Single-tissue proteomics in Caenorhabditis elegans reveals proteins resident in intestinal lysosome-related organelles.

The nematode intestine is the primary site for nutrient uptake and storage as well as the synthesis of biomolecules; lysosome-related organelles known as gut granules are important for many of these functions. Aspects of intestine biology are not well understood, including the export of the nutrients it imports and the molecules it synthesizes, as well as the complete functions and protein content of the gut granules. Here, we report a mass spectrometry (MS)-based proteomic analysis of the intestine of the Caenorhabditis elegans and of its gut granules. Overall, we identified approximately 5,000 proteins each in the intestine and the gonad and showed that most of these proteins can be detected in samples extracted from a single worm, suggesting the feasibility of individual-level genetic analysis using proteomes. Comparing proteomes and published transcriptomes of the intestine and the gonad, we identified proteins that appear to be synthesized in the intestine and then transferred to the gonad. To identify gut granule proteins, we compared the proteome of individual intestines deficient in gut granules to the wild type. The identified gut granule proteome includes proteins known to be exclusively localized to the granules and additional putative gut granule proteins. We selected two of these putative gut granule proteins for validation via immunohistochemistry, and our successful confirmation of both suggests that our strategy was effective in identifying the gut granule proteome. Our results demonstrate the practicability of single-tissue MS-based proteomic analysis in small organisms and in its future utility.

Perspectives on tracking data reuse across biodata resources.

Motivation: Data reuse is a common and vital practice in molecular biology and enables the knowledge gathered over recent decades to drive discovery and innovation in the life sciences. Much of this knowledge has been collated into molecular biology databases, such as UniProtKB, and these resources derive enormous value from sharing data among themselves. However, quantifying and documenting this kind of data reuse remains a challenge. Results: The article reports on a one-day virtual workshop hosted by the UniProt Consortium in March 2023, attended by representatives from biodata resources, experts in data management, and NIH program managers. Workshop discussions focused on strategies for tracking data reuse, best practices for reusing data, and the challenges associated with data reuse and tracking. Surveys and discussions showed that data reuse is widespread, but critical information for reproducibility is sometimes lacking. Challenges include costs of tracking data reuse, tensions between tracking data and open sharing, restrictive licenses, and difficulties in tracking commercial data use. Recommendations that emerged from the discussion include: development of standardized formats for documenting data reuse, education about the obstacles posed by restrictive licenses, and continued recognition by funding agencies that data management is a critical activity that requires dedicated resources. Availability and implementation: Summaries of survey results are available at: https://docs.google.com/forms/d/1j-VU2ifEKb9C-sW6l3ATB79dgHdRk5v_lESv2hawnso/viewanalytics (survey of data providers) and https://docs.google.com/forms/d/18WbJFutUd7qiZoEzbOytFYXSfWFT61hVce0vjvIwIjk/viewanalytics (survey of users).

Sensory integration of food availability and population density during the diapause exit decision involves insulin-like signaling in Caenorhabditis elegans.

Decisions made over long time scales, such as life cycle decisions, require coordinated interplay between sensory perception and sustained gene expression. The Caenorhabditis elegans dauer (or diapause) exit developmental decision requires sensory integration of population density and food availability to induce an all-or-nothing organismal-wide response, but the mechanism by which this occurs remains unknown. Here, we demonstrate how the ASJ chemosensory neurons, known to be critical for dauer exit, perform sensory integration at both the levels of gene expression and calcium activity. In response to favorable conditions, dauers rapidly produce and secrete the dauer exit-promoting insulin-like peptide INS-6. Expression of ins-6 in the ASJ neurons integrate population density and food level and can reflect decision commitment since dauers committed to exiting have higher ins-6 expression levels than those of non-committed dauers. Calcium imaging in dauers reveals that the ASJ neurons are activated by food, and this activity is suppressed by pheromone, indicating that sensory integration also occurs at the level of calcium transients. We find that ins-6 expression in the ASJ neurons depends on neuronal activity in the ASJs, cGMP signaling, a CaM-kinase pathway, and the pheromone components ascr#8 and ascr#2. We propose a model in which decision commitment to exit the dauer state involves an autoregulatory feedback loop in the ASJ neurons that promotes high INS-6 production and secretion. These results collectively demonstrate how insulin-like peptide signaling helps animals compute long-term decisions by bridging sensory perception to decision execution.

