Bessel beam illumination reduces random and systematic errors in quantitative functional studies using light-sheet microscopy

Light-sheet microscopy (LSM), in combination with intrinsically transparent zebrafish larvae, is a choice method to observe brain function with high frame rates at cellular resolution. Inherently to LSM, however, residual opaque objects cause stripe artifacts, which obscure features of interest and, during functional imaging, modulate fluorescence variations related to neuronal activity. Here, we report how Bessel beams reduce streaking artifacts and produce high-fidelity quantitative data demonstrating a fivefold increase in sensitivity to calcium transients and a 20 fold increase in accuracy in the detection of activity correlations in functional imaging. Furthermore, using principal component analysis, we show that measurements obtained with Bessel beams are clean enough to reveal in one-shot experiments correlations that can not be averaged over trials after stimuli as is the case when studying spontaneous activity. Our results not only demonstrate the contamination of data by systematic and random errors through conventional Gaussian illumination and but,furthermore, quantify the increase in fidelity of such data when using Bessel beams

Designer rhamnolipid production

Rhamnolipids are biosurfactants featuring surface-active properties that render them suitable for a broad range of applications, e.g., in detergents, food, bioremediation, medicine/pharmacology, and crop science. These properties include their emulsification and foaming capacities and their ability to lower the surface tension. Further, aspects like biocompatibility and environmental friendliness, both features of rhamnolipids [1] are becoming increasingly important. Rhamnolipids thus constitute suitable substitutes for synthetic surfactants produced from fossil resources. Native producers of rhamnolipids are mainly pathogenic bacteria like Pseudomonas aeruginosa. We previously designed and constructed a recombinant Pseudomonas putida KT2440, which synthesizes rhamnolipids by decoupling production from host-intrinsic regulations and cell growth [2]. As most biosurfactants, rhamnolipids are synthesized in mixtures. We here show our approach to alter the native mixture of surfactant molecules to produce specific new-to-nature combinations. The molecular structure (Figure 1) can on the one hand be altered in the hydrophilic moiety by changing the number of rhamnose molecules. We achieved this by using only distinct genes from the native rhamnolipid synthesis pathway. On the other hand, we were also able to change the length of the fatty acids in the hydrophobic part. This chain length is determined by the acyl-transferase (RhlA). Using rhlA genes from different organisms enables our microbial cell factory to synthesize molecules with different chain lengths [3]. The different molecular structures have further been shown to feature diverse physico-chemical properties [4]. Exploiting the natural structural diversity will thus allow for the synthesis of designer rhamnolipids tailormade for specific applications. We thus present a novel approach to use biochemical engineering to create tailormade products for a more sustainable future. Please click Additional Files below to see the full abstract

Optical mapping of neuronal activity during seizures in zebrafish

Mapping neuronal activity during the onset and propagation of epileptic seizures can provide a better understanding of the mechanisms underlying this pathology and improve our approaches to the development of new drugs. Recently, zebrafish has become an important model for studying epilepsy both in basic research and in drug discovery. Here, we employed a transgenic line with pan-neuronal expression of the genetically-encoded calcium indicator GCaMP6s to measure neuronal activity in zebrafish larvae during seizures induced by pentylenetretrazole (PTZ). With this approach, we mapped neuronal activity in different areas of the larval brain, demonstrating the high sensitivity of this method to different levels of alteration, as induced by increasing PTZ concentrations, and the rescuing effect of an anti-epileptic drug. We also present simultaneous measurements of brain and locomotor activity, as well as a high-throughput assay, demonstrating that GCaMP measurements can complement behavioural assays for the detection of subclinical epileptic seizures, thus enabling future investigations on human hypomorphic mutations and more effective drug screening methods. Notably, the methodology described here can be easily applied to the study of many human neuropathologies modelled in zebrafish, allowing a simple and yet detailed investigation of brain activity alterations associated with the pathological phenotype

BEL \u3b2\u2010Trefoil Reduces the Migration Ability of RUNX2 Expressing Melanoma Cells in Xenotransplanted Zebrafish

