Genetic Tractability Research Articles

Self-DNA in Caenorhabditis elegans Affects the Production of Specific Metabolites: Evidence from LC-MS and Chemometric Studies

The worm Caenorhabditis elegans, with its short lifecycle and well-known genetic and metabolic pathways, stands as an exemplary model organism for biological research. Its simplicity and genetic tractability make it an ideal system for investigating the effects of different conditions on its metabolism. The chemical analysis of this nematode was performed to identify specific metabolites produced by the worms when fed with either self- or nonself-DNA. A standard diet with OP50 feeding was used as a control. Different development stages were sampled, and their chemical composition was assessed by liquid chromatography–mass spectrometry combined with chemometrics, including both principal component analysis and orthogonal partial least squares discriminant analysis tools. The obtained data demonstrated that self-DNA-treated larvae, when arrested in their cycle, showed significant decreases in dynorphin, an appetite regulator of the nematode, and in N-formyl glycine, a known longevity promoter in C. elegans. Moreover, a substantial decrease was also recorded in the self-DNA-fed adults for the FMRF amide neuropeptide, an embryogenesis regulator, and for a dopamine derivative modulating nematode locomotion. In conclusion, this study allowed for the identification of key metabolites affected by the self-DNA diet, providing interesting hints on the main molecular pathways involved in its biological inhibitory effects.

Zebrafish: A Versatile Animal Model in Biomedical Research

Zebrafish (Danio rerio) has emerged as a powerful animal model in biomedical research, offering a unique combination of genetic tractability, rapid development, and conserved gene function with humans. This review highlights the significance of zebrafish in understanding various human diseases, including neurodegenerative disorders, metabolic disorders, cancer, and more. The zebrafish genome, with over 70% similarity to the human genome, allows for the modelling of human diseases with high fidelity. The ease of genetic manipulation, high fecundity, and low maintenance costs make zebrafish an attractive model for large-scale genetic screens and drug discovery. Zebrafish models of neurodegenerative diseases, such as Parkinson's disease, Alzheimer's disease, and Huntington's disease, have been developed, providing insights into disease mechanisms and potential therapeutic targets. Similarly, zebrafish models of metabolic disorders, including diabetes, dyslipidaemia, and atherosclerosis, have been established, enabling the study of disease pathogenesis and prevention strategies.The zebrafish has also been used to model various types of cancer, including leukaemia, melanoma, and pancreatic cancer, facilitating the discovery of novel oncogenes and tumor suppressors. The transparent nature of zebrafish embryos and larvae allows for in vivo imaging of cancer cells, enabling the study of cancer cell behavior and response to therapy. Furthermore, zebrafish has been used to investigate brain-to-organ communication, cell transplantation, and cell-cell interactions, providing insights into the complex interactions between different tissues and organs. The development of novel tools, such as CRISPR/Cas9 genome editing and single-cell RNA sequencing, has further expanded the capabilities of zebrafish research.

GATA1-deficient human pluripotent stem cells generate neutrophils with improved antifungal immunity that is mediated by the integrin CD18.

Neutrophils are critical for host defense against fungi. However, the short life span and lack of genetic tractability of primary human neutrophils has limited in vitro analysis of neutrophil-fungal interactions. Human induced pluripotent stem cell (iPSC)-derived neutrophils (iNeutrophils) are a genetically tractable alternative to primary human neutrophils. Here, we show that deletion of the transcription factor GATA1 from human iPSCs results in iNeutrophils with improved antifungal activity against Aspergillus fumigatus. GATA1 knockout (KO) iNeutrophils have increased maturation, antifungal pattern recognition receptor expression and more readily execute neutrophil effector functions compared to wild-type iNeutrophils. iNeutrophils also show a shift in their metabolism following stimulation with fungal β-glucan, including an upregulation of the pentose phosphate pathway (PPP), similar to primary human neutrophils in vitro. Furthermore, we show that deletion of the integrin CD18 attenuates the ability of GATA1-KO iNeutrophils to kill A. fumigatus but is not necessary for the upregulation of PPP. Collectively, these findings support iNeutrophils as a robust system to study human neutrophil antifungal immunity and has identified specific roles for CD18 in the defense response.

