Many psychiatric drugs modulate the nervous system through multitarget mechanisms. However, systematic identification of multitarget compounds has been difficult using traditional in vitro screening assays. New approaches to phenotypic profiling in zebrafish can help researchers identify novel compounds with complex polypharmacology. For example, large-scale behavior-based chemical screens can rapidly identify large numbers of structurally diverse and phenotype-related compounds. Once these compounds have been identified, a systems-level analysis of their structures may help to identify statistically enriched target pathways. Together, systematic behavioral profiling and multitarget predictions may help researchers identify new behavior-modifying pathways and CNS therapeutics.

Several molecular platforms can identify bacteria associated with bloodstream infections but require positive culture bottles as starting material. Here, we describe results of screening 1140 blood cultures at 8h postinoculation, from 918 eligible adults being evaluated for bloodstream infection. DNA was extracted and analyzed by 16S and/or 23S rRNA real-time PCR/pyrosequencing. Compared to culture, PCR/pyrosequencing displayed 90.9% sensitivity, 99.6% specificity, 95.7% positive predictive value, and 99.1% negative predictive value. Overall concordance rate was 98.9% (1127/1140). In 4 cases with molecular-positive/culture-negative results, medical chart reviews provided evidence of identical bacteria from subsequent blood or concomitant urine/sputum cultures. Nine culture-positive/molecular-negative cases were associated with either polymicrobial growth, grew only in the anaerobic bottle of the clinical pair, and/or were detected by PCR/pyrosequencing after 8h. In summary, this approach accurately detected and identified bacteria in ~91% of culture-confirmed cases significantly sooner than the phenotypic identification was available, having the potential to improve antibiotic stewardship.

Behavioral larval zebrafish screens leverage a high-throughput small molecule discovery format to find neuroactive molecules relevant to mammalian physiology. We screen a library of 650 central nervous system active compounds in high replicate to train deep metric learning models on zebrafish behavioral profiles. The machine learning initially exploited subtle artifacts in the phenotypic screen, necessitating a complete experimental re-run with rigorous physical well-wise randomization. These large matched phenotypic screening datasets (initial and well-randomized) provide a unique opportunity to quantify and understand shortcut learning in a full-scale, real-world drug discovery dataset. The final deep metric learning model substantially outperforms correlation distance-the canonical way of computing distances between profiles-and generalizes to an orthogonal dataset of diverse drug-like compounds. We validate predictions by prospective in vitro radio-ligand binding assays against human protein targets, achieving a hit rate of 58% despite crossing species and chemical scaffold boundaries. These neuroactive compounds exhibit diverse chemical scaffolds, demonstrating that zebrafish phenotypic screens combined with metric learning achieve robust scaffold hopping capabilities.

Wide field of view microscopy that can resolve 3D information at high speed and spatial resolution is highly desirable for studying the behaviour of freely moving model organisms. However, it is challenging to design an optical instrument that optimises all these properties simultaneously. Existing techniques typically require the acquisition of sequential image snapshots to observe large areas or measure 3D information, thus compromising on speed and throughput. Here, we present 3D-RAPID, a computational microscope based on a synchronized array of 54 cameras that can capture high-speed 3D topographic videos over an area of 135 cm2, achieving up to 230 frames per second at spatiotemporal throughputs exceeding 5 gigapixels per second. 3D-RAPID employs a 3D reconstruction algorithm that, for each synchronized snapshot, fuses all 54 images into a composite that includes a co-registered 3D height map. The self-supervised 3D reconstruction algorithm trains a neural network to map raw photometric images to 3D topography using stereo overlap redundancy and ray-propagation physics as the only supervision mechanism. The resulting reconstruction process is thus robust to generalization errors and scales to arbitrarily long videos from arbitrarily sized camera arrays. We demonstrate the broad applicability of 3D-RAPID with collections of several freely behaving organisms, including ants, fruit flies, and zebrafish larvae.

