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Hybridization chain reaction (HCR) and DNA‐templated silver nanoclusters (AgNCs) have emerged as powerful tools in biosensing. HCR enables cascade amplification through programmable DNA interactions, while DNA‐AgNCs serve as transducing units with unique fluorogenic and electrochemical properties. Integrating these components into a hybrid sensor could significantly enhance sensing capabilities across various fields. Nonetheless, limited studies and the lack of systematic guidelines for HCR‐AgNCs systems have hindered research progress, despite their potential. This review aims to address this gap by providing a comprehensive overview of HCR‐AgNCs biosensors, facilitating further innovation in this field. The working principles, performance factors, and complementary features are discussed. Thereafter, reported HCR‐AgNCs studies are assessed, emphasizing their distinct sensing mechanisms (e.g., fluorogenic, electrochemical), applications across various fields, and challenges in adopting the hybrid sensors. Drawing from the experience developing multiple HCR‐AgNCs sensors, insights and guidelines for designing and developing HCR‐AgNCs systems are provided for future researchers. Finally, prospective directions in HCR‐AgNCs research, including multiplex assays and integration with emerging technologies, are explored to guide future advancements. The synergistic combination of HCR and AgNCs as a hybrid biosensor holds promise for addressing pressing challenges in healthcare, environmental monitoring, and beyond, paving the way for next‐generation biosensing technologies. [ABSTRACT FROM AUTHOR]

The p53 gene is an important tumour suppressor gene, which has an important impact on the early diagnosis of cancer. Here, inspired by the fact that single-stranded DNA (ss-DNA) can be nonspecifically adsorbed on the surface of nanogold, we report on a sensitive and cost-effective new method to detect the p53 gene by combining hybridization chain reaction (HCR) with gold nanoparticles. The long double helix structure generated by HCR could not be adsorbed on the nanogold surface after the addition of the p53 gene. When an appropriate amount of Na+ is introduced into the solution, the nanogold aggregates and the color of the solution changes from red to blueviolet. The sensor has a high sensitivity with a detection limit of 2 nM visible to the naked eye and a quantitative detection limit of 0.2 nM using a UV-visible spectrophotometer. Notably, by combining fluorescence spectroscopy and gel electrophoresis, the science and specificity of p53 gene-induced HCR is systematically validated. The use of an enzyme-free, label-free colorimetric method to detect the p53 gene greatly reduces the complexity and cost of the experiment. This study has a broad market application prospect and provides a new method for early mass screening of cancer genes. [ABSTRACT FROM AUTHOR]

The association between microRNA (miRNA) and various diseases has been established; miRNAs have the potential to be biomarkers for these diseases. Nevertheless, the challenge of correctly quantifying an miRNA arises from its low abundance and a high degree of family homology. Therefore, in the present study, we devised a chemiluminescence (CL) detection method for miRNAs, known as the hybridization chain reaction (HCR)-CL, utilizing the enzyme-free signal amplification technology of HCR. The proposed methodology obviates the need for temperature conversion and offers a straightforward procedure owing to the absence of enzymatic participation, and the lumino-H2O2-mediated CL reaction occurs at a high rate. The technique successfully detected 2.5 amol of the target analyte and 50 amol of miR-146b in a 1% concentration of human serum. In summary, the method developed in this study is characterized by its ease of operation, cost-effectiveness, remarkable analytical prowess, and ability to detect miRNA without the need for total RNA extraction from serum samples. This method is expected to be widely used for biological sample testing in clinical settings. [ABSTRACT FROM AUTHOR]