A critical appraisal of geroprotective activities of flavonoids in terms of their bio-accessibility and polypharmacology

Flavonoids, a commonly consumed natural product, elicit health-benefits such as antioxidant, anti-inflammatory, antiviral, anti-allergic, hepatoprotective, anti-carcinogenic and neuroprotective activities. Several studies have reported the beneficial role of flavonoids in improving memory, learning, and cognition in clinical settings. Their mechanism of action is mediated through the modulation of multiple signalling cascades. This polypharmacology makes them an attractive natural scaffold for designing and developing new effective therapeutics for complex neurological disorders like Alzheimer's disease and Parkinson's disease. Flavonoids are shown to inhibit crucial targets related to neurodegenerative disorders (NDDs), including acetylcholinesterase, butyrylcholinesterase, β-secretase, γ-secretase, α-synuclein, Aβ protein aggregation and neurofibrillary tangles formation. Conserved neuro-signalling pathways related to neurotransmitter biogenesis and inactivation, ease of genetic manipulation and tractability, cost-effectiveness, and their short lifespan make Caenorhabditis elegans one of the most frequently used models in neuroscience research and high-throughput drug screening for neurodegenerative disorders. Here, we critically appraise the neuroprotective activities of different flavonoids based on clinical trials and epidemiological data. This review provides critical insights into the absorption, metabolism, and tissue distribution of various classes of flavonoids, as well as detailed mechanisms of the observed neuroprotective activities at the molecular level, to rationalize the clinical data. We further extend the review to critically evaluate the scope of flavonoids in the disease management of neurodegenerative disorders and review the suitability of C. elegans as a model organism to study the neuroprotective efficacy of flavonoids and natural products.

An improved CRISPR and CRISPR interference (CRISPRi) toolkit for engineering the model methanogenic archaeon Methanococcus maripaludis

BackgroundThe type II based CRISPR-Cas system remains restrictedly utilized in archaea, a featured domain of life that ranks parallelly with Bacteria and Eukaryotes. Methanococcus maripaludis, known for rapid growth and genetic tractability, serves as an exemplary model for studying archaeal biology and exploring CO2−based biotechnological applications. However, tools for controlled gene regulation remain deficient and CRISPR-Cas tools still need improved in this archaeon, limiting its application as an archaeal model cellular factory.ResultsThis study not only improved the CRISPR-Cas9 system for optimizing multiplex genome editing and CRISPR plasmid construction efficiencies but also pioneered an effective CRISPR interference (CRISPRi) system for controlled gene regulation in M. maripaludis. We developed two novel strategies for balanced expression of multiple sgRNAs, facilitating efficient multiplex genome editing. We also engineered a strain expressing Cas9 genomically, which simplified the CRISPR plasmid construction and facilitated more efficient genome modifications, including markerless and scarless gene knock-in. Importantly, we established a CRISPRi system using catalytic inactive dCas9, achieving up to 100-fold repression on target gene. Here, sgRNAs targeting near and downstream regions of the transcription start site and the 5′end ORF achieved the highest repression efficacy. Furthermore, we developed an inducible CRISPRi-dCas9 system based on TetR/tetO platform. This facilitated the inducible gene repression, especially for essential genes.ConclusionsTherefore, these advancements not only expand the toolkit for genetic manipulation but also bridge methodological gaps for controlled gene regulation, especially for essential genes, in M. maripaludis. The robust toolkit developed here paves the way for applying M. maripaludis as a vital model archaeal cell factory, facilitating fundamental biological studies and applied biotechnology development of archaea.

A series of vectors for inducible gene expression in multidrug-resistant Acinetobacter baumannii.

Clinical isolates of bacterial pathogens often harbor resistance to multiple antibiotics, with Acinetobacter baumannii being a prime example. The drug-resistance phenotypes associated with these pathogens represent a significant hurdle to researchers who wish to study modern isolates due to the limited availability of plasmid tools. Here, we present a series of freely replicating and Tn7-insertion vectors that rely on selectable markers to less frequently encountered antibiotics, apramycin, and hygromycin. We demonstrate the utility of these plasmid tools through a variety of experiments looking at a multidrug-resistant strain of A. baumannii, strain AB5075. Strain AB5075 is an established model strain for present-day A. baumannii, due in part to its genetic tractability and because it is a representative isolate of the globally disseminated multidrug-resistant clade of A. baumannii, global clone 1. In addition to the drug-selection markers facilitating use in strains resistant to more commonly used antibiotics, the vectors allow for controllable expression driven by several regulatory systems, including isopropyl β-D-1-thiogalactopyranoside (IPTG), arabinose, anhydrotetracycline, and toluic acid.