WormBase 2024: status and transitioning to Alliance infrastructure.

WormBase has been the major repository and knowledgebase of information about the genome and genetics of Caenorhabditis elegans and other nematodes of experimental interest for over 2 decades. We have 3 goals: to keep current with the fast-paced C. elegans research, to provide better integration with other resources, and to be sustainable. Here, we discuss the current state of WormBase as well as progress and plans for moving core WormBase infrastructure to the Alliance of Genome Resources (the Alliance). As an Alliance member, WormBase will continue to interact with the C. elegans community, develop new features as needed, and curate key information from the literature and large-scale projects.

Enrichment on steps, not genes, improves inference of differentially expressed pathways.

Enrichment analysis is frequently used in combination with differential expression data to investigate potential commonalities amongst lists of genes and generate hypotheses for further experiments. However, current enrichment analysis approaches on pathways ignore the functional relationships between genes in a pathway, particularly OR logic that occurs when a set of proteins can each individually perform the same step in a pathway. As a result, these approaches miss pathways with large or multiple sets because of an inflation of pathway size (when measured as the total gene count) relative to the number of steps. We address this problem by enriching on step-enabling entities in pathways. We treat sets of protein-coding genes as single entities, and we also weight sets to account for the number of genes in them using the multivariate Fisher's noncentral hypergeometric distribution. We then show three examples of pathways that are recovered with this method and find that the results have significant proportions of pathways not found in gene list enrichment analysis.

FAIR Header Reference genome: a TRUSTworthy standard.

The lack of interoperable data standards among reference genome data-sharing platforms inhibits cross-platform analysis while increasing the risk of data provenance loss. Here, we describe the FAIR bioHeaders Reference genome (FHR), a metadata standard guided by the principles of Findability, Accessibility, Interoperability and Reuse (FAIR) in addition to the principles of Transparency, Responsibility, User focus, Sustainability and Technology. The objective of FHR is to provide an extensive set of data serialisation methods and minimum data field requirements while still maintaining extensibility, flexibility and expressivity in an increasingly decentralised genomic data ecosystem. The effort needed to implement FHR is low; FHR's design philosophy ensures easy implementation while retaining the benefits gained from recording both machine and human-readable provenance.

An improved solid medium-based culturing method for Steinernema hermaphroditum.

Steinernema hermaphroditum is the only identified entomopathogenic nematode that is consistently hermaphroditic and thus offers a great opportunity to use genetic approaches to probe symbiosis. Evolutionarily, ecologically, and morphologically distinct from laboratory nematodes commonly used in the laboratory, with both forward and reverse genetics tools available, this species also provides an opportunity to explore other areas of biology, especially using comparative studies. Here, we describe an improved solid medium-based culturing method for S. hermaphroditum that we found particularly helpful for phenotypic analysis and genetic manipulation. We document the rapid increase in the size of the worm; and show that the uniform growth of the worm on this medium provides a good basis for developmental studies. Finally, we measure the brood size of individual animals, which, although far larger, has a very similar trajectory to that of the hermaphroditic Caenorhabditis elegans, suggesting common reproductive restraints.

AI-aided geometric design of anti-infection catheters.

Bacteria can swim upstream in a narrow tube and pose a clinical threat of urinary tract infection to patients implanted with catheters. Coatings and structured surfaces have been proposed to repel bacteria, but no such approach thoroughly addresses the contamination problem in catheters. Here, on the basis of the physical mechanism of upstream swimming, we propose a novel geometric design, optimized by an artificial intelligence model. Using Escherichia coli, we demonstrate the anti-infection mechanism in microfluidic experiments and evaluate the effectiveness of the design in three-dimensionally printed prototype catheters under clinical flow rates. Our catheter design shows that one to two orders of magnitude improved suppression of bacterial contamination at the upstream end, potentially prolonging the in-dwelling time for catheter use and reducing the overall risk of catheter-associated urinary tract infection.

Molecular identification of a peroxidase gene controlling body size in the entomopathogenic nematode Steinernema hermaphroditum.