An advanced electrochemiluminescence (ECL) biosensor was developed that integrates T7 RNA polymerase amplification, ladder-branch hybridization chain reaction (HCR), and the precise targeting capabilities of CRISPR/Cas13a technology. The novelty of this research lies in the unique combination of these three cutting-edge technologies, which has not been previously utilized together in biosensing platforms, enabling highly sensitive and specific detection of biomolecules with exceptional precision. This innovative biosensor addresses the critical need for sensitive and specific detection of matrix metalloproteinase-2 (MMP-2), a key biomarker in cancer diagnostics. Through meticulous optimization of amplification and reaction conditions, the biosensor demonstrated remarkable sensitivity and specificity, achieving a detection limit as low as 6.34 aM, surpassing existing methodologies. The biosensor also exhibited excellent stability and reproducibility across multiple scans and maintained consistent functionality over an extended period, highlighting its reliability for practical applications. The effectiveness of the biosensor was validated using real samples, demonstrating its capability to accurately quantify MMP-2 in complex biological matrices with high recovery and minimal interference. The integration of isothermal amplification and CRISPR/Cas13a within the ECL biosensor platform represents a significant advancement in molecular diagnostics, offering a powerful tool for real-time monitoring of MMP-2. This combination of technologies sets the platform apart from traditional methods, marking a significant step forward in biosensor innovation. This biosensor holds substantial promise for revolutionizing cancer diagnostics and facilitating personalized treatment strategies, ultimately aiming to improve patient outcomes in cancer care. Future research may explore further enhancements and applications of this biosensor in various clinical settings. [ABSTRACT FROM AUTHOR]

Accurate signal amplification in living cells is highly important in biomedical research and medical diagnostics. Benefiting from its enzyme-free, efficient isothermal signal amplification ability, hybridization chain reaction (HCR) plays an important role in intracellular signal amplification; however, HCR fails the accurate signal amplification in the situation when the properties of some biological targets and analogues are too similar. Particularly, their signal amplification accuracy for mature miRNAs is unsatisfactory due to the signal interference of precursor microRNAs (abbreviated as pre-miRNAs), which also contain the sequence of mature miRNAs. Herein, we develop the first example of size-selective hybridization chain reaction probe for accurate signal amplification, which achieved accurate and sensitive biosensing of mature miRNAs in living cancer cells. Our probe, termed as qTcage, consists of a DNA nanocage for size-selective responsive to mature miRNAs, as well as a quadrivalent tetrahedral DNA structure for HCR signal amplification. Benefiting from the size-selectivity of DNA nanocage, shorter mature miRNAs (19–23 nt) rather than longer pre-miRNAs (60–70 nt) could enter the cavity to release triggers strand, which activates HCR reaction for fluorescence signal recovery. The probe efficiently reduces signal interference of pre-miRNAs and improves the imaging sensitivity for intracellular mature miRNAs, which was successfully applied for mature miRNAs imaging during drug treatment. Overall, this strategy provides the hybridization chain reaction with the feature of size-selective ability, which holds promise for further accurate signal amplification in biological processes study and clinical diagnostics. [ABSTRACT FROM AUTHOR]

Live‐cell imaging of RNA in specific subcellular compartments is essential for elucidating the rich repertoire of cellular functions, but it has been limited by a lack of simple, precisely controlled methods. Here such an approach is presented via the combination of hybridization chain reaction and spatially restricted enzymatic activation with organelle‐targeted delivery. The system can localize engineered DNA hairpins in the mitochondria, where target RNA‐initiated chain reaction of hybridization events is selectively activated by a specific enzyme, enabling amplified RNA imaging with high precision. It is demonstrated that the approach is compatible with live cell visualization and enables the regulatable imaging of microRNA in mitochondria. Since in situ activation of the signal amplification with enzyme eliminates the need for genetically encoded protein overexpression, it is envisioned that this simple platform will be broadly applicable for precise RNA imaging with subcellular resolution in a variety of biological processes. [ABSTRACT FROM AUTHOR]