Advanced Neural Functional Imaging in C. elegans Using Lab-on-a-Chip Technology.

The ability to perceive and adapt to environmental changes is crucial for the survival of all organisms. Neural functional imaging, particularly in model organisms, such as Caenorhabditis elegans, provides valuable insights into how animals sense and process external cues through their nervous systems. Because of its fully mapped neural anatomy, transparent body, and genetic tractability, C. elegans serves as an ideal model for these studies. This review focuses on advanced methods for neural functional imaging in C. elegans, highlighting calcium imaging techniques, lab-on-a-chip technologies, and their applications in the study of various sensory modalities, including chemosensation, mechanosensation, thermosensation, photosensation, and magnetosensation. We discuss the benefits of these methods in terms of precision, reproducibility, and ability to study dynamic neural processes in real time, ultimately advancing our understanding of the fundamental principles of neural activity and connectivity.

An actomyosin network organizes niche morphology and responds to feedback from recruited stem cells

Stem cells often rely on signals from a niche, which in many tissues adopts a precise morphology. What remains elusive is how niches are formed and how morphology impacts function. To address this, we leverage the Drosophila gonadal niche, which affords genetic tractability and live-imaging. We have previously shown mechanisms dictating niche cell migration to their appropriate position within the gonad and the resultant consequences on niche function. Here, we show that once positioned, niche cells robustly polarize filamentous actin (F-actin) and non-muscle myosin II (MyoII) toward neighboring germ cells. Actomyosin tension along the niche periphery generates a highly reproducible smoothened contour. Without contractility, niches are misshapen and exhibit defects in their ability to regulate germline stem cell behavior. We additionally show that germ cells aid in polarizing MyoII within niche cells and that extrinsic input is required for niche morphogenesis and function. Our work reveals a feedback mechanism where stem cells shape the niche that guides their behavior.

Expanding the transgene expression toolbox of the malaria vector Anopheles stephensi.

Anopheles stephensi Liston, 1901 (Diptera: culicidae) is a competent vector of Plasmodium falciparum (Haemosporida: plasmodiidae) malaria, and its expansion in the African continent is of concern due to its viability in urban settings and resistance to insecticides. To enhance its genetic tractability, we determined the utility of a ~2 kb An. stephensi lipophorin (lp) promoter fragment in driving transgene expression. Lipophorin genes are involved in lipid transport in insects, and an orthologous promoter in An. gambiae (AGAP001826) was previously demonstrated to successfully express a transgene. In the present study, we qualitatively characterised the expression of a ZsYellow fluorescent marker protein, expressed by An. stephensi lp promoter fragment. Our study indicated that the lp promoter fragment was effective, generating a distinct expression pattern in comparison to the commonly utilised 3xP3 promoter. The lp:ZsYellow fluorescence was largely visible in early instar larvae and appeared more intense in later instar larvae, pupae and adults, becoming especially conspicuous in adult females after a blood meal. Different isolines showed some variation in expression pattern and intensity. Aside from general transgene expression, as the lp promoter produces a suitable fluorescent protein marker expression pattern, it may facilitate genotypic screening and aid the development of more complex genetic biocontrol systems, such as multi-component gene drives. This study represents an expansion of the An. stephensi genetic toolbox, an important endeavour to increase the speed of An. stephensi research and reach public health milestones in combating malaria.

An actomyosin network organizes niche morphology and responds to feedback from recruited stem cells.

