TANGO1 inhibitors reduce collagen secretion and limit tissue scarring.

Uncontrolled secretion of ECM proteins, such as collagen, can lead to excessive scarring and fibrosis and compromise tissue function. Despite the widespread occurrence of fibrotic diseases and scarring, effective therapies are lacking. A promising approach would be to limit the amount of collagen released from hyperactive fibroblasts. We have designed membrane permeant peptide inhibitors that specifically target the primary interface between TANGO1 and cTAGE5, an interaction that is required for collagen export from endoplasmic reticulum exit sites (ERES). Application of the peptide inhibitors leads to reduced TANGO1 and cTAGE5 protein levels and a corresponding inhibition in the secretion of several ECM components, including collagens. Peptide inhibitor treatment in zebrafish results in altered tissue architecture and reduced granulation tissue formation during cutaneous wound healing. The inhibitors reduce secretion of several ECM proteins, including collagens, fibrillin and fibronectin in human dermal fibroblasts and in cells obtained from patients with a generalized fibrotic disease (scleroderma). Taken together, targeted interference of the TANGO1-cTAGE5 binding interface could enable therapeutic modulation of ERES function in ECM hypersecretion, during wound healing and fibrotic processes.

Dynamics of macrophage polarization support Salmonella persistence in a whole living organism.

Numerous intracellular bacterial pathogens interfere with macrophage function, including macrophage polarization, to establish a niche and persist. However, the spatiotemporal dynamics of macrophage polarization during infection within host remain to be investigated. Here, we implement a model of persistent Salmonella Typhimurium infection in zebrafish, which allows visualization of polarized macrophages and bacteria in real time at high resolution. While macrophages polarize toward M1-like phenotype to control early infection, during later stages, Salmonella persists inside non-inflammatory clustered macrophages. Transcriptomic profiling of macrophages showed a highly dynamic signature during infection characterized by a switch from pro-inflammatory to anti-inflammatory/pro-regenerative status and revealed a shift in adhesion program. In agreement with this specific adhesion signature, macrophage trajectory tracking identifies motionless macrophages as a permissive niche for persistent Salmonella. Our results demonstrate that zebrafish model provides a unique platform to explore, in a whole organism, the versatile nature of macrophage functional programs during bacterial acute and persistent infections.

Volumetric Temperature Mapping Using Light-Sheet Microscopy and Upconversion Fluorescence from Micro- and Nano-Rare Earth Composites.

We present a combination of light-sheet excitation and two-dimensional fluorescence intensity ratio (FIR) measurements as a simple and promising technique for three-dimensional temperature mapping. The feasibility of this approach is demonstrated with samples fabricated with sodium yttrium fluoride nanoparticles co-doped with rare-earth ytterbium and erbium ions (NaYF4:Yb3+/Er3+) incorporated into polydimethylsiloxane (PDMS) as a host material. In addition, we also evaluate the technique using lipid-coated NaYF4:Yb3+/Er3+ nanoparticles immersed in agar. The composite materials show upconverted (UC) fluorescence bands when excited by a 980 nm near-infrared laser light-sheet. Using a single CMOS camera and a pair of interferometric optical filters to specifically image the two thermally-coupled bands (at 525 and 550 nm), the two-dimensional FIR and, hence, the temperature map can be readily obtained. The proposed method can take optically sectioned (confocal-like) images with good optical resolution over relatively large samples (up to the millimetric scale) for further 3D temperature reconstruction.

Three-Photon Infrared Stimulation of Endogenous Neuroreceptors in Vivo.