In the field of neonatal infections nursing, methicillin-resistant Staphylococcus aureus (MRSA) is a major bacterial pathogen. Here, we present a portable biosensor for MRSA detection that is both highly sensitive and portable, owing to its implementation on the personal glucose meter (PGM) platform. The H probe was fixed on the magnetic bead for mecA gene analysis. A blunt 3′ terminus appeared in the MBs-H probe when the mecA gene was present. Exonuclease-III (Exo-III) recognized the blunt terminus and cleaved it, freeing the mecA gene and so facilitating target recycling. In the meantime, the remaining H probe-initiated hybridization chain reaction (HCR) led to the desired signal amplification. Portable quantitative detection of mecA gene is possible because PGM can read the quantity of invertase tagged on HCR product. After optimizing several experimental parameters, such as the concentration of Exo-III and incubation time, the constructed sensor is extremely sensitive, with a detection limit of 2 CFU/mL. The results from this sensitive PGM-based sensor are in agreement with those obtained from plate counting methods, suggesting that it can be used to accurately assess the MRSA content in artificial clinical samples. In addition, the PGM sensor can significantly cut down on time spent compared to plate counting techniques. The manufactured sensor provides a promising option for accurate identification of pathogenic bacteria. [ABSTRACT FROM AUTHOR]

In recent years, microRNAs have been used as cancer markers in clinical testing. In this study, an electrochemical biosensor was developed based on nanomaterials and the hybridization chain reaction (HCR) for highly sensitive and selective detection of the target miR-378 using K3Fe(CN)6/K4Fe(CN)6 as a redox indicator. MiR-378 is located on human chromosome 5q32 and serves as an important gene regulatory locus. Recent studies have found that aberrant expression of miR-378 is associated with cervical cancer, breast cancer, lung cancer, and other diseases. HCR is a simple and efficient isothermal amplification technique that does not require enzyme mediation and can be performed at room temperature. Evaluations of the specificity, stability, and sensitivity of the developed sensor revealed its ability to rapidly and specifically detect miR-378 in two hours with a detection limit as low as 100 cells in a volume of 400 µL, thereby indicating that this device could provide a novel approach for clinical diagnosis. [ABSTRACT FROM AUTHOR]

Circulating microRNAs (miRNAs) play a pivotal role in the occurrence and development of acute myocardial infarction (AMI), and precise detection of them holds significant clinical implications. The development of luminol-based luminophores in the field of electrochemiluminescence (ECL) for miRNA detection has been significant, while their effectiveness is hindered by the instability of co-reactant hydrogen peroxide (H2O2). In this work, an iron single-atom catalyst (Fe-PNC) was employed for catalyzing the luminol-O2 ECL system to achieve ultra-sensitive detection of myocardial miRNA. Target miRNA triggers a hybridization chain reaction (HCR), resulting in the generation of a DNA product featuring multiple sticky ends that facilitate the attachment of Fe-PNC probes to the electrode surface. The Fe-PNC catalyst exhibits high promise and efficiency for the oxygen reduction reaction (ORR) in electrochemical energy conversion systems. The resulting ECL biosensor allowed ultrasensitive detection of myocardial miRNA with a low detection limit of 0.42 fM and a wide linear range from 1 fM to 1.0 nM. Additionally, it demonstrates exceptional performance when evaluated using serum samples collected from patients with AMI. This work expands the application of single-atom catalysis in ECL sensing and introduces novel perspectives for utilizing ECL in disease diagnosis. [ABSTRACT FROM AUTHOR]

The sensitive detection of cancer biomarkers is crucial for early accurate diagnostics and therapy of cancer patients. Carcinoembryonic antigen (CEA) is a tumor‐associated antigen derived from colon cancer and embryonic tissues. In this study, we have developed a label‐free fluorescence biosensing platform for the quantification of CEA with the "turn‐on" signal output. This platform employs a label‐free strategy that incorporates an aptamer‐modified gold nanoparticle (Apt@AuNP) probe for the recognition of CEA, in combination with hybridization chain reaction (HCR) amplification. In the presence of target CEA, Apt@AuNPs selectively capture CEA, resulting in a reduction of subsequent complementary chains (CP) binding on Apt@AuNPs. The remaining CP, acting as the initiator sequence for HCR, triggers the HCR, leading to the formation of abundant G‐quadruplex structures. By employing Thioflavin T (ThT) for the formation of G‐quadruplex/ThT complexes, the biosensor exhibits a significant enhancement of the fluorescence signal. Under optimized conditions, the biosensor platform demonstrates a limit of detection of 0.03 nM and a linear range from 0.1 to 2.5 nM. Additionally, the specificity investigation reveals the high selectivity of this fluorescent biosensor. Finally, the performance of this method has been validated by successfully detecting CEA in real‐life samples, highlighting its potential for clinical applications. [ABSTRACT FROM AUTHOR]

