Seamless knockins in Drosophila via CRISPR-triggered single-strand annealing.

CRISPR-Cas greatly facilitated the integration of exogenous sequences into specific loci. However, knockin generation in multicellular animals remains challenging, partially due to the complexity of insertion screening. Here, we describe SEED/Harvest, a method to generate knockins in Drosophila, based on CRISPR-Cas and the single-strand annealing (SSA) repair pathway. In SEED (from "scarless editing by element deletion"), a switchable cassette is first integrated into the target locus. In a subsequent CRISPR-triggered repair event, resolved by SSA, the cassette is seamlessly removed. Germline excision of SEED cassettes allows for fast and robust knockin generation of both fluorescent proteins and short protein tags in tandem. Tissue-specific expression of Cas9 results in somatic cassette excision, conferring spatiotemporal control of protein labeling and the conditional rescue of mutants. Finally, to achieve conditional protein labeling and manipulation of short tag knockins, we developed a genetic toolbox by functionalizing the ALFA nanobody.

Protocol for generating in-frame seamless knockins in Drosophila using the SEED/Harvest technology.

The generation of knockins is fundamental to dissect biological systems. SEED/Harvest, a technology based on CRISPR-Cas9, offers a powerful approach for seamless genome editing in Drosophila. Here, we present a protocol to tag any gene in the Drosophila genome using SEED/Harvest technology. We describe knockin design, plasmid preparation, injection, and insertion screening. We then detail procedures for germline harvesting. The technique combines straightforward cloning and robust screening of insertions, while still resulting in scarless gene editing. For complete details on the use and execution of this protocol, please refer to Aguilar et al.1.

Effect of lung volume preservation during spontaneous breathing trial on successful extubation in patients receiving mechanical ventilation: protocol for a multicenter clinical trial.

BACKGROUND: In standard weaning from mechanical ventilation, a successful spontaneous breathing test (SBT) consisting of 30 min 8 cmH2O pressure-support ventilation (PSV8) without positive end-expiratory pressure (PEEP) is followed by extubation with continuous suctioning; however, these practices might promote derecruitment. Evidence supports the feasibility and safety of extubation without suctioning. Ultrasound can assess lung aeration and respiratory muscles. We hypothesize that weaning aiming to preserve lung volume can yield higher rates of successful extubation. METHODS: This multicenter superiority trial will randomly assign eligible patients to receive either standard weaning [SBT: 30-min PSV8 without PEEP followed by extubation with continuous suctioning] or lung-volume-preservation weaning [SBT: 30-min PSV8 + 5 cmH2O PEEP followed by extubation with positive pressure without suctioning]. We will compare the rates of successful extubation and reintubation, ICU and hospital stays, and ultrasound measurements of the volume of aerated lung (modified lung ultrasound score), diaphragm and intercostal muscle thickness, and thickening fraction before and after successful or failed SBT. Patients will be followed for 90 days after randomization. DISCUSSION: We aim to recruit a large sample of representative patients (N = 1600). Our study cannot elucidate the specific effects of PEEP during SBT and of positive pressure during extubation; the results will show the joint effects derived from the synergy of these two factors. Although universal ultrasound monitoring of lungs, diaphragm, and intercostal muscles throughout weaning is unfeasible, if derecruitment is a major cause of weaning failure, ultrasound may help clinicians decide about extubation in high-risk and borderline patients. TRIAL REGISTRATION: The Research Ethics Committee (CEIm) of the Fundació Unió Catalana d'Hospitals approved the study (CEI 22/67 and 23/26). Registered at ClinicalTrials.gov in August 2023. Identifier: NCT05526053.

Unveiling Non-Covalent Interactions in Novel Cooperative Photoredox Systems for Efficient Alkene Oxidation in Water.