RUNX2, a master osteogenic transcript ion factor, is overexpressed in several cancer cells; in melanoma it promotes cells migration and invasion as well as neoangiogenesis. The annual mortality rates related to metastatic melanoma are high and novel agents are needed to improve melanoma patients\u2019 survival. It has been shown that lectins specifically target malignant cells since they present the Thomsen\u2013Friedenreich antigen. This disaccharide is hidden in normal cells, while it allows selective lectins binding in transformed cells. Recently, an edible lectin named BEL \u3b2-trefoil has been obtained from the wild mushroom Boletus edulis. Our previous study showed BEL \u3b2-trefoil effects on transcription factor RUNX2 downregulation as well as on the migration ability in melanoma cells treated in vitro. Therefore, to better understand the role of this lectin, we investigated the BEL \u3b2-trefoil effects in a zebrafish in vivo model, transplanted with human melanoma cells expressing RUNX2. Our data showed that BEL \u3b2-trefoil is able to spread in the tissues and to reduce the formation of metastases in melanoma xenotransplanted zebrafish. In conclusion, BEL \u3b2-trefoil can be considered an effective biomolecule to counteract melanoma disease

Capturing the facets of evolvability in a mechanistic framework

‘Evolvability’ – the ability to undergo adaptive evolution – is a key concept for understanding and predicting the response of biological systems to environmental change. Evolvability has various facets and is applied in many ways, easily leading to misunderstandings among researchers. To clarify matters, we first categorize the mechanisms and organismal features underlying evolvability into determinants providing variation, determinants shaping the effect of variation on fitness, and determinants shaping the selection process. Second, we stress the importance of timescale when studying evolvability. Third, we distinguish between evolvability determinants with a broad and a narrow scope. Finally, we highlight two contrasting perspectives on evolvability: general evolvability and specific evolvability. We hope that this framework facilitates communication and guides future research

Comparison between control-based continuation and phase-locked loop methods for the identification of backbone curves and nonlinear frequency responses

Control-based continuation (CBC) and phase-locked loops (PLL) are two experimental testing methods that have demonstrated great potential for the non-parametric identification of key nonlinear dynamic features such as nonlinear frequency responses and backbone curves. Both CBC and PLL exploit stabilizing feedback control to steer the dynamics of the tested system towards the responses of interest and overcome important difficulties experienced when applying conventional testing methods such as sine sweeps to nonlinear systems. For instance, if properly designed, the feedback controller can prevent the system from exhibiting untimely transitions between coexisting responses or even losing stability due to bifurcations. This contribution aims to highlight the similarities that exist between CBC and PLL and present the first thorough comparison of their capabilities. Comparisons are supported by numerical simulations as well as experimental data collected on a conceptually simple nonlinear structure primarily composed of a thin curved beam. The beam is doubly clamped and exhibits nonlinear geometric effects for moderate excitation amplitudes

Comparison between control-based continuation and phase-locked loop methods for the identification of backbone curves and nonlinear frequency responses

Control-based continuation (CBC) and phase-locked loops (PLL) are two experimental testing methods that have demonstrated great potential for the non-parametric identification of key nonlinear dynamic features such as nonlinear frequency responses and backbone curves. Both CBC and PLL exploit stabilizing feedback control to steer the dynamics of the tested system towards the responses of interest and overcome important difficulties experienced when applying conventional testing methods such as sine sweeps to nonlinear systems. For instance, if properly designed, the feedback controller can prevent the system from exhibiting untimely transitions between coexisting responses or even losing stability due to bifurcations. This contribution aims to highlight the similarities that exist between CBC and PLL and present the first thorough comparison of their capabilities. Comparisons are supported by numerical simulations as well as experimental data collected on a conceptually simple nonlinear structure primarily composed of a thin curved beam. The beam is doubly clamped and exhibits nonlinear geometric effects for moderate excitation amplitudes

Development and first operation of a Cavity Ring Down Spectroscopy diagnostic in the negative ion source SPIDER