To interrogate neural circuits and crack their codes, in vivo brain activity imaging must be combined with spatiotemporally precise stimulation in three dimensions using genetic or pharmacological specificity. This challenge requires deep penetration and focusing as provided by infrared light and multiphoton excitation, and has promoted two-photon photopharmacology and optogenetics. However, three-photon brain stimulation in vivo remains to be demonstrated. We report the regulation of neuronal activity in zebrafish larvae by three-photon excitation of a photoswitchable muscarinic agonist at 50 pM, a billion-fold lower concentration than used for uncaging, and with mid-infrared light of 1560 nm, the longest reported photoswitch wavelength. Robust, physiologically relevant photoresponses allow modulating brain activity in wild-type animals with spatiotemporal and pharmacological precision. Computational calculations predict that azobenzene-based ligands have high three-photon absorption cross-section and can be used directly with pulsed infrared light. The expansion of three-photon pharmacology will deeply impact basic neurobiology and neuromodulation phototherapies.

Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas.

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the impaired light-sensing photoreceptor cells in the degenerated retina, aiming to partially restore vision by inducing visual percepts. One common avenue of investigation involves the design and production of implantable devices with a flexible physical structure, housing a high number of electrodes. This enables the efficient and precise generation of visual percepts. However, with each technological advancement, there arises a need for a reliable and manageable ex vivo method to verify the functionality of the device before progressing to in vivo experiments, where factors beyond the device's performance come into play. This article presents a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer (GCL) following electrical stimulation. Specifically, the following steps are outlined: (1) fluorescently labeling the rat retina using genetically encoded calcium indicators, (2) capturing the fluorescence signal using an inverted fluorescence microscope while applying distinct patterns of electrical stimulation, and (3) extracting and analyzing the calcium traces from individual cells within the GCL. By following this procedure, researchers can efficiently test new stimulation protocols prior to conducting in vivo experiments.

Crop rotation with Meloidogyne-resistant germplasm is useful to manage and revert the (a)virulent populations of Mi1.2 gene and reduce yield losses.

A rotation sequence of ungrafted and grafted tomato-melon-pepper-watermelon on resistant rootstocks 'Brigeor', Cucumis metuliferus, 'Oscos' and Citrullus amarus, respectively, was carried out in a plastic greenhouse, ending with a susceptible or resistant tomato crop. The rotation was conducted in plots infested by an avirulent (Avi) or a partially virulent (Vi) Meloidogyne incognita population to the Mi1.2 gene. At the beginning of the study, the reproduction index (RI, relative reproduction in the resistant respect susceptible tomato) of Avi and Vi populations was 1.3% and 21.6%, respectively. Soil nematode density at transplanting (Pi) and at the end (Pf) of each crop, disease severity and crop yield were determined. Moreover, the putative virulence selection and fitness cost were determined at the end of each crop in pot tests. In addition, a histopathological study was carried out 15 days after nematode inoculation in pot test. The volume and number of nuclei per giant cell (GC) and the number of GC, their volume and the number of nuclei per feeding site in susceptible watermelon and pepper were compared with C. amarus and resistant pepper. At the beginning of the study, the Pi of Avi and Vi plots did not differ between susceptible and resistant germplasm. At the end of the rotation, the Pf of Avi was 1.2 the Pi in susceptible and 0.06 in resistant, the cumulative yield of grafted crops was 1.82 times higher than that of the ungrafted susceptible ones, and the RI in resistant tomato less than 10% irrespective of the rotation sequence. Concerning the Vi, Pf was below the detection level at the end of the rotation in resistant and 3 times Pi in the susceptible. The cumulative yield of grafted crops was 2.83 times higher than that of the ungrafted and the RI in resistant tomato was 7.6%, losing the population's virulence. In the histopathological study, no differences in number of GC per feeding site were observed in watermelon compared to C. amarus, but they were more voluminous and contained higher number of nuclei per GC and per feeding site. Regarding pepper, Avi population did not penetrate resistant rootstock.

Sphingolipid desaturase DEGS1 is essential for mitochondria-associated membrane integrity.