A new cooperative photoredox catalytic system, [RuII(trpy)(bpy)(H2O)][3,3'-Co(8,9,12-Cl3-1,2-C2B9H8)2]2, 5, has been synthesized and fully characterized for the first time. In this system, the photoredox catalyst [3,3'-Co(8,9,12-Cl3-1,2-C2B9H8)2]-[Cl6-1]-, a metallacarborane, and the oxidation catalyst [RuII(trpy)(bpy)(H2O)]2+, 2 are linked by non-covalent interactions. This compound, along with the one previously synthesized by us, [RuII(trpy)(bpy)(H2O)][(3,3'-Co(1,2-C2B9H11)2]2, 4, are the only examples of cooperative molecular photocatalysts in which the catalyst and photosensitizer are not linked by covalent bonds. Both cooperative systems have proven to be efficient photocatalysts for the oxidation of alkenes in water through Proton Coupled Electron Transfer processes (PCETs). Using 0.05 mol% of catalyst 4, total conversion values were achieved after 15 min with moderate selectivity for the corresponding epoxides, which decreases with reaction time, along with the TON values. However, with 0.005 mol% of catalyst, the conversion values are lower, but the selectivity and TON values are higher. This occurs simultaneously with an increase in the amount of the corresponding diol for most of the substrates studied. Photocatalyst 4 acts as a photocatalyst in both the epoxidation of alkenes and their hydroxylation in aqueous medium. The hybrid system 5 shows generally higher conversion values at low loads compared to those obtained with 4 for most of the substrates studied. However, the selectivity values for the corresponding epoxides are lower even after 15 min of reaction. This is likely due to the enhanced oxidizing capacity of CoIV in catalyst 5, resulting from the presence of more electron-withdrawing substituents on the metallacarborane platform.

Hedgehog on the Move: Glypican-Regulated Transport and Gradient Formation in Drosophila.

Glypicans (Glps) are a family of heparan sulphate proteoglycans that are attached to the outer plasma membrane leaflet of the producing cell by a glycosylphosphatidylinositol anchor. Glps are involved in the regulation of many signalling pathways, including those that regulate the activities of Wnts, Hedgehog (Hh), Fibroblast Growth Factors (FGFs), and Bone Morphogenetic Proteins (BMPs), among others. In the Hh-signalling pathway, Glps have been shown to be essential for ligand transport and the formation of Hh gradients over long distances, for the maintenance of Hh levels in the extracellular matrix, and for unimpaired ligand reception in distant recipient cells. Recently, two mechanistic models have been proposed to explain how Hh can form the signalling gradient and how Glps may contribute to it. In this review, we describe the structure, biochemistry, and metabolism of Glps and their interactions with different components of the Hh-signalling pathway that are important for the release, transport, and reception of Hh.

Predictive model for cytoneme guidance in Hedgehog signaling based on Ihog- Glypicans interaction.

During embryonic development, cell-cell communication is crucial to coordinate cell behavior, especially in the generation of differentiation patterns via morphogen gradients. Morphogens are signaling molecules secreted by a source of cells that elicit concentration-dependent responses in target cells. For several morphogens, cell-cell contact via filopodia-like-structures (cytonemes) has been proposed as a mechanism for their gradient formation. Despite of the advances on cytoneme signaling, little is known about how cytonemes navigate through the extracellular matrix and how they orient to find their target. For the Hedgehog (Hh) signaling pathway in Drosophila, Hh co-receptor and adhesion protein Interference hedgehog (Ihog) and the glypicans Dally and Dally-like-protein (Dlp) interact affecting the cytoneme behavior. Here, we describe that differences in the cytoneme stabilization and orientation depend on the relative levels of Ihog and glypicans, suggesting a mechanism for cytoneme guidance. Furthermore, we have developed a mathematical model to study and corroborate this cytoneme guiding mechanism.

Hedgehog on track: Long-distant signal transport and transfer through direct cell-to-cell contact.

The function of Hedgehog (Hh) as a morphogen results from its long-distance distribution from producing to neighboring receiving cells within the developing tissue. This signal distribution enables, for example, the formation of a concentration gradient eliciting distinct cellular responses that will give rise to spatial patterning. Hh is a lipid modified protein and its dispersion is better guaranteed through cytonemes, cell protrusions that allow direct cell membrane contact and signal transfer at a distance. Hh and its receptor Patched (Ptc) meet at cytoneme contacts in a way that reminds synapses. Both Hh and Ptc require a recycling process prior to presentation in cytonemes. Increasing research on the role of cytonemes in Hh signaling is revealing cellular mechanisms that link signal transport through dynamic cytonemes with concurrent regulation of cell adhesion. The equilibrium between these two processes is being unveiled as crucial to both patterned morphogen distribution and signal transfer. In addition, these discoveries are pushing forward our understanding of the role of extracellular elements involved in the Hh pathway, such as the Hh coreceptors Ihog and Boi and the glypicans Dally and Dally-like protein (Dlp).

Improving the understanding of cytoneme-mediated morphogen gradients by in silico modeling.