Anesthetics are generally associated with sedation, but some anesthetics can also increase brain and motor activity-a phenomenon known as paradoxical excitation. Previous studies have identified GABA_A receptors as the primary targets of most anesthetic drugs, but how these compounds produce paradoxical excitation is poorly understood. To identify and understand such compounds, we applied a behavior-based drug profiling approach. Here, we show that a subset of central nervous system depressants cause paradoxical excitation in zebrafish. Using this behavior as a readout, we screened thousands of compounds and identified dozens of hits that caused paradoxical excitation. Many hit compounds modulated human GABA_A receptors, while others appeared to modulate different neuronal targets, including the human serotonin-6 receptor. Ligands at these receptors generally decreased neuronal activity, but paradoxically increased activity in the caudal hindbrain. Together, these studies identify ligands, targets, and neurons affecting sedation and paradoxical excitation in vivo in zebrafish.

Delivering cargo molecules across the plasma membrane is critical for biomedical research, and the need to develop molecularly well-defined tags that enable cargo transportation is ever-increasing. We report here a hydrophilic endocytosis-promoting peptide (EPP6) rich in hydroxyl groups with no positive charge. EPP6 can transport a wide array of small-molecule cargos into a diverse panel of animal cells. Mechanistic studies revealed that it entered the cells through a caveolin- and dynamin-dependent endocytosis pathway, mediated by the surface receptor fibrinogen C domain-containing protein 1. After endocytosis, EPP6 trafficked through early and late endosomes within 30 min. Over time, EPP6 partitioned among cytosol, lysosomes, and some long-lived compartments. It also demonstrated prominent transcytosis abilities in both in vitro and in vivo models. Our study proves that positive charge is not an indispensable feature for hydrophilic cell-penetrating peptides and provides a new category of molecularly well-defined delivery tags for biomedical applications.

The promise of single-objective light-sheet microscopy is to combine the convenience of standard single-objective microscopes with the speed, coverage, resolution and gentleness of light-sheet microscopes. We present DaXi, a single-objective light-sheet microscope design based on oblique plane illumination that achieves: (1) a wider field of view and high-resolution imaging via a custom remote focusing objective; (2) fast volumetric imaging over larger volumes without compromising image quality or necessitating tiled acquisition; (3) fuller image coverage for large samples via multi-view imaging and (4) higher throughput multi-well imaging via remote coverslip placement. Our instrument achieves a resolution of 450 nm laterally and 2 μm axially over an imaging volume of 3,000 × 800 × 300 μm. We demonstrate the speed, field of view, resolution and versatility of our instrument by imaging various systems, including Drosophila egg chamber development, zebrafish whole-brain activity and zebrafish embryonic development - up to nine embryos at a time.

The psychedelic alkaloid ibogaine has anti-addictive properties in both humans and animals¹. Unlike most medications for the treatment of substance use disorders, anecdotal reports suggest that ibogaine has the potential to treat addiction to various substances, including opiates, alcohol and psychostimulants. The effects of ibogaine-like those of other psychedelic compounds-are long-lasting², which has been attributed to its ability to modify addiction-related neural circuitry through the activation of neurotrophic factor signalling^3,4. However, several safety concerns have hindered the clinical development of ibogaine, including its toxicity, hallucinogenic potential and tendency to induce cardiac arrhythmias. Here we apply the principles of function-oriented synthesis to identify the key structural elements of the potential therapeutic pharmacophore of ibogaine, and we use this information to engineer tabernanthalog-a water-soluble, non-hallucinogenic, non-toxic analogue of ibogaine that can be prepared in a single step. In rodents, tabernanthalog was found to promote structural neural plasticity, reduce alcohol- and heroin-seeking behaviour, and produce antidepressant-like effects. This work demonstrates that, through careful chemical design, it is possible to modify a psychedelic compound to produce a safer, non-hallucinogenic variant that has therapeutic potential.