The entomopathogenic nematode Steinernema hermaphroditum was recently rediscovered and is being developed as a genetically tractable experimental system for the study of previously unexplored biology, including parasitism of its insect hosts and mutualism with its bacterial endosymbiont Xenorhabdus griffiniae. Through whole-genome re-sequencing and genetic mapping we have for the first time molecularly identified the gene responsible for a mutationally defined phenotypic locus in an entomopathogenic nematode. In the process we observed an unexpected mutational spectrum following ethyl methansulfonate mutagenesis in this species. We find that the ortholog of the essential Caenorhabditis elegans peroxidase gene skpo-2 controls body size and shape in S. hermaphroditum. We confirmed this identification by generating additional loss-of-function mutations in the gene using CRISPR-Cas9. We propose that the identification of skpo-2 will accelerate gene targeting in other Steinernema entomopathogenic nematodes used commercially in pest control, as skpo-2 is X-linked and males hemizygous for loss of its function can mate, making skpo-2 an easily recognized and maintained marker for use in co-CRISPR.

Distinct neurogenetic mechanisms establish the same chemosensory valence state at different life stages in Caenorhabditis elegans.

An animal's preference for many chemosensory cues remains constant despite dramatic changes in the animal's internal state. The mechanisms that maintain chemosensory preference across different physiological contexts remain poorly understood. We previously showed that distinct patterns of neural activity and motor output are evoked by carbon dioxide (CO2) in starved adults vs dauers of Caenorhabditis elegans, despite the two life stages displaying the same preference (attraction) for CO2. However, how the distinct CO2-evoked neural dynamics and motor patterns contribute to CO2 attraction at the two life stages remained unclear. Here, using a CO2 chemotaxis assay, we show that different interneurons are employed to drive CO2 attraction at the two life stages. We also investigate the molecular mechanisms that mediate CO2 attraction in dauers vs adults. We show that insulin signaling promotes CO2 attraction in dauers but not starved adults and that different combinations of neurotransmitters and neuropeptides are used for CO2 attraction at the two life stages. Our findings provide new insight into the distinct molecular and cellular mechanisms used by C. elegans at two different life stages to generate attractive behavioral responses to CO2.

DATA RESOURCES AND ANALYSES FAIR Header Reference genome: A TRUSTworthy standard.

The lack of interoperable data standards among reference genome data-sharing platforms inhibits cross-platform analysis while increasing the risk of data provenance loss. Here, we describe the FAIR-bioHeaders Reference genome (FHR), a metadata standard guided by the principles of Findability, Accessibility, Interoperability, and Reuse (FAIR) in addition to the principles of Transparency, Responsibility, User focus, Sustainability, and Technology (TRUST). The objective of FHR is to provide an extensive set of data serialisation methods and minimum data field requirements while still maintaining extensibility, flexibility, and expressivity in an increasingly decentralised genomic data ecosystem. The effort needed to implement FHR is low; FHR's design philosophy ensures easy implementation while retaining the benefits gained from recording both machine and human-readable provenance.

A cGAL-UAS bipartite expression toolkit for Caenorhabditis elegans sensory neurons.

Animals integrate sensory information from the environment and display various behaviors in response to external stimuli. In Caenorhabditis elegans hermaphrodites, 33 types of sensory neurons are responsible for chemosensation, olfaction, and mechanosensation. However, the functional roles of all sensory neurons have not been systematically studied due to the lack of facile genetic accessibility. A bipartite cGAL-UAS system has been previously developed to study tissue- or cell-specific functions in C. elegans. Here, we report a toolkit of new cGAL drivers that can facilitate the analysis of a vast majority of the 60 sensory neurons in C. elegans hermaphrodites. We generated 37 sensory neuronal cGAL drivers that drive cGAL expression by cell-specific regulatory sequences or intersection of two distinct regulatory regions with overlapping expression (split cGAL). Most cGAL-drivers exhibit expression in single types of cells. We also constructed 28 UAS effectors that allow expression of proteins to perturb or interrogate sensory neurons of choice. This cGAL-UAS sensory neuron toolkit provides a genetic platform to systematically study the functions of C. elegans sensory neurons.

Semantic representation of neural circuit knowledge in Caenorhabditis elegans.