Stem cells often rely on signals from a niche, which in many tissues adopts a precise morphology. What remains elusive is how niches are formed, and how morphology impacts function. To address this, we leverage the Drosophila gonadal niche, which affords genetic tractability and live-imaging. We have previously shown mechanisms dictating niche cell migration to their appropriate position within the gonad, and the resultant consequences on niche function. Here, we show that once positioned, niche cells robustly polarize filamentous actin (F-actin) and Non-muscle Myosin II (MyoII) towards neighboring germ cells. Actomyosin tension along the niche periphery generates a highly reproducible smoothened contour. Without contractility, niches are misshapen and exhibit defects in their ability to regulate germline stem cell behavior. We additionally show that germ cells aid in polarizing MyoII within niche cells, and that extrinsic input is required for niche morphogenesis and function. Our work reveals a feedback mechanism where stem cells shape the niche that guides their behavior.

Reprogramming macrophages with R848-loaded artificial protocells to modulate skin and skeletal wound healing.

After tissue injury, inflammatory cells are rapidly recruited to the wound where they clear microbes and other debris, and coordinate the behaviour of other cell lineages at the repair site in both positive and negative ways. In this study, we take advantage of the translucency and genetic tractability of zebrafish to evaluate the feasibility of reprogramming innate immune cells in vivo with cargo-loaded protocells and investigate how this alters the inflammatory response in the context of skin and skeletal repair. Using live imaging, we show that protocells loaded with R848 cargo (which targets TLR7 and TLR8 signalling), are engulfed by macrophages resulting in their switching to a pro-inflammatory phenotype and altering their regulation of angiogenesis, collagen deposition and re-epithelialization during skin wound healing, as well as dampening osteoblast and osteoclast recruitment and bone mineralization during fracture repair. For infected skin wounds, R848-reprogrammed macrophages exhibited enhanced bactericidal activities leading to improved healing. We replicated our zebrafish studies in cultured human macrophages, and showed that R848-loaded protocells similarly reprogramme human cells, indicating how this strategy might be used to modulate wound inflammation in the clinic.

High-resolution Cell Transplantation in Embryonic and Larval Zebrafish.

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation between animals to track cell fate and to induce mismatches in the genetic, spatial, or temporal properties of donor and host cells is a powerful means of examining the nature of these interactions. Organisms such as chick and amphibians have made crucial contributions to our understanding of development and regeneration, respectively, in large part because of their amenability to transplantation. The power of these models, however, has been limited by low genetic tractability. Likewise, the major genetic model organisms have lower amenability to transplantation. The zebrafish is a major genetic model for development and regeneration, and while cell transplantation is common in zebrafish, it is generally limited to the transfer of undifferentiated cells at the early blastula and gastrula stages of development. In this article, we present a simple and robust method that extends the zebrafish transplantation window to any embryonic or larval stage between at least 1 and 7 days post fertilization. The precision of this approach allows for the transplantation of as little as one cell with near-perfect spatial and temporal resolution in both donor and host animals. While we highlight here the transplantation of embryonic and larval neurons for the study of nerve development and regeneration, respectively, this approach is applicable to a wide range of progenitor and differentiated cell types and research questions.

Orsay Virus Infection in Caenorhabditis elegans.

Orsay virus infection in the nematode Caenorhabditis elegans presents an opportunity to study host-virus interactions in an easily culturable, whole-animal host. Previously, a major limitation of C. elegans as a model for studying antiviral immunity was the lack of viruses known to naturally infect the worm. With the 2011 discovery of the Orsay virus, a naturally occurring viral pathogen, C. elegans has emerged as a compelling model for research on antiviral defense. From the perspective of the host, the genetic tractability of C. elegans enables mechanistic studies of antiviral immunity while the transparency of this animal allows for the observation of subcellular processes in vivo. Preparing infective virus filtrate and performing infections can be achieved with relative ease in a laboratory setting. Moreover, several tools are available to measure the outcome of infection. Here, we describe workflows for generating infective virus filtrate, achieving reproducible infection of C. elegans, and assessing the outcome of viral infection using molecular biology approaches and immunofluorescence. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of Orsay virus filtrate Support Protocol: Synchronize C. elegans development by bleaching Basic Protocol 2: Orsay virus infection Basic Protocol 3: Quantification of Orsay virus RNA1/RNA2 transcript levels by qRT-PCR Basic Protocol 4: Quantification of infection rate and fluorescence in situ hybridization (FISH) fluorescence intensity Basic Protocol 5: Immunofluorescent labeling of dsRNA in virus-infected intestinal tissue.