Tandem repeat (TR) proteins are abundant in nature and show diverse biochemical and mechanical properties. However, these proteins can be difficult to synthesize in a precise way because of their repetitive nature. Using a supramolecular polymerization scheme known as hybridization chain reaction, we show how to generate the DNA sequences encoding such TR proteins. The polymerization mechanism allows for identical "monomers" to selectively create TR proteins of different molecular weight. We demonstrate this strategy with examples from elastin‐like polypeptides (ELPs) and mussel foot proteins (mfps). Moreover, the scheme can be adapted to create protein libraries through the use of multiple DNA hairpin "monomers." [ABSTRACT FROM AUTHOR]

Detecting RNA molecules within their natural environment inside intact arthropods has long been challenging, particularly in small organisms covered by a tanned and pigmented cuticle. Here, we have developed a methodology that enables high‐resolution analysis of the spatial distribution of transcripts of interest without having to dissect tiny organs or tissues, thereby preserving their integrity. We have combined an in situ amplification approach based on hybridization chain reaction, which enhances the signal‐to‐noise ratio, and a clearing approach that allows the visualization of inner organs beneath the cuticle. We have implemented this methodology for the first time in Hemiptera, mapping two salivary aphid (Acyrthosiphon pisum) transcripts, the effector c002 and the salivary sheath protein SHP. With a multiplex approach, we could simultaneously detect different mRNAs in mounted pea aphid head‐thorax samples and show that they were distributed in distinct secretory cells of salivary glands. Research Highlights: Combining hybridisation chain reaction and clearing allows the detection of transcripts in intact aphids heads.The transcripts of the two salivary proteins c002 and SHP are compartmentalized in distinct secretory cells of the principal glands. [ABSTRACT FROM AUTHOR]

An ultrasensitive electrochemical biosensor to detect trace Hg2+ in environmental samples was developed utilizing nanogold-decorated magnetic reduced graphene oxide (MrGO-AuNPs), exonuclease III-assisted target cycle (Exo III-ATC) and hybridization chain reaction (HCR) synergistic triple signal amplification. The MrGO-AuNPs is a superior carrier for capture DNA (cDNA) and acts as magnetic media for automatic separation and adsorption. This innovative utilization of the magnetism and improved sensing efficiency obviates the need for direct modification and repeated polishing of the working electrode. Additionally, the three DNA hairpins (cDNA, methylene blue (MB) labeled HP1 and HP2) further contribute to biosensor specificity and selectivity. When cDNA captures Hg2+, it activates Exo III-ATC due to the formation of a sticky end in the cDNA stem via thymine-Hg2+-thymidine (T-Hg2+-T), this leads to the hydrolysis of self-folded cDNA by Exo III-ATC to form "key" DNA (kDNA). The kDNA subsequently initiates HCR, resulting in massive super-sandwich structures (kDNA-[HP1/HP2]n carrying signaling molecules on MrGO-AuNPs, and this overall structure serves as a signal probe (SP). Leveraging magnetic adsorption, the SP was automatically adsorbed onto the magneto-glass carbon electrode (MGCE), generating an amplified signal. This biosensor's detection limit (LOD) was 3.14 pmol/L, far below the limit of 10 nmol/L for mercury in drinking water set by the US EPA. The biosensor also showed excellent selectivity when challenged by interfering ions, and the results of its application in actual samples indicate that it has good potential for practical applications in environmental monitoring. [ABSTRACT FROM AUTHOR]