In modern biology, new knowledge is generated quickly, making it challenging for researchers to efficiently acquire and synthesise new information from the large volume of primary publications. To address this problem, computational approaches that generate machine-readable representations of scientific findings in the form of knowledge graphs have been developed. These representations can integrate different types of experimental data from multiple papers and biological knowledge bases in a unifying data model, providing a complementary method to manual review for interacting with published knowledge. The Gene Ontology Consortium (GOC) has created a semantic modelling framework that extends individual functional gene annotations to structured descriptions of causal networks representing biological processes (Gene Ontology-Causal Activity Modelling, or GO-CAM). In this study, we explored whether the GO-CAM framework could represent knowledge of the causal relationships between environmental inputs, neural circuits and behavior in the model nematode C. elegans [C. elegans Neural-Circuit Causal Activity Modelling (CeN-CAM)]. We found that, given extensions to several relevant ontologies, a wide variety of author statements from the literature about the neural circuit basis of egg-laying and carbon dioxide (CO2) avoidance behaviors could be faithfully represented with CeN-CAM. Through this process, we were able to generate generic data models for several categories of experimental results. We also discuss how semantic modelling may be used to functionally annotate the C. elegans connectome. Thus, Gene Ontology-based semantic modelling has the potential to support various machine-readable representations of neurobiological knowledge.

Fluorescence dynamics of lysosomal-related organelle flashing in the intestinal cells of Caenorhabditis elegans.

The biological roles of the autofluorescent lysosome-related organelles ("gut granules") in the intestinal cells of many nematodes, including Caenorhabditis elegans, have been shown to play an important role in metabolic and signaling processes, but they have not been fully characterized. We report here a previously undescribed phenomenon in which the autofluorescence of these granules increased and then decreased in a rapid and dynamic manner that may be associated with nutrient availability. We observed that two distinct types of fluorophores are likely present in the gut granules. One displays a "flashing" phenomenon, in which fluorescence decrease is preceded by a sharp increase in fluorescence intensity that expands into the surrounding area, while the other simply decreases in intensity. Gut granule flashing was observed in the different life stages of C. elegans and was also observed in Steinernema hermaphroditum, an evolutionarily distant nematode. We hypothesize that the "flashing" fluorophore is pH-sensitive, and the fluorescence intensity change results from the fluorophore being released from the lysosome-related organelles into the relatively higher pH environment of the cytosol. The visually spectacular dynamic fluorescence phenomenon we describe might provide a handle on the biochemistry and genetics of these lysosome-related organelles.

Trends in Imaging Utilization Among United States Medicare Beneficiaries and the Impact of the COVID-19 Pandemic.

This study characterizes trends in ophthalmic imaging volume and utilization in the United States among and assesses the potential impact of the COVID-19 pandemic using data from the Centers for Medicare and Medicaid Services. Utilization of macular optical coherence tomography (OCT), optic nerve OCT, and fundus photography steadily increased from 2013 to 2020 and was not impacted by the COVID-19 pandemic. At the same time, there was minimal adoption of anterior segment OCT and a decline in the utilization of dye-based angiography. Utilization patterns may be impacted by the advent of new technologies, role of the clinician, and alignment with treatment paradigms. [Ophthalmic Surg Lasers Imaging Retina 2023;54:661-665.].

The alliance of genome resources: transforming comparative genomics.

Comparing genomic and biological characteristics across multiple species is essential to using model systems to investigate the molecular and cellular mechanisms underlying human biology and disease and to translate mechanistic insights from studies in model organisms for clinical applications. Building a scalable knowledge commons platform that supports cross-species comparison of rich, expertly curated knowledge regarding gene function, phenotype, and disease associations available for model organisms and humans is the primary mission of the Alliance of Genome Resources (the Alliance). The Alliance is a consortium of seven model organism knowledgebases (mouse, rat, yeast, nematode, zebrafish, frog, fruit fly) and the Gene Ontology resource. The Alliance uses a common set of gene ortholog assertions as the basis for comparing biological annotations across the organisms represented in the Alliance. The major types of knowledge associated with genes that are represented in the Alliance database currently include gene function, phenotypic alleles and variants, human disease associations, pathways, gene expression, and both protein-protein and genetic interactions. The Alliance has enhanced the ability of researchers to easily compare biological annotations for common data types across model organisms and human through the implementation of shared programmatic access mechanisms, data-specific web pages with a unified "look and feel", and interactive user interfaces specifically designed to support comparative biology. The modular infrastructure developed by the Alliance allows the resource to serve as an extensible "knowledge commons" capable of expanding to accommodate additional model organisms.