foci in their soft tissues. Our results also suggest that the impairment of abcc6b.1 does not affect this biological process.

Tissue-Specific Requirement for the GINS Complex During Zebrafish Development.

Efficient and accurate DNA replication is particularly critical in stem and progenitor cells for successful proliferation and survival. The replisome, an amalgam of protein complexes, is responsible for binding potential origins of replication, unwinding the double helix, and then synthesizing complimentary strands of DNA. According to current models, the initial steps of DNA unwinding and opening are facilitated by the CMG complex, which is composed of a GINS heterotetramer that connects Cdc45 with the mini-chromosome maintenance (Mcm) helicase. In this work, we provide evidence that in the absence of GINS function DNA replication is cell autonomously impaired, and we also show that gins1 and gins2 mutants exhibit elevated levels of apoptosis restricted to actively proliferating regions of the central nervous system (CNS). Intriguingly, our results also suggest that the rapid cell cycles during early embryonic development in zebrafish may not require the function of the canonical GINS complex as neither zygotic Gins1 nor Gins2 isoforms seem to be present during these stages.

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.