Monitoring neuronal activity at single-cell resolution in freely moving Drosophila engaged in social behaviors is challenging because of their small size and lack of transparency. Extant methods, such as Flyception, are highly invasive. Whole-brain calcium imaging in head-fixed, walking flies is feasible but the animals cannot perform the consummatory phases of social behaviors like aggression or mating under these conditions. This has left open the fundamental question of whether neurons identified as functionally important for such behaviors using loss- or gain-of-function screens are actually active during the natural performance of such behaviors, and if so during which phase(s). Here, we perform brain-wide mapping of active cells expressing the Immediate Early Gene hr38 using a high-sensitivity/low background fluorescence in situ hybridization (FISH) amplification method called HCR-3.0. Using double-labeling for hr38 mRNA and for GFP, we describe the activity of several classes of aggression-promoting neurons during courtship and aggression, including P1a cells, an intensively studied population of male-specific interneurons. Using HI-FISH in combination with optogenetic activation of aggression-promoting neurons (opto-HI-FISH), we identify candidate downstream functional targets of these cells in a brain-wide, unbiased manner. Finally, we compare the activity of P1a neurons during sequential performance of courtship and aggression, using intronic vs. exonic hr38 probes to differentiate newly synthesized nuclear transcripts from cytoplasmic transcripts synthesized at an earlier time. These data provide evidence suggesting that different subsets of P1a neurons may be active during courtship vs. aggression. HI-FISH and associated methods may help to fill an important lacuna in the armamentarium of tools for neural circuit analysis in Drosophila.

A fluorogenic aptamer (FA)-based hybridization chain reaction (HCR) could provide a sensitive and label-free signal amplification method for imaging molecules in living cells. However, existing FA-HCR methods usually face some problems, such as a complicated design and significant background leakage, which greatly limit their application. Herein, we developed an FA-centered HCR (FAC-HCR) method based on a remote toehold-mediated strand displacement reaction. Compared to traditional HCRs mediated by four hairpin probes (HPs) and two HPs, the FAC-HCR displayed significantly decreased background leakage and improved sensitivity. Furthermore, the FAC-HCR was used to test a non-nucleic acid target, apurinic/apyrimidinic endonuclease 1 (APE1), an important BER-involved endonuclease. The fluorescence analysis results confirmed that FAC-HCR can reach a detection limit of 0.1174 U/mL. By using the two HPs for FAC-HCR with polyetherimide-based nanoparticles, the activity of APE1 in living cells can be imaged. In summary, this study could provide a new idea to design an FA-based HCR and improve the performance of HCRs in live cell imaging. [ABSTRACT FROM AUTHOR]

The ultrasensitive detection of hepatitis C virus (HCV) nucleic acid is crucial for the early diagnosis of hepatitis C. In this study, by combining Ag@Au core/shell nanoparticle (Ag@AuNP)-based surface-enhanced Raman scattering (SERS) tag with hybridization chain reaction (HCR), a novel SERS-sensing method was developed for the ultrasensitive detection of HCV nucleic acid. This SERS-sensing system comprised two different SERS tags, which were constructed by modifying Ag@AuNP with a Raman reporter molecule of 4-ethynylbezaldehyde, two different hairpin-structured HCR sequences (H1 or H2), and a detection plate prepared by immobilizing a capture DNA sequence onto the Ag@AuNP layer surface of the detection wells. When the target nucleic acid was present, the two SERS tags were captured on the surface of the Ag@AuNP-coated detection well to generate many "hot spots" through HCR, forming a strong SERS signal and realizing the ultrasensitive detection of the target HCV nucleic acid. The limit of detection of the SERS-sensing method for HCV nucleic acid was 0.47 fM, and the linear range was from 1 to 105 fM. [ABSTRACT FROM AUTHOR]