Morphogen gradients are crucial for the development of organisms. The biochemical properties of many morphogens prevent their extracellular free diffusion, indicating the need of an active mechanism for transport. The involvement of filopodial structures (cytonemes) has been proposed for morphogen signaling. Here, we describe an in silico model based on the main general features of cytoneme-meditated gradient formation and its implementation into Cytomorph, an open software tool. We have tested the spatial and temporal adaptability of our model quantifying Hedgehog (Hh) gradient formation in two Drosophila tissues. Cytomorph is able to reproduce the gradient and explain the different scaling between the two epithelia. After experimental validation, we studied the predicted impact of a range of features such as length, size, density, dynamics and contact behavior of cytonemes on Hh morphogen distribution. Our results illustrate Cytomorph as an adaptive tool to test different morphogen gradients and to generate hypotheses that are difficult to study experimentally.

Glypicans define unique roles for the Hedgehog co-receptors boi and ihog in cytoneme-mediated gradient formation.

The conserved family of Hedgehog (Hh) signaling proteins plays a key role in cell-cell communication in development, tissue repair, and cancer progression, inducing distinct concentration-dependent responses in target cells located at short and long distances. One simple mechanism for long distance dispersal of the lipid modified Hh is the direct contact between cell membranes through filopodia-like structures known as cytonemes. Here we have analyzed in Drosophila the interaction between the glypicans Dally and Dally-like protein, necessary for Hh signaling, and the adhesion molecules and Hh coreceptors Ihog and Boi. We describe that glypicans are required to maintain the levels of Ihog, but not of Boi. We also show that the overexpression of Ihog, but not of Boi, regulates cytoneme dynamics through their interaction with glypicans, the Ihog fibronectin III domains being essential for this interaction. Our data suggest that the regulation of glypicans over Hh signaling is specifically given by their interaction with Ihog in cytonemes. Contrary to previous data, we also show that there is no redundancy of Ihog and Boi functions in Hh gradient formation, being Ihog, but not of Boi, essential for the long-range gradient.

Aqueous Persistent Noncovalent Ion-Pair Cooperative Coupling in a Ruthenium Cobaltabis(dicarbollide) System as a Highly Efficient Photoredox Oxidation Catalyst.

An original cooperative photoredox catalytic system, [RuII(trpy)(bpy)(H2O)][3,3'-Co(1,2-C2B9H11)2]2 (C4; trpy = terpyridine and bpy = bipyridine), has been synthesized. In this system, the photoredox metallacarborane catalyst [3,3'-Co(1,2-C2B9H11)2]- ([1]-) and the oxidation catalyst [RuII(trpy)(bpy)(H2O)]2+ (C2') are linked by noncovalent interactions and not through covalent bonds. The noncovalent interactions to a large degree persist even after water dissolution. This represents a step ahead in cooperativity avoiding costly covalent bonding. Recrystallization of C4 in acetonitrile leads to the substitution of water by the acetonitrile ligand and the formation of complex [RuII(trpy)(bpy)(CH3CN)][3,3'-Co(1,2-C2B9H11)2]2 (C5), structurally characterized. A significant electronic coupling between C2' and [1]- was first sensed in electrochemical studies in water. The CoIV/III redox couple in water differed by 170 mV when [1]- had Na+ as a cation versus when the ruthenium complex was the cation. This cooperative system leads to an efficient catalyst for the photooxidation of alcohols in water, through a proton-coupled electron-transfer process. We have highlighted the capacity of C4 to perform as an excellent cooperative photoredox catalyst in the photooxidation of alcohols in water at room temperature under UV irradiation, using 0.005 mol % catalyst. A high turnover number (TON = 20000) has been observed. The hybrid system C4 displays a better catalytic performance than the separated mixtures of C2' and Na[1], with the same concentrations and ratios of Ru/Co, proving the history relevance of the photocatalyst. Cooperative systems with this type of interaction have not been described and represent a step forward in getting cooperativity avoiding costly covalent bonding. A possible mechanism has been proposed.

Noncovalently Linked Metallacarboranes on Functionalized Magnetic Nanoparticles as Highly Efficient, Robust, and Reusable Photocatalysts in Aqueous Medium.