The Neutral Beam Injectors of the ITER experiment will rely on negative ion sources to produce 16.7 MW beams of H/D particles accelerated at 1 MeV. The prototype of these sources was built and is currently operated in the SPIDER experiment (Source for the Production of Ions o Deuterium Extracted from an RF plasma), part of the Neutral Beam Test Facility of Consorzio RFX, Padua. In SPIDER, the H-/D- ion source is coupled to a three grids 100 kV acceleration system. One of the main targets of the experimentation in SPIDER is to uniformly maximize the extracted current density; to achieve this it is important to study the density of negative ions available in proximity of the ion acceleration system. In SPIDER, line-integrated measurements of negative ion density are performed by a Cavity Ring Down Spectroscopy (CRDS) diagnostic. Its principle of operation is based on the absorption of the photons of a laser beam pulse by H-/D- photo-detachment; the absorption detection is enhanced by trapping the laser pulse in an optical cavity, containing the absorbing medium (i.e. negative ions). The paper presents and discusses the CRDS diagnostic setup in SPIDER, including the first measurements of negative ion density, correlated to the main source parameters.Comment: 5 pages, 7 figures. Contributer paper for the HTPD 2020 conference. Accepted manuscrip

Mutations in the Cardiac Ryanodine Receptor Gene (hRyR2) Underlie Catecholaminergic Polymorphic Ventricular Tachycardia

BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia is a genetic arrhythmogenic disorder characterized by stress-induced, bidirectional ventricular tachycardia that may degenerate into cardiac arrest and cause sudden death. The electrocardiographic pattern of this ventricular tachycardia closely resembles the arrhythmias associated with calcium overload and the delayed afterdepolarizations observed during digitalis toxicity. We speculated that a genetically determined abnormality of intracellular calcium handling might be the substrate of the disease; therefore, we considered the human cardiac ryanodine receptor gene (hRyR2) a likely candidate for this genetically transmitted arrhythmic disorder. METHODS AND RESULTS: Twelve patients presenting with typical catecholaminergic polymorphic ventricular tachycardia in the absence of structural heart abnormalities were identified. DNA was extracted from peripheral blood lymphocytes, and single-strand conformation polymorphism analysis was performed on polymerase chain reaction-amplified exons of the hRyR2 gene. Four single nucleotide substitutions leading to missense mutations were identified in 4 probands affected by the disease. Genetic analysis of the asymptomatic parents revealed that 3 probands carried de novo mutations. In 1 case, the identical twin of the proband died suddenly after having suffered syncopal episodes. The fourth mutation was identified in the proband, in 4 clinically affected family members, and in none of 3 nonaffected family members in a kindred with 2 sudden deaths that occurred at 16 and 14 years, respectively, in the sisters of the proband. CONCLUSIONS: We demonstrated that, in agreement with our hypothesis, hRyR2 is a gene responsible for catecholaminergic polymorphic ventricular tachycardia

A GFP-Tagged Gross Deletion on Chromosome 1 Causes Malignant Peripheral Nerve Sheath Tumors and Carcinomas in Zebrafish

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive soft-tissue sarcomas, characterized by complex karyotypes. The molecular bases of such malignancy are poorly understood and efficient targeted molecular therapies are currently lacking. Here we describe a novel zebrafish model of MPNSTs, represented by the transgenic mutant line Tg(-8.5nkx2.2a:GFP)ia2. ia2 homozygous animals displayed embryonic lethality by 72 hpf, while the heterozygotes develop visible tumor masses with high frequency in adulthood. Histological and immunohistochemical examination revealed aggressive tumors with either mesenchymal or epithelial features. The former (54% of the cases) arose either in the abdominal cavity, or as intrathecal/intraspinal lesions and is composed of cytokeratin-negative spindle cells with fascicular/storiform growth pattern consistent with zebrafish MPNSTs. The second histotype was composed by polygonal or elongated cells, immunohistochemically positive for the pan-cytokeratin AE1/AE3. The overall histologic and immunohistochemical features were consistent with a malignant epithelial neoplasm of possible gastrointestinal/pancreatic origin. With an integrated approach, based on microsatellite (VNTR) and STS markers, we showed that ia2 insertion, in Tg(-8.5nkx2.2a:GFP)ia2 embryos, is associated with a deletion of 15.2 Mb in the telomeric portion of chromosome 1. Interestingly, among ia2 deleted genes we identified the presence of the 40S ribosomal protein S6 gene that may be one of the possible drivers for the MPNSTs in ia2 mutants.Thanks to the peculiar features of zebrafish as animal model of human cancer (cellular and genomic similarity, transparency and prolificacy) and the GFP tag, the Tg(-8.5nkx2.2a:GFP)ia2 line provides a manageable tool to study in vivo with high frequency MPNST biology and genetics, and to identify, in concert with the existing zebrafish MPNST models, conserved relevant mechanisms in zebrafish and human cancer development