Conventional spherical nucleic acid enzymes (SNAzymes), made with gold nanoparticle (AuNPs) cores and DNA shells, are widely applied in bioanalysis owing to their excellent physicochemical properties. Albeit important, the crowded catalytic units (such as G-quadruplex, G4) on the limited AuNPs surface inevitably influence their catalytic activities. Herin, a hybridization chain reaction (HCR) is employed as a means to expand the quantity and spaces of G4 enzymes for their catalytic ability enhancement. Through systematic investigations, we found that when an incomplete G4 sequence was linked at the sticky ends of the hairpins with split modes (3:1 and 2:2), this would significantly decrease the HCR hybridization capability due to increased steric hindrance. In contrast, the HCR hybridization capability was remarkably enhanced after the complete G4 sequence was directly modified at the non-sticky end of the hairpins, ascribed to the steric hindrance avoided. Accordingly, the improved SNAzymes using HCR were applied for the determination of AFB1 in food samples as a proof-of-concept, which exhibited outstanding performance (detection limit, 0.08 ng/mL). Importantly, our strategy provided a new insight for the catalytic activity improvement in SNAzymes using G4 as a signaling molecule. [ABSTRACT FROM AUTHOR]

Although immobilization-free and label-free electrochemical DNA (E-DNA) biosensors have engaged tremendous interest due to their superior properties, such as easy operation, time-saving and cost-saving, most of them are fabricated in homogeneous modes and usually produce high background current. In the present work, we proposed a new immobilization-free and label-free heterogeneous E-DNA assay based on a dual-blocker-aided multibranched hybridization chain reaction (HCR) for one-pot nucleic acid detection with zero background. The target nucleic acid triggers the HCR involving cascaded hybridization between two metastable hairpins, resulting in the generation of HCR products with multibranched arms, which can be captured onto the electrode via π-π stacking interactions between multibranched arms and reduced graphene oxide (rGO). Prior to the incubation process with an electrode, two blockers are designed to prohibit the nonspecific absorption of unreacted hairpin probes. Thus, an immobilization-free and label-free heterogeneous electrochemical assay for one-pot nucleic acid detection with zero background is readily realized. This strategy also presents additional merits of simplicity and cheap cost, since probe immobilization, signal tag labeling, and multiple incubation processes are avoided. Therefore, the as-proposed effective and versatile biosensor has great potential to be applied in nucleic acid-related practical biosensing.

ABSTRACT Microbial gene expression measurements derived from infected organs are invaluable to understand pathogenesis. However, current methods are limited to “bulk” analyses that neglect microbial cell heterogeneity and the lesion's spatial architecture. Here, we report the use of hybridization chain reaction RNA fluorescence in situ hybridization (HCR RNA-FISH) to visualize and quantify Candida albicans transcripts at single-cell resolution in tongues of infected mice. The method is compatible with fixed-frozen and formalin-fixed paraffin-embedded tissues. We document cell-to-cell variation and intriguing spatiotemporal expression patterns for C. albicans mRNAs that encode products implicated in oral candidiasis. The approach provides a spatial dimension to gene expression analyses of host-Candida interactions.IMPORTANCECandida albicans is a fungal pathobiont inhabiting multiple mucosal surfaces of the human body. Immunosuppression, antibiotic-induced microbial dysbiosis, or implanted medical devices can impair mucosal integrity enabling C. albicans to overgrow and disseminate, causing either mucosal diseases such as oropharyngeal candidiasis or life-threatening systemic infections. Profiling fungal genes that are expressed in the infected mucosa or in any other infected organ is paramount to understand pathogenesis. Ideally, these transcript profiling measurements should reveal the expression of any gene at the single-cell level. The resolution typically achieved with current approaches, however, limits most gene expression measurements to cell population averages. The approach described in this report provides a means to dissect fungal gene expression in infected tissues at single-cell resolution.

Abstract Background In the past few years, there has been an explosion in single-cell transcriptomics datasets, yet in vivo confirmation of these datasets is hampered in plants due to lack of robust validation methods. Likewise, modeling of plant development is hampered by paucity of spatial gene expression data. RNA fluorescence in situ hybridization (FISH) enables investigation of gene expression in the context of tissue type. Despite development of FISH methods for plants, easy and reliable whole mount FISH protocols have not yet been reported. Results We adapt a 3-day whole mount RNA-FISH method for plant species based on a combination of prior protocols that employs hybridization chain reaction (HCR), which amplifies the probe signal in an antibody-free manner. Our whole mount HCR RNA-FISH method shows expected spatial signals with low background for gene transcripts with known spatial expression patterns in Arabidopsis inflorescences and monocot roots. It allows simultaneous detection of three transcripts in 3D. We also show that HCR RNA-FISH can be combined with endogenous fluorescent protein detection and with our improved immunohistochemistry (IHC) protocol. Conclusions The whole mount HCR RNA-FISH and IHC methods allow easy investigation of 3D spatial gene expression patterns in entire plant tissues.