A successful homogeneous photoredox catalyst has been fruitfully heterogenized on magnetic nanoparticles (MNPs) coated with a silica layer, keeping intact its homogeneous catalytic properties but gaining others due to the easy magnetic separation and recyclability. The amine-terminated magnetic silica nanoparticles linked noncovalently to H[3,3'-Co(1,2-C2B9H11)2]- (H[1]), termed MSNPs-NH2@H[1], are highly stable and do not produce any leakage of the photoredox catalyst H[1] in water. The magnetite MNPs were coated with SiO2 to provide colloidal stability and silanol groups to be tethered to amine-containing units. These were the MSNPs-NH2 on which was anchored, in water, the cobaltabis(dicarbollide) complex H[1] to obtain MSNPs-NH2@H[1]. Both MSNPs-NH2 and MSNPs-NH2@H[1] were evaluated to study the morphology, characterization, and colloidal stability of the MNPs produced. The heterogeneous MSNP-NH2@H[1] system was studied for the photooxidation of alcohols, such as 1-phenylethanol, 1-hexanol, 1,6-hexanediol, or cyclohexanol among others, using catalyst loads of 0.1 and 0.01 mol %. Surfactants were introduced to prevent the aggregation of MNPs, and cetyl trimethyl ammonium chloride was chosen as a surfactant. This provided adequate stability, without hampering quick magnetic separation. The results proved that the catalysis could be speeded up if aggregation was prevented. The recyclability of the catalytic system was demonstrated by performing 12 runs of the MSNPs-NH2@H[1] system, each one without loss of selectivity and yield. The cobaltabis(dicarbollide) catalyst supported on silica-coated magnetite nanoparticles has proven to be a robust, efficient, and easily reusable system for the photooxidation of alcohols in water, resulting in a green and sustainable heterogeneous catalytic system.

Dally-like Is Unlike Dally in Assisting Wingless Spread.

Lipidated morphogens can spread within tissues to regulate cell fate during development or tissue repair. How these insoluble molecules reach distant target cells remains unclear. Reporting in Nature, McGough et al. (2020) reveal the secret of how the cell-surface proteoglycan Dally-like-protein (Dlp) promotes long-range signaling of the palmitoylated morphogen Wingless.

Polarized sorting of Patched enables cytoneme-mediated Hedgehog reception in the Drosophila wing disc.

Hedgehog (Hh) signal molecules play a fundamental role in development, adult stem cell maintenance and cancer. Hh can signal at a distance, and we have proposed that its graded distribution across Drosophila epithelia is mediated by filopodia-like structures called cytonemes. Hh reception by Patched (Ptc) happens at discrete sites along presenting and receiving cytonemes, reminiscent of synaptic processes. Here, we show that a vesicle fusion mechanism mediated by SNARE proteins is required for Ptc placement at contact sites. Transport of Ptc to these sites requires multivesicular bodies (MVBs) formation via ESCRT machinery, in a manner different to that regulating Ptc/Hh lysosomal degradation after reception. These MVBs include extracellular vesicle (EV) markers and, accordingly, Ptc is detected in the purified exosomal fraction from cultured cells. Blockage of Ptc trafficking and fusion to basolateral membranes result in low levels of Ptc presentation for reception, causing an extended and flattened Hh gradient.

Metallacarboranes as Photoredox Catalysts in Water.

Metallacarboranes with the shape of the Greek letter θ, such as [Co(C2 B9 H11 )2 ]- , were tested, for the first time, as efficient photoredox catalysts in the oxidation of aromatic and aliphatic alcohols in water. Their efficiency is linked to their high solubility in water, their high oxidizing power (Co4+/3+ ), and their absence of fluorescence on excitation, among others. In most of the studied examples, using a catalyst load of 0.4 mol % gave high yields of 90-95 % with selectivity greater than 99 %. By reducing the catalyst load to 0.01 mol %, quantitative conversion of reactants to products was achieved, in some cases with greater than 99 % yield, high catalyst efficiency reaching a turnover number of 10 000, and a higher yield with a 45 times lower concentration of catalyst. The metallacarboranes can be recovered easily by precipitation on addition of [NMe4 ]Cl. A pathway for the photoredox-catalyzed oxidation of alcohols is proposed.

Extracellular Vesicles From Liver Progenitor Cells Downregulates Fibroblast Metabolic Activity and Increase the Expression of Immune-Response Related Molecules.

Extracellular vesicles (EVs) mediate cell-to-cell crosstalk whose content can induce changes in acceptor cells and their microenvironment. MLP29 cells are mouse liver progenitor cells that release EVs loaded with signaling cues that could affect cell fate. In the current work, we incubated 3T3-L1 mouse fibroblasts with MLP29-derived EVs, and then analyzed changes by proteomics and transcriptomics. Results showed a general downregulation of protein and transcript expression related to proliferative and metabolic routes dependent on TGF-beta. We also observed an increase in the ERBB2 interacting protein (ERBIN) and Cxcl2, together with an induction of ribosome biogenesis and interferon-related response molecules, suggesting the activation of immune system signaling.

Drosophila Zic family member odd-paired is needed for adult post-ecdysis maturation.