Sphingolipids function as membrane constituents and signaling molecules, with crucial roles in human diseases, from neurodevelopmental disorders to cancer, best exemplified in the inborn errors of sphingolipid metabolism in lysosomes. The dihydroceramide desaturase Δ4-dihydroceramide desaturase 1 (DEGS1) acts in the last step of a sector of the sphingolipid pathway, de novo ceramide biosynthesis. Defects in DEGS1 cause the recently described hypomyelinating leukodystrophy-18 (HLD18) (OMIM #618404). Here, we reveal that DEGS1 is a mitochondria-associated endoplasmic reticulum membrane-resident (MAM-resident) enzyme, refining previous reports locating DEGS1 at the endoplasmic reticulum only. Using patient fibroblasts, multiomics, and enzymatic assays, we show that DEGS1 deficiency disrupts the main core functions of the MAM: (a) mitochondrial dynamics, with a hyperfused mitochondrial network associated with decreased activation of dynamin-related protein 1; (b) cholesterol metabolism, with impaired sterol O-acyltransferase activity and decreased cholesteryl esters; (c) phospholipid metabolism, with increased phosphatidic acid and phosphatidylserine and decreased phosphatidylethanolamine; and (d) biogenesis of lipid droplets, with increased size and numbers. Moreover, we detected increased mitochondrial superoxide species production in fibroblasts and mitochondrial respiration impairment in patient muscle biopsy tissues. Our findings shed light on the pathophysiology of HLD18 and broaden our understanding of the role of sphingolipid metabolism in MAM function.

Infrared Laser Effects on Cell Projection Depend on Irradiation Intermittence and Cell Activity.

Highly focused near-infrared (NIR) lasers have been used to induce fibroblast and neuron protrusions in a technique called optical guidance. However, little is known about the biochemical and biophysical effects that the laser provokes in the cell and optimal protocols of stimulation have not yet been established. Using intermittent NIR laser radiation and multivariate time series representations of cell leading edge movement, we analyzed the direction and velocity of cell protrusions. We found that the orientation and advance of PC12 neuron phenotype cells and 3T3 fibroblasts protrusions remain after the laser is turned off, but the observed increase in velocity stops when radiation ceases. For an increase in the speed and distance of cell protrusions by NIR laser irradiation, the cell leading edge needs to be advancing prior to the stimulation, and NIR irradiation does not enable the cell to switch between retracting and advancing states. Using timelapse imaging of actin-GFP, we observed that NIR irradiation induces a faster recruitment of actin, promoting filament formation at the induced cell protrusions. These results provide fresh evidence to understand the phenomenon of the optical guidance of cell protrusions.

Volumetric imaging of fast cellular dynamics with deep learning enhanced bioluminescence microscopy.

Bioluminescence microscopy is an appealing alternative to fluorescence microscopy, because it does not depend on external illumination, and consequently does neither produce spurious background autofluorescence, nor perturb intrinsically photosensitive processes in living cells and animals. The low photon emission of known luciferases, however, demands long exposure times that are prohibitive for imaging fast biological dynamics. To increase the versatility of bioluminescence microscopy, we present an improved low-light microscope in combination with deep learning methods to image extremely photon-starved samples enabling subsecond exposures for timelapse and volumetric imaging. We apply our method to image subcellular dynamics in mouse embryonic stem cells, epithelial morphology during zebrafish development, and DAF-16 FoxO transcription factor shuttling from the cytoplasm to the nucleus under external stress. Finally, we concatenate neural networks for denoising and light-field deconvolution to resolve intracellular calcium dynamics in three dimensions of freely moving Caenorhabditis elegans.

Few-cycle all-fiber supercontinuum laser for ultrabroadband multimodal nonlinear microscopy.

Temporally coherent supercontinuum sources constitute an attractive alternative to bulk crystal-based sources of few-cycle light pulses. We present a monolithic fiber-optic configuration for generating transform-limited temporally coherent supercontinuum pulses with central wavelength at 1.06 µm and duration as short as 13.0 fs (3.7 optical cycles). The supercontinuum is generated by the action of self-phase modulation and optical wave breaking when pumping an all-normal dispersion photonic crystal fiber with pulses of hundreds of fs duration produced by all-fiber chirped pulsed amplification. Avoidance of free-space propagation between stages confers unequalled robustness, efficiency and cost-effectiveness to this novel configuration. Collectively, the features of all-fiber few-cycle pulsed sources make them powerful tools for applications benefitting from the ultrabroadband spectra and ultrashort pulse durations. Here we exploit these features and the deep penetration of light in biological tissues at the spectral region of 1 µm, to demonstrate their successful performance in ultrabroadband multispectral and multimodal nonlinear microscopy.