Zebrafish as a model organism for virus disease research: Current status and future directions

Zebrafish (Danio rerio) have emerged as valuable models for investigating viral infections, providing insights into viral pathogenesis, host responses, and potential therapeutic interventions. This review offers a comprehensive synthesis of research on viral infections using zebrafish models, focusing on the molecular mechanisms of viral action and host-virus interactions. Zebrafish models have been instrumental in elucidating the replication dynamics, tissue tropism, and immune evasion strategies of various viruses, including Chikungunya virus, Dengue virus, Herpes Simplex Virus type 1, and Influenza A virus. Additionally, studies utilizing zebrafish have evaluated the efficacy of antiviral compounds and natural agents against emerging viruses such as SARS-CoV-2, Zika virus, and Dengue virus. The optical transparency and genetic tractability of zebrafish embryos enable real-time visualization of viral infections, facilitating the study of viral spread and immune responses. Despite challenges such as temperature compatibility and differences in host receptors, zebrafish models offer unique advantages, including cost-effectiveness, high-throughput screening capabilities, and conservation of key immune pathways. Importantly, zebrafish models complement existing animal models, providing a platform for rapid evaluation of potential therapeutics and a deeper understanding of viral pathogenesis. This review underscores the significance of zebrafish research in advancing our understanding of viral diseases and highlights future research directions to combat infectious diseases effectively.

JNK Signaling Positively Regulates Acute Ethanol Tolerance in C. elegans.

Alcohol use disorder (AUD) is a chronic neurobehavioral condition characterized by a cycle of tolerance development, increased consumption, and reinstated craving and seeking behaviors during withdrawal. Understanding the intricate mechanisms of AUD necessitates reliable animal models reflecting its key features. Caenorhabditis elegans (C. elegans), with its conserved nervous system and genetic tractability, has emerged as a valuable model organism to study AUD. Here, we employ an ethanol vapor exposure model in Caenorhabditis elegans, recapitulating AUD features while maintaining high-throughput scalability. We demonstrate that ethanol vapor exposure induces intoxication-like behaviors, acute tolerance, and ethanol preference, akin to mammalian AUD traits. Leveraging this model, we elucidate the conserved role of c-jun N-terminal kinase (JNK) signaling in mediating acute ethanol tolerance. Mutants lacking JNK signaling components exhibit impaired tolerance development, highlighting JNK's positive regulation. Furthermore, we detect ethanol-induced JNK activation in C. elegans. Our findings underscore the utility of C. elegans with ethanol vapor exposure for studying AUD and offer novel insights into the molecular mechanisms underlying acute ethanol tolerance through JNK signaling.

A Pretty Kettle of Fish: A Review on the Current Challenges in Mediterranean Teleost Reproduction.

The Mediterranean region is facing several environmental changes and pollution issues. Teleosts are particularly sensitive to these challenges due to their intricate reproductive biology and reliance on specific environmental cues for successful reproduction. Wild populations struggle with the triad of climate change, environmental contamination, and overfishing, which can deeply affect reproductive success and population dynamics. In farmed species, abiotic factors affecting reproduction are easier to control, whereas finding alternatives to conventional diets for farmed teleosts is crucial for enhancing broodstock health, reproductive success, and the sustainability of the aquaculture sector. Addressing these challenges involves ongoing research into formulating specialized diets, optimizing feeding strategies, and developing alternative and sustainable feed ingredients. To achieve a deeper comprehension of these challenges, studies employing model species have emerged as pivotal tools. These models offer advantages in understanding reproductive mechanisms due to their well-defined physiology, genetic tractability, and ease of manipulation. Yet, while providing invaluable insights, their applicability to diverse species remains constrained by inherent variations across taxa and oversimplification of complex environmental interactions, thus limiting the extrapolation of the scientific findings. Bridging these gaps necessitates multidisciplinary approaches, emphasizing conservation efforts for wild species and tailored nutritional strategies for aquaculture, thereby fostering sustainable teleost reproduction in the Mediterranean.