Endocrine-disrupting chemicals (EDCs) represent a global threat to human health and the environment. In vertebrates, lipophilic EDCs primarily act by mimicking endogenous hormones, thus interfering with the transcriptional activity of nuclear receptors (NRs). The demonstration of the direct translation of these mechanisms into perturbation of NR-mediated physiological functions in invertebrates, however, has rarely proven successful, as the modes of action of EDCs in vertebrates and invertebrates seem to be distinct. In the present work, we investigated the members of the NR superfamily in a bivalve mollusk, the Mediterranean mussel Mytilus galloprovincialis. In addition to annotating the M. galloprovincialis NR complement, we assessed the potential developmental functions and susceptibility to EDC challenge during early development by gene expression analyses. Our results indicate that a majority of mussel NRs are dynamically expressed during early development, including receptors characterized by a potential susceptibility to EDCs. This study thus indicates that NRs are major regulators of early mussel development and that NR-mediated endocrine disruption in the mussel could be occurring at a larger scale and at earlier stages of the life cycle than previously anticipated. Altogether, these findings will have significant repercussions for our understanding of the stability of natural mussel populations. This article is part of the theme issue 'Endocrine responses to environmental variation: conceptual approaches and recent developments'. [ABSTRACT FROM AUTHOR]

A model organism in developmental biology is defined by its experimental amenability and by resources created for the model system by the scientific community. For the most powerful invertebrate models, the combination of both has already yielded a thorough understanding of developmental processes. However, the number of developmental model systems is still limited, and their phylogenetic distribution heavily biased. Members of one of the largest animal lineages, the Spiralia, for example, have long been neglected. In order to remedy this shortcoming, we have produced a detailed developmental transcriptome for the bivalve mollusk Mytilus galloprovincialis, and have expanded the list of experimental protocols available for this species. Our high-quality transcriptome allowed us to identify transcriptomic signatures of developmental progression and to perform a first comparison with another bivalve mollusk: the Pacific oyster Crassostrea gigas. To allow co-labelling studies, we optimized and combined protocols for immunohistochemistry and hybridization chain reaction to create high-resolution co-expression maps of developmental genes. The resources and protocols described here represent an enormous boost for the establishment of Mytilus galloprovincialis as an alternative model system in developmental biology. [ABSTRACT FROM AUTHOR]

Multicellular eukaryotic organisms are hosts to communities of bacteria that reside on or inside their tissues. Often the eukaryotic members of the system contribute to high proportions of metagenomic sequencing reads, making it challenging to achieve sufficient sequencing depth to evaluate bacterial ecology. Stony corals are one such complex community; however, separation of bacterial from eukaryotic (primarily coral and algal symbiont) cells has so far not been successful. Using a combination of hybridization chain reaction fluorescence in situ hybridization and fluorescence activated cell sorting (HCR-FISH + FACS), we sorted two populations of bacteria from five genotypes of the coral Acropora loripes , targeting (i) Endozoicomonas spp, and (ii) all other bacteria. NovaSeq sequencing resulted in 67–91 M reads per sample, 55%–90% of which were identified as bacterial. Most reads were taxonomically assigned to the key coral-associated family, Endozoicomonadaceae, with Vibrionaceae also abundant. Endozoicomonadaceae were 5x more abundant in the ' Endozoicomonas ' population, highlighting the success of the dual-labelling approach. This method effectively enriched coral samples for bacteria with <1% contamination from host and algal symbionts. The application of this method will allow researchers to decipher the functional potential of coral-associated bacteria. This method can also be adapted to accommodate other host-associated communities. [ABSTRACT FROM AUTHOR]