Modeling Biochemical Gradients In Vitro to Control Cell Compartmentalization in a Microengineered 3D Model of the Intestinal Epithelium.

Gradients of signaling pathways within the intestinal stem cell (ISC) niche are instrumental for cellular compartmentalization and tissue function, yet how are they sensed by the epithelium is still not fully understood. Here a new in vitro model of the small intestine based on primary epithelial cells (i), apically accessible (ii), with native tissue mechanical properties and controlled mesh size (iii), 3D villus-like architecture (iv), and precisely controlled biomolecular gradients of the ISC niche (v) is presented. Biochemical gradients are formed through hydrogel-based scaffolds by free diffusion from a source to a sink chamber. To confirm the establishment of spatiotemporally controlled gradients, light-sheet fluorescence microscopy and in-silico modeling are employed. The ISC niche biochemical gradients coming from the stroma and applied along the villus axis lead to the in vivo-like compartmentalization of the proliferative and differentiated cells, while changing the composition and concentration of the biochemical factors affects the cellular organization along the villus axis. This novel 3D in vitro intestinal model derived from organoids recapitulates both the villus-like architecture and the gradients of ISC biochemical factors, thus opening the possibility to study in vitro the nature of such gradients and the resulting cellular response.

Control of up-to-down/down-to-up light-induced ferroelectric polarization reversal.

Light control of ferroelectric polarization is of interest for the exploitation of ferroelectric thin films in ultrafast data storage and logic functionalities. The rapidly oscillating electric field of light absorbed in a ferroelectric layer can suppress its polarization but cannot selectively reverse its direction. Here we take advantage of the built-in asymmetry at ferroelectric/electrode interfaces to break the up/down symmetry in uniaxial ferroelectrics to promote polarization reversal under illumination. It is shown that appropriate ferroelectric/metal structures allow the direction of the imprint electric field to be selected, which is instrumental for polarization reversal. This ability is further exploited by demonstrating the optical control of the resistance states in a ferroelectric capacitor.

The Capillary Morphogenesis Gene 2 Triggers the Intracellular Hallmarks of Collagen VI-Related Muscular Dystrophy.

Collagen VI-related disorders (COL6-RD) represent a severe form of congenital disease for which there is no treatment. Dominant-negative pathogenic variants in the genes encoding α chains of collagen VI are the main cause of COL6-RD. Here we report that patient-derived fibroblasts carrying a common single nucleotide variant mutation are unable to build the extracellular collagen VI network. This correlates with the intracellular accumulation of endosomes and lysosomes triggered by the increased phosphorylation of the collagen VI receptor CMG2. Notably, using a CRISPR-Cas9 gene-editing tool to silence the dominant-negative mutation in patients' cells, we rescued the normal extracellular collagen VI network, CMG2 phosphorylation levels, and the accumulation of endosomes and lysosomes. Our findings reveal an unanticipated role of CMG2 in regulating endosomal and lysosomal homeostasis and suggest that mutated collagen VI dysregulates the intracellular environment in fibroblasts in collagen VI-related muscular dystrophy.

Focus variation due to near infrared laser in a confocal microscope.