Larval development of Holothuria tubulosa, a new tractable system for evo-devo

To explore animal diversity, new experimentally tractable organisms must be established. Echinoderms include five groups of marine animals that have been used as developmental models for over a century thanks to their low costs, high fecundity, optically clear larvae and genetic tractability. An additional advantage of echinoderms is that their larval forms display diverse morphologies. This rich diversity enables comparative studies to investigate the evolutionary relationships among cell types, tissues, and organs. However, reproducible protocols to obtain gametes, detailed information on embryogenesis, and genomic tools have been optimized only for selected species of sea urchins and sea stars. To address this gap, we established the abundant Mediterranean sea cucumber Holothuria tubulosa as a new experimental system. Here we describe a method to reliably obtain gametes and make embryonic cultures multiple times from the same animal and characterize unique larval tissues combining immunohistochemistry and high-resolution microscopy. This work represents a step forward in our understanding of holothurian development and establishes H. tubulosa as an emerging experimental system for evo-devo and other biological disciplines.

Nourseothricin as a novel drug for selection of transgenic Giardia lamblia

Functional gene and protein characterizations in parasitic protists are often limited by their genetic tractability. Despite the development of CRISPR-Cas9-derived or inspired approaches for a handful of protist parasites, the overall genetic tractability of these organisms remains limited. The intestinal parasite Giardia lamblia is one such species, with the added challenge of a paucity of reliable selection markers.To address this limitation, we tested the feasibility of using Nourseothricin as an effective selection agent in Giardia. Here, we report that axenically-grown WB Giardia cells are sensitive to Nourseothricin and that engineering expression of the streptothricin acetyltransferase (SAT-1) gene from Streptomyces rochei in transgenic parasites confers resistance to this antibiotic. Furthermore, we determine that SAT-1-expressing parasites are cross-resistant neither to Neomycin nor Puromycin, which are widely used to select for transgenic parasites. Consequently, we show that Nourseothricin can be used in sequential combination with both Neomycin and Puromycin to select for dual transfection events.This work increases the number of reliable selection agents and markers for Giardia genetic manipulation, expanding the limited molecular toolbox for this species of global medical importance.

The effect of common paralytic agents used for fluorescence imaging on redox tone and ATP levels in Caenorhabditis elegans.

One aspect of Caenorhabditis elegans that makes it a highly valuable model organism is the ease of use of in vivo genetic reporters, facilitated by its transparent cuticle and highly tractable genetics. Despite the rapid advancement of these technologies, worms must be paralyzed for most imaging applications, and few investigations have characterized the impacts of common chemical anesthetic methods on the parameters measured, in particular biochemical measurements such as cellular energetics and redox tone. Using two dynamic reporters, QUEEN-2m for relative ATP levels and reduction-oxidation sensitive GFP (roGFP) for redox tone, we assess the impact of commonly used chemical paralytics. We report that no chemical anesthetic is entirely effective at doses required for full paralysis without altering redox tone or ATP levels, and that anesthetic use alters the detected outcome of rotenone exposure on relative ATP levels and redox tone. We also assess the use of cold shock, commonly used in combination with physical restraint methods, and find that cold shock does not alter either ATP levels or redox tone. In addition to informing which paralytics are most appropriate for research in these topics, we highlight the need for tailoring the use of anesthetics to different endpoints and experimental questions. Further, we reinforce the need for developing less disruptive paralytic methods for optimal imaging of dynamic in vivo reporters.

Light Control in Microbial Systems.

Light is a key environmental component influencing many biological processes, particularly in prokaryotes such as archaea and bacteria. Light control techniques have revolutionized precise manipulation at molecular and cellular levels in recent years. Bacteria, with adaptability and genetic tractability, are promising candidates for light control studies. This review investigates the mechanisms underlying light activation in bacteria and discusses recent advancements focusing on light control methods and techniques for controlling bacteria. We delve into the mechanisms by which bacteria sense and transduce light signals, including engineered photoreceptors and light-sensitive actuators, and various strategies employed to modulate gene expression, protein function, and bacterial motility. Furthermore, we highlight recent developments in light-integrated methods of controlling microbial responses, such as upconversion nanoparticles and optical tweezers, which can enhance the spatial and temporal control of bacteria and open new horizons for biomedical applications.