Specific neuropeptides regulate in arthropods the shedding of the old cuticle (ecdysis) followed by maturation of the new cuticle. In Drosophila melanogaster, the last ecdysis occurs at eclosion from the pupal case, with a post-eclosion behavioural sequence that leads to wing extension, cuticle stretching and tanning. These events are highly stereotyped and are controlled by a subset of crustacean cardioactive peptide (CCAP) neurons through the expression of the neuropeptide Bursicon (Burs). We have studied the role of the transcription factor Odd-paired (Opa) during the post-eclosion period. We report that opa is expressed in the CCAP neurons of the central nervous system during various steps of the ecdysis process and in peripheral CCAP neurons innerving the larval muscles involved in adult ecdysis. We show that its downregulation alters Burs expression in the CCAP neurons. Ectopic expression of Opa, or the vertebrate homologue Zic2, in the CCAP neurons also affects Burs expression, indicating an evolutionary functional conservation. Finally, our results show that, independently of its role in Burs regulation, Opa prevents death of CCAP neurons during larval development.

The cytoneme connection: direct long-distance signal transfer during development.

During development, specialized cells produce signals that distribute among receiving cells to induce a variety of cellular behaviors and organize tissues. Recent studies have highlighted cytonemes, a type of specialized signaling filopodia that carry ligands and/or receptor complexes, as having a role in signal dispersion. In this Primer, we discuss how the dynamic regulation of cytonemes facilitates signal transfer in complex environments. We assess recent evidence for the mechanisms for cytoneme formation, function and regulation, and postulate that contact between cytoneme membranes promotes signal transfer as a new type of synapse (morphogenetic synapsis). Finally, we reflect on the fundamental unanswered questions related to understanding cytoneme biology.

From intra- to extracellular vesicles: extracellular vesicles in developmental signalling.

Signalling from cell-to-cell is fundamental for determining differentiation and patterning. This communication can occur between adjacent and distant cells. Extracellular vesicles (EVs) are membrane-based structures thought to facilitate the long-distance movement of signalling molecules. EVs have recently been found to allow the transport of two major developmental signalling pathways: Hedgehog and Wnt. These signalling molecules undergo crucial post-translational lipid modifications, which anchor them to membranes and impede their free release into the extracellular space. Preparation of these ligands in EVs involves intracellular vesicle sorting in an endocytosis-dependent recycling process before secretion. In the present review, we discuss the most recent advances with regard to EV involvement in developmental signalling at a distance. We focus on the role of the protein complexes involved in EV genesis, and provide a comprehensive perspective of the contribution of these complexes to intracellular vesicle sorting of developmental signals for their extracellular secretion, reception and transduction.

Cytoneme-mediated cell-cell contacts for Hedgehog reception.

Morphogens regulate tissue patterning through their distribution in concentration gradients. Emerging research establishes a role for specialized signalling filopodia, or cytonemes, in morphogen dispersion and signalling. Previously we demonstrated that Hedgehog (Hh) morphogen is transported via vesicles along cytonemes emanating from signal-producing cells to form a gradient in Drosophila epithelia. However, the mechanisms for signal reception and transfer are still undefined. Here, we demonstrate that cytonemes protruding from Hh-receiving cells contribute to Hh gradient formation. The canonical Hh receptor Patched is localized in these cellular protrusions and Hh reception takes place in membrane contact sites between Hh-sending and Hh-receiving cytonemes. These two sets of cytonemes have similar dynamics and both fall in two different dynamic behaviours. Furthermore, both the Hh co-receptor Interference hedgehog (Ihog) and the glypicans are critical for this cell-cell cytoneme mediated interaction. These findings suggest that the described contact sites might facilitate morphogen presentation and reception.

Perspectives on Intra- and Intercellular Trafficking of Hedgehog for Tissue Patterning.

Intercellular communication is a fundamental process for correct tissue development. The mechanism of this process involves, among other things, the production and secretion of signaling molecules by specialized cell types and the capability of these signals to reach the target cells in order to trigger specific responses. Hedgehog (Hh) is one of the best-studied signaling pathways because of its importance during morphogenesis in many organisms. The Hh protein acts as a morphogen, activating its targets at a distance in a concentration-dependent manner. Post-translational modifications of Hh lead to a molecule covalently bond to two lipid moieties. These lipid modifications confer Hh high affinity to lipidic membranes, and intense studies have been carried out to explain its release into the extracellular matrix. This work reviews Hh molecule maturation, the intracellular recycling needed for its secretion and the proposed carriers to explain Hh transportation to the receiving cells. Special focus is placed on the role of specialized filopodia, also named cytonemes, in morphogen transport and gradient formation.