Focus precision and stability is crucial in confocal microscopy not only for image sharpness but also to avoid radiometric fluctuations that can wrongly be interpreted as variations of the fluorescence intensity in the sample. Here we report a focus variation provoked by a continuous wave laser of 810-nm wavelength introduced along the optical path of an inverted confocal microscope with an oil immersion ×60 objective. When the laser is turned on or off, the focus position drifts toward lower or high values of the vertical coordinate z, respectively. The maximum drift observed was 2.25 ðœ‡m for a laser power of 40 mW at the sample and over a 600-s exposure time. The temporal evolution of the focus position is well fitted by exponential curves that mimic temperature variations due to a heat source. Our analysis strongly suggests that the focus drift is due to heating of the immersion oil.

Hippocampal Neuronal Cultures to Detect and Study New Pathogenic Antibodies Involved in Autoimmune Encephalitis.

Over the last 15 years, a new category of antibody-mediated diseases of the central nervous system (CNS) has been characterized and is now defined as "autoimmune encephalitis" (AE). There are currently 17 known AE syndromes, and all are associated with antibodies against the neuronal cell surface or synaptic proteins. The clinical syndromes are complex and vary according to the type of associated antibody. The best-known of these diseases is anti-N-methyl D-aspartate receptor (NMDAR) encephalitis, which is a prominent neuropsychiatric disorder associated with severe memory and behavioral impairments. The associated antibodies react with the GluN1 subunit of the NMDAR at the N-terminal domain. The approach most frequently used for the discovery and characterization of AE antibodies includes the culture of dissociated, fetal, rodent hippocampal neurons. During the process of antibody characterization, live neurons in culture are exposed to patients' serum or CSF, and the detection of reactivity indicates that the serum or CSF samples of the patient contain antibodies against neuronal surface antigens. Hippocampal cultures can also be used to determine whether the antibodies in patients are potentially pathogenic by examining if they cause structural or functional alterations of the neurons. The level of success of these studies depends on the quality of the cultures and on the protocols used to obtain and detect the reactivity of patient samples. This article provides an optimized protocol for primary cell culture of fetal rat hippocampal neurons combined with immunostaining to determine the presence of antibodies in the serum or CSF of patients. An example of how to examine the potential pathogenic effects of NMDAR antibodies using cultured neurons and calcium imaging is also presented.

Multi-modal and multi-scale clinical retinal imaging system with pupil and retinal tracking.

We present a compact multi-modal and multi-scale retinal imaging instrument with an angiographic functional extension for clinical use. The system integrates scanning laser ophthalmoscopy (SLO), optical coherence tomography (OCT) and OCT angiography (OCTA) imaging modalities and provides multi-scale fields of view. For high resolution, and high lateral resolution in particular, cellular imaging correction of aberrations by adaptive optics (AO) is employed. The entire instrument has a compact design and the scanning head is mounted on motorized translation stages that enable 3D self-alignment with respect to the subject's eye by tracking the pupil position. Retinal tracking, based on the information provided by SLO, is incorporated in the instrument to compensate for retinal motion during OCT imaging. The imaging capabilities of the multi-modal and multi-scale instrument were tested by imaging healthy volunteers and patients.

Deficiency of the ywhaz gene, involved in neurodevelopmental disorders, alters brain activity and behaviour in zebrafish.

Genetic variants in YWHAZ contribute to psychiatric disorders such as autism spectrum disorder and schizophrenia, and have been related to an impaired neurodevelopment in humans and mice. Here, we have used zebrafish to investigate the mechanisms by which YWHAZ contributes to neurodevelopmental disorders. We observed that ywhaz expression was pan-neuronal during developmental stages and restricted to Purkinje cells in the adult cerebellum, cells that are described to be reduced in number and size in autistic patients. We then performed whole-brain imaging in wild-type and ywhaz CRISPR/Cas9 knockout (KO) larvae and found altered neuronal activity and connectivity in the hindbrain. Adult ywhaz KO fish display decreased levels of monoamines in the hindbrain and freeze when exposed to novel stimuli, a phenotype that can be reversed with drugs that target monoamine neurotransmission. These findings suggest an important role for ywhaz in establishing neuronal connectivity during development and modulating both neurotransmission and behaviour in adults.

Human CASPR2 Antibodies Reversibly Alter Memory and the CASPR2 Protein Complex.

OBJECTIVE: The encephalitis associated with antibodies against contactin-associated proteinlike 2 (CASPR2) is presumably antibody-mediated, but the antibody effects and whether they cause behavioral alterations are not well known. Here, we used a mouse model of patients' immunoglobulin G (IgG) transfer and super-resolution microscopy to demonstrate the antibody pathogenicity. METHODS: IgG from patients with anti-CASPR2 encephalitis or healthy controls was infused into the cerebroventricular system of mice. The levels and colocalization of CASPR2 with transient axonal glycoprotein 1 (TAG1) were determined with stimulated emission depletion microscopy (40-70µm lateral resolution). Hippocampal clusters of Kv1.1 voltage-gated potassium channels (VGKCs) and GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) were quantified with confocal microscopy. Behavioral alterations were assessed with standard behavioral paradigms. Cultured neurons were used to determine the levels of intracellular CASPR2 and TAG1 after exposure to patients' IgG. RESULTS: Infusion of patients' IgG, but not controls' IgG, caused memory impairment along with hippocampal reduction of surface CASPR2 clusters and decreased CASPR2/TAG1 colocalization. In cultured neurons, patients' IgG led to an increase of intracellular CASPR2 without affecting TAG1, suggesting selective CASPR2 internalization. Additionally, mice infused with patients' IgG showed decreased levels of Kv1.1 and GluA1 (two CASPR2-regulated proteins). All these alterations and the memory deficit reverted to normal after removing patients' IgG. INTERPRETATION: IgG from patients with anti-CASPR2 encephalitis causes reversible memory impairment, inhibits the interaction of CASPR2/TAG1, and decreases the levels of CASPR2 and related proteins (VGKC, AMPAR). These findings fulfill the postulates of antibody-mediated disease and provide a biological basis for antibody-removing treatment approaches. ANN NEUROL 2022;91:801-813.

Multiple asters organize the yolk microtubule network during dclk2-GFP zebrafish epiboly.

It is known that the organization of microtubule (MT) networks in cells is orchestrated by subcellular structures named MT organizing centers (MTOCs). In this work, we use Light Sheet Fluorescence and Confocal Microscopy to investigate how the MT network surrounding the spherical yolk is arranged in the dclk2-GFP zebrafish transgenic line. We found that during epiboly the MT network is organized by multiple aster-like MTOCS. These structures form rings around the yolk sphere. Importantly, in wt embryos, aster-like MTOCs are only found upon pharmacological or genetic induction. Using our microscopy approach, we underscore the variability in the number of such asters in the transgenic line and report on the variety of global configurations of the yolk MT network. The asters' morphology, dynamics, and their distribution in the yolk sphere are also analyzed. We propose that these features are tightly linked to epiboly timing and geometry. Key molecules are identified which support this asters role as MTOCs, where MT nucleation and growth take place. We conclude that the yolk MT network of dclk2-GFP transgenic embryos can be used as a model to organize microtubules in a spherical geometry by means of multiple MTOCs.

Modular multimodal platform for classical and high throughput light sheet microscopy.

Light-sheet fluorescence microscopy (LSFM) has become an important tool for biological and biomedical research. Although several illumination and detection strategies have been developed, the sample mounting still represents a cumbersome procedure as this is highly dependent on the type of sample and often this might be time consuming. This prevents the use of LSFM in other promising applications in which a fast and straightforward sample-mounting procedure and imaging are essential. These include the high-throughput research fields, e.g. in drug screenings and toxicology studies. Here we present a new imaging paradigm for LSFM, which exploits modularity to offer multimodal imaging and straightforward sample mounting strategy, enhancing the flexibility and throughput of the system. We describe its implementation in which the sample can be imaged either as in any classical configuration, as it flows through the light-sheet using a fluidic approach, or a combination of both. We also evaluate its ability to image a variety of samples, from zebrafish embryos and larvae to 3D complex cell cultures.